TABLE 1.
Bacterial strains and plasmids used in the study
| Strain or plasmid | Characteristics | Source or reference |
|---|---|---|
| Strains | ||
| S. erythraea NRRL2338 | Used as parental strain, wild type | DSM 40517 |
| S. erythraea ΔpccD | S. erythraea SACE_3396 null mutant, thiostrepton resistant | 29 |
| S. erythraea WT/pIB-pccD | SACE_3396 overexpression strain, WT carrying pIB-pccD | 29 |
| S. erythraea WT/pIB139 | WT carrying vector pIB139 | This study |
| S. erythraea ΔpccD/pIB-pccD | SACE_3396 complementation strain, ΔpccD carrying pIB-pccD, which is under the control of pccD promoter | This work |
| S. erythraea ΔpccD/pIB139 | Empty plasmid control, ΔpccD carrying pIB139 | This study |
| S. erythraea ΔgdhA1 | S. erythraea SACE_3882 null mutant | This study |
| S. erythraea ΔgdhA1/pIB-gdhA1 | gdhA1 complementation strain, ΔgdhA1 carrying pIB-gdhA1 | This study |
| S. erythraea ΔgdhA1/pIB139 | Empty plasmid control, ΔgdhA1 carrying pIB139 whose ermE* promoter was replaced by SACE_3880–3884 promoter | This study |
| S. erythraea ΔbcdhE1 | S. erythraea SACE_3884 null mutant | This study |
| S. erythraea ΔbcdhE1/pIB-bcdhE1 | bcdhE1 complementation strain, ΔBcdhE1 carrying pIB-BcdhE1 gene | This study |
| S. erythraea ΔbcdhE1/pIB139 | The empty plasmid control, ΔBcdhE1 gene carrying pIB139 whose ermE* promoter was replaced by SACE_3880–3884 promoter | This study |
| S. erythraea WT/pIB-bkd | SACE_3880–3884 overexpression strain, WT carrying pIB-bkd | This study |
| E. coli Rosetta (DE3) | F− ompT hsdSB(rB− mB−) gal dcm λ(DE3)pRARE2(Camr) | Novagen |
| S. erythraea E3 | Industrial high-erythromycin-producing strain | SKLBE, ECUST |
| S. erythraea E3ΔpccD | S. erythraea E3 SACE_3396 null mutant, thiostrepton resistant | This study |
| S. erythraea E3ΔpccD/pIB-bkd | SACE_3880–3884 overexpression strain, E3ΔpccD carrying pIB-bkd | This study |
| S. erythraea WT/pKC-egfp | egfp overexpression strain, WT carrying pKC-egfp | This study |
| Plasmids | ||
| pET19b | Expression vector, Ampr | Novagen |
| pET-pccD | pET19b derivative carrying SACE_3396 | 29 |
| pKC1139 | acc(3)IV, pSG5, pBR322, oriTRK2 | 49 |
| pIB139 | E. coli-S. erythraea integrative shuttle vector containing a strong constitutive ermE* promoter, apramycin resistance | 50 |
| pIB-pccD | pIB139 carrying an extra SACE_3396 for overexpression (under the control of the ermE* promoter)/complementation (under the control of pccD promoter) | 29; this study |
| pIB-gdhA1 | pIB139 carrying an extra SACE_3882 for complementation under the control of SACE_3880–3884 promoter | This study |
| pIB-BcdhE1 | pIB139 carrying an extra SACE_3884 for complementation under the control of SACE_3880–3884 promoter | This study |
| pIB-bkd | pIB139 carrying an extra SACE_3880–3884 for gene overexpression | This study |
| pIB-egfp | pIB139 carrying an extra egfp gen for gene overexpression | This study |
| pUC-fd term | pUC57 carrying a synthetic fd terminator gene originating from bacteriophage | This study |
| pUC19-tsr | pUC19 derivative containing a 1.36-kb fragment of a thiostrepton resistance cassette in the BamHI/SmaI sites | 51 |
| pUC-pccD | pUC19-tsr, with the 1.5-kb DNA fragments upstream and downstream of the pccD gene inserted upstream and downstream of tsr correspondingly | This study |
| pKC-egfp | pKC1139 containing PermE-egfp-fd cassette for egfp overexpression | This study |
| pUC-Sencas9 | pUC57 carrying a synthetic codon-optimized cas9 gene | This study |
| pKC-cas9 | pKC1139 containing PermE-Sencas9-fd cassette | This study |
| pKC-cas9-sgRNA | pKC1139 containing PermE-Sencas9-fd cassette and PermE-sgRNA cassette | This study |
| pkC-CRISPR/Cas9 | pKC1139 containing PermE-Sencas9-fd cassette, PermE-sgRNA cassette, and homologous arm for gene deletion | This study |