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Applied and Environmental Microbiology, June 1999, p. 2781-2783, Vol. 65, No. 6
Tokyo Research Laboratories, Kyowa Hakko
Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan
Received 19 January 1999/Accepted 23 March 1999
Arthrobacter oxydans HAP-1 hyperproduces
DL-alanine in a non-growth-associated manner. We found that
decreased activities of pyruvate dehydrogenase and of the enzyme
catalyzing NADH oxidation in the stationary phase are paralleled by a
shift of pyruvate metabolism to alanine synthesis by
L-alanine dehydrogenase. We propose that this enzyme
functions as an electron sink even under aerobic conditions.
Since the discovery of bacterial
glutamic-acid production in 1957 (11, 12), several
L-amino acids have been produced by fermentative processes
(14). However, the commercial production of
L-alanine, the simplest L-amino acid, is not
carried out in this manner (3). We previously studied the
fermentative production of L-alanine and found a
DL-alanine-hyperproducing strain of Arthrobacter oxydans HAP-1 (4). Subsequently, mutants lacking
alanine racemase were isolated from this strain, and one of them was
shown to produce L-alanine at a high yield with high
optical purity (5). Furthermore, A. oxydans
HAP-1 possessed the activity of L-alanine dehydrogenase (EC
1.4.1.1) (ALD) (4), which catalyzes the reversible,
NADH-dependent reductive amination of pyruvate to
L-alanine. The finding that ALD-deficient mutants derived
from HAP-1 no longer excrete alanine (5) indicated that
alanine overproduction by A. oxydans HAP-1 depends on
ALD. Although several ALD-possessing microorganisms have been reported
(15, 16, 21), this was the first demonstration of the
functioning of ALD in L-alanine synthesis in a natural isolate by genetic analysis.
In addition to these traits, alanine production by A. oxydans HAP-1 has another significant characteristic: its growth
and amino acid production phases are clearly separated. During the initial stage of cultivation, the bacterium grows vigorously but excretes no alanine. On the contrary, upon cessation of growth, abrupt
accumulation of alanine in the medium occurs. This pattern is in marked
contrast to the fermentation profiles of the production of other amino
acids, such as lysine (7, 20), threonine (8), arginine (22), tryptophan (9), proline
(13), or histidine (1), in which production of an
amino acid is associated with cell growth. In this study, we
investigated the biochemical mechanisms involved in alanine
hyperproduction in a non-growth-associated manner.
A. oxydans HAP-1 was cultivated in a 5-liter jar
fermentor containing an initial glucose concentration of 5%, to which
glucose was fed to a final concentration of 15.5% (5). The
pH was automatically maintained at 6.8 by the addition of 10 N
NH4OH. Cell growth and metabolites in the medium were
measured as previously reported (5). The dissolved oxygen
concentration of the medium was continuously monitored with an oxygen
electrode (model OE8270G; Toa Electronics, Tokyo, Japan). The exhaust
gas of the fermentor was cooled and analyzed by a gas analyzer (model
Ex-1562; AIBL, Tokyo, Japan). CO2 production and
O2 consumption rates were calculated according to the
equations described by Postma et al. (17).
Fermentation profile.
In the growth phase (0 to 20 h),
the rate of CO2 production increased while little alanine
was accumulated. It was calculated that 0.55 mol of glucose was
consumed and 48.8% of carbon derived from glucose was released as
CO2 in this phase. Once cell growth was arrested, the rate
of CO2 production decreased and alanine production started.
In the stationary phase (20 to 80 h), 2.02 mol of glucose was
consumed, and CO2 and alanine formation accounted for 21.2 and 51.6% of the carbon from glucose, respectively. Thus, sugar
metabolism clearly shifted from CO2 production to alanine synthesis after the shift to the stationary phase. The O2
uptake rate (Fig. 1a) and the changes in
dissolved oxygen concentration (Fig. 1b) paralleled the decrease in
CO2 production (Fig. 1a). Therefore, cells in the late
stage of cultivation (after 40 h) utilize less O2
despite the aerobic conditions.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mechanism of Alanine Hyperproduction by Arthrobacter
oxydans HAP-1: Metabolic Shift to Fermentation under
Nongrowth Aerobic Conditions
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FIG. 1.
Time course of fed-batch cultivation of A. oxydans HAP-1 (a through d) and A. oxydans DAN 89 (e through h). Cultivations were carried out under aerobic conditions
(throughout the cultivation, the airflow and the stirrer speed were
maintained at 1.0 liter · min 
1 and 600 rpm,
respectively). (a and e) Rates of CO2 evolution (
) and
O2 consumption (
); (b and f) concentration of dissolved
oxygen in the medium; (c and g) growth of cells (dry weight) () and
concentrations of DL-alanine (
), pyruvate (
), and
glucose (×) in the medium. Arrows indicate the point at which
additional glucose solution started to be fed continuously to the
medium. Feeding for A. oxydans HAP-1 was stopped at
70 h. Feeding for A. oxydans DAN 89 was stopped at
60 h because its glucose consumption stopped earlier (around
50 h). (d and h) Changes in enzymatic activities. Activities are
expressed as percentages of the activities observed at the beginning of
cultivation. Symbols: , ALD (100% corresponds to 0.34 U · mg
of protein1; the ALD activity of DAN 89 was below the
detection limit [0.005 U · mg of protein1]);
, PDH (100% corresponds to 0.30 and 0.29 U · mg of
protein1 for HAP-1 and DAN 89, respectively); 
, NADH
oxidase (100% corresponds to 0.9 µmol · min1 · mg of protein1 for both
strains).
1. The cells were disrupted as described previously
(5), and cellular debris was removed by centrifugation (at
10,500 × g for 45 min) at 4°C. The supernatants were
dialyzed against PED buffer (at 4°C for 6 h) and used for the
enzyme assays. The amount of protein was determined by the method of
Bradford (2) with a Bio-Rad kit.
We measured three enzyme activities: ALD and pyruvate dehydrogenase
(PDH), both of which compete for pyruvate, and NADH oxidase, which is
essential for aerobic respiration by A. oxydans
(10). ALD activity was determined as previously described
(4, 5). PDH activity was assayed by the method described by
Reed and Willms (18), in which the reaction was coupled to
phosphotransacetylase to yield acetylphosphate, which reacted with
ferric chloride and was measured spectrophotometrically. One unit of
enzyme activity was defined as the amount of protein required to form 1 µmol of acetylphosphate per min at 30°C. The assay for NADH oxidase
was performed by essentially the same method as that reported by
Sakamoto et al. (19). The dialyzed cell extracts were
suspended in 100 mM potassium phosphate buffer (pH 7.0) and flushed
with air. The reaction was started by the addition of 5 mM NADH and was
immediately monitored with a Hitachi 100-60 spectrophotometer set at
340 nm. The activity was determined from the decrease in the amount of NADH in the first 15 s.
Changes in the three activities during cultivation are shown in Fig.
1d. The activities of PDH and NADH oxidase decreased in the stationary
phase and reached 30% of their initial levels at the end of
cultivation, whereas the level of ALD activity was essentially
unchanged throughout the cultivation. Based on these results, the shift
in sugar metabolism was likely not the result of an ALD induction but
resulted from the decline in PDH activity and the reduction in
respiratory activity due to decreased NADH oxidase activity.
To clarify whether ALD plays a role in sugar metabolism, A. oxydans DAN 89, an ALD-deficient mutant derived from
A. oxydans HAP-1 (5), was cultivated
and analyzed in the same way as strain HAP-1 (Fig. 1e through h).
Cell growth, CO2 production, O2 consumption, and activities of PDH and NADH oxidation of the mutant were similar to
those of the parental strain. Significant differences, however, were
found in that (i) DAN 89 excreted pyruvate but not alanine (Fig. 1g),
as reported previously (5), and (ii) the rate of glucose
consumption of the mutant strain was markedly decreased in the
stationary phase and reached 0 at around 60 h (Fig. 1g). These
results indicate that alanine synthesis by ALD and the
maintenance of glycolytic flow are closely related during
the stationary phase of A. oxydans HAP-1. Since
NADH-oxidizing activity is reduced in the stationary phase, reoxidation
of NADH through respiration is apparently insufficient to
maintain glycolytic flow. Therefore, the reductive amination of
pyruvate by ALD may function as an alternative NADH-reoxidizing
reaction, and thus, alanine may be an electron sink. A similar function
of ALD has recently been assumed in Mycobacterium smegmatis
during adaptation from aerobic growth to the anaerobic dormant state
(6), although it remains unclear whether the bacterium
overproduces alanine. In the case of A. oxydans HAP-1,
the coupling between glycolysis and alanine synthesis, which may be
considered a fermentative mechanism, likely results in the high-yield
alanine production.
In summary, we propose the following mechanism for alanine
overproduction by A. oxydans HAP-1: (i) during
the growth phase, the glycolytic end product, pyruvate, is
metabolized by the tricarboxylic-acid cycle and respiration because of
sufficiently high activities of PDH and NADH oxidase; (ii) when cell
growth is arrested, the activities of PDH and NADH oxidase decrease,
yielding an excess of pyruvate and NADH. Under these conditions, NADH
is oxidized by the reductive amination of ALD. This metabolic shift to
a fermentative pathway even under aerobic conditions causes the
overproduction of alanine.
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ACKNOWLEDGMENTS |
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We thank Naoko Kodama-Oda for her skillful technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan. Phone: 81-427-25-2555. Fax: 81-427-26-8330. E-mail: shashimoto{at}kyowa.co.jp.
Present address: Laboratory of Animal Microbiology, Faculty of
Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiya-machi, Aobaku,
Sendai-shi, 981, Japan.
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Applied and Environmental Microbiology, June 1999, p. 2781-2783, Vol. 65, No. 6
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