Biosynthesis Of Healthy Sugar By Metabolic Engineered Bacillus Subtilis

D-tagatose is an isomer of D-fructose which is approximately 90% sweeter than sucrose,but only 30% calories after ingest.D-Tagatose is similar to the sucrose in having a low caloric value and tooth-friendly property,which attracted much attention for applications in the food industry because of their ability to prevent and treat these chronic diseases.It is also a rare sugar that was generally recognized as safe by the Food and Drug Administration in 2002.Biological manufactures of D-tagatose mainly conducted by L-arabinose isomerase using D-galactose as substrate.The high cost of substrates,low conversion rate and complicated purification procedures have limited the biotransformation of D-tagatose.Thus,a cost-effective synthetic pathway for Dtagatose biosynthesis from renewable,cheap and readily available bulk substrates is urgently needed.In this study,a recombinant Bacillus subtilis strain have been constructed by metabolic engineering strategies to produce D-tagatose.The main research conclusions are as follows:Firstly,the encoding genes of uracil phosphoribosyltransferase,RNA polymerase sigma-F factor,surfactin synthase subunit 3,alpha-amylase,PTS system glucose-specific EIICBA component,serine/threonine protein kinase,6-phosphofructokinase and alphaphosphoglucomutase（upp,spoⅡAC,srf AC,amy E,pts G,hprk,pfk A and pgc A,respectively）were knocked-out sequentially from the genome of Bacillus subtilis strain SCK6,while the glucokinase encoding gene glk from E.coli were knocked-in at the location of gene pts G in the genome of SCK6.The resulting strain SCK15 has better properties in fermentation and metabolic flux direction.Then,the D-tagatose 6-phosphate 4-epimerase from Agrobacterium tumefaciens and the phosphoglycolate phosphatase from Archaeoglobus profundus were co-expressed in SCK15 using high copy number plasmid and constitutive promoter.The resulting strain SCK15 T has the ability to convert glucose to tagatose during fermentation process with a titer of 2.64 g?L^-1 tagatose.At last,we optimized the culture temperature and the concentration of glucose in the medium,resulting a best culture conditions of 42℃ and 100 g?L^-1 glucose,with a titer of 5.26 g?L^-1 tagatose.It is nearly 2 times of the titer before optimization.