The interplay between transport and metabolism in fungal itaconic acid production

Abstract

Besides enzymatic conversions, many eukaryotic metabolic pathways also involve transport proteins that shuttle molecules between subcellular compartments, or into the extracellular space. Fungal itaconate production involves two such transport steps, involving an itaconate transport protein (Itp), and a mitochondrial tricarboxylate transporter (Mtt). The filamentous actinomycete Aspergillus terreus and the unicellular basidiomycete Ustilago maydis both produce itaconate, but do so via very different molecular pathways, and under very different cultivation conditions. In contrast, the transport proteins of these two strains are assumed to have a similar function. This study aims to investigate the roles of both the extracellular and mitochondrial transporters from these two organisms by expressing them in the corresponding U. maydis knockouts and monitoring the extracellular product concentrations. Both transporters from A. terreus complemented their corresponding U. maydis knockouts in mediating itaconate production. Surprisingly, complementation with Mtt of A. terreus (At_MfsA) led to a partial switch from itaconate to (S)-2-hydroxyparaconate secretion. Apparently, the export protein from A. terreus has a higher affinity for (S)-2-hydroxyparaconate than for itaconate, even though this species is classically regarded as an itaconate producer. Complementation with At_MttA increased itaconate production by 2.3-fold compared to complementation with Um-Mtt1, indicating that the mitochondrial carrier from A. terreus supports a higher metabolic flux of itaconic acid precursors than its U. maydis counterpart. The biochemical implications of these differences are discussed in the context of the biotechnological application in U. maydis and A. terreus for the production of itaconate and (S)-2-hydroxyparaconate. Keywords Itaconate (S)-2-hydroxyparaconate Ustilago maydis Aspergillus terreus Transporter Metabolism

Mitochondrial carriers of Ustilago maydis and Aspergillus terreus involved in itaconate production: same physiological role but different biochemical features

“…Interestingly, genes encoding Adi1 and Tad1 were found in all species with Um_Mtt1 ortholog while CadA‐encoding genes were found only in those expressing At_MttA (Table S2). These results suggest that the different IA biosynthetic pathways found in U. maydis and A. terreus may be connected to a different flux of substrates across the inner mitochondrial membrane catalyzed by Um_Mtt1 and At_MttA, as observed by transporter exchange experiments . The envisaged transport mechanism obtained in this paper can be helpful in the design of new metabolic engineering approaches with the aim of providing an increased amount of Mtt countersubstrates and thus improve itaconate production.…”

“…The coding sequences for Um_Mtt1, optimized for its expression in Escherichia coli , and that for At_MttA, optimized for its expression in the fungus U. maydis , were amplified by PCR using the plasmids pMK‐UM05079 (GeneArt) and pETEF‐At_MttA , respectively, as templates. The Bam HI /Hind III Um_Mtt1 and At_MttA coding fragments were cloned into the E. coli expression vectors pET21b and pQE30 (QIAGEN Inc., Hilden, Germany), respectively, and transformed into E. coli DH5α‐T1 cells (Invitrogen, Carlsbad, CA, USA).…”

“…Ustilago maydis and A. terreus genomes contain a gene cluster encoding enzymes for IA biosynthesis, a regulatory protein, a plasma membrane transporter, and a mitochondrial carrier, called Mtt1 and MttA, respectively [7]. Although physiological studies suggested that At_MttA and Um_Mtt1 had a similar biochemical function [5,6,10], their percentage of amino acid identity (32.4%) was much lower than that found between other U. maydis and A. terreus orthologs; for example, the identity of the two putative mitochondrial citrate/oxoglutarate carriers (Yhm2) in U. maydis and A. terreus is 58.6%, that of the ADP/ATP carrier is 73.4%, and that of the phosphate carrier is 61.8%. This pointed out the possibility that At_MttA might not be necessarily the ortholog of Um_Mtt1.…”

AnUstilago maydischassis for itaconic acid production without by‐products

“…In the previous experiment, the pH decreased from 6.5 to 3.68 for the ITA chassis. This likely affects itaconate productivity as U. maydis is not particularly tolerant to low pH (Hosseinpour Tehrani et al , ). In order to eliminate this potential pH inhibition, the buffer system was changed from MES to CaCO 3 and the substrate concentration was increased.…”

“…U. maydis represents a promising organism for application in industrial itaconate production. Recent efforts have achieved considerable improvements in itaconate production with Ustilago , including characterization and upregulation of the itaconate gene cluster and associated pathway (Geiser et al , ,c, ), engineering of transporters involved in itaconate production (Hosseinpour Tehrani et al , ), and morphological and metabolic engineering of the acid‐tolerant U. cynodontis (Hosseinpour Tehrani et al , ). However, the potpourri of metabolites naturally synthesized by U. maydis results in suboptimal specificity, productivity and yield of itaconate as a product.…”