Bengelsdorf Frank R, Poehlein Anja, Linder Sonja, Erz Catarina, Hummel Tim, Hoffmeister Sabrina, Daniel Rolf, Dürre Peter
Institut für Mikrobiologie und Biotechnologie, Universität Ulm Ulm, Germany.
Genomic and Applied Microbiology and Göttingen Genomics Laboratory, Georg-August University Göttingen Göttingen, Germany.
Front Microbiol. 2016 Jul 7;7:1036. doi: 10.3389/fmicb.2016.01036. eCollection 2016.
Synthesis gas (syngas) fermentation by anaerobic acetogenic bacteria employing the Wood-Ljungdahl pathway is a bioprocess for production of biofuels and biocommodities. The major fermentation products of the most relevant biocatalytic strains (Clostridium ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii) are acetic acid and ethanol. A comparative metabolic and genomic analysis using the mentioned biocatalysts might offer targets for metabolic engineering and thus improve the production of compounds apart from ethanol. Autotrophic growth and product formation of the four wild type (WT) strains were compared in uncontrolled batch experiments. The genomes of C. ragsdalei and C. coskatii were sequenced and the genome sequences of all four biocatalytic strains analyzed in comparative manner. Growth and product spectra (acetate, ethanol, 2,3-butanediol) of C. autoethanogenum, C. ljungdahlii, and C. ragsdalei were rather similar. In contrast, C. coskatii produced significantly less ethanol and its genome sequence lacks two genes encoding aldehyde:ferredoxin oxidoreductases (AOR). Comparative genome sequence analysis of the four WT strains revealed high average nucleotide identity (ANI) of C. ljungdahlii and C. autoethanogenum (99.3%) and C. coskatii (98.3%). In contrast, C. ljungdahlii WT and C. ragsdalei WT showed an ANI-based similarity of only 95.8%. Additionally, recombinant C. ljungdahlii strains were constructed that harbor an artificial acetone synthesis operon (ASO) consisting of the following genes: adc, ctfA, ctfB, and thlA (encoding acetoacetate decarboxylase, acetoacetyl-CoA:acetate/butyrate:CoA-transferase subunits A and B, and thiolase) under the control of thlA promoter (P thlA ) from C. acetobutylicum or native pta-ack promoter (P pta-ack ) from C. ljungdahlii. Respective recombinant strains produced 2-propanol rather than acetone, due to the presence of a NADPH-dependent primary-secondary alcohol dehydrogenase that converts acetone to 2-propanol. Furthermore, the ClosTron(TM) system was used to construct an adhE1 integration mutant. These results provide extensive insights into genetic features of industrially relevant bacterial biocatalysts and expand the toolbox for metabolic engineering of acetogenic bacteria able to ferment syngas.
利用伍德-龙格达尔途径的厌氧产乙酸细菌进行合成气发酵是一种生产生物燃料和生物制品的生物过程。最相关的生物催化菌株（Ljungdahlii梭菌、自养乙醇梭菌、Ragsdalei梭菌和Coskatii梭菌）的主要发酵产物是乙酸和乙醇。使用上述生物催化剂进行比较代谢和基因组分析可能为代谢工程提供靶点，从而提高除乙醇外的化合物产量。在不受控制的分批实验中比较了四种野生型（WT）菌株的自养生长和产物形成。对Ragsdalei梭菌和Coskatii梭菌的基因组进行了测序，并以比较的方式分析了所有四种生物催化菌株的基因组序列。自养乙醇梭菌、Ljungdahlii梭菌和Ragsdalei梭菌的生长和产物谱（乙酸盐、乙醇、2,3-丁二醇）相当相似。相比之下，Coskatii梭菌产生的乙醇明显较少，其基因组序列缺少两个编码醛：铁氧化还原蛋白氧化还原酶（AOR）的基因。对四种WT菌株的比较基因组序列分析显示，Ljungdahlii梭菌和自养乙醇梭菌的平均核苷酸同一性（ANI）较高（99.3%），与Coskatii梭菌的ANI为98.3%。相比之下，Ljungdahlii WT和Ragsdalei WT基于ANI的相似性仅为95.8%。此外，构建了重组Ljungdahlii菌株，其携带一个人工丙酮合成操纵子（ASO），该操纵子由以下基因组成：adc、ctfA、ctfB和thlA（分别编码乙酰乙酸脱羧酶、乙酰乙酰辅酶A：乙酸/丁酸：辅酶A转移酶亚基A和B以及硫解酶），受丙酮丁醇梭菌的thlA启动子（P thlA ）或Ljungdahlii梭菌的天然pta-ack启动子（P pta-ack ）控制。由于存在一种将丙酮转化为2-丙醇的依赖NADPH的伯-仲醇脱氢酶，相应的重组菌株产生的是2-丙醇而不是丙酮。此外，使用ClosTron(TM)系统构建了adhE1整合突变体。这些结果为工业相关细菌生物催化剂的遗传特征提供了广泛的见解，并扩展了能够发酵合成气的产乙酸细菌代谢工程的工具箱。
