Supplementary information for:
Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae
Zhubo Dai1†, Yi Liu1,3†, Luqi Huang2*, Xueli Zhang1*
1
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences
2
Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Science
3
College of Biotechnology, Tianjin University of Science & Technology
Materials and methods
Plasmids construction
Cloning of genes, promoters and transcriptional terminators
DNA fragments containing tHMGR gene, UPC2 gene, ERG20 gene, BTS1 gene,
TEF promoter (PTEF1), PGK promoter (PPGK1), ADH promoter (PADH1), ADH
transcriptional terminator (TADH1), upstream (1-234 bp) and downstream (235 To 474
bp) region of δDNA (δDNA1 and δDNA2) were PCR amplified from the genomic
DNA of S. cerevisiae BY4742 using corresponding primer pairs, which were then
cloned into pEASY-Blunt (TransGen Biotech, Beijing, China), resulting in p-tHMGR,
p-UPC2, p-ERG20, p-BTS1, p-TEF1, p-PGK1, p-ADH1, p-ADH1t, p-δDNA1 and
p-δDNA2 (Table 1), respectively.
For creating a mutation in the UPC2 gene, a DNA fragment was PCR amplified
from p-UPC2 using primer pair 5/6, digested with DpnI and self-ligated with T4
ligase, resulting in p-UPC2.1. This plasmid was sequenced for verification of the
mutation. For creating a fusion ERG20/BTS1 gene, the ERG20 and BTS1 genes were
PCR amplified from plasmids p-ERG20 and p-BTS1 using primer pairs 13/14 and
15/16 respectively, which were then used as templates for amplification of the
ERG20/BTS1 fusion gene using primer pair 13/16 as described previously (Tokuhiro
et al., 2009). The amplified ERG20/BTS1 fusion gene was cloned into pEASY-Blunt,
resulting in p-ERG20/BTS1.
A heterologous GGPP synthase gene from Sulfolobus acidocaldarius (SaGGPS)
was synthesized by Biosunne (Shanghai, China) with codon optimization for
improved expression in S. cerevisiae. The SmCPS and SmKSL genes were PCR
amplified from pET32a(+) vectors harboring the open reading frame of SmCPS and
SmKSL (Gao et al., 2009) using primer pairs 42/43 and 44/45, and cloned into
pEASY-Blunt, resulting in p-SmCPS and p-SmKSL respectively.
Construction of plasmids containing the tHMGR-UPC2.1 cassette
For expression of tHMGR and UPC2.1 in a plasmid with low copy number,
plasmid pLLeu-tHMGR-UPC2.1 was constructed by the following steps.
Construction of pHUra-δDNA. p-δDNA1 was digested with KpnI and BstEI,
and p-δDNA2 was digested with BstEI and SacI. These two digested DNA fragments
were ligated with T4 ligase and used as a template for PCR amplification using primer
pair 7/9. The amplified product was cloned into pRS406 at KpnI and SacI sites,
resulting in plasmid pRS406-δDNA. The auxotrophic marker URA3 was PCR
amplified from plasmid pYES2.0/CT using primer pair 11/12 and cloned into
pRS406-δDNA at BstEI sites, resulting in pHUra-δDNA (Fig. S1).
Construction of pHUra-δDNA-tHMGR-A, pHUra-δDNA-tHMGR-T and
pHUra-δDNA-tHMGR-P. p-ADH1 was digested with SexAI and SacII, and
p-tHMGR was digested with SexAI and AscI. These two digested DNA framgents
were ligated with T4 ligase and used as a template for PCR amplification using primer
pair 23/2. The amplified product was cloned into pEASY-Blunt, resulting in
p-ADH1-tHMGR. p-ADH1t was digested with AscI and PmeI, and p-ADH1-tHMGR
was digested with SacII and AscI. These two digested DNA framgents were ligated
with T4 ligase and used as a template for PCR amplification using primer pair 23/26.
The amplified product was cloned into pEASY-Blunt, resulting in plasmid
p-ADH1-tHMGR-ADH1t. The PADH1-tHMGR-TADH1 cassette was then cloned into
pRS406-δDNA at SacII sites, resulting in pHUra-δDNA-tHMGR-A (Fig. S1). TEF1
promoter from plasmid p-TEF1 was cloned into pHUra-δDNA-tHMGR-A at SexAI
and PacI sites, resulting in pHUra-δDNA-tHMGR-T (Fig. S1). PGK1 promoter from
plasmid p-PGK1 was cloned into pHUra-δDNA-tHMGR-A at SexAI and PacI sites,
resulting in pHUra-δDNA-tHMGR-P.
Construction of pHUra-δDNA-tHMGR-UPC2.1. The UPC2.1 gene was
cloned into pHUra-δDNA-tHMGR-T at SexAI and AscI sites, resulting in
pHUra-δDNA-UPC2.1. The PTEF1-UPC2.1-TADH1 cassette was PCR amplified from
pHUra-δDNA-UPC2.1
using
primer
pair
28/26,
and
cloned
into
pHUra-δDNA-tHMGR-A at PmeI sites, resulting in pHUra-δDNA-tHMGR-UPC2.1
(Fig. S1).
Construction of pLLeu-tHMGR-UPC2.1. The tHMGR-UPC2.1 cassette was
obtained by digesting pHUra-δDNA-tHMGR-UPC2.1 with SacII, which was then
cloned into pRS313 at SacII sites, resulting in pLHis-tHMGR-UPC2.1. The
auxotrophic selection marker Leu was PCR amplified from pRS425 using primer pair
37/38, phosphorylated by T4 Polynucleotide kinase and ligated with a DNA fragment
amplified from plasmid pLHis-tHMGR-UPC2.1 using primer pair 31/32, resulting in
pLLeu-tHMGR-UPC2.1 (Fig. S2).
For expression of tHMGR and UPC2.1 in a plasmid with high copy number,
plasmid pHLeu-tHMGR-UPC2.1 was constructed. The tHMGR-UPC2.1 cassette
was obtained by digesting pHUra-δDNA-tHMGR-UPC2.1 with SacII, which was then
cloned into pRS425 at SacII sites, resulting in pHLeu-tHMGR-UPC2.1 (Fig. S2).
For expression of tHMGR and UPC2.1 in plasmids with antibiotic markers,
plasmids pLKanMX-tHMGR-UPC2.1 and pHKanMX-tHMGR-UPC2.1 were
constructed (Fig. S3). The antibiotic marker KanMX was PCR amplified from
pRS41K using primer pair 39/40, phosphorylated by T4 Polynucleotide kinase and
ligated with a DNA fragment amplified from plasmid pLLeu-tHMGR-UPC2.1 using
primer pair 31/32, resulting in pLKanMX-tHMGR-UPC2.1. This phosphorylated
DNA fragment was also ligated with a DNA fragment amplified from plasmid
pHLeu-tHMGR-UPC2.1
using
primer
pair
33/34,
resulting
in
pHKanMX-tHMGR-UPC2.1.
Construction of plasmids containing the ERG20/BTS1-SaGGPS cassette
For expression of ERG20/BTS1 and SaGGPS in a plasmid with low copy number
and antibiotic marker, plasmid pLhphNT1-ERG20/BTS1-SaGGPS was constructed
by the following steps. The ERG20/BTS1 fusion gene was cloned into
pHUra-δDNA-tHMGR-A
at
SexAI
pHUra-δDNA-ERG20/BTS1-A.
The
pHUra-δDNA-tHMGR-P
SexAI
at
and
AscI
SaGGPSS
and
gene
AscI
sites,
was
sites,
resulting
cloned
resulting
in
into
in
pHUra-δDNA-SaGGPS-P. The PPGK1-SaGGPS-TADH1 cassette was obtained by PCR
amplification from pHUra-δDNA-SaGGPS-P using primer pair 27/26, which was then
cloned
into
pHUra-δDNA-ERG20/BTS1-A
at
PmeI
sites,
resulting
in
pHUra-δDNA-ERG20/BTS1-SaGGPS (Fig. S4). The ERG20/BTS1-SaGGPS cassette
was obtained by digesting pHUra-δDNA-ERG20/BTS1-SaGGPS with SacII, which
was
then
cloned
into
pRS316
at
SacII
sites,
resulting
in
pLUra-ERG20/BTS1-SaGGPS. The antibiotic selection marker hphNT1 was PCR
amplified from pRS42H using primer pair 39/41, phosphorylated by T4
Polynucleotide kinase and ligated with a DNA fragment amplified from plasmid
pLUra-ERG20/BTS1-SaGGPS
using
primer
pair
29/30,
resulting
in
pLhphNT1-ERG20/BTS1-SaGGPS (Fig. S5).
For expression of ERG20/BTS1 and SaGGPS in a plasmid with high copy
number and antibiotic marker, plasmid pHhphNT1-ERG20/BTS1-SaGGPS was
constructed. The ERG20/BTS1-SaGGPS cassette was obtained by digesting
pHUra-δDNA-ERG20/BTS1-SaGGPS with SacII, which was then cloned into
pRS426 at SacII sites, resulting in pHUra-ERG20/BTS1-SaGGPS. The antibiotic
selection marker hphNT1 was PCR amplified from pRS42H using primer pair 39/41,
phosphorylated by T4 Polynucleotide kinase and ligated with a DNA fragment
amplified from plasmid pHUra-ERG20/BTS1-SaGGPS using primer pair 29/30,
resulting in pHhphNT1-ERG20/BTS1-SaGGPS (Fig. S5).
For expression of ERG20/BTS1 and SaGGPS in plasmids with auxotrophic
markers,
plasmids
pLHis-ERG20/BTS1-SaGGPS
and
pHHis-ERG20/BTS1-SaGGPS were constructed. The auxotrophic marker His3 was
PCR amplified from pRS313 using primer pair 35 and 36, phosphorylated by T4
Polynucleotide kinase and ligated with a DNA fragment amplified from plasmid
pLhphNT1-ERG20/BTS1-SaGGPS
using
primer
pair
29/30,
resulting
in
pLHis-ERG20/BTS1-SaGGPS (Fig. S6). This phosphorylated DNA fragment was
also
ligated
with
a
DNA
pHhphNT1-ERG20/BTS1-SaGGPS
fragment
using
primer
amplified
pair
from
29/30,
pHHis-ERG20/BTS1-SaGGPS (Fig. S6).
Construction of plasmids for integration of SmCPS and SmKSL
plasmid
resulting
in
For integration of an expression cassette for SmCPS and SmKSL, plasmid
pHUra-δDNA-SmCPS-SmKSL was constructed by the following steps (Fig. S7).
The SmCPS gene was truncated to a pseudomature construct by PCR amplification
from plasmid p-SmCPS using primer pair 42/43, removing the first 222 bp of the gene
and adding an initiating ATG codon, as described previously (Gao et al., 2009). This
truncated SmCPS gene was then cloned into pHUra-δDNA-tHMGR-T at SexAI and
AscI sites, resulting in pHUra-δDNA-SmCPS-T. The SmKSL gene was PCR amplified
from
plasmid
p-SmKSL
pHUra-δDNA-tHMGR-P
using
at
primer
SexAI
and
pair
44/45,
AscI
and
sites,
cloned
into
resulting
in
pHUra-δDNA-SmKSL-P. The PPGK1-SmKSL-TADH1 cassette was obtained by PCR
amplification from pHUra-δDNA-SmKSL-P using primer pair 27/26, and cloned into
pHUra-δDNA-SmCPS-T at PmeI sites, resulting in pHUra-δDNA-SmCPS-SmKSL.
Table S1. Primers used for plasmids construction
Primer
number
Sequence (5' to 3')
1
TCGCGACCWGGTAAAACAATGGCTGCAGACCAATTGG
2
TCGCGGCGCGCCTTAGGATTTAATGCAGGTGACGGAC
3
TCGCGACCWGGTAAAACAATGAGCGAAGTCGGTATACAG
4
TCGCGGCGCGCCTCATAACGAAAAATCAGAGAAATTTG
5
TGGAGGTGGTGATATGCATATGATG
6
CATCATATGCATATCACCACCTCCA
7
GCGGAGCTCCCTAGGATGAAGCAGGTGTTGTTGTCTGT
8
TCGCGGGTCACCGAGGAGAACTTCTAGTATATTC
9
TCGCGGGTACCCCTAGG GAGGATATAGGAATCCTCA
10
TCGCGGGTCACCTGTTGGAATAGAAATCAACTATC
11
GCGGGTCACCGCGG TCCGGAACTCTTCCTTTTTCAATGGGTA
12
GGGTCACC TCCGGAGCTAGCTTTTCAATTCAATTCATC
13
TCGGTGGTGGTTCTATGGCTTCAGAAAAAGAAATTAG
14
TCGCGGCGCGCCCTATTTGCTTCTCTTGTAAACTTTG
15
GCG ACCWGGTAAAACAATGGAGGCCAAGATAGATGAG
16
TCTGTTCATAGAACCACCACCCAATTCGGATAAGTGGTCTAT
17
GTGCGACCWGGTAAAACAATGTCATACTTCGATAACTACT
18
TGTCGCGGCGCGCCTTATTTTCTTCTTCTGATAGTGA
19
GTGCGTTAATTAAACGCACAGATATTATAACATC
20
TGGCGACCWGGTTGTTTTATATTTGTTGTAAAAAGTA
21
GTGCGTTAATTAAAGTGATCCCCCACACACCATAG
22
TGGCGACCWGGTTTTGTAATTAAAACTTAGATTAGA
23
TCGCGCCGCGG TTAATTAAGGGATCGAAGAAATGATGGTAAATGA
24
GCGACCWGGTTGTATATGAGATAGTTGATTGTATG
25
GCGGCGCGCCAGTTATAAAAAAAATAAGTGTATAC
26
GCGCCGCGGGTTTAAACTCGGCATGCCGGTAGAGGTGTGGTC
27
GTGCGGTTTAAACACGCACAGATATTATAACATC
28
GTGCGGTTTAAACAGTGATCCCCCACACACCATAG
29
GATTTATCTTCGTTTCCTGCAG
30
AAACTCACAAATTAGAGCTTCA
31
CTTTGCCTTCGTTTATCTTGC
32
TATATGTATACCTATGAATGTCAG
33
GAAATTCATAATAGAAACGACACGA
34
TGGCAAAACGACGATCTTCTTAGGG
35
TGGCGTCCGGATCGCGCGTTTCGGTGATGACGG
36
TGGCGTCCGGAGTGTCACTACATAAGAACACCT
37
TGGCGTCCGGATTAAGCAAGGATTTTCTTAACTTCTTC
38
TGGCGTCCGGAGATGCGGTATTTTCTCCTTACGCA
39
GTGCGATAACTTCGTATAGCATACATTATACGA
-AGTTATCAGCGACATGGAGGCCCAGAATACC
40
GTGCGGTTTAAACATAACTTCGTATAATGTATGCTAT
-ACGAAGTTATTCGACACTGGATGGCGGCGTTAGTA
41
GTGCG GTTTAAACATAACTTCGTATAATGTATGCTA
-TACGAAGTTATCGTCCCAAAACCTTCTCAAGCAAG
42
GCGACCWGGTAAAACAATGCATCAAGGCCATGATGC
43
GCGGCGCGCCTTACGCGACTGGCTCGAAAAGCACT
44
GCGACCWGGTAAAACAATGTCGCTCGCCTTCAACC
45
GCGGCGCGCCTCATTTCCCTCTCACATTATTAG
46
ATGAAGCAGGTGTTGTTGTCTGT
47
CCTAGG GAGGATATAGGAATCCTCA
1 Nucleotides
indicating restriction sites are underlined and bold.
Figure legends.
Fig. S1 Construction of plasmid pHUra-δDNA-tHMGR-UPC2.1
Fig.
S2
Construction
pHLeu-tHMGR-UPC2.1
Fig.
S3
Construction
of
pHKanMX-tHMGR-UPC2.1
of
plasmids
plasmids
pLLeu-tHMGR-UPC2.1
and
pLKanMX-tHMGR-UPC2.1
and
Fig. S4 Construction of ERG20/BTS1-SaGGPS cassette
Fig. S5 Construction of plasmids
pHhphNT1-ERG20/BTS1-SaGGPS
Fig. S6 Construction of
pHHis-ERG20/BTS1-SaGGPS
pLhphNT1-ERG20/BTS1-SaGGPS
plasmids
pLHis-ERG20/BTS1-SaGGPS
Fig. S7 Construction of plasmid pHUra-δDNA-SmCPS-SmKSL
and
and