Biochemical production from engineered Bacillus species

Abstract

Microbial production of biochemicals has gained significant attention due to increasing global concern regarding the depletion of fossil resources. As prominent workhorses in microbial fermentations, Bacillus species have been extensively investigated and harnessed for their industrial potential in generating valuable biochemicals. Notably, 2,3-butanediol (2,3-BD), a versatile compound with diverse industrial applications, has emerged as a focal point of research. While substantial genetic modification tools are available for some Bacillus species, particularly Bacillus subtilis, efficient genetic manipulations of other Bacillus strains possessing unique characteristics are limited. In this dissertation work, we carried out the analysis of the whole genome sequence and genetic functionality of Bacillus velezensis FJ-4, which can produce optically pure D-2,3-BD. Subsequently, we applied metabolic engineering techniques to B. subtilis 168 to enable the production of chirally pure D-2,3-BD. Furthermore, we achieved a significant milestone by introducing the CRISPR-Cas9 system into Bacillus velezensis FZB42, demonstrating its utility in biocontrol and the manipulation of various biochemicals and bioproducts, including lactate, biofilm, and bacilysin. These advancements broaden the applications of this strain across various fields. The genomic analysis of FJ-4 revealed a guanine-cytosine (GC) content of 46.46%, encompassing a total of 3,867 genes, with 3,749 protein coding sequences (CDSs). Phylogenetic analysis based on whole-genomic amino acid sequences indicated a remarkably close genetic relationship between FJ-4 and B. velezensis 160. A comparative genomic analysis with B. amyliquefaciens ATCC 23350 was also conducted, allowing for the identification and elucidation of core genes, including bdhA, encoding 2,3-BD dehydrogenase. These core genes were subsequently introduced into the bdhA-deficient B. subtilis 168. Additionally, key genes relevant to D-2,3-BD biosynthesis were identified and characterized. Building upon the optically pure D-2,3-BD producing mutant B. subtilis strain BS02, a series of metabolic engineering strategies including blocking competing metabolic pathways, enhancing carbon flux, and manipulating cofactor, were implemented to further enhance the production titer and productivity. Ultimately, our engineered strain, designated as BS09, accomplished an impressive final D-2,3-BD titer of 32.2 g/L with a yield of 0.4 g/g glucose and productivity of 0.22 g/(L·h). Furthermore, B. velezensis FZB42 plays a crucial role as a biofertilizer and biocontrol agent for the biosynthesis of antimicrobial compounds. The development of an efficient genome editing tool for this strain shows great potential for improving its performance in various applications. Through the effective genome editing tool employed in our prior work, we marked the first introduction of the CRISPR-Cas9 system into B. velezensis FZB42 with an impressive lactate dehydrogenase coding ldh gene deletion efficiency of 95.6%. The impact of knocking out the biofilm formation regulator and overexpression of bac gene clusters associated with the biosynthesis of bacilysin in FZB42 were explored. These endeavors provided valuable insights into biofilm development and anti-bacterial compound production manipulation, benefiting the widespread applications of strain FZB42 across diverse fields.