Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.Escherichia coli accumulates acetic acid when growing at a high rate of glucose consumption even in the presence of ample oxygen (2,11,30). This phenomenon is known as overflow metabolism. Acetate is generated when carbon flux from acetyl-coenzyme A (CoA) is directed to acetate instead of entering the tricarboxylic acid (TCA) cycle (11). This by-product induces a stress response even at extremely low concentrations (21), hinders growth (26), and reduces the production of recombinant proteins (37). Overflow metabolism has been attributed to an enzymatic limitation in the TCA cycle (27). In E. coli the complete oxidation of 1 mol of glucose in glycolysis and the TCA cycle generates 10 mol of NAD(P)H and 2 mol of FADH 2 (31):If the rate of oxygen utilization is sufficiently high, the reduced cofactors generated by glucose consumption are reoxidized in the electron transport chain, which serves the dual purpose of maintaining an optimal redox environment and generating energy by oxidative phosphorylation. In the absence of oxygen glucose cannot be completely oxidized, and metabolic intermediates accumulate to maintain the redox balance. Even in the presence of oxygen, if the rate of glucose consumption is greater than the capacity to reoxidize the reduced equivalents generated...

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In Escherichia coli secreted proteins must be maintained in an export-competent state before translocation across the cytoplasmic membrane. This function is carried out by a group of proteins called chaperones. SecB is the major chaperone that interacts with precursor proteins before their secretion. We report results indicating that the DnaK and DnaJ heat shock proteins are also involved in the export of several proteins, most likely by acting as their chaperones. Translocation of alkaline phosphatase, a SecB-independent protein, was inhibited in dnaK-and dnaJ-mutant strains, suggesting that export of this protein probably involves DnaK and DnaJ. In addition, DnaK and DnaJ play a critical role in strains lacking SecB. They are required both for viability and for the residual processing of the SecB-dependent proteins LamB and maltose-binding protein (MBP) seen in secB null strains. Furthermore, overproduction of DnaK and DnaJ permits strains lacking SecB to grow in rich medium and accelerates the processing of LamB and MBP. These results suggest that under conditions where SecB becomes limiting, DnaK and DnaJ probably substitute for SecB and facilitate protein export. This provides the cell with a mechanism to overcome a temporary imbalance in the secretion process caused by an abrupt expansion in the pool of precursor proteins.

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.

We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation. The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient) strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates.

The growth of Megasphaera elsdenii on lactate with acrylate and acrylate analogues was studied under batch and steady-state conditions. Under batch conditions, lactate was converted to acetate and propionate, and acrylate was converted into propionate. Acrylate analogues 2-methyl propenoate and 3-butenoate containing a terminal double bond were similarly converted into their respective saturated acids (isobutyrate and butyrate), while crotonate and lactate analogues 3-hydroxybutyrate and (R)-2-hydroxybutyrate were not metabolized. Under carbon-limited steady-state conditions, lactate was converted to acetate and butyrate with no propionate formed. As the acrylate concentration in the feed was increased, butyrate and hydrogen formation decreased and propionate was increasingly generated, while the calculated ATP yield was unchanged. M. elsdenii metabolism differs substantially under batch and steady-state conditions. The results support the conclusion that propionate is not formed during lactate-limited steady-state growth because of the absence of this substrate to drive the formation of lactyl coenzyme A (CoA) via propionyl-CoA transferase. Acrylate and acrylate analogues are reduced under both batch and steady-state growth conditions after first being converted to thioesters via propionyl-CoA transferase. Our findings demonstrate the central role that CoA transferase activity plays in the utilization of acids by M. elsdenii and allows us to propose a modified acrylate pathway for M. elsdenii. Megasphaera elsdenii is an ecologically important rumen bacterium whose genome has recently been sequenced (20) and which metabolizes DL-lactate principally to propionate and acetate (5,7,19). Lactate conversion to propionate occurs via the acrylate pathway with acrylyl coenzyme A (CoA) serving as an intermediate (32), a pathway also used by several other organisms, including Clostridium propionicum. The dead-end reduction of lactate to propionate allows the cell to balance the anaerobic oxidation of lactate to acetate and carbon dioxide (16), steps which appear to be the primary means of ATP generation (26). M. elsdenii also produces butyrate, and several strains also accumulate longer-chain fatty acids from the fermentation of lactate (9). The generation of butyrate from lactate relies on the presence of acetate, and M. elsdenii also has the flexibility to generate hydrogen from reduced ferredoxin as another means to balance redox (11). While M. elsdenii continues to be of great interest as a member of the rumen microbial community (21, 31), the organism and its enzymes also have potential biotechnological applications (28).Key steps in the metabolic pathway of M. elsdenii reduction of lactate are mediated by propionyl-CoA transferase (24), lactylCoA dehydratase (2, 14, 17), and acrylyl-CoA reductase (3, 10). Propionyl-CoA transferase (EC 2.8.3.1; systematic name, acetylCoA:propionate-CoA-transferase) is typically implicated in the interconversion of propionate/propionyl-CoA and DL-lactate/DLlactyl-CoA (26). However, th...

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h ؊1 . Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h ؊1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g.Pyruvic acid (pyruvate) is widely used in food, chemicals, and pharmaceuticals. The chemical is a precursor for the enzymatic production of L-tryptophan, L-tyrosine, D-/L-alanine, and L-dihydroxyphenylalanine (22), and it also serves in several health-related roles, including weight loss (25,33,34), exercise endurance (32), cholesterol reduction (35), and acne treatment (10). Recently, pyruvate has been used as the key metabolic precursor to the second-generation biofuels isobutanol and 3-methyl-1-butanol (2). By applying metabolic engineering strategies to them, microorganisms such as Escherichia coli and yeasts can be used to produce significant quantities of pyruvate from glucose and other renewable resources (22). In general, such approaches must delete or repress pathways which metabolize pyruvate. For example, pyruvate accumulates readily in E. coli strains having mutations in aceEF, encoding components of the pyruvate dehydrogenase complex (37). Additional mutations, of the ldhA, poxB, pfl, and pps genes, further improve pyruvate formation (48). Figure 1 shows the principal metabolic pathways and enzymes involved in the formation of pyruvate.Because pyruvate resides biochemically at the end of glycolysis, pyruvate production is directly related to the glycolytic flux. Metabolic engineering strategies to form pyruvate therefore also aim to enhance glycolysis (8,45,46). Glycolysis is not transcriptionally limited, and control principally resides outside the pathway in cellular demand for global cofactors, such as ATP and NADH (19,23,40). Glycolytic flux is substantially increased by disrupting oxidative phosphorylation or by increasing ATP hydrolysis (8,19). Increased glycolytic flux assists pyruvate accumulation: for example, an F 1 -ATPase-defective mutant (E. coli lipA2 bgl ϩ atpA401) generated pyruvate more quickly than its parent (45,46). Similarly, E. coli atpFH (strain TC44), defi...

The accumulation of secretory protein precursors, caused either by mutations in secB or secA or by the overproduction of export-defective proteins, results in a two-to fivefold increase in the synthesis of heat shock proteins. In such strains, &.32, the alternative sigma factor responsible for transcription of the heat shock genes, is stabilized. The resultant increase in the level of &32 leads to increased transcription of heat shock genes and increased synthesis of heat shock proteins. We have also found that although a secB null mutant does not grow on rich medium at a temperature range of 30 to 42°C, it does grow at 44°C. In addition, we found that a secB null mutant exhibits greater thermotolerance than the wild-type parental strain. Elevated levels of heat shock proteins, as well as some other non-heat shock proteins, may account for the partial heat resistance of a SecB-lacking strain.The Escherichia coli SecB protein participates in the export of a subset of proteins destined for the periplasm or outer membrane. In its function as a chaperone, it maintains target precursor proteins in a partially unfolded state competent for translocation. Moreover, because of its interaction with SecA, the peripheral membrane domain of the translocase, SecB helps to position precursor proteins at membrane sites containing the integral SecY/E components of translocase (for a review, see references 24, 25, and 30). Strains lacking the SecB chaperone are viable, but they have a number of pleiotropic defects. In such strains, export of the secretory proteins LamB, OmpA, OmpF, PhoE, and maltose-binding protein is defective, leading to an accumulation of precursor forms of these proteins. In addition, although secB deletion strains are viable on minimal glycerol medium, they are unable to grow on rich medium (16, 17).We have recently shown that the DnaK and DnaJ heat shock proteins can partially substitute for the SecB chaperone function. Strains lacking SecB require DnaK and DnaJ both for residual transport of several SecB-dependent proteins and for viability on minimal medium. Furthermore, overproduction of DnaK and DnaJ permits secB null mutants to grow on rich medium, albeit at a reduced rate, and enhances the rate of export of some SecB-dependent proteins (31). These observations led us to wonder whether mutants lacking SecB might have a higher level of heat shock proteins than wild-type strains. This seemed possible because the cytoplasmic precursor proteins that accumulate in secB deletion strains may be seen as abnormal proteins by the cell. A variety of abnormal proteins, including puromycyl fragments (11), induce the heat shock response. In addition, unfolded proteins that are not degraded (23) and the MalE-LacZ hybrid protein (14) also induce the heat shock response. In this report, we show that in a secB null mutant and a secA(Ts) mutant, synthesis of heat shock proteins is also induced. Moreover, we show that the signal for induction of heat shock protein synthesis is accumulation of cytoplasmic precursors of sec...