Itaconic acid degradation in Aspergillus niger: the role of unexpected bioconversion pathways

Abstract

BackgroundItaconic acid (IA), a C5-dicarboxylic acid, has previously been identified as one of the top twelve biochemicals that can be produced by biotechnological means. IA is naturally produced by Aspergillus terreus, however, heterologous production in the related species Aspergillus niger has been proposed earlier. Remarkably, we observed that during high producing conditions and elevated titers A. niger detoxifies the extracellular medium of IA. In order to determine the genes responsible for this decline in IA titers a transcriptome analysis was performed.ResultsTranscriptome analysis has led to the identification of two novel and previously unknown IA bioconversion pathways in A. niger. One pathway is proposed to convert IA into pyruvate and acetyl-CoA through the action of itaconyl-CoA transferase (IctA), itaconyl-CoA hydratase (IchA) and citramalyl-CoA lyase, similar to the pathway identified in A. terreus. Another pathway putatively converts IA into 1-methyl itaconate through the action of trans-aconitate methyltransferase (TmtA). Upon deleting the key genes ictA and ichA we have observed increased IA production and titers and cessation of IA bioconversion. Surprisingly, deletion of tmtA lead to strong reduction of heterologous IA production.ConclusionHeterologous IA production in A. niger induces the expression of IA bioconversion pathways. These pathways can be inhibited by deleting the key genes ictA, ichA and tmtA. Deletion of ictA and ichA resulted in increased IA production. Deletion of tmtA, however, resulted in almost complete cessation of IA production.Electronic supplementary materialThe online version of this article (10.1186/s40694-018-0062-5) contains supplementary material, which is available to authorized users.

Metabolic engineering with ATP-citrate lyase and nitrogen source supplementation improves itaconic acid production in Aspergillus niger

“…Simultaneously with the findings on NO 3 − /NH 4 + availability in relation to IA degradation, we have observed upregulation of the IA bioconversion pathway in A. niger responsible for detoxifying the extracellular medium of IA during high producing conditions, resembling the IA degrading pathway of A. terreus described by Chen et al [45, 54]. Knock-out of the key pathway-associated genes in CitB#99 resulted in cessation of IA bioconversion and elevated titers [45].…”

“…Simultaneously with the findings on NO 3 − /NH 4 + availability in relation to IA degradation, we have observed upregulation of the IA bioconversion pathway in A. niger responsible for detoxifying the extracellular medium of IA during high producing conditions, resembling the IA degrading pathway of A. terreus described by Chen et al [45, 54]. Knock-out of the key pathway-associated genes in CitB#99 resulted in cessation of IA bioconversion and elevated titers [45]. However, as these high IA titers have shown to result in stronger growth effects possibly also due to weak-acid stress, combining this knock-out of the IA degradation pathway in combination with NH 4 + /NO 3 − balancing conditions is a clear target for ongoing research to improve IA production in A. niger .…”

“…In our previous experiments, we have observed that CitB#99 and acl12 overexpressing transformants start to degrade IA at titers between 15 and 20 g/L (Fig. 3) [45]. Interestingly, by maintaining NO 3 − and NH 4 + availability, both cessation of IA degradation and elevated titers could be established as shown by the cultivations in MRA medium as well as pulse-feeding experiments (Figs.…”

Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production

“…In another Aspergillus species from the section Nigri, Aspergillus luchuensis the two putative CTP homologues ctpA and ctpB have been studied in some detail [18, 19]. The ctpB gene was shown to be non-expressed under citric acid production conditions, whereas ctpA was constitutively expressed in both A. luchuensis and A. niger [18, 20]. Although deletion of the ctpA gene resulted in serious growth effects of the resulting mutant strains, the effects on citric acid production were marginally [18, 19].…”

“…In S. cerevisiae this antiporter is encoded by the yhm2 gene responsible for exporting citrate from mitochondria to cytosol in exchange of oxoglutarate [22]. Also in this case one of the homologues, yhmB , was shown to be hardly expressed, while the closest homologue to yhm2 (cocA) , yhmA was also expressed constitutively in A. luchuensis and A.niger, [18, 20]. Deletion of yhmB showed no effect on citrate production, whereas yhmA and cocA deletion had a clear effect on citrate production, which was further aggravated in a conditional ctpA / yhmA double mutant, where malate and oxo-glutarate production was increased [19].…”