Identification and Investigation of Autolysin Genes in Clostridium saccharoperbutylacetonicum Strain N1-4 for Enhanced Biobutanol Production

Abstract

Biobutanol is a valuable biochemical and one of the most promising biofuels. Clostridium saccharoperbutylacetonicum N1-4 is a hyperbutanol-producing strain. However, its strong autolytic behavior leads to poor cell stability, especially during continuous fermentation, thus limiting the applicability of the strain for long-term and industrial-scale processes. In this study, we aimed to evaluate the role of autolysin genes within the C. saccharoperbutylacetonicum genome related to cell autolysis and further develop more stable strains for enhanced butanol production. First, putative autolysin-encoding genes were identified in the strain based on comparison of amino acid sequence with homologous genes in other strains. Then, by overexpressing all these putative autolysin genes individually and characterizing the corresponding recombinant strains, four key genes were pinpointed to be responsible for significant cell autolysis activities. Further, these key genes were deleted using CRISPR-Cas9. Fermentation characterization demonstrated enhanced performance of the resultant mutants. Results from this study reveal valuable insights concerning the role of autolysins for cell stability and solvent production, and they provide an essential reference for developing robust strains for enhanced biofuel and biochemical production.IMPORTANCE Severe autolytic behavior is a common issue in Clostridium and many other microorganisms. This study revealed the key genes responsible for the cell autolysis within Clostridium saccharoperbutylacetonicum, a prominent platform for biosolvent production from lignocellulosic materials. The knowledge generated in this study provides insights concerning cell autolysis in relevant microbial systems and gives essential references for enhancing strain stability through rational genome engineering.

Keywords: CRISPR-Cas9; Clostridium; autolysis; biobutanol; biofuel; fermentation.

FIG 1

Growth profiles of the recombinant strains with overexpression of the putative autolysin genes compared to the control strain. The P value at a 90% confidence level from the repeated-measures ANOVA during the stationary/death phases (36 h to 72 h) has been put at the top of each profile. The error bar represents the standard error at a 95% confidence interval.

FIG 2

Agarose gel electrophoresis showed the colony PCR results confirming the gene deletion in the mutant strain (St) versus the control strain (Ctrl). (A) Deletion of gene 26 in N1-4; (B) deletion of gene 26 in Δ1234; (C) deletion of gene 26 in Δ12345; (D) deletion of gene 30 in N1-4; (E) deletion of gene 30 in Δ1234; (F) deletion of gene 30 in Δ12345. The positive mutant for the deletion of gene 26 should have a PCR band of 2,186 bp (versus 4,166 bp in the control), and the positive mutant for the deletion of gene 30 should have a PCR band of 2,125 bp (versus 3,070 bp in the control). The NEB 1-kb DNA ladder was used as the marker, with numbers on the left representing the band length in kilobases.

FIG 3

Profiles of serum bottle fermentation using the gene 26 and gene 30 single-deletion mutants compared to the mother strains (C. saccharoperbutylacetonicum N1-4, Δ1234, and Δ12345, respectively). The error bar represents the standard error at a 95% confidence interval.

FIG 4

Profiles of batch fermentation in bioreactors with pH controlled for Δ1234Δ26 compared to Δ1234 and the wild type (N1-4). The error bar represents the standard error at a 95% confidence interval.