Study On The Key Genes Involved In Glycerol Catabolism Pathway Of Candida Glycerinogenes And Its Application

Glycerol is the main by-product in the production of biodiesel.Compared with S.cerevisiae,C.glycerinogenes is a potential excellent cell factory that can utilize glycerol as carbon source to generate high level biomass without organic acids and alcohol accumulations.In order to investigate the glycerol catabolism in C.glycerinogenes,the fermentation performance with glycerol and glucose as sole carbon source were examined.The results suggested that glycerol as carbon source generated high level biomass without organic acids and alcohol accumulations;When the cells were cultivated with glycerol and xylose as co-substrates,the titer and conversion rate of xylitol reached 39.4 g·L^-1 and 88%,respectively,which were 1.79 and 1.32-fold of that of the cells cocultivated by xylose and glucose.Genes involved in glycerol catabolism were cloned and function verified.The regulation of glycerol catabolism was assessed by qRT-PCR and enzyme activity assays.the results showed that the gluconeogenesis and HMP pathways were up-regulated to generate cell skeleton,the EMP and byproducts synthesis pathways were downregulated to reduce the production of organic acids and alcohol,the TCA cycle was also upregulated to compensate the loss of reducing power and energy resulting from the reduced EMP pathway activity,when the cell was cultivated with glycerol as sole carbon source.These changes led to the improved biomass by 25% without the accumulation of organic acids and alcohol.To understand glycerol catabolism in C.glycerinogenes,the mutant strains C.g gcy1?,C.g gcy2?,C.g gcy1?/gcy2? and C.g dak? were constructed by "Ura-Blaster" knockout box,respectively.The results showed that the deletions of CgGCY1 or CgGCY2 reduced but not elimimated the glycerol utilization.The mutant strains C.g gcy1?/gcy2? and C.g dak? cannot grow on the glycerol-based medium,indicating that the glycerol catabolism in C.glycerinogenes was solely depended on the DHA pathway.Besides,the gene CgGCY2 played a more important role than CgGCY1 during glycerol catabolism in C.glycerinogenes.To explore the application of glycerol catabolic pathway,the effects on the synthesis of xylitol as the target product through enhanced glycerol catabolic pathway were assessed.The recombinant strains C.g-pUR-CgGCY1-CgDAK and C.g-pUR-CgGCY2-CgDAK accelerated glycerol and xylose consumptions.Overexpression of the key genes involved in the DHA pathway increased the titer and conversion of xylitol,the xylitol production of recombinant strains reached 44.0 g·L^-1 and 45.0 g·L^-1,which was 12% and 13% higher than the wild-type strain,respectively.Delection of the xylose reductase gene CgXYL2 blocked downstream of xylitol metabolism and increased xylitol conversion rate,leading to accumulated xylitol efficiently.The xylitol production of recombinant strains C.g xyl2?-pUR-CgGCY1-CgDAK and C.g xyl2?-pUR-CgGCY2-CgDAK reached 49.2 g·L^-1 and 49.5 g·L^-1,which was 23% and 24% higher than the wild-type strain,respectively.The conversion rate of xylitol reached 97% and 98%,increased 9% and 10% compared with the wild-type strain.The xylitol yield per cell of recombinant strains up to 2.89 g·g^-1 and 2.91 g·g^-1,was 34% and 35% higher than the wild-type strain.The results present here provided new information to further produce high value chemicals by engineered C.glycerinogenes using glycerol as co-substrate.