Several lines of evidence suggesting that conversion of pyruvate to acetyl-CoA limits ethanol titer. In many thermophilic bacteria, including C. thermocellum, this conversion is mediated by the pyruvate ferredoxin oxidoreductase (PFOR) enzyme. To identify metabolic bottlenecks in enzymes from C. thermocellum, we replaced the T. saccharolyticum enzymes with C. thermocellum enzymes. Of the three enzymes we tested, AdhE (which provides both acetaldehyde dehydrogenase and alcohol dehydrogenase activity) and NfnAB (which transfers electrons from ferredoxin and NADH to NADP+) did not limit ethanol titer in T. saccharolyticum (ethanol production was > 60 g/L). However, Pfor reduced ethanol production by 7-fold, from 60 g/L to 9 g/L (Cui et al. in review). In a second line of evidence, expressing the T. saccharolyticum Pfor enzyme in C. thermocellum increased ethanol titer from 10 to 20 g/L (Hon et al. in review).Pyruvate decarboxylase (PDC) is an enzyme used by Saccharomyces cerevisiae and Zymomonas mobilis, two organisms known for their ability to produce ethanol at high titer (Olson et al., 2015). Although this enzyme has been successfully transferred to several mesophilic organisms, it does not seem to function well above 45°C in vivo. There have been several attempts to introduce pdc genes into C. thermocellum, however the maximum titer we achieved was 21 g/L, and the enzyme activity was 100-fold lower than what we observed in E. coli (0.3 U/mg vs 31 U/mg) (Tian et al., 2017). We think this problem is due to folding (unpublished data) and therefore the challenge is to increase the thermostability of the Pdc protein. We propose to build a single-site saturation mutagenesis library of the Acetobacter pasteurianus Pdc protein (10,621 members) using the CREATE system (Garst et al., 2016) to ensure even coverage of the experimental space. We will then screen this library in C. thermocellum for increased ethanol production. Promising mutations will also be used to develop computational models of protein stability.
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