Metabolic Engineering of Histidine Kinases in Clostridium beijerinckii for Enhanced Butanol Production

Abstract

Clostridium beijerinckii, a promising industrial microorganism for butanol production, suffers from low butanol titer and lack of high-efficiency genetical engineering toolkit. A few histidine kinases (HKs) responsible for Spo0A phosphorylation have been demonstrated as functionally important components in regulating butanol biosynthesis in solventogenic clostridia such as C. acetobutylicum, but no study about HKs has been conducted in C. beijerinckii. In this study, six annotated but uncharacterized candidate HK genes sharing partial homologies (no less than 30%) with those in C. acetobutylicum were selected based on sequence alignment. The encoding region of these HK genes were deleted with CRISPR-Cas9n-based genome editing technology. The deletion of cbei2073 and cbei4484 resulted in significant change in butanol biosynthesis, with butanol production increased by 40.8 and 17.3% (13.8 g/L and 11.5 g/L vs. 9.8 g/L), respectively, compared to the wild-type. Faster butanol production rates were observed, with butanol productivity greatly increased by 40.0 and 20.0%, respectively, indicating these two HKs are important in regulating cellular metabolism in C. beijerinckii. In addition, the sporulation frequencies of two HKs inactivated strains decreased by 96.9 and 77.4%, respectively. The other four HK-deletion (including cbei2087, cbei2435, cbei4925, and cbei1553) mutant strains showed few phenotypic changes compared with the wild-type. This study demonstrated the role of HKs on sporulation and solventogenesis in C. beijerinckii, and provided a novel engineering strategy of HKs for improving metabolite production. The hyper-butanol-producing strains generated in this study have great potentials in industrial biobutanol production.

Sporulation in solventogenic and acetogenic clostridia

“…The disruption of cac0323, cac0903, and cac3319 resulted in a decreased sporulation, in contrast to cac0437 deficient strains hyper-sporulated (earlier sporulation and a fifteen-fold increase in heat resistant spores). In C. beijerinckii NCIM 8052, six homologs of these kinases were disrupted in a recent study (Xin et al 2020). A significant decrease in sporulation efficiency (between 70 and 90%) coupled with an increase in solvent production of 38% and 14% were reported only for the Δcbei2073 and Δcbei4484 mutants, respectively.…”

“…Unlike regular TCS kinases, orphan histidine kinases can modulate several response regulators. In clostridia, orphan histidine kinases are integrated for signal transduction to the sporulation regulation network (Freedman et al 2019 ; Paredes-Sabja et al 2014 ; Steiner et al 2011 ; X. Xin et al 2020 ). In C. acetobutylicum , the impact of four histidine kinases (Cac0323, Cac0437, Cac0903 and Cac3319) on sporulation has been studied (Steiner et al 2011 ).…”

“…Unlike regular TCS kinases, orphan histidine kinases can modulate several response regulators. In clostridia, orphan histidine kinases are integrated for signal transduction to the sporulation regulation network (Freedman et al 2019;Paredes-Sabja et al 2014;Steiner et al 2011;X. Xin et al 2020).…”

Identification and Validation of Reference Genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR Using RNA-Seq Data

“…However, thus far, no evaluation study of reference genes has been performed for this species. C. beijerinckii has been found to be a promising microorganism for industrial production of biobutanol, and efforts to increase butanol productivity by means of genetic and metabolic engineering of its strains have been recently reported ( Agu et al, 2019 ; Xin et al, 2020 ). However, the strain engineering of C. beijerinckii is hindered by insufficient understanding of cellular physiology and regulatory mechanisms of gene expression.…”

Co-feeding glucose with either gluconate or galacturonate during clostridial fermentations provides metabolic fine-tuning capabilities

“…This excess input can lead to a production of undesired products 16,17 . Current strategies at re-direction of these excess inputs have focused on the generation of biosynthetic pathways [18][19][20] and the reconfiguration of bioreactors 21 . However, less consideration has gone into the optimization of precursor production in cells.…”