The Journal of Antibiotics (2020) 73:711–720 https://doi.org/10.1038/s41429-020-00362-6 SPECIAL FEATURE: ARTICLE The Laetiporus polyketide synthase LpaA produces a series of antifungal polyenes 1 1 1 1 Paula Sophie Seibold ● Claudius Lenz ● Markus Gressler ● Dirk Hoffmeister Received: 30 April 2020 / Revised: 19 July 2020 / Accepted: 21 July 2020 / Published online: 21 August 2020 © The Author(s) 2020. This article is published with open access Abstract The conspicuous bright golden to orange-reddish coloration of species of the basidiomycete genus Laetiporus is a hallmark feature of their fruiting bodies, known among mushroom hunters as the “chicken of the woods”. This report describes the identification of an eight-domain mono-modular highly reducing polyketide synthase as sole enzyme necessary for laetiporic acid biosynthesis. Heterologous pathway reconstitution in both Aspergillus nidulans and Aspergillus niger verified that LpaA functions as a multi-chain length polyene synthase, which produces a cocktail of laetiporic acids with a methyl- branched C26–C32 main chain. Laetiporic acids show a marked antifungal activity on Aspergillus protoplasts. Given the multiple products of a single biosynthesis enzyme, our work underscores the diversity-oriented character of basidiomycete 1234567890();,: 1234567890();,: natural product biosynthesis. Introduction functionally reconstituted in Aspergillus niger as hetero- logous host [4]. Non-terpenoid polyenes are a remarkable class of biolo- The intense, conspicuous orange color is the signature gically active basidiomycete natural products. These feature of specimens of the Laetiporus sulphureus species compounds with up to ten conjugated carbon–carbon complex, i.e., the “chicken of the woods” fungi. These are double bonds have been attributed to chemical defense: brown-rotting bracket mushrooms that have a European and piptoporic acid (Fig. 1), a polyene from Piptoporus aus- North American distribution and which are commonly traliensis with seven double bonds in conjugation deters found on oak, eucalypt, or willow trees. The coloration is fungivorous larvae from feeding on the fruiting bodies conferred by a blend of polyenes. In previous works, Weber [1, 2]. More recently, 18-methyl-19-oxoicosaoctaenoic et al. elucidated the structure of laetiporic acid A and its 2 acid and 20-methyl-21-oxodocosanonaenoic acid (Fig. 1) dehydro-3-deoxy derivative (Fig. 1)[5, 6], i.e., two non- of a taxonomically undescribed stereaceous basidiomy- terpenoid polyenes that possess a C26 main chain and cete, preliminarily referred to as BY1, were shown to share the 1-methyl-2-oxo-propylidene moiety with the inhibit pupation of larvae [3]. Biosynthetically, the above basidiomycete polyenes yet show ten conjugated respective compounds are polyketides and were instru- double bonds. The same authors detected even longer mental in gaining first insight in polyene biogenesis putative polyene products, laetiporic acids B and C (with in Basidiomycota as the BY1 multi-domain highly redu- C28 and C30 main chains, respectively) by liquid chroma- cing polyketide synthase (HR-PKS) PPS1 was tography and mass spectrometry. To learn more about the structural diversity of fungal polyenes, including the as yet largely uninvestigated bio- synthesis of non-aromatic polyketides in basidiomycetes, Supplementary information The online version of this article (https:// we built upon the above previous Laetiporus-related results. doi.org/10.1038/s41429-020-00362-6) contains supplementary material, which is available to authorized users. Herein, we describe the L. sulphureus enzyme LpaA as a multi-chain length polyene synthase. Heterologous pathway * Dirk Hoffmeister reconstitution in two independent recombinant lpaA- [email protected] expressing Aspergillus species led to a polyene profile 1 Pharmaceutical Microbiology, Friedrich Schiller University, similar to that found in L. sulphureus mycelium and fruiting Jena, Germany bodies. We demonstrate that LpaA, i.e., a single HR-PKS, 712 P. S. Seibold et al. O transformation were A. niger ATNT16ΔpyrGx24 [7]and HO O A. nidulans FGSC A4. Piptoporic acid A. niger transformants tPS01 and tPS02 were cultivated O on Aspergillus minimal medium agar plates (AMM) [8] HO O supplemented with 5 mM L-glutamine at 30 °C for 18-methyl-19-oxoicosaoctaenoic acid 5–7 days. Media for ATNT16ΔpyrGx24 were supple- O mented with 10 mM uridine. To produce laetiporic acids in HO O recombinant A. niger, transformants tPS01 (vector control) 20-methyl-21-oxodocosanonaenoic acid and tPS02 (polyene producer) were pre-cultivated overnight O OH in 30 Erlenmeyer flasks, each filled with 50 ml YPD med- HO O ium, at 30 °C and 140 rpm. The main culture was a 30 × 1 l Laetiporic acid A fermentation (AMM containing 200 mM D-glucose and O 50 mM L-glutamine), inoculated with 50 ml pre-culture HO O each. To induce lpaA expression, 30 mg l−1 doxycycline 2-Dehydro-3-deoxylaetiporic acid A was added after 18 h, and cultivation was continued Fig. 1 Structures of basidiomycete polyenes. For laetiporic acid A, the for additional 48 h. A. nidulans FGSC A4 and mutant fi – predominant isomer is shown (cis-con gured double bond C7 C8) tMG01 were maintained on AMM plates supplemented with 5 mM L-glutamine at 37 °C for 3 days. Plates for tMG01 were supplemented with 0.1 μg ml−1 pyrithiamine produces a series of compounds with C26–C32 main chain hydrobromide. To produce laetiporic acids in A. nidulans, lengths. the strains were cultivated in 100 l auto-inducing AMM, prepared with 200 mM ethanol and 10 mM D-glucose as carbon sources, at 30 °C and 140 rpm for 72 h. Details on Materials and methods fungal strains are given in Supplementary Table S1. Aspergillus conidia were harvested with 10 ml sterile water General experimental procedures and the suspension was filtered by a cell strainer (40 μm, EASYstrainer). Media were inoculated at a titer of 1 × 106 Semipreparative HPLC was performed on an Agilent 1260 conidia per milliliter. instrument, equipped with a diode-array detector, UHPLC- MS runs were done on an Agilent 1290 Infinity II chroma- cDNA cloning and construction of lpaA expression tograph, interfaced to an Agilent 6130 single quadrupole plasmids mass detector. HR-ESI-MS spectra were recorded in posi- tive mode on a Thermo Scientific Exactive Orbitrap L. sulphureus mycelium was grown in liquid YPD medium instrument. UV/Vis spectra were recorded from λ = at 20 °C and 140 rpm for 7 days, harvested, and ground 200–700 nm with diode array detectors connected with the under liquid nitrogen. RNA was isolated using the SV Total respective chromatographs, chromatograms were extracted RNA Isolation Kit (Promega). Residual genomic DNA was at λ = 450 nm. digested by Baseline-ZERO DNase (Biozym). Reverse transcription was carried out with anchored oligo-dT18 pri- Microbial strains and growth conditions mers and RevertAid Reverse Transcriptase (ThermoFisher). The lpaA coding sequence was PCR-amplified from the first Escherichia coli XL1-blue was used for routine cloning strand reaction, using the oligonucleotides oCL46 and and was cultivated in LB supplemented with 50 μg ml−1 oCL47 (Supplementary Table S2), using method A (Sup- carbenicillin, if required. Laetiporus sulphureus (s.l.) plementary Table S3). The gel-purified fragment was liga- JMRC SF012599 was provided by the Jena Microbial ted into pJET1.2 (Thermo) to yield plasmid pCL10 Resource Collection (JMRC) and was routinely main- (Supplementary Table S4) which was sequenced (GenBank tained on MEP medium (per liter: malt extract 30 g, accession number MT304701) to verify accurate amplifi- soytone peptone 3 g, agar 18 g) for 14 days at 25 °C. To cation and then served as template for subsequent PCRs. A produce laetiporic acids, L. sulphureus was cultivated on tag-free version (8190 bp) was expressed in A. nidulans, 150 YPD agar plates (per liter: yeast extract 10 g, soytone while in A. niger, a gene for a hexahistidine fusion protein peptone 20 g, D-glucose 20 g, agar 18 g) at 20 °C for was used (8229 bp). 14–21 days. Fruiting bodies of L. sulphureus were col- The lpaA coding sequence was PCR-amplified lected in Jena, Germany, on willow trees along the Saale (method B, Supplementary Table S3) from pCL10 using river, in September 2019. Aspergillus strains used for oMG459 and oMG460 (Supplementary Table S2) in order The Laetiporus polyketide synthase LpaA produces a series of antifungal polyenes 713 to introduce PacI sites at either end of the fragment. The A. Mycelia were ground to a fine powder and extracted six niger expression vector pSMX2-URA [7], allowing for times with methanol and subsequently twice with acetone doxycycline-inducible gene expression, was modified by (50 ml per 1 g dry biomass and extraction). PCR-mediated ligation (oligonucleotides oMG457/ Aspergillus and Laetiporus extracts were filtered through oMG458) to incorporate a PacI restriction site in the mul- cellulose round filters and evaporated to dryness. The dry tiple cloning site to create vector pPS01 (Supplementary residue was dissolved in 2 l water and repeatedly extracted Tables S2, S4). Both the insert and pPS01 were restricted with a total of 12 l ethyl acetate. The organic phase was with PacI and ligated to create the lpaA expression vector evaporated. The residue was dissolved in 400 ml methanol, pPS03. Plasmids pPS01 (vector) and pPS03 (lpaA expres- and 40 ml aliquots were subjected to size exclusion chro- sion plasmid) were used to transform A. niger. matography on Sephadex LH-20 (60 × 4 cm) with methanol To construct an alcohol-inducible lpaA expression vec- as eluent. Three fractions (FI-III) were obtained, containing tor, the vector backbone of plasmid pMD03 [9] as well as laetiporic acid (LA)-A, LA-B in F-I, LA-C in F-II, and the lpaA coding sequence inserted in plasmid pCL10 were LA-D and traces of other derivatives in F-III. All fractions amplified (method B, Supplementary Table S3) using oli- were subjected to reversed phase semi-preparative HPLC, gonucleotides oMG468/oMG469 and oMG471/oMG472, using methods C (F-I and F-II) and D (F-III) (Supplemen- respectively.