Construction Of A System For The Conversion Of Glucose To Mannitol In Leuconostoc

Mannitol,a C6 sugar alcohol,is widely used in many fields such as medicine,food,and chemical industry.At present,the methods for producing mannitol at home and abroad mainly include chemical synthesis,plant extraction,enzyme conversion and microbial fermentation.The first three methods all have their shortcomings.In recent years,the production of mannitol by microbial fermentation has gradually become the mainstream.Many microorganisms can be fermented to produce mannitol,and this method has many advantages such as mild production conditions,high yield,no by-products such as sorbitol,and the ease of purification.As a FDA-recognized GRAS strain,Leuconostoc mesenteroides can produce mannitol with sucrose as a substrate.It has the advantages of small genome,simple and easy metabolism,short fermentation cycle,etc.Leuconostoc mesenteroides is the dominant strain for the fermentation production of mannitol.The exogenous gene mannitol-1-phosphate dehydrogenase gene(mt1d)can produce 6-phosphate-fructose to form 1-phospho-mannitol.And then,mannitol can be produced by the action of the mannitol-1-phosphatase gene(m1p).The two exogenous genes were tandemly introduced into the chromosome of Leuconostoc mesenteroides for expression,and the obtained mutant strain could produce mannitol from glucose as a substrate.So far,the system of conversion of glucose to mannitol in Leuconostoc mesenteroides has been successfully constructed,laying a foundation for genetic engineering breeding.In this study,two gene knock-out strains of aldehyde dehydrogenase gene(aldh)and glucan sucrase gene(dts)were constructed by homologous recombination.Using the suicide-type homologous recombination vector carrying the α-amylase gene,the aldh gene-inactivated strain and the dts gene-inactivated strain were obtained,respectively.Then the exogenous gene mt1d-m1 p tandem expression cassette was introduced into the chromosome of the above-mentioned gene-inactivated strain to obtain the target mutant strain.The mutant strains L.mesteneroides Δaldh::(mt1d-m1p)and L.mesenteroides Δaldh::(mt1d-m1p)Δdts::(mt1d-m1p)were respectively fermented at the substrate concentration of 20 g/L glucose and 90 g/L sucrose,and then the fermentation broth was detected by an evaporative light scattering detector.The yields of mannitol were 2.52 g/L and 45.74 g/L,respectively.The latter conversion rate was 50.82%,and the yield was 45.3% higher than that of the original strain.