Augmented production of alginate oligosaccharides by the Pseudomonas mendocina NK-01 mutant

Abstract

Pseudomonas mendocina NK-01 can simultaneously synthesize medium-chain-length polyhydroxyalkanoate (PHA(MCL)) and alginate oligosaccharides (AO) from glucose under conditions of limited nitrogen. In this study, the PHA(MCL) synthesis pathway was blocked by a deletion of approximately 57% of the sequence of PHA synthase operon mediated by the suicide plasmid, pEX18TcC1ZC2Amp. Deletion of the PHA synthase operon in P. mendocina NK-01 was confirmed by polymerase chain reaction (PCR) and antibiotic resistance assays to form the gene knockout mutant, P. mendocina C7. Shake-flask and 30 L fermentor cultures of P. mendocina C7 showed a 2.21-fold and 2.64-fold accumulation of AO from glucose, respectively, compared with the wild-type strain. Mass spectrometry and gel permeation chromatography characterization revealed that P. mendocina C7 and P. mendocina NK-01 produced AO were identical in terms of monomer composition and average molecular weight (M(W)). Thus, the mutant P. mendocina C7 has potential use in large scale fermentation of AO. Furthermore, it was demonstrated that the PHA(MCL) and AO synthesis pathways compete for the use of carbon sources in P. mendocina NK-01.