Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, inefficient conversion of glycerol to ethanol by natural Saccharomyces cerevisiae limits its use in these processes. In this study, we have developed an efficient glycerol-converting yeast strain by genetically modifying the oxidation of cytosolic NAD (NADH) by an O₂-dependent dynamic shuttle and abolishing both glycerol phosphorylation and biosynthesis in S. cerevisiae strain D452-2, as well as by vigorous expression of whole genes in the dihydroxyacetone (DHA) pathway (Candida utilis glycerol facilitator, Ogataea polymorpha glycerol dehydrogenase, endogenous dihydroxyacetone kinase, and triosephosphate isomerase). The engineered strain showed conversion efficiencies (CE) up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with a production rate of >1 g liter^-1 h^-1 when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain converted a mixture of glycerol and glucose into bioethanol (>86 g/liter) with 92.8% CE. To the best of our knowledge, this is the highest reported titer of bioethanol produced from glycerol and glucose. Notably, we developed a glycerol-utilizing transformant from a parent strain which cannot utilize glycerol as a sole carbon source. The developed strain converted glycerol to ethanol with a productivity of 0.44 g liter^-1 h^-1 on minimal medium under semiaerobic conditions. Our findings will promote the utilization of glycerol in eco-friendly biorefineries and integrate bioethanol and plant oil industries. IMPORTANCE With the development of efficient lignocellulosic biorefineries, glycerol has attracted attention as an eco-friendly biomass-derived solvent that can enhance the dissociation of lignin and cell wall polysaccharides during the pretreatment process. Coconversion of glycerol with the sugars released from biomass after glycerolysis increases the resources for ethanol production and lowers the burden of component separation. However, low conversion efficiency from glycerol and sugars limits the industrial application of this process. Therefore, the generation of an efficient glycerol-fermenting yeast will promote the applicability of integrated biorefineries. Hence, metabolic flux control in yeast grown on glycerol will lead to the generation of cell factories that produce chemicals, which will boost biodiesel and bioethanol industries. Additionally, the use of glycerol-fermenting yeast will reduce global warming and generation of agricultural waste, leading to the establishment of a sustainable society.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Khattab SMR, Watanabe T

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