Mechanism Of Regulation Ethanol Fermentation And Metabolic Engineering In Thermoanaerobacter Ethanolicus

Fermentation Thermoanaerobacter ethanolicus offers the potential to produce ethanol from lignocellulose and to separate ethanol in continuous cultures during thermophilic growth. The practical application of this strategy, however, has been hindered by the fact that ethanol fermentation by these strains is limited to relatively low final ethanol concentrations. Thus, it is important to elucidate at the molecular level how ethanol metabolism is regulated in these systems so that strategies can be developed to increase the final ethanol concentration (or ethanol titer) for an economical fermentation process. The aims of this study were to determine the ethanol pathway of the T. ethanolicus and the mechanism for regulating ethanol fermentation and glycolysis by RSP.1. Under the simulated physiological conditions, AdhB and AdhE did not act as typical bifunctional aldehyde/alcohol dehydrogenases as observed in enzymology assays. AdhB exhibited only a very weak ALDH activity, AdhB was highly active to catalyze ethanol formation from acetaldehyde when the substrate concentration was 2 mM. However, its activity for ethanol consumption was much higher than that for ethanol production when the ethanol concentration was increased to 1% (v/v). AdhE had a high ALDH activity with no ADH activity detected for reducing acetaldehyde to ethanol. Its activity for Ac-CoA production (45.05±0.93 U/mg) was higher than that for Ac-CoA consumption (35.61±2.51 U/mg) at pH 7.2. But its activity for Ac-CoA consumption (113.35±4.76 U/mg) was much higher than that for Ac-CoA production (29.33±2.09 U/mg) at pH 6.6. These results have already indicated that AdhE should be crucial for ethanol formation, and AdhB favored ethanol consumption when ethanol concentration was high e.g. 1%.2. Proteins (P_adhE-1 and RSP) interacting with the transcriptional regulatory regions (TRRs) of adhB and adhE were for the first time isolated and identified. Using the web service software, we predicted the structure of the P_adhE-1 and RSP. P_adhE-1 belongs to the CcpA family. RSP, a homolog of Rex family, that responds to the NADH/NAD+ ratio, was involved in the metabolism of ethanol in T. ethanolicus JW200.3. Through electromobility shift assays, it was determined that NADH, other than pyridine nucleotides, inhibited the DNA-binding activity of RSP. However, NAD⁺ competed with NADH for RSP binding, allowing RSP to sense redox poise of the NADH/NAD⁺ pool. After the RSP was incubated with NADH, its secondary structure contents changed with an increase ofβ-sheets and decreases ofα-helixes and turns. In an in vitro transcription system of T. ethanolicus, RSP repressed the transcription of an alcohol dehydrogenase, whereas the repression was reversed by adding NADH.4. Base substitutes in the repeats of the palindrome reduced the affinity between RSP and the operator, and thus delicate regulation could be achieved. The typical operator was identified as a palindromic sequence -ATTGTTANNNNNNTAACAAT-. Delicate regulation could be achieved by substituting certain bases in the inverted repeats. It is significant that the study has, for the first time, revealed a repressor/operator system and the mechanism responsible for the regulation of ethanol fermentation by redox levels, the system couples a redox signal with an ethanol metabolic pathway.5. The ethanol production was improved by transforming adhE into T. ethanolicus. By designing the promoter of adhE to eliminate the RSP binding sites, the ethanol production does not further increase. Thus, to further improve the ethanol production lies in increasing the catalytic efficiency of acetaldehyde to ethanol, while reducing the consumption rate of ethanol.