A) the Sequences of ADH2 (RHTO 03062) Promoter Region

Supporting information a) The sequences of ADH2 (RHTO_03062) promoter region ggctgaggcttccccgacgcccctcctcccctccctcctcgccctcctcctcgtcctcctcgtgatgccat cagtcgcgccggcggggtgctgcggtttgcgacggtgttctttgagggtgtgtgtgcgctgagcaagcagt agcaggcagtccagcaggtgagcgcggtgtgggaacagctcgagcttgttgcgggcggtgtactgatcaag taggacctcggagactacggtcagagcctacgacgacgtcgttgcagtcgtgtacactgggaatggcagct aggctgatccccgccgctctccaccacactttactcgtcttccgtcctcgcccgcagtcgtgaattcatgg gtggagacgtcgaagggtgggctttgcttcggggtggctgggtcccgtgctcgagtggctgtgggcgtcaa ttaagcgtgtctcgtgcgagagcgagccgacttgcgagtcgtcccgagcaggagtggccttcctcgctctt cgccttccccacccatcgaccgtcccgcaaccttgcgctcgctctctctcttacagcttgctcttcacttg cacacactgctacacaccctcgcacagcactcgcactcagcccataccggtccagccttcgcagccctctc tcttcgtgcgttcccattcccggctgttcactctcgcaccgtcatttcccgtctagctgacagctgccacg tctccgacagtcaca b) The sequences of GPD (RHTO_3746) promoter region

TGGAGTTCGACGTTCTCCTCGCTCCGCAAGCATTGGAATGAACCTTGCTCTCTAGTTCCCTCCTCCGTGAC CTCGTTTCGTCCTTTAGACGGCACGATGGAAGGAAGAAATCTCTGCGGACAAGCAAATCTGCTGGCTCGCC TTGTAGGTCGCCTACCGGAGCAAGCCTTGTGCCGCCGGGATGCCAACGTCGTTTTTTGACGTTTGCAAGAC GTAGAGGACGCTTCGGACGACGAAACAAGCTGTGAGGACATGGAAGTCGTGGGAGGAACGGCGCAGAGCGG CGCCGCGGGAGCATAAGGCAAGCGAGATAGTCCAGAAATCGCGGCGCCAAGTACAGTAATTTATTGGAGCA GGCACCAGAAGCGGGCAGCAGTATGCGCAGGCTTGGGGTCGACGAGAGACGACTCCCTCATACTCGGTTAC CTCGAGCAATACAATCAATCGAAGCTGCGCGAATCTCGGCTTGTAAGGGTCGGAAAGGAACCTCGGAGATG GCCACGTCACATCACCAACTTATCGATCTCAGCCGACGTCGCAGAGAGGGCGAGCGAAGCGGTGAAGGAGG GAAACAATCCCTCGAGAGCATGATCCGTCTGAATCTGCAGCGCAGGAAGCCGTCACACGCCCGCCTCGAGC GCAGGTCGGGTCCAGCCGGGGGACGAAACGCGCGAGGGCTGATTTCGTGAGCGAAGGAAGCCGCATCGACA AGTTCGCGTCCCTTTGCCCTCTTTCCCATCACCCGCTCTCGCTCTACCCGCTCAGAACAACACCAGATCAG TCACA c) The sequences of the RHTO_04602 locus (exon sequences shown in bold): 2577 bp

ATGCGCCCGCTTGCACGTGAAGAGGAGCTTCGATGCGATGCCGAGCAACGCAAGGTGAAGGTGATCGCTTC CGGGAACTCATCCGCTCGATCGCCGATGCCAGGCGGGGCGGTGCGATGCTTGACGGGGTTGCTCTTGTCCT GCCACCAACTCGTTAGCACAGCAAGGATGGCAGCTGCGAACGGACACGGCAAGGGAAAGCCCTCGGTGCTC ATCGTCGGAGCGGGCGTCGGAGGGACTGCCTCCGCCGCTCGCCTCGCCCAGTCCGGGTTCGACGTGACAGG TGCGCACACTCTGTCTCCTGCGTCGTCCTGCCTCGATCGGGGGACGTGGGAGCACTCGTAGAACCTGGTTG GAGGCACTGACGAGTTCAACCTCGTCGACAGTCCTCGAGAAGAACGACTTTGCCGGCGGACGATGCTCCCT CTTCACCGATCCGACCAAGTCCTTCCGCTTCGACCAGGGCCCGAGCCTGTTCCTCATCCCGCGACTGTTTG ACGAGACCTTTAACGATCTTGGGACGAGCCTCGAGAACGAGGGCATCAAGCTCGTCAAGTGCGAGCCAAAC TACCGGATCGTCTTCCCCGACAAGGAGGTCGTCGAGATGAGCAGCGACTTGACAAGGATGAAGAAGCAGGT CGAGCGGTGGGAGGGAGAGAAGGGCTTTGAAGGGTGAGTGGCTTGGAACCTCGGAGCGAGACGACGAGCGG AGATGCCCTTTCGCTTGTGCTCTCTTCCCGCTTCCTTTCCAGTTACTGACGCTCATGTTGTATGCAGATTC CTTGGCTTCTTGAAGGAGGGACACGCGCACTACGAGCTGTCGATGGTCCACGTCCTGCACCGCAACTTCAC

1 CTCGCTCCTCTCGATGGTCCGCCCGTCTCTCATCATCCAGCTCCGCAAGCTCCATCCCTTTGTCTCTGTCG TACGTCACCTCATCAGTGCTGATACGACTCGCTGGGAACGCAGGATTGCTGATGAGCGTCGCCTCGACTCA GTACTCGCGCGCGACCAAGTACTTCAAGACGGACCGCATGCGGAGAGCGTTCACTTTCGCCTCGATGTATC TCGTGAGTCGGAGAGCGTCGCGATTCGAGCGCCAGCTCTGGCTTTAATCGCCGGCCAAGTTCCTCCTCCGC TCACCCGCACCTCCAATCTCGCAGGGCATGTCCCCCTTCGACGCTCTCGGCGCCTACAACCTCCTCCAGTA CACCGAGCACTGCGAAGGCATCCTCTACCCTCTCGGCGGCTTCGGCCGCATCCCGCAAACCCTCCAACAGC TCGCCGAAAAGAGCGGCGCCAAGTTCCGCTTCAACAGTCCCGTCAAGCGCGTCACGGTCGAGAACGGCACG GCTAAGGGTGTCGAGCTCGAGAGCGGCGAGAAGTTGACCGCCGACATCGTCCTCGTCAATGCGGATTTGGT GTGGAGTATGGCGCACTTGTACGAGGAGACGAGCTACTCGAAGCGCCTTGAGGAGCGGCCCGTCAGCTGCT CGTCCATCTCGCTCTACTGGTCAATGAACCGGTGCGTGAATGTTCACTCGTCCTCGATCGCGAATGGCGGA TACTGACTCCCCCTTGACCGCTCTACAGCAAGATACCCCAGCTCGACTCGCATACCATTTTCCTCGCAGAG GAGTACCGAGAGTGAGCACACCTGCGCTACTCGCCGCTCTCGAAGCGGTCGCTGATGAAGCCTCTCCCGTC CAGGTCCTTCGACTCGATCTTCCGCGAACACCGCATCCCGCATGAGCCTTCCTTCTACGTCAACGTTCCCA GCCGTCACGACCCTTCGTATGTCCCACTCATGTCGCTAGCAGCCAGCCATCGCTGACTCCGCATGCGCTCG CAGCGCCGCACCCGCCGACAAAGACGCCGTCATCGTCCTCGTTCCCGTCGGGCACATCTCCGCCGCCCTCC CCTCCTCTTCCGACTGGGACAAAGTCGTCGAAGAGACGCGCAACAAGATCATCGGCGAGATTGAGCGCCGC CTCGACATCGAGGACCTCCGGAGCTGCATCGAGCACGAGACGATCAACACGCCCATCACTTGGGGCGAGAA GTTCAACTTGCACCGCGGCAGCATTCTCGGACTCAGTCACGACTTCTTGTGAGTTCCGCCAGCCTGTCTTG TCCTGCTTGCTCTTGAAGCTTGCGCTGACCTTCCCAACCTTTCGCTTCTTCGCAGCAACGTCCTCTCTTTC CGCCCCAAGACCCGCCACCCGAGCGTCAAGAACGCTTACTTTGTCGGCGCGTCGGCGCACCCGGGAACGGG GTGATTTGTGGTGGTCTTCGCTCGCCTCGCTTGTCGCTGCCTCTCCAGCGTACCGCATGCGTCGAGTTCAA GGAATGGCTGCTAATTCGTATTGTCCTCGCAGCGTCCCCATCGTCCTCGCCGGCGCCCGCCTCGTCGCAAC CCAAATCCTCAACGACCTCGGGATGCCCATCCCCTCGCGTTGGAACGTCTCCTCGTCGGAACTCGCGACGC ACAAGACGATCCGCGATGCAGCAGGAGGGTTTACGCTCCTCTCGGTGTTGTTTGGGTTGATTGCTTTGTTG GTGATGTACCTGCGCGGTTGA

2 d) Homologous arm amplification sequences CRTI-3225-R

CRTI-5695-R

3 Supplementary Table 1. Strains and plasmids used

Strain or plasmid Relevant characteristics Source or reference

Strain

NP11 MAT A of Rhodosporidium toruloides Zhu et al. (2012)

Agrobacterium tumefaciens AGL1 AGL0 recA::bla pTiBo542DT Mop+ CbR Lazo et al. (1991)

E. coli DH5α F-, φ80dlacZ, ΔM15, Δ(lacZYA-argF) U169, Takara deoR, recA1, endA1, hsdR17 (rK-, mK+), phoA , supE44, λ-, thi-1, gyrA96 , relA1

AGL1-CRT-HYG-CRT AGL1/pZPK-CRT-PPGK-HYG-Tnos-CRT This study

AGL1-NAT- ADH2-gCRT AGL1/pZPK -PPGK-NAT-Tnos-PADH2-CRT- This study Thsp

AGL1- HYG-PGK-gCRT AGL1/pZPK-PPGK-HYG-Tnos-PGPD-CRT-Thsp This study

Δ-CRTI NP11 CRTI::hyg This study

ADH2-CRT NP11-CRT/pZPK- N- PADH2-CRT-Thsp This study

GPD-CRT NP11-CRT/pZPK- H- PGPD-CRT-Thsp This study

Plasmid pZPK-mcs KanR with multiple cloning site Lin et al. (2014) pZPK-PPGK-HYG-Tnos HYG-expression cassette Wang et al. (2016a) pZPK-H-PPHO89-MCS-Thsp Two expresion cassette vector, KanR with multiple Ma et al. (2015) cloning site pMD19-CRT CRTI in pMD19 This study pZPK-CRT CRTI in pZPK-mcs This study pZPK-CRT-PPGK-HYG-Tnos-CRT CRT-PPGK-HYG-Tnos-CRT This study pZPK-N-PADH2-CRT-Thsp NATR with CRTI This study pZPK-H-PGPD-CRT-Thsp HYGR with CRTI This study

4 Supplementary Table 2. Primers used

Primer Sequence(5`→3`) Description

CRT-RF1-F GTGAATTCGAGCTCGGTACCATGCGCCCGCTTGCA Amplification of the CRT CGTGAAG gene

CRT-RF1-R GCCTGCAGGTCGACTCTAGATCAACCGCGCAGGTA CATCAC

CRT-NcoI-PGK- GTCGAGAACGGCACGGCTAAGGGTGTCGAccatggCC Amplification of pPGK- F AGACGGACCTTGAGAACCCTCA HYG-Tnos fragment for RF cloning Tnos-NdeI-CRT- CGGCGGTCAACTTCTCGCCGCTCTCGAGCCATATG YH-R AGGCCCGATCTAGTAACATAGATGACAC

NAT-RF-F CTCCCACCCTCCCCCGTGCAGCCCACCATGGGTAC Amplification of NAT CACTCTTGACGACAC fragment for RF cloning

NAT-RF-R TGAGCATGCCCTGCCCCTAGGATCGTTCAAACATTT GGCAATAAAGTTTC

ADH2-RF-F GAGCTTGAGCTTGGATCAGATTGTCGTTTCGGCTG Amplification of ADH2 AGGCTTCCCCGACG fragment for RF cloning

ADH2-RF-R CGGAATCGTACTAGTCCATGGGATATCTGTGACTGT CGGAGACGTGGCAGC

GPD-RF-F GAGCTTGAGCTTGGATCAGATTGTCGTTTTGGAGTT Amplification of GPD CGACGTTCTCCTCGCTC fragment for RF cloning

GPD-RF-R CGGAATCGTACTAGTCCATGGGATATCTGTGACTGA TCTGGTGTTGTTCTG

NcoI-CRT-F CGGCCATGGtcATGCGCCCGCTTGCACGTGAAGAGG Amplification of the CRT gene with endonuclease sites CRT-SpeI-R GCCACTAGTTCAACCGCGCAGGTACATCACC