Arbutin is a plant-derived glycoside with potential antioxidant, antibacterial and anti-inflammatory activities. Currently, it is mainly produced by plant extraction or enzymatic processes, which suffers from expensive processing cost and low product yield. Metabolic engineering of microbes is an increasingly powerful method for the high-level production of valuable biologicals. Since Pseudomonas chlororaphis has been widely engineered as a phenazine-producing platform organism due to its well-characterized genetics and physiology, and faster growth rate using glycerol as a renewable carbon source, it can also be engineered as the cell factory using strong shikimate pathway on the basis of synthetic biology. In this work, a plasmid-free biosynthetic pathway was constructed in P. chlororaphis P3 for elevated biosynthesis of arbutin from sustainable carbon sources. The arbutin biosynthetic pathway was expressed under the native promoter Pphz using chromosomal integration. Instead of being plasmid and inducer dependent, the metabolic engineering approach used to fine-tune the biosynthetic pathway significantly enhanced the arbutin production with a 22.4-fold increase. On the basis of medium factor optimization and mixed fed-batch fermentation of glucose and 4-hydroxybenzoic acid, the engineered P. chlororaphis P3-Ar5 strain led to the highest arbutin production of 6.79 g/L with the productivity of 0.094 g/L/h, with a 54-fold improvement over the initial strain. The results suggested that the construction of plasmid-free synthetic pathway displays a high potential for improved biosynthesis of arbutin and other shikimate pathway derived biologicals in P. chlororaphis.

DAHP synthase catalyzes the first step in the shikimate pathway, deriving the biosynthesis of aromatic amino acids (Trp, Phe and Tyr), phenazine-1-carboxamide, folic acid, and ubiquinone in Pseudomonas chlororaphis. In this study, we identified and characterized one DAHP synthase encoding gene phzC, which differs from the reported DAHP synthase encoding genes aroF, aroG and aroH in E. coli. PhzC accounts for approximately 90% of the total DAHP synthase activities in P. chlororaphis HT66 and plays the most critical role in four DAHP synthases in the shikimate pathway. Inactivation of phzC resulted in the reduction of PCN production by more than 90%, while the absence of genes aroF, aroG and aroH reduced PCN yield by less than 15%, and the production of PCN was restored after the complementation of gene phzC. Moreover, the results showed that phzC in P. chlororaphis HT66 is not sensitive to feedback inhibition. This study demonstrated that gene phzC is essential for PCN biosynthesis. The expression level of both phzC and phzE genes are not inhibited in feedback by PCN production due to the absence of a loop region required for allosteric control reaction. This study highlighted the importance of PhzC and applying P. chlororaphis for shikimate pathway-derived high-value biological production.