title: [Construction of co-expression SHMT and TPase recombinant vector and dual-enzymatic synthesis of L-tryptophan].
abstract: Hydroxymethyltransferase (SHMT) and tryptophanase (TPase) are key enzymes in biosynthesis of L-tryptophan. We constructed three recombinant plasmids, including pET-SHMT, pET-TPase, and pET-ST for over-expression or co-expression of SHMT and TPase in Escherichia coli BL21 (DE3). The SDS-PAGE analysis showed that the recombinant proteins of 47 kDa and 50 kDa were expressed of pET-SHMT and pET-TPase, respectively. As compared to the host stain, the enzyme activity of SHMT and TPase was increased by 6.4 and 8.4 folds, respectively. Co-expression of both recombinant proteins, 47 kDa and 50 kDa, was also successful by using pET-ST and the enzyme activities were enhanced by 6.1 and 6.9 folds. We designed two pathways of dual-enzymatic synthesis of L-tryptophan by using these recombinant strains as source of SHMT and TPase. In the first pathway, the pET-SHMT carrying strain was used to catalyze synthesis of L-serine, which was further transformed into L-tryptophan by the pET-TPase expressing strain. These two steps sequentially took place in different bioreactors. In the second pathway, the pET-ST carrying strain, in which two enzymes were co-expressed, was used to catalyze simultaneously two steps in a single bioreactor. HPLC analysis indicated a high yield of 41.5 g/L of L-tryptophan was achieved in the first pathway, while a lower yield of 28.9 g/L was observed in the second pathway. In the first pathway, the calculated conversion rates for L-glycine and indole were 83.3% and 92.5%, respectively. In the second pathway, a comparable conversion rate, 82.7%, was achieved for L-glycine, while conversion of indole was much lower, only 82.9%.
authors.affiliation: College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Qinqin
authors.initials: Q
authors.lastname: Zhao
authors.forename: Aicai
authors.initials: A
authors.lastname: Xu
mesh_terms: D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics*; D012696:Glycine Hydroxymethyltransferase / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D011995:Recombination, Genetic / Q000235:genetics*; D014364:Tryptophan / Q000096:biosynthesis*; D014368:Tryptophanase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D012696:Glycine Hydroxymethyltransferase; D014368:Tryptophanase