*4.3. Gene Cluster Deletion*

Strain 177205 was reported to produce the main product enterocin and its biosynthesis gene cluster was located in genome [9]. Hence, strain 172205Δ*enc* was constructed by the whole enterocin biosynthetic gene cluster deletion with double-crossover homologous recombination. To construct the plasmid for gene cluster deletion, two DNA fragments, an 1870 bp *Avr* II-*Hin*d III homologous arm and another 1829 bp *Nde* I-*Hin*d III homologous arm were cloned from strain 17225 genome DNA covering both ends of the enterocin biosynthetic gene cluster. DNA fragments were ligated into pMD19-T simple and fragment sequences were identified by DNA sequencing. Two recycled DNA fragments were inserted into the delivery vector pYH7 [14] by *Nde* I-*Hin*d III restriction sites to yield pWHU2343 (Supplementary Figure S1). Intergeneric conjugation of plasmid pWHU2343 into strain 172205 by *E. coli* ET12657/pUZ8002 were carried out as protocol described in Practical Streptomyces Genetics [15]. The donor ET12657/pUZ8002 containing plasmid and the recipient spores were mixed and spread on Mannitol-Soy-agar (MS) plates with 10 mM MgCl2 and grown for 14 h at 28 ◦C. Then the plates were overlaid with 1 mL sterile water contained 4 μg/mL apramycin and 25 μg/mL nalidixic acid. Single colonies were transferred to a new MS plate with same antibiotics for further confirmation of antibiotic resistance. To screen the double-crossover mutants, single clones from no antibiotics plate were replicated on a MS plates with apramycin. Genome DNA of all candidates that had no apramycin resistance were extracted for PCR identification. Four pair of primers, *enc*-U, *enc*-D, *enc*-UD and *enc*-M, were used for screening (Supplementary Table S1), and a specific 1080 bp product for *enc*-UD was only amplified in mutant clones with absence of 21.6 kb gene cluster, but specific products for *enc*-U, *enc*-D and *enc*-M were only amplified in genome of wild-type clones. The mutant was also confirmed by detecting enterocin production in fermentation.
