共表达SHMT和TPase载体的构建及双酶法合成L-色氨酸

Construction of co-expression SHMT and TPase recombinant vector and dual-enzymatic synthesis of L-tryptophan

利用重组大肠杆菌表达丝氨酸羟甲基转移酶(SHMT)和色氨酸酶(TPase),并利用双酶法合成L-色氨酸。采用PCR从大肠杆菌K12基因组中扩增上述两种酶的基因,利用pET-28a载体,构建单表达重组质粒pET-SHMT、pET-TPase和共表达重组质粒pET-ST。将上述3种重组质粒转入大肠杆菌BL21(DE3)进行表达。SDS-PAGE结果表明,单表达基因工程菌BL21(DE3)/pET-SHMT和BL21(DE3)/pET-TPase分别在47kDa(SHMT)和50kDa(TPase)处有蛋白表达带;共表达基因工程菌BL21(DE3)/pET-ST在上述两处均有蛋白表达带。与宿主菌相比,单表达SHMT基因工程菌产酶活性提高了6.4倍;单表达TPase基因工程菌产酶活性提高了8.4倍;共表达SHMT和TPase基因工程菌产酶活性分别提高了6.1和6.9倍。利用工程菌所产酶进行双菌双酶法和单菌双酶法合成L-色氨酸。两菌双酶合成L-色氨酸的累积量达到41.5g/L,甘氨酸转化率为83.3%,吲哚转化率为92.5%;单菌双酶合成L-色氨酸的累积量达到28.9g/L,甘氨酸转化率为82.7%,吲哚转化率为82.9%。

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%.