Biosynthesis of Alkylcitric Acids in Aspergillus niger Involves Both Co-localized and Unlinked Genes

Abstract

Filamentous fungi are an abundant source of bioactive secondary metabolites (SMs). In many cases, the biosynthetic processes of SMs are not well understood. This work focuses on a group of SMs, the alkylcitric acids, each of which contains a saturated alkyl "tail," and a citrate-derived "head." We initially identified their biosynthetic gene cluster and the transcriptional regulator (akcR) involved in the biosynthesis of alkylcitrates in the filamentous fungus Aspergillus niger by examining the functional annotation of SM gene clusters predicted from genomic data. We overexpressed the transcription regulator gene akcR and obtained from one liter of culture filtrate 8.5 grams of extract, which are represented by seven alkylcitric acids as determined by NMR. Hexylaconitic acid A comprised 94.1% of the total production, and four of the seven identified alkylcitrates have not been reported previously. Analysis of orthologous alkylcitrate gene clusters in the Aspergilli revealed that in A. oryzae and A. flavus an in-cluster gene displays sequence similarity to cis-aconitate decarboxylase, the orthologue of which in A. niger, NRRL3_00504, is located on a different chromosome. Overexpression of the A. niger NRRL3_00504 and akcR genes together shifted the profile of alkylcitrates production from primarily hexylaconitic acids to mainly hexylitaconic acids, suggesting that NRRL3_00504 encodes an enzyme with hexyl aconitate decarboxylase activity. We also detected two additional, previously unreported, alkylcitric acids in the double overexpression strain. This study shows that phylogenomic analysis together with experimental manipulations can be used to reconstruct a more complete biosynthetic pathway in generating a broader spectrum of alkylcitric compounds. The approach adopted here has the potential of elucidating the complexity of other SM biosynthetic pathways in fungi.

Caprylic acid enhances hydroxyhexylitaconic acid production in Aspergillus niger S17‐5

“…Previously, we reported that 9-HHIA and 10-HHIA produced by A. niger S17-5 have cytotoxic and polymer building block properties (Aso et al 2020;Sano et al 2020). Related studies have predicted that 9-HHIA and 10-HHIA are synthesized by the condensation of octanoyl-CoA, which is derived from caprylic acid, with oxaloacetic acid (Guti errez et al 2002;Cao et al 2019;Palys et al 2020). However, there have been no studies regarding the effects of the addition of caprylic acid on 9-HHIA and 10-HHIA production.…”

“…2019; Palys et al . 2020). However, there have been no studies regarding the effects of the addition of caprylic acid on 9‐HHIA and 10‐HHIA production.…”

“…This revealed that the production ratio of 9-HHIA : 10-HHIA is approximately 1 : 3. Palys et al (2020) reported that A. niger PY11 overexpressing cadA and akcR produced 9-HHIA and 10-HHIA at concentrations of 0Á8 and 1Á6 g l À1 respectively. The production ratio shown by the recombinant A. niger has a similar tendency to that observed in this study.…”

“…However, no enhancement effect of 9-HHIA or 10-HHIA production by the addition of palmitic acid was observed. A previous study suggested that 9-HHIA and 10-HHIA are synthesized by the condensation of octanoyl-CoA with oxaloacetic acid (Palys et al 2020). Therefore, we performed the same experiment with the addition of oxaloacetic acid instead of caprylic acid.…”

“…Given these findings, alkylitaconic acids are generally synthesized by the condensation of acyl-CoAs derived from fatty acids with oxaloacetic acid via similar biosynthetic pathways. More recently, Palys et al (2020) predicted a biosynthetic pathway for various hexylitaconic acid derivatives, including 9-HHIA and 10-HHIA, by heterologous overexpression of the cis-aconitate decarboxylase gene (cadA) and the alkylcitrates biosynthetic transcriptional regulator gene (akcR) in A. niger PY11. They suggested that hexylitaconic acid is first synthesized from octanoyl-CoA and oxaloacetic acid, then hydroxylated by endogenous P450 monooxygenase(s), leading to the generation of 9-HHIA and 10-HHIA.…”