Succinic acid has great potential to be a new bio-based building block for deriving a number of value-added chemicals in industry. Bio-based succinic acid production from renewable biomass can provide a feasible approach to partially alleviate the dependence of global manufacturing on petroleum refinery. To improve the economics of biological processes, we attempted to explore possible solutions with a fungal cell platform. In this study, Aspergillus niger, a well-known industrial production organism for bio-based organic acids, was exploited for its potential for succinic acid production. With a ribonucleoprotein (RNP)-based CRISPR–Cas9 system, consecutive genetic manipulations were realized in engineering of the citric acid-producing strain A. niger ATCC 1015. Two genes involved in production of two byproducts, gluconic acid and oxalic acid, were disrupted. In addition, an efficient C4-dicarboxylate transporter and a soluble NADH-dependent fumarate reductase were overexpressed. The resulting strain SAP-3 produced 17 g/L succinic acid while there was no succinic acid detected at a measurable level in the wild-type strain using a synthetic substrate. Furthermore, two cultivation parameters, temperature and pH, were investigated for their effects on succinic acid production. The highest amount of succinic acid was obtained at 35 °C after 3 days, and low culture pH had inhibitory effects on succinic acid production. Two types of renewable biomass were explored as substrates for succinic acid production. After 6 days, the SAP-3 strain was capable of producing 23 g/L and 9 g/L succinic acid from sugar beet molasses and wheat straw hydrolysate, respectively. In this study, we have successfully applied the RNP-based CRISPR–Cas9 system in genetic engineering of A. niger and significantly improved the succinic acid production in the engineered strain. The studies on cultivation parameters revealed the impacts of pH and temperature on succinic acid production and the future challenges in strain development. The feasibility of using renewable biomass for succinic acid production by A. niger has been demonstrated with molasses and wheat straw hydrolysate.

中文翻译：

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通过基于核糖核蛋白的CRISPR–Cas9系统对黑曲霉进行代谢工程，从而从可再生生物质生产琥珀酸

琥珀酸具有巨大的潜力，可以成为一种新的生物基构件，可用于衍生工业中的多种增值化学品。由可再生生物质生产生物基琥珀酸可提供一种可行的方法，以部分缓解全球制造业对石油精炼厂的依赖。为了提高生物过程的经济性，我们尝试使用真菌细胞平台探索可能的解决方案。在这项研究中，黑曲霉是一种著名的生物基有机酸的工业生产生物，被利用来生产琥珀酸。借助基于核糖核蛋白（RNP）的CRISPR–Cas9系统，在生产柠檬酸菌株A.niger ATCC 1015的工程中实现了连续的遗传操作。两个基因参与了两个副产物葡萄糖酸和草酸的生产，被打乱了。另外，有效的C4-二羧酸盐转运蛋白和可溶性NADH依赖的富马酸酯还原酶被过表达。所得菌株SAP-3产生17g / L的琥珀酸，而在使用合成底物的野生型菌株中未检测到可测量水平的琥珀酸。此外，研究了两个培养参数，温度和pH对琥珀酸生产的影响。3天后在35°C时可获得最高量的琥珀酸，低培养pH对琥珀酸的产生具有抑制作用。探索了两种类型的可再生生物质作为琥珀酸生产的底物。6天后，SAP-3菌株能够分别从甜菜糖蜜和小麦秸秆水解产物中产生23g / L和9g / L的琥珀酸。在这个研究中，我们已经成功地将基于RNP的CRISPR–Cas9系统应用于黑曲霉的基因工程中，并显着提高了工程菌株中的琥珀酸产量。栽培参数的研究揭示了pH和温度对琥珀酸生产的影响以及菌株开发中的未来挑战。用糖蜜和麦秸水解物证明了将可再生生物质用于黑曲霉生产琥珀酸的可行性。