Enhanced trans‐2,3‐dihydro‐3‐hydroxyanthranilic acid production by pH control and glycerol feeding strategies in engineered Pseudomonas chlororaphis GP72

Abstract BACKGROUND Trans ‐2, 3‐dihydro‐3‐hydroxyanthranilic acid (DHHA) is a valuable metabolic intermediate for the biosynthesis of wide‐ranging benzoic acid derivatives with enormous biological or pharmaceutical activities. Pseudomonas chlororaphis GP72 is a non‐pathogenic biocontrol strain that displays unique capability to produce phenazines. Nevertheless, DHHA production is quite low by the wild type strains, which necessitates yield improvement by constructing engineered strains for large‐scale biotechnological applications. RESULTS In this study, two negative regulatory genes namely rsmE and lon were successively deactivated in the DA4 strain. The resulting engineered DA6 strain produced a significantly high titer of DHHA (20.6% higher than that of DA4). The influence of varying pH from 6.2 to 8.2 on DHHA production was studied in a 6‐L benchtop fermenter. A controlled broth pH of 7.2 stimulated maximum DHHA production by the engineered DA6 strain. A DO‐stat based fed‐batch system maintaining a constant DO level (about 20%) accompanied by a glycerol feeding strategy was applied. Highest DHHA production using this approach was recorded to be 10.06 g L ‐1 , which was 31% higher than with traditional batch cultivation. CONCLUSION The production of DHHA in metabolically engineered GP72 was considerably improved by inactivating two negative regulatory genes in a fed‐batch mode. It is concluded that fed‐batch fermentation with intermittent glycerol feeding and pH‐control was an effective approach to improve DHHA production yield. © 2017 Society of Chemical Industry