Comparative Genomics Unveils the Habitat Adaptation and Metabolic Profiles of <i>Clostridium</i> in an Artificial Ecosystem for Liquor Production

Abstract

<i>Clostridium</i> inhabiting pit mud (PM) is one of the important bacterial populations for synthesizing flavor compounds of Chinese strong-flavor baijiu. The long-term cereal fermentation with sorghum as the main raw material creates an environment rich in starch, ethanol, and organic acids (mainly lactic acid). However, the genetic factors underpinning <i>Clostridium</i>'s adaptation to PM remain poorly understood. Here, we performed comparative genomic analysis between 30 pit mud-associated (PMA) and 100 non-pit mud-associated (NPMA) <i>Clostridium</i> strains. Comparison analysis of the enrichment of KEGG pathways between PMA and NPMA <i>Clostridium</i> strains showed two-component system, flagellar assembly, and bacterial chemotaxis pathways related to environmental adaptation were enriched in PMA strains. The number of genes encoding alcohol dehydrogenase and l-lactate dehydrogenase in PMA <i>Clostridium</i> strains was significantly higher than that in NPMA, which is helpful for them to adapt to the ethanol- and lactic acid-rich environment. The analysis of carbohydrate-active enzymes demonstrated that glycoside hydrolases (GHs) was the most abundant family in all <i>Clostridium</i> strains, and genes encoding GH4 and GH13, involved in starch and sucrose metabolism, were enriched in PMA <i>Clostridium</i>. Horizontal gene transfer analysis revealed that multiple genes encoding the enzymes involved in carbohydrate and amino acid metabolism were transferred from <i>Bacillus</i> to <i>Clostridium</i> in pit mud. Most of the PMA <i>Clostridium</i> strains had good potential for butyric acid synthesis from ethanol, lactic acid, and starch. Collectively, this study furthers our understanding of the habitat adaptation and metabolic potential of PMA <i>Clostridium</i> strains. <b>IMPORTANCE</b> Pit mud is a typical artificial ecosystem for Chinese liquor production. <i>Clostridium</i> inhabiting pit mud plays essential roles in the flavor formation of strong-flavor baijiu. The relative abundance of <i>Clostridium</i> increased with pit mud quality, further influencing the quality of baijiu. So far, the ecological adaptation of <i>Clostridium</i> to a pit mud-associated lifestyle is largely unknown. Here, comparative genomic analysis of pit mud-associated (PMA) and non-pit mud-associated (NPMA) <i>Clostridium</i> strains was performed. We found genes related to the metabolism of starch, ethanol, and lactic acid were enriched in PMA <i>Clostridium</i> strains, which facilitated their adaptation to the unique brewing environment. In addition, horizontal gene transfer contributed to the adaptation of <i>Clostridium</i> to pit mud. Our findings provide genetic insights on PMA <i>Clostridium</i> strains' ecological adaptation and metabolic characteristics.