Supplementary Table 1. Strains and plasmids used

Strains & plasmids / Relevant characteristics / Source
S. cerevisiae strains
BY4741 / MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0 / (Brachmann et al. 1998)
NK-L70 / BY4741; pSP-G1; pLC42; pLCm7 / This study
NK-L71 / BY4741; PGPD1: ENO2; PGPD1: TAL1 / This study
NK-L72 / NK-L71; pSP-G1; pLC42; pLC-m7 / This study
NK-L73 / NK-L71; pLC-m1 / This study
NK-L74 / NK-L71; pLC-m1; pLC42; pLC-m7 / This study
NK-M1 / NK-L71; pLC-m1; pLC-m2; pLC-m7 / This study
NK-M2 / NK-L71; pLC-m1; pLC-m3; pLC-m7 / This study
NK-M3 / NK-L71; pLC-m1; pLC-m4; pLC-m7; / This study
NK-M4 / NK-L71; pLC-m1; pLC-m5; pLC-m7; / This study
NK-M5 / NK-L71; pLC-m1; pLC-m6; pLC-m7; / This study
NK-L107 / NK-L71; PGPD1: ARO1 / This study
NK-M6 / NK-L107; pLC-m1; pLC-m6; pLC-m7; / This study
Plasmids
pSP-G1 / 2 μm ori, URA3, PTEF1- TADH1, PPGK1-TCYC1, Ampr / (Partow et al. 2010)
pLC41 / 2 μm ori, HIS3, PTEF1- TADH1, PPGK1-TCYC1, Ampr / This study
pLC42 / 2 μm ori, LEU2, PTEF1- TADH1, PPGK1-TCYC1, Ampr / This study
pGEM-T Easy / Clone vector, Ampr / Promega (Beijing, China )
pT-ARO4WT / pGEM-T::ARO4wt / This study
pT-ARO7WT / pGEM-T:: ARO7wt / This study
pLC-m1 / pSP-G1:: PTEF1- ARO4fbr -TADH1, PPGK1- ARO7fbr-TCYC1 / This study
pLC-m2 / pLC42:: PTEF1- ARO2 -TADH1, PPGK1-TCYC1 / This study
pLC-m3 / pLC42:: PTEF1- TYR1 -TADH1, PPGK1-TCYC1 / This study
pLC-m4 / pLC42:: PTEF1- ARO8 -TADH1, PPGK1-TCYC1 / This study
pLC-m5 / pLC42:: PTEF1- ARO9 -TADH1, PPGK1-TCYC1 / This study
pLC-m6 / pLC42:: PTEF1- TYR1 -TADH1, PPGK1- ARO2-TCYC1 / This study
pLC-m7 / pLC41:: PTEF1- TADH1, PPGK1-TAL-TCYC1 / This study
pGREG506 / Containing the HIS3 expression cassette, Ampr / (Jansen et al. 2005)
pUG73 / Containing the LEU2 expression cassette, Ampr / (Hegemann and Heick 2011)
pUG72 / Containing the loxP-URA3-loxP cassette, Ampr / (Hegemann and Heick 2011)
pYM-N14 / Template for PGPD1 / (Janke et al. 2004)

Supplementary Table 2. Primers used

Primers / Sequences (5’-3’) / Applications
Promoter replacement in situ
1 / TTCCACCACCGCCAGAAGA / Amplify TAL upstream homologous region for TAL promoter replacement from S. cerevisiae genome, forward primer.
2 / CCTGCAGCGTACGAAGCTTCAGCTGTATGTACACGTATATGTGACGAGTT / Amplify TAL upstream homologous region for TAL promoter replacement from S. cerevisiae genome, reverse primer.
3 / AACTCGTCACATATACGTGTACATACAGCTGAAGCTTCGTACGC / Amplify URA3 selection marker for TAL promoter replacement from pUG72, forward primer.
4 / ATAAACTGAGCTCGTTTTCGACACTGCATAGGCCACTAGTGGATCTG / Amplify URA3 selection marker for TAL promoter replacement from pUG72, forward primer, reverse primer.
5 / TATCAGATCCACTAGTGGCCTATGCAGTGTCGAAAACGAGCTCAGT / Amplify GPD1 promoter for TAL promoter replacement from pYM-N14, forward primer.
6 / GTGTACATAATGTCTGAACCAGCTCGATCCACTAGTTCTAGAATCCG / Amplify GPD1 promoter for TAL promoter replacement from pYM-N14, reverse primer.
7 / CGACGGATTCTAGAACTAGTGGATCGTGTACATAATGTCTGAACCAGCT / Amplify TAL downstream homologous region for TAL promoter replacement from S. cerevisiae genome, forward primer.
8 / TGAAAGAAGCACCCATAACA / Amplify TAL downstream homologous region for TAL promoter replacement from S. cerevisiae genome, reverse primer.
9 / GATGAAAACACTAAACGAAGG / Amplify ENO2 upstream homologous region for TAL promoter replacement from S. cerevisiae genome, forward primer.
10 / CCTGCAGCGTACGAAGCTTCAGCTGTATTATTGTATGTTATAGTATTAGTTGCT / Amplify ENO2 upstream homologous region for TAL promoter replacement from S. cerevisiae genome, reverse primer.
11 / ACTAATACTATAACATACAATAATACAGCTGAAGCTTCGTACGC / Amplify URA3 selection marker for ENO2 promoter replacement from pUG72, forward primer.
12 / ATAAACTGAGCTCGTTTTCGACACTGCATAGGCCACTAGTGGATCTG / Amplify URA3 selection marker for ENO2 promoter replacement from pUG72, reverse primer.
13 / TATCAGATCCACTAGTGGCCTATGCAGTGTCGAAAACGAGCTCAGT / Amplify GPD1 promoter for ENO2 promoter replacement from pYM-N14, forward primer.
14 / CTTTAGAGACAGCCATTATTATTGTGATCCACTAGTTCTAGAATCCG / Amplify GPD1 promoter for ENO2 promoter replacement from pYM-N14, reverse primer.
15 / CGACGGATTCTAGAACTAGTGGATCACAATAATAATGGCTGTCTCTAAAG / Amplify ENO2 downstream homologous region for ENO2 promoter replacement from S. cerevisiae genome, forward primer.
16 / GATAGCGTCAACAATCAAGTC / Amplify ENO2 downstream homologous region for ENO2 promoter replacement from S. cerevisiae genome, reverse primer.
17 / ACTGCATCGGAAAGGGAAAC / Amplify ARO1 upstream homologous region for ARO1 promoter replacement from S. cerevisiae genome, forward primer.
18 / CCTGCAGCGTACGAAGCTTCAGCTGCGTAATATACAATTATCTTACGTAGG / Amplify ARO1 upstream homologous region for ARO1 promoter replacement from S. cerevisiae genome, reverse primer.
19 / CTACGTAAGATAATTGTATATTACGCAGCTGAAGCTTCGTACGC / Amplify URA3 selection marker for ARO1 promoter replacement from pUG72, forward primer.
20 / ATAAACTGAGCTCGTTTTCGACACTGCATAGGCCACTAGTGGATCTG / Amplify URA3 selection marker for ARO1 promoter replacement from pUG72, forward primer, reverse primer.
21 / TATCAGATCCACTAGTGGCCTATGCAGTGTCGAAAACGAGCTCAGT / Amplify GPD1 promoter for ARO1 promoter replacement from pYM-N14, forward primer.
22 / CTTTGGCTAACTGCACCATTGTTTTGATCCACTAGTTCTAGAATCCG / Amplify GPD1 promoter for ARO1 promoter replacement from pYM-N14, reverse primer.
23 / CGACGGATTCTAGAACTAGTGGATCAAAACAATGGTGCAGTTAGCCAAAGTC / Amplify ARO1 downstream homologous region for ARO1 promoter replacement from S. cerevisiae genome, forward primer.
24 / CAGCGTTCCAAATACAAGCA / Amplify ARO1 downstream homologous region for ARO1 promoter replacement from S. cerevisiae genome, reverse primer.
For site-directed mutagenesis of ARO4 and ARO7 genes
25 / AATCTAAGTTTTAATTACAAATGAGTGAATCTCCAATGTTCG / Amplify ARO4 for pGEM-T::ARO4WT construction from S. cerevisiae genome, forward primer.
26 / TGTAATCCATCGATACTAGTCACAATTAAATAATACCGAATTGG / Amplify ARO4 for pGEM-T::ARO4WT construction from S. cerevisiae genome, reverse primer.
27 / GGTGTTACTTTGCATGGTGTTGCTG / ARO4 site-directed mutations primers, forward primer.
28 / AGCAACACCATGCAAAGTAACACCC / ARO4 site-directed mutations primers, reverse primer.
29 / TTACAACAAATATAAAACAAATGGATTTCACAAAACCAGAAAC / Amplify ARO7 for pGEM-T::ARO4WT construction from S. cerevisiae genome, forward primer.
30 / CCTATAGTGAGTCGTATTACGAATTGTTACCGTGATAGCCTTC / Amplify ARO7 for pGEM-T::ARO4WT construction from S. cerevisiae genome, reverse primer.
31 / AAGAATAACTTCTCATCTGTTGCCA / ARO7 site-directed mutations primers, forward primer.
32 / GTGGCAACAGATGAGAAGTTATTCT / ARO7 site-directed mutations primers, reverse primer.
Vectors pLC41 and pLC42 construction
33 / GAGCACAGACTTAGATTGGTATATATACGCATATGGCTGCACGGTCCTGTTCCTA / Amplify HIS3 selective marker for pLC41 construction from pGREG506, forward primer.
34 / CCTTGCATGACAATTCTGCTAACATCAAAAGGCCTACTGCTGTCGATTCGATACTAACGC / Amplify HIS3 selective marker for pLC41 construction from pGREG506, reverse primer.
35 / GAGCACAGACTTAGATTGGTATATATACGCATATGCAGCTGAAGCTTCGTACGC / Amplify LEU2 selective marker for pLC42 construction from pUG73, forward primer.
36 / CCTTGCATGACAATTCTGCTAACATCAAAAGGCCTGCATAGGCCACTAGTGGATCTG / Amplify LEU2 selective marker for pLC42 construction from pUG73, reverse primer.
Cloning and overexpression of L- tyrosine pathway genes
37 / TAGCAATCTAATCTAAGTTTTAATTACAAGCGGCCAAAACAATGTCAACGTTTGGGAAACTG / Amplify ARO2 for overexpression from S. cerevisiae genome, forward primer.
38 / CGTCATCCTTGTAATCCATCGATACTAGTGCGGCCACATAACTCTTGAGGGGTTTTG / Amplify ARO2 for overexpression from S. cerevisiae genome, reverse primer.
39 / TAGCAATCTAATCTAAGTTTTAATTACAAGCGGCCAAAACAATGGTATCAGAGGATAAGATTGAG / Amplify TYR1 for overexpression from S. cerevisiae genome, forward primer.
40 / CGTCATCCTTGTAATCCATCGATACTAGTGCGGCCGAAGGCCTAATATTATAGGAAATCA / Amplify TYR1 for overexpression from S. cerevisiae genome, reverse primer.
41 / TAGCAATCTAATCTAAGTTTTAATTACAAGCGGCCAAAACAATGACTTTACCTGAATCAAAAGAC / Amplify ARO8 for overexpression from S. cerevisiae genome, forward primer.
42 / CGTCATCCTTGTAATCCATCGATACTAGTGCGGCCGCAATACAAAAATACGTACGTCC / Amplify ARO8 for overexpression from S. cerevisiae genome, reverse primer.
43 / TAGCAATCTAATCTAAGTTTTAATTACAAGCGGCCAAAACAATGACTGCTGGTTCTGCC / Amplify ARO9 for overexpression from S. cerevisiae genome, forward primer.
44 / CGTCATCCTTGTAATCCATCGATACTAGTGCGGCCACACATAGGTTTAATCTTCACTGTT / Amplify ARO9 for overexpression from S. cerevisiae genome, reverse primer.
Quantitative real-time PCR primers
45 / CGTTTTGCCAGTTCCATTC / Amplify partial cDNA of S. cerevisiae ENO2 gene for real-time PCR, forward primer.
46 / CGGCAGAAGCACCGTATC / Amplify partial cDNA of S. cerevisiae ENO2 gene for real-time PCR, reverse primer.
47 / CAAGCAACCAACTTACGCC / Amplify partial cDNA of S. cerevisiae TAL1 gene for real-time PCR, forward primer.
48 / GTGGAGACTCTGCCTGGAAC / Amplify partial cDNA of S. cerevisiae TAL1 gene for real-time PCR, reverse primer.
49 / CCATCTCCAATCGTGCTTTA / Amplify partial cDNA of S. cerevisiae ARO1 gene for real-time PCR, forward primer.
50 / TACCGTCTCACCATTATCTTCC / Amplify partial cDNA of S. cerevisiae ARO1 gene for real-time PCR, reverse primer.
51 / GGTGCCATTGCTGAGAAGTT / Amplify partial cDNA of S. cerevisiae ARO2 gene for real-time PCR, forward primer.
52 / ACGGAGGCGTCTGGACAT / Amplify partial cDNA of S. cerevisiae ARO2 gene for real-time PCR, reverse primer.
53 / TGGTATCCAGGCAAAGCG / Amplify partial cDNA of S. cerevisiae TYR1 gene for real-time PCR, forward primer.
54 / AATGCACGGTAATGATGTCG / Amplify partial cDNA of S. cerevisiae TYR1 gene for real-time PCR, reverse primer.
55 / GGGTCAAAAGGGTTACTTGG / Amplify partial cDNA of S. cerevisiae ARO8 gene for real-time PCR, forward primer.
56 / AACTCAGGGTGGACAGATGC / Amplify partial cDNA of S. cerevisiae ARO8 gene for real-time PCR, reverse primer.
57 / CCCAAAGACACGATTTCCTC / Amplify partial cDNA of S. cerevisiae ARO9 gene for real-time PCR, forward primer.
58 / GGCATCTGTATCCAAAGTCAAG / Amplify partial cDNA of S. cerevisiae ARO9 gene for real-time PCR, reverse primer.
59 / ATCGCTAACGGTGAAAACG / Amplify partial cDNA of S. cerevisiae ARO4 gene for real-time PCR, forward primer.
60 / TCTGACAGCAGCAGCCAAT / Amplify partial cDNA of S. cerevisiae ARO4 gene for real-time PCR, reverse primer.
61 / ATGAGGCAAACCATCCAGG / Amplify partial cDNA of S. cerevisiae ARO7 gene for real-time PCR, forward primer.
62 / GGGTAGTTAATGCTCGGTAAGA / Amplify partial cDNA of S. cerevisiae ARO7 gene for real-time PCR, reverse primer.
63 / CGCTATCTGTCCACTGGGTC / Amplify partial cDNA of S. cerevisiae ALG9 gene for real-time PCR, forward primer.
64 / CAGTCGAATGCGGTTCTGA / Amplify partial cDNA of S. cerevisiae ALG9 gene for real-time PCR, reverse primer.

Supplementary Fig. 1. Schematic representation of the engineered strains. Native promoters were replaced with GPD1 promoter via chromosomal integration. ARO4fbr, ARO7fbr (feedback-resistant variants of ARO4,ARO7) and other genes of L-tyrosine pathway were overexpressed in the dual-promoter vectors pSP-G1, pLC41 and pLC42.

Supplementary Fig. 2. Schematic representation of markless promoter replacement in S. cerevisaie. Promoter replacement cassettes firstly were individually amplified via PCR and co-transformed into yeast cell to form the functional cassette via chromosomal integration; secondly, Cre recombinase was induced with galactose to remove URA3 selection marker; Eventually, Native promoters were replaced with GPD1 promoter.

Supplementary Fig. 3. The schematic maps of dual-promoter vectors pLC41 and pLC42 construction by marker substitution.

Supplementary Fig.4. The qPCR analysis of gene expression levels of the engineered strains compared with NK-L70 strain. Average ± standard deviations were calculated from three biological replicates. Total RNA extraction from yeast cell cultures during exponential growth phase was performed using the RNApure Yeast Kit (CWBIO, Beijing, China). The PrimeScriptTM 1st strand cDNA Synthesis Kit (Takara, Dalian, China) was used to synthesize the first-strand cDNA by reverse transcription PCR. The qRT-PCR was performed using the FS Universal SYBR Green Master (Roche, Mannheim, Germany). The housekeeping gene ALG9 was used as the reference gene.

Reference

Brachmann CB, Davies A, Cost GJ, Caputo E, Li JC, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115-132

Hegemann JH, Heick SB (2011) Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae. Methods in molecular biology 765:189-206

Janke C et al. (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21:947-962

Jansen G, Wu CL, Schade B, Thomas DY, Whiteway M (2005) Drag&Drop cloning in yeast. Gene 344:43-51

Partow S, Siewers V, Bjorn S, Nielsen J, Maury J (2010) Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast 27:955-964

1