System Construction For Biosynthesis Of Rare Ketohexoses From Glycerol Based On DHAP-dependent Aldolases

Rare ketohexoses,which exist rarely in nature,have wide applications in food,medicine and health care due to their unique properties.However,the synthesis of rare ketoses is still costly and limited.Therefore,it is of great importance to construct an efficient and low-cost platform for the synthesis of rare ketoses in large scale.In this dissertation,the production of dihydroxyacetone phosphate(DHAP)and glyceraldehyde from cheap glycerol were studied.A system for synthesis of rare ketoses from glycerol was established based on DHAP-dependent aldolases which catalyze aldol reaction of DHAP and glyceraldehyde to form rare ketoses-1-phosphate.DHAP was synthesized from glycerol by glycerol kinase(GK)and L-?-glycerophosphate oxidase(GPO),and then,an efficient system for whole-cell cascade synthesis of rare ketoses from glycerol and glyceraldehyde was built by coupling aldolase and phosphatase.Subsequently,in order to avoid the direct use of glyceraldehyde,alditol oxidase(AldO)and horse liver alcohol dehydrogenase(HLADH)were used to oxidize glycerol to generate D-glyceraldehyde and L-glyceraldehyde,which were combined with the synthesis pathway of rare ketoses.As a result,a system for the synthesis of rare ketoses using glycerol as the sole substrate was set up.Finally,in order to produce rare ketoses safely and effectively,ClearColi was used to express key genes ald O,rha D and yqa B for the synthesis of D-sorbose and D-psicose from glycerol by fermentation.The main results were described as follows:(1)The genes from DHAP pathway was studied,and a system for whole-cell cascade synthesis of rare ketoses from glycerol and glyceraldehyde based on GK,GPO,aldolase and phosphatase were constructed.It was found that overexpression of GK and GPO could significantly improve the conversion rate of rare ketoses.The related phosphatase genes for the synthetic pathway of rare ketose were screened.It was found that overexpression of phosphatases could improve the utilization rate of D-glyceraldehyde,and fructose-1-phosphotase(Yqa B)could significantly increase the yield of rare ketoses.On this basis,a more efficient system for synthesis of rare ketoses was built.The total yield of D-sorbose and D-psicose was 21.7 g/L,and the yield of L-fructose was 16.8 g/L.(2)The enzymatic properties of AldO from Streptomyces coelicolor A3 was studied,it was found that glycerol could be oxidized by AldO to produce D-glyceraldehyde.Then AldO was integrated into the synthesis pathway of rare ketoses,a system for the synthesis of rare ketoses using glycerol as the sole substrate was constructed.Moreover,the effect of catalase overexpression on the yield of rare ketoses was also studied.It was found that catalase could significantly increase the yield of rare ketoses.The total yield of D-sorbose and D-psicose was 16.8 g/L.(3)The engineered strain overexpressing HLADH and NADH oxidase(NOX)was studied.It was found that this recombinant strain could produce L-glyceraldehyde from glycerol without the addition of cofactor NAD~+.Then the engineered strain was combined with the engineered strain constructed in chapter 2 to establish a system for the synthesis of L-fructose from glycerol.The effects of the factors(temperature,p H,wet cell amounts,glycerol concentration)on the production of L-fructose were investigated.It was found that the order of the influence of the four factors on L-fructose production was glycerol concentration(D)>p H(B)>wet cell amounts(C)>temperature(A),and glycerol concentration had the most significant effect on the synthesis of L-fructose.The total yield of L-fructose was 2.67 g/L.(4)By the analysis of 6 genes related in the synthesis pathway of D-sorbose and D-psicose from glycerol,it was found that only expression of three key genes ald O,rha D and yqa B could achieve the synthesis of D-sorbose and D-psicose from glycerol by fermentation.Single regulation and combined regulation of RBS mutant library strategy were used to control the expression of key genes in the synthesis pathway.It was found that the above strategy was more effective for the synthesis of rare ketoses compared with the original RBS regulation,and the strategy of combined regulation of RBS mutant library showed better performance.The total yield of D-sorbose and D-psicose was 6.29 g/L and the conversion rate was 62.9%.The self-assembly of Spytag-AldO/Spy Catcher-Rha D to form self-assembly complex and the ability to produce rare ketoses were studied.The production of rare ketoses was improved significantly based on Spy Tag/Spy Catcher self-assembly complex.The total yield of D-sorbose and D-psicose was 5.79 g/L.Phosphotase could significantly increase the yield of rare ketoses.and the total yield was 7.12 g/L.