Lignocellulosic biomass is a promising resource of renewable biochemicals and biofuels. However, the presence of inhibitors existing in lignocellulosic hydrolysates (LCH) is a great challenge to acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. In particular, phenolic compounds (PCs) from LCH severely block ABE production even at low concentrations. Thus, it is urgent to gain insight into the intracellular metabolic disturbances caused by phenolic inhibitors and elucidate the underlying mechanisms to identify key industrial bottlenecks that undermine efficient ABE production. In this study, a time-course of ABE fermentation by C. acetobutylicum in the presence of four typical PCs (syringaldehyde, vanillin, ferulic acid, and p-coumaric acid) was characterized, respectively. Addition of PCs caused different irreversible effects on ABE production. Specifically, syringaldehyde showed the greatest inhibition to butanol production, followed by vanillin, ferulic acid, and p-coumaric acid. Subsequently, a weighted gene co-expression network analysis (WGCNA) based on RNA-sequencing data was applied to identify metabolic perturbations caused by four LCH-derived PCs, and extract the gene modules associated with extracellular fermentation traits. The hub genes in each module were subjected to protein–protein interaction analysis and enrichment analysis. The results showed that functional modules were PC-dependent and shared some unique features. Specifically, p-coumaric acid caused the most extensive transcriptomic disturbances, particularly affecting the gene expressions of ribosome proteins and the assembly of flagella, DNA replication, repair, and recombination; the addition of syringaldehyde caused significant metabolic disturbances on the gene expressions of ribosome proteins, starch and sucrose metabolism; vanillin mainly disturbed purine metabolism, sporulation and signal transduction; and ferulic acid caused a metabolic disturbance on glycosyl transferase-related gene expressions. This study uncovers novel insights into the inhibitory mechanisms of PCs for the first time and provides guidance for future metabolic engineering efforts, which establishes a powerful foundation for the development of phenol-tolerant strains of C. acetobutylicum for economically sustainable ABE production with high productivity from lignocellulosic biomass.

中文翻译：

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基因共表达网络分析揭示了丙酮丁醇梭菌响应木质纤维素水解产物酚类抑制剂的新代谢机制

木质纤维素生物质是可再生生物化学品和生物燃料的有前景的资源。然而，木质纤维素水解产物 (LCH) 中存在的抑制剂对丙酮丁醇梭菌发酵丙酮-丁醇-乙醇 (ABE) 是一个巨大挑战。特别是 LCH 中的酚类化合物 (PC) 即使在低浓度下也会严重阻碍 ABE 的产生。因此，迫切需要深入了解酚类抑制剂引起的细胞内代谢紊乱，并阐明潜在的机制，以确定破坏高效 ABE 生产的关键工业瓶颈。在这项研究中，分别表征了在四种典型 PC（丁香醛、香草醛、阿魏酸和对香豆酸）存在下由丙酮丁醇梭菌进行 ABE 发酵的时间过程。PC 的添加对 ABE 生产造成了不同的不可逆影响。具体而言，丁香醛对丁醇产生的抑制作用最大，其次是香草醛、阿魏酸和对香豆酸。随后，应用基于 RNA 测序数据的加权基因共表达网络分析 (WGCNA) 来识别由四种 LCH 衍生的 PC 引起的代谢扰动，并提取与细胞外发酵性状相关的基因模块。对每个模块中的中心基因进行蛋白质-蛋白质相互作用分析和富集分析。结果表明，功能模块依赖于PC，并具有一些独特的功能。具体来说，对香豆酸引起最广泛的转录组学紊乱，特别是影响核糖体蛋白的基因表达和鞭毛的组装，DNA复制、修复和重组；丁香醛的添加对核糖体蛋白、淀粉和蔗糖代谢的基因表达造成显着的代谢紊乱；香草醛主要干扰嘌呤代谢、孢子形成和信号转导；阿魏酸引起糖基转移酶相关基因表达的代谢紊乱。这项研究首次揭示了对 PC 抑制机制的新见解，并为未来的代谢工程工作提供了指导，这为开发耐酚的丙酮丁醇梭菌菌株奠定了坚实的基础，从而实现经济可持续的 ABE 生产，并从木质纤维素生物质。丁香醛的添加对核糖体蛋白、淀粉和蔗糖代谢的基因表达造成显着的代谢紊乱；香草醛主要干扰嘌呤代谢、孢子形成和信号转导；阿魏酸引起糖基转移酶相关基因表达的代谢紊乱。这项研究首次揭示了对 PC 抑制机制的新见解，并为未来的代谢工程工作提供了指导，这为开发耐酚的丙酮丁醇梭菌菌株奠定了坚实的基础，从而实现经济可持续的 ABE 生产，并从木质纤维素生物质。丁香醛的添加对核糖体蛋白、淀粉和蔗糖代谢的基因表达造成显着的代谢紊乱；香草醛主要干扰嘌呤代谢、孢子形成和信号转导；阿魏酸引起糖基转移酶相关基因表达的代谢紊乱。这项研究首次揭示了对 PC 抑制机制的新见解，并为未来的代谢工程工作提供了指导，这为开发耐酚的丙酮丁醇梭菌菌株奠定了坚实的基础，从而实现经济可持续的 ABE 生产，并从木质纤维素生物质。阿魏酸引起糖基转移酶相关基因表达的代谢紊乱。这项研究首次揭示了对 PC 抑制机制的新见解，并为未来的代谢工程工作提供了指导，这为开发耐酚的丙酮丁醇梭菌菌株奠定了坚实的基础，从而实现经济可持续的 ABE 生产，并从木质纤维素生物质。阿魏酸引起糖基转移酶相关基因表达的代谢紊乱。这项研究首次揭示了对 PC 抑制机制的新见解，并为未来的代谢工程工作提供了指导，这为开发耐酚的丙酮丁醇梭菌菌株奠定了坚实的基础，从而实现经济可持续的 ABE 生产，并从木质纤维素生物质。