Recycling CO₂ For Enhanced Bioethanol Production By Engineered Saccharomyces Cerevisiae

Global climate change caused by anthropogenic emission of greenhouse gases (GHGs) is one of the grand challenges jeopardizing the human society. The consumption of fossil fuels needs to be limited to mitigate the emission of carbon dioxide (CO2) and global warming. Therefore, an alternative energy source is necessary to decrease the reliance on fossil fuels. Biofuel has been considered a promising solution. However, the utilization of edible biomass for producing first generation of biofuels inevitably faces the dilemma between "food versus fuel". Lignocellulosic biomass is a better feedstock for bioethanol production without disturbing the food supply and arable land, but this bioprocess cannot directly assimilate atmopheric CO2. Biofuel production from phototrophic microorganisms offers a promising future to capture the lost carbon through direct fixation of atmospheric CO2. Nevertheless, the titers of usable biofuels produced from the engineered autotrophic microorganisms have so far failed to match the industrial requirements. Therefore, it will be ideal if the bioethanol production from lignocellulosic biomass becomes carbon conservative via re-assimilating the carbon from the carbon dioxide released in the ethanol-producing pathway.To do so, we first established a heterologous expression platform for bacterial gene expression, and two key enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK), were introduced into an engineered Saccharomyces cerevisiae strain SR8 to enable CO2 recycling through a synthetic reductive pentose phosphate pathway during xylose fermentation. The main conclusions are as follow.(1) We demonstrated the functional expression of Escherichia coli xylose isomerase and arabinose isomerase in yeast both in vitro and in vivo together with the expression of the GroE chaperonins, as well as the functional expression of the Agrobacterium tumefaciens D-psicose epimerase in S. cerevisiae. The results suggested that the mismatching of the HSP60 chaperone systems between bacterial and eukaryotic cells is the reason these bacterial enzymes cannot be functionally expressed in yeast. The results showed that the co-expression of E. coli GroE in S. cerevisiae is a promising post-translational strategy for the functional expression of bacterial enzymes in yeast.(2) RuBisCO from Rhodospirillum rubrum and PRK from Spinacia oleracea were introduced into the SR8 strain. The resulting strain with a synthetic reductive pentose phosphate pathway was able to exhibit a higher yield of ethanol and lower yields of byproducts, including xylitol and glycerol, than a control strain. In addition, the reduced release of CO2 by the engineered strain was observed during xylose fermentation, suggesting that the CO2 generated by pyruvate decarboxylase was partially re-assimilated through the synthetic reductive pentose phosphate pathway. These results demonstrated that the recycling of carbon dioxide from the ethanol fermentation pathway in yeast can be achieved during lignocellulosic bioethanol production through a synthetic carbon conservative routine.(3) Initial CO2 concentration influences the fermentation profiles of S. cerevisiae strains. The results showed that purging with CO2 before fermentation would significnatly increase the yield of ethanol and decrease the accumulation of byproducts. These effects have been observed in the fermentation using either glucose, xylose or galactose as carbon source regardless of cultivation media. We further discovered that the enhancing effects of CO2 on yeast fermentationwere partially regulated by pyruvate carboxylase and CO2 may serve as or interact with global regulation factors, thus influencing the cell physiology.