Overexpression of a C4-dicarboxylate transporter is the key for rerouting citric acid to C4-dicarboxylic acid production in Aspergillus carbonarius

Abstract

BackgroundC4-dicarboxylic acids, including malic acid, fumaric acid and succinic acid, are valuable organic acids that can be produced and secreted by a number of microorganisms. Previous studies on organic acid production by Aspergillus carbonarius, which is capable of producing high amounts of citric acid from varieties carbon sources, have revealed its potential as a fungal cell factory. Earlier attempts to reroute citric acid production into C4-dicarboxylic acids have been with limited success.ResultsIn this study, a glucose oxidase deficient strain of A. carbonarius was used as the parental strain to overexpress a native C4-dicarboxylate transporter and the gene frd encoding fumarate reductase from Trypanosoma brucei individually and in combination. Impacts of the introduced genetic modifications on organic acid production were investigated in a defined medium and in a hydrolysate of wheat straw containing high concentrations of glucose and xylose. In the defined medium, overexpression of the C4-dicarboxylate transporter alone and in combination with the frd gene significantly increased the production of C4-dicarboxylic acids and reduced the accumulation of citric acid, whereas expression of the frd gene alone did not result in any significant change of organic acid production profile. In the wheat straw hydrolysate after 9 days of cultivation, similar results were obtained as in the defined medium. High amounts of malic acid and succinic acid were produced by the same strains.ConclusionsThis study demonstrates that the key to change the citric acid production into production of C4-dicarboxylic acids in A. carbonarius is the C4-dicarboxylate transporter. Furthermore it shows that the C4-dicarboxylic acid production by A. carbonarius can be further increased via metabolic engineering and also shows the potential of A. carbonarius to utilize lignocellulosic biomass as substrates for C4-dicarboxylic acid production.

Contrasting P acquisition strategies of the bacterial communities associated with legume and grass in subtropical orchard soil

“…Recently, the genes (gcd, gad, pqq) encoding the related enzymes have been investigated (de Werra et al, 2009;Hsu et al, 2015;An and Moe, 2016). In addition to OA biosynthesis, some genes encoding OA transporters have been identified and characterized in bacteria, such as ttdT, dct, JEN, oxlT (Kim and Unden, 2007;Iyalomhe et al, 2014;Becker, 2015;de Lima et al, 2016;Yang L et al, 2017), although their involvement in solubilizing sparingly soluble Pi is largely unknown. For the mobilization of Po, P mineralizing bacteria exude nonspecific phosphatase (phosphomonoesterase, phosphodiesterase, phosphotriesterase), phytase, phosphonatase or C-P lyase to hydrolyze Po and release orthophosphate (Eivazi and Tabatabai, 1977;Nannipieri et al, 2011).…”

Evaluation of pyruvate decarboxylase‐negative Saccharomyces cerevisiae strains for the production of succinic acid

“…The expression of the mae1 gene was critical in both strain backgrounds and led to an almost three times higher succinate titer in the wildtype (REF‐6) and a twofold increase in the TAM strain (TAM‐6) compared with the strains carrying the succinate synthesis pathway but lacking the transporter. In several other studies, introduction of a heterologous transporter function has been shown to be critical for the extracellular accumulation of several organic acids including succinate …”

“…The expression of the mae1 gene was critical in both strain backgrounds and led to an almost three times higher succinate titer in the wildtype (REF-6) and a twofold increase in the TAM strain (TAM-6) compared with the strains carrying the succinate synthesis pathway but lacking the transporter. In several other studies, introduction of a heterologous transporter function has been shown to be critical for the extracellular accumulation of several organic acids including succinate [5,18,36] The TAM strain did not produce ethanol, the main byproduct formed by the wild-type strain in concentrations as F I G U R E 2 Comparison of the reference and TAM strains engineered for succinate production. The strains were cultivated in mineral salt medium with 5% glucose as substrate and supplemented with 22 mM formate under microaerobic conditions (shaking frequency 90 rpm).…”

In silico evolution of Aspergillus niger organic acid production suggests strategies for switching acid output

“…Expression of NADH-dependent fumarate reductase from Trypanosoma brucei increased succinic production from 3.8 to 16.2 g/L while negatively affecting malic and citric acid production [37]. When the same fumarate reductase gene was expressed in an engineered strain of Aspergillus carbonarius lacking the glucose oxidase gene the acid production profile was unchanged, yet increased succinic production up to 16 g/L when a C4-dicarboxylate transporter was overexpressed [38]. The studies highlight the need to consider organic acid transport in organisms that do not naturally secrete the target acid.…”