Institut für Mikrobiologie und Biotechnologie, Universität Ulm, Albert-Einstein-Allee 11, D-89081, Ulm, Germany.
Institut für Mikrobiologie und Biotechnologie, Universität Ulm, Albert-Einstein-Allee 11, D-89081, Ulm, Germany.
Metab Eng. 2021 Jul;66:296-307. doi: 10.1016/j.ymben.2021.04.010. Epub 2021 Apr 21.
Gas fermentation is a technology for producing platform chemicals as well as fuels and one of the most promising alternatives to petrochemicals. Medium-chained acids and alcohols such as hexanoate and hexanol are particularly interesting due to their versatile application. This study elucidated the pathway of chain elongation in native C6 compound-producing acetogens. Essential genes of Clostridium carboxidivorans for synthesis of medium-chained acids and alcohols were identified in order to demonstrate their catalytic activity in the acetogenic model organism Acetobacterium woodii. Two such gene clusters were identified, which are responsible for conversion of acetyl-CoA to butyryl-CoA by reverse β-oxidation. Using RT-PCR it could be demonstrated that only genes of cluster 1 are expressed constitutively with simultaneous formation of C6 compounds. Based on genes from C. carboxidivorans, a modular hexanoyl-CoA synthesis (hcs) plasmid system was constructed and transferred into A. woodii. With the recombinant A. woodii strains AWO [pPta_hcs1], AWO [pPta_hcs2], AWO [pTet_hcs1], and AWO [pTet_hcs2] butyrate and hexanoate production under heterotrophic (1.22-4.15 mM hexanoate) and autotrophic conditions (0.48-1.56 mM hexanoate) with both hcs clusters could be detected. hcs Cluster 1 from C. carboxidivorans was transferred into the ABE-fermenting strain Clostridium saccharoperbutylacetonicum as well. For further analysis, genes were also cloned into the hcs plasmid system individually. The resulting recombinant C. saccharoperbutylacetonicum strains with just individual genes neither produced hexanoate nor hexanol, but the strains containing the entire gene cluster were capable of chain elongation. A production of 0.8 mM hexanoate and 5.2 mM hexanol in the fermentation with glucose could be observed.
气体发酵是一种生产平台化学品、燃料的技术，是替代石化产品最有前途的方法之一。由于其应用广泛，中链酸和醇（如己酸酯和己醇）特别有趣。本研究阐明了产 C6 化合物的天然乙酰生成菌的链延伸途径。为了在产乙酰的模式生物伍德氏醋酸杆菌中展示其催化活性，鉴定了产中链酸和醇的梭菌的必需基因。鉴定了两个这样的基因簇，它们负责通过反向β-氧化将乙酰辅酶 A 转化为丁酰辅酶 A。通过 RT-PCR 可以证明，只有簇 1 的基因在同时形成 C6 化合物的情况下组成型表达。基于来自 C. carboxidivorans 的基因，构建了一个模块化的己酰辅酶 A 合成（hcs）质粒系统，并将其转移到 A. woodii 中。利用重组 A. woodii 菌株 AWO[pPta_hcs1]、AWO[pPta_hcs2]、AWO[pTet_hcs1]和 AWO[pTet_hcs2]，在异养（1.22-4.15 mM 己酸酯）和自养条件下（0.48-1.56 mM 己酸酯）都可以检测到丁酸盐和己酸酯的生成，并且这两个 hcs 簇都可以检测到。来自 C. carboxidivorans 的 hcs 簇 1 也被转移到 ABE 发酵菌株丙酮丁醇梭菌中。为了进一步分析，基因也分别被克隆到 hcs 质粒系统中。含有单个基因的重组 C. saccharoperbutylacetonicum 菌株既不能生产己酸酯也不能生产己醇，但含有整个基因簇的菌株则能够进行链延伸。在葡萄糖发酵中可以观察到 0.8 mM 己酸酯和 5.2 mM 己醇的生成。
