In order to improve the ability of Escherichia coli to synthesize 5-hydroxytryptophan, the overexpression plasmid pSTV28-TPH150 was designed and constructed to enhance the expression of hydroxylase gene TPH150.Using HTP10 as the original strain and electrotransforming it with the pSTV28-TPH150 plasmid to obtain the strain HTP10-1. The fermentation results showed that the by-product L-tryptophan accumulated too much and the plasmid was unstable. Therefore, on the basis of the initial fermentation process, four pH gradients were designed to carry out fermentation contrast experiments and four different KH2PO4 flow addition strategies to explore the optimal fermentation process. The experimental results showed that the optimal growth rate of the bacteria was achieved when the fermentation pH of strain HTP10-1 was 6.7 and 1 g/L KH2PO4 was added to the fermentation medium and 3 g/L KH2PO4 was added to the glucose replenishment bottle. After the fermentation, the accumulation of 5-hydroxytryptophan reached the highest level of 5.46 g/L. The accumulation of L-tryptophan, a by-product, was reduced to 1.37 g/L, and the plasmid loss rate was reduced by 32% compared with the control group, which greatly improved the plasmid stability of strain HTP10-1. The feasibility of regulating the growth of bacteria by phosphate and fermentation pH was verified, which has important reference significance for the fermentation control of plasmid engineering bacteria.
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