Table 2.
Strains and plasmids used in this study
| Strain or plasmid | Relevant marker | Source or references |
|---|---|---|
| Escherichia coli | ||
| DH5α | F−, ϕ80lacZ∆M15, ∆(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(r−K m+K), phoA, supE44, λ−, thi-1, gyrA96, relA1 | Lab stock |
| BL21(DE3) | E. coli B, F−, dcm, ompT, hsdS(r−B m−B), gal, λ(DE3) | Novagen |
| S17-1 | thi pro hsdR recA chromosomal RP4; Tra+; Tmpr Str/Spcr | [43] |
| Ralstonia eutropha | ||
| H16 | Wild type | DSM 428 |
| NSDG | H16 derivative; ΔphaC::phaCNSDG | [33] |
| NSDG-GG | NSDG derivative; ΔnagR, nagE(G793C), PA2858-glpFKEc-h16_A2858 | This study |
| NSDG-GGΔB1 | NSDG-GG derivative; ΔphaB1 | This study |
| NSDG-GG-BNV | NSDG-GG∆B1 derivative; phaB1NV | This study |
| NSDG-GG-BNVYF | NSDG-GG∆B1 derivative; phaB1NVYF | This study |
| NSDG-GG-B2 | NSDG-GG∆B1 derivative; ∆phaB1::phaB2 | This study |
| NSDG-GG-HC | NSDG-GG∆B1 derivative; ∆phaB1::had-crt2 | This study |
| Plasmids | ||
| pEE32 | pUC18 derivative; phaPCJAc | [10] |
| pBPP | pBBR ori (broad host range), mob, Kanr, PphaP1 | [44] |
| pBPP-ccrMeJ4a-emd | pBPP derivative; ccrMe, phaJ4a, emdMm | [23] |
| pBPP-ccrMeJAc-emd | pBPP derivative; ccrMe, phaJAc, emdMm | This study |
| pColdII | ColE1 ori, Ampr, PcspA | Takara Bio |
| pColdII-phaB1 | pColdII derivative, phaB1 | This study |
| pColdII-phaB2 | pColdII derivative, phaB2 | This study |
| pET15b-phaB3 | pET15b derivative, phaB3 | This study |
| pColdII-phaB1NV | pColdII derivative, phaB1NV | This study |
| pColdII-phaB1NVYF | pColdII derivative, phaB1NVYF | This study |
| pK18mobsacB-AR | pK18mobsacB derivative; phaB1 del | [33] |
| pK18mobsacB-ABNVYFR | pK18mobsacB-AR derivative; phaB1NVYF ins | This study |
| pK18mobsacB-ABNVR | pK18mobsacB-AR derivative; phaB1NV ins | This study |
| pK18mobsacB-AB2R | pK18mobsacB-AR derivative; phaB2 ins | This study |
| pK18mobsacB-AHCR | pK18mobsacB-AR derivative; had-crt2 ins | This study |
The postfix del and ins indicate constructs for targeted gene deletion and insertion, respectively
Ac, Aeromonas caviae; Me, Methylorubrum extorquens; Mm, Mus musculus. phaCNSDG, a gene encoding N149S/D171G mutant of PHA synthase from A. caviae. phaB1NV and phaB1NVYF, genes encoding N142V and N142V/Y185F mutants of NADPH-acetoacetyl-CoA reductase PhaB1 from R. eutropha, respectively