Construction Of Co-expression SHMT And TPase Recombinant Vector And Dual-enzymatic Synthesis Of L-tryptophan

Three recombinant strains were constructed including two single-expression strains of SHMT or TPase and a co-expression strain of SHMT and TPase. Lactose was used to induce the expression of recombinant strains, and the induction conditions on the expression of recombinant strains were optimized when lactose was used as an inducer. Two pathways of dual-enzymatic synthesis of L-tryptophan were designed: one was a two-step pathway that SHMT catalyzed synthesis of L-serine and TPase catalyzed synthesis of L-tryptophan orderly in two reactors; another was also a two-step pathway, but the reaction of these two syntheses processed simultaneously in the same reactor. Advantage and disadvantage of these two pathways were analysed and compared.Using genome of Escherichia coli K-12 as templates, gene coding hydroxymethyltransferase(SHMT) and gene coding tryptophanase(TPase) were cloned by PCR. Both products of PCR were cut by NcoⅠ/BamHⅠ, and ligated with vector pET28a which was also cut by NcoⅠ/BamHⅠ. Product of ligation was transformed into E. coli BL21(DE3) to obtain the single-expression recombinant strains BL21(DE3)/pET-SHMT and BL21(DE3)/pET-TPase. Recombinant plasmid pET-TPase was cut by BglⅡ/HindⅢ, and reserved theDNA segment which contained T7 promoter and gene coding TPase. This DNA segment was ligated with segment of recombinant plasmid pET-SHMT which was cut by BamHⅠ/HindⅢ. Product of ligation was transformed into E. coli BL21(DE3) to obtain the co-expression SHMT and TPase recombinant strain BL21(DE3)/pET-ST.The induction conditions on the expression of three recombinant strains were optimized. As for BL21(DE3)/pET-SHMT, the best lactose concentration was 8g/L; the optimum initial seed age was when OD600 reached about 1.0; the best inducation temperature was 37℃; the optimum inducation time was 6 hours. When the recombinant straint was cultured under the optimum conditions, the enzyme activity of SHMT reached 220.7U/mL. Compared with host stain, the enzyme activity of SHMT was increased by 6.4 folds in recombinant strain.As for BL21(DE3)/pET-TPase, the best lactose concentration was 12g/L; the optimum initial seed age was when OD600 reached about 1.0; the best inducation temperature was 37℃; the optimum inducation time was 6 hours. When the recombinant straint was cultured under the optimum conditions, the enzyme activity of TPase reached 113.7U/mL. Compared with host stain, the enzyme activity of TPase was increased by 8.4 folds in recombinant strain.As for BL21(DE3)/pET-ST, the best lactose concentration was 12g/L; the optimum initial seed age was when OD600 reached about 1.0; the best inducation temperature was 30℃; the optimum inducation time was 6 hours. When the recombinant straint was cultured under the optimum conditions, the enzyme activity of SHMT and TPase respectively reached 210.2 U/mL and 93.5 U/mL. Compared with host stain, the enzyme activity of SHMT and TPase was respectively increased by 6.1 and 6.9 folds in co-expression recombinant strain.The first pathway applied two single-expression recombinant strains as source of SHMT and TPase. Reaction that used glycin and formaldehyde to synthesize L-serine was catalyzed by SHMT. The quantities of glycin were 30g; formaldehyde that was feed into the reactor was used to control the pH bwteen 6.8~7.2. Then the products of L-serine synthesis reaction and 9g indole were used to synthesize L-tryptophan catalyzed by TPase. In this pathway, the conversion rate for L-glycin and for indole was 83.3% and 92.5%, the concentration of L-tryptophan in reactor arrived at 41.5g/L.The second pathway applied the co-expression recombinant as source of SHMT and TPase. Reaction of synthesis L-serine and synthesis L-tryptophan was happened at same time in same reactor. Three groups of different quantities of substrates were designed in order to find out which group is the best. The result indicated that when quantities of glycin and formaldehyde were respectively 25g and 7g, both conversion rate of substrates and concentration of L-tryptophan reached the highest. The conversion rate for L-glycin and for indole was 82.7% and 82.9%, the concentration of L-tryptophan in reactor arrived at 28.9g/L.