Effect Of The Pat,fk,stpk Knock-out And Mdh Knock-in On Mannitol Production In Leuconostoc Mesenteroides

Mannitol,a C6 sugar alcohol,is widely used in chemical,medical and food industries.At present,the methods for producing mannitol include chemical synthesis,plant extraction,enzyme conversion and microbial fermentation.The method of chemical synthesis requires high pressure at a high temperature with poor selectivity catalyst and produces a mixture of mannitol and sorbitol;the method of plant extraction has some disadvantages such as low yield,serious waste of water resources,and the products refinement is difficult.The method of enzyme conversion requires expensive cofactors,thence the cost is high and uneconomic;The method of microbial fermentation has the advantages of high product quality,such as no production of sorbitol,and high conversion rate.In order to increase mannitol productivity of Leuconostoc mesenteroides,gene knock-out strains and gene knock-in strains were constructed by a two-step homologous recombination method.In this study,the phosphate acetyltransferase gene pat,fructokinase gene fk and serine-threonine protein kinase gene stpk of Leuconostoc mesenteroides were knocked out respectively,resulting in the corresponding knock-out strains.Then the mannitol dehydrogenase gene mdh was knocked in,resulting in the knock-in strains.Then compared with the original strains for physiological and biochemical characteristics,thereby studying the changing rules of carbon metabolism and laying a foundation for genetic engineering breeding.In order to further study the effects of pat,fk,stpk and mdh on mannitol production by Leuconostoc mesenteroides CGMCC1.10327,double gene knock-out and mdh knock-in strains,multiple gene knock-out and knock-in strains were constructed.In this study,the mutant strains,complementary strains and wild strains that had been constructed were subjected to a 20 h fermentation to test the productivity of mannitol under the sucrose as a substrate.The results showed that the pat gene had the most significant effect on the production of mannitol.The mutant with the highest mannitol yield of 97.3% was the multi-mutant strain ?dts?ldh?pat::mdh?stpk::mdh?fk::mdh with 24.7% increase over the wild strain.