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工程化柠檬酸外排蛋白可提高黑曲霉中的柠檬酸产量。

Engineering of the citrate exporter protein enables high citric acid production in Aspergillus niger.

机构信息

Austrian Centre of Industrial Biotechnology (ACIB GmbH), Muthgasse 18, Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria.

Austrian Centre of Industrial Biotechnology (ACIB GmbH), Muthgasse 18, Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria; CD Laboratory for Biotechnology of Glycerol, Muthgasse 18, Vienna, Austria.

出版信息

Metab Eng. 2019 Mar;52:224-231. doi: 10.1016/j.ymben.2018.12.004. Epub 2018 Dec 13.

Abstract

Aspergillus niger was engineered using a gene responsible for citric acid transport, which has a significant impact on citric acid secretion when overexpressed. The transport gene was identified by a homology search using an itaconic acid transporter from Ustilago maydis as template. The encoding homologous protein CexA belongs to the major facilitator superfamily subclass DHA1 and members of this family work as drug-H antiporter. The disruption of this gene completely abolishes citric acid secretion, which indicates that this protein is the main citric acid transporter in A. niger. In the disruption strain, the metabolism is re-routed mainly to oxalic acid, which is a known by-product during citric acid production. The gene can be heterologously expressed in Saccharomyces cerevisiae, which leads to the secretion of citric acid during the growth on glucose. These results confirm the functionality of CexA as the main transporter for citric acid of A. niger. Overexpression of cexA leads to a significant increase in secreted citric acid. Thereby, striking differences between a strong constitutive expression system using pmbfA as a promoter and an inducible expression system using ptet-on can be observed. The inducible system significantly outcompetes the constitutive expression system yielding up to 109 g/L citric acid, which is 5 times higher compared to the parental wild-type strain and 3 times higher compared to the constitutive expression system. These results demonstrate the importance of the cellular transport system for an efficient production of metabolites. By overexpressing a single gene, it is possible to significantly improve the citric acid secretion capability of a moderately producing parental strain.

摘要

黑曲霉使用负责柠檬酸运输的基因进行了工程改造,当过度表达时,这对柠檬酸分泌有重大影响。通过使用构巢曲霉的异柠檬酸转运蛋白作为模板进行同源性搜索,鉴定出了该转运基因。编码同源蛋白 CexA 属于主要易化因子超级家族亚类 DHA1,该家族的成员作为药物-H 反向转运体工作。该基因的破坏完全消除了柠檬酸的分泌,这表明该蛋白是黑曲霉中主要的柠檬酸转运蛋白。在破坏菌株中,代谢主要重新定向为草酸,草酸是柠檬酸生产过程中的已知副产物。该基因可以在酿酒酵母中异源表达,这导致在葡萄糖生长过程中分泌柠檬酸。这些结果证实了 CexA 作为黑曲霉主要柠檬酸转运蛋白的功能。cexA 的过表达导致分泌的柠檬酸显著增加。因此,可以观察到强组成型表达系统(使用 pmbfA 作为启动子)和诱导型表达系统(使用 ptet-on)之间的明显差异。诱导型系统明显优于组成型表达系统,可产生高达 109 g/L 的柠檬酸,比亲本野生型菌株高 5 倍,比组成型表达系统高 3 倍。这些结果表明细胞运输系统对代谢物的高效生产非常重要。通过过表达单个基因,可以显著提高中等产亲本菌株的柠檬酸分泌能力。

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