Combined methods of mutation breeding and genetice modifications enhanced rhamnolipids production of oil well Indigenous Pseusdomonas aeruginosa

Abstract

Rhamnolipids (RLs) are considered as the most popular and efficient biosurfactant, which exhibit superior thermal stability, chemical resistance, low ecotoxicity, biodegradability and good antimicrobial activity. The RLs-producing wild-type strains cannot produce high-yield rhamnolipids to meet with the demand of industrial applications. In view of the problems existing in the practical application of RLs, atmospheric and room temperature plasma-mediated (ARTP) mutagenesis, ultraviolet mutagenesis, ethyl methane sulfonate (EMS) were used alone and combined to mutate and screen Pseudomonas aeruginosa SH6 for many rounds, and a mutant strain P. aeruginosa EZD3 that produced 5.05 g/L of RLs was obtained. Then the original promoter of the rhamnosyl transferase genes was replaced with a strong promoter in strain EZD3, a recombinant strain P. aeruginosa APA1 produced 7.32 g/L of RLs, which showed a 4.67 times increase in RLs production than the original strain P. aeruginosa SH6. Finally, after the optimization of the carbon source of for RLs production, the RLs yield of the recombinant strain P. aeruginosa APA1 reached 7.59 g/L.

Keywords: rhlABRI; Mutagenesis; Over-expression; Rhamnolipids; Strong promoter.

Fig. 1

Synthetic pathway of RLs in P. aeruginosa.

Fig. 2

Genetic stability of high-yield mutant EZD3 and optimization of RLs-producing medium. (A) The RLs production of the mutant strain EZD3 from the first day to the sixth day; (B) Effects of different carbon sources on RLs production of mutant strain EZD3 (Gly: glycerol; G: Glucose; C oil: cottonseed oil); (C) RLs production of the mutant strain EDZ3 under different carbon and nitrogen ratios.

Fig. 3

RLs production of recombinant strains from P. aeruginosa D1 with integrated plasmid pEX18ACA through triparental hybrid (A, B, C, D: Cultures were added 300, 400, 600, 800 µM ZnSO4, respectively).

Fig. 4

(A) RLs production of recombinant strains of P. aeruginosa SH6 containing pET28RAS; (B) RLs production of recombinant strain P. aeruginosa D1 containing pTrc99RAS; (C) RLs production of recombinant strain P. aeruginosa EDZ3 containing pEX18APA; (D) RLs production of recombinant strains No. 1, 3, 7, 10, 11 containing pEX18APA; (E) Effects of different carbon sources on RLs production of high-yield recombinant strain P. aeruginosa APA1 (R oil rapeseed oil, C oil corn oil, G glucose, Fl oil linseed oil, Gly glycerol).