3-1-3　Com prehensive engineering and redirection of glycerol paths to fermentative pathway in Saccharomyces cerevisiae for promoting biofuels and chemicals production from lignocellulosic biomass

抄録

For decades, the thresholds of converting lignocellulosic biomass to biofuels and chemicals are the higher costs of pretreatments and consolidated bioprocess that accompanied with low productivity. On the other hand, glycerol is inevitable product of several vast growing industries e.g. biodiesel, which had led to a significant overproduction of crude glycerol resulting in a remarkable drops of its market price.

Our perspective for sustainability is dual utilizing of glycerol as delignifing and hydrolysis agent of biomass and then converted it to biofuel or chemical with its released sugars. We had been initiated an advanced scenario for pretreatment and convert biomass via glycerolysis catalyzed by alum (1). Therefore, evolving new generation of glycerol-fermenting' Saccharomyces cerevisiae is a magnificent start point for this standpoint. In the current study, we succeeded via genetic engineering technologies, metabolic engineering, and rewriting NADH between introduced genes to convert glycerol to bioethanol with reach > 95%.

The outset of evolves the glycerol fermentation, via S. cerevisiae with replaces NAD-dependent glycerol-3-phosphate dehydrogenase by NADH oxidase gene. This replacement not only to restrain the glycerol production under normal fermentation of glucose, but also, for oxidizing the further excesses of NADH under the expected higher influx of glucose and/or glycerol in the recombinant strains. Thereafter, synthetic fermentable metabolic pathway for the glycerol was homologouslly recombined to genomic DNA of S. cerevisiae D452-2, composed of; glycerol facilitator (2 copy), glycerol dehydrogenase (2 copy), dihydroxyacetone kinase (3 copy), and triose phosphate isomerase (2 copy). During these recombination, native glycerol kinase GUT1 (assimilative pathway) was knocked out in order to save the ATP for that synthetic path and solidarity of substrates.

Hitherto, the glycerol fermenting recombinant strain able to ferment initial mixtures of glucose-20g/glycerol-80g and further fed batch of pure glycerol to produce 9% ethanol with production rate 1 g-l/h-l in the shaking flasks. Furthermore, the evolved strain able to ferment that soluble fraction from pretreatment of alum-catalyzed glycerolysis of bagasse and produce up to 7% ethanol. For our knowledge, this is our first trial and also the first report discussing direct conversion of either pure glycerol or glycerol catalyzed glycerolysis of biomass to produce bioethanol using S. cerevisiae. A lot of ways to improve the production rate and produce other valuable products are in progress.