Table 1.
Overview of constructs used to engineer terpene production in R. toruloides
| Genotype/features | Name | Registry ID |
|---|---|---|
| Plasmids | ||
| PGAPDH-BIS-TNOS, NAT | GPD-BIS-NOS | JPUB_017422 |
| PANT-BIS-TNOS, HYG | pGEN-295 | JPUB_017423 |
| PANT-HYP3-TNOS, HYG | pGEN-299 | ABFPUB_000356 |
| PTEF1-tAgGPPS2-TNOS, KAN | pGEN-376 | ABFPUB_000335 |
| PANT-HYP3-TNOS, PTEF1-AgGPPS2-T35S, KAN | pGEN-308 | ABFPUB_000348 |
| PGAPDH-HYP3-TNOS, NAT | pGEN-300 | ABFPUB_000357 |
| PTEF1-AgGPPS2-T35S, BLE | pGEN-305 | ABFPUB_000351 |
| PANT-HYP3-TNOS, PTEF1-tAgGPPS2-T35S, HYG | pGEN-309 | ABFPUB_000353 |
| PTEF1-RtERG20(F91W)-TNOS, KAN | pGEN-374 | ABFPUB_000331 |
| PTEF1-RtERG20(F91W, N122W)-TNOS, KAN | pGEN-375 | ABFPUB_000333 |
| PTEF1-ScERG20(F96W)-TNOS, KAN | pGEN-372 | ABFPUB_000327 |
| PTEF1-ScERG20(F96W, N127W)-TNOS, KAN | pGEN-373 | ABFPUB_000329 |
| PANT-HYP3-TNOS, PTEF1-GgFPS(N144W)-T35S, NAT | pGEN-307 | ABFPUB_000344 |
| PGAPDH-HYP3-GgFPS(N144W)-TNOS (fusion), NAT | pGEN-379 | ABFPUB_000339 |
| PGAPDH-GgFPS(N144W)-HYP3-TNOS (fusion), NAT | pGEN-378 | ABFPUB_000337 |
| PTEF1-GgFPS(N144W)-TNOS, KAN | pGEN-304 | ABFPUB_000341 |
| PGAPDH-tCgHMGR-TNOS, KAN | GPD-tHMGR | ABFPUB_000355 |
| PANT-SpHMGR-TNOS, PSKP1-McMK-TSKP1, PDUF-ScPMK-TDUF (ECO), KAN | pGEN-446 | ABFPUB_000313 |
| PANT-SpHMGR-TNOS, PSKP1-McMK-TSKP1, PDUF-ScPMK-TDUF (HC), KAN | pGEN-484 | ABFPUB_000315 |
| PANT-SpHMGR-TNOS, PSKP1-McMK-TSKP1, PDUF-ScPMK-TDUF (GO), KAN | pGEN-494 | ABFPUB_000325 |
| PANT-SpHMGR-TNOS, PSKP1-McMK-TSKP1, PDUF-SpPMK-TDUF (HC), KAN | pGEN-485 | ABFPUB_000318 |
| PANT-DaHMGR-TNOS, PSKP1-McMK-TSKP1, PDUF-ScPMK-TDUF (HC), KAN | pGEN-486 | ABFPUB_000321 |
| PANT-DaHMGR-TNOS, PSKP1-McMK-TSKP1, PDUF-SpPMK-TDUF (HC), KAN | pGEN-487 | ABFPUB_000323 |
| Strains | ||
| R. toruloides IFO0880, mating type A2 (WT) | ABFPUB_000014 | |
| IFO0880/PGAPDH-BIS-TNOS (1 copy) | BIS18 | JPUB_013664 |
| IFO0880/PGAPDH-BIS-TNOS (7 copies) | BIS14 | JPUB_017424 |
| IFO0880/PGAPDH-BIS-TNOS (10 copies) | BIS3 | JPUB_009679 |
| IFO0880/PGAPDH-BIS-TNOS, PANT-BIS-TNOS | GB2 | ABFPUB_000311 |
| IFO0880/PANT-HYP3-TNOS, PTEF1-tAgGPPS2-T35S | 336 | ABFPUB_000336 |
| IFO0880/PANT-HYP3-TNOS_PTEF1-tAgGPPS2-T35S | 350 | ABFPUB_000350 |
| IFO0880/PGAPDH-HYP3-TNOS, PTEF1-tAgGPPS2-T35S | 352 | ABFPUB_000352 |
| IFO0880/PGAPDH-HYP3-TNOS, PTEF1-tAgGPPS2-T35S_PANT-HYP3-TNOS | 354 | ABFPUB_000354 |
| IFO0880/PGAPDH-HYP3-TNOS, PTEF1-AgGPPS2-T35S_PANT-HYP3-TNOS | 349 | ABFPUB_000349 |
| IFO0880/PANT-HYP3-TNOS, PTEF1-RtERG20(F91W)-TNOS | 332 | ABFPUB_000332 |
| IFO0880/PANT-HYP3-TNOS, PTEF1-RtERG20(F91W, N122W)-TNOS | 334 | ABFPUB_000334 |
| IFO0880/PANT-HYP3-TNOS, PTEF1-ScERG20(F96W)-TNOS | 328 | ABFPUB_000328 |
| IFO0880/PANT-HYP3-TNOS, PTEF1-ScERG20(F96W, N127W)-TNOS | 330 | ABFPUB_000330 |
| IFO0880/PANT-HYP3-TNOS_PTEF1-GgFPS(N144W)-T35S | 345 | ABFPUB_000345 |
| IFO0880/PGAPDH-HYP3-GgFPS(N144W)-TNOS (fusion) | 340 | ABFPUB_000340 |
| IFO0880/PGAPDH-GgFPS(N144W)-HYP3-TNOS (fusion) | 338 | ABFPUB_000338 |
| IFO0880/PGAPDH-HYP3-TNOS, PTEF1-GgFPS(N144W)-TNOS | 343 | ABFPUB_000343 |
| IFO0880/PANT-HYP3-TNOS, PGAPDH-HYP3-TNOS, PTEF1-GgFPS(N144W)-TNOS | 342 | ABFPUB_000342 |
| IFO0880/PGAPDH-BIS-TNOS, PANT-BIS-TNOS, PGAPDH-tCgHMGR-TNOS | 312 | ABFPUB_000312 |
| 345/PANT-SpHMGR-TNOS_PSKP1-McMK-TSKP1_PDUF-ScPMK-TDUF (ECO) | 314 | ABFPUB_000314 |
| 345/PANT-SpHMGR-TNOS_PSKP1-McMK-TSKP1_PDUF-ScPMK-TDUF (HC) | 316 | ABFPUB_000316 |
| 345/PANT-SpHMGR-TNOS_PSKP1-McMK-TSKP1_PDUF-ScPMK-TDUF (GO) | 326 | ABFPUB_000326 |
| 345/PANT-SpHMGR-TNOS_PSKP1-McMK-TSKP1_PDUF-SpPMK-TDUF (HC) | 320 | ABFPUB_000320 |
| 345/PANT-DaHMGR-TNOS_PSKP1-McMK-TSKP1_PDUF-ScPMK-TDUF (HC) | 322 | ABFPUB_000322 |
| 345/PANT-DaHMGR-TNOS_PSKP1-McMK-TSKP1_PDUF-SpPMK-TDUF (HC) | 324 | ABFPUB_000324 |
| 345/PANT-SpHMGR-TNOS_PSKP1-McMK-TSKP1_PDUF-ScPMK-TDUF (HC) | 317 | ABFPUB_000317 |
| GB2/PANT-SpHMGR-TNOS_PSKP1-McMK-TSKP1_PDUF-SpPMK-TDUF (HC) | 319 | ABFPUB_000319 |
Strains and plasmids used in this study are available upon request through strain registries of the Agile BioFoundry (http://public-registry.agilebiofoundry.org/) and the Joint BioEnergy Institute (https://public-registry.jbei.org/), designated by ABF and JBx, respectively [16]. Unless indicated, sequences are from R. toruloides, with the exception of antibiotic resistance genes. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; BIS, α-bisabolene synthase from Abies grandis (NCBI Accession Number, O81086), NOS, nopaline synthase from A. tumefaciens (MK078637); ANT, adenine nucleotide translocase; TEF1, translational elongation factor; NAT, nourseothricin resistance cassette; HYG, Hygromycin B resistance cassette; KAN, kanamycin (G418) resistance cassette; HYP3, 1,8-cineole synthase from Hypoxylon sp. E7406B (AHY23922); AgGPPS2, GPP synthase from A. grandis (AAN01134); tAgGPPS2, AgGPPS2 with the N-terminal plastid transit peptide (84 amino acids) removed; 35S, 35S mRNA gene from cauliflower mosaic virus (KJ716236); RtERG20, FPP synthase from R. toruloides (PRQ75922); ScERG20, FPP synthase from S. cerevisiae (NP_012368); GgFPS, FPP synthase from Gallus gallus (P08836.2); tCgHMGR, HMGR from Cricetulus griseus, truncated by removal of 351 N-terminal amino acids (XP_027257481), SpHMGR, HMGR from Silicibacter pomeroyi (WP_011241944); SKP1, S-phase kinase-associated protein 1 from R. toruloides (PRQ77980); McMK, MK from Methanosaeta concilii (WP_013720012); DUF, domain of unknown function from R. toruloides (PRQ75822); ScPMK, PMK from S. cerevisiae (AJS65138); SpPMK, PMK from Streptococcus pneumoniae (WP_044791288); and DaHMGR, HMGR from Delftia acidovorans (WP_099752490). Codon optimization was performed by Genscript, using a R. toruloides codon table, except where indicated by HC (high-CAI method) and ECO (expression cassette optimization). In the strain genotype description, a comma indicates sequential insertion (stacking) while an underscore indicates that the cassettes are on the same construct