Abstract

Since halophile Halomonas spp can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low cost chemicals in an economic way. New metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed outer membrane protein porin as a site for external gene integration. The gene(s) of interest was/were inserted downstream the porin gene, forming an artificial operon porin-inserted gene(s). The feasibility of the artificial operon to strongly and stably express external gene(s) was proven by three examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, when genes encoding methylmalonyl-CoA mutase and methylmalonyl-CoA decarboxylase were chromosomally expressed, 2-methylcitrate synthase deleted Halomonas TD08 synthesized copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) from glucose as the sole carbon source. The chromosome engineered strain produced PHBV consisting of stably 5-12 mol% 3-hydroxyvalerate (3HV) compared with 7-25 mol% 3HV by a medium copy number plasmid-based expression system. Third, an inducible expression system was constructed in phaC deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp was established.