E coli dh5α cells

Chemically competent E. coli DH5α cells (TransGen Biotech, Beijing, China) were used for amplification of pYES2 plasmid (Invitrogen). Growth and selection were carried out in Luria-Bertani medium supplemented with 100 mg/L ampicillin. Commercial S. cerevisiae strain INVSc1 (Invitrogen) was used as a host strain for the expression of STR3. Yeasts were cultivated at 30 °C in either a rich medium YPD (Yeast Extract Peptone Dextrose medium, containing 1% (w/v) yeast extract, 2% peptone, and 2% glucose), or a synthetic minimal defined medium SC (synthetic complete medium, containing 2% glucose or raffinose, 0.67% yeast nitrogen base without amino acids (Biosharp)). Uracil (SC-U) was omitted to obtain selective plates for growing pYES2 transformants.
Thirty isolated fungal strains were grown on potato-agar-dextrose slants, precultured in fermentation medium (sucrose, 35 g/L; peptone, 2.5 g/L; yeast extract, 2.5 g/L; KH₂PO₄ ·H₂O, 1 g/L; MgSO₄·7H₂O 0.5 g/L; Vitamin B1, 0.05 g/L), and then their ability to degrade Met was tested by inoculating them in fermentation medium added with 5 g/L Met. Met transamination product KMBA was only detected in the culture inoculated with Clonostachys rosea Tang 19, which was identified by ITS sequence (Genbank accession number: KT007105). This strain was selected for further study.

T4 DNA polymerase, which has strong 3′-5′ exonuclease, was as an exonuclease to generate complementary single-stranded DNA ends for IOEP to enhance its transformation efficiency. Briefly, in a 10 μL reaction, a total amount of 100 ng DNA fragment(s) obtained by IOEP was mixed in equal molar ratio in 1X T4 DNA polymerase buffer (Sangon) or 1X CutSmart buffer (NEB), 1 U of T4 DNA polymerase (Sangon) was added before incubating at 20 °C for 2 min to recess the DNA for generating of single-stranded regions at the DNA ends, followed by heating to 75 °C for 5 min to inactivate T4 DNA polymerase, then the reaction was cooled to 50 °C for 10 min to allow the annealing of complementary DNA ends^{16 (link),17 (link)}. An aliquot (5 μl) of each T4 DNA polymerase treated product was used to transform 50 μl of commercial chemically competent E. coli DH5α cells (Transgen Biotech) using standard protocol.

DNA cloning was performed with chemically competent E. coli DH5α cells (TransGen, Beijing). All plaque assays, bacterial migration tests, in vivo transcriptional assays, and SPACE experiments were performed using E. coli FM15 strain. This strain was derived from E. coli MG1655‐mCherry (Liu et al, 2019 (link)) by the following steps: (i) rendered F⁺ by conjugation with E. coli K12 ER2738 (NEB); (ii) deletion of the α‐fragment of LacZ gene with the λ Red recombineering system. The genotype of the resulting strain FM15 is F′ proA + B+ lacI^qΔ(lacZ)M15 zzf::Tn10 (TetR)/λ– ilvG– rfb‐50 rph‐1 attB::Kan^RΔ(lacZ)M15. Information about bacterial strains used in the study is listed in Appendix Table S1. Cells were cultured in Luria‐Bertani medium (LB: 10 g/l NaCl, 10 g/l tryptone, 5 g/l yeast extract). LB containing 0.25% (w/v) agar (Huankai, Guangdong) was used for SPACE experiments. Antibiotics including chloramphenicol (25 μg/ml), tetracycline (15 μg/ml), carbenicillin (50 μg/ml), and spectinomycin (100 μg/ml) were added where appropriate. For in vivo transcriptional activity measurement, M9 medium (6.78 g/l Na₂HPO₄, 3 g/l KH₂PO₄, 1 g/l NH₄Cl, 0.5 g/l NaCl) was used, with casamino acid (CAA) and glucose supplemented where necessary.