Efficient production of a cyclic dipeptide (cyclo-TA) using heterologous expression system of filamentous fungus Aspergillus oryzae

Abstract

Background Cyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce. Results We isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an l-tryptophan-l-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from l-tryptophan and l-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production. Conclusions In this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.

Discovery of Uncommon Tryptophan-Containing Diketopiperazines from Aspergillus homomorphus CBS 101889 Using an Aspergillus nidulans Heterologous Expression System

“…Furthermore, the BGC is surrounded by genes that are clearly not related to secondary metabolite biosynthesis, suggesting that the cluster is small and is composed of only these four genes. BLASTP search results (Table S1) revealed that the closest NRPS homologues described in the scientific literature were the NRPSs from the echinulin family of BGCs, EchPS in Aspergillus ruber CBS 135680 (46% amino acid identity) 11 and CriC in Eurotium cristatum NWAFU-1 (46% identity) 16 as well as the NRPS from the acu-dioxomorpholine cluster in Aspergillus aculeatus ATCC 16872 (42% identity). 17 The acu-dioxomorpholine cluster in A. aculeatus ATCC 16872 contains a gene encoding a phenylpyruvate reductase, AdxB, that converts phenylpyruvate into phenyllactate, which the NRPS then combines with tryptophan.…”

A Flavin-Dependent Oxygenase Catalyzes Hydroxylation and Simultaneous Pyrrolidine Ring Formation in Protubonine Biosynthesis in Aspergillus ustus

“…21 The putative cluster consists of four genes, pboA−pboD, encoding an NRPS, two acetyltransferases, and one flavin-dependent monooxygenase (Figure 1A). The NRPS PboA (KIA75848) with two A-T-C modules (A: adenylation, T: thiolation, C: condensation) shows 44% amino acid sequence identity to CirC for the formation of cyclo-L-Trp-L-Ala. 22 Sequence analysis of the first A domain revealed 50% sequence identity with that for tryptophan activation in CirC. 22 The two putative acetyltransferases PboB (KIA75847) and PboC (KIA75846) share sequence identities of 37% with the N-acetyltransferase AnaAT and 39% with the O-acetyltransferase AusP, respectively.…”

“…The putative cluster consists of four genes, pboA – pboD , encoding an NRPS, two acetyltransferases, and one flavin-dependent monooxygenase (Figure A). The NRPS PboA (KIA75848) with two A-T-C modules (A: adenylation, T: thiolation, C: condensation) shows 44% amino acid sequence identity to CirC for the formation of cyclo - l -Trp- l -Ala . Sequence analysis of the first A domain revealed 50% sequence identity with that for tryptophan activation in CirC .…”

“…The NRPS PboA (KIA75848) with two A-T-C modules (A: adenylation, T: thiolation, C: condensation) shows 44% amino acid sequence identity to CirC for the formation of cyclo - l -Trp- l -Ala . Sequence analysis of the first A domain revealed 50% sequence identity with that for tryptophan activation in CirC . The two putative acetyltransferases PboB (KIA75847) and PboC (KIA75846) share sequence identities of 37% with the N -acetyltransferase AnaAT and 39% with the O -acetyltransferase AusP, respectively. , The flavin-dependent oxygenase PboD (KIA75845) displays low amino acid sequence identity to the known flavin-dependent oxygenases NotB (31%) and AspB (39%) in the biosynthesis of notoamide and asperlicin E, respectively. , This is consistent with the requirement for the biosynthesis of (−)-protubonine B.…”

High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae

“…[25]. To achieves efficient biopreparation of OA and its derivatives, the filamentous fungus A. oryzae was selected as the heterologous expression host, which can synthesize polyketides, terpenoids, nonribosomal peptides, and their post-modification products [26][27][28]. Through sequence similarity networks (SNNs) analysis, we found a gene herA in the genome of the basidiomycete H. erinaceus, that prediction involved in the synthesis of OA.…”