原文链接：Metabolite-Based Mutualistic Interaction between Two Novel Clostridial Species from Pit Mud Enhances Butyrate and Caproate Production | Applied and Environmental Microbiology (asm.org)

ABSTRACT

Pit mud microbial consortia play crucial roles in the formation of Chinese strong-flavor baijiu’s key flavor-active compounds, especially butyric and caproic acids. Clostridia, one of the abundant bacterial groups in pit mud, were recognized as important butyric and caproic acid producers. Research on the interactions of the pit mud microbial community mainly depends on correlation analysis at present. Interaction between Clostridium and other microorganisms and its involvement in short/medium-chain fatty acid (S/MCFA) metabolism are still unclear. We previously found coculture of two clostridial strains isolated from pit mud, Clostridium fermenticellae JN500901 (C.901) and Novisyntrophococcus fermenticellae JN500902 (N.902), could enhance S/MCFA accumulation. Here, we investigated their underlying interaction mechanism through the combined analysis of phenotype, genome, and transcriptome. Compared to monocultures, coculture of C.901 and N.902 obviously promoted their growth, including shortening the growth lag phase and increasing biomass, and the accumulation of butyric acid and caproic acid. The slight effects of inoculation ratio and continuous passage on the growth and metabolism of coculture indicated the relative stability of their interaction. Transwell coculture and transcriptome analysis showed the interaction between C.901 and N.902 was accomplished by metabolite exchange, i.e., formic acid produced by C.901 activated the Wood-Ljungdahl pathway of N.902, thereby enhancing its production of acetic acid, which was further converted to butyric acid and caproic acid by C.901 through reverse β-oxidation. This work demonstrates the potential roles of mutually beneficial interspecies interactions in the accumulation of key flavor compounds in pit mud.

摘要

窖泥微生物群落对中国浓香型白酒关键风味活性化合物的形成起着至关重要的作用，尤其是丁酸和己酸。梭菌是窖泥中丰富的细菌群落之一，被认为是重要的丁酸和己酸生产者。目前对窖泥微生物群落相互作用的研究主要依赖于相关性分析。梭菌与其他微生物之间的相互作用及其在短链/中链脂肪酸 (S/MCFA) 代谢中的参与尚不清楚。我们之前发现从窖泥中分离的两株梭菌菌株，发酵梭菌 JN500901 (C.901) 和发酵新营球菌 JN500902 (N.902) 的共培养可以增强 S/MCFA 的积累。在这里，我们通过表型、基因组和转录组的综合分析研究了它们的潜在相互作用机制。与单培养相比，C.901和N.902共培养明显促进了它们两个的生长，包括缩短了生长滞后期、增加了生物量，以及增加了丁酸和己酸的积累。接种比例和连续传代对共培养物生长代谢的影响较小，表明它们之间的相互作用相对稳定。Transwell共培养和转录组分析表明，C.901和N.902之间的相互作用是通过代谢物交换完成的，即C.901产生的甲酸激活了N.902的Wood-Ljungdahl途径，从而促进了其乙酸的产生，而乙酸进一步在C.901通过逆向β-氧化转化为丁酸和己酸。这项工作证明了互利的跨物种相互作用在窖泥关键风味化合物积累中的潜在作用。

IMPORTANCE Microbial interactions played crucial roles in influencing the assembly, stability, and function of the microbial community. The metabolites of pit mud microbiota are the key to flavor formation of Chinese strong-flavor baijiu. So far, researches on the interactions of the pit mud microbial community have been mainly based on the correlation analysis of sequencing data, and more work needs to be performed to unveil the complicated interaction patterns. Here, we identified a material exchange-based mutualistic interaction system involving two fatty acid-producing clostridial strains (Clostridium fermenticellae JN500901 and Novisyntrophococcus fermenticellae JN500902) isolated from pit mud and systematically elucidated their interaction mechanism for promoting the production of butyric acid and caproic acid, the key flavor-active compounds of baijiu. Our findings provide a new perspective for understanding the complicated interactions of pit mud microorganisms.

重要性微生物相互作用在影响微生物群落的组装、稳定性和功能方面起着至关重要的作用。窖泥微生物代谢产物是中国浓香型白酒风味形成的关键。目前对窖泥微生物群落相互作用的研究主要基于测序数据的相关性分析，其复杂的相互作用模式还有待进一步研究。本研究从窖泥中分离得到两株产脂肪酸梭菌（Clostridium fermenticellae JN500901和Novisyntrophococcus fermenticellae JN500902）并鉴定出一个基于物质交换的互利相互作用系统，并系统阐明了它们促进白酒关键风味活性物质丁酸和己酸产生的相互作用机制。该研究结果为理解窖泥微生物复杂的相互作用提供了新的视角。

图 1 C.901 和 N.902 单独培养和共培养中生长和代谢物随时间的变化。（A）OD600。（B）葡萄糖。（C）pH。（D）甲酸。（E）乙酸。（F）丁酸。（G）己酸。每条折线周围的彩色阴影表示误差带。

图 2 共培养中 C.901 和 N.902 的形态和个体生物量。（A）36 小时后对细菌的扫描电子显微镜观察。从上到下依次为 C.901、N.902 和 C.901-N.902 共培养。（B）单一培养和共培养中 C.901 和 N.902 的绝对定量。每条折线周围的彩色阴影表示误差带。

图3 不同初始接种比例共培养效果。（A）高初始比例C.901共培养。数字1、2.5、5、7.5、10分别代表C.901和N.902的接种比例。（B）高初始比例N.902共培养。数字1、2.5、5、7.5、10分别代表N.902和C.901的接种比例。从上到下分别为过程中OD600、pH、葡萄糖的变化以及48小时时脂肪酸（乙酸、丁酸、己酸）含量的变化。

图 4 无物理接触共培养的特点。（A）transwell 中五种培养模式示意图。（B）48 小时时 C.901 和 N.902 单独培养和共培养的 OD600。（C）48 小时时 C.901 和 N.902 单独培养和共培养的脂肪酸（乙酸、丁酸和己酸）产量。分别测定 transwell 上室和下室的发酵液。N = 6，其中 N 代表每种培养模式的生物学重复数。

图 5 C.901-N.902 共培养在连续传代过程中的表现。（A）共培养中 C.901 和 N.902 的生物量变化。（B）共培养中的脂肪酸产量（乙酸、丁酸和己酸）。每条曲线周围的彩色阴影代表误差带。

图6 根据转录组中的DEGs预测C.901与N.902之间的相互作用机制。（A）C.901的糖酵解、丙酮酸代谢和逆β-氧化途径以及N.902的Wood-Ljungdahl途径、糖酵解和丙酮酸代谢。（B）上述途径中DEGs的log2(FPKM)和log2(fold change)。DEGs代表差异表达基因（q值<0.05）。*表示显著差异表达基因[|log2(fold change)|≥1.5，q值<0.05]。

图7 RT-qPCR验证转录组中基因的相对表达量。（A）C.901的pflB1和cat1。（B）N.902的fhs和ackA。时间点分别为24h、36h（同时进行RNA-Seq）和48h。（C）RT-qPCR与转录组分析（36h）获得的四个基因的倍数变化Spearman相关性分析。Pearson相关系数为0.86。pflB1编码甲酸C-乙酰转移酶，cat1编码酰基辅酶A转移酶，fhs编码甲酸-四氢叶酸连接酶，ackA编码乙酸激酶。*，P < 0.05；**，P < 0.01；***，P < 0.001。

图8 C.901与N.902相互作用机制验证。（A）培养基中额外添加0.3g/L和0.6g/L甲酸钠后N.902单菌OD600和乙酸产量。（B）去除培养基中原有乙酸钠后N.902单菌OD600和乙酸产量。（C）培养基中额外添加1g/L和3g/L乙酸钠后C.901单菌OD600及丁酸和己酸产量。*，P < 0.05；**，P < 0.01；***，P < 0.001。

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