Department of Microbiology, University of Massachusetts-Amherst, Amherst, MA, United States of America.
Departamento de Biología, Facultad de Ciencias Naturales y Exactas, Universidad de Playa Ancha, Playa Ancha, Valparaíso, Chile.
PLoS One. 2020 Sep 17;15(9):e0233823. doi: 10.1371/journal.pone.0233823. eCollection 2020.
Lignin is the second most abundant carbon polymer on earth and despite having more fuel value than cellulose, it currently is considered a waste byproduct in many industrial lignocellulose applications. Valorization of lignin relies on effective and green methods of de-lignification, with a growing interest in the use of microbes. Here we investigate the physiology and molecular response of the novel facultative anaerobic bacterium, Tolumonas lignolytica BRL6-1, to lignin under anoxic conditions. Physiological and biochemical changes were compared between cells grown anaerobically in either lignin-amended or unamended conditions. In the presence of lignin, BRL6-1 accumulates higher biomass and has a shorter lag phase compared to unamended conditions, and 14% of the proteins determined to be significantly higher in abundance by log2 fold-change of 2 or greater were related to Fe(II) transport in late logarithmic phase. Ferrozine assays of the supernatant confirmed that Fe(III) was bound to lignin and reduced to Fe(II) only in the presence of BRL6-1, suggesting redox activity by the cells. LC-MS/MS analysis of the secretome showed an extra band at 20 kDa in lignin-amended conditions. Protein sequencing of this band identified a protein of unknown function with homology to enzymes in the radical SAM superfamily. Expression of this protein in lignin-amended conditions suggests its role in radical formation. From our findings, we suggest that BRL6-1 is using a protein in the radical SAM superfamily to interact with the Fe(III) bound to lignin and reducing it to Fe(II) for cellular use, increasing BRL6-1 yield under lignin-amended conditions. This interaction potentially generates organic free radicals and causes a radical cascade which could modify and depolymerize lignin. Further research should clarify the extent to which this mechanism is similar to previously described aerobic chelator-mediated Fenton chemistry or radical producing lignolytic enzymes, such as lignin peroxidases, but under anoxic conditions.
木质素是地球上第二丰富的碳聚合物，尽管其燃料价值高于纤维素，但在许多工业木质纤维素应用中，它目前仍被视为废物副产物。木质素的增值依赖于有效和绿色的脱木质素方法，微生物的应用越来越受到关注。在这里，我们研究了新型兼性厌氧细菌 Tolumonas lignolytica BRL6-1 在缺氧条件下对木质素的生理学和分子反应。比较了在木质素添加或未添加条件下厌氧生长的细胞的生理和生化变化。在木质素存在的情况下，BRL6-1 积累的生物量更高，与未添加条件相比，迟滞期更短，通过 log2 倍数变化 2 或更大确定的 14%的蛋白质丰度显著更高，这些蛋白质与对数后期铁(II)运输有关。上清液的 Ferrozine 测定证实，只有在 BRL6-1 的存在下，Fe(III)才与木质素结合并还原为 Fe(II)，这表明细胞具有氧化还原活性。分泌组的 LC-MS/MS 分析显示，在木质素添加条件下出现了 20 kDa 的额外条带。该条带的蛋白质测序鉴定出一种具有未知功能的蛋白质，与自由基 SAM 超家族中的酶具有同源性。在木质素添加条件下表达这种蛋白质表明其在自由基形成中的作用。根据我们的发现，我们认为 BRL6-1 正在使用自由基 SAM 超家族中的一种蛋白质与结合在木质素上的 Fe(III)相互作用，并将其还原为 Fe(II)以供细胞使用，从而增加木质素添加条件下的 BRL6-1 产量。这种相互作用可能会产生有机自由基，并引发自由基级联反应，从而修饰和解聚木质素。进一步的研究应该阐明这种机制与先前描述的需氧螯合剂介导的芬顿化学或产生自由基的木质素降解酶（如木质素过氧化物酶）在多大程度上相似，但在缺氧条件下。
