Abstract
BackgroundSuccinic 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.ResultsWith 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.ConclusionsIn 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.
Highlights
Succinic acid has great potential to be a new bio-based building block for deriving a number of value-added chemicals in industry
Strain engineering via RNP‐based CRISPR–Cas9 system In this study, all the genetic modifications were introduced via the CRISPR–Cas9 RNP-based system
Null mutations were introduced into the gox and oah genes via non-homologous end joining (NHEJ) repair pathway after CRISPR–Cas9-mediated DNA double-strand breaks
Summary
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 has been one of the research hotspots in sustainable biotechnology due to the great potentials of succinic acid to be a building block for deriving varieties of commodity and specialty chemicals [1]. Bio-based succinic acid production is considered as a promising paradigm demonstrating the feasibility of using sustainable biological processes to replace petrol-based processes in the chemical industry [2]. If the further application of succinic acid aims at becoming a new bio-based building block in chemical industry to produce commodity chemicals, such as 1,4-butanediol and biodegradable polymers, the current price is still too expensive, which demands further improvement on the economics of biological processes. In order to break through the technical bottlenecks, more research attention has been directed on exploitation of new microbial platforms for bio-succinic acid production [7, 8]
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