Engineering electron metabolism to increase ethanol production in Clostridium thermocellum.

The NfnAB (NADH-dependent reduced ferredoxin: NADP+ oxidoreductase) and Rnf (ion-translocating reduced ferredoxin: NAD+ oxidoreductase) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P)+. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes had little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.

Determining the roles of the three alcohol dehydrogenases (AdhA, AdhB and AdhE) in Thermoanaerobacter ethanolicus during ethanol formation.

Thermoanaerobacter ethanolicus is a promising candidate for biofuel production due to the broad range of substrates it can utilize and its high ethanol yield compared to other thermophilic bacteria, such as Clostridium thermocellum. Three alcohol dehydrogenases, AdhA, AdhB and AdhE, play key roles in ethanol formation. To study their physiological roles during ethanol formation, we deleted them separately and in combination. Previously, it has been thought that both AdhB and AdhE were bifunctional alcohol dehydrogenases. Here we show that AdhE has primarily acetyl-CoA reduction activity (ALDH) and almost no acetaldehyde reduction (ADH) activity, whereas AdhB has no ALDH activity and but high ADH activity. We found that AdhA and AdhB have similar patterns of activity. Interestingly, although deletion of both adhA and adhB reduced ethanol production, a single deletion of either one actually increased ethanol yields by 60-70%.

Deletion of the hfsB gene increases ethanol production in Thermoanaerobacterium saccharolyticum and several other thermophilic anaerobic bacteria.

BACKGROUND: With the discovery of interspecies hydrogen transfer in the late 1960s (Bryant et al. in Arch Microbiol 59:20-31, 1967), it was shown that reducing the partial pressure of hydrogen could cause mixed acid fermenting organisms to produce acetate at the expense of ethanol. Hydrogen and ethanol are both more reduced than glucose. Thus there is a tradeoff between production of these compounds imposed by electron balancing requirements; however, the mechanism is not fully known. RESULTS: Deletion of the hfsA or B subunits resulted in a roughly 1.8-fold increase in ethanol yield. The increase in ethanol production appears to be associated with an increase in alcohol dehydrogenase activity, which appears to be due, at least in part, to increased expression of the adhE gene, and may suggest a regulatory linkage between hfsB and adhE. We studied this system most intensively in the organism Thermoanaerobacterium saccharolyticum; however, deletion of hfsB also increases ethanol production in other thermophilic bacteria suggesting that this could be used as a general technique for engineering thermophilic bacteria for improved ethanol production in organisms with hfs-type hydrogenases. CONCLUSION: Since its discovery by Shaw et al. (JAMA 191:6457-64, 2009), the hfs hydrogenase has been suspected to act as a regulator due to the presence of a PAS domain. We provide additional support for the presence of a regulatory phenomenon. In addition, we find a practical application for this scientific insight, namely increasing ethanol yield in strains that are of interest for ethanol production from cellulose or hemicellulose. In two of these organisms (T. xylanolyticum and T. thermosaccharolyticum), the ethanol yields are the highest reported to date.

Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485.

BACKGROUND: Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30-70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. RESULTS: It was found that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. CONCLUSION: PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.