Supplementary Information
1
2
3
Engineering of chromosomal wax ester synthase integrated
4
Saccharomyces cerevisiae mutants for improved biosynthesis of
5
fatty acid ethyl esters
6
Shuobo Shi1, 2, Juan Octavio Valle-Rodríguez 1, Verena Siewers1 and Jens Nielsen1, *
7
8
Supplementary Text S1. Details of plasmid construction and deletion of RAD52.
9
Supplementary Text S2. Procedures for fermentation and details of analytics.
10
Supplementary Table SI List of primers used in this study.
11
Supplementary Table SII The sequence of codon optimized gapN from Streptococcus
12
mutans for expression in a yeast host. The following sequence was synthesized and provided
13
by DNA2.0 (Menlo Park, CA, USA).
14
Supplementary Figure S1 Schematic construction of the chromosomal integration cassette,
15
which is obtained by fusion PCR and contains the wax ester synthase gene (ws2) controlled
16
by the PGK1 promoter and a selectable marker (neor). This cassette is flanked with
17
homologous sequences to delta-sites to facilitate genome integrations through homologous
18
recombination.
19
Supplementary Figure S2 Schematic presentation of the construction of delta-integration
20
strains. Multiple gene copies are obtained by selection on plates with increasing antibiotics
21
concentration after repeated transformation with integration cassette that is flanked with
22
homologous sequences to delta-sites. Each integration event leads to integration of one
1
antibiotic resistance gene and one gene of interest. Therefore, strains with desired gene copy
2
numbers (× 𝑁) are generated through plating on certain antibiotic concentrations.
3
Supplementary Figure S3 Photographs of agarose gels containing the PCR products
4
confirming the gene deletion of RAD52. Primer pairs used: RAD52-Con-For and RAD52-
5
Con-Rev; DNA templates were genomic DNA of: 1) RAD52 deletion mutant, 2) CB2I20.
6
PCR product for amplification and expected sizes were as follows: 1) 1370 bps for RAD52
7
deletion mutant, 2) 1538 bps for wild-type RAD52.
8
9
Supplementary Text S1. Details of plasmid construction and deletion of RAD52.
10
A DNA fragment containing a delta-sequence element with the neor gene was amplified by
11
PCR using Phusion DNA polymerase (Finnzymes, Vantaa, Finland) from plasmid pJEF1105
12
(Wang et al. 1996) with primers listed in Supplementary Table SI and restricted with
13
AscI/AfeI. The AscI/AfeI digested DNA fragment was ligated into the AscI/AfeI sites of vector
14
pSP-GM2 (Partow et al. 2010) to construct pCIChE. The codon optimized wax ester synthase
15
gene (ws2) from M. hydrocarbonoclasticus DSM 8798 was amplified by PCR using Phusion
16
DNA polymerase from CB2 (Shi et al. 2012) with primers listed in Supplementary Table SI
17
and restricted with NotI/SacI. The NotI/SacI digested DNA fragments were ligated into the
18
NotI/SacI sites of vector pCIChE and pSP-GM2 to construct pCIChE-WS and pSP-B2N,
19
respectively. Plasmid pSP-B2N was used for generating the control FAEE producing strain
20
based on plasmid-mediated gene expression. The ACT1 gene was amplified from genomic
21
DNA of S. cerevisiae with primers listed in Supplementary Table SI and ligated to the
22
SalI/NheI sites of vector pCIChE-WS. The resulting plasmid was named pCWA and was
23
used for generating the standard curve for determination of the gene copy number.
1
The ACB1 gene was amplified by PCR using Phusion polymerase from genomic DNA of S.
2
cerevisiae with the oligonucleotide primers listed in Supplementary Table SI and restricted
3
with BamHI/KpnI. The BamHI/KpnI digested DNA fragment was ligated into the
4
BamHI/KpnI sites of vector pSP-GM2 to construct pSP-A. The sequence of the gapN gene,
5
encoding
6
dehydrogenase (GAPN), from Streptococcus mutans was codon optimized for expression in S.
7
cerevisiae. It was synthesized and provided by DNA2.0 (Menlo Park, CA, USA). The gene
8
sequence is listed in Supplementary Table SII. The gapN gene was restricted with NotI/SacI,
9
and ligated into the NotI/SacI sites of vector pSP-A to construct pSP-GA.
non-phosphorylating,
NADP+-dependent
glyceraldehyde-3-phosphate
10
Deletion of RAD52 was performed by replacement with a nourseothricin resistance cassette.
11
The cassette containing the nat1 gene from Streptomyces noursei encoding nourseothricin N-
12
acetyl-transferase was amplified from pAG25 (Goldstein and McCusker 1999) with primers
13
RAD52-Del-For and RAD52-Del-Rev listed in Supplementary Table SI. The 5' ends of the
14
primers contained sequences homologous to RAD52. Transformants were picked from agar
15
plates containing the nourseothricin antibiotic and confirmed by PCR with primers RAD52-
16
Con-For and RAD52-Con-Rev listed in Supplementary Table SI (PCR results are presented
17
in the Supplementary Figure S3).
18
19
20
Supplementary Text S2. Procedures for fermentation and analytics.
21
Growth conditions
22
E. coli recombinant cells were grown in LB (Luria Bertani) medium in the presence of
23
ampicillin (100 mg/l) at 37 °C.
1
A series of YPD plates (1% bacto-yeast extract, 2% bacto-peptone, 2% agar, and 2% glucose)
2
supplemented with G418 (Sigma-Aldrich, USA) at different concentrations (from 0.2 to 20
3
mg/ml) was used for the selection of transformed integration strains. For FAEE detection,
4
strains were cultured in 500 ml shake flasks and 100 ml synthetic dextrose (SD) medium
5
containing 2% (w/v) glucose, 6.7 g l-1 yeast nitrogen base without amino acids (Formedium,
6
Hunstanton, UK), and complete supplement mixture (0.750 g l-1; CSM, Formedium) at 30°C
7
with reciprocal shaking at 120 rpm, which were inoculated at an optical density at 600 nm
8
(OD600) of 0.02 from pre-cultures. G418 was added as required. The growth was measured by
9
OD600 and samples for FAEE analysis were taken in stationary phase.
10
Analytical methods
11
Cell growth was monitored by OD600. The dry cell weight (DCW), the concentrations of
12
residual glucose and external metabolites were determined as described previously (Shi et al.
13
2012). The exponential growth rate was calculated by log-linear regression analysis of
14
biomass versus incubation time. Yields on substrate and specific rates were calculated
15
according to Nielsen et al (Nielsen et al. 2003).
16
Details of quantitative PCR (qPCR) for determination of gene copy number
17
The yeast DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) was employed for isolating
18
total DNA from fast frozen cell samples. The wax ester synthase gene ws2 and the G418
19
resistance gene neor were selected as target genes, while ACT1 encoding actin was selected as
20
reference gene. ws2 and neor are present in a single copy on the integration cassette. The
21
ACT1 gene exists as a single copy per genome (Ng and Abelson 1980). Plasmid pCWA
22
containing one copy each of neor, ACT1 and ws2 was adopted as the template for standard
23
curves. The sizes of haploid S. cerevisiae genome and plasmid pCWA are 12’495,682 bp and
24
8,137 bp, respectively.
1
According to the following equation modified from Whelan et al. (Whelan et al. 2003)
2
3
𝑔
𝑑𝑠𝐷𝑁𝐴 𝑀𝑊𝑎𝑣 ( ⁄𝑚𝑜𝑙 )
𝐷𝑁𝐴 𝑒𝑛𝑡𝑖𝑡𝑦 𝑚𝑎𝑠𝑠 (𝑔) = 𝐷𝑁𝐴 𝑒𝑛𝑡𝑖𝑡𝑦 𝑠𝑖𝑧𝑒 (𝑏𝑝) [
]
𝑏𝑝
𝐴𝑣𝑜𝑔𝑎𝑑𝑟𝑜′ 𝑠 𝑁𝑜. ( ⁄𝑚𝑜𝑙 )
4
where DNA entity represents the genome or plasmid, dsDNA MWav the average double-
5
stranded DNA molecular weight (617 g/mol) and Avogadro’s No. 6.022 x 1023 bp/mol the
6
mass of S. cerevisiae genomic DNA is 0.0128 pg and it contains one copy of the ACT1 gene
7
and an unknown number of neor and ws2; the mass of plasmid pCWA is 8.34 × 10-6 pg and it
8
contains one copy of the three genes neor, ACT1 and ws2. Oligonucleotide primers for qPCR
9
are listed in Supplementary Table S1. They were designed and chosen from DNA fragments
10
utilizing the program Primer 3 Version 0.4.0 (Rozen and Skaletsky) and their properties were
11
confirmed with NetPrimer software (PREMIER Biosoft International, Palo Alto, CA, USA,
12
http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html). They all have a
13
predicted melting temperature (Tm) of 60°C and a product amplicon length between 230 and
14
245 bp. Agarose gel electrophoresis and melting curve analysis were employed to exclude
15
any possible primer-dimer and unspecific amplicons. A Stratagene Mx3005P instrument
16
(Agilent Technologies Division, La Jolla, CA, USA) was used for qPCR analysis. qPCRs
17
were prepared in triplicates in Agilent optical 96-well reaction plates. Each experimental
18
reaction consisted of 20 µl containing 10 µl of 2× Brilliant III Ultra-Fast SYBR® Green
19
QPCR Master Mix (Agilent Technologies), 4 µl of 2 µM aliquots of each forward and reverse
20
primer (for a final concentration of 400 nM) and 2 µl of a series of diluted plasmid or
21
genomic DNA as template. DNA concentrations were determined utilizing a NanoDrop TM
22
2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA).
23
Serial 10-fold dilutions, ranging from 60 to 6’000,000 copy numbers, of both plasmid pCWA
1
and genomic DNA were analyzed in triplicates to establish the standard curves. A negative
2
control reaction was set for each pair of primers by substituting the template DNA with
3
nuclease-free water, these Cq values were found above 33. The PCR program consisted of an
4
initial denaturation at 95°C for 3 min and then 40 cycles of two steps: 95°C for 20 sec and
5
60°C for 20 sec. ACT1 was used as reference, and its absolute copy numbers were calculated
6
from the amount of initial genomic DNA. The copy numbers of ws2 and neor were calculated
7
using a modified version of a relative quantification equation (Pfaffl 2001):
8
𝐶𝑞
𝑁0 𝐴𝑡𝑎𝑟 (1 + 𝐸𝐵𝑟𝑒𝑓 ) 𝐵𝑟𝑒𝑓 (1 + 𝐸𝐴𝑡𝑎𝑟 )𝐶𝑞𝐴𝑡𝑎𝑟
=
𝑁0 𝐴𝑟𝑒𝑓 (1 + 𝐸𝐴𝑟𝑒𝑓 )𝐶𝑞𝐴𝑟𝑒𝑓 (1 + 𝐸𝐵𝑡𝑎𝑟 )𝐶𝑞𝐵𝑡𝑎𝑟
9
10
where N0 represents the initial number of DNA molecules, Atar refers to a target gene (ws2 or
11
neor) in a mutant strain, Bref refers to the reference gene (ACT1) in plasmid pCWA, E
12
represents PCR efficiency and Cq represents the threshold quantification cycle.
13
14
PCR efficiencies (E) were obtained from the slopes of standard curves. Each standard curve
15
consisted of a linearly fitted plot of threshold quantification cycles (Cq) vs. the decimal
16
logarithm of relative copy numbers. Cq values were determined after setting the threshold line
17
of baseline substracted fluorescence reading normalized to the reference dye (from 1,000 to
18
7,000) in a semi-logarithmic scale for each standard curve. E values ranged between 85 and
19
98%.
20
Details of quantitative RT-PCR (qRT-PCR) for determination of relative gene expression
21
levels
1
Samples for determination of relative gene expression levels utilizing quantitative RT-PCR
2
were taken from yeast cells during exponential growth phase cultivated at 30°C in shake
3
flasks containing 100 ml SD medium and stored at -80°C until processing. Total RNA was
4
extracted from cells employing mechanical disruption with glass beads and using the
5
RNeasy® Mini Kit (QIAGEN GmbH, Hilden, Germany). Its quality was assayed using an
6
Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA was used to synthesize cDNA
7
using the GeneChip® 3’ IVT Express Kit (Affymetrix, Santa Clara, CA, USA) by adding 100
8
ng of total RNA to a final reaction volume of 20 µl. 2 µl of the cDNA were used as template
9
with 10 µl of 2× Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent
10
Technologies) and 4 µl of 2 µM aliquots of each forward and reverse primer (for a final
11
concentration of 400 nM). RNA and DNA concentrations were determined utilizing a
12
NanoDropTM 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). Reactions were
13
processed in a Stratagene Mx3005P instrument (Agilent Technologies). Primers were the
14
same as the primers for determination of gene copy number (Supplementary Table S1). The
15
qPCR protocol followed was as the one stated in the previous section. The relative abundance
16
of each transcript was calculated as relative to S. cerevisiae ACT1 expression levels using the
17
same method as for determination of gene copy number.
18
Enzyme activity assay
19
Cell-free extracts were prepared using a previously reported fast prep method for enzyme
20
analysis (Shi et al. 2012). Protein concentration was measured using a BCA protein assay kit
21
(Pierce Chemical Company, Rockford, IL, USA). WS activities in the extracts were measured
22
in vitro using [1-14C] palmitoyl-CoA and ethanol as substrates (Shi et al. 2012).
23
24
1
2
Supplementary Table SI. List of primers used in this study
Primer
For plasmid construction
Wax
ester Forward
synthase
gene
Reverse
(ws2)
ACB1
Forward
Reverse
Delta-sequence
Forward
element with a
neor gene
Reverse
ACT1
Forward
RAD52
Del-For
Sequence 5'→3'
ACAACAAATATAAAACAAGCGGCCGCACTATGA
AGAGATTAGGTACTCTA
GGCGAAGAATTGTTAATTAAGAGCTCGGTACCC
CAAGCTTGGGTTA
AGTTTTAATTACAAGGATCCACTATGGTTTCCCA
ATTATTCG
GCGGATCTTAGCTAGCCGCGGTACC
CTAAGAGGAGTACTTGGCA
TTGTTAGGGCGCGCCCTAACAAATGGATTCATT
AGATC
GAAACAAGCGCTCATGAGCCCGAAGTGGCG
CTATAGGGCCCGGGCGTCGACACTATTTTTCACT
CTCCCATAACC
Reverse
GTTAGAGCGGATCTTAGCTAGCCAGATTCCAAA
CCCAAAACAG
For deletion of RAD52 and confirmation
Con-For
GCTTTGGTGTGTTGTTGATGAATGAAATTATGGA
TATGGATGAGAAGAGCTTGCCTTGTCCC
GTTTCGGCCAGGAAGCGTTTCAAGTAGGCTTGC
GTGCATGCAGGGTCGACACTGGATGGC
AAGGAGGTTGCCAAGAACTGCTG
Con-Rev
GAGTAACTAGAGGATTTTGGAG
Del-Rev
For delta-integration cassette construction
Primer 1
GTTGGGATTCCATTGTTGATAAAGGCGGAAGTA
CCTTCAAAGAATGGGGTC
CTTGTGCAATGTAGAGCGACCTCATGCTATACCT
GAG
ATGAGGTCGCTCTACATTGCACAAGATAAAAAT
ATATCATCATGAAC
GCCTTTATCAACAATGGAATCCCAACCGCCGTC
CCGTCAAGT
Primer 2
Primer 3
Primer 4
For qPCR and qRT-PCR
Wax
synthase
(ws2)
ACT1
neor
ester q-Forward
gene
q-Reverse
CGAAGGAGCATCTCCAAAAG
ATGTGATGTTCAAGGCACCA
q-Forward
ATCAAACAGAGAAAAGATGACTCAAAT
q-Reverse
ATCAAGTAGTCAGTCAAATCTCTACCG
q-Forward
CAGTTTAGTCTGACCATCTCATCTGTA
q-Reverse
AAACAGTAATACAAGGGGTGTTATGAG
1
Supplementary Table SII. The sequence of codon optimized gapN from Streptococcus
2
mutans for expression in a yeast host. The following sequence was synthesized and provided
3
by DNA2.0 (Menlo Park, Calif.)
4
5
Name
Optimized gapN from Streptococcus mutans
Sequence
ATGACAAAACAATACAAAAACTACGTTAATGGTGAATGGAAACTAA
GTGAGAATGAAATCAAGATATACGAACCTGCCTCAGGCGCAGAACT
GGGTTCTGTTCCTGCAATGTCCACTGAGGAAGTGGATTACGTGTATG
CTTCAGCCAAAAAGGCTCAGCCTGCATGGAGATCCCTAAGTTACATT
GAAAGAGCTGCCTATTTGCATAAAGTCGCAGACATATTGATGAGGGA
TAAAGAGAAGATTGGCGCTGTGCTTTCTAAGGAAGTCGCTAAGGGAT
ACAAATCTGCAGTATCTGAGGTAGTTAGAACAGCAGAGATTATCAAT
TACGCTGCCGAGGAAGGTCTTAGAATGGAGGGAGAGGTACTTGAAG
GAGGATCATTTGAAGCAGCATCCAAAAAGAAGATCGCTGTAGTAAG
GAGAGAACCAGTAGGCCTTGTTCTAGCCATCAGTCCTTTCAACTATC
CAGTCAACTTAGCTGGCTCCAAAATCGCTCCTGCCTTAATCGCTGGTA
ATGTCATTGCTTTCAAGCCACCTACTCAAGGGTCTATTTCAGGTTTGT
TGTTGGCCGAGGCTTTTGCTGAAGCAGGTCTGCCAGCTGGTGTTTTCA
ATACAATTACAGGTAGAGGATCTGAAATTGGAGACTACATTGTCGAA
CATCAAGCTGTCAACTTTATCAATTTCACAGGTTCAACAGGAATTGG
CGAGAGAATAGGGAAAATGGCAGGTATGCGTCCAATCATGTTAGAA
CTAGGCGGGAAAGACTCTGCAATCGTGTTGGAAGATGCTGATTTGGA
ACTTACCGCCAAAAACATCATTGCCGGTGCATTCGGTTATTCTGGAC
AAAGATGTACTGCAGTTAAACGTGTTTTAGTAATGGAATCAGTGGCA
GATGAACTTGTGGAAAAGATCAGGGAAAAAGTCCTTGCACTGACTAT
CGGTAATCCAGAAGATGATGCTGACATCACCCCACTTATTGACACTA
AGTCAGCTGATTATGTTGAAGGTTTGATCAATGATGCAAATGATAAG
GGTGCCGCTGCCTTAACAGAGATCAAAAGAGAAGGTAACTTAATCTG
CCCAATCCTGTTTGATAAGGTTACTACTGATATGAGATTGGCTTGGG
AGGAACCATTTGGTCCTGTTTTGCCTATCATAAGAGTTACCTCTGTTG
AGGAAGCTATAGAGATATCTAACAAATCAGAATACGGCTTACAAGC
CTCTATCTTTACTAATGATTTCCCAAGAGCATTTGGAATAGCTGAACA
ACTAGAAGTAGGTACAGTTCACATTAACAACAAAACCCAGAGAGGC
ACAGACAATTTCCCATTTCTAGGGGCCAAAAAGTCAGGGGCTGGAAT
TCAAGGCGTGAAATACTCCATTGAAGCTATGACTACAGTGAAAAGTG
TTGTCTTTGACATAAAGTGA
1
2
3
Supplementary Figure S1
1
2
3
Supplementary Figure S2
1
2
3
4
5
6
7
8
9
10
11
12
Supplementary Figure S3
1
Reference
2
Goldstein AL, McCusker JH. 1999. Three new dominant drug resistance cassettes for gene
3
4
5
6
7
8
9
10
11
12
13
disruption in Saccharomyces cerevisiae. Yeast 15:1541-1553.
Ng R, Abelson J. 1980. Isolation and sequence of the gene for actin in Saccharomyces
cerevisiae. Proc. Natl. Acad. Sci. U. S. A. 77(7):3912-3916.
Nielsen J, Villadsen J, Lidén G. 2003. Bioreaction Engineering Principles. 2nd ed. New York,
USA: Kluwer Academic/Plenum Publishers.
Partow S, Siewers V, Bjørn S, Nielsen J, Maury J. 2010. Characterization of different
promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast
27(11):955-964.
Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT–
PCR. Nucleic Acids Res. 29(9):e45-e45.
Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers.
14
In: Bioinformatics Methods and Protocols: Methods in Molecular Biology. Edited by
15
Krawetz S, Misener S. Humana Press, Totowa, NJ, 2000, pp 365-386.
16
Shi S, Valle-Rodríguez JO, Khoomrung S, Siewers V, Nielsen J. 2012. Functional expression
17
and characterization of five wax ester synthases in Saccharomyces cerevisiae and
18
their utility for biodiesel production. Biotechnol. Biofuels 5(1):7.
19
Wang X, Wang Z, Da Silva NA. 1996. G418 selection and stability of cloned genes
20
integrated at chromosomal δ sequences of Saccharomyces cerevisiae. Biotechnol.
21
Bioeng. 49(1):45-51.
22
23
24
Whelan JA, Russell NB, Whelan MA. 2003. A method for the absolute quantification of
cDNA using real-time PCR. J. Immunol. Methods 278(1):261-269.