The molecular steps of citrinin biosynthesis in fungi

Heterologous expression of the citrinin polyketide synthase, CitS, plus the tailoring enzymes CitA–CitE from Monascus ruber has fully elucidated the biosynthetic pathway to citrinin for the first time, showing relationships to tropolone, azaphilone and sorbicillinoid biosynthetic pathways in fungi.


Gene
Size (

Comparision of citrinin biosynthesis gene clusters from M. ruber, M. aurantiacus, M. purpureus and Penicillium expansum
The genes inclosed by the red dotted line between Monascus spp. and P. expansum are highly homologous. The nucleotide identity of these marked genes reaches 99% among these three different Monascus strains, and their nucleotide identity with P. expansum reaches 81%. Note that the orf4 gene in M. aurantiacus and M. purpureus was annotated as oxidoreductase according to the published papers, actually it is a homolog of mrl1. Histidine phosphatase --ctnF -mrr3 Unknown protein Carbonic anhydrase --orf8 -mrr6 Unknown protein --ctnG -mrr7 Reductase Transporter --ctnI - 7 was isolated by rapid mass-directed reverse-phase purification. Solvent was evaporated, NMR solvent (DMSO-d 6 ) was added and NMR spectra gained as quickly as possible. However spectra always contain traces of 6 and other degradation products. 7 was identified as the only component of the mixture consistent with the measured HRMS data indicating a molecular formula of C 15 H 19 NO 6 . In particular HMBC correlations confirmed the skeleton as the same as 6, and also the location of the CH 2 CH 2 OH group (HMBC from H-1 to C-13).

Semi-Preparative LCMS and compound purification.
Purification of compounds was generally achieved using a Waters mass-directed autopurification system comprising of a Waters 2767 autosampler, Waters 2545 pump system, a Phenomenex Kinetex Axia column (5µ, C 18 , 100 Å, 21.2 × 250 mm) equipped with a Phenomenex Security Guard precolumn (Luna C 5 300 Å) eluted at 20 mL/min at ambient temperature. Solvent A, HPLC grade H 2 O + 0.05% formic acid; Solvent B, HPLC grade CH 3 CN + 0.045% formic acid. The post-column flow was split (100:1) and the minority flow was made up with HPLC grade MeOH + 0.045% formic acid to 1 mL·min-1 for simultaneous analysis by diode array (Waters 2998), evaporative light scattering (Waters 2424) and ESI mass spectrometry in positive and negative modes (Waters SQD-2). Detected peaks were collected into glass test tubes. Combined tubes were evaporated under a flow of dry N 2 gas, weighed, and residues dissolved directly in NMR solvent for NMR analysis.

General techniques for DNA manipulation
Polymerase chain reactions were performed with PrimeSTAR ® HS DNA Polymerase (TaKaRa Bio Inc.). Restriction digests were carried out according to the manufacturer's protocols (NEB, Fermentas, Promega). The primers used to amplify each fragment were synthesized by Sigma, and are listed in Table S2.

Cloning procedures for heterologous expression of citrinin genes in A. oryzae NSAR1
8.4.1 exp. 1: The citS gene was amplified from M. ruber M7 genomic DNA as four fragments using primers pks-1-F/pks-1-R, pks-2-F/pks-2-R, pks-3-F/pks-3-R, pks-4-F/pks-4-R according to the strategy described in Fig. S8.1. These fragments with overlaps with each other were reassembled by homologous recombination in S. cerevisiae with NotI and AscI cut pE-YA vector and shuttled back into E. coli to create pE-YA·citS (the only 56-bp intron in citS based on bioinformatics analysis was removed). The cloned citS gene with the only 56-bp intron removed was then transferred into pTYGS·arg by Gateway LR recombination (Invitrogen) to create pTYGS·arg·citS. Thus, the citS gene was placed under the control of P amyB and T amyB . Transformation of A. oryzae NSAR1 with this plasmid yielded 8 transformants.

exp. 2:
The long 942-bp mrl1 gene was amplified from M. ruber M7 cDNA as a single fragment using primers mrl1-F-long and mrl1-R which flanked the PCR product with sequences overlapping the 3´ terminal of alcohol dehydrogenase promoters (P adh ) and the 5´ terminal of enolase terminator (T eno ). This fragment was cloned into AscI-cut pTYGS·arg·citS under the control of P adh and T eno by homologous recombination in S. cerevisiae and shuttled back into E. coli to create pTYGS·arg·citS·mrl1·long. Transformation of A. oryzae NSAR1 with this plasmid yielded 13 transformants.

exp. 2´:
The short mrl1 gene with first 156 bp nucleotides removed (define as mrl1 gene in this MS) was amplified from M. ruber M7 cDNA as a single fragment using primers mrl1-F and mrl1-R which flanked the PCR product with sequences overlapping the 3´ terminal of P adh and the 5´ terminal of T eno . The same method was used to create pTYGS·arg·citS·mrl1. Transformation of A. oryzae NSAR1 with this plasmid yielded 10 transformants.

exp. 3:
The mrl2 gene was amplified from M. ruber M7 cDNA as a single fragment using primers mrl2-F and mrl2-R-b which flanked the PCR product with sequences overlapping P adh and T eno . This fragment was cloned into AscI-cut pTYGS·ade under the control of P adh and T eno by homologous recombination in S. cerevisiae and shuttled back into E. coli to create pTYGS·ade·mrl2. Transformation of A. oryzae NSAR1 harboring pTYGS·arg·citS·mrl1 with this plasmid yielded 10 transformants.

exp. 4:
The mrl2 and mrl4 genes were amplified from M. ruber M7 cDNA as single fragment using primers mrl2-F/mrl2-R and mrl4-F/mrl4-R-b respectively. The cloned mrl2 gene was 5´ flanked by a 30-bp nucleotides overlap with P adh and 3´ flanked by a 30-bp nucleotides overlap with the alcohol dehydrogenase terminator (T adh ). The cloned mrl4 gene was 5´ flanked by a 30-bp nucleotides overlap with the glyceraldehyde-3-phosphate dehydrogenase promoter (P gpdA ) and 3´ flanked by a 30-bp nucleotides overlap with T eno . These two fragments together with a patch fragment T adh -P gpdA (gel purified from AscI digested pTYGS·ade vector) were cloned into AscI-cut pTYGS·ade by homologous recombination in S. cerevisiae and shuttled back into E. coli to create pTYGS·ade·mrl2·mrl4. Transformation of A. oryzae NSAR1 harboring pTYGS·arg·citS·mrl1 with this plasmid yielded 22 transformants.

exp. 5:
The mrl7 gene was amplified from M. ruber M7 cDNA as a single fragment using primers mrl7-F and mrl7-R which flanked the PCR product with sequences overlapping the upstream and downstream of NotI and AscI cut pE-YA vector. This fragment was reassembled by homologous recombination in S. cerevisiae with NotI and AscI cut pE-YA vector and shuttled back into E. coli to create pE-YA·mrl7. The cloned mrl7 gene was then transferred into pTYGS·ade·mrl2 by Gateway LR recombination to create pTYGS·ade·mrl2·mrl7. Thus, the mrl7 gene was placed under the control of P amyB and T amyB . Transformation of A. oryzae NSAR1 harboring pTYGS·arg·citS·mrl1 with this plasmid yielded 16 transformants.

exp. 6:
The mrl2 and mrl6 genes were amplified from M. ruber M7 cDNA as single fragment using primers mrl2-F/mrl2-R and mrl6-F-b/mrl6-R respectively. The cloned mrl2 gene was 5´ flanked by a 30-bp nucleotides overlap with P adh and 3´ flanked by a 30-bp nucleotides overlap with the T adh . The cloned mrl6 gene was 5´ flanked by a 30-bp nucleotides overlap with the P gpdA and 3´ flanked by a 30-bp nucleotides overlap with T eno . These two fragments together with a patch fragment T adh -P gpdA (gel purified from AscI digested pTYGS·ade vector) were cloned into AscI-cut pTYGS·ade by homologous recombination in S. cerevisiae and shuttled back into E. coli to create pTYGS·ade·mrl2·mrl6. Transformation of A. oryzae NSAR1 harboring pTYGS·arg·citS·mrl1 with this plasmid yielded 27 transformants.

Gene knock-out procedures, schemes in M. ruber M7
8.5.1 exp. 11: gene knock-out of citS. The citS gene knock-out strategy was designed to insert the 1.2 kb neomycin phosphotransferase resistance gene (neo) which was amplified by PCR from pKN1 using the primers G418F and G418R into the internal of citS. A 721 bp of 5´ fragment and a 954 bp of 3´ fragment of citS were amplified from M. ruber M7 genomic DNA using primers pksCT-5F/pksCT-5R and pksCT-3F/pksCT-3R respectively and served as homologous arms for recombination event (Fig. S8.2A). These three fragments were reassembled by homologous recombination in S. cerevisiae with NotI and AscI cut pE-YA vector and shuttled back into E. coli to create pE-YA-citS-KO. Then both pE-YA-citS-KO and pCAMBIA3300 were digested with SalI and HindIII and ligated by T4 DNA ligase to create pC3300-citS. This plasmid was transformed into Agrobacterium tumefaciens EHA105 afterwards. Subsequently, A. tumefaciens mediated transformation (ATMT) was performed with MrΔku80 strain (a ku80 knock-out mutant of M. ruber M7). A citS knock-out mutant (ΔcitS::neo) was confirmed by analytical PCR (Fig. S8.2B). A 1410 bp fragment was expected to amplify from the WT (MrΔku80) genomic DNA using primers pksCT-VF and pksCT-VR, while nothing was obtained from the citS knock-out mutant. A 1.2 kb fragment of the neo gene could be amplified using primers G418F and G418R from the citS knock-out mutant, while no PCR product got from the WT sample. Lanes 1/3 and 2/4 were PCR results to amplify partial citS gene and neo with primers pksCT-VF/pksCT-VR and G418F/G418R respectively. Lane 1 and 2 were using gDNA of ΔcitS::neo mutant as template, lane 3 and 4 were using gDNA of MrΔku80 strain as template. M: NEB 1 kb DNA ladder. 8.5.2 exp. 12: gene knock-out of mrl1. The similar strategy was used to inactivate mrl1. A 611 bp of 5´ fragment and a 594 bp of 3´ fragment of mrl1 were amplified from M. ruber M7 genomic DNA using primers mrl1-5F/mrl1-5R and mrl1-3F/mrl1-3R respectively and served as homologous arms for recombination event (Fig. S8.3A). These two fragments together with the neo gene were reassembled as a knock-out cassette by homologous recombination in S. cerevisiae with NotI and AscI cut pE-YA vector and shuttled back into E. coli to create pE-YA-mrl1-KO. KpnI and XbaI digestion and T4 DNA ligation of pE-YA-mrl1-KO and pCAMBIA3300 yielded pC3300-mrl1. The same ATMT method was used to obtain mrl1 knock-out mutant (Δmrl1::neo). Analytical PCR verified the homologous recombination at the right position in the mrl1 knock-out mutant (Fig. S8.3B). A 633 bp fragment was expected to amplify from the WT (MrΔku80) genomic DNA using primers mrl1-VF and mrl1-VR, while nothing was obtained from the mrl1 knock-out mutant. A 1.2 kb fragment of the neo gene could be amplified using primers G418F and G418R from the mrl1 knock-out mutant, while no PCR product got from the WT sample. Lanes 1/3 and 2/4 were PCR results to amplify partial mrl1 gene and neo with primers mrl1-VF/mrl1-VR and G418F/G418R respectively. Lane 1 and 2 were using gDNA of Δmrl1::neo mutant as template, lane 3 and 4 were using gDNA of MrΔku80 strain as template. M: NEB 1 kb DNA ladder. 8.5.3 exp. 13: gene knock-out of mrl2. The similar strategy was used to inactivate mrl2. A 520 bp of 5´ fragment and a 489 bp of 3´ fragment of mrl2 were amplified from M. ruber M7 genomic DNA using primers mrl2-5F/mrl2-5R and mrl2-3F/mrl2-3R respectively and served as homologous arms for recombination event (Fig. S8.4A). These two fragments together with the neo gene were reassembled as a knock-out cassette by homologous recombination in S. cerevisiae with NotI and AscI cut pE-YA vector and shuttled back into E. coli to create pE-YA-mrl2-KO. KpnI and XbaI digestion and T4 DNA ligation of pE-YA-mrl2-KO and pCAMBIA3300 yielded pC3300-mrl2. The same ATMT method was used to obtain mrl2 knock-out mutant (Δmrl2::neo). Analytical PCR verified the homologous recombination at the right position in the mrl2 knock-out mutant (Fig. S8.4B). Briefly, part of mrl2 (537 bp) could only be amplified from the WT and neo could only be amplified from the mrl2 knock-out mutant using primers mrl2-VF/mrl2-VR and G418F/G418R respectively, while the other two situations yielded no PCR products. Lanes 1/3 and 2/4 were PCR results to amplify partial mrl2 gene and neo with primers mrl2-VF/mrl2-VR and G418F/G418R respectively. Lane 1 and 2 were using gDNA of Δmrl2::neo mutant as template, lane 3 and 4 were using gDNA of MrΔku80 strain as template. M: NEB 1 kb DNA ladder. 8.5.4 exp. 14: gene knock-out of mrl4. The similar strategy was used to inactivate mrl4. A 839 bp of 5´ fragment and a 520 bp of 3´ fragment of mrl4 were amplified from M. ruber M7 genomic DNA using primers mrl4-5F/mrl4-5R and mrl4-3F/mrl4-3R respectively and served as homologous arms for recombination event (Fig. S8.5A). These two fragments together with the neo gene were reassembled as a knock-out cassette by homologous recombination in S. cerevisiae with NotI and AscI cut pE-YA vector and shuttled back into E. coli to create pE-YA-mrl4-KO. XbaI and HindIII digestion and T4 DNA ligation of pE-YA-mrl4-KO and pCAMBIA3300 yielded pC3300-mrl4. The same ATMT method was used to obtain mrl4 knock-out mutant (Δmrl4::neo). Analytical PCR verified the homologous recombination at the right position in the mrl4 knock-out mutant (Fig. S8.5B). Briefly, part of mrl4 (740 bp) could only be amplified from the WT and neo could only be amplified from the mrl4 knock-out mutant using primers mrl4-VF/mrl4-VR and G418F/G418R respectively, while the other two situations yielded no PCR products. Figure S8.5 Scheme to knock-out mrl4 (A) and PCR verification of Δmrl4::neo mutant (B) Lanes 1/3 and 2/4 were PCR results to amplify partial mrl4 gene and neo with primers mrl4-VF/mrl4-VR and G418F/G418R respectively. Lane 1 and 2 were using gDNA of Δmrl4::neo mutant as template, lane 3 and 4 were using gDNA of MrΔku80 strain as template. M: NEB 1 kb DNA ladder. 8.5.5 exp. 15: gene knock-out of mrl6. The similar strategy was used to inactivate mrl6. A 542 bp of 5´ fragment and a 576 bp of 3´ fragment of mrl6 were amplified from M. ruber M7 genomic DNA using primers mrl6-5F/mrl6-5R and mrl6-3F/mrl6-3R respectively and served as homologous arms for recombination event (Fig. S8.6A). These two fragments together with the neo gene were reassembled as a knock-out cassette by homologous recombination in S. cerevisiae with NotI and AscI cut pE-YA vector and shuttled back into E. coli to create pE-YA-mrl6-KO. KpnI and XbaI digestion and T4 DNA ligation of pE-YA-mrl6-KO and pCAMBIA3300 yielded pC3300-mrl6. The same ATMT method was used to obtain mrl6 knock-out mutant (Δmrl6::neo). One mutant was confirmed by analytical PCR (Fig. S8.6B). Part of mrl6 (829 bp) could only be amplified from the WT and neo could only be amplified from the mrl6 knock-out mutant using primers mrl6-VF/mrl6-VR and G418F/G418R respectively, while the other two situations yielded no PCR products. Lanes 1/3 and 2/4 were PCR results to amplify partial mrl6 gene and neo with primers mrl6-VF/mrl6-VR and G418F/G418R respectively. Lane 1 and 2 were using gDNA of Δmrl6::neo mutant as template, lane 3 and 4 were using gDNA of MrΔku80 strain as template. M: NEB 1 kb DNA ladder. 8.5.6 exp. 16: gene knock-out of mrl7. The similar strategy was used to inactivate mrl7. A 878 bp of 5´ fragment and a 536 bp of 3´ fragment of mrl7 were amplified from M. ruber M7 genomic DNA using primers mrl7-5F/mrl7-5R and mrl7-3F/mrl7-3R respectively and served as homologous arms for recombination event (Fig. S8.7A). These two fragments together with the neo gene were reassembled as a knock-out cassette by homologous recombination in S. cerevisiae with NotI and AscI cut pE-YA vector and shuttled back into E. coli to create pE-YA-mrl7-KO. KpnI and XbaI digestion and T4 DNA ligation of pE-YA-mrl7-KO and pCAMBIA3300 yielded pC3300-mrl7. The same ATMT method was used to obtain mrl7 knock-out mutant (Δmrl7::neo). One mutant was confirmed by analytical PCR (Fig. S8.7B). Part of mrl7 (491 bp) could only be amplified from the WT and neo could only be amplified from the mrl7 knock-out mutant using primers mrl7-VF/mrl7-VR and G418F/G418R respectively, while the other two situations yielded no PCR products. Figure S8.7 Scheme to knock-out mrl7 (A) and PCR verification of Δmrl7::neo mutant (B) Lanes 1/3 and 2/4 were PCR results to amplify partial mrl7 gene and neo with primers mrl7-VF/mrl7-VR and G418F/G418R respectively. Lane 1 and 2 were using gDNA of Δmrl7::neo mutant as template, lane 3 and 4 were using gDNA of MrΔku80 strain as template. M: NEB 1 kb DNA ladder.

Cloning procedures to mutate catalytic triad in mrl1 protein
8.8.1 exp. 23: construction of pTYGS·arg·citS·mrl1·S 122 A (citS, mrl1 with mutation of S 122 to A). The mrl1 gene was amplified from M. ruber cDNA as two fragments using primers mrl1-F/mrl1-R-519 and mrl1-F-490/mrl1-R. The cloned two fragments were used to do yeast recombination in S. cerevisiae with AscI-cut pTYGS·arg·citS and shuttled back into E. coli to create pTYGS·arg·citS·mrl1·S 122 A. Transformation of A. oryzae M-2-3 with this plasmid yielded 11 transformants.
The MPM fermentation broth was filtered to remove the mycelium and acidified to pH 4.0 using 37% HCl and then transferred into a separating funnel. An equal volume of ethyl acetate was added into the separating funnel and shaken vigorously. The mixture was allowed to stand to separate the layers. After taking out the organic layer, the rest water layer was extracted with equal volume ethyl acetate again. The organic phase from two extractions was dried (MgSO 4 ), filtered and evaporated to dryness. The crude extract was dissolved in 2 mL HPLC grade MeOH and analysed by LC-MS.

M. ruber and mutants
The wild-type M. ruber M7 or MrΔku80 strains were grown on PDA (2.4%(w/v) potato dextrose broth, 1.5% (w/v) agar) plates for 7-10 days at 28 °C for spores production. The mutants obtained through ATMT method were selected on PDA plates with 15 µg/mL G418. For extraction, the spores were collected from 10 days old growing MrΔku80 or mutants strains and inoculated in each 100 mL PDB (2.4%(w/v) potato dextrose broth) liquid media contained in 500 mL Erlenmeyer flask. The spores were allowed to grow in the liquid culture for 10 days on shakers at 160 rpm at 28 °C. The extraction method of PDB fermentation broth was the same with MPM fermentation broth.

Transformation of A. oryzae NSAR1
Plasmid DNA for fungal transformation was prepared using Fermentas Miniprep kits. A. oryzae NSAR1 or A. oryzae NSAR1 harboring pTYGS·arg·citS·mrl1 strains were grown on DPY plates for 10 days. Spores washed by 4 mL sterile water were inoculated into 100 mL DPY liquid medium and cultivated for 2 day at 28 °C. Collect the mycelia on a sterile filter paper (autoclaved with a filter funnel) and wash with sterile water, then 0.8 M NaCl. Put the mycelia in a sterile falcon centrifuge tube. Add 10 mL of filter-sterilized TF buffer 1 (10 mg/mL Yatalase (Takara), 0.6 M (NH 4 ) 2 SO 4 , 50 mM maleic acid, pH 5.5) and incubate at 30 °C, 100rpm for 2 hours. Filter the protoplasting solution through a syringe with glasswool inside. Centrifuge the filtrate at 3000 rpm for 10 min. Wash the pelleted protoplasts with 15 mL TF buffer 2 (1.2 M sorbitol, 50 mM CaCl 2 , 35 mM NaCl, 10 mM Tris HCl pH 7.5). Resuspend the protoplasts in TF buffer 2 to final concentration of 2.5 ×10 8 /mL. Put 0.2 mL portions into Falcon tubes. Add 20 µL plasmid DNA and place on ice for 30 min. Add 250 µL, 250 µL and 850 µL TF buffer 3 (PEG 4000 (60% w/v), 50 mM CaCl 2 , 10 mM Tris HCl pH 7.5), mix well gently and place at room temperature for 20 min. 10 mL soft agar (0.8% agar containing 5% NaCl) was added to the transformation mixtures, and then poured onto MPM selection plates supplemented with sorbitol (1 M) and incubated at 28˚C for 5-7 days.

Transformation of A. oryzae M-2-3
Plasmid DNA for fungal transformation was prepared using Fermentas Miniprep kits. A. oryzae M-2-3 was grown on MEA plates for 10 days. Spores washed by 4 mL sterile water were inoculated into 100 mL GNB liquid medium (2% glucose, 1% nutrient broth number 2 (from Thermo Scientific)) and cultivated for 2 day at 28 °C. Collect the mycelia on a sterile filter paper (autoclaved with a filter funnel) under vacuum and wash with sterile water, then 0.8 M NaCl. Put the mycelia in a sterile falcon centrifuge tube. Add 10 mL of filter-sterilized protoplasting solution (20 mg/mL lysing enzyme (Sigma L1412), 10 mg/mL driselase (Sigma D9515), 0.8 M NaCl, 10 mM Na phosphate buffer pH 6) and incubate at 30 °C, 100rpm for no longer than 3 hours. Filter the protoplasting solution through a syringe with glasswool inside. Centrifuge the filtrate at 3000 rpm for 10 min. Wash the pelleted protoplasts once with 0.8 M NaCl (ca. 15 mL) and then once with Solution 1 (0.8 M NaCl, 10 mM CaCl 2 , 50 mM Tris HCl pH 7.5). Resuspend the protoplasts in Solution 1 to final concentration of 2.5 ×10 8 /mL and add 1/5 volume of Solution 2 (PEG 4000 (60% w/v) in Solution 1 but 50 mM CaCl 2 ). Put 0.2 mL portions into Falcon tubes. Add 20 µL plasmid DNA and place on ice for 30 min. Add 1 mL of Solution 2, mix well gently and place at room temperature for 20 min. 10 ml soft agar (0.8% agar containing 5% NaCl) was added to the transformation mixtures, and then poured onto Czapek-Dox plates supplemented with sorbitol (1 M) and incubated at 28˚C for 5-7 days.

Transformation of S. cerevisiae for yeast recombination
A single colony of S. cerevisiae YPH499 was inoculated into a 10 mL YPAD (1% (w/v) yeast extract, 2% (w/v) bactotryptone, 2% (w/v) glucose, 0.04% (w/v) adenine sulphate) starter culture and grown overnight at 28 °C with shaking at 200 rpm. The starter culture was then added to 40 mL of YPAD in a 250 mL flask and incubated at 28 °C with shaking at 200 rpm for 5 hours, after which the culture was centrifuged at 3000 g for 5 min and the supernatant discarded. The cells were washed with 25 mL sterile H 2 O and the centrifugation repeated, the pellet was then resuspended in 1 mL 0.1 M LiOAc and transferred to a 1.5 mL microfuge tube. The cells were then pelleted at 14500 rpm for 15 sec and the supernatant discarded, after which the cells were resuspended in 400 µL 0.1 M LiOAc. For each transformation to be performed 50 µL of the suspension was transferred to a new 1.5 mL microfuge tube and pelleted again at 14500 rpm for 15 sec and the supernatant discarded. 240 µL of PEG solution (50% (w/v) polyethylene glycol 3350), 36 µL 1 M LiOAc, 20 µl SS-DNA (Salmon Sperm DNA, 5 mg/ml in TE buffer, Rockland MB-103-0025) and up to 34 µL of DNA were added to the pelleted cells in order. Approximately 0.5 -1 µg of each DNA fragment was added, with linear DNA fragments to be joined containing at least 30 bp overlap. Cells were resuspended in the transformation mixture by vortexing, and incubated at 30 °C for 30 min and then 42 °C for 30 min. The cells were pelleted at 6000 rpm for 15 sec then gently resuspended in 1 mL of sterile water. 200 µL aliquots were spread on SM-URA plates (0.17% (w/v) yeast nitrogen base, 0.5% (w/v) ammonium sulphate, 2% (w/v) glucose, 0.077% (w/v) complete supplement mixture minus uracil (Q-biogene), 1.5% (w/v) agar) and incubated at 28 °C for 3-4 days until colonies appeared.   Figure S9.2 Biosynthetic route to Citrinin proposed by Li and coworkers. 2 N.b. ctnB is also incorrectly referred to as encoding an oxidoreductase in this paper.