Effect of long-term adaptation of an acetogenic bacterium Clostridium sp. AWRP under acetate stress

Acetate is a simple carboxylic acid that is synthesized in various microorganisms. Although acetate toxicity and tolerance have been studied in many microorganisms, little is known about the effects of exogenous acetate on the cell growth of acetogenic bacteria. In this study, we report the phenotypic changes that occurred in the acetogenic bacterium Clostridium sp. AWRP as a result of an adaptive laboratory evolution under acetate challenge. When compared with the wild-type strain, the acetate-adapted strain displayed a tolerance to acetate up to 10 g L-1 and higher biomass yields in batch cultures, although the metabolite profiles greatly varied depending on culture conditions. Interestingly, genome sequencing revealed that the adapted strain harbored three point mutations in the genes encoding an electron-bifurcating hydrogenase, which is crucial to its autotrophic growth on CO2 + H2, in addition to one in the dnaK gene. Transcriptome analysis revealed the global change in the gene expression profile of the acetate-adapted strain. Strikingly, most genes involved in CO2-fixing Wood-Ljungdahl pathway and auxiliary pathways for energy conservation (e.g., Rnf complex, Nfn, etc.) were significantly down-regulated. In addition, we observed that a couple of metabolic pathways associated with dissimilation of nucleosides and carbohydrates were significantly up-regulated in the acetate-adapted strain as well as several amino acid biosynthetic pathways, indicating that the strain might increase its fitness by utilizing organic substrates in response to the down-regulation of carbon fixation. Further investigation into the carbon fixation degeneration of the acetate-adapted strain will provide practical implications in CO2 + H2 fermentation using acetogenic bacteria for long-term continuous fermentation. The transcriptome profiles of the wild-type Clostridium sp. AWRP and its acetate-tolerant derivative 46T-a were compared. Overall design: Two strains were cultivated using bioreactors with continous gas flow. The samples were taken at exponential and stationary phases and subjected to transcriptome analysis.

乙酸盐（acetate）是一类可在多种微生物中合成的简单羧酸。尽管学界已针对多种微生物中的乙酸盐毒性与耐受性开展了大量研究，但目前对外源性乙酸盐对产乙酸菌（acetogenic bacteria）细胞生长的影响仍所知有限。
本研究报道了产乙酸菌梭菌属菌种AWRP（Clostridium sp. AWRP）在乙酸盐胁迫下经适应性实验室进化后出现的表型变化。与野生型菌株相比，经乙酸盐适应性进化的菌株可耐受最高10 g·L⁻¹的乙酸盐，且在批量培养中具备更高的生物量得率；不过其代谢产物谱会随培养条件发生显著改变。
有趣的是，基因组测序结果显示，该适应性菌株除在dnaK基因（dnaK gene）中存在一处点突变外，在编码电子歧化氢化酶（electron-bifurcating hydrogenase，该酶对其以CO₂+H₂为底物的自养生长至关重要）的基因中携带有三处点突变。
转录组分析揭示了该乙酸盐适应性菌株的基因表达谱发生了全局性改变。值得注意的是，绝大多数参与CO₂固定伍德-隆德哈尔途径（Wood-Ljungdahl pathway）以及能量守恒辅助途径（如Rnf复合物（Rnf complex）、Nfn等）的基因均显著下调。
此外，研究还观察到，与核苷、糖类异化相关的多条代谢途径，以及数条氨基酸生物合成途径，在该乙酸盐适应性菌株中均显著上调，这表明该菌株可能通过利用有机底物提升自身适配性，以应对碳固定途径的下调。
后续针对该乙酸盐适应性菌株的碳固定退化机制开展深入研究，将为利用产乙酸菌进行CO₂+H₂发酵的长期连续发酵工艺提供重要的实践参考。
本研究对比了野生型梭菌属菌种AWRP（Clostridium sp. AWRP）与其乙酸盐耐受性衍生菌株46T-a的转录组谱。整体实验设计：采用带有连续气流的生物反应器培养两种菌株，分别在指数生长期与稳定期采集样本并进行转录组分析。