Identification of novel citramalate biosynthesis pathways in Aspergillus niger

Abstract

Background: The filamentous fungus Aspergillus niger is frequently used for industrial production of fermentative products such as enzymes, proteins and biochemicals. Notable examples of industrially produced A. niger fermentation products are glucoamylase and citric acid. Most notably, the industrial production of citric acid achieves high titers, yield and productivities, a feat that has prompted researchers to propose A. niger to serve as heterologous production host for the industrial production of itaconic acid (IA), a promising sustainable chemical building-block for the fabrication of various synthetic resins, coatings, and polymers. Heterologous production of IA in A. niger has resulted in unexpected levels of metabolic rewiring that has led us to the identification of IA biodegradation pathway in A. niger. In this study we have attempted to identify the final product of the IA biodegradation pathway and analyzed the effect of metabolic rewiring on the bioproduction of 9 industrially relevant organic acids.

Results: IA biodegradation manifests in diminishing titers of IA and the occurrence of an unidentified compound in the HPLC profile. Based on published results on the IA biodegradation pathway, we hypothesized that the final product of IA biodegradation in A. niger may be citramalic acid (CM). Based on detailed HPLC analysis, we concluded that the unidentified compound is indeed CM. Furthermore, by transcriptome analysis we explored the effect of metabolic rewiring on the production of 9 industrially relevant organic acids by transcriptome analysis of IA producing and WT A. niger strains. Interestingly, this analysis led to the identification of a previously unknown biosynthetic cluster that is proposed to be involved in the biosynthesis of CM. Upon overexpression of the putative citramalate synthase and a genomically clustered organic acid transporter, we have observed CM bioproduction by A. niger.

Conclusion: In this study, we have shown that the end product of IA biodegradation pathway in A. niger is CM. Knock-out of the IA biodegradation pathway results in the cessation of CM production. Furthermore, in this study we have identified a citramalate biosynthesis pathway, which upon overexpression drives citramalate bioproduction in A. niger.

Keywords: Aspergillus niger; Citramalate; Citramalate synthase; Itaconic acid biodegradation; Metabolic engineering; Organic acid transport; Transcriptome analysis.

Fig. 1

Unidentified peak next to the glucose peak on RI detector in sample after 14 days cultivation of CitB#99 performed in 500 ml non-baffled flasks [10]

Fig. 2

Previous fermentation data revisited. a Controlled batch-cultivation of CitB#99 in 5 l bioreactors [8]. CM can be detected after 180 h EFT when IA titers start to diminish. b Non-shaking 500 ml flask cultivation [10]. CM detected in cultivations with CitB#99 after 264 h EFT when IA titer starts to drop. No CM detected in cultivations with CitB#99 ΔICT and CitB#99 ΔICH

Fig. 3

Shake flask cultivation of cimA overexpressing strains. Experiments were performed in duplicate. Samples were taken and measured after 280 h incubation

Fig. 4

Putative organic acid biosynthesis pathways in A. niger. The enzymes facilitating the biochemical conversions are given with numbers and are linked to Table 2. Industrially relevant organic acids are indicated in dark blue. Figure adapted from Li et al. [13]

Fig. 5

Shake flask experiment to compare the organic acid production of cimA and mfsB overexpressing strains with WT strain. a Production of CM, citric acid (CA) and citraconic acid (CC). b Consumption of glucose