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Table 1 Overview of constructs used to engineer terpene production in R. toruloides

From: Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass

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

  1. 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
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Biotechnology for Biofuels and Bioproducts

ISSN: 2731-3654

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