Metabolic engineering of Aspergillus niger via ribonucleoprotein-based CRISPR–Cas9 system for succinic acid production from renewable biomass

Abstract

Background 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. Results 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. Conclusions 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.

Something old, something new: challenges and developments in Aspergillus niger biotechnology

“…By overexpressing key genes in the cexA disruption mutant, including a low affinity glucose membrane transporter mstC, hexokinase hxkA and pyruvate kinase pkiA, among other genes, malic acid titres could be increased by ∼23% compared with the progenitor control [11]. Taken together, recent functional characterizations of CexA and other membrane transporters can be viewed as breakthroughs in A. niger biotechnology, which may soon be applied to generate organic acid hyper-producing isolates at an industrial level, especially when combined with other recent genetic strategies for maximizing productivity [13,38]. More generally, discovery and characterization of all A. niger organic acid organelle and plasma membrane transporters, and their detailed mechanistic functions, are crucial future objective for biotechnologists.…”

CRISPR-Cas9: Role in Processing of Modular Metabolic Engineered Bio-Based Products

“…As a result, Ozaki et al modified the S. pombe strain using the CRISPR-Cas9 system and synthesized D-lactic acid from both glucose and cellobiose. The active genes for pyruvate decarboxylases, dehydrogenase, and glycerol-3-phosphate dehydrogenase were deleted in this research, and the D-lactate dehydrogenase gene from Lactobacillus plantarum was incorporated into the S. pombegenome [75]. The applications of CRISPR biotechnology to specified host species are outlined below in order to generate varied metabolic products.…”

Biomass to fuels and chemicals: A review of enabling processes and technologies

“…97 A genetically modified Aspergillus niger strain has been investigated for improving SA yield, and SA yields of 3 and 9 g L −1 were reported from sugar beet molasses and wheat straw hydrolysate, respectively. 98 In another report, the concept of consolidated bioprocessing was demonstrated using a microbial consortium of Thermoanaerobacterium thermosaccharolyticum and Actinobacillus succinogenes, which resulted in 32.50 g L −1 succinic acid with a yield of 0.39 g g −1 from xylan. In addition, 12.51 g L −1 succinic acid was also obtained from untreated corn cob.…”