TABLE 1.
Plasmids used in this study.
| Plasmid | Relevant characteristic | Reference/Origin |
|---|---|---|
| pKD4 | R6K γ ori, FRT-flanked KanR, and AmpR | Datsenko and Wanner (2000) |
| pKD46 | pSC101 ori, araC-ParaB, λ-Red recombinase system (γ, β, exo, λ tL3 terminator), and AmpR | Datsenko and Wanner (2000) |
| pCP20 | pSC101 ori, AmpR, CmR, and Flp | Cherepanov and Wilfried (1995) |
| In vitro studies | ||
| pET28a(+) | f1 origin, KanR, and T7 promoter | Novagen™ |
| pET28-Ec-Mdh 5Q | pET28a(+) derivative N-terminal His-tagged Ec.mdh R81A:M85Q:I12V:G179D:D86S | Frazão et al. (2018) |
| pET28-Ec-Mdh 5Q -D34G | pET28a(+) derivative with N-terminal His-tagged Ec.mdh R81A:M85Q:I12V:G179D:D86S:D34G | This study |
| pET28-Ec-Mdh 5Q -D34G:I35K | pET28a(+) derivative with N-terminal His-tagged Ec.mdh R81A:M85Q:I12V:G179D:D86S:D34G:I35K | This study |
| pET28-Ec-Mdh 5Q -D34G:I35R | pET28a(+) derivative with N-terminal His-tagged Ec.mdh R81A:M85Q:I12V:G179D:D86S:D34G:I35R | This study |
| pET28-Ec-Mdh 5Q -D34G:I35S | pET28a(+) derivative with N-terminal His-tagged Ec.mdh R81A:M85Q:I12V:G179D:D86S:D34G:I35S | This study |
| pET28-Ec-Mdh 5Q -D34G:I35T | pET28a(+) derivative with N-terminal His-tagged Ec.mdh R81A:M85Q:I12V:G179D:D86S:D34G:I35T | This study |
| In vivo DHB production | ||
| pZA23 | p15A origin, KanR, and PA1lacO-1 promoter | Expressys |
| pZA23-HS1-5Q | pZA23 derivative carrying Ec.thrA S345F , Ec.aspC, Ec.mdh 5Q , and Ec.ppc K620S | Frazão et al. (2018) |
| pZA23-HS1-7Q | pZA23 derivative harboring Ec.thrA S345F , Ec.aspC, Ec.mdh 7Q , and Ec.ppc K620S | This study |
| pZA23-HS2-5Q | pZA23 derivative harboring Ec.thrA S345F , Ec.alaC A142P:Y275D , Ec.Mdh 5Q , and Ec.ppc K620S | Frazao (2019) |
| pZA23-HS2-7Q | pZA23 derivative harboring Ec.thrA S345F , Ec.alaC A142P:Y275D , Ec.mdh 7Q , and Ec.ppc K620S | This study |