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.

Keywords: CRISPR-Cas9n; Clostridium beijerinckii; butanol; histidine kinases; sporulation.

FIGURE 1

Comparison of HK phosphodonor active site domains. The DHp/HisKA domains of several putative orphan HKs in C. beijerinckii were compared to known orphan kinases Cac3319, Cac0903, and Cac0323 (C. acetobutylicum), respectively. Identical residues conserved with Cac3319 or Cac0903 and Cac0323 are shaded black, identical residues conserved with Cac0903 are shaded dark gray, and identical residues conserved with Cac0323 are shaded light gray. The phosphorylated histidine residue is denoted by an asterisk.

FIGURE 2

Fermentation results of the wild-type (WT) and mutant strains in batch fermentation. Symbols: glucose (filled squares), OD₆₀₀ (open squares), butanol (filled circles), acetone (filled triangles), ethanol (filled diamonds); butyrate (open circles), acetate (open diamonds).

FIGURE 3

Sporulation frequencies of the wild-type (WT) and mutant strains. The heat-resistant colony-forming units (CFU) were evaluated by treating the cell cultures (5 days) in 80°C for 10 min, after which 100 μL of them were plated on CGM agar directly (A) or following diluting for 10 times (B). (C) The numbers of heat-resistant colonies.