24

Noda Institute for Scientific Research GRANT
2014 Research Grant
Outline of Research Result

Enhanced central carbon metabolism and its application tometabolite production in pdhR-defective Escherichia coli
Atsushi Yokota
Laboratory of Microbial Physiology, Research Group of Biosceince and Chemistry, Division of Fundamental AgriScience
Research, Research Faculty of Agriculture, Hokkaido University

Research aims

Methods

The production of useful chemicals by microbial fermentation has attracted significant attention due to the sustainable nature of bioprocessing, in which biomass is used as
the starting material. Thus, improved bioprocessing productivity is an ongoing goal. Our approach to meeting this goal
is to enhance cellular central carbon metabolism. In thisproject, we focused on pyruvate dehydrogenase complex
regulator (PdhR), which regulates pyruvate dehydrogenase
complex (PDHc) expression (Fig. 1). PdhR is a negativetranscription factor that de-represses transcription of thepdh operon encoding PDHc in response to an increase in
the intracellular concentration of pyruvic acid, resulting inenhanced pyruvic acid metabolism.(1) As PDHc is an enzyme connecting glycolysis and the TCA cycle, deletion of
the gene encoding PdhR (pdhR) is expected to enhance
central carbon metabolism. Moreover, PdhR reportedly not
only regulates the transcription of the pdh operon but alsothat of ndh, which encodes NADH dehydrogenase-II(NDH-II), and the cyo operon, which encodes cytochromebo3 oxidase (Cyt bo3).(2) Therefore, we investigated the possible activation of central carbon metabolism by deletion ofpdhR and its application to metabolite production. In thisstudy, the metabolism of glucose in wild-type and pdhR-deleted (ΔpdhR) Escherichia coli cells was compared to explore alterations in fermentation characteristics of the mutant.

The ΔpdhR mutant was constructed from the wild-type
E. coli strain W1485 according to the method of Datsenko.(3)The wild-type strain and ΔpdhR mutant were cultured inminimal fermentation medium containing 50 g/L glucose ina 2-L jar fermentor under batch fermentation conditions.
Cultures were prepared under STAT conditions (the agitation speed was automatically controlled to maintain the dissolved oxygen [DO] concentration above 2 ppm) or fixedagitation conditions (the agitation speed was fixed such thatall DO was depleted after the late stationary phase). Growthstatus, residual glucose level, organic acid formation, andrespiration rate were monitored and compared between thetwo strains. Furthermore, the enzymatic activities of PDHc,
citrate synthase (CS), NDH, and Cyt, which play importantroles in central carbon metabolism and the respiratory
chain, were measured using cells harvested at the late stationary phase. Changes in the levels of transcription of Cyt
genes were also assessed by quantitative real-time PCR. Inaddition, the intracellular NAD+/NADH ratio, an indicatorof intracellular redox balance, was measured using cycling
assay methods.

Fig. 1. Enzymes under the control of PdhR in Escherichia coli. All
enzyme abbreviations are given in the text. ―|:Repression.

Results① Construction of the ΔpdhR mutant
Deletion of the pdhR region (~600 bp) in the ΔpdhR mutant was confirmed by PCR analysis. Dramatic increases in
the activities of both PDHc and NDH-II in the ΔpdhR mutant compared to the wild-type strain were also confirmed(see Fig. 3), as reported previously.(1)② Fermentation analysis
The wild-type strain and ΔpdhR mutant were cultured inminimal salts medium in 2-L jar fermentors. Under fixedagitation at 400, 600, or 800 rpm, DO was depleted after 4,6, or 8 h of culture, respectively, while under STAT conditions, the DO concentration was maintained above 2 ppm.
These results indicate that the oxygen supply was adequately controlled by the agitation speed.
Under STAT conditions, both the wild-type strain andΔpdhR mutant showed good and comparable growth (Fig.
2A). On the other hand, under fixed agitation conditions at		25400, 600, and 800 rpm, both the wild-type strain and ΔpdhR
mutant showed reduced growth; however, the ΔpdhR mutant showed greater growth than did the wild-type strain. As
shown in Fig. 2A, the difference was remarkable, especially in the culture agitated at 600 rpm (600 rpm conditions); the maximum growth level, indicated by the opticaldensity of the culture broth at 660 nm, of the ΔpdhR mutant
reached 20, while that of the wild-type strain was 12.
The total time to consume all glucose in the medium wasapproximately 14 h in both strains under STAT conditions.
In contrast, under 600 rpm conditions, more than 30 h wererequired by the wild-type strain, while the ΔpdhR mutant
consumed all available glucose in about 24 h (Fig. 2B). The
specific respiration rate of the ΔpdhR mutant was increased
1.2-fold compared to that of the wild-type strain. Therefore,
the ΔpdhR mutant showed greater metabolic activity than
the wild-type strain under oxygen-depleted conditions.
The concentrations of 2-oxoglutarate, pyruvate, and acetate in the culture medium were measured. Under STAT
conditions, both strains produced around 2 g/L of 2-oxoglutarate, and little pyruvate and acetate. Under 600 rpm conditions, however, the ΔpdhR mutant produced more 2-oxoglutarate (1.71 g/L) than did the wild-type strain (0.85 g/L).
Pyruvate production was observed in the wild-type strain(maximum level, ~4 g/L), while the ΔpdhR mutant produced little pyruvate. Acetate formation was observed inFig. 2. Growth and glucose consumption under the two culture conditions (STAT and 600 rpm). W, wild-type strain; D, ΔpdhR mutant; STAT,STAT conditions; 600, 600 rpm conditions.

both strains; however, the maximum yield in the ΔpdhR
mutant (~5 g/L) was lower than that in the wild-type strain(~10 g/L). These results suggest that, in the ΔpdhR mutant,the carbon flux was maintained beyond pyruvate and agreater amount of carbon flowed into the TCA cycle.
③ Enzymatic activities of the central carbon metabolic
pathway and the respiratory chain
To investigate the mechanisms underlying the metabolicchanges observed in the ΔpdhR mutant, the activities ofPDHc, CS, NDH, and Cyt were measured in cell extracts
prepared from log phase cells (Fig. 3). PDHc activity wassignificantly increased in the ΔpdhR mutant up to 6.1- and
3.0-fold the levels in the wild-type strain under both STAT
and 600 rpm conditions, respectively. CS activity was relatively constant (~300 nmol/min/mg) in both strains irrespective of culture conditions (data not shown). NDH activity was measured separately in terms of NDH-I and -II activity. The level of NDH-I activity was similar and constantin both strains under both culture conditions. In contrast,
the levels of NDH-II activity in the ΔpdhR mutant underboth the STAT and 600 rpm conditions were increased 5.1and 4.2-fold compared to those in the wild-type strain, respectively. Cyt activity was measured as the total activity of
three enzymes, Cyt bo3, Cyt bd-I, and Cyt bd-II. The total
Cyt activity level did not differ significantly between thetwo strains under either culture condition. Compared to theSTAT conditions, however, both strains showed a higher
activity level with agitation at 600 rpm. This may havebeen caused not by the deletion of pdhR, but by someglobal regulators that respond to anaerobic conditions.
④ Analysis of terminal oxidase transcription
In E. coli, Cyt consists of three isozymes, Cyt bo3 (encoded by cyo), Cyt bd-I (encoded by cyd), and Cyt bd-II(encoded by app). These three enzymes could not be evaluated separately by enzymatic assays. Therefore, transcriptional levels of the three genes (cyo, cyd, and app) were independently quantified and compared by real-time PCR
analysis. Our results revealed no significant difference between the wild-type strain and ΔpdhR mutant under either
culture condition. These results indicate that the effect ofthe deletion of pdhR on the expression of Cyt bo3 was limited.

Fig. 3. Activity levels of PDHc and respiratory enzymes in the wild-type strain and ΔpdhR mutant under both culture conditions (STAT and 600 rpm).
Error bars represent the standard deviation (n=3). *, significant t-test differences (p<0.05). For NDH activity, the difference in total activity levels of NDH-Iand -II was evaluated. The abbreviations are the same as those in Fig. 2.

26⑤ Intracellular NAD+/NADH ratio
Based on the measured enzyme activity levels, PDHc
and NDH-II activities were significantly increased in theΔpdhR mutant compared to that in the wild-type strain. As
these enzymes are related to NADH production and consumption in cells, respectively, the intracellular NAD+/
NADH ratio was determined and compared in both strains.
Under STAT conditions, the NAD+/NADH ratio of theΔpdhR mutant was similar to or slightly higher than (~1.2fold increase) that in the wild-type strain, suggesting that aslightly oxidative environment existed in the ΔpdhR mutantcells. On the other hand, the NAD+/NADH ratio in theΔpdhR mutant was significantly decreased 0.6-fold that in
the wild-type strain under the 600 rpm conditions. Theseresults suggest that the intracellular environment of theΔpdhR mutant was more reductive than that of the wildtype strain under 600 rpm conditions.
Conclusions
The ΔpdhR mutant showed both a higher glucose consumption rate and increased growth under 600 rpm conditions. This may be due to the increased availability of precursor metabolites for growth, which originated from thehigher carbon flux into the TCA cycle in the ΔpdhR mutant.
Therefore, we conclude that the ΔpdhR mutation enabledenergy-saving fermentation processes in E. coli, in whichagitation or aeration costs were reduced.
In the ΔpdhR mutant, the formation of pyruvate and acetate was remarkably relieved under 600 rpm conditions, although the by-production of these organic acids is generallyobserved in E. coli cultures along with insufficient aeration;
this interferes with normal growth and metabolite production. The disappearance of pyruvate formation in the ΔpdhR
mutant is believed to be due to increased PDHc activity.
This must have increased the intracellular concentration ofacetyl-CoA. However, in the ΔpdhR mutant, the excretionof acetate, a typical anaerobic metabolite of acetyl-CoA,
was also decreased, while 2-oxoglutarate formation was increased. One possible explanation for the decrease in ace-tate formation is the activation of CS activity by acetylCoA, a known activator of E. coli CS, leading to the increased flow of acetyl-CoA into the TCA cycle.
Our enzyme activity measurements revealed dramatic increases in the activities of both PDHc and NDH-II, which
are under the control of PdhR, in the ΔpdhR mutant underboth STAT and 600 rpm conditions. However, the total Cyt
activity level and transcriptional level of cyo, which arealso controlled by PdhR, did not differ between the twostrains, while an increase in respiration rate was observedin the ΔpdhR mutant. These results indicate that the NDHII reaction is the rate-limiting step in the respiratory chain.
Indeed, we observed that the total NDH activity level was
only one-tenth that of Cyt in the wild-type strain under 600
rpm conditions, suggesting that NDH-II controls the rate ofrespiration in E. coli.
The much lower NAD+/NADH ratio observed in theΔpdhR mutant compared to the wild-type strain under 600rpm conditions indicates a reduced intracellular environment in the mutant compared to the parent. However, thisresult is unexpected as the ΔpdhR mutant exhibited a higher
respiration rate, which enabled enhanced NAD+/NADH recycling. In the ΔpdhR mutant, enhanced formation ofNADH caused by the increase in PDHc activity was notbalanced even by the increased re-oxidation of NADH
driven by the increased activity of NDH, resulting in
NADH accumulation in the mutant.
References
(1)

(2)
(3)

Ogasawara, H., Ishida, Y., Yamada, K., Yamamoto, K., and Ishihama, A. (2007) PdhR (pyruvate dehydrogenase complex regulator)
controls the respiratory electron transport system in Escherichia coli.
J. Bacteriol. 189: 5534–5541.
Göhler A. K. et al. (2011) More than just a metabolic regulator-elucidation and validation of new targets of PdhR in Escherichia coli.
BMC Syst. Biol. 5: 197. doi: 10.1186/1752-0509-5-197.
Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products.
Proc. Natl. Acad. Sci. USA. 97: 6640–6645.