Enabling and enhancing anaerobic synthesis of rhamnolipids from glucose by an oil reservoir-derived Pseudomonas aeruginosa strain

Pseudomonas aeruginosa , the rhamnolipids-producing bacterial strain, is a facultative anaerobe widely distributed in oil reservoirs. Given the anoxic conditions in oil reservoirs, the high production of rhamnolipids by P. aeruginosa under anaerobic conditions is of great significance for the development of microbial enhanced oil recovery (MEOR). Under anaerobic conditions, Pseudomonas aeruginosa exhibits low biomass and low rhamnolipid production using glycerol as a carbon source. When glucose is used as a carbon source, P. aeruginosa exhibits high biomass under anaerobic conditions, but it cannot synthesize rhamnolipids anaerobically. Can genetic modification enable P. aeruginosa to anaerobically synthesize rhamnolipids from glucose? Comparative analysis of transcriptomes of P. aeruginosa SG revealed that the anaerobic expression of genes rmlBDAC (m), rhlABRI (h) and rhlC (c) involved in rhamnolipids synthesis was down-regulated when using glucose as a carbon source. Among these three sets of genes, only the overexpression of the rmlBDAC (m) genes enables P. aeruginosa to synthesize rhamnolipids from glucose under anaerobic conditions. HPLC-MS analysis confirmed that genetically engineered strains synthesized rhamnolipids from glucose under anaerobic conditions. Genetically engineered strain P. aeruginosa SGm anaerobically produced 0.8 g/L rhamnolipids using glucose as a carbon source. In addition to increasing the copy number of genes rmlBDAC (m), further increasing the copy number of genes rhlABRI can further enhance the anaerobic yield of rhamnolipids from glucose. Genetically engineered strain P. aeruginosa SGmh anaerobically produced 1.5 g/L rhamnolipids utilizing glucose. Notably, both genetically engineered strains SGm and SGmh grew better and produced more rhamnolipids utilizing glucose than glycerol under anaerobic conditions. Overexpression of genes rmlBDAC enables oil reservoir-derived P. aeruginosa to anaerobically synthesize rhamnolipids using glucose as a carbon source. Genes rmlBDAC determine whether P. aeruginosa can anaerobically synthesize rhamnolipids from glucose. Genes rhlABRI is crucial to the high production of rhamnolipids from glucose under anaerobic conditions. This study makes glucose a superior alternative carbon source for anaerobic biosynthesis of rhamnolipids by P. aeruginosa through key genes modification.