Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation in Aspergillus luchuensis mut. kawachii
Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation in Aspergillus luchuensis mut. kawachii
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DOI：
10.1128/aem.03136-18
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发表时间：
2019-04-01
影响因子：
4.4
通讯作者：
Futagami, Taiki
中科院分区：
文献类型：
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作者：
Kadooka, Chihiro;Izumitsu, Kosuke;Futagami, Taiki
葡萄曲霉。川崎（a . kawachii）在发酵烧酒（日本传统的蒸馏酒）的过程中会产生大量的柠檬酸。在这项研究中，我们对A. kawachii CtpA和YhmA进行了表征，它们分别与酵母Saccharomyces cerevisiae线粒体柠檬酸转运体Ctp1和Yhm2同源。从川崎菌中纯化CtpA和YhmA并重组为脂质体。蛋白脂质体仅表现出反交换转运活性；CtpA转运柠檬酸盐使用反底物，尤其是顺式乌头酸盐和苹果酸盐，而YhmA转运柠檬酸盐使用更广泛的反底物，包括柠檬酸盐、2-氧戊二酸盐、苹果酸盐、顺式乌头酸盐和琥珀酸盐。ctpA和yhmA的破坏导致菌丝生长和分生孢子形成不足，菌丝减少！重量标准化的柠檬酸盐产量。由于我们无法获得Delta ctpA Delta yhmA菌株，我们使用Tet-On启动子系统在Delta yhmA背景下构建了s标记的ctpA （ctpA- s）条件表达菌株。敲低ctpA- s在Delta yhmA中导致了严重的生长缺陷，乙酰辅酶a （acetyl- coa）和赖氨酸水平显著降低，表明ctpA和yhmA的双重破坏导致了合成致死；然而，我们随后发现，添加乙酸或赖氨酸可以缓解严重的生长缺陷，这可以弥补乙酰辅酶a水平。我们的研究结果表明CtpA和YhmA是参与柠檬酸生成的线粒体柠檬酸转运蛋白，并且柠檬酸从线粒体转运到细胞质在川芎乙酰辅酶a的生物生成中起重要作用。重要性：柠檬酸运输被认为在丝状真菌的柠檬酸生产中起着重要作用；然而，这一过程的细节仍不清楚。本研究对两种柠檬酸转运体进行了表征。kawachii。生化和基因破坏分析表明，CtpA和YhmA是线粒体柠檬酸转运体，是正常菌丝生长、分生孢子形成、胞质乙酰辅酶a合成和柠檬酸生产所必需的。本研究阐明的真菌柠檬酸转运体的特性将有助于扩大我们对柠檬酸生产机制的认识，并促进工业有机酸发酵工艺的开发和优化。
Aspergillus luchuensis mut. kawachii (A. kawachii) produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especially cis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate, cis-aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidium formation with reduced mycelia! weight-normalized citrate production. Because we could not obtain a Delta ctpA Delta yhmA strain, we constructed an S-tagged ctpA (ctpA-S) conditional expression strain in the Delta yhmA background using the Tet-On promoter system. Knockdown of ctpA-S in Delta yhmA 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 ctpA and yhmA 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 A. kawachii.IMPORTANCE 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 Aspergillus luchuensis mut. kawachii. 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.