Table 2 Strains and plasmids used in this study

Strain

E. coli DH5α

Host for plasmids construction and subcloning

New England Biolabs

E. coli ET12567/pUZ8002

Non-methylating ET12567 containing non-transmissible RP4 derivative plasmid pUZ8002

Kieser et al. [28]

S. hygroscopicus subsp. aureolacrimosus NRRL 5739

The milbemycin-producing strain

Takiguchi et al. [6]

S. avermitilis SA-01

Industrial avermectin-producing S. avermitilis

This study

S. avermitilis SAMA1

Mutant strain of S. avermitilis SA-01 with AveA1 replaced by MilA1

This study

S. avermitilis SAMA13

Mutant strain of SAMA1 with AveA3 replaced by MilA3

This study

S. avermitilis SAMA1M7

Mutant strain of SAMA1 with AveA3-module7 replaced by MilA3-module7

This study

S. avermitilis SAMA1M7ΔD

AveD disruption mutant of SAMA1M7

This study

Plasmid

pLitmus28

E. coli vector for subcloning, Amp^R

New England Biolabs

pGEM T easy

E. coli vector for cloning PCR products, Amp^R

Promega

pCR2.1 TOPO

E. coli vector for cloning PCR products, Amp^R

Invitrogen

pKC1139

Temperature-sensitive E. coli-Streptomyces shuttle vector containing oriT for gene substitution and disruption, Apr^R

Bierman et al. [27]

pSAMA1

pKC1139-based plasmid containing aveA1 upstream fragment, entire milA1 fragment, and aveA1 downstream fragment

This study

pSAMA13

pKC1139-based plasmid containing aveA3 upstream fragment, entire milA3 fragment, and aveA3 downstream fragment

This study

pSAMA1M7

pKC1139-based plasmid containing aveA3-module 7 upstream fragment, milA3-module 7 fragment, and aveA3-module 7 downstream fragment

This study

pΔAveD

pKC1139-based plasmid containing inactivated aveD fragment

This study