Construction of Recombinant Corynebacterium glutamicum for L-tryptophan Production
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Abstract:
In the aromatic amino acid synthesis pathway, chorismic acid produces two branches. One is to synthetize tryptophan under the catalyze of anthranilate isomerase (ANS), anthranilate phosphoribosyl transferase (PRT) and tryptophan synthase (TS). And the other is to generate prephenic acid (PPA) under the catalyze of chorismic acid mutase (CM), which further produces tyrosine and phenylalanine. Meanwhile, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS) is the key enzyme in this pathway. We recently engineered Corynebacterium glutamicum SPT9 (Phe-, Tyr-, 4-FPr and 6-FTr) for improved production of L-tryptophan. Inactivation of gene csm encoding chorismate mutase was firstly conducted by homologous recombination to get auxotrophic tyrosine and phenylalanine, which was different from the classical mutagenesis method. Then aroⅡ, trpEGD and ts genes encoding DS, ANS and PRT, TS were over-expressed alone or together to obtain series of recombinant strains SPT9?csm-XK99E-aroⅡ-trpEGD, SPT9?csm-XK99E-ts, SPT9?csm-XK99E-cspB-ts, SPT9?csm-mob2 -ts and SPT9?csm-mob2-ts-aroⅡ-trpEGD. After 72 hr fermentation, the tryptophan yield in these five strains increased by 21.4%, 57.1%, 100%, 121.4% and 178.6%, respectively compared with SPT9. qPCR was employed to evaluate the transcript quantification of the target genes. The expressions of ts, aroⅡ and trpEGD genes in SPT9?csm-mob2-ts-aroⅡ-trpEGD were 9.2, 13.0 and 10.6 times, respectively of that in strain SPT9.
