Development of Artificial Synthetic Pathway of Endophenazines in Pseudomonas chlororaphis P3

Abstract

Endophenazine A is a terpenoid phenazine with phenazine-1-carboxylic acid (PCA), and dimethylallyl diphosphate (DMAPP) derived from the 2-methyl-D-erythritol-4-phosphate (MEP) pathway as the precursor, which shows good antimicrobial activity against several Gram-positive bacteria and fungi. However, the highest yield of endophenazine A was about 20 mg/L in Streptomyces, limiting its large-scale industrial development. Pseudomonas chlororaphis P3, possessing an efficient PCA synthesis and MEP pathways, is a suitable chassis to synthesize endophenazine A. Herein, we designed an artificial biosynthetic pathway for the synthesis of endophenazine A in P. chlororaphis P3. Primarily, the prenyltransferase PpzP from Streptomyces anulatus 9663 was introduced into P. chlororaphis P3 and successfully synthesized endophenazine A. Another phenazine compound, endophenazine A1, was discovered and identified as a leakage of the intermediate 4-hydroxy-3-methyl-2-butene pyrophosphate (HMBPP). Finally, the yield of endophenazine A reached 279.43 mg/L, and the yield of endophenazine A1 reached 189.2 mg/L by metabolic engineering and medium optimization. In conclusion, we successfully synthesized endophenazine A and endophenazine A1 in P. chlororaphis P3 for the first time and achieved the highest titer, which provides a reference for the heterologous synthesis of terpenoid phenazines.

Development of the Static and Dynamic Gene Expression Regulation Toolkit in Pseudomonas chlororaphis

“…Currently, only some elements derived from E. coli (e.g., P lac and P trc ) or endogenous known P phz promoters are commonly used. − In order to solve this dilemma, it is necessary to construct a native PUTR library of P. chlororaphis.…”

“…In addition, P. chlororaphis has also been developed as a host for heterologous synthesis of other complex phenazine compounds due to its powerful phenazine synthetic pathway. − The engineering modifications required for these application scenarios of P. chlororaphis require a more precise synthetic biology toolbox for gene expression regulation.…”

Brevibacterium spongiae sp. nov., isolated from marine sponge Hymeniacidon sp.

“…Other strains of the genus Brevibacterium , with the exception of strain B. limosum o2 T , had the potential to produce endophenazine A1, endophenazine F and endophenazine G. Natural phenazine compounds showed antitumour and antibacterial activities and were potential cancer therapeutic agents [29, 30].…”

“…As predicted by the antiSMASH 6.0 program [28], the genome of strain WHS-Z9 T harboured eight secondary metabolite biosynthetic gene clusters (Table S1). Other strains of the genus Brevibacterium, with the exception of strain B. limosum o2 T , had the potential to produce endophenazine A1, endophenazine F and endophenazine G. Natural phenazine compounds showed antitumour and antibacterial activities and were potential cancer therapeutic agents [29,30].…”

Enhanced Biosynthesis of Phenazine-1,6-dicarboxylic Acid in Streptomyces coelicolor

“…Biorefinery and biomanufacturing processes for producing value-added biofuel, biobased materials, and chemicals are widely investigated with the development of metabolic engineering. Genetic engineering strategies for the high production of phenazines mainly include byproduct pathway elimination, , transcription factor engineering, , reinforcement of rate-limiting enzymes, ,, and efflux pump engineering . The production of PCA and its derivatives has been widely studied.…”