Exploiting Type I-B CRISPR Genome Editing System in Thermoanaerobacterium Aotearoense SCUT27 And Engineering The Strain For High-Level Ethanol Production
Abstract Background: Thermophilic microbes for biofuels and chemicals have attracted great attention due to their tolerance of high temperature and wide range of substrate utilization. Thermoanaerobacterium aotearoense SCUT27 has the ability of glucose and xylose co-utilization in lignocellulosic biomass. Polygene manipulation was a bottleneck since it was hindered by available markers for selection. In this study, the endogenous Type I-B CRISPR/Cas system was developed for multiplex genome editing in SCUT27. Results: The protospacer-adjacent motif (PAM) was identified by in silico and orotidine-5’-phosphate decarboxylase (pyrF) and then lactate dehydrogenase (ldh) were chosen as the editing target to assess the toxicity of this immune system and gene editing efficiency. The mutants could be repeatedly obtained with an editing efficiency of 58.3-100%. Higher transformation efficiency was observed after optimization of some editing strategies. Furthermore, a new method was performed for screening mutants of plasmid curing (recycling of the editing plasmid) for multiplex genome editing based on the negative selection marker tdk, and then ldh and arginine repressor (argR) were knocked out successively. The mutant SCUT27/Δldh/ΔargR had the prominent advantages over SCUT27 for ethanol production with enhanced ability to metabolize xylose. When cultured under various lignocellulosic hydrolysates, the mutant showed a satisfactory performance with the ethanol titer and yield improved by 147.42–739.40% and 112.67–267.89%, respectively, compared with SCUT27, as well as the enhanced tolerance to inhibitors.Conclusion: The multi-gene editing by native CRISPR/Cas system is a promising strategy to engineer SCUT27 for higher ethanol production with lignocellulosic hydrolysates.