Volume 24, Issue 12, Pages 1479-1489.e4 (December 2017) Fungal Cordycepin Biosynthesis Is Coupled with the Production of the Safeguard Molecule Pentostatin  Yongliang Xia, Feifei Luo, Yanfang Shang, Peilin Chen, Yuzhen Lu, Chengshu Wang  Cell Chemical Biology  Volume 24, Issue 12, Pages 1479-1489.e4 (December 2017) DOI: 10.1016/j.chembiol.2017.09.001 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 1479-1489. e4DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Chemical Structures of Adenosine Analogs and Phosphorylated Adenosines Identified in this Study COR, cordycepin; PTN, pentostatin; 3′-dI, 3′-deoxyinosine; 2′-C-3′-dA, 2′-carbonyl-3′-deoxyadenosine; 3′-AMP, adenosine-3′-monophosphate; 2′,3′-cAMP, 2′,3′-cyclic monophosphate. Cell Chemical Biology 2017 24, 1479-1489.e4DOI: (10.1016/j.chembiol.2017.09.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Prediction of the COR Biosynthetic Gene Cluster and Functional Verification (A) The highly syntenic gene cluster present in divergent fungal species. In A. nidulans, AN3332 could be a mis-annotated gene. Cm, C. militaris; An, A. nidulans; Ac, Ac. chrysogenum. (B) Schematic structure of the proteins. Cns1 belongs to the GFO/IDH/MOCA family of oxidoreductases or the MviM family of dehydrogenases. Cns2 is a member of the putative metal-dependent HDc family of phosphohydrolases. Cns3 contains two domains: an N-terminal (9–101 aa) nucleoside/nucleotide kinase (NK) and a C-terminal (681-851 aa) HisG family of ATP phosphoribosyltransferases. Cns4 is a member of the putative pleiotropic drug resistance (PDR) family of ATP-binding cassette (ABC) transporters. (C) Functional verification of the gene cluster for COR biosynthesis. The WT and null mutants were grown in SDB for 10 days, and the culture filtrates were collected for HPLC analysis. (D) Deletion of the cns1–cns2 homologous genes in A. nidulans (An) abolished COR (red peak) production when compared with that in the WT. (E) COR production in transgenic yeast cells. The cells of S. cerevisiae (Sc) were grown in synthetic medium supplemented with 20% galactose for 24 hr. After centrifugation, the supernatants were used for HPLC analysis. Vector, yeast cells transformed with the blank vector were used as a control. (F) HPLC verification of COR production after heterologous expression of the cns1–cns3 gene cassette in M. robertsii (Mr) enabled the transformants to produce COR (red peak). The fungi were grown in SDB for 7 days, and the culture filtrates were used for HPLC analysis. Cell Chemical Biology 2017 24, 1479-1489.e4DOI: (10.1016/j.chembiol.2017.09.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Protein Localization and Interaction Assays (A and B) Both Cns1 and Cns2 were individually fused with the GFP protein. Microscopic examinations indicated that both Cns1 (A) and Cns2 (B) were localized on the lipid droplets (stained with Nile red) in the mycelia of C. militaris. Scale bar, 5 μm. (C) Protein interaction assay. Yeast two-hybrid analysis indicating an interaction between Cns1 and Cns2 and a weak self-interaction of Cns2. The compound 3-amino-1,2,4-triazole (3-AT) was added to SD/-Trp/-Leu/-His (SD/-T/-L/-H) for a high-affinity binding assay involving the two proteins. PC, positive control; AD, activation domain of Gal4; BD, DNA-binding domain of Gal4. Cell Chemical Biology 2017 24, 1479-1489.e4DOI: (10.1016/j.chembiol.2017.09.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Fruiting-Body Induction, Identification, and Verification of PTN Production in C. militaris (A and B) The induction of FB formation on a rice medium (A) and silkworm pupae (B) for 60 days revealed no obvious phenotypic differences between the WT and various mutants of C. militaris. (C) HILIC-HPLC analysis of the WT and mutant FB samples. The presence of the blue peak for PTN is associated with the cns3 gene. The FB samples were harvested from the silkworm pupae 60 days after inoculation and then extracted with water for analysis. (D) LC-MS analysis of the extracted ion chromatogram (EIC) showing the detection of PTN in different samples. Cell Chemical Biology 2017 24, 1479-1489.e4DOI: (10.1016/j.chembiol.2017.09.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Identification and Verification of 3′-dI Production in C. militaris (A) HPLC analysis indicates that a peak labeled in blue with an asterisk could be detected in the FB samples from the WT grown on silkworm pupae but not on the rice medium. (B) HPLC analysis of the compound (identified as 3′-dI; blue peak with an asterisk) in the WT and various mutants of C. militaris. (C) MS analysis of 3′-dI. (D) NMR analytic data for structural identification of 3′-dI. Cell Chemical Biology 2017 24, 1479-1489.e4DOI: (10.1016/j.chembiol.2017.09.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Delineation of the Coupled Biosynthetic Pathway of COR and PTN Dual biosynthesis of COR and PTN is initiated by Cns3 from the precursor adenosine. The compound 3′-AMP is either formed through adenosine phosphorylation mediated by NK activity of Cns3 or from 2′,3′-cAMP during mRNA degradation. The conversion of 3′-AMP to 2′-C-3′-dA is catalyzed by Cns2, and will be further converted to COR by Cns1 through reduction reaction. In particular, the function of either Cns1 or Cns2 requires the formation of a protein complex. PTN production is mediated by the HisG domain of Cns3 to inhibit the activity of ADA to prevent COR deamination to 3'-dI. Cns4 potentially contributes to cell detoxification of COR to 3′-dI by outpumping PTN to release ADA inhibition. PRPP, phosphoribosyl pyrophosphate. Cell Chemical Biology 2017 24, 1479-1489.e4DOI: (10.1016/j.chembiol.2017.09.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Examination of Fungal Species that Can Jointly Produce COR and PTN (A) Phylogenetic analysis of different fungal species. The consensus neighbor-joining tree was generated using the β-tubulin gene DNA sequences. The species highlighted in red and solid red lines can produce COR while the species with the dashed lines cannot produce COR. (B) HPLC analysis of COR (red peak) production by different fungi. The fungi were grown in SDB for 10 days and the culture filtrates were used for HPLC analysis. The yeast cells were grown in the complete synthetic medium for 24 hr. (C) MS verification of COR production in different fungi by reference to the COR standard. m/z, [M + H]+. The peaks each highlighted with an asterisk are for COR. (D) LC-MS analysis of the extracted ion chromatogram (EIC) showing the production or non-production of PTN in different fungi. The fungi were grown in SDB for 10 days, and the mycelia were harvested for PTN extraction and LC-MC analysis. m/z, [M + H]+. Cell Chemical Biology 2017 24, 1479-1489.e4DOI: (10.1016/j.chembiol.2017.09.001) Copyright © 2017 Elsevier Ltd Terms and Conditions