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,
3College 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: ;
*Co-corresponding author at: No. 16 Nanxiaojie, Dongzhimennei Ave, Beijing, 100700, China. Tel.: +86 10 64014411. Fax: +86 10 64013996. Email address:
Methods
Plasmid 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 ofpM13-LYS2:The LYS2 gene was amplified from the genomic DNA of S. cerevisiae BY4742 (usingprimer 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 (usingprimer setsX-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 clonedintopM3-ERG9 at its SexAI and AscI sites, resulting inpM3-MtOAS.
Construction ofpM8-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 (usingprimer 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 ofpHis-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 plasmidsin this work were summarised in Table S1.
Table S1. Plasmids used in this study
Name / Description / SourcepRS314 / CEN6/ARSH4, TRP1 / Sikorski et al.1{Sikorski, 1989 #215}
pRS313 / CEN6/ARSH4, HIS3 / Sikorski et al.1{Sikorski, 1989 #215}
pSH47 / CEN6/ARSH4,PGAL1-Cre-TCYC1cassette, 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 SynPgPPTSgene into pUC57 / This study
p-ΔTrp1 / Trp1DNA site, HIS3 / This study
pM2-tHMG1 / Cloning PPGK1-tHMG1-TADH1 cassette into pEASY-Blunt / Daiet al.3
pδ-tHMG1 / Cloning PPGK1-tHMG1-TADH1 cassette into pδ-UB / Daiet al.3
pM13-LYS2 / Cloning PTEF1-LYS2-TCYC1cassette into pEASY-Blunt simple / This study
pM3-ERG9 / Cloning PTEF1-ERG9-TCYC1cassette into pEASY-Blunt simple / Daiet 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-TTPI1cassette into pEASY-Blunt simple / Daiet al.3
prDNA-LEU / Cloning rDNA Site and LEU2 marker into pEASY-Blunt / Daiet al.3
pM3-MtOAS / Cloning PTEF1-MtOAS-TCYC1cassette into pEASY-Blunt simple / This study
pM11-AtCPR1 / Cloning PTDH3-AtCPR1-TTPI1cassette into pEASY-Blunt simple / Daiet al.3
pTrp-HIS / Cloning Trp1 Siteand HIS3 marker into pEASY-Blunt / Daiet al.3
pM3-SynPgPPDS / Cloning PTEF1-SynPgPPDS-TCYC1 cassette into pEASY-Blunt simple / Daiet al.3
pM14-PgDDS / Cloning PPGK1-PgDDS-TADH1 cassette into pEASY-Blunt simple / Daiet 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. Genetics122,19-27(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 Res24,2519-2524(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'to3')SexA1-PgPNY / GCGACCWGGTAAAACAATGTGGAAGCTTAAGATAGCGGAAGGGA
PgPNY-Asc1 / GCGGCGCGCCTTAGGTGCCTAGGGACGGTAATGGGA
PAC1-LYS-2 / GCGTTAATTAAATGACTAACGAAAAGGTCTGGATAGAG
LYS-ASC1 / GCGGCGCGCCTTAAGCTGCTGCGGAGCTTCCACGAGC
X-Only-pTEF1-F / AGTGATCCCCCACACACCATAGCTTC
X-pTEF1-Pac-R / GCGTTAATTAATTTGTAATTAAAACTTAGATTAGATTGC
Asc1-CYC1t / GCGGCGCGCCCCGCTGATCCTAGAGGGCCGCATCA
CYC1t-Pme1 / GCGGTTTAAACGCGCGTTGGCCGATTCATTAATGCA
Pac-pFBA / GCGTTAATTAAGATCCAACTGGCACCGCTGGCTTGA
pFBA-SexA / GCGACCWGGTTTTGAATATGTATTACTTGGTTATG
ASC-tTDH2 / GCGGCGCGCCATTTAACTCCTTAAGTTACTTTAAT
tTDH2-Pme1 / GCGGTTTAAACGGCGAAAAGCCAATTAGTGTGATAC
Bsp-TRP-F / TGGCGTCCGGATACAATCTTGATCCGGAGCT
Pme1-TRP-R / GCGGTTTAAACCACAAACAATACTTAAATAAATAC
Loxp-His-p / GTGCGATAACTTCGTATAGCATACATTATACGAAGTTA
-TTCGCGCGTTTCGGTGATGACGG
Pme-Loxp-His-t / GTGCGGTTTAAACATAACTTCGTATAATGTATGCTATA
-CGAAGTTATGTGTCACTACATAAGAACACCT
ZD-TRP1-int-up(400) / CTTATGGCATGTCTGGCGATGAT
ZD-TRP1-int-down(450） / TTGATATTACTGTCAGCGTAGAAG
ZD-His3interg.-up / TCTCCTTTAGCTTCTCGACGTGGGC
ZD-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 / CTCACTATTTTTTACTGCGGAAGCGG
1-M-pEASY-PGK1-F / CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCTTAATTAAACGCACAGATATTATAAC
3G -1-M-ADHt-TDH3-R / CCTCCGCGTCATTAAACTTCTTGTTGTTGACGCTAACATTCAACGCTAGTATTCGGCATGCCGGTAGAGGTGTGG
3G -3-M-ADHt-TDH3-F / CAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCCGAATACTAGCGTTGAATGTTAGCGTC
3G -3-M-TPI1t-TEF1-R / AGGAGTAGAAACATTTTGAAGCTATGGTGTGTGGGGGATCACTTTAATTAA TCTATATAACAGTTGAAATTTGGA
3G -2-M-TPI1t-TEF1-F / GTCATTTTCGCGTTGAGAAGATGTTCTTATCCAAATTTCAACTGTTATATAGATTAATTAAAGTGATCCCCCACAC
M-CYC1t-pEASY-R / CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGCGTTGGCCGATTCATTAATGC
X2-r-t-F-rDNA / GAACTGGGTTACCCGGGGCACCTGTC
X2-M-pEASY-r-t-R / CGAAGGCTTTAATTTGCAAGCTGCGGCCCTGCATTAATGAATCGGCCAACGCGCCAGGGTTTTCCCAGTCACGACGTTG
X1-r-t-R-Trp1 / GCAAGAATACCAAGAGTTCCTCGGT
X2-r-t-F-Trp1 / CACGACTCATCTCCATGCAGTTGGACGA
1-M-HisG-PGK1-F / GCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCATGAGCTTCAATACCCTGATTGACTGGAAGTTAATTAAACGCACAGATAT
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 / TCGAGGAGAACTTCTAGTATATTC
ZD-His3 interg.-1 / TTTATATAATGTATAATTCATT
ZD-His3 interg.-2 / CATTCTATACGTGTCATTCTGA
4G-1-M-ADHt-FBA1-R / CAGAAGTTGGAAGGCTGGTATTGTTGTTCAAGCCAGCGGTG-CCAGTTGGATCTCGGCATGCCGGTAGAGGTGTGGTC
4G-4-M-ADHt-FBA1-F / CTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTA-CCGGCATGCCGA GATCCAACTGGCACCGCTGGCTTGA
4G-4-M-TDH2t-TDH3-R / GGCCTCCGCGTCATTAAACTTCTTGTTGTTGACGCTAACATT
-CAACGCTAGTATGGCGAAAAGCCAATTAGTGTGATAC
4G-3-M-TDH2t-TDH3-F / GGCATCACGGATTTTCGATAAAGCACTTAGTATCACACTAAT
-TGGCTTTTCGCCATACTAGCGTTGAATGTTAGCGTCAAC
Fig. S1 Identification of fermentation products of strain BY-T1and 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 ofβ-amyrin (4) standard; (C) GC-MS analysis ofthe cell extraction of strain BY-T1; (D) GC-MS analysis of the cell extraction of strain BY-βA-G; (E) Mass spectra ofβ-amyrin.
Fig. S2Production of squalene, lanosterol, ergosterol and β-amyrin by engineered S. cerevisiae strainsBY-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 acidthat 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 protopanaxadiolthat 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 ofprotopanaxadiol.
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.