Construction Of Engineered Serratia Marcescens MG1 For (2R,3R)-2,3-butanediol Production

As an important bio-based product,2,3-butanediol has attracted researchers' attention.2,3-butanediol has many practical applications,such as preparation of plasticizers,synthetic rubber,fumigants,antifreeze agents,and other additives in the production of energy life,especially for optically pure 2,3-butanediol,it can be used as pharmaceutical intermediates and aviation materials.This research used Serratia marcescens MG1 as the original strain,2,3-butanediol isomers produced by the strain were studied,and the genetic engineering method was use of for the transformation of MG1 and it was foundation for the production of optically active(2R,3R)-2,3-butanediol.The main research contents and results are as follows:1.Proposed formation pathway of 2,3-butanediol isomers in S.marcescens MG1.The fermentation products of S.marcescens MG1 was analysised by gas chromatographic,the strain was found to produce three of 2,3-butanediol isomers:meso-2,3-butanediol,(2S,3S)-2,3-butanediol and(2R,3R)-2,3-butanediol,and the concentration was 30.58 g/1,0.92 g/l and 0.67 g/l,respectively.The optical purity of meso-2,3-butanediol was 95.08%.In slaC mutant strain MG1-slaCm,there was still a small amount of meso-2,3-butanediol and(2R,3R)-2,3-butanediol,and the content of(2R,3R)-2,3-butanediol was slightly higher,while the content of acetoin became much higher.Therefore,there could be inferred that another dehydrogenase existed in MG1 and the activity of that enzyme in MG1 was not high and it was responsible for the production of meso-2,3-butanediol and(2R,3R)-2,3-butanediol.Finally,the formation pathway of 2,3-butanediol isomers was proposed.2.Construction of genetically engineered strain for(2R,3R)-2,3-butanediol production.First,the(2R,3R)-2,3-butanediol dehydrogenase gene of Bacillus subtilis 168 was cloned successfully.Under the promoter of 2,3-butanediol dehydrogenase in MG1,the gene was successfully build into pUC19 and was transformed into MG1-slaCm then got MG1-slaCm-dBDH strains.After detection of the fermentation products of the engineered strain,it was founded that it could produce(2R,3R)-2,3-butanediol and meso-2,3-butanediol with a concentration of 27.42g/l and 0.75g/l,respectively,while a much lower levels of acetoin.The optical purity of(2R,3R)-2,3-butanediol was over 97%,indicating that the engineered strain was successfully constructed and it could be used for the production of optically active(2R,3R)-2,3-butanediol.