Metabolic engineering of Aspergillus niger via ribonucleoprotein-based CRISPR-Cas9 system for succinic acid...

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.