Mating-type genes control sexual reproduction, conidial germination and virulence in Cochliobolus lunatus

Cochliobolus lunatus (anamorph: Curvularia lunata) is a major pathogenic fungus that causes the Curvularia leaf spot of maize. ClMAT1-1-1 and ClMAT1-2-1, the C. lunatus orthologs of Cochliobolus heterostrophus ChMAT1-1-1 and ChMAT1-2-1, were investigated in the present study to uncover their functions in C. lunatus. Southern blot analysis showed that these mating-type MAT genes exist in the C. lunatus genome as a single copy. ClMAT1-1-1 and ClMAT1-2-1 were knocked out and complemented to generate ΔClmat1-1-1 and ΔClmat1-2-1, ΔClmat1-1-1-C and ΔClmat1-2-1-C, respectively. The mutant strains had defective sexual development and failed to produce pseudothecia. There were no significant differences in growth rate or conidia production between the mutant and wild-type strains. However, the aerial mycelia and mycelial dry weight of ΔClmat1-1-1 and ΔClmat1-2-1 were lower than that of wild type, suggesting that MAT genes affect asexual development. ClMAT genes were involved in the responses to cell wall integrity and osmotic adaptation. ΔClmat1-2-1 had a lower conidial germination rate than the wild-type strain CX-3. The virulence of ΔClmat1-2-1 and ΔClmat1-1-1 was also reduced compared to the wild type. Complementary strains could restore all the phenotypes.

Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis

14α-hydroxylated steroids are starting materials for the synthesis of contraceptive and anti-inflammatory compounds in the steroid industry. A synthetic bacterial operon containing the cytochrome P450 CYP103168 and the reductase CPR64795 of the fungus Cochlioboluslunatus able to hydroxylate steroids has been engineered into a shuttle plasmid named pMVFAN. This plasmid was used to transform two mutants of Mycolicibacterium smegmatis named MS6039-5941 and MS6039 that accumulate 4-androstene-3,17-dione (AD), and 1,4-androstadiene-3,17-dione (ADD), respectively. The recombinant mutants MS6039-5941 (pMVFAN) and MS6039 (pMVFAN) were able to efficiently express the hydroxylating CYP system of C.lunatus and produced in high yields 14αOH-AD and 14αOH-ADD, respectively, directly from cholesterol and phytosterols in a single fermentation step. These results open a new avenue for producing at industrial scale these and other hydroxylated steroidal synthons by transforming with this synthetic operon other Mycolicibacterium strains currently used for the commercial production of steroidal synthons from phytosterols as feedstock.

Lipopeptide Epimers and a Phthalide Glycerol Ether with AChE Inhibitory Activities from the Marine-Derived Fungus Cochliobolus Lunatus SCSIO41401

A pair of novel lipopeptide epimers, sinulariapeptides A (1) and B (2), and a new phthalide glycerol ether (3) were isolated from the marine algal-associated fungus Cochliobolus lunatus SCSIO41401, together with three known chromanone derivates (4–6). The structures of the new compounds, including the absolute configurations, were determined by comprehensive spectroscopic methods, experimental and calculated electronic circular dichroism (ECD), and Mo2 (OAc)4-induced ECD methods. The new compounds 1–3 showed moderate inhibitory activity against acetylcholinesterase (AChE), with IC50 values of 1.3–2.5 μM, and an in silico molecular docking study was also performed.