Effect Of The AcrB Knock-in On Mannitol Production In Leuconostoc

Mannitol is an important hexitol,which has the characteristics of suitable sweetness,low calorie,low moisture absorption and no toxic side effects.It is widely used in food,medicine,chemical and other fields.At present,methods for industrially producing mannitol at home and abroad include biological extraction methods,chemical hydrogenation methods,and electrochemical methods,but the above methods have drawbacks.Recent studies have shown that many microorganisms in nature can synthesize mannitol,and the production of mannitol by microbial fermentation has obvious advantages.The reaction conditions are mild and easy to control,no by-product sorbitol produced.Leuconostoc is a type of Gram-positive bacterium.It is a recognized GRAS strain in the United States.It can be fermented to produce mannitol by using sucrose and fructose.And it has easy growth control conditions,simple metabolic pathway,short fermentation cycle,and can secrete specific protein.These characteristics are the advantage of being the strain of mannitol production.In order to further improve the mannitol yield of Leuconostoc,and solve the problem of insufficient substrate utilization due to excessive accumulation of intracellular products during fermentation,this study knocked in and expressed the Acr B gene on the chromosome of Leuconostoc.The Acr B gene is an important part of the E.coli multi-drug efflux pump system,which has a wide range of substrates,and can transport a variety of drugs directly,detergents and dyes to the extracellular space.Acr B is a drug transporter among them,playing an important role.In order to explore the effect of knock-in of Acr B gene on the production of mannitol by Leuconostoc,this study selected acetaldehyde dehydrogenase and glucan sucrase genes as knock-in sites,and constructed Acr B gene single copy knock-in and double copy knock-in mutant strains.By constructing a homologous recombination vector of acetaldehyde dehydrogenase gene,using the ?-amylase gene as a mutation screening marker,introducing the vector into the recipient strain,and performing a homologous recombination to obtain a mutant strain carrying the ?-amylase gene marker.On the basis of this mutant,an aldh homologous recombination vector containing the exogenous gene Acr B was introduced,and a second homologous recombination occurred to obtain a singlecopy knock-in mutant of Acr B gene.On the basis of the single-copy knock-in mutant,Acr B was knocked in again by constructing a homologous recombinant vector of glucan sucrase dts gene,and a double-copy knock-in mutant strain was obtained by the same method.The fermentation results of the original strain and the mutant strains indicated that the knock-in of the Acr B gene increased the ability of Leuconostoc to utilize the sucrose,and the optimum substrate concentration was increased from 90 g/L to 110 g/L.And the sucrose conversion rate of Acr B gene double-copied strain was 44.41%.The mannitol yield of which reached 48.85 g/L.