谷氨酸棒杆菌产L-色氨酸重组菌株的构建

Construction of Recombinant Corynebacterium glutamicum for L-tryptophan Production

摘要:

在芳香族氨基酸合成途径中，由分支酸产生两个分支：一方面，在邻氨基苯甲酸异构酶（ANS）、邻氨基苯甲酸磷酸核糖转移酶（PRT）和色氨酸合成酶（TS）的催化下合成色氨酸；另一方面，分支酸在分支酸变位酶（CM）的催化下生成预苯酸（PPA），由预苯酸又产生两个分支，最后生成酪氨酸或苯丙氨酸。在该途径中，3-脱氧-D-阿拉伯庚酮糖酸-7磷酸合成酶（DS）是关键酶。以谷氨酸棒杆菌SPT9（Phe-、Tyr-、4-FPr、6-FTr）为出发菌株，通过同源重组敲除分支酸变位酶基因csm获得菌株SPT9?csm。将编码DS、ANS和PRT、TS的基因aroⅡ、trpEGD及ts在SPT9?csm中分别过量表达和串联过量表达，获得一系列重组菌：SPT9?csm-XK99E-aroⅡ-trpEGD、SPT9?csm-XK99E-ts、SPT9?csm-XK99E-cspB-ts、SPT9?csm-mob2-ts和SPT9?csm-mob2-ts- aroⅡ-trpEGD。这五株菌摇瓶发酵72 h后，色氨酸产量相对于出发菌株SPT9分别提高了21.4%、57.1%、100%、121.4%和178.6%。通过实时荧光定量PCR检测表明，ts、aroⅡ和trpEGD基因在重组菌SPT9?csm-mob2-ts-aroⅡ-trpEGD中的表达量是它们在菌株SPT9中的9.2倍、13.0倍以及10.6倍。

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.