Exploiting Hydrogenophaga pseudoflava for aerobic syngas-based production of chemicals

Gasification is a suitable technology to generate energy-rich synthesis gas (syngas) from biomass or waste streams, which can be utilized in bacterial fermentation processes for the production of chemicals and fuels. Established microbial processes currently rely on acetogenic bacteria which perform an energetically inefficient anaerobic CO oxidation and acetogenesis potentially hampering the biosynthesis of complex and ATP-intensive products. Since aerobic oxidation of CO is energetically more favorable, we exploit in this study the Gram-negative beta-proteobacterium Hydrogenophaga pseudoflava DSM1084 as novel host for the production of chemicals from syngas. We sequenced and annotated the genome of H. pseudoflava and established a genetic engineering toolbox, which allows markerless chromosomal modification via the pk19mobsacB system and heterologous gene expression on pBBRMCS2-based plasmids. The toolbox was extended by identifying strong endogenous promotors such as P-gapA2 which proved to yield high expression under heterotrophic and autotrophic conditions. H. pseudoflava showed relatively fast heterotrophic growth in complex and minimal medium with sugars and organic acids which allows convenient handling in lab routines. In autotrophic bioreactor cultivations with syngas, H. pseudoflava exhibited a growth rate of 0.06 h(-1) and biomass specific uptakes rates of 14.2 +/- 0.3 mmol H(2)g(CDW)(-1) h(-1), 73.9 +/- 1.8 mmol CO g(CDW)(-1) h(-1), and 31.4 +/- 0.3 mmol O-2 g(CDW)(-1) h(-1). As proof of concept, we engineered the carboxydotrophic bacterium for the aerobic production of the C-15 sesquiterpene (E)-alpha-bisabolene from the C-1 carbon source syngas by heterologous expression of the (E)-alpha-bisabolene synthase gene agBIS. The resulting strain H. pseudoflava (pOCEx1:agBIS) produced 59 +/- 8 mu g (E)-alpha-bisabolene L-1 with a volumetric productivity Q(p) of 1.2 +/- 0.2 mu g L-1 h(-1) and a biomass-specific productivity q(p) of 13.1 +/- 0.6 mu g g(CDW)(-1) h(-1). The intrinsic properties and the genetic repertoire of H. pseudoflava make this carboxydotrophic bacterium a promising candidate for future aerobic production processes to synthesize more complex or ATP-intensive chemicals from syngas.