Constructing and culturing P1-P6-P7 point mutants
The second of the 2 plasmids encoding theP1 -P6 -P7 triketide lactone synthase, pTM5, was used as a template to generate 20P1 -P6 -P7 point mutants.24 Two halves of the plasmid were amplified using 2 pairs of mutagenic primers and joined through Gibson assembly (Table S3). The plasmids encodingP1 -P6 -P7 and each of its point mutants were co-transformed into E. coli K207-3.35 A single colony was used to inoculate 5 mL of Luria–Bertani (LB) media containing 100 mg L-1 kanamycin and 100 mg L-1 streptomycin. After the starter culture grew for 16 h at 37 °C and 250 rpm, 400 µL of it was used to innoculate 250-mL Erlenmeyer flasks containing 40 mL production media [5 g L-1 yeast extract, 10 g L-1 casein, 15 g L-1 glycerol, 10 g L-1 NaCl, 100 mg L-1 kanamycin, 100 mg L-1streptomycin, 100 mM potassium phosphate buffer (pH 7.6)].24 When OD600 = 1.0, cells were cooled to 19 °C and both 20 mM sodium propionate and 100 μM IPTG were added. Cultures were incubated at 19 °C at 240 rpm for 8 d.