In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Metabolically engineered E. coli has the potential to further improve the production of DHS.
3-Deoxy-7-Phosphoheptulonate Synthase ; genetics ; Amino Acids, Aromatic ; biosynthesis ; Biosynthetic Pathways ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Metabolic Engineering ; Shikimic Acid ; analogs & derivatives ; metabolism ; Transketolase ; genetics

To improve tryptophan production in Escherichia coli, key genes in the tryptophan biosynthesis pathway -aroG, trpED, trpR and tnaA were manipulated. TrpR gene was knocked out to eliminate the repression on the key genes controlling tryptophan biosynthesis and transportation on bacteria chromosome, and the tryptophan degradation was blocked by tnaA gene knockout. Then the bottleneck in tryptophan biosynthesis pathway was removed by co-expressing aroGfbr gene and trpEDfbr gene. Compared with the MG1655, the tryptophan production of trpR knockout and double-genes knockout strains was improved 10-folds and about 20-folds, respectively. After the trpEDfbr was expressed, the tryptophan production increased to 168 mg/L, and when the aroGfbr and trpEDfbr were co-expressed, the tryptophan production increased to 820 mg/L. This work laid the foundation for further construction of higher-efficient engineered strain for tryptophan production.
3-Deoxy-7-Phosphoheptulonate Synthase ; metabolism ; Amino Acid Transport Systems ; genetics ; Bacterial Proteins ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Gene Knockout Techniques ; Genetic Engineering ; Repressor Proteins ; genetics ; Tryptophan ; biosynthesis