<i>C</i>-Methylation controls the biosynthetic programming of alternapyrone

Phakeovilay, Jaiyfungkhong; Imaram, Witcha; Vuttipongchaikij, Supachai; Bunnak, Waraporn; Lazarus, Colin M.; Wattana-Amorn, Pakorn

doi:10.1039/d2ob00947a

Cited by 3 publications

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“…Deconstruction studies of single domains of the PKS PksCT producing the highly (C2,C4,C6)-trimethylated pentaketide citrinin showed that in absence of the C-MT domain, the minimal PKS solely produced an unmethylated triketide pyrone [ 52 ]. Similar to our results on the LpaA C-MT, single mutations in the catalytic dyad of the C-MT of the PKS MtaltA in the alternapyrone biosynthetic pathway completely abolished product formation [ 83 ]. Since no intermediates are detectable in the lpaA C-MT mutant, C4-methylation occurs most likely at the thioester α-carbon following the first extension cycle and not on the fully elongated hexacosaketide.…”

Section: Discussionsupporting

confidence: 88%

A genetic tool to express long fungal biosynthetic genes

Kirchgaessner

Wurlitzer

Seibold

et al. 2023

Fungal Biol Biotechnol

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Background Secondary metabolites (SMs) from mushroom-forming fungi (Basidiomycota) and early diverging fungi (EDF) such as Mucoromycota are scarcely investigated. In many cases, production of SMs is induced by unknown stress factors or is accompanied by seasonable developmental changes on fungal morphology. Moreover, many of these fungi are considered as non-culturable under laboratory conditions which impedes investigation into SM. In the post-genomic era, numerous novel SM genes have been identified especially from EDF. As most of them encode multi-module enzymes, these genes are usually long which limits cloning and heterologous expression in traditional hosts. Results An expression system in Aspergillus niger is presented that is suitable for the production of SMs from both Basidiomycota and EDF. The akuB gene was deleted in the expression host A. niger ATNT∆pyrG, resulting in a deficient nonhomologous end-joining repair mechanism which in turn facilitates the targeted gene deletion via homologous recombination. The ∆akuB mutant tLK01 served as a platform to integrate overlapping DNA fragments of long SM genes into the fwnA locus required for the black pigmentation of conidia. This enables an easy discrimination of correct transformants by screening the transformation plates for fawn-colored colonies. Expression of the gene of interest (GOI) is induced dose-dependently by addition of doxycycline and is enhanced by the dual TetON/terrein synthase promoter system (ATNT) from Aspergillus terreus. We show that the 8 kb polyketide synthase gene lpaA from the basidiomycete Laetiporus sulphureus is correctly assembled from five overlapping DNA fragments and laetiporic acids are produced. In a second approach, we expressed the yet uncharacterized > 20 kb nonribosomal peptide synthetase gene calA from the EDF Mortierella alpina. Gene expression and subsequent LC–MS/MS analysis of mycelial extracts revealed the production of the antimycobacterial compound calpinactam. This is the first report on the heterologous production of a full-length SM multidomain enzyme from EDF. Conclusions The system allows the assembly, targeted integration and expression of genes of > 20 kb size in A. niger in one single step. The system is suitable for evolutionary distantly related SM genes from both Basidiomycota and EDF. This uncovers new SM resources including genetically intractable or non-culturable fungi.

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Section: Discussionsupporting

confidence: 88%

A genetic tool to express long fungal biosynthetic genes

Kirchgaessner

Wurlitzer

Seibold

et al. 2023

Fungal Biol Biotechnol

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“…The biosynthetic programming of highly methylated polyene α-pyrones has been investigated using the polyketide phytotoxin, alternapyrone 23 . 18 Heterologous expression of the mtalt gene cluster from Menisporopsis theobromae BCC 4162 in Aspergillus oryzae NSAR1 and subsequent mutations of catalytic residues of the C -methyltransferase domain significantly reduced the production of alternapyrone.…”

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Hill

Sutherland

2022

Nat. Prod. Rep.

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Anthranilic Acid Accumulation in Saccharomyces cerevisiae Induced by Expression of a Nonribosomal Peptide Synthetase Gene from Paecilomyces cinnamomeus BCC 9616

et al. 2022

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Heterologous expression of nrps33, a nonribosomal peptide synthetase gene, from Paecilomyces cinnamomeus BCC 9616 in Saccharomyces cerevisiae unexpectedly resulted in the accumulation of anthranilic acid, an intermediate in tryptophan biosynthesis. Based on transcriptomic and real‐time quantitative polymerase chain reaction (RT‐qPCR) results, expression of nrps33 affected the transcription of tryptophan biosynthesis genes especially TRP1 which is also the selectable auxotrophic marker for the expression vector used in this work. The product of nrps33 could inhibit the activity of Trp4 involved in the conversion of anthranilate to N‐(5′‐phosphoribosyl)anthranilate and therefore caused the accumulation of anthranilic acid. This accumulation could in turn result in down‐regulation of downstream tryptophan biosynthesis genes. Anthranilic acid is typically produced by chemical synthesis and has been used as a substrate for synthesising bioactive compounds including commercial drugs; our results could provide a new biological platform for production of this compound.

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C-Methylation controls the biosynthetic programming of alternapyrone

Abstract: Alternapyrone is a highly methylated polyene α-pyrone biosynthesised by a highly reducing polyketide synthase. Mutations of the catalytic dyad residues, H1578A/Q and E1604A, of the C-methyltransferase domain resulted in either...

Cited by 3 publications

References 24 publications

A genetic tool to express long fungal biosynthetic genes

A genetic tool to express long fungal biosynthetic genes

Hot off the Press

Anthranilic Acid Accumulation in Saccharomyces cerevisiae Induced by Expression of a Nonribosomal Peptide Synthetase Gene from Paecilomyces cinnamomeus BCC 9616

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