Organic acid production in Aspergillus niger: Rewiring endogenous metabolic pathways by introducing and modifying the itaconic acid pathway from Aspergillus terreus

Abeer Hossain 1,2 Roy van Gerven 1 Peter S. Lubeck 3 Peter Punt 1
1., Dutch DNA Biotech, Utrecht, Netherlands
2SILS, University of Amsterdam, Amsterdam, Netherlands
3Department of Chemistry and Bioscience, Aalborg University, Kopenhagen, Denmark

Rising carbon emissions due to increased industrialization and its effect on the global climate are raising awareness to move from a fossil fuel-based economy to a bio-based economy. Organic acids have huge potential as alternative for petrochemicals and concomitantly its derivatives as commodities [1]. Filamentous fungi are widely known as efficient organic acid producers, in particular members of the genus Aspergillus.

Itaconic acid (IA), a C5-dicarboxylic acid, has been identified as one of the top twelve building block chemicals that can be produced by biotechnological means. The potential applications of IA in green chemistry are numerous and IA is already naturally produced by Aspergillus terreus. However, for several reasons heterologous production in the related species Aspergillus niger has been proposed. Previously we have shown that rewiring of a non-canonicla citrate synthase gene (citB) derived from an A. niger secondary metabolism cluster has led to an increased yield, titer and productivity of IA, reaching to the highest levels reported for heterologous IA production.

In our research we have now performed a RNA-Seq analysis of high, medium and low IA producing strains to further improve our optimized IA pathway and understand the effect of heterologous IA production on A. niger metabolism. It was found that apart from citB, another non-canonical citrate synthase displayed a similar role in itaconic acid production upon overexpression. Further rewiring of metabolic pathways was seen by specific gene deletion of pathways involved in byproduct formation and overexpression of canonical primary metabolic pathways genes. Several of these strain modifications were found to improve production of itaconic acid. Finally, our research also showed a hitherto unknown involvement of N-metabolism on prolonged itaconic acid production to achieve higher titers and yields.