United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan.
Education and Research Center for Fermentation Studies, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan.
Appl Environ Microbiol. 2019 Apr 4;85(8). doi: 10.1128/AEM.03136-18. Print 2019 Apr 15.
mut. () produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized CtpA and YhmA, which are homologous to the yeast mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from and reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especially -aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate, -aconitate, and succinate. Disruption of and caused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a Δ Δ strain, we constructed an S-tagged () conditional expression strain in the Δ background using the Tet-On promoter system. Knockdown of in Δ resulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption of and leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in acetyl-CoA biogenesis in Citrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters from mut. Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidium formation, cytosolic acetyl-CoA synthesis, and citric acid production. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.
mut. () 在酿造日本传统蒸馏酒清酒的过程中会产生大量柠檬酸。在这项研究中，我们鉴定了 CtpA 和 YhmA，它们分别与酵母线粒体柠檬酸转运蛋白 Ctp1 和 Yhm2 同源。从 中纯化了 CtpA 和 YhmA，并将其重构成脂质体。这些蛋白脂质体仅表现出反向交换转运活性；CtpA 利用反底物，特别是顺乌头酸和苹果酸，来转运柠檬酸，而 YhmA 则利用更广泛的反底物，包括柠檬酸、2-氧代戊二酸、苹果酸、顺乌头酸和琥珀酸来转运柠檬酸。 和 的缺失导致菌丝生长和分生孢子形成缺陷，菌丝体重量归一化的柠檬酸产量降低。由于我们无法获得 Δ Δ 菌株，我们使用 Tet-On 启动子系统在 Δ 背景中构建了一个 S 标记的 () 条件表达菌株。Δ 中 的敲低导致在最小培养基上的严重生长缺陷，乙酰辅酶 A (acetyl-CoA) 和赖氨酸水平显著降低，表明 和 的双重缺失导致合成致死；然而，我们随后发现，通过添加乙酸盐或赖氨酸可以缓解严重的生长缺陷，这可以补救乙酰辅酶 A 水平。我们的结果表明 CtpA 和 YhmA 是参与柠檬酸生成的线粒体柠檬酸转运蛋白，柠檬酸从线粒体向细胞质的转运在 乙酰辅酶 A 生物合成中起着重要作用。柠檬酸转运被认为在丝状真菌的柠檬酸生成中起着重要作用；然而，其过程细节仍不清楚。本研究从 mut. 中鉴定了两种柠檬酸转运蛋白。生化和基因敲除分析表明，CtpA 和 YhmA 是线粒体柠檬酸转运蛋白，对于正常的菌丝生长、分生孢子形成、细胞质乙酰辅酶 A 合成和柠檬酸生成是必需的。本研究中阐明的真菌柠檬酸转运蛋白的特性将有助于我们扩展对柠檬酸生成机制的理解，并促进工业有机酸发酵工艺的开发和优化。
