Nwaokorie Ugochi Jennifer, Reinmets Kristina, de Lima Lorena Azevedo, Pawar Pratik Rajendra, Shaikh Kurshedaktar Majibullah, Harris Audrey, Köpke Michael, Valgepea Kaspar
ERA Chair in Gas Fermentation Technologies, Institute of Technology, University of Tartu, Tartu, Estonia.
LanzaTech Inc., Skokie, IL, United States.
Front Bioeng Biotechnol. 2023 May 15;11:1167892. doi: 10.3389/fbioe.2023.1167892. eCollection 2023.
Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions-hypothetical protein ( LABRINI_07945) and CooT nickel binding protein ( LABRINI_07950)-directly adjacent and expressed at similar levels to the C-fixing gene cluster in the gas-fermenting model-acetogen Targeted deletion of either the or gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both ∆ and ∆ strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.
气体发酵已成为一种可持续的途径，通过利用气体发酵微生物从气态和固态废物中回收廉价的一碳（C）原料来生产燃料和化学品。目前，利用伍德-龙格达尔途径将碳氧化物（CO和CO₂）转化为有价值产品的产乙酸菌是气体发酵中最先进的生物催化剂。然而，我们对参与碳固定基因簇及其紧密相连基因的功能的了解并不完整。在这里，我们研究了两个功能不明的基因——假设蛋白（LABRINI_07945）和CooT镍结合蛋白（LABRINI_07950）——在气体发酵模型产乙酸菌中与碳固定基因簇直接相邻且表达水平相似的作用。使用CRISPR/nCas9对LABRINI_07945或LABRINI_07950基因进行靶向缺失，并在异养和自养分批培养以及自养生物反应器连续培养中进行表型表征，结果表明，ΔLABRINI_07945和ΔLABRINI_07950菌株均存在显著的生长缺陷且副产物谱发生改变。基因缺失对丰富或基本培养基上自养分批生长的影响各不相同，这表明这两个基因都会影响复杂营养物质的利用。自养恒化器培养显示，两种缺失菌株的乙酸和乙醇生产率较低，而向CO₂和生物质的碳通量较高。此外，蛋白质组分析表明，任一基因的破坏都会影响碳固定基因簇和乙醇合成途径中蛋白质的表达。我们的工作有助于更好地理解产乙酸菌的基因型-表型关系，并为提高气体发酵中的碳固定效率提供了工程靶点。
