Genetic Engineering Of Saccharomyces Cerevisiae For Optimization Of Glycerol Production

Glycerol is an important green chemical and, due to its cleanness and safety, production of glycerol by microbial fermentation has drawn more and more attention. In this study the following 3 genetic strategies were employed to increase the glycerol production in Saccharomyces cerevisiae: (1) knocking out the N-terminal domain of FPS1, which encodes a channel protein mediating glycerol export; (2) knocking out GUT2, which encodes a key enzyme of the glycerol-3-phosphate shuttle; (3) knocking out NDE1 and NDE2, which encode the two isoenzymes of mitochondrial external NADH dehydrogenase. Deletion of N-terminal domain of FPS1 resulted in a constitutive open channel and strategy (2) and (3) cut off the mitochondrial oxidation of cytosolic NADH in Saccharomyces cerevisiae.Starting with strain 12-1, a haploid progeny of an industrial yeast strain, we created a set of recombinant strains that carries different or different combination of the null allele of the genes described above. Growth experiments and aerobic fermentation of 12-1, 12-1-f(fps1?), 12-1-fg(fps1?, gut2?::repeat), 12-1-fn12(fps1?, nde1?::repeat, nde2?::repeat) and 12-1-fgn12 (fps1?,gut2?::repeat,nde1?::repeat,nde2?::repeat) were performed. The results showed that 12-1-fgn12 can not utilize glycerol for its growth but grew very well on YP-Ethanol and even better than the control 12-1. Meanwhile, when cultured in YPD, 12-1-fgn12 could give successive glycerol yield while the control could not after glucose exhausted. On the other hand, 12-1-f could utilize glycerol better than control, and in fermentation, it dissimilate glycerol more than control after glucose exhaustion. Its glycerol yield peaked at 12h and decreased dramatically after 24h. Additionally, 12-1-fg, 12-1-fn12 increased the glycerol production to some extent.In a word, the deletion of 75-230aa domain of Fps1p could facilitate glycerol export and play some positive role in the uptake and utilization of glycerol. And the quadruple deletion of GUT2, NDE1, NDE2 and N-domain of FPS1 could cut off the pathway of glycerol uptake and utilization.