Beyond rational-biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes.

BACKGROUND: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis. RESULTS: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant. CONCLUSIONS: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

Engineering and application of a biosensor with focused ligand specificity.

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

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 ß-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 PgapA2 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 ±â€¯0.3 mmol H2 gCDW-1 h-1, 73.9 ±â€¯1.8 mmol CO gCDW-1 h-1, and 31.4 ±â€¯0.3 mmol O2 gCDW-1 h-1. As proof of concept, we engineered the carboxydotrophic bacterium for the aerobic production of the C15 sesquiterpene (E)-α-bisabolene from the C1 carbon source syngas by heterologous expression of the (E)-α-bisabolene synthase gene agBIS. The resulting strain H. pseudoflava (pOCEx1:agBIS) produced 59 ±â€¯8 µg (E)-α-bisabolene L-1 with a volumetric productivity Qp of 1.2 ±â€¯0.2 µg L-1 h-1 and a biomass-specific productivity qp of 13.1 ±â€¯0.6 µg gCDW-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.