Isolation, Characterization and Metabolic Engineering of a Thermophilic Bacillus for Green Chemical Production

Biomass is an attractive renewable resource as alternative to fossil-fuel-based chemicals for the production of green chemicals such as organic acids that can be processed into for example bio-plastics. Major challenges for the transition to a bio-based economy are to develop cost-effective production processes and to avoid the use of food substrates. Therefore, we are developing a new generation of microbial platform organisms based on moderately thermophilic, facultatively anaerobic Bacilli. Industrial advantages compared to today’s mesophilic white biotechnology work horses are the thermophilic nature, the anaerobic fermentation capacities and the possibility to use simultaneous saccharification and fermentation (SSF) of lignocellulosic substrates at the optimum temperature of commercial hydrolytic enzymes. The thermophilic nature will lower production costs via more efficient SSF, less contamination risk and less cooling costs.

We have isolated a collection of thermophilic Bacilli from environmental samples using several different enrichment techniques. An initial selection of strains was made based on product formation and sugar utilization, after which the selected isolates were tested for genetic accessibility. Genetic accessibility was found to be highly strain-specific and only a very limited number of strains was reproducibly transformable. These strains were subsequently tested in lab-scale fermentations, resulting in one strain that performed best and was selected for further studies. For this strain, the transformation protocol was optimized and its genome was sequenced. We have developed an advanced genetic toolbox for this strain which allowed us to make multiple clean gene deletions using a counter-selection system, as well as overexpress genes from a plasmid. As the wild type strain produces mainly lactate, the first target was to remove the ldh-gene, which resulted in a viable mutant. Results of the metabolic engineering of multiple knockouts and overexpression of genes in central metabolism, as well as biochemical analyses of related enzyme activities will be discussed.