DECHEMA-Forschungsinstitut, Microbial Biotechnology, Theodor-Heuss-Allee 25, 60486, Frankfurt am Main, Germany.
Department of Life Sciences of the Goethe University Frankfurt am Main, Max-von-Laue-Str. 9, 60438, Frankfurt am Main, Germany.
Appl Microbiol Biotechnol. 2022 Oct;106(19-20):6713-6731. doi: 10.1007/s00253-022-12161-0. Epub 2022 Sep 15.
The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids. KEY POINTS: • 2,2-Difluorosuccinic acid is used to select for dicarboxylic acid uptake mutations. • Deletion of dctA2 leads to reduction of dicarboxylic acid uptake. • Transporter-deficient strains show improved production of citramalic acid.
甲基营养型细菌甲基沼泽红假单胞菌 AM1 具有成为甲醇驱动生物技术的平台生物的潜力。其乙基丙二酰辅酶 A 途径（EMCP）在生长过程中对 C1 化合物必不可少，并且含有几种辅酶 A 激活的二羧酸。这些酸可以作为各种聚合物的前体分子。过去，两种二羧酸产物，即衣康酸和 2-甲基琥珀酸，成功地使用来自大肠杆菌的异源硫酯酶 YciA 生产，但产量因产物再摄取而降低。在我们的研究中，我们对这些二羧酸产物的摄取机制进行了广泛的研究。通过使用 2,2-二氟琥珀酸作为选择剂，我们分离出一种二羧酸导入突变体。该菌株基因组的分析表明基因 dctA2 缺失，该基因可能编码一种酸转运蛋白。通过测试额外的单、双和三重缺失，我们排除了其他两种 DctA 转运蛋白同源物和酮戊二酸转运蛋白 KgtP 的参与。dctA2 突变体中 2-甲基琥珀酸的摄取显著减少，而衣康酸的摄取则完全被阻止。此外，我们还证明了在基于 M. extorquens 的柠檬酸和进一步提高 1.4 倍产物产量的合成中，使用转运缺陷型菌株。这项工作是开发用于二羧酸的稳健 M. extorquens AM1 生产菌株的重要一步。关键点：• 2,2-二氟琥珀酸用于选择二羧酸摄取突变体。• dctA2 的缺失导致二羧酸摄取减少。• 转运缺陷型菌株显示 citramalic 酸产量提高。
