Allemann Marco N, Kato Ryo, Carper Dana L, Hochanadel Leah H, Alexander William G, Giannone Richard J, Kamimura Naofumi, Masai Eiji, Michener Joshua K
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
Department of Materials Science and Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
Appl Environ Microbiol. 2025 Feb 19;91(2):e0208124. doi: 10.1128/aem.02081-24. Epub 2025 Jan 23.
Lignin contains a variety of interunit linkages, leading to a range of potential decomposition products that can be used as carbon and energy sources by microbes. β-O-4 linkages are the most common in native lignin, and associated catabolic pathways have been well characterized. However, the fate of the mono-aromatic intermediates that result from β-O-4 dimer cleavage has not been fully elucidated. Here, we used experimental evolution to identify mutant strains of with improved catabolism of a model aromatic dimer containing a β-O-4 linkage, guaiacylglycerol-β-guaiacyl ether (GGE). We identified several parallel causal mutations, including a single nucleotide polymorphism in the promoter of an uncharacterized gene that roughly doubled the growth yield with GGE. We characterized the associated enzyme and demonstrated that it oxidizes an intermediate in GGE catabolism, β-hydroxypropiovanillone, to vanilloyl acetaldehyde. Identification of this enzyme and its key role in GGE catabolism furthers our understanding of catabolic pathways for lignin-derived aromatic compounds.IMPORTANCELignin degradation is a key step for both carbon cycling in nature and biomass conversion to fuels and chemicals. Bacteria can catabolize lignin-derived aromatic compounds, but the complexity of lignin means that full mineralization requires numerous catabolic pathways and often results in slow growth. Using experimental evolution, we identified an uncharacterized enzyme for the catabolism of a lignin-derived aromatic monomer, β-hydroxypropiovanillone. A single nucleotide polymorphism in the promoter of the associated gene significantly increased bacterial growth with either β-hydroxypropiovanillone or a related lignin-derived aromatic dimer. This work expands the repertoire of known aromatic catabolic genes and demonstrates that slow catabolism of lignin-derived aromatic compounds may be due to misregulation under laboratory conditions rather than inherent catabolic challenges.
木质素含有多种单元间连接方式，会产生一系列潜在的分解产物，这些产物可被微生物用作碳源和能源。β-O-4连接是天然木质素中最常见的，与之相关的分解代谢途径已得到充分表征。然而，β-O-4二聚体裂解产生的单芳族中间体的去向尚未完全阐明。在此，我们利用实验进化来鉴定能够更好地分解含有β-O-4连接的模型芳族二聚体——愈创木基甘油-β-愈创木基醚（GGE）的突变菌株。我们鉴定出了几个平行的因果突变，包括一个未表征基因启动子中的单核苷酸多态性，该突变使GGE的生长产量大致提高了一倍。我们对相关酶进行了表征，并证明它将GGE分解代谢中的中间体β-羟基松柏醛氧化为香草酰乙醛。鉴定出这种酶及其在GGE分解代谢中的关键作用，进一步加深了我们对木质素衍生芳族化合物分解代谢途径的理解。
重要性
木质素降解是自然界碳循环以及生物质转化为燃料和化学品的关键步骤。细菌能够分解代谢木质素衍生的芳族化合物，但木质素的复杂性意味着完全矿化需要众多分解代谢途径，且往往导致生长缓慢。利用实验进化，我们鉴定出一种用于分解代谢木质素衍生芳族单体β-羟基松柏醛的未表征酶。相关基因启动子中的单核苷酸多态性显著提高了细菌利用β-羟基松柏醛或相关木质素衍生芳族二聚体的生长速度。这项工作扩展了已知芳族分解代谢基因的范围，并表明木质素衍生芳族化合物分解代谢缓慢可能是由于实验室条件下的调控不当，而非固有的分解代谢挑战。
