Flaiz Maximilian, Ludwig Gideon, Bengelsdorf Frank R, Dürre Peter
Institute of Microbiology and Biotechnology, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany.
Biotechnol Biofuels. 2021 May 10;14(1):117. doi: 10.1186/s13068-021-01966-2.
The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.
In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth.
The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.
近年来，使用甲醇作为底物培养产乙酸菌的兴趣有所增加，因为它可以由合成气可持续生产，并且具有减少温室气体排放的额外好处。黏滞真杆菌是少数能够利用甲醇的产乙酸菌之一，其基因易于获取，因此是利用这种C1碳源重组生产生物商品的有前途的候选菌株。尽管已经有几种遗传工具可用于某些产乙酸菌，包括黏滞真杆菌，但明亮荧光报告蛋白的使用仍然有限。
在本研究中，我们通过引入荧光激活和吸收转移标签（FAST）作为荧光报告蛋白，扩展了黏滞真杆菌的遗传工具箱。构建了以诱导型和组成型方式表达编码FAST基因的重组黏滞真杆菌菌株。即使在厌氧条件下培养这些重组菌株，也能产生明亮荧光的细胞。此外，我们利用重组黏滞真杆菌菌株从甲醇中生产了生物商品丁醇和丙酮。因此，我们分别使用产生双功能乙醛/醇脱氢酶或乙酰乙酸脱羧酶的FAST标签融合蛋白的黏滞真杆菌培养物，并在生长过程中测定荧光强度和产物浓度。
添加FAST作为不依赖氧气的荧光报告蛋白扩展了黏滞真杆菌的遗传工具箱。此外，我们的结果表明，可以构建FAST标签融合蛋白，而不会对所得酶的稳定性、功能和生产力产生负面影响。最后，可以使用表达编码荧光FAST标签融合蛋白基因的重组黏滞真杆菌菌株从甲醇中生产丁醇和丙酮。
