Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment

Verification of the genes involved in d-Ala conversion and Bmt biosynthesis. (A) HPLC analysis of CsA production by WT and different mutants with or without the addition of d-Ala. The inset shows the mass spectra detected for the CsA and ΔsimB samples. m/z, [M+H]⁺; ΔSimBΔ6009, ΔSimB ΔTINF06009 double mutant. (B) Quantification analysis of CsA production by WT and different mutants. The strains were grown in fructose CSN induction medium with or without the supplementation of d-Ala (at a final concentration of 20 mM) for 10 days. The mycelia were then harvested for CSN extraction. Values are means plus standard errors (SE) (error bars). DW, mycelium dry weight. (C) LC-MS analysis of the extracted ion chromatography (EIC) showing the production or nonproduction of Bmt by WT and mutant strains. m/z, [M+H]⁺. (D) Chemical structure of Bmt. (E) Supplementation of Bmt (at a final concentration of 85 μM) in the growth medium enabled the null mutants to produce CsA (peaks shown in blue).

Functional verification of the pathway-specific transcription factor SimL. (A) RT-PCR analysis of gene expression. The WT, ΔSimL, and WT::SimL strains were grown in fructose CSN induction medium for 10 days, and the mycelia were harvested for RNA extraction and gene expression analysis. TINF00183 and TINF07874 are indicated as 183 and 7874, respectively. β-Tub, β-Tubulin. (B) In silico analysis of the putative binding motif by the bZIP-type TF SimL. (C) Comparative quantification of CsA production. The WT and WT::SimL strains were grown in fructose CSN induction medium for 10 days, and the mycelia were harvested for cyclosporine extraction. There were three replicates for each sample. Values are means plus SE.

Proposed pathway for CsA biosynthesis. (A) Bmt biosynthesis by the PKS pathway. The PKS SimG domains include the following: β-ketoacyl synthase (KS), acyltransferase (AT), dehydrogenase (DH), methyltransferase (MT), enoylreductase (ER), ketoreductase (KR), acyl carrier protein (ACP), S-adenosylmethionine (SAM). The chemical structure of compounds b1 to b3 and Bmt are shown. (B) Schematic structure of NRPS SimA and the machinery of CsA biosynthesis. There are 11 modules of SimA, and each module contains the condensation (C), adenylation (A), thiolation (T), and/or N-methylation (NM) domains. The terminal C domain (C_T) is implicated in cyclization of the peptidyl chains to form CsA and its analogs. The cyclophilin SimC and exporter SimD may jointly contribute to cell tolerance of CSNs. Abu, aminobutyric acid; Sar, sarcocine; Nva, norvaline; MeLeu, methylleucine; aa, amino acids.

Antifungal effect of CsA production. (A) Fungal cocultivation tests. The WT and mutants of T. inflatum were inoculated on PDA plates in parallel for 3 days, and the strain of A. flavus was then inoculated between the two T. inflatum colonies for 4 days. (B) Schematic diagram showing how the colony edge distances between the WT T. inflatum and Aspergillus (D1) or between the T. inflatum mutant and Aspergillus (D2) were measured. (C) Comparison of the colony edge distances between strains. Values are means plus SE. Values that are significantly different (P < 0.001 by two-tailed t test) are indicated by a bar and three asterisks. (D) Comparison of the colony edge distances between WT and WT::SimL strains after different incubation times (7 or 14 days [d]). Values are means plus SE. ***, P < 0.001. (E) Representative phenotypes of a fungal pair after inoculation and 3 weeks of growth of T. inflatum.