Effect Of Inulinase Encoding Gene Expression Cassette On Mannitol Production In Leuconostocs

Mannitol is a kind of hexacarbohydrate alcohol,which has been widely used in many fields such as food,medicine and chemical industry.At present,the production methods of mannitol mainly include plant extraction method,catalytic hydrogenation method,enzyme conversion method and microbial fermentation method.Among them,microbial fermentation method has the advantages of mild conditions,no sorbitol production,high product quality and high conversion rate,which has developed into the main trend.There are many kinds of microorganisms known to produce mannitol in nature.Leuconostoc is a GRAS bacteria recognized by FDA in the United States.It can produce mannitol dehydrogenase in cells by heterotypic lactic acid fermentation,which has obvious advantages in the research of fermentation to produce mannitol.Inulin is a kind of natural fructan which exists in a large number of plants.It can be hydrolyzed into fructooligosaccharides or fructose by inulinase,which can become a low-cost fructose resource.Therefore,the transformation from inulin to mannitol in Leuconostoc is of great significance for the study of mannitol production.In this study,the inulinase genes from Aspergillus niger was codon optimized and linked to the expression element of Leuconostoc mesenteroide CGMCC1.10327.An endo-inulinase InuA gene expression cassette and an exo-inulinase InuE gene expression cassette were constructed.Using the lactate dehydrogenase D-ldh,0503-ldh,0373-ldh genes and alcohol dehydrogenase adh gene as the gene knock-in sites,the InuA expression cassette and the InuE expression cassette were integrated into Leuconostocs for exogenous expression by two homologous recombinations.Several gene knock-in strains were constructed.In order to study the synergistic effect of endo-and exo-inulinase genes,the mutant strain which both knock in at the same time was constructed.In order to achieve better expression effect,the InuE gene was multiple copies and the optimal copy number was obtained.The most suitable fermentation conditions of mutant strains were studied by changing the type,concentration of fermentation substrates and fermentation time.The results showed: Both InuA gene and InuE gene were successfully expressed in Leuconostoc mesenteroide CGMCC1.10327,but the activity of endo-inulinase produced by InuA gene expression was not high in Leuconostoc,so it did not play a great role.The multi-gene knock-out and three copies of InuE gene cumulative knock-in had a great improvement in mannitol production.When the substrate concentration(g/L)ratio of sucrose/inulin was 50/20 and the fermentation time was28 h,the mannitol yield of the mutant strain ?W?adh::InuE?0503::InuE?0373::InuE was the highest at 42.93 g/L.Compared with the original strain,the yield increased by 36.4 %.