Valk Laura C, Diender Martijn, Stouten Gerben R, Petersen Jette F, Nielsen Per H, Dueholm Morten S, Pronk Jack T, van Loosdrecht Mark C M
Department of Biotechnology, Delft University of Technology, Delft, Netherlands.
Laboratory of Microbiology, Wageningen University and Research, Wageningen, Netherlands.
Front Microbiol. 2020 Jan 29;11:63. doi: 10.3389/fmicb.2020.00063. eCollection 2020.
Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of " Galacturonibacter soehngenii," from a novel genus within the , suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaHCO fed to chemostat-grown, galacturonate-limited enrichment cultures of " G. soehngenii" was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the " G. soehngenii" MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of " G. soehngenii" enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of " G. soehngenii."
产乙酸菌能够在发酵过程中通过伍德-Ljungdahl途径（WLP）固定碳，该途径在细菌和古菌中高度保守。在之前的一项研究中，来自一个新属的“索氏半乳糖醛酸杆菌”在以半乳糖醛酸为限的厌氧富集培养物中的产物化学计量表明，半乳糖醛酸发酵的改良Entner-Doudoroff途径和产乙酸的WLP同时运行。然而，基于短读长的宏基因组组装基因组（MAG）草图并未揭示编码经典WLP一氧化碳脱氢酶/乙酰辅酶A合酶（CODH/ACS）复合物的基因同源物。在本研究中，向恒化器培养的、以半乳糖醛酸为限的“索氏半乳糖醛酸杆菌”富集培养物中添加的NaHCO₃被证明掺入到了乙酸中。乙酸羧基的优先标记与通过WLP进行的产乙酸过程一致，在该过程中，乙酸的甲基主要来源于甲酸。假定的丙酮酸-甲酸裂解酶基因的高转录水平和甲酸脱氢酶候选基因的极低转录水平进一步支持了这一解释。在长读长纳米孔测序数据的支持下对“索氏半乳糖醛酸杆菌”MAG进行重新组装，得到了一个单支架MAG，这证实了经典CODH/ACS复合物基因同源物的缺失。然而，在“索氏半乳糖醛酸杆菌”富集培养物的细胞提取物中检测到了高CO脱氢酶活性，这与MAG中缺乏相应同源物相矛盾。基于与厌氧镍-CO脱氢酶蛋白相关的高度保守氨基酸基序，鉴定出了一个可能负责观察到的活性的新候选物。这些结果表明，在厌氧、以半乳糖醛酸为限的“索氏半乳糖醛酸杆菌”培养物中存在一条产乙酸途径，很可能是伍德-Ljungdahl途径的一种尚未解析的变体。
