Table 1.
Overview of constructs used to engineer ent-kaurene production in R. toruloides
| Plasmids | Genotypes/features | ABFPUB registry ID |
|---|---|---|
| Construct 1 | PANT-GfKS-HYGR | ABFPUB_1 |
| Construct 2 | PGAPDH-GfKS-NATR | ABFPUB_3 |
| Construct 3 | PTEF1-RtERG20(F91C)-KanMXR | ABFPUB_6 |
| Construct 4 | PTEF1-TcGGPPS(S239C,G29D)-KanMXR | ABFPUB_7 |
| Construct 5 | PTEF1-GgFPS(F112A)-KanMXR | ABFPUB_8 |
| Construct 6 | PTEF1-RtBTS1-KanMXR | ABFPUB_9 |
| Construct 7 | PANT-GfKS-PTEF1-RtERG20(F91C)-NATR | ABFPUB_10 |
| Construct 8 | PANT-GfKS-PTEF1-TcGGPPS(S239C, G295D)-NATR | ABFPUB_11 |
| Construct 9 | PANT-GfKS-PTEF1-GgFPS(F112A)-NATR | ABFPUB_12 |
| Construct 10 | PANT-GfKS-PTEF1-RtBTS1-NATR | ABFPUB_13 |
| Strains | IFO0880 (WT) | ABFPUB_14 |
| IFO0880/PGAPDH-GfKS-NATR | ABFPUB_15–17 | |
| IFO0880/PANT-GfKS-HYGR | ABFPUB_18, 30, 34 | |
| IFO0880/PGAPDH-GfKS-NATR/PTEF1-RtERG20(F91C)-KanMXR | ABFPUB_47–49 | |
| IFO0880/PGAPDH-GfKS-NATR/PTEF1-TcGGPPS(S239C,G29D)-KanMXR | ABFPUB_19, 20, 31 | |
| IFO0880/PGAPDH-GfKS-NATR/PTEF1-GgFPS(F112A)-KanMXR | ABFPUB_32, 33, 35 | |
| IFO0880/PGAPDH-GfKS-NATR/PTEF1-RtBTS1-KanMXR | ABFPUB_38–40 | |
| IFO0880/PANT-GfKS-PTEF1-RtERG20(F91C)-NATR | ABFPUB_41–43 | |
| IFO0880/PANT-GfKS-PTEF1-TcGGPPS(S239C,G295D)-NATR | ABFPUB_21, 22, 29 | |
| IFO0880/PANT-GfKS-PTEF1-GgFPS(F112A)-NATR | ABFPUB_23, 26, 36 | |
| IFO0880/PANT-GfKS-PTEF1-RtBTS1-NATR | ABFPUB_44–46 |
Strains and plasmids used in this study are available upon request through the Agile BioFoundry Strain Registry (http://public-registry.agilebiofoundry.org// [37])
GAPDH glyceraldehyde 3-phosphate dehydrogenase, ANT, adenine nucleotide translocase, TEF1 translational elongation factor, GfKS, Gibberella fujikuroi kaurene synthase (NCBI Accession Number, Q9UVY5), RtERG20(F91C) mutant farnesyl pyrophosphate synthase from R. toruloides (PRQ75922.1), TcGGPPS(S239C,G295D) mutant geranylgeranyl pyrophosphate synthase from Taxus canadensis (AAD16018), GgFPS(F112A) mutant farnesyl pyrophosphate synthase from Gallus gallus (P08836.2) and RtBTS1 geranylgeranyl pyrophosphate synthase BTS1 from R. toruloides (PRQ72579)