SupplementaryInformation for
In Vitro Reconstitution of Mevalonate Pathway and Targeted Engineering
of Farnesene Overproduction in Escherichia coli
Fayin Zhua, Xiaofang Zhonga, Mengzhu Hua, Lei Lua, Zixin Denga,bandTiangang
Liua,b,*
a
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan
University), Ministry of Education, and Wuhan University School of Pharmaceutical
Sciences, Wuhan 430071, P. R. China.
b
Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of
Biotechnology,Wuhan 430075, P. R. China.
*To whom correspondence should be addressed. E-mail:liutg@whu.edu.cn
Methods and Materials
Plasmid construction
Plasmid construction for protein overexpression and purification
Genomic DNAs from Saccharomyces cerevisiae INVSC1 and Escherichia coli
BL21(DE3) were obtained using the Blood and Cell Culture DNA Mini Kit (Qiagen)
following the instructions of the manufacturer. The atoB, idi, and ispA genes were
individually amplified by PCR from E. coli genomic DNA using primers
AtoB-BamHI, AtoB-XhoI; IDI-NdeI, IDI-XhoI; IspA-BamHI, and IspA-EcoRI-SpeI,
separately. The erg13, thmg1, erg12, erg8, and mvd1 genes were individually
amplified by PCR from S. cerevisiae genomic DNA using primers ERG13-NdeI,
ERG13-XhoI;
tHMG1-EcoRI,
tHMG1-XhoI;
ERG12-NdeI,
ERG12-XhoI;
ERG8-EcoRI, ERG8-XhoI; and MVD1-NdeI, MVD1-XhoI, separately (primers used
are shown in suppl. Table I). The afs gene from Malus x domestica was codon
optimized for E. coli expression and synthesized by Genescript (DNA sequence is
shown in suppl. Table V). All genes were digested with the corresponding restriction
enzymes (New England Biolabs) and individually inserted into pET28a(+) (Novagen)
using T4 DNA ligase (Fermentas), yielding pJW1, pGZI, pFZ32, pFZ12, pJW2,
pFZ13,pFZ14, pFZ15 and pFZ31(suppl. Table Ⅱ ), respectively, for protein
overexpression and purification. E. coli XL1-blue was used as cloning host. For ALF
overexpression, pFZ36 was constructed first. IspA was amplified by PCR from E. coli
genomic DNA using primers IspA-NdeI and IspA-EcoRI, which introduce 40 bp
overlapping with pET28a(+) at each end (suppl. Table I). pET28a(+) was amplified
with primers pET28-NdeI and pET28-EcoRI and the Simple Cloning method was
used to obtain pFZ36. Then ispA was amplified from pFZ36 by using the primers
IspAL-NcoI and IspAL-BamHI, and inserted into pET28a (+); at the same time the afs
gene was amplified with primers AFSL-BamHI and AFSL-XhoI and ligated into the
BamHI-XhoI site to give pFZ37.
Construction of mevalonate pathway overexpression plasmids
pMH1 and pFZ81 were constructed for overexpressing the entire set ofmevalonate
pathway genesby using the Gibson method which can assemble multiple genes
dependent on the overlap of DNA(primers used are shownin suppl. TableI). A
detailed description of the construction is as follows:
The origin of pBBR1MCS-1 was replaced by p15A before insertion of the
indicated genes. The p15A origin was amplified from pMSD15 using primers p15A-f
and p15A-r, at the same time pBBR1MCS1 was amplified using primers
pBBR1MCS1-p15A-f and pBBR1MCS1-p15A-r. The two PCR fragments were
combined using the Simple Cloning method, resulting in plasmid pBBR1MCS1/p15A.
The Gibson method was then used to assemble the indicated genes into
pBBR1MCS1/p15A to yield pMH1. First, the three primer pairs atoB-f, atoB-r;
ERG13-f, ERG13-r; and thMG1-f, tHMG1-r were used to amplify atoB, erg13 and
thmg1, respectively, from plasmid pJW1, pFZ12 or pJW2 separately, and primers
pBBR1MCS1-f and pBBR1MCS1-r were used to amplify the pBBR1MCS1/p15A
plasmid backbone. Fifty nanograms of pBBR1MCS1/p15A PCR fragment and
equimolar amounts of atoB, erg13, and thmg1 were combined (volume adjusted to 5
μL by ddH2O) and added to 15μL Gibson working buffer; after incubation at 50 °C
for 1 h, 5 μL was used for transformation. Gibson working buffer has been previously
described, and contains 100 mM Tris-Cl, 10 mM MgCl2, 10 mM DTT, 0.2 mM of
each dNTP, 1 mM NAD, 5% PEG8000, 5 U/mL T5 exo (Epicenter), 3.3 U/mL
Phusion DNA polymerase (NEB), and 5330 U/mL Taq DNA ligase (NEB). pFZ81
was constructed by the same method as described for pMH1. Erg12, erg8, mvd1, and
idi were amplified from plasmid pFZ13, pFZ14, pFZ15 or pGZI separately, by using
primers ERG12-f, ERg12-r; ERG8-f, ERG8-r; MVD1-f, MVD1-r, and Idi-f, Idi-r,
respectively. pBBR1MCS2 was amplified by PCR using primers pBBR1MCS2-f and
pBBR1MCS2-r. The Gibson procedure was then used to combine erg12, erg8, mvd1,
and idi into pBBR1MCS2 to give plasmid pFZ81.
Farnesene overproduction plasmid construction
pFZ38 containing separate ispA and afs genes was constructed for farnesne
production. The XbaI-XhoI fragment from pFZ31, which contains the synthetic afs
gene, was inserted into pET21a(+) to give pFZ35, and the XbaI-XhoI fragment of ispA
from pFZ32 was ligated into the SpeI-XhoI fragment of pFZ35 to give pFZ38; thus,
the ispA and afs genes were controlled by the T7 promoterinthe pET21a(+) backbone.
The derivative plasmids from pFZ38 were constructed by the same method using
the isocaudarner SpeI and XbaI to splice together the desired genes. An additional
copy of erg8, erg13, or idi was introduced into pFZ38 by inserting the XbaI-XhoI
fragment of erg8, erg13, or idi into the SpeI-XhoI fragment of pFZ38 to give pFZ65,
pFZ69, and pFZ71, respectively. All the inserted genes were controlled by the single
T7 promoter from pET21a(+).
Modified targeted proteomic analysis in engineered strains
Sample preparation
The engineered strains (Fig. 4A) were cultured in 500-mL flasks containing 150 mL
2×TY medium at 30°C with 2% glycerol, 100 μg/mL ampicillin, 50 μg/mL
kanamycin and 34 μg/mL chloramphenicol. When the OD600 reached 0.6 to 0.8, IPTG
was added to a final concentration of 0.1 mM. After 3 hrs induction, the medium was
overlaid with 30 mL decane and cells were harvested 20 hrs after IPTG addition for
targeted proteomics analysis. Cell pellets were resuspended in 100 mM ammonium
bicarbonate and lysed by sonication in a sonic bath. After centrifugation at 4 °C at
10,000 g for 30 min, the supernatant was additionally centrifuged at 4 °C at 200,000 g
for 2 hrs; the supernatant was used for protein quantification after the concentrations
of total proteins were measured by the BCA protein assay kit (Pierce). The individual
concentrations of all nine biosynthetic proteins were then simultaneously analyzed by
targeted proteomics analysis using a modification of thepreviously described methods.
First, one hundred micrograms of total proteins from each sample was used for trypsin
digestion and 1μg bovine serum albumin (0.2 μg/μL) was spiked into the solution to
serve as an internal standard.After the reaction mixture wasadjusted to 60μL with 100
mM ammonium bicarbonate,5 mM tris (2-carboxyethyl) phosphine (TCEP) was
added and incubated at 30 °C for 1h to reduce the disulfide bonds in each sample. The
proteins were alkylated with 10mM iodoacetamide (IAA, 200 mM prepared in 100
mM NaOH)at 30 °C for 30 min in the dark. Five micrograms of trypsin (0.2 μg/μL)
was then added to each sample and incubated at 37 °C for 14 hrs. Finally, the solution
was dried in a vacuum concentrator and redissolved in 100 μL LC-MS grade water
with 0.3% formic acid.
Mass spectrometric targeted proteomic analysis:
Targeted proteomic identification was performed using an AB SCIEX Triple TOF
5600+ high resolution LC-MS/MS system. The MS/MS spectra were processed using
the Paragon algorithm (ProteinPilot 4.5, AB SCIEX) and searched against a database,
which contained the entire protein sequence database of E. coli obtained from the
UniProt database (www.uniprot.org) plus the exogenous protein sequences of selected
proteins for farnesene synthesis through the MVA pathway. The data were searched
with settings as follows: sample type--identification, protease digestion with trypsin,
cysteine alkylation with iodoacetamide, and confidence level set to 99%
(ProtScore=2.0). When the critical False Discovery Rate was set to 1.0%, all
targeted proteins were identified.
Targeted proteomic quantification was performed on an AB SCIEX Q-Trap 4500
mass spectrometer running with Analyst 1.6 software (AB SCIEX), coupled with a
LC-MS/MS system consisting of an Eksigent NanoLC-Ultra 1Dplus system and a
cHiPLC-nanoflex system. The peptides were loaded onto a Nano cHiPLC Trap
column (200 μm×0.5 mm ChromXP C18-CL 3 μm 120 Å, Eksigent) and washed with
100% buffer A (2% v/v ACN, 0.1% formic acid, balance H2O) at the flow rate of 2
μL/min for 15 min. Peptides were eluted over a Nano cHiPLC column (75 μm×15 cm
ChromXP C18-CL 3 μm 120 Å, Eksigent) with 5% to 80% buffer B (98% v/v ACN,
0.1% formic acid, balance H2O) at a flow rate of 300 nL/min in 45 min. Following the
elution of peptides, the column was washed with 80% buffer B for 5 min and then
re-equilibrated with 5% buffer B for 10 min. The analytical system was interfaced to
the mass spectrometer using the NanoSpray III source and a 10 μm PicoTip emitter
(New Objective) operating in the positive mode.
Peptide transitions for each targeted protein were selected in a Multiple Reaction
Monitoring-Initiated Detection And Sequencing (MIDAS) workflow, in which a
Multiple Reaction Monitoring (MRM) survey scan was followed by an Enhanced
Product Ion (EPI) scan with the Information Dependent Acquisition (IDA) criteria.
The experiments were divided into two rounds using Skyline 1.4 software. In the first
round, based on the results from the ProteinPilot, five to eight credible peptides were
selected for each protein; and four to six MRM transitions were selected for each
peptide. The MS/MS spectra scan was set to a mass range of 100-1500 m/z at the scan
rate of 1000 Da/s with rolling collision energy, dynamic fill time, and Q1 resolution
set to unit. In combination with the retention time, peptide transitions were verified by
acquiring full MS/MS scans of each target peptide. Based on the preliminary results,
two to three best peptides of each target protein, and three to four most suitable MRM
transitions of each peptide were selected for the following scheduled MRM method.
For calculating optimum collision energy and declustering potential of each transition,
the default equations from Skyline were utilized. In the second round, a scheduled
MRM assay was further designed based on the selected transitions and the retention
time. To provide higher multiplexing with good analytical precision, the detection
window of 8 min and the target scan time of 1.5 s were optimized. Once all MRM
transitions were validated and optimized, the MRM-only acquisition method was used
for the quantification of targeted proteins. The MS parameters of peptides used in the
targeted proteomic analysis are summarized in suppl. Table IV.
Data analysis of targeted proteomics
The data were acquired in four replicatesand then imported into MultiQuantTM 2.1
software (AB SCIEX) to determine the peak area for all MRM transitions. Automatic
peak integration using the MQ4 algorithm was processed with peak smoothing set to
3.0. Three to four transitions and two to three peptides were selected for each protein,
all peak areas were averaged for each peptide (Apep), and then all peptide areas Apep
were averaged for each protein (Apro). The Apro values were then normalized by the
BSA internal standard (Apro of indicated protein/Apro of BSA), to give a normalized
average peak area for each protein (NApro). Normalized average peak area (NApro)
was averaged for four replicate analyses to get final average peak area of each protein
(FApro). The average peak area (FApro) was then divided by the corresponding molar
weight of each protein to obtain the relative protein concentration; the data in Table I
has been multiplied by 1,000,000 to show the data more clearly.
Analysis of intermediates
Sample preparation
The engineered strainsF1 toF4 (Fig. 4A) were cultured in 500-mL flasks containing
150 mL 2×TY medium at 30°C with 2% glycerol, 100 μg/mL ampicillin, 50 μg/mL
kanamycin, and 34 μg/mL chloramphenicol. When the OD600 reached 0.6 to 0.8,
IPTG was added to a final concentration of 0.1 mM. Twenty hours after IPTG
addition, 200 OD600 cells were transferred into 15 mL quench solution (methanol: 0.5%
NaCl=2:1) and mixed thoroughly. The mixture was then immediately centrifuged at
5,000 g for 3 min at 4 °C. After centrifugation, the supernatant was discarded and 1.5
mL extraction solution (methanol: acetonitrile: water=2:2:1) was added to the cell
pellets. After flash freezing with liquid nitrogen for 5 min, the mixture was thawed on
ice for 4 min and vortexed for 30 s; the freeze-thaw cycle was repeated three times.
The extracts were then centrifuged at 12, 000 g for 10 min at 4 °C and 1.1 mL of
supernatant was transferred to 2 mL eppendorf tubes and dried in a Speedvac. The
residue was dissolvedwith 60 μL buffer A-buffer B (4:1, v/v) followed by vortex
mixing for 3 min. The tube was centrifuged for 10 min at 10,000 g, and the
supernatant was transferred to a clean tube ready for LC-MS/MS analysis.
Mass spectrometric analysis of intermediates:
Analysis of intermediates was performed using an ultra-fast liquid chromatography
(Shimadzu Corporation UFLC XR; Kyoto, Japan) apparatus coupled with a triple
time-of-flight mass spectrometer (AB SCIEX TripleTOF 5600+; Foster City, CA)
system with a duo spray ionization source. All samples were separated byAcclaim
Mixed-Mode HILIC-1 LC analytical column (150mm× 4.6mm × 5 μm; Thermo
Scientific). The mobile phases were composed of water/acetonitril/NH4AC (A,
95/5/5mM; B, 5/95/5mM). To achieve a better separation, the gradient was initially
maintained at 100% B for 2 min, ramped to 45% B within 11 min, held for 1 min at
45% B, and then returned to 100% B for the following 5 min. The flow rate was kept
at 1.0 mL/min, and the injection volume was set at 10 μL. The mass spectrometer was
operated in negative ESI mode. The following MS condition were used: ionspray
voltage, -4500 V; curtain gas, 35 psi; nebulizer gas (Gas 1), 60 psi; heater gas (Gas 2),
60 psi; source temperature, 600 °C. A typical IDA method was also created to acquire
both MS and MS/MS data in a single run analysis. The IDA criteria were as follows:
peak intensity more than 200 cps, a maximum number of four monitoring ions,
TOF/MS spectra scan set at m/z 100–1,000, and the mass range for selection of
candidate ions for dependent MS/MS scan was m/z 50–1,000. Both CE and CES were
set at 30 and 15 eV, respectively, (meaning 30±15 eV). The mass spectra were
acquired at high sensitivity mode with a 250 ms ion accumulation time for MS and
100 ms for MS/MS to identify more ions of higher intensity signal in the survey scan.
Dynamic background subtraction (DBS) by IDA was applied to facilitate the selection
of those ions that had an LC peak profile with the greatest intensity rate against
background ions.
Data analysis of intermediates
The high-resolution MS data was analyzed by using the PeakView software (AB
SCIEX, Foster City, CA) with the XIC Manager, Formula Finder, and Fragment
Interpretation tool to assist in data interpretation. XIC Manager was used to screen the
presence of metabolites by importing their names and chemical formulas and it was
also applied to display information for non-target compounds; this application was
used, therefore, for the identification of target and non-target metabolites. The
quantitation of the all targeted intermediates was performed with the MultiQuantTM
2.1 software (AB SCIEX) to calculate the peak area of the candidates. Each peak area
was averaged for four replicate analyses.
Supplementary figure legends
Supplementary Figure 1. Overview of the Targeted Engineering process. All
proteins for farnesene production through the mevalonate pathway were
overexpressed in E. coli BL21(DE3) and purified by a Histap QHP column (GE
Healthcare). The product produced in the in vitro reconstitution assay was confirmed
by GC-MS and radioactive TLC. After confirmation, titrations of substrate, cofactors,
metal ions, and individual proteins were performed by in vitro radioactive assays
measured by liquid scintillation analyzer. The synergistic effects of two or more
proteins were investigated. The in vitro assays guided engineering work in vivo; the
modified targeted proteomic analysis and intermediate analysis was used to verify
protein expression and determine the accumulation of intermediates in the mutants.
This information directly guided further engineering work.