Effect Of Aldh And Stpk Gene Knockout On Mannitol Production In Leuconostoc

Mannitol is widely applied to the food,pharmaceutical,electron and chemical industries.The current methods of producting mannitol were divided into four categories,the method of plant extraction has some disadvantages such as low yield,serious waste of water resources,and the products refinement is difficult.Chemical synthesis has some disadvantages such as the higer reaction conditions,serious environmental pollution and purification of products is more difficult.The method of enzyme catalysis needs to add expensive coenzyme factor,it has the higher cost of production.the microorganism fermentation has some advantages such as the high quality,high conversion rate and doesn't produce sorbitol.Leuconostoc is a gram positive lactic acid bacteria,it is recognized as safe strain(GRAS)and doesn't produce endotoxin,Leuconostoc has the higher production capacity of mannitol.In the study,we knocked out aldehyde dehydrogenase gene and serine/threonine protein kinase gene in Leuconostoc strains respectively.We compared physiological characterization of mutant strains with wild-type strain to study carbon metabolism and lay the foundation for genetic engineering breeding.To explore the effects of acetaldehyde dehydrogenase on the metabolic of L.mesenteroides CGMCC1.10327,the aldh gene knockout strain was constructed by twice homologous recombination.By using the suicide homologous recombination vector with the tetracycline marker,the aldh gene inactivation strain was constructed.By using the suicide homologous recombination vector without the tetracycline marker,then the vectors was transferred to L.mesenteroides by electrotransformation,the aldh gene knockout strain was constructed.A mutant strain was constructed by knocking out glucan sucrase,aldehyde dehydrogenase and lactate dehydrogenase genes.Compared with the original strain,mannitol production of the mutant strain is increased by 18.8%,ethanol production of the mutant strain is decreased by 62%.To reveal the effects of serine/threonine protein kinase on the metabolic of L.mesenteroides CGMCC1.10327,the stpk gene knockout strain was constructed by twice homologous recombination.By using the suicide homologous recombination vector with ?-amy marker,the stpk gene inactivation strain was constructed.By using the suicide homologous recombination vector without ?-amy marker,the stpk gene knockout strain was constructed.In the process of screening the mutant strain,the blue white screening method was used.Compared with the original strain,dextran production of the mutant strain is decreased by 12.8%,mannitol production of the mutant strain is increased by 6%.