Department of Biosystems Engineering, Auburn University, Auburn, Alabama, USA.
School of Chemistry, National University (UNA), Heredia, Costa Rica.
Appl Environ Microbiol. 2021 Mar 11;87(7). doi: 10.1128/AEM.02442-20.
Biobutanol is a valuable biochemical and one of the most promising biofuels. 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 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. Severe autolytic behavior is a common issue in and many other microorganisms. This study revealed the key genes responsible for the cell autolysis within , 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.
丁醇是一种有价值的生化物质，也是最有前途的生物燃料之一。N1-4 是一种高产正丁醇的菌株。然而，其强烈的自溶行为导致细胞稳定性差，尤其是在连续发酵过程中，从而限制了该菌株在长期和工业规模过程中的应用。在这项研究中，我们旨在评估与细胞自溶相关的基因组中自溶素基因的作用，并进一步开发更稳定的菌株以提高丁醇产量。首先，根据与其他菌株同源基因的氨基酸序列比较，在该菌株中鉴定出潜在的自溶素编码基因。然后，通过单独过表达所有这些潜在的自溶素基因，并对相应的重组菌株进行表征，确定了四个关键基因负责显著的细胞自溶活性。进一步使用 CRISPR-Cas9 删除这些关键基因。发酵特性表明，所得突变体的性能得到了增强。本研究结果揭示了自溶素在细胞稳定性和溶剂生产中的作用的有价值的见解，并为开发用于增强生物燃料和生物化学生产的稳健菌株提供了重要参考。严重的自溶行为是 和许多其他微生物的共同问题。本研究揭示了 中导致细胞自溶的关键基因， 是木质纤维素材料生物溶剂生产的重要平台。本研究中获得的知识提供了有关相关微生物系统中细胞自溶的见解，并为通过合理的基因组工程增强菌株稳定性提供了重要参考。
