bit25323-sm-0001-SuppData-S1

advertisement
SUPPLEMENTARY METHODS AND DATA
A kinetic-based approach to understanding heterologous mevalonate pathway
function in E. coli
Lane J. Weaver1,2, Mirta M. L. Sousa1,5,6, George Wang1,3, Edward Baidoo1,3, Christopher
J. Petzold1,3, Jay D. Keasling1,2,3,4
1
Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608
2
UCB-UCSF Joint Graduate Group in Bioengineering, Berkeley, California 94720
3
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA
94720
4
Department of Chemical & Biomolecular Engineering, University of California,
Berkeley, Berkeley, CA 94720
5
Department of Cancer Research and Molecular Medicine, Faculty of Medicine,
Norwegian University of Science and Technology (NTNU), Trondheim, NO-7489
6
The Proteomics and Metabolomics Core Facility (PROMEC) at NTNU, Trondheim,
Norway
Correspondence:
Jay D Keasling
Address: Joint BioEnergy Institute, 5885 Hollis Avenue, Fourth Floor, Emeryville, CA
94608
Email: jdkeasling@lbl.gov
Phone: 510.495.2620
Fax: 510.495.2630
Supplementary table I. Peptides used in synthetic Mev2.0 QconCat protein
Gene
Peptide sequence
E. coli atoB
LGDGQVYDVILR
S. cerevisiae HMGS
GLVSDPAGSDALNVLK
S. aureus HMGS
SGYEDAVDYNR
S. cerevisiae HMGR
SVVAEATIPGDVVR
S. aureus HMGR
LIGTIEVPMTLAIVGGGTK
S. cerevisiae MK
SLVFQLFENK
M. mazei MK
ASYDFLGK, QAVEDVHK
S. aureus MK
IGEQLVLSGDYASIGR
S. cerevisiae PMK
VQWLDVTQADWGVR
S. cerevisiae PMD
LYQLPQSTSEISR
E. coli IDI
YELGVEITPPESIYPDFR, LSAFTQLK
E. coli NUDB
WLDAPAAAALTK
E. coli ISPA
STYPALLGLEQAR
A. grandis GPPS
AAPLLGLADYVAFR
M. spicata LS
LADDLGTSVEEVSR
A. grandis BIS
LLIVDNIVR
A. annua ADS
VPGEIILEDALGFTR
Supplementary table II. Enzyme constants used in modeling studies
Enzyme
kcat (s-1)
Km (M)
S. cerevisiae mevalonate
kcat1
Km1
38
131×10-6
Ki (M)
Reference
1.9×10-6
(Primak et al.,
kinase (scMK)
S. aureus mevalonate
2011)
kcat1
6
Km1
41×10-6
46×10-6
kinase (saMK)
S. cerevisiae
(Voynova et al.,
2003)
kcat2
10
Km2
885×10-6
Garcia et al.
kcat3
4.9
Km3
123×10-6
(Krepkiy and
phosphomevalonate
kinase (scPMK)
S. cerevisiae mevalonate
diphosphate
Miziorko,
decarboxylase (scPMD)
2004)
E. coli Isopentenyl-
kcat4
0.33
Km4
8×10-6
(Hahn et al.,
diphosphate Isomerase
1999)
(ecIDI)
E. coli farnesyl
kcat5
0.21
Km5-IPP
1.3×10-6
(Ku et al.,
Km5-DMAPP
29.3×10-6
Km6-IPP
10.3×10-6
Km6-GPP
5.5×10-6
2005)
Km7
3.3×10-6
(Picaud et al.,
pyrophosphate synthase
2005)
(ecISPA, reaction 1)
E. coli farnesyl
kcat6
0.47
pyrophosphate synthase
74×10-6
(Ku et al.,
(ecISPA, reaction 2)
A. annua Amorphadiene
Synthase (aaADS)
kcat7
6.8×10-3
2005)
Supplementary table III. Enzyme concentrations used in modeling studies
Strain
mbis3 (μM)
saMK3 (μM)
10kADS (μM)
Enzyme
scMK
66.3
saMK
23.2
scPMK
2.9
4.9
14.1
scPMD
2.2
2.7
2.3
ecIDI
18.4
15.3
21.6
ecISPA
35.0
36.8
24.2
aaADS
39.6
25.6
678
Equations used in modeling studies
(1)
𝑑[𝑀𝑒𝑣𝑃]
𝑑𝑑
=
π‘˜π‘π‘Žπ‘‘ 1 [𝑀𝐾][𝑀𝑒𝑣]
[𝐹𝑃𝑃]
πΎπ‘š1 (1+
𝐾𝐼,𝐹𝑃𝑃
)+[𝑀𝑒𝑣]
−
π‘˜π‘π‘Žπ‘‘ 2 [𝑃𝑀𝐾][𝑀𝑒𝑣𝑃]
πΎπ‘š2 +[𝑀𝑒𝑣𝑃]
(2)
𝑑[𝑀𝑒𝑣𝑃𝑃]
𝑑𝑑
(3)
=
197
π‘˜π‘π‘Žπ‘‘ 2 [𝑃𝑀𝐾][𝑀𝑒𝑣𝑃]
πΎπ‘š2 +[𝑀𝑒𝑣𝑃]
−
π‘˜π‘π‘Žπ‘‘ 3 [𝑃𝑀𝐷][𝑀𝑒𝑣𝑃𝑃]
πΎπ‘š3 +[𝑀𝑒𝑣𝑃𝑃]
𝑑[𝐼𝑃𝑃]
𝑑𝑑
=
π‘˜π‘π‘Žπ‘‘ 3 [𝑃𝑀𝐷][𝑀𝑒𝑣𝑃𝑃]
πΎπ‘š3 +[𝑀𝑒𝑣𝑃𝑃]
−
π‘˜π‘π‘Žπ‘‘ 4 [𝐼𝐷𝐼][𝐼𝑃𝑃]
πΎπ‘š4 +[𝐼𝑃𝑃]
+
π‘˜π‘π‘Žπ‘‘ 5 [𝐼𝑆𝑃𝐴][𝐼𝑃𝑃][𝐷𝑀𝐴𝑃𝑃]
πΎπ‘š5,𝐼𝑃𝑃 [𝐷𝑀𝐴𝑃𝑃]+πΎπ‘š5,𝐷𝑀𝐴𝑃𝑃 [𝐼𝑃𝑃]+[𝐷𝑀𝐴𝑃𝑃][𝐼𝑃𝑃]
π‘˜π‘π‘Žπ‘‘ 4 [𝐼𝐷𝐼][𝐷𝑀𝐴𝑃𝑃]
πΎπ‘š4 +[𝐷𝑀𝐴𝑃𝑃]
−
−
π‘˜π‘π‘Žπ‘‘ 6 [𝐼𝑆𝑃𝐴][𝐼𝑃𝑃][𝐺𝑃𝑃]
𝐾𝐼,𝐺𝑃𝑃 πΎπ‘š6,𝐼𝑃𝑃 +πΎπ‘š6,𝐼𝑃𝑃 [𝐺𝑃𝑃]+πΎπ‘š6,𝐺𝑃𝑃 [𝐼𝑃𝑃]+[𝐺𝑃𝑃][𝐼𝑃𝑃]
(4)
𝑑[𝐷𝑀𝐴𝑃𝑃]
𝑑𝑑
=
π‘˜π‘π‘Žπ‘‘ 4 [𝐼𝐷𝐼][𝐼𝑃𝑃] π‘˜π‘π‘Žπ‘‘ 4 [𝐼𝐷𝐼][𝐷𝑀𝐴𝑃𝑃]
−
πΎπ‘š4 + [𝐼𝑃𝑃]
πΎπ‘š4 + [𝐷𝑀𝐴𝑃𝑃]
π‘˜π‘π‘Žπ‘‘ 5 [𝐼𝑆𝑃𝐴][𝐼𝑃𝑃][𝐷𝑀𝐴𝑃𝑃]
πΎπ‘š5,𝐼𝑃𝑃 [𝐷𝑀𝐴𝑃𝑃] + πΎπ‘š5,𝐷𝑀𝐴𝑃𝑃 [𝐼𝑃𝑃] + [𝐷𝑀𝐴𝑃𝑃][𝐼𝑃𝑃]
−
(5)
𝑑[𝐺𝑃𝑃]
𝑑𝑑
=
π‘˜π‘π‘Žπ‘‘ 5 [𝐼𝑆𝑃𝐴][𝐼𝑃𝑃][𝐷𝑀𝐴𝑃𝑃]
πΎπ‘š5,𝐼𝑃𝑃 [𝐷𝑀𝐴𝑃𝑃]+πΎπ‘š5,𝐷𝑀𝐴𝑃𝑃 [𝐼𝑃𝑃]+[𝐷𝑀𝐴𝑃𝑃][𝐼𝑃𝑃]
−
π‘˜π‘π‘Žπ‘‘ 6 [𝐼𝑆𝑃𝐴][𝐼𝑃𝑃][𝐺𝑃𝑃]
𝐾𝐼,𝐺𝑃𝑃 πΎπ‘š6,𝐼𝑃𝑃 +πΎπ‘š6,𝐼𝑃𝑃 [𝐺𝑃𝑃]+πΎπ‘š6,𝐺𝑃𝑃 [𝐼𝑃𝑃]+[𝐺𝑃𝑃][𝐼𝑃𝑃]
(6)
𝑑[𝐹𝑃𝑃]
𝑑𝑑
π‘˜π‘π‘Žπ‘‘ 6 [𝐼𝑆𝑃𝐴][𝐼𝑃𝑃][𝐺𝑃𝑃]
π‘˜π‘π‘Žπ‘‘ 7 [𝐴𝐷𝑆][𝐹𝑃𝑃]
−
𝐾𝐼,𝐺𝑃𝑃 πΎπ‘š6,𝐼𝑃𝑃 + πΎπ‘š6,𝐼𝑃𝑃 [𝐺𝑃𝑃] + πΎπ‘š6,𝐺𝑃𝑃 [𝐼𝑃𝑃] + [𝐺𝑃𝑃][𝐼𝑃𝑃]
πΎπ‘š7 + [𝐹𝑃𝑃]
=
(7)
𝑑[𝐴𝑀𝑂]
𝑑𝑑
=
π‘˜π‘π‘Žπ‘‘ 7 [𝐴𝐷𝑆][𝐹𝑃𝑃]
πΎπ‘š7 +[𝐹𝑃𝑃]
Supplementary Methods
Plasmid Construction
To construct the pMBIS-saMK plasmid, the S. cerevisiae mevalonate kinase
(scMK) gene was replaced with the S. aureus mevalonate kinase (saMK) gene as follows:
saMK was first amplified by PCR with 30-bp, non-annealing 5’ ends that overlapped
with the regions flanking the scMK in the pMBIS3 plasmid (Nowroozi et al., 2013).
These regions were also used to generate primers to amplify the pMBIS3 vector
(excluding scMK), which was used as the backbone for a Circular Polymerase Extension
Cloning (CPEC)-based insertion of saMK (Quan, 2009). The pMBIS3-10kADS plasmid
was constructed by round-the-horn mutagenesis with a forward primer containing a
10,000-strength ribosome binding site (as calculated by the RBScalculator (Salis et al.,
2009) and a reverse primer binding just upstream of the RBS. Prior to PCR, the primers
were phosphorylated with Polynucleotide Kinase (PNK), which provided phosphates for
the blunt-end, circularization ligation reaction performed on the amplicon following a gel
purification clean-up.
The mevalonate pathway QconCAT gene (Mev2.0) encodes a non-natural,
synthetic protein used as a standard for quantifying proteins in the Mev pathway. It was
designed to include peptides from enzymes of the mevalonate pathway listed in
Supplementary Table I. The gene was synthesized (Genscript) with flanking 5’ NdeI
and 3’ XhoI sites and cloned into a pET29b vector, generating pET-Mev2.0, for
expression.
Conversions to cellular concentrations
To convert concentrations measured in culture volume to intracellular
concentrations, empiric relationships (Volkmer and Heinemann, 2011) were used to
generate the following formulae for intracellular metabolites (Supplementary Equation
1) and proteins (Supplementary Equation 2):
(1)
Cintracellular,metabolite [μM] = 2.78 × 103 [OD600 ] ×
CLC−MS [μM]
OD600
(2)
OD600
Cintracellular,protein [μM] = 52.8[
μM
]×
CMS [μM]×Ctotal protein [μM]
OD600
where
Cintracellular,metabolite:
Intracellular concentration of metabolites
Cintracellular,protein:
Intracellular concentration of proteins
CLC-MS:
Concentration of metabolites measured by LC-MS
CMS:
Intracellular concentration of proteins measured by
SRM-MS
Ctotal protein:
Concentration of total extracted protein
OD600:
Optical density of E. coli at 600nm
QconCAT purification and standard curve generation
pET-Mev2.0 was transformed into E. coli BL21 for protein expression. The
resulting transformant was grown to an OD600~0.2 in LB medium, at which point it was
induced with 500 μM IPTG and grown at 20°C for 12 hours. The cells were lysed via
homogenization and the Mev2.0 protein was purified with an immobilized nickel affinity
column under denaturing conditions with urea (Qiagen, 2003); the protein was judged to
be >95% pure by SDS-PAGE analysis.
To generate a standard curve for quantitation of enzymes in the mevalonate
pathway, the Mev2.0 protein was reconstituted in 100 mM ammonium bicarbonate with
10% (v/v) methanol at 1 mg/mL and trypsinized as described in the previous section. A
dilution series containing a total of 1000 fmol, 750 fmol, 500 fmol, 250 fmol, and 125
fmol of the trypsinized Mev2.0 protein was generated and analyzed on a 6460QQQ
operating in selective-reaction monitoring mode as described above.
References
Hahn FM, Hurlburt AP, Poulter CD. 1999. Escherichia coli open reading
frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate
isomerase. J Bacteriol 181:4499–4504.
Krepkiy D, Miziorko HM. 2004. Identification of active site residues in
mevalonate diphosphate decarboxylase: implications for a family of
phosphotransferases. Protein Sci 13:1875–1881.
Ku B, Jeong J-C, Mijts BN, Schmidt-Dannert C, Dordick JS. 2005.
Preparation, characterization, and optimization of an in vitro C30
carotenoid pathway. Applied Environ Microbiol 71:6578–6583.
Nowroozi FF, Baidoo EEK, Ermakov S, Redding-Johanson AM, Batth TS, Petzold CJ,
Keasling JD. 2013. Metabolic pathway optimization using ribosome binding site
variants and combinatorial gene assembly. Appl Microbiol Biotechnol. 58: 15671581.
Primak YA, Du M, Miller MC, Wells DH, Nielsen AT, Weyler W, Beck ZQ.
2011. Characterization of a feedback-resistant mevalonate kinase from
the archaeon
Qiagen. 2003. QIAexpressionist: Fifth edition:1–128.
Quan J, Tian J 2009. Circular Polymerase Extension Cloning of Complex Gene Libraries
and Pathways. PLoS One 4 (7): e6441
Salis HM, Mirsky EA, Voigt CA. 2009. Automated design of synthetic ribosome binding
sites to control protein expression. Nat Biotechnol 27:946–950.
Volkmer B, Heinemann M. 2011. Condition-dependent cell volume and concentration of
Escherichia coli to facilitate data conversion for systems biology modeling. PLoS
ONE 6:e23126.
Download