Construction of recombinant Saccharomyces cerevisiae producing 1,3-propanediol by one step method

Abstract:

1，3-Propanediol is one of the most important industrial chemicals for its highly desired properties and its wide applications as a key component of an emerging polymer business. Biological production of 1，3-propanediol has been a novel and competitive way. In our previous job，the gene dahB encoding for glycerol dehydratase from Klebsiella and the gene yqhD encoding for 1，3-propanediol oxidoreductase isoenzyme from E. coli were cloned respectively. The two genes were then tandemly ligated and expressed successfully in E. coli. The recombinant E. coli strain could produce 1，3-propanediol from D-glycerol. In the current research，the expression vectors including pGAPZB-yqhD，pGAPZB-dhaB and pYX212-zeocin-pGAP-yqhD-pGAP-dhaB were furtherly constructed on the basis of our previous job. Then the vector pYX212-zeocin-pGAP-yqhD-pGAP-dhaB was introduced into Saccharomyces cerevisiae W303-1A using LiAc transformation method successfully. D-glucose is used as substrate to produce 1，3-propanediol with the recombinant strain after fermentation for 72h. SDS-PAGE analysis showed recombinant products at about 61kD、43kD、21kD、15kD，consistent with the molecular weight from the report. The specific enzymatic activity of the glycerol dehydratase and 1，3-propanediol oxidoreductase isoenzyme of the recombinant yeast strain S. cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD -pGAP-dhaB were 24U/mg protein and 15U/mg protein，respectively，while those of the control were undetectable. In contrast to the wild strain without 1，3-propanediol output，1，3-propanediol concentration of the recombinant yeast strain S. cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB reaches about 1.5g/L. The above results showed that the engineered S. cerevisiae strain which can convert the D-glucose as substrate to produce 1，3-propanediol by one-step fermentation was constructed successfully. This accomplishment bodes well for future construction of recombinant yeast strain which could overproduce 1，3-propanediol with the lower cost feedstock D-glucose by introducing the two genes yqhD and dhaB into the yeast strain overproducing glycerol with D-glucose (e.g. Candida glycerinogenes WL2002-5，which is capable of producing glycerol more than 120g/L with D-glucose as substrate and has been used for the commercial production of glycerol).