Abstract
Shikimate is a key intermediate in the synthesis of neuraminidase inhibitors. Compared with traditional methods, microbial production of shikimate has the advantages of environmental friendliness, low cost, feed stock renewability, and product selectivity and diversity. Despite these advantages, shikimate kinase I and II respectively encoded by aroK and aroL are inactivated in most shikimate microbial producers, thus requiring the addition of aromatic compounds during the fermentation process. To overcome this problem, we constructed a non-auxotrophic, shikimate-synthesising strain of Escherichia coli. By inactivation of repressor proteins, blocking of competitive pathways and overexpression of key enzymes, we increased the shikimate production of wild-type E. coli BW25113 to 1.73 g/L. We then designed a tunable switch that can conditionally decrease gene expression and substituted it for the original aroK promoters. Expression of aroK in the resulting P-9 strain was maintained at a high level during the growth phase and then reduced at a suitable time by addition of an optimal concentration of inducer. In 5-L fed-batch fermentation, strain P-9 produced 13.15 g/L shikimate without the addition of any aromatic compounds. The tunable switch developed in this study is an efficient tool for regulating indispensable genes involved in critical metabolic pathways.
Highlights
Metabolic engineering approaches used to construct a shikimate-producing strain mainly focus on the central carbon metabolic pathway and the shikimate pathway
When no inducer was incorporated into the culture, the expression of TetR controlled by PBAD promoter was repressed, with consequent normal expression of the target gene under the regulation of PLtetO1
When 1.00 g/L L-arabinose was incorporated into the medium at 0, 4, or 8 h after inoculation of seed cultures, the relative fluorescence intensities of super-folding green fluorescent protein (sfGFP) were all obviously decreased compared with the control strain lacking inducer (Fig. 3)
Summary
Metabolic engineering approaches used to construct a shikimate-producing strain mainly focus on the central carbon metabolic pathway and the shikimate pathway. The expression of aroK was conditionally repressed after accumulation of adequate biomass, demonstrating the successful generation of a non-auxotrophic, shikimate-synthesising E. coli. Encoded by aroK is crucial for increasing shikimate accumulation and maintaining normal cell growth, a tunable switch was first constructed to allow appropriate regulation of AroK (Fig. 2).
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