在明串珠菌构建高产甘露醇的细胞工厂

The construction of cell factory with high mannitol yield in Leuconostoc

甘露醇是一种具有多种功效的六糖醇,被广泛应用于医药、食品、化工和电子等行业。为构建高产甘露醇的明串珠菌,建立了将底盘细胞转化为高产目标产物菌株的理论:细胞工厂的“源-库”学说。通过2次同源重组,打破染色体中基因的操纵子和重叠基因模式,并将甘露醇外排泵蛋白(mannitol efflux pump,MEP)基因序列和再生NADH的酶(甲酸脱氢酶)基因序列定点插入到染色体上。蔗糖为90 g/L时,打破mep基因的操纵子和重叠基因模式的菌株甘露醇产量比原始菌株(CGMCC1.10327)提高了9.5%。原始菌株染色体上敲入一个拷贝mep基因序列的菌株,底物蔗糖能添加到120 g/L,甘露醇产量也达到55.18 g/L。CCTCC M2020762菌株染色体上敲入一个拷贝甲酸脱氢酶基因序列的菌株,底物蔗糖能添加到145 g/L,甘露醇产量也达到101.6 g/L。使细胞工厂具备更强的“源”能(NADH再生等)和更大的“库”容(提高MEP活性),实现目标产物的超高产。

Mannitol is a hexasugar alcohol with many functions,which is widely used in medicine,food,chemical industry and electronics.In order to construct Leuconostoc with high yield of mannitol,the theory of transforming chassis cells into high-yield target product strain—the“source-sink”theory of cell factory was built.Through two homologous recombination,the operon and overlapping gene patterns were broken,and the mannitol efflux pump(MEP)gene sequence and the NADH-regenerating enzyme(formate dehydrogenase)gene sequence were site-direct inserted into the chromosome.When sucrose was 90 g/L,the mannitol yield of the strain that broke the operon and overlapping gene pattern of mep gene was 9.5%higher than that of the original strain(CGMCC1.10327).A strain with a copy of mep gene expression cassette was knocked into the chromosome of the original strain,and the substrate sucrose could be added to 120 g/L,and the output of mannitol reached 55.18 g/L.A strain with a copy of formate dehydrogenase gene expression cassette was knocked into the chromosome of CCTCC M2020762[Δdts1ΔldhΔpat::mdhΔstpk::mdhΔfk::mdhΔaldh::(mtld-mlp)0385/0386::ldhA-eeΔadh::mepΔldh(0503)::mepΔldh(0373)::mep],and the substrate sucrose could be added to 145 g/L,and the output of mannitol reached 101.6 g/L.Make the cell factory have stronger“source”energy(NADH regeneration,etc.)and larger“sink”capacity(improve MEP activity),and obtain super-high yield of the target product.