Table 2.
Strains and plasmids used in this study
| Name | Description | Reference | Remarks |
|---|---|---|---|
| E. coli BL21-Gold (DE3) | F– ompT hsdS (rB– mB–) dcm+ Tetr gal λ(DE3) endA Hte | Stratagene | Base strain for genome editing |
| B2 | E. coli BL21-Gold (DE3) ΔaroA ΔaroB ΔaroC ΔserC ΔzapB | This study | Auxotrophic strain with increased size |
| B3 | B2 ΔdgkA | This study | Strain to increase TAG production |
| B4 | B3 ΔadhE | This study | Strain to increase acetyl-coA |
| B5 | B3 ΔldhA | This study | Strain to increase acetyl-coA |
| B6 | B3 ΔadhE ΔldhA | This study | Strain to increase acetyl-coA |
| pTarget | Plasmid used to express sgRNA under J23119 promoter. SacB gene under its native promoter is inserted between aadA and pMB1 origin of replication | This study | Modified based on [6] |
| pCas-V5 | Plasmid to express cas9 under its native promoter, λRed from pBAD promoter. It carries a temperature sensitive origin of replication and is kanamycin resistent. The sgRNA targeting pMB1 origin of replication is removed | This study | Modified based on [6] |
| pCas-V2 | Similar to pCas-v5 except λRed is controlled by T7 promoter | This study | Modified based on [6] |
| pCas-V6 | Similar to pCas-v5 with inactive recA56 being overexpressed with cas9 in a polycistronic manner | This study | Modified based on [6] |
| p15A-spec-Tm1-hmgS-atoB-hmgR | Plasmid for overexpression of hmgs, atoB, thmgR genes, controlled by mutated Tm1 promoter. It carries spectromycin resistance gene | [21] | Module 1 |
| p15A-cam-Tm2-mevK-pmk-pmd-idi | Plasmid for overexpression of mevK, pmK, pmd and idi genes, controlled by Tm2 promoter. It carries chloramphenicol resistance gene | [21] | Module 2 |
| p15A-kan-Tm1-crtEBI-ispA | Plasmid for overexpression of crtE, crtB, crtI and ispA genes, controlled by Tm1 promoter. It carries kanamycine resistance gene | [21] | Module 3 |
| p15A-amp-Tm1-Ec.fadD (D1) | Plasmid for overexpression of fadD gene from E. coli, controlled by Tm1 promoter. It carries ampicilin resistance gene | This study | Module 4 |
| p15A-amp-Tm1-Ec.fadD-a.DGT-Ro.PAP (DTP1a) | Plasmid for overexpression of fadD gene from E. coli, WS/DGAT from Acinetobacter baylyi and PAP from Rhodococcus opacus, controlled by Tm1 promoter. It carries ampicilin resistance gene | This study | Module 4 |
| p15A-amp-Tm1-Ec.fadD-t.DGT-Ro.PAP (DTP1b) | Plasmid for overexpression of fadD gene from E. coli, WS/DGAT from Thermomonospora curvata and PAP from Rhodococcus opacus, controlled by Tm1 promoter. It carries ampicilin resistance gene | This study | Module 4 |
| p15A-amp-Tm1-Ro.fadD (D2) | Plasmid for overexpression of fadD gene from Rhodococcus opacus, controlled by Tm1 promoter. It carries ampicilin resistance gene | This study | Module 4 |
| p15A-amp-Tm1-Ro.fadD-a.DGT-Ro.PAP (DTP2a) | Plasmid for overexpression of fadD gene from Rhodococcus opacus, WS/DGAT from Acinetobacter baylyi and PAP from Rhodococcus opacus, controlled by Tm1 promoter. It carries ampicilin resistance gene | This study | Module 4 |
| p15A-amp-Tm1-Ro.fadD-t.DGT-Ro.PAP (DTP2b) | Plasmid for overexpression of fadD gene from Rhodococcus opacus, WS/DGAT from Thermomonospora curvata and PAP from Rhodococcus opacus, controlled by Tm1 promoter. It carries ampicilin resistance gene | This study | Module 4 |