Strains and sequences

Rhodosporidium toruloides (a.k.a. Rhodotorula toruloides, a.k.a Rhodotorula graciis) strain IFO 0880 (a.k.a NBRC 0880) was obtained from the Biological Resource Center, NITE (NBRC), Japan. All strains named in this work are available to order through the Agile BioFoundry parts registry at https://public-registry.agilebiofoundry.org. The registry website also hosts all applicable plasmid sequences. Applicable strains and plasmid sequences are listed by figure in Additional file 2. Protein identification numbers used in this manuscript are from the R. toruloides genome version 4, available on Mycocosm, the US Department of Energy Joint Genome Institute fungal genome repository [46]. Selection markers used in R. toruloides were hygromycin and G418 resistance cassettes using the R. toruloides Tub2 promoter and terminator (see sequences on the Agile BioFoundry parts registry). Some selectable markers had a C-terminal fused sequence of thymidine kinase from herpes simplex virus, a useful construct for counter-selection and marker recovery [47], though no markers were recycled in this study.

For strains constructed by homologous recombination (e.g., full-deletion mutants), the parental strain was either a deletion mutant of the non-homologous end-joining factor Ku70 [48] or wild type. Homologous recombination and non-homologous end-joining (i.e., for generating randomly integrated mutants) was achieved by transforming R. toruloides with linearized plasmid by a lithium acetate transformation protocol as described in [49], or TDNA insertion by Agrobacterium tumefaciens-mediated transformation as described in [50]. Strain construction methods are listed for each strain in Additional file 2. For all deletion mutants, successful deletion was confirmed by diagnostic PCR at the altered locus. For targeted insertion of OE-Pnt1 at the RTO4_11990 locus, insertion was confirmed by diagnostic PCR followed by sequence verification of the inserted sequence. For construction of the OE-Pnt1 strain by random insertion, 24 randomly selected transformants were screened for growth in liquid culture with 600 µg/mL of the selective agent G418 (100 µg/mL is a common concentration for routine selection with G418). Significant variation was observed in growth rates amongst transformants, likely a consequence of different levels of expression of the selective marker due to local chromatin structure, number of integrations, and some rate of incomplete partial TDNA integrations. The three fastest growing strains were tested for growth on xylose plus glycerol. All three of these strains exhibited faster growth than WT in this condition and the fastest growing colony was selected for further characterization as the OE-Pnt1 strain.

For fatty alcohol production, the parental strain was R. toruloides IFO 0880 with a single copy of the Marinobacter aquaeolei fatty-acyl-CoA reductase (Maqu FAR 2220, [12]) integrated at the Ku70 locus, further engineered to reduce fatty alcohol catabolism (manuscript in preparation). To create the ∆11990 OE-Pnt1 fatty alcohol production strain, the parental strain was transformed with a linearized plasmid containing the OE-Ptn1 expression cassette flanked by homologous sequences to the RTO4_11990 locus. Outside the RTO4_11990 sequence was a GFP fluorescence cassette. A non-GFP expressing transformant was confirmed for OE-Pnt1 integration at the RTO4_11990 locus with diagnostic PCR. Three independent GFP positive transformants were also selected as random integration mutants, with a presumably intact RTO4_11990 locus, though that sequence was not confirmed.