Increased production of pyocyanin in recombinant Pseudomonas aeruginosa PS39-phzMS strain harboring the pUCP24-phzMS plasmid

Abstract

PhzM and PhzS are two “core” enzymes that are necessary for conversion of phenazine-1-carboxylic acid (PCA) into pyocyanin (PYO) phenazine in Gram (-) bacterium Pseudomonas aeruginosa. Apparently, the raise in copy number of their genes could increase the amount of pyocyanin phenazine in the microbe. In previous research, two genes phzM and phzS originated from Pseudomonas aeruginosa PS39 strain had been inserted into a Pseudomonas – Escherichia coli shutlle vector pUCP24 to generate a plasmid pUCP24-phzMS. The obtained plasmid had been transformed into P. aeruginosa PS39 strain to create the recombinant P. aeruginosa PS39-phzMS strain. In this study, pUCP24-phzMS was sequenced to verify the polycistronic expression cassette containing both phzM and phzS genes. The results demonstrated that the recombinant plasmid comprised the ori of Pseudomonas and E. coli, gentamicin resistance-cassette, and polycistronic expression cassette for expression of PhzM and PhzS. In which, both genes will be transcripted together in one mRNA strand by the regulation of Lac promoter and operator. The translation from the mRNA to the corresponding proteins will be started by binding ribosome into RBS located upstream of each gene. The nucleotide sequence of these genes were completely homologous (100%) to the submitted sequences MF673740 (phzM) and MF770713 (phzS) on the NCBI GenBank database. Result on assessing the synthesis of pyocyanin in the recombinant strain with the presence of pUCP24-phzMS plasmid showed that pyocyanin concentration in the recombinant strain increased significantly over 2 times (31.22 mg/mL) more than that in the wild strain (13.47 mg/mL). The absorbance at 360 nm of PCA from the P. aeruginosa PS39-phzMS (OD367 = 0.03) strain decreased significantly compared to the one from wild type strain (OD367 = 0.39). Therefore, the plasmid with phzM and phzS genes was proved to improve the pyocyanin biosynthesis through a better conversion of PCA phenazine into PYO via PhzM and PhzS enzymes.

Effect of culture conditions on pyocyanin production by recombinant pyocyanin-producing strain Pseudomonas aeruginosa PS39-phzMS

“…In the combination of the production medium GM and the optimized culture conditions of pH 8, at 30 °C under shaking at 200 rpm for 120 h of cultivation, the yield of pyocyanin production rose to 49.57 ± 1.71 µg/mL (Figure 4B). The quantity was 159% higher than that of pyocyanin obtained before optimization of pH 7.2, at 30 °C with the agitated condition at 200 rpm for 120 h of cultivation in KingA medium (Vinh et al, 2022). Abo-Zaid et al (2015) optimized the pyocyanin production of P. aeruginosa isolate JY21 and proposed that the optimal conditions for JY21 were pH 8, 30 °C, which is similar to P. aeruginosa PS39-phzMS.…”

“…P. aeruginosa PS39-phzMS is a recombinant strain constructed by wild-type P. aeruginosa PS39 harboring plasmid pUCP24-phzMS (Vinh et al, 2022). The P. aeruginosa PS39 was isolated from a shrimp pond and can produce pyocyanin at a relatively low level.…”