During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H ؉ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.All living systems generate the needed energy for cell maintenance and growth by catalyzing a set of coupled oxidationreduction reactions. A critical component of these oxidationreduction reactions is the redox balance of the sum of all metabolic reactions. With external input of an oxidant, such as dioxygen, the carbon and energy source can be completely oxidized to carbon dioxide with concomitant reduction of the terminal electron acceptor. However, under strict anaerobic conditions and in the absence of an external input of an oxidant, such as nitrate, metabolic intermediates serve the role of oxidant to maintain the overall redox balance. For many heterotrophic fermentative bacteria, the primary oxidant is pyruvate or derivatives of pyruvate, the terminal product of glycolysis. The redox state of total carbon in all final end products produced by the anaerobic cell should equal the redox state ...

Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher K i for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.Escherichia coli, a facultative heterotroph, grows under aerobic and anaerobic conditions. During aerobic growth, this bacterium metabolizes glucose through the reactions of glycolysis, pyruvate dehydrogenase (PDH), and the tricarboxylic acid cycle. The NADH generated during these enzyme-catalyzed reactions is oxidized ultimately by oxygen. Under anaerobic conditions and in the absence of external electron acceptors, organic compounds generated from glucose during glycolysis serve as the electron acceptors to maintain the redox balance and continued growth of the bacterium. Due to the differences in electron acceptors between the two growth modes, the reported [NADH]/[NAD ϩ ] ratio of an anaerobic cell is severalfold higher (about 0.75) than that of an aerobic cell (about 0.03) (13, 33).The PDH complex that connects glycolysis and tricarboxylic acid cycle enzymes is composed of multiple subunits of three enzymes, pyruvate decarboxylase (dehydrogenase; enzyme 1 [E1]; EC 1.2.4.1), dihydrolipoamide acetyltransferase (enzyme 2 [E2]; EC 2.3.1.12), and dihydrolipoamide dehydrogenase (LPD) (enzyme 3 [E3]; EC 1.8.1.4) (14). NADH, a product of the PDH reaction, is a competitive inhibitor of the PDH complex (15,30,31). The NADH sensitivity of the PDH complex has been demonstrated to reside in LPD, the enzyme that interacts with NAD ϩ as a substrate (29,30,38). Although...

Conversion of lignocellulosic feedstocks to ethanol requires microorganisms that effectively ferment both hexose and pentose sugars. Towards this goal, recombinant organisms have been developed in which heterologous genes were added to platform organisms such as Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli. Using a novel approach that relies only on native enzymes, we have developed a homoethanologenic alternative, Escherichia coli strain SE2378. This mutant ferments glucose or xylose to ethanol with a yield of 82% under anaerobic conditions. An essential mutation in this mutant was mapped within the pdh operon (pdhR aceEF lpd), which encodes components of the pyruvate dehydrogenase complex. Anaerobic ethanol production by this mutant is apparently the result of a novel pathway that combines the activities of pyruvate dehydrogenase (typically active during aerobic, oxidative metabolism) with the fermentative alcohol dehydrogenase.