Supplementary information

Producing aglycons of ginsenosides in bakers’ yeast

Zhubo Dai1,2§, Beibei Wang 1,2,3§, Yi Liu1,2,3, Mingyu Shi1,2,3, Dong Wang1,2,3, Xianan Zhang4,5, Tao Liu1,2, Luqi Huang4*, Xueli Zhang1,2*

1 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences,2 Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences,3 College of Biotechnology, Tianjin University of Science and Technology,4 National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences,5 School of Traditional Chinese Medicine, Capital Medical University.

§ These authors contribute equally to this work. *Corresponding author at: 32 XiQiDao, Tianjin Airport Economic Park, Tianjin, 300308, China. Tel and Fax: 86-22-84861983. Email address: zhang_xl@tib.cas.cn; *Co-corresponding author at: No. 16 Nanxiaojie, Dongzhimennei Ave, Beijing, 100700, China. Tel.: +86 10 64014411. Fax: +86 10 64013996. Email address: huangluqi@263.net

MethodsPlasmid construction

Construction of p-ΔTrp1: The homologous recombination region of Trp1 was amplified from the genomic DNA of S. cerevisiae BY4742 (using primer sets ZD-TRP1-int-up(400)/ZD-TRP1-int-down(450)) and cloned into pEASY-Blunt, which resulted in p-Trp1. Upstream and downstream regions of the Trp1 gene were amplified from p-Trp1 (using primer sets ZD-TRP1interg.-1/ZD-TRP1interg.-2), and the auxotrophic marker HIS3 was PCR amplified from plasmid pRS313 (using primer sets Loxp-His-p/Pme-Loxp-His-t) and was phosphorylated by T4 Polynucleotide kinase. These two DNA fragments were ligated with T4 ligase, which resulted in p-ΔTrp1.

Construction of pM13-LYS2: The LYS2 gene was amplified from the genomic DNA of S. cerevisiae BY4742 (using primer sets PAC1-LYS-2 /LYS-ASC1) and digested with PacI and AscI. The TEF1 promoter (PTEF1) and CYC1 terminator (TCYC1) were amplified from the genomic DNA of S. cerevisiae BY4742 (using primer sets X-Only-pTEF1-F/X-pTEF1-Pac-R and Asc1-CYC1t/CYC1t-Pme1), and digested with PacI and AscI, respectively. These three digested DNA fragments were ligated using T4 ligase and used as templates for PCR amplification of the PTEF1-LYS2-TCYC1 cassette (using primer sets X-Only-pTEF1-F/CYC1t-Pme1). The amplified product was cloned into pEASY-Blunt simple, resulting in pM13-LYS2.

Construction of pM2-PgbAS: Plasmid p-PgbAS was digested with SexAI and AscI, and cloned into pM2-tHMG1 at its SexAI and AscI sites, resulting in pM2-PgbAS.

Construction of pM2-GgbAS: Plasmid p-GgbAS was digested with SexAI and AscI, and cloned into pM2-tHMG1 at the SexAI and AscI sites, resulting in pM2-GgbAS.

Construction of pM3-MtOAS: MtOAS gene from plasmid p-MtOAS was cloned into pM3-ERG9 at its SexAI and AscI sites, resulting in pM3-MtOAS.

Construction of pM8-SynPgPPTS: Plasmid p-SynPgPPTS was digested with SexAI and AscI. The FBA1 promoter (PFBA1) and TDH2 terminator (T TDH2) were amplified from the genomic DNA of S. cerevisiae BY4742 (using primer sets Pac-pFBA/pFBA-SexA and ASC-tTDH2/tTDH2-Pme1) and digested with SexAI and AscI, respectively. These three digested DNA fragments were ligated with T4 ligase and used as templates for PCR amplification of the PFBA1-SynPgPPTS-TTDH2 cassette (using primer sets Pac-pFBA/ tTDH2-Pme1). The amplified product was cloned into pEASY-Blunt simple, resulting in pM8-SynPgPPTS.

Construction of pHis-TRP: The auxotrophic marker TRP1 was PCR amplified from plasmid pRS314 (using primer sets Bsp-TRP-F/Pme1-TRP-R) and cloned into pEASY-Blunt, resulting in p-Trp1. This plasmid was then digested with PmeI and treated with CIP. The homologous recombination region of HIS3 was amplified from the genomic DNA of S.cerevisiae BY4742 (using primer sets ZD-His3 interg.-up/ZD-His3 interg.-down) and was phosphorylated by T4 Polynucleotide kinase. These two DNA fragments were ligated with T4 ligase, resulting in pHis-TRP.

All the plasmids in this work were summarised in Table S1.

Table S1. Plasmids used in this study

Name Description Source

pRS314 CEN6/ARSH4, TRP1 Sikorski et al.1

pRS313 CEN6/ARSH4, HIS3 Sikorski et al.1

pSH47 CEN6/ARSH4,PGAL1-Cre-TCYC1 cassette, URA3 Guldener et al.2

pEASY-Blunt Cloning vector with multiple cloning sites, Amp, Km

TransGen Biotech

pEASY-Blunt Simple

Cloning vector for blunt ligation, Amp, Km TransGen Biotech

pUC57 Cloning vector with multiple cloning sites, Amp GenScript

p-PgbAS Cloning PgbAS gene into pEASY-Blunt This study

p-GgbAS Cloning GgbAS gene into pUC57 This study

p-MtOAS Cloning MtOAS gene into pUC57 This study

p-SynPgPPTS Cloning SynPgPPTS gene into pUC57 This study

p-ΔTrp1 Trp1DNA site, HIS3 This study

pM2-tHMG1 Cloning PPGK1-tHMG1-TADH1 cassette into pEASY-Blunt

Dai et al.3

pδ-tHMG1 Cloning PPGK1-tHMG1-TADH1 cassette into pδ-UB Dai et al.3

pM13-LYS2 Cloning PTEF1-LYS2-TCYC1 cassette into pEASY-Blunt simple

This study

pM3-ERG9 Cloning PTEF1-ERG9-TCYC1 cassette into pEASY-Blunt simple

Dai et al.3

pM2-PgbAS Cloning PPGK1-PgbAS-TADH1 cassette into pEASY-Blunt

This study

pM2-GgbAS Cloning PPGK1-GgbAS-TADH1 cassette into pEASY-Blunt

This study

pM11-ERG1 Cloning PTDH3-ERG1-TTPI1 cassette into pEASY-Blunt simple

Dai et al.3

prDNA-LEU Cloning rDNA Site and LEU2 marker into pEASY-Blunt

Dai et al.3

pM3-MtOAS Cloning PTEF1-MtOAS-TCYC1 cassette into pEASY-Blunt simple

This study

pM11-AtCPR1 Cloning PTDH3-AtCPR1-TTPI1 cassette into pEASY-Blunt simple

Dai et al.3

pTrp-HIS Cloning Trp1 Site and HIS3 marker into pEASY- Dai et al.3

BluntpM3-SynPgPPDS Cloning PTEF1-SynPgPPDS-TCYC1 cassette into

pEASY-Blunt simpleDai et al.3

pM14-PgDDS Cloning PPGK1-PgDDS-TADH1 cassette into pEASY-Blunt simple

Dai et al.3

pM8-SynPgPPTS Cloning PFBA1-SynPgPPTS-TTDH2 cassette into pEASY-Blunt simple

This study

pHis-TRP Cloning HIS3 Site and Trp1 marker into pEASY-Blunt

This study

1. Sikorski, R.S.& Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122,19-27(1989).

2. Guldener, U., Heck, S., Fielder, T., Beinhauer, J.& Hegemann, J.H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24,2519-2524(1996).

3. Dai, Z. et al. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metab Eng 20,146-156(2013).

Table S2. Primers used for plasmids construction

Primer name Sequence(5' to 3')SexA1-PgPNY GCGACCWGGTAAAACAATGTGGAAGCTTAAGATAGCGGAAGGGAPgPNY-Asc1 GCGGCGCGCCTTAGGTGCCTAGGGACGGTAATGGGAPAC1-LYS-2 GCGTTAATTAAATGACTAACGAAAAGGTCTGGATAGAGLYS-ASC1 GCGGCGCGCCTTAAGCTGCTGCGGAGCTTCCACGAGCX-Only-pTEF1-F AGTGATCCCCCACACACCATAGCTTCX-pTEF1-Pac-R GCGTTAATTAATTTGTAATTAAAACTTAGATTAGATTGCAsc1-CYC1t GCGGCGCGCCCCGCTGATCCTAGAGGGCCGCATCACYC1t-Pme1 GCGGTTTAAACGCGCGTTGGCCGATTCATTAATGCAPac-pFBA GCGTTAATTAAGATCCAACTGGCACCGCTGGCTTGApFBA-SexA GCGACCWGGTTTTGAATATGTATTACTTGGTTATGASC-tTDH2 GCGGCGCGCCATTTAACTCCTTAAGTTACTTTAATtTDH2-Pme1 GCGGTTTAAACGGCGAAAAGCCAATTAGTGTGATACBsp-TRP-F TGGCGTCCGGATACAATCTTGATCCGGAGCTPme1-TRP-R GCGGTTTAAACCACAAACAATACTTAAATAAATACLoxp-His-p GTGCGATAACTTCGTATAGCATACATTATACGAAGTTA

-TTCGCGCGTTTCGGTGATGACGGPme-Loxp-His-t GTGCGGTTTAAACATAACTTCGTATAATGTATGCTATA

-CGAAGTTATGTGTCACTACATAAGAACACCTZD-TRP1-int-up(400) CTTATGGCATGTCTGGCGATGAT

ZD-TRP1-int-down(450） TTGATATTACTGTCAGCGTAGAAGZD-His3interg.-up TCTCCTTTAGCTTCTCGACGTGGGCZD-His3interg.-down GACATGTTTGCCATCGATCCATCTA

1 Nucleotides indicating restriction sites were underlined and bold.

Table S3. Primers used in DNA assembly

Primer name Sequence (5' to 3')

X1-M-pEASY-r-t-F CTTGCAAATGCCTATTGTGCAGATGTTATAATATCTGTGCGTTTAATTAAGGCTCGTATGTTGTGTGGAATTGT

X1-r-t-R-rDNA CTCACTATTTTTTACTGCGGAAGCGG1-M-pEASY-PGK1-F CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA

ACATACGAGCCTTAATTAAACGCACAGATATTATAAC3G -1-M-ADHt-TDH3-R CCTCCGCGTCATTAAACTTCTTGTTGTTGACGCTAACATTCAA

CGCTAGTATTCGGCATGCCGGTAGAGGTGTGG3G -3-M-ADHt-TDH3-F CAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCG

GCATGCCGAATACTAGCGTTGAATGTTAGCGTC3G -3-M-TPI1t-TEF1-R AGGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCAC

TTTAATTAA TCTATATAACAGTTGAAATTTGGA3G -2-M-TPI1t-TEF1-F GTCATTTTCGCGTTGAGAAGATGTTCTTATCCAAATTTCAACT

GTTATATAGATTAATTAAAGTGATCCCCCACACM-CYC1t-pEASY-R CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTG

GGAAAACCCTGGCGCGTTGGCCGATTCATTAATGCX2-r-t-F-rDNA GAACTGGGTTACCCGGGGCACCTGTCX2-M-pEASY-r-t-R CGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAAT

CGGCCAACGCGCCAGGGTTTTCCCAGTCACGACGTTGX1-r-t-R-Trp1 GCAAGAATACCAAGAGTTCCTCGGTX2-r-t-F-Trp1 CACGACTCATCTCCATGCAGTTGGACGA1-M-HisG-PGK1-F GCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCATGA

GCTTCAATACCCTGATTGACTGGAAGTTAATTAAACGCACAGATAT

1-M-ADHt-TEF1-R GGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCACTTTAATTAATCGGCATGCCGGTAGAGGTG

2-M-ADHt-TEF1-F GGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCCGATTAATTAAAGTGATCCCCCA

2-M-CYC1t-δ2-R CGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAAGCGCGTTGGCCGATTCATTA

3-M-CYC1t-δ2-F GGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCTTGTAAAACGACGGCCAGTG

3-δ2-R TCGAGGAGAACTTCTAGTATATTCZD-His3 interg.-1 TTTATATAATGTATAATTCATTZD-His3 interg.-2 CATTCTATACGTGTCATTCTGA4G-1-M-ADHt-FBA1-R CAGAAGTTGGAAGGCTGGTATTGTTGTTCAAGCCAGCGGTG-

CCAGTTGGATCTCGGCATGCCGGTAGAGGTGTGGTC4G-4-M-ADHt-FBA1-F CTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTA -

CCGGCATGCCGA GATCCAACTGGCACCGCTGGCTTGA4G-4-M-TDH2t-TDH3-R GGCCTCCGCGTCATTAAACTTCTTGTTGTTGACGCTAACATT

-CAACGCTAGTATGGCGAAAAGCCAATTAGTGTGATAC4G-3-M-TDH2t-TDH3-F GGCATCACGGATTTTCGATAAAGCACTTAGTATCACACTAAT

-TGGCTTTTCGCCATACTAGCGTTGAATGTTAGCGTCAAC

Fig. S1 Identification of fermentation products of strain BY-T1 and BY-βA-G. This strain was cultivated in YPD medium with 2% glucose for 7 days. (A) GC-MS analysis of squalene (1), ergosterol (2) and lanosterol (3) standards; (B) GC-MS analysis of β-amyrin (4) standard; (C) GC-MS analysis of the cell extraction of strain BY-T1; (D) GC-MS analysis of the cell extraction of strain BY-βA-G; (E) Mass spectra of β-amyrin.

Fig. S2 Production of squalene, lanosterol, ergosterol and β-amyrin by engineered S. cerevisiae strains BY-βA-G and BY-βA-CK. Titres were obtained after fermentation for 7 days. Three replicates were performed, and the error bars represented standard deviation.

Fig. S3 Identification of oleanolic acid that was produced by strain BY-OA. This strain was cultivated in YPD medium with 2% glucose for 7 days. (A) LC-MS analysis of the oleanolic acid standard; (B) LC-MS analysis of the strain BY-βA-G; (C) LC-MS analysis of strain BY-OA; (D) Mass spectra of oleanolic acid.

Fig. S4 Identification of protopanaxadiol that was produced by strain GY-1. This strain was cultivated in YPD medium with 2% glucose for 5 days. (A) LC-MS analysis of the protopanaxadiol standard; (B) LC-MS analysis of the strain BY-OA; (C) LC-MS analysis of strain GY-1; (D) Mass spectra of protopanaxadiol.

Fig. S5 Identification of protopanaxatriol that was produced by strain GY-1. This strain was cultivated in YPD medium with 2% glucose for 5 days. (A) LC-MS analysis of the protopanaxatriol standard; (B) LC-MS analysis of the strain BY-OA; (C) LC-MS analysis of strain GY-1; (D) Mass spectra of protopanaxatriol.