Genetic Improvement of Processes Yielding Microbial Products

Genetic improvement of processes yielding microbial products
Jose L. Adrio1 & Arnold L. Demain2

1
Department of Biotechnology, Puleva Biotech, S.A., Granada, Spain; and 2Charles A. Dana Institute for Scientists Emeriti, Drew University, Madison, NJ, USA
Correspondence: Jose L. Adrio, Department of Biotechnology, Puleva Biotech, S.A., Camino de Purchil, 66, 18004 Granada, Spain. Tel.:134 958 24 02 27; fax:134 958 24 01 60; e-mail: jladrio@pulevabiotech.es Received 3 March 2005; revised 18 August 2005; accepted 19 August 2005 First published online 17 October 2005. doi:10.1111/j.1574-6976.2005.00009.x Editor: Alexander Boronin Keywords strain improvement; genetic recombination; primary metabolites; secondary metabolites; directed evolution; combinatorial biosynthesis.
Abstract Although microorganisms are extremely good in presenting us with an amazing array of valuable products, they usually produce them only in amounts that they need for their own benet; thus, they tend not to overproduce their metabolites. In strain improvement programs, a strain producing a high titer is usually the desired goal. Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by mutagenesis and screening/selection for higher producing microbial strains and the application of recombinant DNA technology.
Introduction
Microorganisms can generate new genetic characters (genotypes) by two means: mutation and genetic recombination. In mutation, a gene is modied either unintentially (spontaneous mutation) or intentially (induced mutation). Although the change is usually detrimental and eliminated by selection, some mutations are benecial to the microorganism. Even if it is not benecial to the organism, but benecial to humans, the mutation can be detected by screening and can be preserved indenitely. This is indeed what the fermentation microbiologists did in the strain development programs that led to the great expansion of the fermentation industry in the second half of the twentieth century. It was fortunate that the intensive development of microbial genetics began in the 1940s when the fermentative production of penicillin became an international necessity. The early studies in basic genetics concentrated on the production of mutants and their properties. The ease with which permanent characteristics of microorganisms could be changed by mutation and the simplicity of the mutation techniques had tremendous appeal to microbiologists. Thus began the cooperative strain-selection program among workers at the U.S. Department of Agriculture Laboratories in Peoria, the Carnegie Institution, Stanford University and the University of Wisconsin, followed by the extensive individual
FEMS Microbiol Rev 30 (2006) 187214
programs that still exist today in industrial laboratories throughout the world. It is clear that mutation has been the major factor involved in the hundred- to thousand-fold increases obtained in production of microbial metabolites and that the ability to modify genetically a microbial culture to higher productivity has been the most important factor in keeping the fermentation industry in its viable, healthy state.
Applications of mutation
Mutation has improved the productivity of industrial cultures (Vinci & Byng, 1999; Parekh et al., 2000). It has also been used to shift the proportion of metabolites produced in a fermentation broth to a more favorable distribution, elucidate the pathways of secondary metabolism, yield new compounds, and for other functions. The most common method used to obtain high yielding mutants is to treat a population with a mutagenic agent until a certain desired kill is obtained, plate out the survivors and test each resulting colony or a randomly selected group of colonies for product formation in asks. The most useful mutagens include nitrosoguanidine (NTG), 4-nitroquinolone-1oxide, methylmethane sulfonate (MMS), ethylmethane sulfonate (EMS), hydroxylamine (HA) and ultraviolet light (UV). The optimum level of kill for increased production of antibiotics is thought to be in the range 7095% (Simpson &
c
2005 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
188
J. L. Adrio & A. L. Demain
Caten, 1979), although some industrial programs use much higher levels, e.g. up to 99.99%. It is incorrect to condemn a mutation and screening procedure because, on average, it decreases production ability; indeed, this is the case for successful mutagenesis. One should only be interested in the frequency of improved mutants. Although single cells or spores are preferred for mutagenesis, non-spore-forming lamentous organisms have been mutated successfully by mutagenizing mycelia, preparing protoplasts and regenerating on solid medium (Keller, 1983). Sonication is sometimes used to break up Streptomyces mycelia after mutagenesis and before screening for improved mutants (Takebe et al., 1989). Poorly sporulating lamentous organisms can be mutagenized after fragmentation or formation of protoplasts (Kim et al., 1983; Kurzatkowski et al., 1986). More detailed information can be found in several authorative reviews on genetics and especially on mutation in actinomycetes (Baltz, 1986, 1995, 1998, 1999; Hopwood, 1999).
1990). High-producing strains contained an amplied region; a 106-kb region amplied ve to six times as tandem repeats was detected in a high-producing strain, whereas wild-type P. chrysogenum and Flemings original strain of P. notatum contained only a single copy (Fierro et al., 1995). Strain improvement has been the main factor involved in the achievement of impressive titers of industrial metabolites. The production titer of tetracycline as far back as 1979 was reported to be over 20 g L 1 (Podojil et al., 1984), mainly due to strain improvement. Later, titers of 3035 g L1 were reached for chlortetracycline and tetracycline. The production titer of penicillin is 70 g L 1 and that of cephalosporin C over 30 g L 1 (Elander, 2003). The production titer of tylosin has been reported to be over 15 g L1 (Chen et al., 2004) and that of salinomycin is 60 g L 1 (Liu, 1982). Morphological and pigment mutants Although almost nothing is known about the mechanisms causing higher production in superior random or morphological mutants, it is likely that many of these mutations involve regulatory genes, especially as regulatory mutants obtained in basic genetic studies are sometimes found to be altered in colonial morphology. Thus, morphological mutants have been very important in strain improvement. These include mutants affected in mycelia formation, which produce colonies with a modied appearance or a new color. Color changes have also been important for pigment producers (Table 1). Auxotrophic mutants Very early in the development of the concepts of regulation, geneticists realized that the end product of a biosynthetic pathway to a primary metabolite excercises strict control over the amount of an intermediate accumulated by an auxotrophic mutant of that pathway. Only at a growthlimiting concentration of the end product would a large accumulation of the substrate of the decient enzyme occur. This principle of decreasing the concentration of an inhibitory or repressive end product to bypass feedback inhibition or repression was best accomplished by the use of auxotrophic mutants. Indeed, auxotrophic mutation has been a major factor in the industrial production of primary products such as amino acids and nucleotides (Table 1). The production of secondary products such as antibiotics is also markedly affected by auxotrophic mutation, even when auxotrophs are grown in nutritionally complete and even complex media. Although the change in product formation is usually in the negative direction, higher-producing auxotrophs are obtained from producers of antibiotics. When several primary metabolites are produced by a single branched pathway, mutation in one branch of the pathway often leads to overproduction of the product of the
Mutants producing increased quantites of metabolites

Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by mutagenesis and screening for higher producing microbial strains. At least ve different classes of genes control metabolite production (Malik, 1979): (i) structural genes coding for product synthases, (ii) regulatory genes determining the onset and expression of structural genes, (iii) resistance genes determining the resistance of the producer to its own antibiotic, (iv) permeability genes regulating entry, exclusion and excretion of the product, and (v) regulatory genes controlling pathways providing precursors and cofactors. Overproduction of microbial metabolites is effected by (i) increasing precursor pools, (ii) adding, modifying or deleting regulatory genes, (iii) altering promoter, terminator and/or regulatory sequences, (iv) increasing copy number of genes encoding enzymes catalyzing bottleneck reactions, and (v) removing competing unnecessary pathways (Strohl, 2001). It is now over 60 years since the rst superior penicillinproducing mutant, Penicillium chrysogenum X-1612, was isolated afer X-ray mutagenesis. This heralded the beginning of a long and successful relationship between mutational genetics and industrial microbiology (Hersbach et al., 1984). The improvement of penicillin production by conventional strain improvement resulted both from enhanced gene expression and from gene amplication (Barredo et al., 1989; Smith et al., 1989). Increased levels of mRNA corresponding to the three enzymes of penicillin G biosynthesis were found in high-penicillin producing strains of P. chrysogenum as compared to wild-type strains (Smith et al.,
c
189
Table 1. Mutations leading to increased product formation Mutation type Organism Mutant characteristics Wrinkled colonies Bald colonies; then white colonies Reddish-orange colonies with no aerial mycelia Pink colonies on agar containing b-ionone or diphenylamine Pink instead of brown mycelia Guanine auxotrophy Leucine auxotrophy Leucine auxotrophy Non-auxotrophic for aspartate Producing ability Producing ability Resistance to ethionine Resistance to thialysine Resistance to thialysine Resistance to 2-ketobutyrate in presence of valine or isoleucine Resistance to valine hydroxamate Resistance to iron, to tubercidin, to 2-DOG Resistance to itaconic acid and aminomethylphosphinic acid Resistance to phenylacetic acid (precursor) Sequential resistance to tryptophan (feedback inhibitor), phydroxybenzoate, and propionate (precursor) Resistance to aurodox Resistance to nocardicin Resistance to leucomycin Resistance to oxytetracycline Resistance to streptomycin, gentamicin, paromomycin, rifamycin and combinations Overproduced compound Histidine Daunorobicin Beromycins Astaxanthin Teichoplanins 5 0 -Inosinic acid (IMP) and hypoxanthine Bacitracin Cephamycin C and penicillin N Tylosin Chlortetracycline Aurodox Methionine Cephamycins Desferrioxamine Monensins A & B Teichoplanins Riboavin Riboavin Penicillin G Rifamycin B Reference Roth & Ames (1966) Blumauerova et al. (1978) Blumaerova et al. (1980, 1980) Lewis et al. (1990); Chumpolkulwong et al. (1997) Lee et al. (2003) Teshiba & Furuya (1983) Haavik & Froyshov (1982) Godfrey (1973) Lee & Lee (1995) Dulaney & Dulaney (1967) Unowsky & Hoppe (1978) Tani et al. (1988) Mendelovitz & Aharonowitz (1983) Smith (1987) Pospisil et al. (1999) Wang et al. (1996); Jin et al. (2002a) Stahmann et al. (2000) Stahmann et al. (2000) Barrios-Gonzalez et al. (1993) Jin et al. (2002)
1. Morphological and/ 1.1. Salmonella or pigment change typhimurium 1.2. Streptomyces coeruleorubidus 1.3. Streptomyces glomeratus 1.4. Phafa rhodozyma 1.5. Actinoplanes teichomyceticus 2. Auxotrophic 2.1. Brevibactetrium ammoniagenes 2.2. Bacillus licheniformis 2.3. Streptomyces lipmanii 3. Reversion of 3.1. Streptomyces auxotrophy fradiae 4. Reversion of 4.1. Streptomyces non-production viridifaciens 4.2. Streptomyces goldiniensis 5. Antimetabolite 5.1. Candida boidinii resistance 5.2. Streptomyces clavuligerus 5.3. Streptomyces pilosus 5.4. Streptomyces cinnamonensis 5.5. Actinoplanes teichomyceticus 5.6. Candida areri 5.7. Ashbya gossypii 5.8. Penicillium chrysogenum 5.9. Amycolatopsis mediterranei
6. Product resistance
6.1 Streptomyces goldiniensis 6.2. Nocardia uniformis 6.3. Streptomyces kitasatoensis 6.4. Streptomyces rimosus 7. Antibiotic resistance 7.1 Streptomyces coelicolor and Streptomyces lividans
Aurodox Nocardicin Leucomycin Oxytetracycline Actinorhodin
Unowsky & Hoppe (1978) Elander & Aoki (1982) Higashide (1984) Gravius et al. (1994) Hosoya et al. (1998); Hesketh & Ochi (1997); Okamoto et al. (2003); Okamoto-Hosoya et al. 2000; Hu & Ochi (2001)
c
190
Table 1. Continued. Mutation type 8. Reversal of carbon source repression Organism 8.1. Saccharomyces cerevisiae 8.2. Schwanniomyces castelli 8.3. Pichia polymorpha 8.4. Penicillium chrysogenum 8.5 Aspergillus niger 8.6. Aspergillus niger 9.1. Streptomyces aureofaciens 9.2. Streptomyces griseus 10.1. Acremonium chrysogenum 10.2. Streptomyces viridifaciens 10.3. Bacillus subtilis 10.4. Aspergillus nidulans 10.5. Aspergillus niger 10.6. Rhyzopus oryzae 10.7. Streptomyces kasugaensis 10.8. Acremoniums chrysogenum 10.9. Streotomyces hygroscopicus 11.1. Brevibacterium avum 11.2. Brevibacterium ammoniagenes 11.3. Escherichia coli 11.4. Corynebacterium glutamicum Mutant characteristics Resistance to 2-deoxyglucose Resistance to 2-deoxyglucose Resistance to 2-deoxyglucose Resistance to 2-deoxyglucose Rapid growth on high sucrose Resistance to 2-deoxyglucose Small colonies on phosphate-limiting agar Production in excess-phosphate medium Increased clear zone around colony Increased clear zone around colony Increased clear zone around colony Increased clear zone around colony Increased clear zone around colony Increased clear zone around colony Increased clear zone around plugs of agar Increased clear zone around plugs of agar Increased clear zone around plugs of agar Inability to grow on glutamate Increased sensitivity to deoxycholate and lysozyme Elimination of active praline uptake Decrease in tryptophan uptake Overproduced compound Cheese whey hydrolysis Isomaltase, amylase Inulinase Penicillin G Citric acid Citric acid Tetracycline Candicidin Cephalosporin C Chlortetracycline Mycobacillin Penicillin Citric acid Lactate Kasugamycin Cephalosporin C Complex 165 Glutamic acid 5 0 -Inosinic acid Proline Tryptophan
Reference Bailey et al. (1982) McCann & Barnett (1984); Sills et al. (1984) Bajon et al. (1984) Chang et al. (1980) Schreferl-Kunar et al. (1989) Kirimura et al. (1992) Colombo et al. (1981) Martin et al. (1979) Elander (1969) Dulaney & Dulaney (1967) Bannerjee & Bose (1964) Ditchburn et al. (1974) Das & Roy (1981) Longacre et al. (1997) Ichikawa et al. (1971) Chang & Elander (1979) Gesheva (1994) Shiio et al. (1982); Mori & Shiio (1983) Teshiba & Furuya (1983) Rancount et al. (1984) Ikeda & Katsumata (1995)
9. Reversal of phosphate inhibition
10. Increased production on agar
11. Change in permeability
other branch. Examples include the overproduction of phenylalanine by tyrosine auxotrophs and vice versa, and the overproduction of lysine by auxotrophs requiring threonine and methionine. In the case of branched pathways leading to a primary metabolite and a secondary metabolite, auxotrophic mutants requiring the primary metabolite sometimes overproduce the secondary metabolite (Table 1). Reversion of an auxotroph to prototrophy sometimes leads to new prototrophs possessing higher enzyme activity than present in the original grandparent prototroph. Such increased enzyme activity was probably the result of a structural gene mutation producing a more active enzyme or an enzyme less subject to feedback inhibition (Table 1). Revertants of non-producing mutants A high proportion of such mutants has been found to produce increased amounts of antibiotics (Table 1).
c
Antimetabolite-resistant mutants Basic studies on regulation have shown that it is possible to select regulatory mutants, which overproduce the end products of primary pathways, using toxic metabolite analogues. Such antimetabolite-resistant mutants often possess enzymes that are insensitive to feedback inhibition, or enzyme-forming systems resistant to feedback repression. The selection of mutants resistant to toxic analogues of primary metabolites has been widely employed by industrial microbiologists (Table 1). A variation of the antimetabolite selection techniques is possible when a precursor is toxic to the producing organism. The principle here is that the mutant most capable of detoxifying the precursor by incorporating it into the antibiotic will be the best grower in the presence of the precursor (Table 1). When the secondary metabolite
191
produced is itself a growth inhibitor of the producing culture, as in the case of certain antibiotics, the metabolite can sometimes be used to select resistant mutants that are improved producers. Certain streptomycin resistance mutations cause increased production of unrelated antibiotics. In addition to improvement in secondary metabolite formation by mutation to streptomycin resistance, resistance to gentamicin or to paromomycin is even more effective. Furthermore, triple mutation to resistance to streptomycin, gentamicin and rifampicin, each of which individually increased actinorhodin formation, was found to be the most effective (Table 1). Mutants resistant to nutritional repression Nutritional repression can also be decreased by mutation to antimetabolite resistance. Examples of selection agents are 2-deoxyglucose (2-DOG) for enzymes and pathways controlled by carbon source regulation (Table 1), methylammonium for those regulated by nitrogen source repression, and arsenate for phosphate regulation. Mutants that use phosphate less efciently for growth sometimes show improved antibiotic production. Thus, screening for small colonies on phosphate-limiting media could be a useful strain improvement technique for phosphate-regulated products (Table 1). Improved production on agar In many cases, fermentation performance on an agar plate is related to production in submerged liquid culture, and the method has application as a means of detecting superior mutants. So-called zone mutants have proven useful for improving several different processes (Table 1). A widely used modication involves the production of antibiotics by colonies on separate plugs of agar followed by placement of these plugs on a seeded assay plate and measurement of the resultant clear zones. The use of this agar piece method resulted in improvement of antibiotic production (Table 1). Agar-piece screening of antibiotic production in the presence of inhibitory levels of phosphate (15 mM) led to the isolation of six markedly improved and stable Streptomyces hygroscopicus strains producing the macrolide antifungal complex 165 (Gesheva, 1994). Permeability mutants Product excretion in overproducing strains often occurs when uptake and/or catabolism is impaired. Thus, genetic lesions eliminating active uptake can be used to specically enhance excretion of metabolites (Table 1). It is often of benet to isolate mutants unable to grow on the
desired product as sole carbon or energy source. Such mutants are often impaired in their ability to takeup the product and they contain lower intracellular levels of the product, thus lessening feedback regulation. In certain improved mutants, there is an increase in sensitivity to deoxycholate and lysozyme, indicating a change in permeability.
Mutants showing qualitative changes in the mix of fermentation products

As many organisms produce secondary metabolites as mixtures of a chemical family or of several chemical families, mutation has been used to eliminate undesirable products in such fermentations. An example is that of Nakatsukasa and Mabe (Nakatsukasa & Mabe, 1978), who found that streaking out a natural single colony isolate from Streptomyces aureofaciens (producing the polyether narasin and the broad-spectrum antibiotic enteromycin) on galactose led to yellow and white sectoring. The effect was specic for galactose. Of the four colony types obtained, one produced only narasin and two produced only enteromycin. Streptomyces griseus ssp. cryophilus makes four R3 sulfated and four R3 unsulfated carbapenems. The sulfated forms are less active than the unsulfated forms. To completely eliminate the R3 sulfated forms, sulfate transport mutants were obtained. These were of two types: (i) auxotrophs for thiosulfate or cysteine; and (ii) selenate-resistant mutants. Each type produced completely unsulfated forms and titers were equivalent to the total titer of the parent (Kitano et al., 1985). Eight avermectins are produced by Streptomyces avermitilis, of which only a small number are desirable. A nonmethylating mutant produced only four of the compounds and a mutant that failed to make the 25-isopropyl substituent (from valine) produced a different mixture of components. By protoplast fusion, a hybrid strain was obtained which made only two components, B2a and B1a (Omura et al., 1991). Stutzman-Engwall and colleagues (StutzmanEngwall et al., 2003) introduced random mutations by PCR into gene aveC and obtained a mutant that produced an avermectin B1 : B2 ratio of 2.5, much improved over the 0.6 ratio of the parent S. avermitilis strain. Mutation was used to eliminate the undesirable polyketides sulochrin and asterric acid from broths of the lovastatin producer, Aspergillus terreus (Vinci et al., 1991). Mutants have also been employed to eliminate undesirable coproducts from the monensin fermentation (Pospisil et al., 1984).
Mutants producing new antibiotics

Mutant methodology has been used to produce new molecules. The medically useful products demethyltetracycline
c
192
and doxorubicin were discovered by simple mutation of the cultures producing tetracycline and daunorubicin, respectively. Later, the technique of mutational biosynthesis (= mutasynthesis) was devised (Shier et al., 1969). In this process, a mutant blocked in secondary metabolism is fed analogs of the moiety whose biosynthesis is blocked. If successful, the mutant (called an idiotroph) produces a new antibiotic derivative (Nagaoka & Demain, 1975). The hybramycins were the rst compounds to be made this way (Shier et al., 1969). Since then, mutational biosynthesis has been used for the discovery of many new secondary metabolites (Lemke & Demain, 1976; Daum & Lemke, 1979; Kitamura et al., 1982). The most well-known is the commercial antihelmintic agent doramectin, the production of which employed a mutant of the avermectin producer S. avermitilis (Cropp et al., 2000). New anthracyclines and aglycones have been isolated from blocked mutants of the daunorubicin and doxorubicin producers (Cassinelli et al., 1980; McGuire et al., 1981). By adding carminomycinone or 13-dihydrocarminomycinone to an idiotroph of Streptomyces galilaeus (the producer of aclacinomycin), the aglycones were glycosylated to form a new trisaccharide anthracycline, trisarubicionol (Yoshimoto et al., 1981). New macrolide antibiotics have been produced from blocked mutants of the tylosin-producer, Streptomyces fradiae (Kirst et al., 1983). Four new hybrid macrolide antibiotics were obtained by feeding erythronolide B to a blocked mutant of the oleandomycin producer, Streptomyces antibioticus (Spagnoli et al., 1983). A blocked-mutant of the mycinamicin producer, Micromonospora polytrota, was fed various rosaramicin precursors and converted them into new rosaramicins (Lee et al., 1983).
Genetic recombination
In contrast to the extensive use of mutation in industry, genetic recombination was not much used at rst, despite early claims of success (Jarai, 1961; Mindlin, 1969), mainly due to the absence or the extremely low frequency of genetic recombination in industrial microorganisms (in streptomycetes, it was usually 106 or even less). Other problems were evident with the b-lactam-producing fungi. Although Aspergillus exhibited sexual and parasexual reproduction, the most commercially interesting genera, Cephalosporium and Penicillium, were the most difcult to work with as they only reproduced parasexually, which rarely resulted in recombination. Recombination was erroneously looked upon as an alternative to mutation instead of a method that would complement mutagenesis programs. The most balanced and efcient strain development strategy would not emphasize one to the exclusion of the other; it would contain both mutagenesis-screening and recombination-screening components. In such a program, strains at different stages of a mutational line, or from lines developed from different ancestors, would be recombined. Such strains would no doubt differ in many genes and by crossing them, genotypes could be generated which would never occur as strictly mutational descendants of either parent. Recombination was also of importance in the mapping of production genes. Studies on the genetic maps of overproducing organisms such as actinomycetes are relatively recent. The model for such investigations was the genetic map of Streptomyces coelicolor (Kieser et al., 1992), which was found to be very similar to those of other Streptomyces species, such as S. bikiniensis, S. olivaceous, S. glaucescens and S. rimosus.
Protoplast fusion
As mentioned above, genetic recombination was virtually ignored in industry, mainly due to the low frequency of recombination. However, use of protoplast fusion changed the situation markedly. After 1980, there was a heightened interest in the application of genetic recombination to the production of important microbial products such as antibiotics. Today, frequencies of recombination have increased to even greater than 101 in some cases (Ryu et al., 1983), and strain improvement programs routinely include protoplast fusion between different mutant lines. The power of recombination was demonstrated by Yoneda (Yoneda, 1980), who recombined individual mutations, each of which increased aamylase production by two- to seven-fold in Bacillus subtilis. A strain constructed by genetic transformation, which contained all ve mutations, produced 250-fold more a-amylase. Recombination is especially useful when combined with conventional mutation programs to solve the problem of sickly organisms produced as a result of accumulated genetic damage over a series of mutagenized generations.
Use of mutants to elucidate biosynthetic pathways

A further use of mutants has been the elucidation of metabolic pathways. This has been exploited for the biosynthesis of tetracyclines (McCormick, 1965), novobiocin (Kominek, 1972), erythromycin (Martin et al., 1966; Martin & Rosenbrook, 1967), neomycin (Pearce et al., 1978), tylosin (Baltz et al., 1983), other aminoglycosides (Penzikova & Levitov M, 1970; Takeda et al., 1978; Fujiwara et al., 1980; Kase et al., 1982; Hanssen & Kirby, 1983), rosaramicin (Vaughn et al., 1982), daunorubicin (McGuire et al., 1981), other anthracyclines (Motamedi et al., 1986; Yue et al., 1986), actinomycin (Troost & Katz, 1979), carbapenems (Nozaki et al., 1984; Kojima et al., 1988), ansamycins (Kibby et al., 1980; Ghisalba et al., 1981), patulin (Gaucher et al., 1981) and phenazines (Byng et al., 1979).
c
193
For example, a cross via protoplast fusion was carried out with strains of Cephalosporium acremonium from a commercial strain improvement program. A low-titer, rapidly-growing, spore-forming strain which required methionine to optimally produce cephalosporin C was crossed with a high-titer, slow-growing, asporogenous strain which could use the less expensive inorganic sulfate. The progeny included a recombinant which grew rapidly, sporulated, produced cephalosporin C from sulfate and made 40% more antibiotic than the high-titer parent (Hamlyn & Ball, 1979). Protoplast fusion was used to modify the characteristics of an improved penicillin-producing strain of P. chrysogenum which showed poor sporulation and poor seed growth. Backcrossing with a low-producing (12 g L1) strain yielded a high-producing (18 g L 1) strain with better sporulation and better growth in seed medium (Lein, 1986). Interspecic protoplast fusion between the osmotolerant Saccharomyces mellis and the highly fermentative S. cerevisiae yielded stable hybrids fermenting high concentrations of glucose (49% w w1) (Legmann & Margalith, 1983). Another application of protoplast fusion is the recombination of improved producers from a single mutagenesis treatment. By recombination, one could combine the yieldincrease mutations and obtain an even more superior producer before carrying out further mutagenesis. Two improved cephamycin-C producing strains from Nocardia were fused and among the recombinants were two cultures that produced 1015% more antibiotic than the best parent (Wesseling & Lago, 1981). Genetic recombination allows the discovery of new antibiotics by fusing producers of different or even the same antibiotics. A recombinant obtained from two different rifamycin-producing strains of Nocardia mediterranei produced two new rifamycins (16,17-dihydrorifamycin S and 16,17-dihydro-17-hydroxy-rifamycin S) (Traxler et al., 1982). However, according to Hopwood (Hopwood, 1983), these examples may reect the different expression of genes from parent A in the cytoplasm of parent B, rather than the formation of hybrid antibiotics. Interspecic protoplast fusion between S. griseus and ve other species (Streptomyces cyaneus, Streptomyces exfoliatus, Streptomyces griseoruber, Streptomyces purpureus and Streptomyces rochei) yielded recombinants of which 60% produced no antibiotics and 24% produced antibiotics different from the parent strains (Okanishi et al., 1996). New antibiotics can also be created by changing the order of the genes of an individual pathway in its native host (Hershberger, 1996). A new antibiotic, indolizomycin, was produced by protoplast fusion between non-antibiotic producing mutants of Streptomyces griseus and Streptomyces tenjimariensis (Gomi et al., 1984). Osmotolerance of food yeasts such as Saccharomyces cerevisiae and S. diastaticus was increased by protoplast fusion with osmotolerant yeasts. Other traits transferred between yeasts by protoplast fusion include
occulation (Panchal et al., 1982), lactose utilization (Farahnak et al., 1986), the killer character (Bortol et al., 1986; Farris et al., 1992), cellobiose fermentation (Pina et al., 1986) and methionine overproduction (Brigidi et al., 1988).
Plasmids, transposons, cosmids and phage

Plasmid DNA has been detected in virtually all antibioticproducing species of Streptomyces. Some are sex plasmids and constitute an essential part of the sexual recombination process and others contain either structural genes or genes somehow inuencing the expression of the chromosomal structural genes of antibiotic biosynthesis. Most plasmids have no apparent effect on metabolite production and only very few antibiotic biosynthesis processes are encoded by plasmid-borne genes. However, the production of methylenomycin A is encoded by genes present on plasmid SCP1 in Streptomyces coelicolor. When the plasmid was transferred to other streptomycetes, the recipients produced the antibiotic. For many years, plasmid SCP1 was never observed or isolated as a circular DNA molecule, because it was a giant linear plasmid. It was initially difcult to separate such giant linear plasmids from chromosomal DNA but this was later accomplished by pulsed eld gel electrophoresis or orthogonal eld alteration gel electrophoresis (OFAGE) (Kinashi & Shimaji, 1987). Some products of unicellular bacteria are plasmid-encoded. These include aerobactin, a hydroxamate siderophore and virulence factor produced by Escherichia coli (McDougall & Neilands, 1984) and other Gram-negative bacteria (Enterobacter aerogenes, Enterobacter cloacae, Vibrio mimicus, and species of Klebsiella, Salmonella and Shigella). Aerobactin is synthesized by a plasmid-borne ve-gene cluster, which is negatively regulated by iron (Roberts et al., 1986); it can also be produced via chromosomal genes (Moon et al., 2004). It also appears that siderophore production by Arizona hinshawii is plasmid-encoded. A microcin, an antimetabolite of methionine, which is produced by E. coli and acts as a competitive inhibitor of homoserine-O-transuccinylase, is encoded by a plasmid that occurs at 20 copies per genome equivalent (Perez-Diaz & Clowes, 1980). The gene coding for the parasporal crystal body (d-endotoxin) of Bacillus thuringiensis is plasmid-borne (Whiteley & Schnepf, 1986; De Maagd et al., 2003) in most species but is on the chromosome in a few other species. Instability in Streptomyces is brought about by environmentally stimulated macrolesions, e.g. deletions, transpositions, rearrangements and DNA amplication. They occur spontaneously or are induced by environmental stresses such as intercalating dyes, protoplast formation and regeneration, and interspecic protoplast fusion. Streptomycetes are the only prokaryotes known to be subject to spontaneous DNA amplication, sometimes amounting to several
c
194
hundred tandem copies, accounting for over 10% of total DNA, in the absence of selection. Amplication seems to be coupled to DNA deletion and may involve insertion sequence (IS)-like elements (Baltz, 1986). Ethidium bromide cures plasmids in streptomycetes but also increases the frequency of deletion mutations, especially in areas of the chromosome that are already unstable (Crameri et al., 1986). Transposable elements, DNA sequences encoding a transposase enzyme (Berg & Berg, 1983) that move from one replicon to another without host recombination functions or extensive homology with the site of integration, have been extremely useful for the following reasons: (i) they usually provide stable, nonreverting mutants; (ii) they can be used to determine the order of genes in an operon; (iii) it is easy to select for mutants because transposons contain antibiotic- or mercury-resistance markers; (iv) they provide portable regions of homology for chromosomal mobilization; (v) they provide markers for non-selectable genes and allow the cloning of such genes which can then be used as hybridization probes to sh out the wild-type gene from a genomic library; and (vi) they often have unique restriction sites, and thus are good markers for isolating dened deletion derivatives or locating the precise position of a gene by heteroduplex mapping. In the daptomycin producer Streptomyces roseosporus, some Tn 5099 transposition mutants produced 5766% more daptamycin than the parent, whereas others produced less or the same (McHenney & Baltz, 1996; Baltz et al., 1997). Transposition increased the rate-limiting step of tylosin biosynthesis in Streptomyces fradiae, i.e. the conversion of macrocin to tylosin. Transposing a second copy of tylF into a neutral site on the S. fradiae chromosome increased its gene product, macrocin O-methyltransferase, and tylosin production, while decreasing the concentration of the nal intermediate (macrocin). Tylosin production was increased by up to 60% and the total macrolide titer was unchanged (Solenberg et al., 1996). Transposon mutagenesis eliminated the production of the toxic oligomycin by the avermectinproducing Streptomyces avermitilis (Ikeda et al., 1993). Cloning a 34-kb fragment from Streptomyces rimosus via a cosmid into Streptomyces lividans and Streptomyces albus resulted in oxytetracycline production by the recipients (Binnie et al., 1989). Contrary to earlier reports, all the oxytetracycline genes were clustered together on the S. rimosus chromosomal map (Butler et al., 1989).
Improvement of microbial processes by genetic engineering

Primary metabolites
New processes for the production of amino acids and vitamins have been developed by recombinant DNA tech c
nology. Escherichia coli strains were constructed with plasmids bearing amino acid biosynthetic operons. Plasmid transformation was accomplished in Corynebacterium, Brevibacterium and Serratia and, as a result, recombinant DNA technology has been used routinely to improve such commercial amino acid-producing strains (Sahm et al., 2000). A recombinant strain of E. coli (made by mutating to isoleucine auxotrophy, cloning in extra copies of the thrABC operon, inactivating the threonine-degrading gene tdh, and mutating to resistance to high concentrations of L-threonine and L-homoserine) produced 80 g L1 L-threonine in 1.5 days at a yield of 50% (Eggeling & Sahm, 1999). Cloning extra copies of threonine export genes into E. coli led to increased threonine production (Kruse et al., 2002). The introduction of the proline 4-hydroxylase gene from Dactylosporangium sp. into a recombinant strain of E. coli producing L-proline at 1.2 g L1 lead to a new strain producing 25 g L 1 of hydroxyproline (trans-4-hydroxy-L-proline) (Shibasaki et al., 2000). When proline was added, hydroxyproline reached 41 g L1, with a yield of 87% from proline. An engineered strain of Corynebacterium glutamicum producing 50 g L1 of L-tryptophan was further modified by cloning in additional copies of its own transketolase gene to increase the level of the erythrose-4-phosphate precursor of aromatic biosynthesis (Ikeda & Katsumata, 1999). A low copy number plasmid increased production to 58 g L1, whereas a high copy number plasmid decreased production. L-Valine production by mutant strain VAL1 of C. glutamicum amounted to 105 g L1 (Radmacher et al., 2002; Lange et al., 2003). The mutant was constructed by overexpressing biosynthetic enzymes via a plasmid, eliminating ilvA encoding threonine dehydratase, and deleting two genes encoding enzymes of pantothenate biosynthesis. The culture was grown with limitation of isoleucine and pantothenate. By introduction of feedback-resistant threonine dehydratases and additional copies of genes encoding branched amino and biosynthetic enzymes, lysine- or threonineproducing strains were converted into L-isoleucine producers with titers up to 10 g L1 (Morbach et al., 1996; Guillouet et al., 1999; Hashiguchi et al., 1999). Amplication of the wild-type threonine dehydratase gene ilvA in a threonine-producing strain of Corynebacterium lactofermentum led to 15 g L1 of isoleucine overproduction (Colon et al., 1995). Biotin has been made traditionally by chemical synthesis but recombinant microbes have approached a competitive economic position. The cloning of a biotin operon (bioABFCD) on a multicopy plasmid allowed E. coli to produce 10 000 times more biotin than did the wild-type strain (Levy-Schil et al., 1993). Sequential mutation of Serratia marcescens to resistance to the biotin antimetabolite acidomycin (= actithiazic acid) led to mutant strain SB412,
195
which produced 20 mg L1 biotin (Sakurai et al., 1994). Further improvements were made by mutating selected strains to ethionine-resistance (strain ET2, 25 mg L1), then mutating ET2 to S-2-aminoethylcysteine resistance (strain ETA23, 33 mg L1) and nally cloning in the resistant bio operon (Sakurai et al., 1994) yielding a strain able to produce 500 mg L1 in fed-batch fermentor culture along with 600 mg L1 of biotin vitamers. Later advances led to production by recombinant S. marcescens of 600 mg L1 of biotin (Masuda et al., 1995). A process for riboavin production in Corynebacterium ammoniagenes (previously Brevibacterium ammoniagenes) was developed by cloning and overexpressing the organisms own riboavin biosynthesis genes (Koizumi et al., 2000) and its own promoter sequences. The resulting culture produced 15.3 g L1 riboavin in 3 days. Genetic engineering of a Bacillus subtilis strain already containing purine analogresistance mutations led to the improved production of riboavin (Perkins & Pero, 1993). An industrial strain of B. subtilis was produced by inserting multiple copies of the rib operon at two different sites in the chromosome, making purine analog-resistance mutations to increase guanosine triphosphate (GTP; a precursor) production and a riboavin analog (roseavin)-resistance mutation in ribC that deregulated the entire pathway (Perkins et al., 1999). Vitamin C (ascorbic acid) has traditionally been made in a ve-step predominantlychemical process by rst converting glucose to 2-keto-L-gulonic acid (2-KGA) with a yield of 50% and then converting the 2-KGA by acid or base to ascorbic acid. A novel process for vitamin C synthesis involved the use of a genetically engineered Erwinia herbicola strain containing a gene from Corynebacterium sp. The engineered organism converted glucose into 1 g L1 of 2KGA (Anderson et al., 1985; Pramik, 1986). A better process was devised independently, which converted 40 g L1 glucose into 20 g L1 2-KGA (Grindley et al., 1988). This process involved cloning and expressing the gene encoding 2,5-diketo-D-gluconate reductase from Corynebacterium sp. into Erwinia citreus. Another process uses a recombinant strain of Gluconobacter oxydans containing genes encoding L-sorbose dehydrogenase and L-sorbosone dehydrogenase from G. oxydans T-100. The new strain was an improved producer of 2-KGA (Saito et al., 1997). Further mutation to suppress the L-idonate pathway and to improve the promoter led to the production of 130 g L1 of 2-KGA from 150 g L1 sorbitol. Carotenoids were overproduced by introducing carotenoid gene clusters from Erwinia uredovora into E. coli and overexpressing E. coli deoxyxylulose phosphate synthase, the key enzyme of the non-mevalonate isoprenoid biosynthetic pathway (Matthews & Wurtzel, 2000). Lycopene accumulated to 1.3 mg g1 dry cell weight and zeaxanthin to 0.6 mg g1.
Cloning of aldehyde dehydrogenase of Acetobacter polyoxogenes on a plasmid vector into Acetobacter aceti ssp. xylinum increased the rate of acetic acid production by over 100% (1.8 g L1 h 1 to 4 g L 1 h1) and titer by 40% (68 g L1 to 97 g L1) (Fukaya et al., 1989). Genetic engineering of the inosine monophosphate (IMP) dehydrogenase gene in a B. subtilis strain producing 7 g L1 of the desirable guanosine and 19 g L1 of the undesirable inosine changed production to 20 g L 1 guanosine and 5 g L1 inosine (Miyagawa et al., 1986). A recombinant E. coli strain was constructed that produced optically active pure D-lactic acid from glucose at virtually the theoretical maximum yield, e.g. two molecules from one molecule of glucose (Zhou et al., 2003). The organism was engineered by eliminating genes of competing pathways encoding fumarate reductase, alcohol/aldehyde dehydrogenase and pyruvate formate lyase and by a mutation in the acetate kinase gene. New technologies that have proven to be very useful for increasing production of primary metabolites include genome-based strain reconstruction, metabolic engineering, and whole genome shufing (see section on Novel genetic technologies).
Secondary metabolites
The application of recombinant DNA technology to the production of secondary metabolites has been of great interest (Baltz & Hosted, 1996; Diez et al., 1997). The tools of the recombinant geneticist for increasing the titers of secondary metabolites have included: (i) transposition mutagenesis, (ii) targeted deletions and duplications by genetic engineering and (iii) genetic recombination by protoplast fusion (Baltz, 2003). Recent additions to these techniques include genomics, transcriptome analysis, proteomics, metabolic engineering, and whole genome shufing (see section on Novel gene technologies). One of the rst indications that rDNA technology could be applied to antibiotics and other secondary metabolites was that it could be carried out in streptomycetes (Thompson et al., 1982). Plasmids were constructed from plasmid SLP 1.2 of Streptomyces lividans and plasmid SCP2 from Streptomyces coelicolor. In mating of plasmid-negative S. lividans, pocks (circular zones of sporulation inhibition associated with plasmid transfer in the lawn of streptomycete growth arising from a regenerated protoplast population) were seen. This was due to looping out of a piece of S. coelicolor DNA, which became a series of small S. lividans plasmids (SLP 1.1 to 1.6) that were good cloning vehicles. The genetic engineering of actinomycetes was limited for a number of years by restriction barriers hindering DNA introduction and by the inhibition of secondary metabolism by self-replicating plasmid-cloning vectors (Baltz & Hosted,
c
196
1996), but these problems were mainly overcome. Early reviews on cloning and expressing antibiotic production genes in Streptomyces were by Martin and Gil (Martin & Gil, 1984) and Liras (Liras, 1988). An interesting possibility was the transfer of operons from one streptomycete to another in the hope that the structural genes might be better able to express themselves in another species. Clustering facilitated the transfer of an entire pathway in a single manipulation. Studies revealed that many antibiotic biosynthesis genes were arranged in clusters including undecylprodigiosin, actinorhodin, chloramphenicol, rifamycin, cephamycin, erythromycin, tetracyclines and tylosin among others. Thus, the entire undecylprodigiosin pathway (red pathway) of S. coelicolor was transferred on a 37-kb fragment into Streptomyces parvulus and the antibiotic was produced (Coco et al., 1991). Similarly, the entire cephamycin C pathway was cloned and expressed from a cephamycin-producing strain of Streptomyces cattleya. When the 29-kb DNA fragment was cloned into the non-b-lactam producer, S. lividans, one transformant (out of 30 000) made cephamycin (Chen et al., 1988). When the fragment was introduced into another cephamycin producer, Streptomyces lactamgens, a two- to three-fold improvement was obtained. In fungi making penicillin G, the three structural genes (ACVS, cyclase and penicillin acyltransferase) are clustered on a single chromosome of Penicillium chrysogenum (Smith et al., 1990) and of Aspergillus nidulans (MacCabe et al., 1990). In these fungi, the genes of the cluster are separately transcribed. By contrast, fungal genes coding for cephalosporin biosynthesis are distributed among different chromosomes. The deacetylcephalosporin C acetyltransferase gene from Cephalosporium acremonium (cefG) is closely linked to the expandase (cefEF) gene (Gutierrez et al., 1992; Matsuda et al., 1992) and both are on chromosome II, whereas the early genes of the pathway (pcbAB, pcbC) are located on chromosome VI.
tion of ACV into isopenicillin N (IPN) by IPN synthase (cyclase; encoded by pcbB). The cloning of the gene encoding ACVS from P. chrysogenum (Diez et al., 1990), C. acremonium (Gutierrez et al., 1991) and Nocardia lactamdurans (Castro et al., 1988) contributed greatly to the elucidation of the biosynthetic pathway. Overexpression of acvA in A. nidulans, by replacing the normal promoter with the ethanol dehydrogenase promoter (Kennedy & Turner, 1996), increased penicillin production up to 30-fold. The cyclase genes from different microorganisms were all cloned (Aharonowitz et al., 1992; Martin et al., 1997) and provided pure enzyme for structural studies. Cloning multiple copies of cyclase into C. acremonium yielded an improved cephalosporin C-producing strain (Skatrud et al., 1987). The hydrophobic branch Producers of penicillin use a single step branch involving penicillin acyltransferase acting on IPN. Its gene penDE (also known as iat, aat and acyA in A. nidulans) was cloned from P. chrysogenum into C. acremonium, which led to the production of penicillin G (in the presence of exogenous phenylacetic acid) along with cephalosporin C (Gutierrez et al., 1991). Without cloning, C. acremonium cannot produce penicillin G. The hydrophilic branch All producers of cephalosporins and cephamycins employ a series of enzymes leading from IPN. First, IPN is epimerized to penicillin N by IPN epimerase (encoded by cefD). The next steps include ring expansion of penicillin N by deacetoxycephalosporin C (DAOC) synthase (expandase, encoded by cefE) and hydroxylation by DAOC 3 0 -hydroxylase (encoded by cefF) to deacetylcephalosporin C (DAC). Although expandase and hydroxylase are separate enzymes encoded by separate genes in bacteria, these two activities are found on the same protein in fungi, which is encoded by one gene cefEF. At the DAC stage, the overall pathway again splits into two branches. In C. acremonium, DAC is acetylated to cephalosporin C by DAC acetyltransferase encoded by cefG. This step is the terminal reaction in cephalosporinproducing fungi. By contrast, actinomycetes carbamoylate DAC using three enzymes, encoded by cmcH, cmcI and cmcJ genes to yield cephamycin C (Brewer et al., 1980). When an industrial production strain of C. acremonium 394-4 was transformed with a plasmid containing the pcbC and the cefEF gene from an early strain of the C. acremonium mutant line, a transformant producing 50% more cephalosporin C than the production strain, as well as less penicillin N, was obtained. Production in pilot plant (150 L) fermentors was further improved by 15% (Skatrud et al., 1989). One copy of the cefEF had been integrated into chromosome III, whereas the native gene is on chromosome II.
b-Lactam antibiotics Cloning has been very important in understanding the biosynthesis of b-lactam antibiotics (Demain & Elander, 1999), its genetics and improving the production processes. Early common pathway All producers of penicillins and cephalosporins, including cephamycins, use the same two enzymes to start the biosynthetic process. The steps involve the condensation of L-a-aminoadipic acid, L-cysteine and L-valine to form the tripeptide, d-(a-aminoadipyl)-L-cysteinyl-D-valine (ACV) by ACV synthetase (ACVS), encoded by gene pcbAB (also known as acvA in A. nidulans). This is followed by cycliza c
197
Transformation of P. chrysogenum with the Streptomyces lipmanii cefD and Streptomyces clavuligerus cefE genes allowed the production of the intermediate DAOC (Cantwell et al., 1992) at titers of 2.5 g L1. DAOC is a valuable intermediate in the commercial production of semi-synthetic cephalosporins. Also, cloning of cefE from S. clavuligerus or cefEF and cefG (see next paragraph) from C. acremonium into P. chrysogenum grown with adipic acid as side-chain precursor (Crawford et al., 1995) resulted in formation of adipyl-6-aminopenicillanic acid (adipyl-6APA) and adipyl-7-aminodeoxycephalosporanic acid (adipyl-7-ADCA) in the case of cefE and adipyl-6APA, adipyl7ADCA, adipyl-7-DAC and adipyl-7-aminocephalosporanic acid (7-ACA) in the case of cefEF and cefG. Disruption and one-step replacement of the cefEF gene of an industrial cephalosporin C production strain of A. chrysogenum yielded strains accumulating up to 20 g L 1 of penicillin N. Cloning and expression of the cefE gene from S. clavuligerus into those high-producing strains yielded recombinant strains producing high titers of DAOC (Velasco et al., 2000). Production levels were nearly equivalent (80%) to the total b-lactams biosynthesized by the parental strain. Weak acetyltransferase promoter activity appears to be the cause of DAC accumulation in broths of C. acremonium. Cloning of cefG increased its copy number and cefG mRNA, tripled acetyltransferase activity, and increased cephalosporin C titers in a dose-dependent manner (Matsuda et al., 1992; Mathison et al., 1993). Cloning of the gene with its own promoter had no effect on the low level of DAC acetyltransferase normally observed in C. acremonium (Gutierrez et al., 1997). However, the use of foreign promoters (the gpd promoter from A. nidulans, the bla promoter from A. niger or the pbcC promoter from P. chrysogenum) had a major effect on the level of cefG transcripts, DAC acetyltransferase protein level and activity, and antibiotic production; cephalosporin C production rose by two- to three-fold. Of the cephalosporins produced, the undesirable DAC decreased from 80% of the total to 3039%, whereas cephalosporin C increased by a similar amount. Transformation of early strain P. chrysogenum Wis541255 with individual genes, pairs of genes, and all three genes of the penicillin pathway showed that the major increases occurred when all three genes were overexpressed (Theilgaard et al., 2001). The best transformant contained three extra copies of pcbAB, one extra copy of pcbC and two extra copies of penDE and produced 299% of control shake ask production and 276% of control productivity in continuous culture.
Microbial enzymes
Genes encoding many microbial enzymes have been cloned and the enzymes expressed at levels hundreds of times
higher than those naturally produced. Recombinant DNA technology has been used (Falch, 1991): (i) to produce in industrial organisms enzymes obtained from microbes that are difcult to grow or handle genetically; (ii) to increase enzyme productivity by use of multiple gene copies, strong promoters, and efcient signal sequences; (iii) to produce in a safe host useful enzymes obtained from a pathogenic or toxin-producing microorganism; and (iv) to improve the stability, activity or specicity of an enzyme by protein engineering. The industrial enzyme business adopted rDNA methods to increase production levels and to produce enzymes from industrially-unknown microorganisms in industrial organisms such as species of Aspergillus and Trichoderma, as well as Kluyveromyces lactis, S. cerevisiae, Yarrowia lipolytica and Bacillus licheniformis. Virtually all laundry detergents contain genetically-engineered enzymes and much cheese is made with genetically-engineered enzymes. Indeed, over 60% of the enzymes used in the detergent, food and starch processing industries are recombinant products (Cowan, 1996). Scientists at Novo Nordisk isolated a very desirable lipase for use in detergents from a species of Humicola. For production purposes, the gene was cloned into Aspergillus oryzae, where it produced 1000-fold more enzyme (Carlsen, 1990) and is now a commercial product. Such lipases are used for laundry cleaning, interesterication of lipids, and esterication of glucosides producing glycolipids which have applications as biodegradable non-ionic surfactants for detergents, skin care products, contact lens cleaners and as food emulsiers. The a-amylase gene from Bacillus amyloliquefaciens was cloned using multicopy plasmid pUB110 in B. subtilis (Palva, 1982). Production was 2500-fold that in wild-type B. subtilis and ve-fold that of the B. amyloliquefaciens donor. An exoglucanase from the cellulolytic Cellulomonas mi was overproduced after cloning in E. coli to a level of over 20% of cell protein (ONeill et al., 1986). The endo-bglucanase components of the cellulase complexes from Thermomonospora and Clostridium thermocellum were cloned in E. coli as was the cellobiohydrolase I gene of Trichoderma reesei (Shoemaker et al., 1983; Teeri et al., 1983). Pichia pastoris, a methanol-utilizing yeast, was engineered to produce S. cerevisiae invertase and to excrete it into the medium at 100 mg L1 (Van Brunt, 1986). Interestingly, in S. cerevisiae, the invertase is periplasmic. Selfcloning of the xylanase gene in S. lividans resulted in sixfold overproduction of the enzyme (Mondou et al., 1986). Many enzymes are made by lamentous organisms, which are slow-growing and difcult to handle in fermentors. The transfer of these genes to rapidly-growing unicellular bacteria means that rapid growth and more reproducible production can be achieved. Other advantages are more rapid nutrient uptake due to a greater surface/ volume ratio, better oxygen transfer, better mixing and thus
c
198
more reliable control of pO2, pCO2 and pH, and a better organism for mutagenesis. Aspartase production was increased by 30-fold by cloning in E. coli (Komatsubara et al., 1986). Captopril esterase of Pseudomonas putida, used in preparing the chiral captopril sidechain, was cloned in E. coli with a 38-fold increase in activity (Elander, 1995). A 1000-fold increase in phytase production was achieved in A. niger using recombinant technology (Van Hartinsveldt et al., 1993). Cloning of the benzylpenicillin acylase gene of E. coli on multicopy (50) plasmids resulted in a 45-fold increase as compared to uninduced wild-type production. Interestingly, the cloned enzyme is constitutive (Mayer et al., 1980). Cloning additional penicillin V amidase genes into wild-type Fusarium oxysporium increased enzyme titer by 130-fold (Komatsubara et al., 1986). The properties of many enzymes have been altered by genetic means. Brute force mutagenesis and random screening of microorganisms over the years led to changes in pH optimum, thermostability, feedback inhibition, carbon source inhibition, substrate specicity, Vmax, Km and Ki. This information was later exploited by the more rational techniques of protein engineering. Single changes in amino acid sequences have yielded similar types of changes in a large variety of enzymes. Today, it is no longer necessary to settle for the natural properties of an enzyme; these can be altered to suit the needs of the investigator or the process. For example, a protease from Bacillus stearothermophilus was increased in heat tolerance from 86 1C to 100 1C, being made resistant to boiling. The enzyme was developed by site-directed mutagenesis (Van den Burg et al., 1998). Only eight amino acids had to be modied. Temperature stability at 100 1C was increased 340-fold and activity at lower temperature was not decreased. All eight mutations were far from the enzymes active site. Washing powders have been improved in activity and low temperature operation by the application of recombinant DNA technology and sitedirected mutagenesis to proteases and lipases (Falch, 1991; Wackett, 1997).
Polymers, fuels, foods and beverages

Microbially-produced xanthan gum is not only an acceptable food-thickener but is one of the most promising agents for enhanced oil recovery in the petroleum industry. Recombinant DNA manipulation of Xanthomonas campestris increased titers of xanthan by two-fold and increased pyruvate content by over 45% (Bigelas, 1989; Tseng et al., 1992). The yield was 0.6 g g1 of sucrose utilized (Letisse et al., 2001). Ten to twenty thousand tons of xanthan are produced annually for use in the oil, pharmaceutical, cosmetic, paper, paint and textile industries (Becker et al., 1998).
c
Escherichia coli was converted into a good ethanol producer (4.3%, v v 1) using recombinant DNA technology (Ingram et al., 1987). Alcohol dehydrogenase II and pyruvate decarboxylase genes from Zymomonas mobilis were inserted in E. coli and became the dominant system for NAD regeneration. Ethanol represented over 95% of the fermentation products in the genetically-engineered strain. By cloning and expressing the same two genes into Klebsiella oxytoca, the recombinant was able to convert crystalline cellulose to ethanol in high yield when fungal cellulase was added (Doran & Ingram, 1993). The maximum theoretical yield was 8186% and titers as high as 47 g L 1 of ethanol were produced from 100 g L 1 of cellulose. Cloning of its ace (acetone) operon gene adc (encoding acetoacetate decarboxylase), ctfA and ctfB (two genes encoding coenzyme A transferase) on a plasmid containing the adc promoter into Clostridium acetobutylicum resulted in a 95% increase in production of acetone, a 37% increase in butanol, a 90% increase in ethanol, a 50% increase in solvent yield from glucose and a 22-fold lower production of undesirable acids (Mermelstein et al., 1993). The introduction of the acetone operon from C. acetobutylicum into E. coli led to high acetone production by the latter (Bermejo et al., 1998). Beer wort contains barley b-glucans which reduce the ltrability of beer and lead to precipitates and haze in the nal product. The gene coding for endoglucanase was transferred from T. reesei to brewers yeast and the engineered yeast strain efciently hydrolyzed the b-glucans (Penttil et al., 1987). Similiar technology created starcha utilizing S. cerevisiae strains and wine yeast strains producing lower acidity and enhanced avor. Brewing yeasts were modied using recombinant DNA technology so that they could produce A. niger amyloglucosidase and break down unfermentable dextrins for light beer production (Van Brunt, 1986; Hammond, 1988). The glucoamylase gene from Aspergillus awamori was cloned and expressed stably in polyploid distillers yeast. A high level of glucoamylase was secreted. Almost all (95%) of the carbohydrates in the 25% starch substrate were utilized and high levels of ethanol were produced. The engineered strain outperformed S. diastaticus (Cole et al., 1988). Brewing yeasts were engineered to produce acetolactate decarboxylase from Enterobacter aerogenes or A. aceti. This enzyme eliminated diacetyl and the requirement for the three- to ve-week avor maturation period which normally follows a one-week fermentation stage (Sone et al., 1988). The resulting beer suffered no loss of quality or avor (Holzman, 1994).
Bioconversions
Recombinant DNA techniques have been useful in developing new bioconversions and improving old ones. Using a
199
plasmid containing tryptophan synthase plus induction with 3-indole acrylate, recombinant E. coli was able to produce 180 g L 1 of L-tryptophan from indole plus L-serine in 8 h (Yukawa et al., 1988). Whereas S. cerevisiae normally produces 2 g L 1 of malic acid from fumaric acid, a recombinant strain containing a cloned fumarase gene was able to produce 125 g L 1 with a yield of almost 90% (Neufeld et al., 1991). An oxidative bioconversion of saturated and unsaturated linear aliphatic 1222 carbon substrates to their terminal dicarboxylic acids was developed by gene disruption and gene amplication (Picataggio et al., 1992). Product concentrations reached 200 g L 1 and problematic side-reactions such as unsaturation, hydroxylation and chainshortening did not occur. 3-0-Acetyl-4 0 0 -0-isovaleryltylosin (AIV) is useful in veterinary medicine against tylosin-resistant Staphylococcus aureus. It is made by rst producing tylosin with Streptomyces fradiae and then using Streptomyces thermotolerans (producer of carbomycin) to bioconvert tylosin into AIV. A new direct fermentation organism was constructed by transforming S. fradiae with S. thermotolerans plasmids containing acyl transferase genes (Arisawa et al., 1996). Recombinant Candida pasteurianum can carry out the conversion of glycerol to 1,3-propanediol (Luers et al., 1997). A more economical process involving conversion of the less expensive glucose to 1,3-propanediol has been achieved with a recombinant E. coli strain (Nakamura & Whited, 2003). The project is a collaborative effort by Genencor International and DuPont (Potera, 1997). The recombinant strain contains two metabolic pathways, one for conversion of glucose to glycerol and the other for conversion of glycerol to 1,3-propanediol (Tong et al., 1991; Laffend et al., 1996). The 1,3-propanediol (also known as trimethylene glycol or 3G) is used as the building block to produce a new biodegradable polyester (3G1).
Novel genetic technologies

A new genomic technique called genome-based strain reconstruction allows one to construct a strain superior to the production strain because it only contains mutations crucial to hyperproduction, but not other unknown mutations which accumulate by brute-force mutagenesis and screening (Ohnishi et al., 2002). This approach was used to improve the lysine production rate of Corynebacterium glutamicum by comparing high producing strain B-6 developed by Hirao and coworkers (Hirao et al., 1989) (production rate slightly less than 2 g L 1 h 1) and a wild-type strain. Comparison of 16 genes from strain B-6, encoding enzymes of the pathway from glucose to lysine, revealed mutations in ve of the genes. Introduction of three of these mutations into the wild-type created a new strain which
produced 80 g L 1 in 27 h, at a rate of 3 g L 1 h 1, the highest rate ever reported for a lysine fermentation. Metabolic engineering is the directed improvement of product formation or cellular properties through the modication of specic biochemical reactions or introduction of new ones using recombinant DNA technology (Stephanopoulos, 1999; Nielsen, 2001). Its essence is the combination of analytical methods to quantify uxes and the control of uxes with molecular biological techniques to implement suggested genetic modications. Flux is the focal point of metabolic engineering. Different means of analyzing ux are: (i) kinetic based models; (ii) control theories; (iii) tracer experiments; (iv) magnetization transfer; (v) metabolite balancing; (vi) enzyme analysis and (vii) genetic analysis (Eggeling et al., 1996). Metabolic control analysis revealed that the overall ux through a metabolic pathway depends on several steps, not just a single rate-limiting reaction (Kacser & Acerenza, 1993). Metabolic engineering has been applied to antibiotic production (Khetan & Hu, 1999, 1999; Thykaer & Nielsen, 2003). The increases in metabolic ux were carried out by enhancing enzymatic activity, manipulating regulatory genes, enhancing antibiotic resistance and heterologous expression of novel genes. Table 2 summarizes several examples of progress on the production of those secondary metabolites. The production of amino acids shows many examples of this approach. A useful review of metabolic engineering in C. glutamicum, especially in relation to L-lysine production, was published by Sahm and colleagues (Sahm et al., 2000). Metabolic flux studies of wild-type C. glutamicum and four improved lysine-producing mutants available from the ATCC showed that yield increased from 1.2% to 24.9% relative to the glucose flux. Other recent examples are on overproduction of aromatic amino acids and derivatives (Bongaerts et al., 2001), L-lysine (Wittmann & Heinzle, 2002) and glutamate (Kimura, 2003). There are many other successful applications of metabolic engineering for products such as 1,3-propanediol (Nakamura & Whited, 2003), carotenoids (Rohlin et al., 2001; Visser et al., 2003; Wang & Keasling, 2003), organic acids (Kramer et al., 2003), ethanol (Nissen et al., 2000), vitamins (Zamboni et al., 2003; Sybesma et al., 2004) and complex polyketides in bacteria (Pfeifer et al., 2001; Khosla & Keasling, 2003). During the last few years, an expanded view of the cell has been possible due to impressive advances in all the omics techniques (genomics, proteomics, metabolomics, etc.) and high-throughput technologies for measuring different classes of key intracellular molecules. Systems biology has recently emerged as a term to describe an approach that considers genome-scale and cell-wide measurements in elucidating processes and mechanisms (Stephanopoulos
c
200
et al., 2004). Progress in strain development will depend, not only on all the technologies mentioned above, but also on the development of mathematical methods that facilitate the elucidation of mechanisms and identication of genetic targets for modication.
Table 2. Metabolic engineering of antibiotics Target 1.Manipulation of structural genes Result
A genome-wide transcript expression analysis called massive parallel signature sequencing (Brenner et al., 2000) was used successfully to discover new targets for further improvement of riboavin production by the fungus A. gossypii (Karos et al., 2004). The authors identied 53
Reference Chen et al. (1988) Otten et al. (1990) Decker et al. (1994) Madduri et al. (2001) Crameri & Davies (1986)
2. Manipulation of regulatory genes
1.1. Amplifying an entire pathway 2.3-fold increase in cephamycin C 1.2. Amplifying a segment of a pathway 7-fold increase in daunorubicin 30% increase in tetracenomycin C 3- to 4-fold increase in spinosyn 1.3. Enhancing resistance 7- fold increase in neomycin 2.1. Amplifying positive regulatory genes 5-fold increase in spiramycin 1.6-fold increase in mithramycin Global regulators Increase in actinorhodin and undecylprodigiosin 2.2. Disrupting negative regulatory genes 1.5-3.5 fold increase in avermectin Pathway specic regulators increase in methylenomycin; in mitomycin C; 7- to 10-fold in lovastatin Global regulators increase in actinorhodin and undecylprodigiosin 3.1. Kinetic analysis 2- to 5-fold increase in cephamycin C 3.2. Increasing expression of rate-limiting 30-fold increase in penicillin increase in enzymes tylosin Pathway specic regulators
Geistlich et al. (1992) Lombo et al. (1999) Voegtli et al. (1994) Hwang et al. (2003); Lee et al. (2000) Chater & Bruton (1985); Mao et al. (1999); Kennedy et al. (1999) Brian et al. (1996) Malmberg et al. (1995) Kennedy & Turner (1996) Cox et al. (1987); Fishman et al. (1987) Skatrud (1992) Ikeda et al. (1993)
3. Engineering of well-known pathways
3.3. Eliminating accumulation and excretion of intermediate 3.4. Deleting gene leading to a side product 3.5. Biosynthesizing compounds previously made semisynthetically 3.6. Biosynthesizing new compounds 3.7. Increasing oxygen availability 3.8. Enhancing precursor uptake
Elimination of excretion of penicillin N; 15% increase in cephalosporin C Elimination of oligomycin production
Production of 7Velasco et al. (2000) aminodeacetoxycephalosporanic acid in Acremonium chrysogenum Production of adipyl-7-ADCA, adipyl-7- Crawford et al. (1995) ACA in P. chrysogenum 60% increase in erythromycin production Brunker et al. (1998); Minas et al. (1998) 4-fold increase in deoxyerythronolide B Lombo et al. (2001) and 8, 8a-deoxyoleoandolide
Integrating transcriptional and metabolite proles from 21 strains of A. terreus producing different levels of lovastatin and another 19 strains with altered (1)-geodin levels led to an improvement in lovastatin production of over 50% (Askenazi et al., 2003). The approach, named association analysis, was used to reduce the complexity of proling data sets to identify those genes in which expression was most tightly linked to metabolite production. Such an application is suitable to all industrially useful organisms for which genome data are limited.
c
genes of known function, some of which could clearly be related to riboavin production. This approach also allowed the nding of sites within the genome with high transcriptional activity during riboavin biosynthesis that are suitable integration loci for the target genes found. Gene expression analysis of wild-type and improved production strains of Saccharopolyspora erythraea and S. fradiae using microarrays of S. coelicolor revealed that regulation of antibiotic biosynthetic enzymes as well as enzymes involved in precursor metabolism were altered in
201
those mutated strains (Lum et al., 2004). The S. erythraea overproducer expressed the entire erythromycin gene cluster for several more days than the wild-type. It seemed that the eryA gene and protein expression differences observed for the overproducer could account for over 50% of the total erythromycin titer increase. The S. fradiae mutant expressed the tylosin biosynthetic genes in a similar way to the wildtype strain; however, two genes, aco (encoding acyl-CoA dehydrogenase) and icmA (encoding isobutyryl-CoA mutase), were expressed more highly than in the wild-type strain. The induction of these two genes could increase the ux of metabolites from fatty acids to tylosin precursors in the overproducer. These recent technologies and mathematical approaches will all contribute to the generation and characterization of microorganisms able to synthesize large quantities of commercially important metabolites. The ongoing sequencing projects involving hundreds of genomes, the availability of sequences corresponding to model organisms, new DNA microarray and proteomics tools, as well as the new techniques for mutagenesis and recombination described above will accelerate strain improvement programs. The development and combined application of these technologies will help to develop what was already succinctly described several years ago as Inverse netabolic engineering (Bailey et al., 1996), which means to identify, construct or calculate a desired phenotype, identify the molecular basis of that desirable property, and incorporate that phenotype into another strain or other species by genetic and environmental manipulations. Directed evolution (= applied molecular evolution = directed molecular evolution) is a rapid and inexpensive way of nding variants of existing enzymes that work better than naturally occurring enzymes under specic conditions (Kuchner & Arnold, 1997; Skandalis et al., 1997; Arnold, 1998). The process involves evolutionary design methods using random mutagenesis, gene recombination and high-throughput screening (Arnold, 2001). Diversity is initially created by in vitro mutagenesis of the parent gene using repeated cycles of mutagenic polymerase chain reaction (error-prone PCR) (Leung et al., 1989), repeated oligonucleotide-directed mutagenesis (Reidhaar-Olson et al., 1991), mutator strains (Bornscheuer et al., 1998) or chemical agents (Taguchi et al., 1998). A key limitation of these strategies is that they introduce random noise mutations into the gene at every cycle and hence improvements are limited to small steps. This strategy has been used successfully in different applications (Zhao et al., 2002). Molecular breeding techniques (DNA shufing, Molecular BreedingTM) come closer to mimicking natural recombination by allowing in vitro homologous recombination (Ness et al., 2000). These techniques not only recombine DNA fragments but also introduce point
mutations at a very low controlled rate (Stemmer, 1994; Zhao & Arnold, 1997). Unlike site-directed mutagenesis, this method of pooling and recombining parts of similar genes from different species or strains has yielded remarkable improvements in enzymes in a very short amount of time (Patten et al., 1997). A step forward in this technique was breeding a population with high genetic variability as a starting point to generate diversity (DNA Family Shufing). This approach led to a 240- to 540-fold improvement in cephalosporinase activity when four cephalosporinase genes were shufed as a starting point (Crameri et al., 1998). When each of these genes was shufed independently, only eight-fold improvements were obtained. Innovations that expand the formats for generating diversity by recombination include formats similar to DNA shufing and others with few or no requirements for parental gene homology (Kurtzman et al., 2001; Lutz et al., 2001). Random redesign techniques are currently being used to generate enzymes with improved properties such as: activity and stability at different pH values and temperatures (Ness et al., 1999), increased or modied enantioselectivity (Jaeger & Reetz, 2000), altered substrate specicity (Suenaga et al., 2001), stability in organic solvents (Song & Rhee, 2001), novel substrate specicity and activity (Raillard et al., 2001), increased biological activity of protein pharmaceuticals and biological molecules (Patten et al., 1997; Kurtzman et al., 2001) as well as novel vaccines (Marshall, 2002; Locher et al., 2004). Proteins from directed evolution work were already on the market by 2000 (Tobin et al., 2000). These were green uorescent protein of Clontech (Crameri et al., 1996) and Novo Nordisks LipoPrimes lipase. Whole genome shufing (WGS) is a novel technique for strain improvement combining the advantage of multiparental crossing allowed by DNA shufing with the recombination of entire genomes. This method was applied successfully to improved tylosin production in S. fradiae (Zhang et al., 2002). Historically, 20 cycles of classical strain improvement at Eli Lilly and Co. carried out over 20 years employing about one million assays improved production six-fold. In contrast, two rounds of WGS with seven early strains each were sufcient to achieve similar results in one year and involved only 24 000 assays. Such recursive genomic recombination has also been used to improve the acid-tolerance of a commercial lactic acid-producing Lactobacillus sp. (Patnaik et al., 2002). Combinatorial biosynthesis is being used for the discovery of new and modied drugs (Hutchinson, 1998; Reeves, 2003). In this technique, recombinant DNA techniques are utilized to introduce genes coding for antibiotic synthases into producers of other antibiotics or into nonproducing strains to obtain modied or hybrid antibiotics. The rst demonstration of this technology involved gene transfer from a streptomycete strain producing the
c
202
isochromanequinone antibiotic actinorhodin into strains producing granaticin, dihydrogranaticin and mederomycin (which are also isochromanequinones). This led to the discovery of two new antibiotic derivatives, mederrhodin A and dihydrogranatirhodin. Since this breakthrough paper by Hopwood and coworkers (Hopwood et al., 1985), many hybrid antibiotics have been produced by recombinant DNA technology. Hundreds of new polyketides have been made by combinatorial biosynthesis (Rodriguez & McDaniel, 2001; Donadio & Sosio, 2003; Kantola et al., 2003). Manipulations include: (i) deletion of one of the domains of a particular module; (ii) addition of a copy of the thioesterase domain to the end of an earlier module resulting in a shortened polyketide; (iii) replacement of an AT domain of a polyketide synthase (PKS) with an AT domain from another PKS, resulting in addition of a methyl group at a particular site or removal of a methyl group; (iv) addition of a reductive domain(s) to a particular module, thus changing a keto group to a double bond or to a methylene group; (v) use of synthetic diketides delivered as N-acetylcysteamine thioesters to load onto the active site of the ketosynthase (KS) in module 2 and to be taken all the way to a novel nal product; (vi) replacement of the loading module of one PKS with the loading module of another PKS, thus changing the starter unit from propionate to acetate, for example; and (vii) replacement of the hydroxylase or glycosylase enzymes from one pathway to another, thus modifying the ring structure with respect to OH groups and/or sugars (Staunton, 1998). As mentioned above, there are many examples of new polyketides been made by combining polyketide biosynthetic genes from different producers (McAlpine et al., 1987; Epp et al., 1989; Donadio et al., 1991, 1993; Weber et al., 1991; Hara & Hutchinson, 1992; Decker & Hutchinson, 1993; Hopwood, 1993; Katz & Donadio, 1993; Khosla et al., 1993; McDaniel et al., 1993a, b, 1999; Hutchinson & Fujii, 1995; Kao et al., 1995; Tsoi & Khosla, 1995; Pacey et al., 1998; Wohlert et al., 1998; Xue et al., 1999; Pfeifer & Khosla, 2001). Some of these novel polyketides contain sugars at normally unglycosylated positions (Trefzer et al., 2002) or as new sugar moieties (Zhao et al., 1999; Mendez & Salas, 2001). New anthracyclines (Bartel et al., 1990; Strohl et al., 1991; Hwang et al., 1995; Niemi & Mantsala, 1995; Kim et al., 1996; Ylihonko et al., 1996) and peptide antibiotics (Stachelhaus et al., 1995) have also been made by combinatorial biosynthesis.
can be smaller molecules that can be essential for vegetative growth or inessential, i.e. primary and secondary metabolites, respectively. The power of the microbial culture in the competitive world of commercial synthesis can be appreciated by the fact that even simple molecules are made by fermentation rather than by chemical synthesis. Most natural products are so complex that they probably will never be made commercially by chemical synthesis. Strains isolated from nature produce only tiny amounts of product. This is because they need these secondary metabolites for their own competitive benet, and they do not overproduce these metabolites. Regulatory mechanisms have evolved in microorganisms which enable a strain to avoid excessive production of its metabolites, thus, strain improvement programs are required for commercial application. The goal is to isolate cultures exhibiting desired phenotypes. Most commonly, the ability of a strain to improve titer is what is desired, although the other traits may also be improved on. The tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by using mutagenesis. In recent years, recombinant DNA technology has also been applied. The promise of the future is via extensive use of new genetic techniques such as: (i) metabolic engineering accomplishing quantication and control of metabolic uxes and including inverse metabolic engineering and transcript expression analyses such as association analysis and massive parallel signature sequencing; (ii) directed evolution; (iii) molecular breeding including DNA shufing and whole genome shufing; and (iv) combinatorial biosynthesis. These efforts will facilitate not only the isolation of improved strains but also the elucidation and identication of new genetic targets to be used in strain improvement programs.
Acknowledgements
The authors thank the following colleagues for supplying information: Richard H. Baltz, Graham S. Byng, Richard P. Elander, David A. Hopwood, Daslav Hranueli, Krishna Madduri, Jaraslav Spizek, William R. Strohl and J. Mark Weber.
References
Aharonowitz Y, Cohen G & Martin JF (1992) Penicillin and cephalosporin biosynthetic genes: structure, organization, regulation and evolution. Ann Rev Microbiol 46: 461495. Anderson S, Marks CB, Lazarus R, Miller J, Stafford K, Seymour J, Light D, Rastetter W & Estell D (1985) Production of 2-ketoL-gulonate: an intermediate in L-ascorbate synthesis by a genetically modied Erwinia herbicola. Science 230: 44149.
Concluding remarks
Microorganisms produce many compounds of industrial interest. These may be very large materials such as proteins, nucleic acids, carbohydrate polymers, or even cells, or they
c
203
Arisawa A, Kawamura N, Narita T, Kojima I, Okamura K, Tsunekawa H, Yoshioka T & Okamoto R (1996) Direct fermentative production of acyltylosins by geneticallyengineered strains of Streptomyces fradiae. J Antibiot 49: 349354. Arnold FH (1998) Design by directed evolution. Acc Chem Res 31: 125131. Arnold FH (2001) Combinatorial and computational challenges for biocatalyst design. Nature 409: 253257. Askenazi M, Driggers EM, Holtzman DA, et al. (2003) Integrating transcriptional and metabolite proles to direct the engineering of lovastatin-producing fungal strains. Nature Biotechnol 21: 150156. Bailey RM, Benitez T & Woodward A (1982) Saccharomyces cerevisiae mutants resistant to catabolite repression: use in cheese whey hydrolysate fermentation. Appl Environ Microbiol 44: 631639. Bailey JE, Sburlati A, Hatzimanikatis V, Lee K, Renner WA & Tsai PS (1996) Inverse metabolic engineering: a strategy for directed genetic engineering of useful phenotypes. Biotechnol Bioeng 52: 109121. Bajon AM, Guiraud JP & Galzy P (1984) Isolation of an inulinase derepressed mutant of Pichia polymorpha for the production of fructose. Biotechnol Bioeng 26: 128133. Baltz RH (1986) Mutagenesis in Streptomyces. Manual of Industrial Microbiology and Biotechnology (Demain AL & Solomon NA, eds), pp. 184190. American Society for Microbiology, Washington, DC. Baltz RH (1995) Gene expression in recombinant Streptomyces. Gene Expression in Recombinant Microorganisms (Smith A, ed), pp. 309381. Marcel Dekker, New York. Baltz RH (1998) Genetic manipulation of antibiotic producing Streptomyces. Trends Microbiol 6: 7683. Baltz RH (1999) Mutagenesis. Encyclopedia of Bioprocessing Technology: Fermentation, Biocatalysis, and Separation (Flickinger MC & Drew SW, eds), pp. 18191822. Wiley, New York. Baltz RH (2003) Genetic engineering solutions for natural products in actinomycetes. Handbook of Industrial Cell Culture: Mammalian, Microbial, and Plant Cells (Vinci VA & Parekh SR, eds), pp. 137170. Humana Press, Totowa, NJ. Baltz RH & Hosted TJ (1996) Molecular genetic methods for improving secondary-metabolite production in actinomycetes. Trends Biotechnol 14: 245250. Baltz RH, McHenney MA, Cantwell CA, Queener SW & Solenberg PJ (1997) Applications of transposition mutagenesis in antibiotic producing streptomyces. Ant v Leeuwenhoek 71: 179187. Baltz RH, Seno ET, Stonesifer J & Wild GM (1983) Biosynthesis of the macrolide antibiotic tylosin: a preferred pathway from tylactone to tylosin. J Antibiot 36: 131141. Bannerjee AB & Bose SK (1964) A rapid method for isolating mutants of Bacillus subtilis producing increased or decreased amounts of the antibiotic, mycobacillin. J Appl Bacteriol 27: 9395.
Barredo JL, Diez B, Alvarez E & Martin JF (1989) Large amplication of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium. chrysogenum Curr Genet 16: 453459. Barrios-Gonzalez J, Montenegro E & Martin JF (1993) Penicillin production by mutants resistant to phenylacetic acid. J Ferm Bioeng 76: 455458. Bartel PL, Zhu CB, Lampel JS, Dosch DC, Connors NC, Strohl WR, Beale JM Jr. & Floss HG (1990) Biosynthesis of anthraquinones by interspecies cloning of actinorhodin biosynthesis genes in streptomycetes; clarication of actinorhodin gene functions. J Bacteriol 172: 48164826. Becker A, Katzen F, Puehler A & Ielpi L (1998) Xanthan gum biosynthesis and application: a biochemical/genetic perspective. Appl Microbiol Biotechnol 50: 145152. Berg DE & Berg CM (1983) The prokaryotic transposable element Tn5. Bio/Technology 1: 417435. Bermejo LL, Welker NE & Papoutsakis ET (1998) Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxication. Appl Environ Microbiol 64: 10791085. Bigelas R (1989) Industrial products of biotechnology: application of gene technology. Biotechnology, Vol. 7b (Jacobson GK & Jolly SO, eds), pp. 230259. VCH, Weinheim. Binnie C, Warren M & Butler MJ (1989) Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis. J Bacteriol 171: 887895. Blumaerova M, Podojil M, Gauze GF, Maksikmova TS, Panos J & Vanek Z (1980) Effect of cultivation conditions on the activity of the beromycin producer Streptomyces glomeratus 3980 and its spontaneous variants. Folia Microbiol 25: 213218. Blumaerova M, Podojil M, Gauze GF, Maksikmova TS, Panos J & Vanek Z (1980) Spontaneous variability of Streptomyces glomeratus, a producer of anthracycline antibiotics beromycins. Folia Microbiol 25: 207212. Blumauerova M, Pokorny V, Stastna J, Hostalek Z & Vanek Z (1978) Developmental mutants of Streptomyces coeruleorubidis, a producer of anthracyclines: isolation and preliminary characterization. Folia Microbiol 23: 177182. Bongaerts J, Kramer M, Muller U, Raeven L & Wubbolts M (2001) Metabolic engineering for microbial production of aromatic amino acids and derived compounds. Metab Eng 3: 289300. Bornscheuer UT, Altenbuchner J & Meyer HH (1998) Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones. Biotechnol Bioeng 58: 554559. Bortol A, Nudel C, Fraile E, de Torres R, Giulietti A, Spencer JFT & Spencer D (1986) Isolation of yeast with killer activity and its breeding with an industrial baking strain by protoplast fusion. Appl Microbiol Biotechnol 24: 414416. Brenner S, Johnson M, Bridgham J, et al. (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nature Biotechnol 18: 630634.
c
204
Brewer SJ, Taylor PM & Turner MK (1980) An adenosine triphosphate-dependent carbamoylphosphate-3hydroxymethylcephem O-carbamoyltransferase from Streptomyces clavuligerus. Biochem J 185: 555564. Brian P, Riggle PJ, Santos RA & Champness WC (1996) Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative transduction system. J Bacteriol 178: 32213231. Brigidi P, Matteuzzi D & Fava F (1988) Use of protoplast fusion to introduce methionine overproduction into Saccharomyces cerevisiae. Appl Microbiol Biotechnol 28: 268271. Brunker P, Minas W, Kallio PT & Bailey JE (1998) Genetic engineering of an industrial strain of Saccharopolyspora erythrea for stable expression of the Vitreoscilla hemoglobin gene (vhb). Microbiology 144: 24412448. van Brunt J (1986) Fungi: the perfect hosts? Bio/Technology 4: 10571062. van den Burg B, Vriend G, Veltman OR, Venema G & Eijsink VGH (1998) Engineering an enzyme to resist boiling. Proc Natl Acad Sci USA 95: 20562060. Butler MJ, Friend EJ, Hunter IS, Kaczmarek FS, Sugden DA & Warren M (1989) Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by Streptomyces rimosus. Molec Gen Genet 215: 231238. Byng GS, Eustice DC & Jensen RA (1979) Biosynthesis of phenazine pigments in mutant and wild-type cultures of Pseudomonas aeruginosa. J Bacteriol 138: 846852. Cantwell C, Beckmann R, Whiteman P, Queener SW & Abraham EP (1992) Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway. Proc R Soc Lond (Biol) 248: 283289. Carlsen S (1990) Molecular biotechnology in the research and production of recombinant enzymes. Industrial Use of Enzymes: Technical and Economic Barriers (Wolnak B & Scher M, eds), pp. 5269. Bernard Wolnak and Associates, Chicago. Cassinelli G, Di Matteo F, Forenza S, Ripamonti MC, Rivola G, Arcamone F, Di Marco A, Cassaza AM, Soranzo C & Pratesi G (1980) New anthracycline glycosides from Micromonospora. II. Isolation, characterization and biological properties. J Antibiot 33: 14681473. Castro JM, Liras P, Laiz L, Cortes J & Martin JF (1988) Purication and characterization of the isopenicillin N synthase of Streptomyces lactandurans. J Gen Microbiol 134: 133141. Chang LT & Elander RP (1979) Rational selection for improved cephalosporin C productivity in strains of Acremonium chrysogenum. Devel Indust Microbiol 20: 367379. Chang LT, McGrory EL & Elander RP (1980) Penicillin production by glucose-derepressed mutants of Penicillium chrysogenum. J Indust Microbiol 6: 165169. Chater KF & Bruton CJ (1985) Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered. EMBO J 4: 18931897.
Chen CW, Lin HF, Kuo CL, Tsai HL & Tsai JFY (1988) Cloning and expression of a DNA sequence conferring cephamycin C production. Bio/Technology 6: 1221224. Chen W-Q, Yu ZN & Zheng Y-H (2004) Expression of Vitreoscilla hemoglobin gene in Streptomyces fradiae and its effect on cell growth and synthesis of tylosin. Chin J Antibiot 29: 516520. Chumpolkulwong N, Kakizono T, Nagai S & Nishio N (1997) Increased astaxanthin production by Phafa rhodozyma mutants isolated as resistant to diphenylamine. J Ferm Bioeng 83: 429434. Coco EA, Narva KE & Feitelson JS (1991) New classes of Streptomyces coelicolor A3(2) mutants blocked in undecylprodigiosin (Red) biosynthesis. Mol Gen Genet 227: 2832. Cole GE, McCabe PC, Inlow D, Gelfand DH, Ben-Bassat A & Innis MA (1988) Stable expression of Aspergillus awamori glucoamylase in distillers yeast. Bio/Technology 6: 417421. Colombo AL, Crespi-Pevellino N, Grein A, Minghetti A & Spalla CJ (1981) Metabolic and genetic aspects of the relationship between growth and tetracycline production in Streptomyces aureofaciens. Biotechnol Lett 3: 7176. Colon GE, Nguyen TT, Jetten MSM, Sinskey AJ & Stephanopoulos G (1995) Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer. Appl Microbiol Biotechnol 43: 482488. Cowan D (1996) Industrial enzyme technology. Trends Biotechnol 14: 177178. Cox KL, Fishman SE, Larson JL, Stanzak R, Reynolds PA, Yeh WK, Van Frank RM, Birmingham VA, Hershberger CL & Seno ET (1987) Cloning and characterization of genes involved in tylosin biosynthesis. Genetics of Industrial Microorganisms, Part B (Alacevic M, Hranueli D & Toman Z, eds), pp. 337346. Pliva, Zagreb. Crameri R & Davies JE (1986) Increased production of aminoglycosides associated with amplied antibiotic resistance genes. J Antibiot 39: 128135. Crameri R, Davies JE & Huetter R (1986) Plasmid curing and generation of mutations induced with ethidium bromide in streptomycetes. J Gen Microbiol 132: 819824. Crameri A, Raillard SA, Bermudez E & Stemmer WP (1998) DNA shufing of a family of genes from diverse species accelerates directed evolution. Nature 391: 288291. Crameri A, Whitehorn A & Stemmer WPC (1996) Improved green uorescent protein by molecular evolution using DNA shufing. Nature Biotechnol 14: 315319. Crawford L, Stepan AM, Mcada PC, Rambosek JA, Conder MJ, Vinci VA & Reeves CD (1995) Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Bio/Technology 13: 5862. Cropp TA, Wilson DJ & Reynolds KA (2000) Identication of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin. Nature Biotechnol 18: 980983.
c
205
Das A & Roy P (1981) Rapid strain selection for citric acid production. Adv Biotechnol 1: 5155. Daum SJ & Lemke JR (1979) Mutational biosynthesis of new antibiotics. Ann Rev Microbiol 33: 241265. De Maagd RA, Bravo A, Berry C, Crickmore N & Schnepf HE (2003) Structure, diversity, and evolution of protein toxins from spore-forming entemopathogenic bacteria. Annu Rev Genet 37: 409433. Decker H & Hutchinson CR (1993) Transcriptional analysis of the Streptomyces glaucescens tetracenomycin biosynthesis gene cluster. J Bacteriol 175: 38873892. Decker H, Summers RG & Hutchinson CR (1994) Overproduction of the acyl carrier protein component of a type II polyketide synthase stimulates production of tetracenomycin biosynthetic intermediates in Streptomyces glaucescens. J Antibiot 47: 5463. Demain AL & Elander RP (1999) The b-lactam antibiotics: past, present, and future. Ant v Leeuwenhoek 75: 519. Diez B, Gutierrez S, Barredo JL, van Solinger P, van der Voort LHM & Martin JF (1990) The cluster of penicillin biosynthetic genes. Identication and characterization of the pcbAB gene encoding the a-aminoadipyl-cysteinyl-valine synthetase and linkage to the pcbC and pcbDE genes. J Biol Chem 265: 1635816365. Diez B, Mellado E, Rodriguez R, Fouces R & Barredo JL (1997) Recombinant microorganisms for industrial production of antibiotics. Biotech Bioeng 55: 216226. Ditchburn P, Giddings B & MacDonald KD (1974) Rapid screening for the isolation of mutants of Aspergillus nidulans with increased penicillin yields. J Appl Bacteriol 37: 515523. Donadio S, McAlpine JB, Sheldon PA, Jackson MA & Katz L (1993) An erythromycin analog produced by reprogramming of polyketide synthesis. Proc Natl Acad Sci USA 90: 71197123. Donadio S & Sosio M (2003) Strategies for combinatorial biosynthesis with modular polyketide synthases. Comb Chem High Throughput Screen 6: 489500. Donadio S, Staver MJ, McAlpine JB, Swanson SJ & Katz L (1991) Modular organization of genes required for complex polyketide biosynthesis. Science 252: 675679. Doran JB & Ingram LO (1993) Fermentation of crystalline cellulose to ethanol by Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes. Biotechnol Prog 9: 533538. Dulaney EL & Dulaney DD (1967) Mutant populations of Streptomyces viridifaciens. Trans N Y Acad Sci 29: 782799. Eggeling L & Sahm H (1999) Amino acid production: principles of metabolic engineering. Metabolic Engineering (Lee SY & Papoutsakis ET, eds), pp. 153176. Marcel Dekker, New York. Eggeling L, Sahm H & de Graaf AA (1996) Quantifying and directing metabolic ux: application to amino acid overproduction. Adv Biochem Eng Biotechnol 54: 130. Elander RP (1995) Genetic engineering applications in the development of selected industrial enzymes and therapeutic proteins. Microbes for Better Living (Sankaran R & Manja KS,
eds), pp. 619628. Defense Food Research Laboratory, Mysore, India. Elander RP (2003) Industrial production of b-lactam antibiotics. Appl Microbiol Biotechnol 61: 385392. Elander RP (1969) Applications of microbial genetics to industrial fermentations. Fermentation Adavances (Perlman D, ed), pp. 89114. Academic Press, New York. Elander RP & Aoki H (1982) b-Lactam producing microorganismstheir biology and fermentation behavior. Chemistry and Biology of b-Lactam Antibiotics, Vol. 3 (Morin RB & Gorman M, eds), pp. 83153. Academic Press, New York. Epp JK, Huber MLB, Turner JR, Goodson T & Schoner BE (1989) Production of hybrid macrolide antibiotic in Streptomyces ambofaciens and Streptomyces lividans by introduction of a cloned carbomycin biosynthetic gene from Streptomyces thermotolerans. Gene 85: 293301. Falch E (1991) Industrial enzymesdevelopments in production and application. Biotech Adv 9: 643658. Farahnak F, Seki T, Ryu DDY & Ogrydziak D (1986) Construction of lactose-assimilating and high-ethanol-producing yeasts by protoplast fusion. Appl Environ Microbiol 51: 362367. Farris GA, Fatichenti F, Bifulco L, Berardi E, Deiana P & Satta T (1992) A genetically improved wine yeast. Biotechnol Lett 14: 219222. Fierro F, Barredo JL, Diez B, Gutierrez S, Fernandez FJ & Martin JF (1995) The penicillin gene cluster is amplied in tandem repeats linked by conserved hexanucleotide sequences. Proc Natl Acad Sci USA 92: 62006204. Fishman SE, Cox K, Larson JL, Reynolds PA, Seno ET, Yeh WK, Van Frank R & Hershberger CL (1987) Cloning genes for the biosynthesis of a macrolide antibiotic. Proc Natl Acad Sci USA 84: 82488252. Fujiwara T, Takahashi Y, Matsumoto K & Kondo E (1980) Isolation of an intermediate of 2-deoxystreptamine biosynthesis from a mutant of Bacillus circulans. J Antibiot 33: 824829. Fukaya M, Tayama K, Tamaki T, Tagami H, Okumura H, Kawamura Y & Beppu T (1989) Cloning of the membranebound aldehyde dehydrogenase gene of Acetobacter polyoxogenes and improvement of acetic acid production by use of the cloned gene. Appl Environ Microbiol 55: 171176. Gaucher GM, Lam KS, Grootwassink JWD, Neway J & Deo YM (1981) The initiation and longevity of patulin biosynthesis. Devel Indust Microbiol 22: 219232. Geistlich M, Losick R, Turner JR & Rao RN (1992) Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens. Mol Microbiol 6: 20192029. Gesheva V (1994) Isolation of spontaneous Streptomyces hygroscopicus 111-81 phosphate-deregulated mutants hyperproducing its antibiotic complex. Biotechnol Lett 16: 443448. Ghisalba O, Fuhrer H, Richter W & Moss S (1981) A genetic approach to the biosynthesis of the rifamycin-chromophore in Nocardia mediterranei. III. Isolation and identication of an
c
206
early ansamycin-precursor containing the seven-carbon amino starter-unit and three initial acetate/propionate-units of the ansa chain. J Antibiot 34: 5863. Godfrey O (1973) Isolation of regulatory mutants of the aspartic and pyruvic acid families and their effect on antibiotic production in Streptomyces lipmanii. Antimicrob Agents Chemother 4: 7379. Gomi S, Ikeda D, Nakamura H, Naganawa H, Yamashita F, Hotta K, Kondo S, Okami Y, Umezawa H & Iitaka Y (1984) Isolation and structure of a new antibiotic, indolizomycin, produced by a strain SK2-52 obtained by interspecies fusion treatment. J Antibiot 37: 14911494. Gravius B, Glocker D, Pigac J, Pandza K, Hranueli D & Cullum J (1994) The 387 kb linear plasmid pPZG101 of Streptomyces rimosus and its interactions with the chromosome. Microbiology 140: 22712277. Grindley JF, Payton MA, van de Pol H & Hardy KG (1988) Conversion of glucose to 2-keto-L-gulonate, an intermediate in L-ascorbate synthesis by a recombinant strain of Erwinia citreus. Appl Environ Microbiol 54: 17701775. Guillouet S, Rodal AA, An G-H, Lessard PA & Sinskey AJ (1999) Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production. Appl Environ Microbiol 65: 31003107. Gutierrez S, Diez B, Alvarez E, Barredo JL & Martin JF (1991) Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzyl penicillin by the transformants. Mol Gen Genet 225: 5664. Gutierrez S, Velasco J, Fernandez FJ & Martin JF (1992) The cefG gene of Cephalosporium acremonium is linked to the cefEF gene and encodes a deacetylcephalosporin C acetyltransferase closely related to homoserine O-acetyltransferase. J Bacteriol 174: 30563064. Gutierrez S, Velasco J, Marcos AT, Fernandez FJ, Fierro F, Barredo JL, Diez B & Martin JF (1997) Expression of the cefG gene is limiting for cephalosporin biosynthesis in Acremonium chrysogenum. Appl Microbiol Biotechnol 48: 606614. Haavik HI & Froyshov O (1982) On the role of L-leucine in the control of bacitracin formation by Bacillus licheniformis. Peptide Antibiotics: Biosynthesis and Functions (Kleinkauf H & von Doehren H, eds), pp. 155159. Walter de Gruyter, Berlin. Hamlyn PF & Ball C (1979) Recombination studies with Cephalosporium acremonium. Genetics of Industrial Microorganisms (Sebek OK & Laskin AI, eds), pp. 185191. American Society for Microbiology, Washington, DC. Hammond JRM (1988) Brewery fermentation in the future. J Appl Bacteriol 65: 169177. Hanssen R & Kirby R (1983) The induction by N-methyl-N 0 nitro-nitrosoguanidine of multiple closely linked mutations in Streptomyces bikiniensis ISP5235 affecting streptomycin resistance and streptomycin biosynthesis. FEMS Microbiol Lett 17: 317320.
Hara O & Hutchinson CR (1992) A macrolide 3-Oacyltransferase gene from the midecamycin-producing species Streptomyces mycarofaciens. J Bacteriol 174: 51415144. van Hartinsveldt W, van Zeijl CM, Harteeld GM, et al. (1993) Cloning, characterization and overexpression of the phytaseencoding gene (phyA) of Aspergillus niger. Gene 127: 8794. Hashiguchi K, Takesada H, Suzuki E & Matsui H (1999) Construction of an L-isoleucine overproducing strain of Escherichia coli K-12. Biosci Biotechnol Biochem 63: 672679. Hersbach GJM, van der Beck CP & van Dijck PWM (1984) The penicillins: properties, biosynthesis, and fermentation. Biotechnology of Industrial Antibiotics (Vandamme EJ, ed), pp. 45140. Marcel Dekker, New York. Hershberger CL (1996) Metabolic engineering of polyketide biosynthesis. Curr Opin Biotechnol 7: 560562. Hesketh A & Ochi K (1997) A novel method for improving Streptomyces coelicolor A3(2) for production of actinorhodin by introduction of rpsL (encoding ribosomal protein S12) mutations conferring resistance to streptomycin. J Antibiot 50: 532535. Higashide E (1984) The macrolides; properties, biosynthesis, and fermentation. Biotechnology of Industrial Antibiotics (Vandamme EJ, ed), pp. 451509. Marcel Dekker, New York. Hirao T, Nakano T, Azuma T, Sugimoto M & Nakanishi T (1989) L-Lysine production in continuous culture of an L-lysine hyperproducing mutant of Corynebacterium glutamicum. Appl Microbiol Biotechnol 32: 269273. Holzman D (1994) Engineered yeasts available but not yet used for brewing. ASM News 60: 585. Hopwood DA (1983) Actinomycete genetics and antibiotic production. Biochemistry and Genetic Regulation of Commercially Important Antibiotics (Vining LC, ed), pp. 123. Addison Wesley, Reading, MA. Hopwood DA (1993) Genetic engineering of Streptomyces to create hybrid antibiotics. Curr Opin Biotechnol 4: 531537. Hopwood DA (1999) Forty years of genetics with Streptomyces: from in vivo to in vitro to in silico. Microbiology 145: 21832202. Hopwood DA, Malpartida F, Kieser HM, Ikeda H, Duncan J, Fujii I, Rudd BAM, Floss HG & Omura S (1985) Production of hybrid antibiotics by genetic engineering. Nature 314: 642644. Hosoya Y, Okamoto S, Muramatsu H & Ochi K (1998) Acquisition of certain streptomycin resistant (str) mutations enhances antibiotic production in bacteria. Antimicrob Agents Chemother 42: 20412047. Hu H & Ochi K (2001) Novel approach for improving the production of antibiotic-producing strains by inducing combined resistant mutations. Appl Environ Microbiol 67: 18851892. Hutchinson CR (1998) Combinatorial biosynthesis for new drug discovery. Curr Opin Microbiol 1: 319329. Hutchinson CR & Fujii I (1995) Polyketide synthase gene manipulation: a structure-function approach in engineering novel antibiotics. Annual Rev Microbiol 49: 201238.
c
207
Hwang Y-S, Kim E-S, Biro S & Choi C-Y (2003) Cloning and analysis of a DNA fragment stimulating avermectin production in various Streptomyces avermitilis strains. Appl Environ Microbiol 69: 12631269. Hwang CK, Kim HS, Hong YS, Kim YH, Hong SK, Kim SJ & Lee JJ (1995) Expresssion of Streptomyces peucetius genes for doxorubicin resistance and aklavinone 11-hydroxylase in Streptomyces galilaeus ATCC 31133 and production of a hybrid aclacinomycin. Antimicrob Agents Chemother 39: 16161620. Ichikawa T, Date M, Ishikura T & Ozaki A (1971) Improvement of kasugamycin-producing strain by agar piece method and the prototroph method. Folia Microbiol 16: 218224. Ikeda M & Katsumata R (1995) Tryptophan production by transport mutants of Corynebacterium glutamicum. Biosci Biotechnol Biochem 59: 16001602. Ikeda M & Katsumata R (1999) Hyperproduction of tryptophan by Corynebacterium glutamicum with the modied pentose pathway. Appl Environ Microbiol 65: 24972502. Ikeda H, Takada Y, Pang C-H, Tanaka H & Omura S (1993) Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis. J Bacteriol 175: 20772082. Ingram LO, Conway T, Clark DP, Sewell GW & Preston JF (1987) Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol 53: 24202425. Jaeger KE & Reetz MT (2000) Directed evolution of enantioselective enzymes for organic chemistry. Curr Opin Chem Biol 4: 6873. Jarai M (1961) Genetic recombination in Streptomyces aureofaciens. Acta Microbiol Acad Sci Hungary 8: 7379. Jin ZH, Lin JP, Xu ZN & Cen PL (2002) Improvement of industry-applied rifamycin B-producing strain, Amycolatopsis mediterranei, by rational screening. J Gen Appl Microbiol 48: 329334. Jin ZH, Wang MR & Cen PL (2002a) Production of teichoplanin by valine-analogue resistant mutant strains of Actinoplanes teichomyceticus. Appl Microbiol Biotechnol 58: 6366. Kacser H & Acerenza L (1993) A universal method for achieving increases in metabolite production. Eur J Biochem 216: 361367. Kantola J, Kunnari T, Mantsala P & Ylihonko K (2003) Expanding the scope of aromatic polyketides by combinatorial biosynthesis. Comb Chem High Throughput Screen 6: 501512. Kao CM, Luo GL, Katz L, Cane DE & Khosla C (1995) Manipulation of macrolide ring size by directed mutagenesis of a modular polyketide synthase. J Amer Chem Soc 117: 91059106. Karos M, Vilarino C, Bollschweiler C & Revuelta JL (2004) A genome-wide transcription analysis of a fungal rivoavin overproducer. J Biotechnol 113: 6976. Kase H, Odakura Y & Nakayama K (1982) Sagamycin and the related aminoglycosides: fermentation and biosynthesis. I. Biosynthetic studies with the blocked mutants of Micromonospora sagamiensis. J Antibiot 35: 19.
Katz L & Donadio S (1993) Polyketide synthesis: prospects for hybrid antibiotics. Annu Rev Microbiol 47: 875912. Keller U (1983) Highly efcient mutagenesis of Claviceps purpurea by using protoplasts. Appl Envir Microbiol 46: 580584. Kennedy J, Auclair K, Kendrew SG, Park C, Vederas JC & Hutchinson CR (1999) Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 284: 13681372. Kennedy J & Turner G (1996) d-(L-a-aminoadipyl)-L-cysteinylD-valine synthetase is a rate limiting enzyme for penicillin production in Aspergillus nidulans. Mol Gen Genet 253: 189197. Khetan A & Hu W-S (1999) Metabolic engineering of antibiotic biosynthetic pathways. Manual of Industrial Microbiology and Biotechnology (Demain AL & Davies JE, eds), pp. 717724. ASM Press, Washington, DC. Khetan A & Hu W-S (1999) Metabolic engineering of antibiotic biosynthesis for process improvement. Metabolic Engineering (Lee SY & Papoutsakis ET, eds), pp. 177202. Marcel Dekker, New York. Khosla C & Keasling JD (2003) Metabolic engineering for drug discovery and development. Nature Rev/Drug Disc 2: 10191025. Khosla C, McDaniel R, Ebert-Khosla S, Torres R, Sherman DH, Bibb MJ & Hopwood DA (1993) Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins. J Bacteriol 175: 21972204. Kibby JJ, McDonald IA & Rickards RW (1980) 3-Amino-5hydroxybenzoic acid as a key intermediate in ansamycin and maytansinoid biosynthesis. J Chem Soc, Chem Comm 768769. Kieser HM, Kieser T & Hopwood DA (1992) A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome. J Bacteriol 174: 54965507. Kim KS, Cho NY, Pai HS & Ryu DDY (1983) Mutagenesis of Micromonospora rosaria by using protoplasts and mycelial fragments. Appl Environ Microbiol 46: 689693. Kim HS, Hong YS, Kim YH, Yoo OJ & Lee JJ (1996) New anthracycline metabolites produced by the aklavinone 11hydroxylase gene in Streptomyces galilaeus ATCC 31133. J Antibiot 49: 355360. Kimura E (2003) Metabolic engineering of glutamate production. Adv Biochem Eng Biotechnol 79: 3757. Kinashi H & Shimaji M (1987) Detection of giant linear plasmids in antibiotic producing strains of Streptomyces by the OFAGE technique. J Antibiot 40: 913916. Kirimura K, Saragbin S, Rugsaseel S & Usami S (1992) Citric acid production by 2-deoxyglucose-resistant mutants of Aspergillus niger. Appl Microbiol Biotechnol 36: 573577. Kirst HA, Wild GM, Baltz RH, Seno ET, Hamill RL, Paschal JW & Dorman DE (1983) Elucidation of structure of novel macrolide antibiotics produced by mutant strains of Streptomyces fradiae. J Antibiot 36: 376382. Kitamura S, Kase H, Odakura Y, Iida T, Shirahata K & Nakayama K (1982) 2-Hydroxysagamicin: a new antibiotic produced by
c
208
mutational biosynthesis of Micromonospora sagamiensis. J Antibiot 35: 9497. Kitano K, Nozaki Y & Imada A (1985) Selective accumulation of unsulfated carbapenem antibiotics by sulfate transportnegative mutants of Streptomyces griseus subsp. cryophilus C19393. Agric Biol Chem 49: 677684. Koizumi S, Yonetani Y, Maruyama A & Teshiba S (2000) Production of riboavin by metabolically engineered Corynebacterium ammoniagenes. Appl Microbiol Biotechnol 51: 674679. Kojima I, Fukagawa Y, Okabe M, Ishikura T & Shibamoto N (1988) Mutagenesis of OA-6129 carbapenem-producing blocked mutants and the biosynthesis of carbapenems. J Antibiot 41: 899907. Komatsubara S, Taniguchi T & Kisumi M (1986) Overproduction of aspartase of Escherichia coli K-12 by molecular cloning. J Biotechnol 3: 281291. Kominek LA (1972) Biosynthesis of novobiocin by Streptomyces niveus. Antimicrob Agents Chemother 1: 123134. Kramer M, Bongaerts J, Bovenberg R, Kremer S, M ller U, Orf S, u Wubbolts M & Raeven L (2003) Metabolic engineering for microbial production of shikimic acid. Metab Eng 5: 277283. Kruse D, Kraemer R, Eggeling L, Rieping M, Pfefferle W, Tchieu JH, Chung YJ, Saier MH Jr & Burkorski A (2002) Inuence of threonine exporters on threonine production in Escherichia coli. Appl Microbiol Biotechnol 59: 205210. Kuchner O & Arnold FH (1997) Directed evolution of enzyme catalysis. Trends Biotechnol 15: 523530. Kurtzman AL, Govindarajan S, Vahle K, Jones JT, Heinrichs V & Patten PA (2001) Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins. Curr Opin Biotechnol 12: 361370. Kurzatkowski W, Kurylowicz W, Solecka J & Penyige A (1986) Improvement of Streptomyces strains by the regeneration of protoplasts. Biological, Biochemical and Biomedical Aspects of Actinomycetes, Part A (Szabo G, Biro S & Goodfellow M, eds), pp. 289292. Academiai Kiado, Budapest. Laffend LA, Nagarajan V & Nakamura CE (1996) Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism. Patent WO 96/53.796 (E. I. DuPont de Nemours and Genencor International). Lange C, Rittmann D, Wendisch VF, Bott M & Sahm H (2003) Global expression proling and physiological characterization of Corynebacterium glutamicum grown in the presence of Lvaline. Appl Environ Microbiol 69: 25212532. Lee JY, Hwang YS, Kim SS, Kim ES & Choi CY (2000) Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis. J Biosci Bioeng 89: 606608. Lee SH & Lee KJ (1995) Threonine dehydratases in different strains of Streptomyces fradiae. J Biotechnol 43: 95102. Lee J-C, Park H-R, Park D-J, Son KH, Yoon K-H, Kim Y-B & Kim C-J (2003) Production of teicoplanin by a mutant of Actinoplanes teicomyceticus. Biotechnol Lett 25: 537540.
Lee BK, Puar MS, Patel M, Bartner P, Lotvin J, Munayyer H & Waitz JA (1983) Multistep bioconversion of 20-deoxo-20dihydro-12, 13-deepoxy-12, 13-dehydrorosaranolide to 22hydroxy-23-o-mycinosyl-20-deoxo-20-dihydro-12, 13deepoxy-rosaramicin. J Antibiot 36: 742743. Legmann R & Margalith P (1983) Interspecic protoplast fusion of Saccharomyces cerevisiae and Saccharomyces mellis. Eur J Appl Microbiol Biotechnol 18: 320322. Lein J (1986) The Panlabs penicillin strain improvement program. Overproduction of Microbial Metabolites; Strain Improvement and Process Control Strategies (Vanek Z & Hostalek Z, eds), pp. 105139. Butterworth Publishers, Boston. Lemke JR & Demain AL (1976) Preliminary studies on streptomutin A. Eur J Appl Microbiol 2: 9194. Letisse F, Chevallereau P, Simon J-L & Lindley ND (2001) Kinetic analysis of growth and xanthan gum production with Xanthomonas campestris on sucrose, using sequentially consumed nitrogen sources. Appl Microbiol Biotechnol 55: 417422. Leung DW, Chen E & Goeddel DV (1989) A method for random mutagenesis of a dened DNA segment using a modied polymerase chain reaction. Technique 1: 1115. Levy-Schil S, Debussche L, Rigault S, Soubrier F, Bacchette F, Lagneaux D, Schleuniger J, Blanche F, Crouzet J & Mayaux JF (1993) Biotin biosynthetic pathway in a recombinant strain of Escherichia coli overexpressing bio genes: evidence for a limiting step upstream from KAPA. Appl Microbiol Biotechnol 38: 755762. Lewis MJ, Ragot N, Berlant MC & Miranda M (1990) Selection of astaxanthin-overproducing mutants of Phafa rhodozyma with b-ionone. Appl Environ Microbiol 56: 29442945. Liras P (1988) Cloning of antibiotic biosynthetic genes. Use of Recombinant DNA Techniques for Improvement of Fermentation Organisms (Thompson JA, ed), pp. 217253. CRC Press, Boca Raton, FL. Liu C-M (1982) Microbial aspects of polyether antibiotics: activity, production and biosynthesis. Polyether antibiotics. Naturally Occurring Acid Ionophores, Vol. 1. Biology (Westley JW, ed), pp. 43102. Marcel Dekker, New York. Locher CP, Soong NW, Whalen RG & Punnonen J (2004) Development of novel vaccines using DNA shufing and screening strategies. Curr Opi Mol Ther 6: 3439. Lombo F, Brana AF, Mendez C & Salas JA (1999) The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster. J Bacteriol 181: 642647. Lombo F, Pfeifer B, Leaf T, Ou S, Kim YS, Cane DE, Licari P & Khosla C (2001) Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering. Biotechnol Prog 17: 612617. Longacre A, Reimers JM, Gannon JE & Wright BE (1997) Flux analysis of glucose metabolism in Rhizopus oryzae for the purpose of increasing lactate yields. Fungal Genet Biol 21: 3039.
c
209
Luers F, Seyfried M, Daniel R & Gottschalk G (1997) Glycerol conversion to 1,3 propanediol by Clostridium pasteurianum: cloning and gene expression of the gene encoding 1,3 propanediol dehydrogenase. FEMS Microbiol Lett 154: 337335. Lum AM, Huang J, Hutchinson CR & Kao CM (2004) Reverse engineering of industrial pharmaceutical-producing actinomycete strains using DNA microarrays. Metab Eng 6: 186196. Lutz S, Ostermeier M, Moore GL, Maranas CD & Benkovic SP (2001) Creating multiple-crossover DNA libraries independent of sequence identity. Proc Natl Acad Sci USA 98: 1124811253. MacCabe AP, Riach MBR, Unkles SE & Kinghorn JR (1990) The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis. EMBO J 9: 279287. Madduri K, Waldrom M, Matsushima P, Broughton MC, Crawford K, Merlo DJ & Baltz RH (2001) Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa. J Indust Microbiol Biotechnol 27: 399402. Malik VS (1979) Genetics of applied microbiology. Adv Genet 29: 37126. Malmberg LH, Hu W-S & Sherman DH (1995) Effects of enhanced lysine epsilon-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus. Appl Microbiol Biotechnol 44: 198205. Mao Y, Varoglu M & Sherman DH (1999) Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564. Chem Biol 6: 251263. Marshall SH (2002) DNA shufing: induced molecular breeding to produce new generation long-lasting vaccines. Biotechnol Avd 20: 229238. Martin JF & Gil JA (1984) Cloning and expression of antibiotic production genes. Bio/Technology 2: 6372. Martin JF, Gutierrez S & Demain AL (1997) b-lactams. Fungal Biotechnology (Anke T, ed), pp. 91127. Chapmam & Hall, Weinheim. Martin JF, Naharro G, Liras P & Villanueva JR (1979) Isolation of mutants deregulated in phosphate control of candicidin biosynthesis. J Antibiot 32: 600606. Martin JR, Perun TJ & Girolami RL (1966) Studies on the biosynthesis of erythromycins. I. Isolation of an intermediate glycoside, 3-alpha-L-mycarosylerythronolide B. Biochemistry 5: 28522856. Martin JR & Rosenbrook WR (1967) Studies on the biosynthesis of erythromycins. II. Isolation and structure of a biosynthetic intermediate, 6-deoxyerythronolide B. Biochemistry 6: 435440. Masuda M, Takahashi K, Sakurai N, Yanagiya K, Komatsubara S & Tosa T (1995) Further improvement of D-biotin production by a recombinant strain of Serratia marcescens. Proc Biochem 30: 553562.
Mathison L, Soliday C, Stepan T, Aldrich T & Rambosek J (1993) Cloning, characterization, and use in strain improvement of the Cephalosporium acremonium gene cefG encoding acetyl transferase. Curr Genet 23: 3341. Matsuda A, Sugiura H, Matsuyama K, Matsumoto H, Ichikawa S & Komatsu K-I (1992) Molecular cloning of acetyl coenzyme A: deacetylcephalosporin C O-acetyltransferase cDNA from Acremonium chrysogenum: sequence and expression of catalytic activity in yeast. Biochem Biophys Res Commun 182: 9951001. Matthews PD & Wurtzel ET (2000) Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase. Appl Microbiol Biotechnol 53: 396400. Mayer H, Collins J & Wagner F (1980) Cloning of the penicillin G-acylase gene of Escherichia coli ATCC 11105 on multicopy plasmids. Enzyme Engineering, Vol. 5 (Weetall HH & Royer GP, eds), pp. 6169. Plenum, New York. McAlpine JB, Tuan JS, Brown DP, Grebner KB, Whittern DN, Buko A & Katz L (1987) New antibiotics from genetically engineered actinomycetes. I. 2-Norerythromycins, isolation and structural determinations. J Antibiot 40: 11151122. McCann AK & Barnett JA (1984) Starch utilization by yeasts: mutants resistant to carbon catabolite repression. Curr Genet 8: 525530. McCormick JRD (1965) Biosynthesis of the tetracyclines. Biogenesis of Antibiotic Substances (Vanek Z & Hostalek Z, eds), pp. 7391. Publishing House of the Czechoslovak Academy of Science, Prague. McDaniel R, Ebert-Khosla S, Hopwood D & Khosla C (1993a) Engineered biosynthesis of novel polyketides: manipulation and analysis of an aromatic polyketide synthase with unproven catalytic specicities. J Amer Chem Soc 115: 1167111675. McDaniel R, Ebert-Khosla S, Hopwood D & Khosla C (1993b) Engineered biosynthesis of novel polyketides. Science 262: 15461550. McDaniel R, Thamchaipenet A, Gustafsson C, Fu H, Betlach M, Betlach M & Ashley G (1999) Multiple genetic modications of the erythromycin polyketide synthase to produce a library of novel unnatural natural products. Proc Natl Acad Sci USA 96: 18461851. McDougall S & Neilands JB (1984) Plasmid- and chromosomecoded aerobactin synthesis in enteric bacteria: insertion sequences ank operon in plasmid-mediated systems. J Bacteriol 159: 300305. McGuire J, Thomas MC, Pandey RC, Toussaint M & White RJ (1981) Biosynthesis of daunorubicin glycosides: analysis with blocked mutants. Advances in Biotechnology, Vol. III, Fermentation Products (Moo-Young M, ed), pp. 117122. Pergammon Press, New York. McHenney MA & Baltz RH (1996) Gene transfer and transposition mutagenesis in Streptomyces roseosporus: mapping of insertions that inuence daptomycin or pigment production. Microbiology 142: 23632373.
c
210
Mendelovitz S & Aharonowitz Y (1983) b-Lactam antibiotic production by Streptomyces clavuligerus mutants impaired in regulation of aspartokinase. J Gen Microbiol 129: 20632069. Mendez C & Salas JA (2001) Altering the glycosylation pattern of bioactive compounds. Trends Biotechnol 19: 449456. Mermelstein LD, Papoutsakis ET, Petersen DJ & Bennett GN (1993) Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic operon. Biotechnol Bioeng 42: 10531060. Minas W, Brunker P, Kallio PT & Bailey JE (1998) Improved erythromycin production in a genetically engineered industrial strain of Saccharopolyspora erythraea. Biotechnol Prog 1: 561566. Mindlin SZ (1969) Genetic recombination in the actinomycete breeding. Genetics and Breeding of Streptomyces (Sermonti G & Alacevic M, eds), pp. 147159. Yugoslav Academy of Sciences and Arts, Zagreb. Miyagawa K, Kimura H, Nakahama K, Kikuchi M, Doi M, Akiyama S & Nakao Y (1986) Cloning of the Bacillus subtilis IMP dehydrogenase gene and its application to increased production of guanosine. Bio/Technology 4: 225228. Mondou F, Shareck F, Morosoli R & Kleupfel D (1986) Cloning of the xylanase gene of Streptomyces lividans. Gene 49: 323329. Moon YH, Tanabe T, Funahashi T, Shiuchi K, Nakao H & Yamamoto S (2004) Identication and characterization of two contiguous operons required for aerobactin transport and biosynthesis in Vibrio mimicus. Microbiol Immunol 48: 389398. Morbach S, Sahm H & Eggeling L (1996) L-Isoleucine production with Corynebacterium glutamicum: further ux increase and limitation of export. Appl Environ Microbiol 62: 43454351. Mori M & Shiio I (1983) Glutamate transport and production in Brevibacterium avum. Agric Biol Chem 47: 983990. Motamedi H, Wendt-Pientowski E & Hutchinson CR (1986) Isolation of tetracenomycin C non-producing Streptomyces glaucescens mutants. J Bacteriol 167: 575580. Nagaoka K & Demain AL (1975) Mutational biosynthesis of a new antibiotic, streptomutin A, by an idiotroph of Streptomyces griseus. J Antibiot 28: 627635. Nakamura CE & Whited GM (2003) Metabolic engineering for the microbial production of 1,3-propanediol. Curr Opin Biotechnol 14: 16. Nakatsukasa WM & Mabe JA (1978) Galactose induced colonial dissociation in Streptomyces aureofaciens. J Antibiot 31: 805808. Ness JE, Welch M, Giver L, Bueno M, Cherry JR, Borchert TV, Stemmmer WP & Minshull J (1999) DNA shufing of subgenomic sequences of subtilisin. Nature Biotechnol 17: 893896. Ness JE, del Cardayre SB, Minshull J & Stemmer WP (2000) Molecular breeding: the natural approach to protein design. Adv Protein Chem 55: 261292.
Neufeld RJ, Peleg Y, Rokem JS, Pines O & Goldberg I (1991) LMalic acid formation by immobilized Saccharomyces cerevisiae amplied for fumarase. Enzyme Microb Technol 13: 991996. Nielsen J (2001) Metabolic engineering. Appl Microbiol Biotechnol 55: 263283. Niemi J & Mantsala P (1995) Nucleotide sequences and expression of genes from Streptomyces purpurescens that cause the production of new anthracyclines in Streptomyces galilaeus. J Bacteriol 177: 29422945. Nissen TL, Kielland-Brandt MC, Nielsen J & Villadsen J (2000) Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng 2: 6977. Nozaki Y, Kitano K & Imada I (1984) Blocked mutants in the biosynthesis of carbapenem antibiotics from Streptomyces griseus subsp. cryophilus. Agric Biol Chem 48: 3744. Ohnishi J, Mitsuhashi S, Hayashi M, Ando S, Yokoi H, Ochiai K & Ikeda M (2002) A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. Appl Microbiol Biotechnol 58: 217223. Okamoto S, Lezhava A, Hosaka T, Okamoto-Hosoya Y & Ochi K (2003) Enhanced expression of S-adenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2). J Bacteriol 185: 601609. Okamoto-Hosoya Y, Sato T & Ochi K (2000) Resistance to paromomycin is conferred by rpsL mutations, accompanied by an enhanced antibiotic production in Streptomyces coelicolor A3(2). J Antibiot 53: 14241427. Okanishi M, Suzuki N & Furuta T (1996) Variety of hybrid characters among recombinants obtained by interspecic protoplast fusion in streptomycetes. Biosci Biotechnol Biochem 60: 12331238. Omura S, Ikeda H & Tanaka H (1991) Selective production of specic components of avermectins in Streptomyces avermitilis. J Antibiot 44: 560563. Otten SL, Stutzman-Engwall J & Hutchinson CR (1990) Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp. caisius. J Bacteriol 172: 34273434. ONeill GP, Kilburn DG, Warren RAJ & Miller RC Jr. (1986) Overproduction from a cellulase gene with a high guanosineplus-cytosine content in Escherichia coli. Appl Environ Microbiol 52: 737743. Pacey MS, Dirlam JP, Geldart RW, Leadlay PF, McArthur HA, McCormick EL, Monday RA, OConnell TN, Staunton J & Winchester TJ (1998) Novel erythromycins from a recombinant Saccharopolyspora erythraea strain NRRL 2338pIG1. I. Fermentation, isolation and biological activity. J Antibiot 51: 10291034. Palva I (1982) Molecular cloning of a-amylase gene from Bacillus amyloliquefaciens and its expression in Bacillus subtilis. Gene 19: 8187.
c
211
Panchal CJ, Harbison A, Russell I & Stewart GG (1982) Ethanol production of genetically modied strains of Saccharomyces. Biotechnol Lett 4: 3338. Parekh S, Vinci VA & Strobel RJ (2000) Improvement of microbial strains and fermentation processes. Appl Microbiol Biotechnol 54: 287301. Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer WPC, Ryan CM & del Cardayre S (2002) Genome shufing of Lactobacillus for improved acid tolerance. Nature Biotechnol 20: 707712. Patten PA, Howard RJ & Stemmer WP (1997) Applications of DNA shufing to pharmaceuticals and vaccines. Curr Opin Biotechnol 8: 724733. Pearce CJ, Akhtar M, Barnett JEG, Mercier D, Sepulchre A-M & Gero S (1978) Sub-unit assembly in the biosynthesis of neomycin. The synthesis of 5-O-b-D-ribofuranosyl and 4-Ob-D-ribofuranosyl-2,6-dideoxystreptamines. J Antibiot 31: 7481. Penttil ME, Suihko M-L, Lehtinen U, Nikkola M & Knowles JKC a (1987) Construction of brewers yeasts secreting fungal endob-glucanase. Curr Genet 12: 413420. Penzikova GA & Levitov MM (1970) A study on transamidinase activity with respect to streptomycin biosynthesis. Biologiya 39: 337342. Perez-Diaz JC & Clowes RC (1980) Physical characterization of plasmids determining synthesis of a microcin which inhibits methionine synthesis in Escherichia coli. J Bacteriol 141: 10151023. Perkins JB & Pero J (1993) Bacillus subtilis and Other Gram Positive Bacteria: Biochemistry, Physiology and Molecular Genetics (Sonenshein AL, ed. in Chief), pp. 319334. ASM Press, Washington, DC. Perkins JB, Sloma A, Hermann T, et al. (1999) Genetic engineering of Bacillus subtilis for the commercial production of riboavin. J Indust Microbiol Biotechnol 22: 818. Pfeifer BA, Admiraal SJ, Gramajo H, Cane DE & Khosla C (2001) Biosynthesis of complex polyketides in a metabolically engineered strain of Escherichia coli. Science 291: 17901792. Pfeifer BA & Khosla C (2001) Biosynthesis of polyketides in heterologous hosts. Microbiol Molec Biol Revs 65: 106118. Picataggio S, Rohver T, Deander K, Lanning D, Reynolds R, Mielenz J & Eirich LD (1992) Metabolic engineering of Candida tropicalis for the production of long-chain dicarboxylic acids. Bio/Technology 10: 894898. Pina A, Calderon IL & Benitez T (1986) Intergeneric hybrids of Saccharomyces cerevisiae and Zygosaccharomyces fermentati obtained by protoplast fusion. Appl Environ Microbiol 51: 9951003. Podojil M, Blumauerova M, Culik K & Vanek Z (1984) The tetracycyclines: properties, biosynthesis and fermentation. Biotechnology of Industrial Antibiotics (Vandamme EJ, ed), pp. 259279. Marcel Dekker, New York. Pospisil S, Kopecky J, Prikrylova V & Spizek J (1999) Overproduction of 2-ketoisovalerate and monensin production by regulatory mutants of Streptomyces
cinnamonensis resistant to 2-ketobutyrate and amino acids. FEMS Microbiol Lett 172: 197204. Pospisil S, Peterkova M, Krumphanzl V & Vanek Z (1984) Regulatory mutants of Streptomyces cinnamonensis producing monensin A. FEMS Microbiol Lett 24: 209213. Potera C (1997) Genencor & DuPont create green polyester. Gen Eng News 17(11): 17. Pramik MJ (1986) Genentech develops recombinant technique for producing vitamin C. Gen Eng News 2(6): 912. Radmacher A, Vaitsikova A, Burger U, Krumbach K, Sahm H & Eggeling L (2002) Linking central metabolism with increased pathway ux: L-valine accumulation by Corynebacterium glutamicum. Appl Environ Microbiol 68: 22462250. Raillard S, Krebber A, Chen Y, et al. (2001) Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes. Chem Biol 8: 891898. Rancount DE, Stephenson JT, Vickell GA & Wood JM (1984) Proline excretion by Escherichia coli K12. Biotechnol Bioeng 26: 7480. Reeves CD (2003) The enzymology of combinatorial biosynthesis. Crit Rev Biotechnol 23: 95147. Reidhaar-Olson J, Bowie J, Breyer RM, Hu JC, Knight KL, Lim WA, Mossing MC, Parsell DA, Shoemaker KR & Sauer RT (1991) Random mutagenesis of protein sequences using oligonucleotide cassettes. Methods Enzymol 208: 564586. Roberts M, Leavitt RW, Carbonetti NH, Ford S, Cooper RA & Williams PH (1986) RNA-DNA hybridization analysis of transcription of the plasmid ColV-K30 aerobactin gene cluster. J Bacteriol 167: 467472. Rodriguez E & McDaniel R (2001) Combinatorial biosynthesis of antimicrobials and other natural products. Curr Opin Microbiol 4: 526534. Rohlin L, Oh MK & Liao JC (2001) Microbial pathway engineering for industrial processes: evolution, combinatorial biosynthesis and rational design. Curr Opin Microbiol 4: 350355. Roth JR & Ames BN (1966) Histidine regulatory mutants in Salmonella typhimurium II. Histidine regulatory mutants having altered histidyl-tRNA synthetase. J Mol Biol 22: 325334. Ryu DDY, Kim KS, Cho NY & Pai HS (1983) Genetic recombination in Micromonospora rosaria by protoplast fusion. Appl Environ Microbiol 45: 18541858. Sahm H, Eggeling L & de Graaf AA (2000) Pathway analysis and metabolic engineering in Corynebacterium glutamicum. Biol Chem 381: 899910. Saito Y, Ishii Y, Hayashi H, et al. (1997) Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-ketogulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain. Appl Environ Microbiol 63: 454460. Sakurai N, Imai Y, Masuda M, Komatsubara S & Tosa T (1994) Improvement of a d-biotin-hyperproducing recombinant strain of Serratia marcescens. J Biotechnol 36: 6373.
c
212
Schreferl-Kunar G, Grotz M, Roehr M & Kubicek CP (1989) Increased citric acid production by mutants of Aspergillus niger with increased glycolytic capacity. FEMS Microbiol Lett 59: 297300. Shibasaki T, Hashimoto S, Mori H & Ozaki A (2000) Construction of a novel hydroxyproline-producing recombinant Escherichia coli by introducing a proline 4-hydroxylase gene. J Biosci Bioeng 90: 522525. Shier WT, Rinehart KL Jr & Gottlieb D (1969) Preparation of four new antibiotics from a mutant of Streptomyces fradiae. Proc Natl Acad Sci USA 63: 198204. Shiio I, Mori M & Ozaki H (1982) Amino acid aminotransferases in an amino acid-producing bacterium, Brevibacterium avum. Agric Biol Chem 46: 29672977. Shoemaker S, Schweickart V, Ladner M, Gelfand D, Kwok S, Myambo K & Innis M (1983) Molecular cloning of exocellobiohydrolase I derived from Trichoderma reesei strain L27. Bio/Technology 1: 691696. Sills AM, Zygora PSJ & Stewart GG (1984) Characterization of Schwanniomyces castelli mutants with increased productivity of amylases. Appl Microbiol Biotechnol 20: 124128. Simpson IN & Caten CE (1979) Induced quantitative variation for penicillin titre in clonal populations of Aspergillus nidulans. J Gen Microbiol 110: 112. Skandalis A, Encell LP & Loeb LA (1997) Creating novel enzymes by applied molecular evolution. Chem Biol 4: 889898. Skatrud PL (1992) Genetic engineering of a beta-lactam antibiotic biosynthetic pathway in lamentous fungi. Trends Biotechnol 10: 324329. Skatrud PL, Fisher DL, Ingolia TD & Queener SW (1987) Improved transformation of Cephalosporium acremonium. Genetics of Industrial Microorganisms, Part B (Alacevic M, Hranueli D & Toman Z, eds), pp. 111119. Pliva, Zagreb. Skatrud PL, Tietz AJ, Ingolia TD, Cantwell CA, Fisher DL, Chapman JL & Queener SW (1989) Use of recombinant DNA to improve production of cephalosporin C by Cephalosporium acremonium. Bio/Technology 7: 477485. Smith A (1987) Enzyme regulation of desferrioxamine biosynthesis: a basis for a rational approach to process development. Fifth International Symposium on the Genetics of Industrial Microorganisms, 1986 (Alacevic M, Hranueli D & Toman Z, eds), pp. 513527. Pliva, Zagreb. Smith DJ, Bull JH, Edwards J & Turner G (1989) Amplication of the isopenicillin N synthetase gene in a strain of Penicillium chrysogenum producing high levels of penicillin. Mol Gen Genet 216: 492497. Smith DJ, Burnham MKR, Bull JH, Hodgson JE, Ward JM, Browne P, Brown J, Barton B, Earl AJ & Turner G (1990) bLactam antibiotic biosynthesis genes have been conserved in clusters in prokaryotes and eukaryotes. EMBO J 9: 741747. Solenberg PJ, Cantwell CA, Tietz AJ, McGilvray D, Queener SW & Baltz RH (1996) Transposition mutagenesis in Streptomyces fradiae: identication of a neutral site for the stable insertion of DNA by transposon exchange. Gene 16: 6772.
Sone H, Fujii T, Kondo K, Shimizu F, Tanaka J-I & Inoue T (1988) Nucleotide sequence and expression of Enterobacter aerogenes alpha-acetolactate decarboxylase gene in brewers yeast. Appl Environ Microbiol 54: 3842. Song JK & Rhee JS (2001) Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution. Biochim Biophys Acta 1547: 370378. Spagnoli R, Cappalletti L & Toscano L (1983) Biological conversion of erythronolide B, an intermediate of erythromycin biogenesis, into new hybrid macrolide antibiotics. J Antibiot 36: 365375. Stachelhaus T, Schneider A & Marahiel MA (1995) Rational design of peptide antibiotics by targeted replacement of bacterial and fungal domains. Science 269: 6972. Stahmann KP, Revuelta JL & Seulberger H (2000) Three biochemical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboavin production. Appl Microbiol Biotechnol 53: 509516. Staunton J (1998) Combinatorial biosynthesis of erythromycin and complex polyketides. Curr Opin Chem Biol 2: 339345. Stemmer WP (1994) Rapid evolution of a protein in vitro by DNA shufing. Nature 370: 389391. Stephanopoulos G (1999) Metabolic uxes and metabolic engineering. Metab Eng 1: 111. Stephanopoulos G, Alper H & Moxley J (2004) Exploiting biological complexity for strain improvement through systems biology. Nature Biotechnol 22: 12611267. Strohl WR (2001) Biochemical engineering of natural product biosynthesis pathways. Metab Eng 3: 414. Strohl WR, Bartel PL, Li Y, Connors NC & Woodman RH (1991) Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes. J Indust Microbiol 7: 163174. Stutzman-Engwall K, Conlon S, Fedechko R, Kaczmarek F, McArthur H, Krebber A, Chen Y, Minshull J, Raillard SA & Gustafsson C (2003) Engineering the aveC gene to enhance the ratio of doramectin to its CHC-B2 analogue produced in Streptomyces avermitilis. Biotechnol Bioeng 82: 359369. Suenaga H, Mitsokua M, Ura Y, Watanabe T & Furukawa K (2001) Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes. J Bacteriol 183: 54415444. Sybesma W, Burgess C, Starrenburg M, van Sinderen D & Hugenholtz J (2004) Multivitamin production in Lactococcus lactis using metabolic engineering. Metab Eng 6: 109115. Taguchi S, Ozaki A & Momose H (1998) Engineering of a coldadapted protease by sequential random mutagenesis and a screening system. Appl Environ Microbiol 64: 492495. Takebe H, Imai S, Ogawa H, Satoh A & Tanaka H (1989) Breeding of bialaphos producing strains from a biochemical engineering viewpoint. J Ferm Bioeng 67: 226232. Takeda K, Aihara K, Furumai T & Ito Y (1978) An approach to the biosynthetic pathway of butirosins and the related antibiotics. J Antibiot 31: 250253.
c
213
Tani Y, Lim W-J & Yang H-C (1988) Isolation of an Lmethionine-enriched mutant of a methylotrophic yeast, Candida boidinii No. 2201. J Ferm Technol 66: 153158. Teeri T, Salovuori I & Knowles J (1983) The molecular cloning of the major cellulase gene from Trichoderma reesei strain L27. Bio/Technology 1: 696699. Teshiba S & Furuya A (1983) Mechanisms of 5 0 -inosinic acid accumulation by permeability mutants of Brevibacterium ammoniagenes. II. Sensitivities of a series of mutants to various drugs. Agric BiolChem 47: 10351041. Theilgaard HA, van den Berg MA, Mulder CA, Bovenberg RAL & Nielsen J (2001) Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes. Biotechnol Bioeng 72: 379388. Thompson CJ, Ward JM & Hopwood DH (1982) Cloning of antibiotic resistance and nutritional genes in streptomycetes. J Bacteriol 151: 668677. Thykaer J & Nielsen J (2003) Metabolic engineering of b-lactam production. Metab Eng 5: 5669. Tobin MB, Gustafsson C & Huisman GW (2000) Directed evolution: the rational basis for irrational design. Curr Opin Struct Biol 10: 421427. Tong I-T, Liao JJ & Cameron DC (1991) 1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha region. Appl Environ Microbiol 57: 35413546. Traxler P, Schupp T & Wehrli W (1982) 16,17-dihydrorifamycin S and 16,17-dihydro-17-hydroxyrifamycin S, two novel rifamycins from a recombinant strain C5/42 of Nocardia mediterranei. J Antibiot 35: 594601. Trefzer A, Blanco G, Remsing L, et al. (2002) Rationally designed glycosylated premithramycins: hybrid aromatic polyketides using genes fom three different biosynthetic pathways. J Amer Chem Soc 124: 60566062. Troost T & Katz E (1979) Phenoxazinone biosynthesis: accumulation of a precursor, 4-methyl-3-hydroxyanthranilic acid, by mutants of Streptomyces parvulus. J Gen Microbiol 11: 121132. Tseng YH, Ting WY, Chou HC, Yang BY & Chun CC (1992) Increase of xanthan production by cloning xps genes into wildtype Xanthomonas campestris. Lett Appl Microbiol 14: 4346. Tsoi CJ & Khosla C (1995) Combinatorial biosynthesis of unnatural natural productsthe polyketide example. Chem Biol 2: 355362. Unowsky J & Hoppe DC (1978) Increased production of the antibiotic aurodox (X-5108) by aurodox-resistant mutants. J Antibiot 31: 662666. Vaughn RV, Lotvin J, Puar MS, Patel M, Kershner A, Kalyanpur MG, Marquez J & Waitz JA (1982) Isolation and characterization of two 16-membered lactones: 20deoxorosaramicin and 20-deoxo-12,13-desepoxy-12,13dehydrorosaramicin aglycones, from a mutant strain of Micromonospora rosaria. J Antibiot 35: 251253. Velasco J, Adrio JL, Moreno MA, Diez B, Soler G & Barredo JL (2000) Environmentally safe production of 7-
aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum. Nature Biotechnol 18: 857861. Vinci VA & Byng G (1999) Strain improvement by nonrecombinant methods. Manual of Industrial Microbiology and Biotechnology, 2nd edn (Demain AL & Davies JE, eds), pp. 103113. ASM Press, Washington, DC. Vinci VA, Hoerner TD, Coffman AD, Schimmel TG, Dabora RL, Kirpekar AC, Ruby CL & Stieber RW (1991) Mutants of a lovaststin-hyperproducing Aspergillus terreus decient in the production of sulochrin. J Indust Microbiol 8: 113120. Visser H, van Ooyen AJ & Verdoes JC (2003) Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous. FEMS Yeast Res 4: 221231. Voegtli M, Chang PC & Cohen SN (1994) afsR2: a previously undetected gene encoding a 63-amino acid protein that stimulates antibiotic production in Streptomyces lividans. Mol Microbiol 14: 643653. Wackett LP (1997) Bacterial biocatalysis: stealing a page from natures book. Nature Biotechnol 15: 415416. Wang MR, Ding H & Hu YJ (1996) The action of arginine and valine in the biosynthesis of teicoplanin. Chin J Antibiot 21(suppl): 7780. Wang GY & Keasling JD (2003) Amplication of HMG-CoA reductase production enhances carotenoid accumulation in Neurospora crassa. Metab Eng 43: 193201. Weber JM, Leung JO, Swanson SJ, Idler KB & McAlpine JB (1991) An erythromycin derivative produced by targeted gene disruption in Saccharopolyspora erythraea. Science 252: 114117. Wesseling AC & Lago B (1981) Strain improvement by genetic recombination of cephamycin producers, Nocardia lactamdurans and Streptomyces griseus. Devel Indust Microbiol 22: 641651. Whiteley HR & Schnepf HE (1986) The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Ann Rev Microbiol 40: 549576. Wittmann C & Heinzle E (2002) Geneology proing through strain improvement using metabolic network analysismetabolic ux geneology of several generations of lysine producing corynebacteria. Appl Environ Microbiol 68: 58435859. Wohlert S-E, Blanco G, Lombo F, et al. (1998) Novel hybrid tetracenomycins through combinatorial biosynthesis using a glycosyltransferase encoded by the elm-genes in cosmid 16F4 and which shows a very broad sugar substrate specicity. J Amer Chem Soc 120: 1059610601. Xue Q, Hutchinson CR & Santi DV (1999) A multi-plasmid approach to preparing large libraries of polyketides. Proc Natl Acad Sci USA 96: 1174011745. Ylihonko K, Hakala J, Kunari T & Mantsala P (1996) Production of hybrid anthracycline antibiotics by heterologous expression of Streptomyces nogalater nogalamycin biosynthesis genes. Microbiology 142: 19651972.
c
214
Yoneda Y (1980) Increased production of extracellular enzymes by the synergistic effect of genes introduced into Bacillus subtilis by stepwise transformation. Appl Environ Microbiol 39: 274276. Yoshimoto A, Matsuzawa Y, Matsuhashi Y, Oki T, Takeuchi T & Umezawa H (1981) Trisarubicinol, new antitumor anthracycline antibiotic. JAntibiot 34: 14921494. Yue S, Motamedi H, Wendt-Pienskowski E & Hutchinson CR (1986) Anthracycline metabolites of tetracenomycinnon-producing Streptomyces glaucescens. J Bacteriol 167: 581586. Yukawa H, Kurusu Y, Shimazu M, Yamagata H & Teresawa M (1988) Stabilization of an Escherichia coli plasmid by a mini-F fragment of DNA. J Indust Microbiol 2: 323328. Zamboni N, Mouncey N, Hohman HP & Sauer U (2003) Reducing maintenance metabolism by metabolic engineering of respiration improves rivoavin production by Bacillus subtilis. Metab Eng 5: 4955.
Zhang Y-X, Perry K, Vinci VA, Powell K, Stemmer WPC & del Cardayre SB (2002) Genome shufing leads to rapid phenotypic improvement in bacteria. Nature 415: 644646. Zhao L, Ahlert J, Xue Y, Thorson JS, Sherman DH & Liu H-W (1999) Engineering a methymycin/pikromycin-calicheamicin hybrid: construction of two new macrolides carrying a designed sugar moiety. J Amer Chem Soc 121: 98819882. Zhao H & Arnold FH (1997) Optimization of DNA shufing for high delity recombination. Nucleic Acids Res 25: 13071308. Zhao H, Chockalingam K & Chen Z (2002) Directed evolution of enzymes and pathways for industrial biocatalysis. Curr Opin Biotechnol 13: 104110. Zhou S, Causey TB, Hasona A, Shanmugam KT & Ingram LO (2003) Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl Environ Microbiol 69: 399407.
c

Genetic Improvement of Processes Yielding Microbial Products

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Genetic Improvement of Processes Yielding Microbial Products

Uploaded by

Copyright:

Available Formats

Genetic improvement of processes yielding microbial products

Jose L. Adrio1 & Arnold L. Demain2

J. L. Adrio & A. L. Demain

Mutants producing increased quantites of metabolites