Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution

Abstract

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A₁B₅ was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP₁ gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A₁B₅P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A₁B₅P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A₁B₅P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A₁B₅P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A₁B₅P and revealed salt-stress related genes of halophilic host strains.

Keywords: ARTP; Ectoine; Halomonas; NGIB; Omics analysis; PHB; Polyhydroxyalkanoates.