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[Agr. Biol. Chem., Vol. 29, No. 2, p.

83-89, 1965]

Studies on Amino Acid Fermentation

Part XI. Effect of Biotin on the Embden-Meyerhof-Parnas Pathway and


the Hexose-Monophosphate Shunt in a Glutamic Acid-Producing
Bacterium, Brevibacterium ammoniagenes 317-1

By Kunio OISHI and Ko AIDA


Instituteof AppliedMicrobiology,
University
of Tokyo,Tokyo
ReceivedNovember9, 1963

The effect of biotin on glucose catabolism in a L-glutamic acid-producing bacterium,


Brevibacterium ammoniagenes, was studied by a modified radiorespirometric method. The
amount of glucose metabolized by way of the hexose-monophosphate shunt (HMP) was
estimated to be 26% in biotin-deficient cells and 3800 in biotin-sufficient cells, respectively.
In both cases, the Embden-Meyerhof-Parnas Pathway (EMP) was found to be the major
route for glucose catabolism. These results will exclude the present theory at least in our
strain that the main route of glucose catabolism is HMP in biotin-deficient cells and EMP
in biotin-sufficient ones, and that this is one of the main effect of biotin on L-glutamic
acid fermentation.

INTRODUCTION cally; namely, the highest yield was obtained

It has been well known that biotin is a key on its suboptimal level, while only very small

substance playing a very important role in amount of L-glutamic acid was accumulated

L-glutamic acid fermentation. Biotin is always on its sufficient one.1,9,13•`24)

required as a growth factor by a number of One of the most important theories on the

bacteria so far as studied which accumulate role of biotin in L-glutamic acid fermentation

a large amount of L-glutamic acid.1•`12) Further which is now generally accepted is as follows:

more, the amount of biotin added changes


13) K. Nakayama, S. Kitada and S. Kinoshita, Amino
the rate of L-glutamic acid formation drasti Acids, 2, 77 (1960).
1) K. Tanaka, T. Iwasaki and S. Kinoshita, J. Agr. Chem. 14) K. Tanaka, S. Akita, K. Kimura and S. Kinoshita,
Soc. Japan, 34, 593 (1960). J. Agr. Chem. Soc. Japan, 34, 600 (1960).
2) T. Chen and F. Chen. Hakko Kogaku Zasshi, 37, 307 15) H. Samejima, T. Nara, C. Fujita and S. Kinoshita,
(1959). ibid., 874 (1960).
3) K. Kobayashi, N. Nunoko, K. Sato and N. Ogawa, 16) K. Nakayama, S. Kitada and S. Kinoshita Amino
ibid., 440 (1959). Acids, 2, 105 (1960).
4) C. Ogawa, M. Oide and Y. Midorikawa, Amino Acids, 17) K. Nakayama, Z. Sato and S. Kinoshita, J. Agr. Chem.
1, 45 (1958). Soc. Japan, 35, 142 (1961).
5) S. Kinoshita and S. Itagaki, ibid., 2, 42 (1960). 18) K. Nakayama, Z. Sato and S. Kinoshita ibid., 146
6) K. C. Chao and J. W. Foster, J. Bacteriol., 77, 715 (1959). (1961.)
7) Y. Hirose and K. Yamada, Amino Acids, 3, 21 (1961). 19) K. Nakayama, S. Kitada and S. Kinoshita, J. Gen.
8) S. Kids, W. Hashida and S. Teramoto, Hakko Kogaku Appl. Microbiol., 7, 52 (1961).
Zasshi, 39, 403 (1961). 20) K. Nakayama, S. Kitada and S. Kinoshita, ibid., 145
9) K. Oishi, K. Aida and T. Asai, J. Agr. Chem. Soc. (1961).
21) H. Okada, I. Kameyama, S. Okumura and T. Tsunoda,
Japan, 35, 855 (1961). ibid., 177 (1961).
10) T. Tsunoda, I. Shiio and K. Mitsugi, J. Gen. Appl.
Microbiol., 7, 18 (1961). 22) Y. Nakagiri, R. Ueda, H. Jose, W. Hashida and S.
11) S. Okumura, R. Tsugawa, T. Tsunoda, K. Kono, T. Teramoto, Hakko Kogaku Zasshi, 39, 649 (1961).
Matsui and N. Miyachi, J. Agr. Chem. Soc. Japan, 36, 141 23) K. Tanaka, Y. Nakajima and S. Kinoshita, Amino
(1962). Acids, 6, 29 (1962).
12) K. Miyai, I. Tsuruo, R. Kodaira, S. Hayakawa, I. Aki 24) K. Ohima, K. Tanaka and S. Kinoshita, ibid., 7, 73
moto and K. Goto, ibid., 37, 32 (1963). (1963).
84 Kunio OISHI and Ko AIDA

Main route of glucose catabolism is HMP in ruvic acid to L-glutamic acid, and the role of

biotin-deficient cells, and NADPH produced biotin was not referred to.

by the action of glucose-6-phosphate and 6- The main object of the present experiments

phosphogluconate dehydrogenases accelerates is to check the above mentioned theory by

NADPH-linked coupling reaction necessary using a modified radiorespirometric method.

for the formation of L-glutamic acid. In the METHODS AND MATERIALS


case of biotin-sufficient cells, main route of
Organism
glucose catabolism is EMP and lactic acid is Brevibacterium ammoniagenes was used throughout the
accumulated instead Of L-glutamic acid.26,27) experiments, which was newly isolated from soil and
But the experiments which support the above identified in our laboratory after Bergey's Manual,
theory do not seem to be so precisely accom 7th Ed
plished. Isotopic experiments will be neces Culture Conditions
sary for the clarification of this subject. The culture medium used contained glucose, 5%;
An isotopic experiment concerning this sub urea, 0.8%; KH2PO4, 0.1%; MgSO4•E7H2O, 0.05%,

ject was presented by Shiio et al.28) They L-cystine•EHCl, 0.01%; FeSO4•E7H2O, 0.0005%; MnSO4

concluded that glucose catabolism by HMP •E nH2O, 0.0005%, and biotin (pH 7.0). Biotin content

was approximately 10% in the case of Brevi was 0.51ƒÊg/l in biotin-deficient medium (DM) and

bacteriumflavum. 20ƒÊg/l in biotin-sufficient one (SM). Incubation was

Their experiment, however, was carried carried out for 20•`24hrs at 30•Ž on a reciprocal

out only in the presence of arsenite, which shaker (120r.p.m.; 7cm stroke). The grown cells were

harvested, washed twice with distilled water, and used


might change the natural catabolism of glucose for the experiments
by stopping the further metabolic flow of py- . 14C-compounds
Glucose-1- and -6-14C were purchased from the

Daiichi Pure Chemicals Co., Ltd. The purity was

estimated chromatographically by using two solvents

(BuOH:AcOH:H2O=4:1:5, water saturated phenol).

Radiorespirometry

The method used was in principle the same as that

developed by Wang et al.29) The apparatus and the

conditions are, however, arranged to be quite similar

to those used in the test tube-scale glutamic acid

fermentation and not as described by them, avoiding

that the results vary with the difference in experi

mental conditions. (Fig. 1) Cells were inoculated to

the fermentation medium or the reaction mixture

containing labeled glucose in the test tubes and in

cubated at 30°C on a shaker aerating at a rate of 25-

30 ml/min. Respiratory CO2 evolved was trapped

with 10% NaOH solution at several intervals and the

radioactivity was determined as Ba14CO3 with a

Geiger-Muller tube.

FIG. 1. The Apparatus of Radiorespirometry Analytical Methods

L-Glutamic acid was determined by the bioassay


25) S. Kinoshita, K. Tanaka, S. Udaka and S. Akita, In
tern. Symp. E„z. Chem., 2, 464 (1957). method using Lactobacillus arabinosus 17-5. Lactic
26) K. Tanaka, S. Akita, K. Kimura and S. Kinoshita, acid was measured by Barker and Summerson's
Amino Acids, 1, 62 (1959).
27) S. Kinoshita, Adv. Appl. Microbiol., 1, 201 (1959). 29) C. H. Wang, 1. Stern, C. M. Gilmour, S. Klungsoyr,
28) I. Shiio, S. Otsuka and T. Tsunoda, J. Bioche,n., 47, D. J. Reed, J. J. Bialy, B. E. Christensen and V. H. Cheldelin,
414 (1960). J. Bacteriol., 76, 207 (1958).
Studies on Amino Acid Fermentation. Part XI 85

method modified by Ishii.30) ƒ¿-Ketoglutaric acid was TABLE II. CO2 EVOLUTION FROM BIOTIN-
estimated by Friedemann and Haugen's method DEFICIENT CULTURE
modified by Shimizu.31)

RESULTS

1. Several Amino and Organic Acids Formation

from Glucose.

Prior to the isotopic experiments, the yields


,of several amino and organic acids produced

from glucose by the cells grown on DM and


'SM were determined
. (Table I) Biotin con-

tents in growth media had a great influence

on the yields of L-glutamic and ƒ¿-ketoglutaric

acids but not on those of pyruvic and lactic

acids.

TABLE I. SEVERAL AMINO AND ORGANIC ACID


S FORMATION FROM GLUCOSE

was formed in 49 hrs. No other product ex

cept a trace of ƒ¿-ketoglutaric acid was detect


ed in the broth.

As shown in Table II, about 17% of carbon


1 (1-C) and 4% of carbon 6 (6-C) of glucose
were evolved as CO, in 49hrs. Time course

determination of the ratio of CO, evolved


from 1-C to that from 6-C of glucose showed
that the ratio was about 5 in initial 7 hrs, but

thereafter it decreased to 3•`4.

In the next place a similar experiment using

SM was carried out. In this experiment glu


II. Radiorespirometry.
cose was quite diminished in 35hrs and the
(1) Radiorespirometry using growing cells.
maximum cell weight, 42 wet mg/ml, was
The cells were inoculated in the DM con
observed at 28 hrs. Glutamic acid was not
taining labeled glucose and the evolution of
formed but lactic acid was formed though
labeled CO, by the growing organism was
very slightly.
investigated. The experimental condition was
Table III shows that the ratio of CO,
the same as that in actual fermentation. (Table
evolved from 1-C to that from 6-C of glucose
II) Under this condition, glucose supplied was
was 3.-4 in initial 14 hrs, which resembled
consumed linearly and diminished to zero
that in the foregoing experiment, and then
after 49 hrs; cell weight reached its maximum,
about 1, which was caused by the increase
13.2 wet mg/ml, at 35 hrs; and glutamic acid
of CO, evolution from 6-C of glucose. Forty
corresponding to 30•`32% of the initial glucose
four % and 40% of 1-C and 6-C of glucose
30) S. Ishii, Igaku to Seibutsugaku, 16, 317 (1950).
were evolved as CO, in 42 hrs.
31) T. Shimizu, I. Biochem., 37, 421 (1950).
86 Kunio OISHI and Ko AIDA

(2) Radiorespirometry using resting cells. in. Then several experiments using resting,
As the above experiments were attended cells were carried out.
with cell growth so the disturbance by other In the first experiment in which cells grown
factors than biotin content would participate on DM were used, glucose entirely disappear

ed in 6 hrs; glutamic acid was produced as


TABLE III. CO2 EVOLUTION FROM BIOTION-
much as 45•`55% of the initial glucose; and
SUFFICIENT CULTURE
no growth was observed. The evolution of
CO2 from 1-C of glucose in 6 hrs was 30%

of the initial and that from 6-C was 5% as


shown in Fig. 2. In the second experiment

cells grown on SM were used. Complete

consumption of glucose, no remarkable amino,


or organic acids production, and no cell growth
were observed in 6 hrs. Differing from the
above experiment, as shown in Fig. 3, the

evolution of CO, from 1-C and 6-C of glucose


rose to 60% and 35% of the initial, respec

tively.
Additionally, avoiding the disturbance by
the metabolism after pyruvate, the following

experiments were carried out in which oxida

tion of pyruvate was inhibited by the addi


tion of arsenite. Under this condition the

FIG. 2. C02 Evolution by Washed Cells Grown FIG. 3. CO2 Evolution by Washed Cells Grown
on Biotin-deficient Medium on Biotin-sufficient Medium
Studies on Amino Acid Fermentation. Part XI 87

formation of ƒ¿-ketoglutaric acid was not ob As shown in Fig. 4, little effect of arsenite
served and the reoxidation of NADPH by re and methylene blue on the CO2 evolution by
ductive amination of ƒ¿-ketoglutaric acid was the cells grown on DM was observed. Evo
eliminated, so the effect of methylene blue lution of CO, from 1-C and 6-C of glucose
which was the hydrogen acceptor from NAD was 25% and zero %o of the initial, respec
PH was also investigated. tively.

FIG. 5. CO2 Evolution by Washed Cells Grown


FIG. 4. CO2 Evolution by Washed Cells Grown
on Biotin-deficient Medium on Biotin-sufficient Medium

FIG. 6. Carbon Dioxide Production from Glucose by Different Pathways


88 Kunio OISHI and Ko AIDA

The evolution of CO2 from 6-C of glucose In the resting cell experiments in which
by the cells grown on SM was strongly in influence of cell growth was removed, the
hibited by the addition of arsenite and only patterns of CO, evolution were similar to
2% was generated as presented in Fig. 5. those in growing cell experiments. Carbon
The evolution of CO, from 1-C was also de dioxide evolution from 6-C of glucose by the;
creased to 40%. The whole pattern of evolu cells grown on DM was very little but con
tion became similar to that of Fig. 4. Any stant (Fig. 2) as seen in the growing cells,
remarkable effect of methylene blue was not suggesting that complete oxidation of glucose
observed in this case, too. occurs at any time and has no connection
DISCUSSION with cell growth. Carbon dioxide evolution
from 6-C of glucose by the cells grown on SM
Fig. 6 shows the pattern of CO, formation
was, resembling that by growing cells, much
from carbons of various positions of glucose
more higher than that by the cells grown on
molecule through EMP, HMP and TCA cycle.
DM. (Fig. 3)
It is apparent that no CO, evolution from
For many years differences in metabolic be
glucose is observed when glucose is cataboliz
ed to pyruvate through EMP and that all the havior of biotin-deficient and biotin-sufficient
1-C of glucose, and 2 or 3-C if the recycle of cells were supposed to be caused, at least
HMP is running on, is evolved when glucose partly, by the difference in supply of oxygen.
catabolism proceeds through HMP. Carbon 6 per cell following different cell growth. But
of glucose is evolved only when TCA cycle in the above experiments cell concentrations,
is operative. were nearly the same and cell growth was
From the results presented in this article, it not seen, so the difference in biotin-deficientt
would be possible to assume as follows. and biotin-sufficient cells would be concluded
The weak evolution of CO, from 6-C of to be the difference in cellular activity itself.
glucose in the experiment using DM (Table Arsenite addition experiments revealed some
II) shows that complete degradation of glucose interesting facts. In the presence of arsenite,
occurs continuously while its rate is very low. CO, evolution from 6-C of glucose by the
On the other hand CO, evolution from 1-C cells grown on both DM and SM was almost
of glucose is several-fold higher than that completely inhibited, indicating that most of
from 6-C, which indicates that HMP takes complete degradation of glucose was not per
part in the glucose catabolism in this organi formed by the extensive pentose cycle in both
sm. Considering the constancy of the ratio cases, while the difference in quantity between
of CO, evolution from 1-C and 6-C of glucose CO2 evolved from 1-C and 6-C of glucose did
except that at an early short stage, the path- not show any sharp contrast to that in the
way of glucose catabolism must be constant. absence of arsenite, which suggests that re
In the next experiment using SM (Table actions succeeding pyruvate oxidation do not
III), the similar result was obtained during have any considerable effect on the ratio of
initial 14 hrs, but afterward a large amount EMP to HMP. This is confirmed by the
of CO2 was generated from 6-C of glucose and findings that the addition of methylene blue
the ratio of CO2 from 1-C and 6-C of glucose had no effect on CO2 evolution in the pre
Fell into 1. From these results it would be sence of arsenite. Therefore NADP reduction
possible to consider that biotin promotes the by glucose-6-phosphate and 6-phosphoglucona
extent of complete degradation of glucose via te dehydrogenases in HMP and NADPH
T CA cycle, which is shown in the extent of CO, oxidation by glutamic dehydrogenase could .
evolution from 6-C of glucose, and does not not be considered to be closely connected
a ffect the ratio of EMP and HMP appreciably. with one another in glutamic acid formation.
Studies on Amino Acid Fermentation. Part XI 89

Wang et al.29) proposed the following equa growing culture the participation of HMP is
tion to calculate the pathways of glucose greater at an early stage than at the follow
catabolism. ing one.
Additionally it became apparent from these
glucose catabolized by HMP=x•\y
results that the Entner-Doudoroff Pathway32)
where x is % of 14C evolved as 14CO2 from is not operative in this organism. In this
glucose-l-14C pathway no CO2 evolution is observed during

y is % of 14C evolved as 14CO2 from glucose- pyruvic acid formation while a large amount
6-14C of CO, evolves from 1-C of glucose at the

This equation is available only when the pyruvic acid oxidation, which would disturb
the calculation of EMP and HMP. How-
value of y is not so high, because the CO2
ever, the calculated values of EMP and
evolution from 1-C of glucose in TCA cycle
HMP in the experiments added with arsenite
is not negligible in this case. In the experi

ment using organisms which have a strong (Fig. 4 and 5), where pyruvic acid oxidation
was not proceeded, were the same as those in
activity of TCA cycle such as used here, a
control experiments (Fig. 2 and 3). These re
slightly modified equation should be used
which is as follows. sults show indirectly that the organism used
lacks the activity of the Entner-Doudoroff
When a (%) of 1-C of glucose is converted
to CO2 through HMP and y (%) of 6-C is pathway.
It is of interest that the values in Table IV
generated by TCA cycle, taltal CO2 evolved
are similar to those in several reports using
from 1-C, x, will be expressed as
Escherichia coli,29) Sarcina lutea,33) Bacillus subti
lis,34•`36) and Arthrobacter globiformis.37) From

these results it is concluded that this organism,


which can be rewritten as follows: one of glutamic acid-producing bacteria, dose

not show an abnormal behavior in glucose


metabolism and that such a difference in the

ratio of EMP to HMP as seen in Table IV


According to this equation, the rate of is not enough to explain the effect of biotin

glucose catabolism by HMP in this organism on fermentation conversion (conversion of L-


was estimated as shown in Table IV. Con glutamic acid fermentation to lactic acid
trary to the expectation, glucose catabolism fermentation) in this organism.
by HMP is somewhat greater in the cells
Acknowledgement The authors wish to thank
grown on SM than in the cells grown on DM.
Miss M. Matsumura and Mr. K. Huang for
This finding is supported by the fact that in
their assistance in the experimental work.
TABLE IV. PATHWAYS OF GLUCOSE CATABOLISM
32) N. Entner and M. Doudoroff, J. Biol. Chem., 196, 853
(1952).
33) E. A. Dawes and W. H. Holms, Biochim. Biophys. Acta,
29, 82 (1959).
34) V. H. Cheldelin, Metabolic Pathways in Microorganisms,
p. 61 (1960).
35) C. H.Wang and J. K. Krackov, J. Biol. Chem., 237,
3614 (1962).
36) M. Goldman and H. J. Blumenthal, J. Bacteriol , 86,
* data after 6hrs 303 (1963).
** calculated 37) J. G. Morris, J. Gen. Microbial., 22, 564 (1960).