Supporting Information
Wiley-VCH 2011
69451 Weinheim, Germany
Design and Application of a Mevalonate-Responsive Regulatory
Protein**
Shuang-Yan Tang and Patrick C. Cirino*
anie_201006083_sm_miscellaneous_information.pdf
Materials and Methods
General
Restriction enzymes and Phusion DNA polymerase were purchased from New England Biolabs (Ipswich, MA). T4 DNA ligase
and oligonucleotides were procured from Invitrogen (Carlsbad, CA). NADPH was purchased from Oriental Yeast CO., LTD
(Tokyo, Japan). DL-mevalonolactone and other chemicals were purchased from Sigma-Aldrich (St.Louis, MO). DNA sequencing
was performed at the Penn State Huck Institutes of the Life Sciences Genomics Core Facility
(http://www.huck.psu.edu/facilities/genomics-core-up). Flow cytometry was performed at the Cytometry Facility (Penn State Huck
Institutes of the Life Sciences, PA) (http://www.huck.psu.edu/facilities/cytometry-up). Mevalonate was prepared from
mevalonolactone as described[1].
The concentrations of antibiotics used for maintaining plasmids are as follows: kanamycin 50 µg/ml, apramycin 50 µg/ml,
chloramphenicol 25 µg/ml.
Plasmid construction
All experiments were performed using E. coli. Strains and plasmids used are listed in Table S1(Supporting Information). Plasmid
maps are depicted in Figure S2 (Supporting Information). Sequences for all primers are listed in Table S7 (Supporting Information).
Plasmid pMevT[1] was purchased from Addgene (http://www.addgene.org/pgvec1). The p15A origin of pMevT was replaced with
the pBR322 origin from pBAD18[2], resulting in plasmid pMevT-PBR. Control plasmid “pMevT-control”, in which the tHMGR
gene is truncated and encodes an inactive protein (Tang SY, unpublished), was prepared by digesting pMevT-PBR with BmgBI
and isolating and re-ligating the resulting large fragment. Plasmid pPCC482 (carrying AraC mutant gene araC-mev and the PBADgfpuv reporter construct) was constructed as follows. The segment of plasmid pPCC423-mev containing lacI and araC-mev under
control of promoter Ptac was PCR-amplified using primers tacNotI and AraC-rev-XbaI. The PCR product was digested with NotI
and XbaI and then ligated into pPCC442 [3], resulting in plasmid pPCC482.
Construction of strain HF22
Chromosomal integration of lacZ was accomplished using the CRIM method[4]. lacZ was PCR-amplified from plasmid pAH125[4]
using primers lacZ-SphI-rev and NheI-lacZ-for. The PCR product was ligated into vector pBAD18[2] after digestion with SphI and
NheI, resulting in pPCC424. pPCC424 was digested with NruI and XmnI and the resulting PBAD-lacZ fragment was ligated into
CRIM plasmid pAH162[4] digested with SmaI, resulting in plasmid pPCC441. The PBAD-lacZ fragment in pPCC441 was then
integrated into strain HF19 [3], together with a tetracycline resistance gene as a screening marker, and the integration was verified
by PCR. The resulting strain is HF22.
Library construction
AraC saturation library construction.
Overlap extension PCR was performed for AraC saturation mutagenesis library construction as follows:
Three parallel PCR reactions were performed to amplify three araC segments using plasmid pPCC423[3] as template using the
following three sets of primers: araC-P8-for and araC-T24-rev (generating fragment “LA”); araC-comp-T24-for and araC-H80Y82-rev (generating fragment “LB”); and araC-H93-for and araC-rev-4 (generating fragment “LC”). The program for these PCR
reactions was as follows: 98 oC for 30 s, then 30 cycles of 98 oC for 10 s, 60 oC for 1 min and 72 oC for 30 s, followed by 72 oC for
10 min. The three amplified segments correspond to site saturation at residue positions 8, 24, 80, 82 and 93. PCR products were
gel-purified and equimolar aliquots of every pair of adjacent DNA fragments (0.15 pmol each) were combined (LA + LB and LB +
LC) and PCR-assembled without primers. The assemblies were performed as follows: 98 oC for 30 s, then 15 cycles of 98 oC for 10
s, 60 oC for 1 min and 72 oC for 40 s, followed by 72 oC for 10 min. These assemblies resulted in PCR products LAB and LBC.
The two fragments were assembled with 20 cycles of PCR without primers: 98 oC for 30 s, 72 oC for 40 s. Finally, outer primers
wt-for-I and araC-rev-4 were added to the assembly reaction and the products were PCR-amplified as follows: 98 oC for 30 s, then
30 cycles of 98 oC for 10 s, 60 oC for 1 min and 72 oC for 40 s, followed by 72 oC for 10 min. Gene libraries were ligated into
pPCC423 after digestion with NdeI and HindIII. Ligation products were transformed into E.coli DH10B (3 × 107 unique
transformants were recovered), and the plasmid library was prepared after amplification by culturing in the presence of antibiotic.
Ten randomly picked clones from the library were sequenced, and these sequences revealed the expected random mutations at the
targeted nucleotide positions, with no additional point mutations.
tHMGR ribosomal binding site (RBS) library construction
A RBS library upstream of tHMGR gene was constructed by overlap extension PCR using pMevT-PBR as template. Two parallel
PCR reactions were performed to amplify two segments of the MevT operon using the the following sets of primers: 488-forBamHI and 488-RBS-rev; and 488-RBS-for and pMevT-rev-SpeI. The PCR reactions were performed as follows: 98 oC for 30 s,
then 30 cycles of 98 oC for 10 s, 60 oC for 1 min and 72 oC for 1 min 30 s, followed by 72 oC for 10 min. The two PCR products
were gel-purified and equimolar aliquots of each fragment (0.00015 nmol) were combined and PCR-assembled without primers as
follows: 98 oC for 30 s, then 15 cycles of 98 oC for 10 s, 60 oC for 1 min and 72 oC for 2 min 30 s, followed by 72 oC for 10 min.
Finally outer primers 488-for-BamHI and pMevT-rev-SpeI were added to the reaction and the products were PCR-amplified as
follows: 98 oC for 30 s, then 30 cycles of 98 oC for 10 s, 60 oC for 1 min and 72 oC for 2 min 30 s, followed by 72 oC for 10 min.
The library was ligated into pMevT-PBR after digestion with BamHI and SpeI. Ligation products were transformed into E.coli
DH10B (1× 106 unique transformants were recovered), and the plasmid library was prepared after amplification by culturing in the
presence of antibiotic. The resulting gene library consists of 6 partly randomized nucleotides [“R” in Table S2 (Supporting
Information) represents G or A] in the RBS region as well as 7 fully randomized nucleotides (“N” represents A, T, G or C) in the
interval sequence between the RBS and the start codon (ATG) of the tHMGR gene, totaling 1.05×106 possible nucleotide
combinations in this upstream region. Ten randomly picked clones from the library were sequenced, and these sequences revealed
the expected random mutations at the targeted nucleotide positions, with no additional point mutations.
Fluorescence-based positive and negative screening for AraC mutant
The araC gene library contained in plasmid pPCC423 was transformed into strain HF19[3] harboring PBAD-gfpuv reporter plasmid
pPCC442[3] (Supporting Information, Figure S2). A 10 mL shake-flask culture inoculated to OD600 0.2 was grown overnight at
37oC in LB medium containing both apramycin and chloramphenicol. The culture was then diluted to OD600=0.2 in LB containing
400 µM IPTG and both apramycin and chloramphenicol, and in the presence or absence of 30 mM mevalonate. The cells were
grown for 14 h at 37oC prior to positive or negative screening performed essentially as previously described[3]. In the first round of
screening, cells were grown in the absence of mevalonate and the least fluorescent 1.5 × 108 cells were collected (represented 82%
of all cells sorted), to eliminate AraC variants having high levels of leaky expression. In the second round, the most fluorescent 1.5
× 106 cells were sorted from a total of 1.5 × 108 cells (i.e., the top 1% were selected) after growth in the presence of 30 mM
mevalonate. This dual screening procedure was repeated five times, followed by a final round of negative screening. Twenty
clones were then selected for rescreening. They were recultured separately as described above, in the presence and absence of 30
mM mevalonate. Three clones showing fluorescence increase in the presence of 30 mM mevalonate were selected for sequencing
and all three contained the same mutations: P8P, T24L, H80L, Y82L and H93R. This mutant was named AraC-mev.
GFP expression fluorescence assays were performed as described[3]. HF19 cells harboring reporter plasmid (pPCC442) and AraC
expression plasmid (pPCC423-mev) were grown overnight at 37oC in LB medium containing chloramphenicol and apramycin and
0.4 mM IPTG, then diluted (to OD600=0.2 for data shown in Figure 2, or to OD600=0.01 for data listed in Supporting Information,
Table S2) in the same medium containing an appropriate concentration of inducer, and allowed to grow at 37oC under inducing
conditions for 14 h. A total of 100 μL of culture was centrifuged, and the cells were washed with 10 mM potassium phosphate
buffer (pH 7.5) and resuspended in 200 μL of the same buffer. The cell suspension optical density (OD600) was measured with a
SPECTRAmax microplate spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA), and fluorescence emission was
measured with a GENios FL fluorescence spectrometer (Tecan Austria GmbH, Groedig, Austria) (360 nm excitation filter, 536/50
nm emission filter). The data were normalized with respect to optical density (OD600). The background fluorescence due to buffer
served as the blank in all measurements.
All reported data in Figure 2 and Table S2 (Supporting Information) represent the mean of three independent data points.
Error bars in Figure 2 represent standard deviations. The coefficient of variation (CV) was always less than 15% and less
than 10% for most data reported.
-galactosidase activity-based solid-phase screening for mevalonate production by tHMGR RBS library clones
The RBS mutant library contained in plasmid pMevT-PBR was transformed into strain HF22 containing reporter plasmid
pPCC482 (Supporting Information, Figure S2). The cells were plated onto LB plates containing 400 µM IPTG, 274 mM glycerol
(as primary carbon source for mevalonate synthesis), 40 µg/ml X-Gal and both chloramphenicol and kanamycin. The plates were
incubated at 37oC and visually inspected at 20 and 24 h. The colonies which were obviously bluer than those containing the
original pMevT-PBR tHMGR RBS were picked for mevalonate production quantification. A total of 1×105 colonies were screened,
from which 6 clones were selected. They were cultured separately for HPLC quantification of mevalonate production and three of
them were found to produce more mevalonate than the precursor pMevT-PBR. The plasmids in these three clones were purified
and retransformed into strain HF22 and the enhanced mevalonate production was confirmed by HPLC. The three RBS mutant
plasmids (RBS2, RBS4, and RBS5) were sequenced (sequences given in Supporting Information, Table S3) and further analyzed.
HPLC quantification of mevalonate concentration in culture broth
A single colony of strain HF22 coexpressing the MevT operon (plasmid pMevT-PBR, RBS2, RBS4, or RBS5) and AraC-mev
(plasmid pPCC482) was grown at 37oC in 3 ml LB containing both chloramphenicol and kanamycin and 20 mM glucose (added to
repress MevT operon expression from the lac promoter during seed cultivation) for 10 hours. The cells were then centrifuged at
4,000 × g for 1 min and washed with LB. The cells were then diluted to OD600=0.2 in 1 mL LB containing 220 mM glycerol (as
carbon source for mevalonate production) and both chloramphenicol and kanamycin and grown at 37oC. At different time points,
100 µl of culture was centrifuged at 10,000 ×g. The supernatant was removed and acidified with 50 mM H2SO4 (forming
mevalonolactone) and filtered through a 0.45 µm filter prior to high performance liquid chromatography (HPLC) quantification of
mevalonate. A Shimadzu LC-10AD HPLC (Shimadzu Corp. Kyoto, Japan) equipped with a refractive index detector (RID) was
used for the quantification. Separation of products was achieved using an Aminex HPX-87H column (Bio-Rad Laboratories,
Hercules, CA) with 4 mM H2SO4 as the mobile phase (flow rate: 0.5 mL min-1, column operated at 45 oC).
All reported data represent the mean of three independent data points. Error bars in Figure 3 and Figure S3 (Supporting
Information) represent standard deviations. The coefficient of variation (CV) was always less than 15% and less than 10%
for most data.
β-galactosidase activity assay
β-galactosidase (LacZ) activity in intact E. coli was quantified using a 4-methylumbelliferyl β-D-galactopyranoside (MUG) assay[5].
The LacZ expression response to exogenous mevalonate in Figure 2 was determined with strain HF22 harboring only plasmid
pPCC482. All other reported data are for strain HF22 harboring both pPCC482 and the indicated MevT operon plasmid (pMevTPBR, RBS2, RBS4 or RBS5). 20 µl of culture prepared as described above for HPLC analysis (at a given time-point) was
transferred to 96-well microplates containing 80 µl of Z-buffer, and the cell density was determined with a SPECTRAmax
microplate spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA) at 595 nm. Then 25 µl of MUG substrate [1
mg/ml in dimethyl sulfoxide (DMSO)] was added to the well and the mixture was incubated at room temperature for 10 min. The
reaction was stopped with 30 µl 1 M Na2CO3. A cell-free LB medium sample was used as reference. The resulting fluorescence
emission was measured with a GENios FL fluorescence spectrometer (Tecan Austria GmbH, Groedig, Austria) (360 nm excitation
filter, 465/50 nm emission filter). MUG units (fluorescence change per minute normalized to cell density) were calculated as
described[5]. All reported data represent the mean of three independent data points. Error bars in Figure 2 and Figure S3
(Supporting Information) represent standard deviations. The coefficient of variation (CV) was always less than 15% and
less than 10% for most data. As shown in Figure S3(c) (Supporting Information), for a given mevalonate-producing clone the galactosidase activity measured on MUG showed a relatively linear correlation with the quantity of secreted mevalonate measured
in the culture broth.
HMG-CoA reductase activity assay
Strain HF22 harboring pPCC482 and the indicated MevT operon plasmid was cultured as decribed above for HPLC analysis.
Following 9h of induced cultivation at 37oC, cells were harvested and cell lysates were assayed for HMG-CoA reductase activity
by monitoring the disappearance of NADPH by measuring the absorbance at 340 nm as described[6]. Briefly, 3 ml of culture were
centrifuged at 4,000 ×g for 5 min at 4oC. The cells were washed and resuspended in 500 µl potassium phosphate buffer (100 mM,
pH 7.0) containing EDTA (1 mM) and dithiothreitol (1 mM). The cells were sonicated using a Sonifier® cell disruptor 350
(Danbury, CT) (Duty Cycle 60%, Output setting 4, 4 × 1 min), and then centrifuged at 18,000 ×g for 10 min at 4 oC. The
supernatant was filtered through a 0.45 µm filter and used for enzyme activity assay and total protein determination. The reaction
mixture (200 µl) for HMG-CoA reductase activity assay contained dithiothreitol (5 mM), potassium phosphate buffer (100 mM,
pH 7.0), NADPH [150 µM, in potassium phosphate buffer (100 mM, pH 8.0)], β-hydroxymethylglutaryl-CoA [150 µM, in acetate
buffer (200 mM, pH 4.6)] and 20 - 50 µl cell lysate. Reactions were initiated by the addition of β-hydroxymethylglutaryl-CoA and
the decrease in absorbance at 340 nm was measured using a SPECTRAmax microplate spectrophotometer (Molecular Devices
Corporation, Sunnyvale, CA). Background NADPH oxidation rates in lysate of HF22 harboring the “pMevT-control” plasmid
were subtracted from all other NADPH oxidation rates. The total protein content in the cell lysate was determined by Quick
StartTM Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA). The HMG-CoA reductase activity was normalized to the
total soluble protein concentration for each sample. All assays were performed in six replicates and standard deviations are
reported with the data presented in Table S4 (Supporting Information).
Figures and Tables
PlacUV5
atoB
Acetyl-CoA
ERG13
Acetoacetyl-CoA
tHMGR
Isoprenoids
IPP
Mevalonate
HMG-CoA
Figure S1. Mevalonate pathway to isoprenoid biosynthesis. Gene symbols and the enzymes they encode are as follows: atoB,
acetoacetyl-CoA thiolase from E. coli; ERG13, HMG-CoA synthase from S. cerevisiae; tHMGR, truncated HMG-CoA reductase
from S. cerevisiae. Pathway intermediates: HMG-CoA, hydroxymethylglutaryl-CoA; IPP, isopentenyl pyrophosphate.
NdeI
EcoRI
AccIII
HindIII
Ptac
araC
(or mutant)
PBAD
KpnI
gfpuv
lacI
cat
pcc423
pPCC423
6432 bps
pcc442
pPCC442
4797 bps
aac
pBR322 origin
a
RSF1030 origin
PBAD
PlacUV5
atoB
gfpuv
kan
cat
araC-mev
pPCC482 Ptac
ERG13
pMevT-PBR
tHMGR
RBS library
PBAD lacZ
lacI
(Integrated in chromosome)
b
Figure S2. Maps of two-plasmid systems used for (a) AraC library screening, and (b) tHMGR RBS library screening.
4
pMevT-control
pMevT-PBR
100
80
MUG unit
[Mevalonate] (mM)
4
3
2
pMevT-control
pMevT-PBR
pMevT-control
pMevT-PBR
Cell Density (OD600)
5
60
40
1
20
3
2
1
0
0
0
4
6
8
10
12
4
14
6
8
Time (h)
10
12
0
14
2
4
6
8
10
12
14
10
12
14
Time (h)
Time (h)
a
12
2500
10
8
6
4
Cell Density (OD600)
RBS2
RBS4
RBS5
3000
MUG unit
[Mevalonate] (mM)
14
4
3500
RBS2
RBS4
RBS5
16
2000
1500
1000
500
2
0
6
8
10
12
14
3
2
1
0
0
4
RBS2
RBS4
RBS5
4
6
8
Time (h)
10
12
14
0
Time (h)
2
4
6
8
Time (h)
b
2500
60
pMevT-PBR
40
MUG unit
MUG unit
RBS2
RBS4
RBS5
2000
50
30
20
10
1500
1000
500
0
0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
0
2
4
6
8
10
12
14
[Mevalonate] (mM)
[Mevalonate] (mM)
c
Figure S3. Time profiles of mevalonate concentration, β-galactosidase activity (expressed as MUG units[5]) and cell density
(OD600) measured from cultures of strain HF22 harboring pPCC482 and (a) plasmid “pMevT-control” (in which tHMGR is
truncated and inactive) or plasmid pMevT-PBR, or (b) the indicated RBS mutant plasmids. (c) β-galactosidase activity is plotted
against the concentration of mevalonate measured at the various time points (4, 7, 9, 11 h).
Table S1. E. coli strains and plasmids used in this study.
HF19
Strain
HF22
pPCC442
pPCC423
pPCC423mev
Plasmid
pMevT-PBR
pPCC482
pMevTcontrol
Relevant features
BW27786 (araFGH, araBAD, lacZ), with araC deleted
HF19 with PBAD-lacZ reporter integrated into chromosome
References
[3, 7]
In this
work
[3]
PBAD-gfpuv reporter construct; Chloramphenicol resistance
araC under the control of promoter tac, Apramycin resistance
araC-mev under the control of promoter tac, Apramycin resistance
[3]
MevT operon under the control of promoter lacUV5; PBR322 origin, Chloramphenicol
resistance
araC-mev under the control of promoter tac; PBAD-gfpuv reporter construct; RSF1030derivative origin, Kanamycin resistance
pMevT-PBR with tHMGR gene truncated, resulting in an inactive HMG-CoA reductase
In this
work
In this
work
In this
work
In this
work
Table S2. Fluorescence of strain HF19 harboring PBAD-gfpuv reporter plasmid (pPCC442) and expressing AraC-mev (pPCC423mev), in the presence of the indicated concentration of small molecule inducers (“effectors”).
Effectors
None
100 mM mevalonate
100 mM succinic acid
100 mM L-arabinose
5 mM Triacetic acid lactone
Fluorescence
2,100
6,500
2,900
1,900
2,400
Induction fold
3.1
1.4
1.0
1.1
Succinic acid
Triacetic acid lactone
Mevalonate
L-arabinose
Table S3. Ribosomal binding site (RBS) region sequences upstream of tHMGR in MevT operon plasmids.
Plasmid
pMevT-PBR (wild-type)
RBS2
RBS4
RBS5
Sequence
GGAGGATTACACT
−
−
G AGGTTTACGG
AGAGGGGGTTAAC
GAGGGAGGGTATG
Table S4. Relative HMG-CoA reductase activities measured from lysates of strain HF22 expressing MevT from the indicated RBS
mutant plasmids, compared to the same activity measured with the original MevT operon plasmid pMevT-PBR (= 37043
µmol/min/mg protein).
pMevT-PBR (wild-type)
RBS2
RBS4
RBS5
1
2
3
4
Randomly
5
picked RBS
6
mutants
7
8
9
10
Relative activity
1.00 ± 0.12
0.45 ± 0.13
0.92 ± 0.17
0.36 ± 0.06
0.59
0.02
0.06
0.0
0.04
0.01
0.03
0.02
0.03
0.06
Table S5. Mevalonate concentrations produced from cultures of selected clones and ten colonies randomly picked from the
tHMGR RBS library. Concentrations were measured by HPLC following 14 hours of cultivation.
pMevT-PBR (wild-type)
RBS2
RBS4
RBS5
1
2
3
4
Randomly
5
picked RBS
6
mutants
7
8
9
10
Mevalonate
(mM)
4.5
17
15
15
5.8
0.80
0.81
0.80
0.79
0.80
0.79
0.79
0.80
0.79
Table S6. Ribosomal binding site (RBS) region sequences upstream of tHMGR in MevT operon plasmids prepared from ten
colonies randomly picked from the tHMGR RBS library.
Plasmid
pMevT-PBR (wild-type)
1
2
3
4
5
6
7
8
9
10
Sequence
GGAGGATTACACT
GGAGGATATGCAC
Not determined
GGGGGACGGGCGT
GAGAGGAATTCAT
AAAGGAATACCTT
GAAAAGGGAAGGT
GGGAAGCTTAGA
AAAGAAAGCCAAG
GAGGAGCGTGTAG
GAGGGGGGGGCGG
Table S7. Primers used in this study. Underlined sequences represent enzyme restriction sites used for cloning. Bold sequences
correspond to mutated nucleotide positions. N represents the nucleotides A, G, C, or T, whereas S represents G or C, and R
represents G or A.
Primer name
araC-P8-for
araC-T24-rev
araC-comp-T24-for
araC-H80-Y82-rev
araC-H93-for
araC-rev-4
wt-for-I
488-for-BamHI
488-RBS-rev
488-RBS-for
pMevT-rev-SpeI
lacZ-SphI-rev
NheI-lacZ-for
Primer sequence (5’- to -3’)
tggctgaagcgcaaaatgatNNSctgctgccg
taaccgttggcctcaatcggSNNtaaacccgc
ccgattgaggccaacggtta
cgagcctccggatgacgaccSNNgtgSNNaatctctcc
ggtcgtcatccggaggctcgcgaatggtatNNScagtgggtt
attgctgtctgccaggtgatc
tatcatatggctgaagcgcaaaatgat
cggggatcctctagagtcgactaggaggaatataaaatg
ttattttttaacatcgtaagatcttctaaat
agaagatcttacgatgttaaaaaataaRRRRRRNNNNNNNatggttttaaccaataaaacagtc
atgactagtttaggatttaatgcaggtgacggacc
cgccgcatgcacatggcctgcccggttatt
gcgcgctagcagaggatcctttatgaccatgattacggattcactg
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