Engineering Halomonas bluephagenesis as a chassis for bioproduction from starch

Abstract

Halomonas bluephagenesis has been successfully engineered to produce multiple products under open unsterile conditions utilizing costly glucose as the carbon source. It would be highly interesting to investigate if H. bluephagenesis, a chassis for the Next Generation Industrial Biotechnology (NGIB), can be reconstructed to become an extracellular hydrolytic enzyme producer replacing traditional enzyme producer Bacillus spp. If successful, cost of bulk hydrolytic enzymes such as amylase and protease, can be significantly reduced due to the contamination resistant and robust growth of H. bluephagenesis. This also allows H. bluephagenesis to be able to grow on low cost substrates such as starch. The modularized secretion machinery was constructed and fine-tuned in H. bluephagenesis using codon-optimized gene encoding α-amylase from Bacillus lichenifomis. Screening of suitable signal peptides and linkers based on super-fold green fluorescence protein (sfGFP) for enhanced expression in H. bluephagenesis resulted in a 7-fold enhancement of sfGFP secretion in the recombinant H. bluephagenesis. When the gene encoding sfGFP was replaced by α-amylase encoding gene, recombinant H. bluephagenesis harboring this amylase secretory system was able to produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), ectoine and L-threonine utilizing starch as the growth substrate, respectively. Recombinant H. bluephagenesis TN04 expressing genes encoding α-amylase and glucosidase on chromosome and plasmid-based systems, respectively, was able to grow on corn starch to approximately 10 g/L cell dry weight containing 51% PHB when grown in shake flasks. H. bluephagenesis was demonstrated to be a chassis for productions of extracellular enzymes and multiple products from low cost corn starch.

Keywords: Amylase; Glucosidase; Halomonas; Hydrolytic enzyme; Next generation industrial biotechnology; PHB; Protein secretion.