Engineering saccharomyces for biofuel production improvement
Date of Issue2017
School of Chemical and Biomedical Engineering
The continuous use of fossil fuels has led to many issues such as energy crisis, environmental change and etc. People are seeking cleaner and more sustainable energy resources to replace traditional fossil fuels. Microbial conversion of renewable feedstock into biofuels and chemicals has been investigated extensively in recent decades. Saccharomyces cerevisiae (S. cerevisiae) is one the most popular microbial factory applied for producing valuable chemical products. Its advantages in industrial application comes from its biological properties such as post-translational modifications, less possibility of contamination, robustness towards harsh industrial condition, good tolerance to inhibitory compounds and etc. Among various biofuels, bioethanol is a natural product of yeast. Moreover, fatty acids and their derivatives are energy-rich molecules and considered as excellent candidates for renewable liquid transport fuels and chemicals. In this study, transcription engineering on RNA polymerase II (RNAP II) was conducted to improve S. cerevisiae ethanol tolerance and production. Error-prone PCR was applied to engineer subunit Rpb7 of RNAP II. Random mutagenesis library of Rpb7 was constructed and subjected to screening under ethanol stress. The isolated variant M1 showed much improved resistance towards 8% and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, about 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response and etc. The systematic engineering approaches for improving S. cerevisiae alcohol tolerance were discussed next. As for fatty acids production improvement, acetyl-CoA carboxylase pathway coupled with malonyl-CoA synthetase pathway were introduced into S. cerevisiae. After 24 h fermentation with the supply of 0.38% (w/v) malonic acids, the engineered strains with enhancement in acetyl-CoA carboxylase pathway and malonyl-CoA synthetase pathway showed much improvement in fatty acids production. Specifically, the accumulated C16:0 and C18:0 in the engineered strain CEN-PAA-AB with two pathways combined reached 86.74 and 86.97 ug/108 cells respectively, which was about five-fold of the wild strain. The research in thesis has successfully improved two kinds of biofuels (ethanol and fatty acids) in S. cerevisiae through different engineering strategies. It firstly demonstrated that eukaryotic RNAP II enzyme could be an alternative gTME target in eliciting improved phenotypes. In addition, the combination of malonyl-CoA pathway with the acetyl-CoA pathway has been proved as a valid platform for improving advanced biofuel production.