Decrease of citric acid produced by <i>Aspergillus niger</i> through disruption of the gene encoding a putative mitochondrial citrate-oxoglutarate shuttle protein

The transporter that exports citric acid (CA) generated in mitochondria to the cytosol is an important component of the CA production machinery of <i>Aspergillus niger</i>. In this report, we cloned and identified the gene <i>cocA</i>, encoding a 33.7-kDa putative mitochondrial citrate-oxoglutarate shuttle protein of the CA hyper-producer <i>A. niger</i> WU-2223L. The amount of CA produced by a representative <i>cocA</i> disruptant (35 g/L) was significantly lower than that produced by strain WU-2223L (63 g/L) after culture for 12 days under CA production conditions, and the phenotype of the <i>cocA</i> disruptant differed in part from that of strain WU-2223L. A <i>cocA</i> disruptant complemented with <i>cocA</i> exhibited the same phenotypes as those of strain WU-2223L. This report is the first to show that <i>cocA</i> and its protein product clearly contribute to substantial CA production by <i>A. niger</i>, and provides a significant insight into microbial organic acid production by fermentation. <b>Abbreviations</b>: CA: citric acid; CD medium: Czapek-Dox medium; CS: citrate synthase; CTP: citrate transport protein; HR: homologous recombination; MCF: mitochondrial carrier family; RT-PCR: reverse-transcription PCR; TCA: tricarboxylic acid The gene <i>cocA</i> encoding a putative mitochondrial citrate-oxoglutarate shuttle protein greatly contributes to hyper-production of citric acid by <i>Aspergillus niger</i>.

将线粒体中生成的柠檬酸（citric acid, CA）转运至细胞质的转运蛋白，是黑曲霉（*Aspergillus niger*）柠檬酸生产机制的重要组成部分。本研究成功克隆并鉴定了基因cocA，其编码黑曲霉柠檬酸高产菌株WU-2223L的一种33.7 kDa的推定线粒体柠檬酸-酮戊二酸穿梭蛋白。在柠檬酸生产条件下培养12天后，代表性cocA基因敲除株的柠檬酸产量为35 g/L，显著低于亲本菌株WU-2223L的63 g/L，且该敲除株的表型与WU-2223L菌株存在部分差异。通过导入cocA基因进行互补的cocA敲除株，其表型与WU-2223L菌株完全一致。本研究首次证实cocA及其编码蛋白对黑曲霉的柠檬酸高产具有实质性贡献，为发酵法生产微生物有机酸提供了重要的研究见解。

**缩写说明**：CA：柠檬酸；CD培养基：察氏培养基（Czapek-Dox medium）；CS：柠檬酸合酶（citrate synthase）；CTP：柠檬酸转运蛋白（citrate transport protein）；HR：同源重组（homologous recombination）；MCF：线粒体载体家族（mitochondrial carrier family）；RT-PCR：反转录PCR（reverse-transcription PCR）；TCA：三羧酸循环（tricarboxylic acid）

编码推定线粒体柠檬酸-酮戊二酸穿梭蛋白的cocA基因，对黑曲霉的柠檬酸高产具有显著促进作用。