Literature Report--- Synthetic Biology and Enzymatic ...
Transcript of Literature Report--- Synthetic Biology and Enzymatic ...
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Literature Report--- Synthetic Biology and
Enzymatic Fluorinations
Wangxiaoying
2013/9/28
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Michelle C. Chang
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Associate Professor
Department of chemistry, University of California, Berkeley
Biography:
B.S., Biochemistry, B.A., French Literature, University of
California, San Diego (1997)
National Science Foundation Predoctoral Fellow (1997-2000)
M.I.T./Merck Foundation Predoctoral Fellow (2000-2002)
Ph.D. Massachusetts Institute of Technology (2004)
Jane Coffin Childs Postdoctoral Fellow, University of California,
Berkeley (2004-2007)
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Michelle C. Chang
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Research Interests:
Biochemistry, Chemical Biology, and Synthetic Biology
(i) the in vivo production of biofuels from plant biomass
(ii)the development of new biosynthetic methods for selective,
catalytic C-F bond formation under mild
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Constructing de Novo Biosynthetic Pathways for
Chemical Synthesis inside Living Cells
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Optimizing flux through synthetic metabolic pathways
Identifying and Overcoming Pathway Bottlenecks
Engineering Pathway Balance Maximizing Pathway Flux through Engineered Spatial Organization
Engineering new or altered enzyme
In Vitro Evolution of New and Altered Enzyme Characteristics
Enzyme Promiscuity and Neutral Drift
Identification of useful chemical transformations
Targeted Gene Identification Integrating Sequence- and Structure-Based
Prediction of Enzyme Function
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Biosynthetic Pathways for Chemical Synthesis
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Pipeline for construction of a de novo metabolic pathway
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Biosynthetic Pathways for Chemical Synthesis
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Chemical phenotypes of interest for de novo
metabolic pathway construction
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Specialized structural motifs and unusual functional
groups in natural products
Biosynthetic Pathways for Chemical Synthesis
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Methods for functional gene annotation
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limit the scope of possible reactions
limit the size of libraries to determine
enzyme function
Functional genomic approaches
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Engineering new or altered enzyme
Neutral drift mechanism of enzyme evolution
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Engineering pathway balance
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(A) Expression of pathway
genes at appropriate levels can be
achieved by adding RNA
regulatory elements.
(B) Control of ribosome binding
site accessibility
(C)ribosome binding site
optimization can be used to tune
protein expression at the
translational level.
(D) Variation of promoter strength
or inducer concentration can be
used to tune protein expression at
the transcriptional level
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The proposed biosynthetic pathway for
fluoroacetate and fluorothreonine in S. cattleya
Lethal synthesis of fluorocitrate and
inactivation of aconitase
Temporal and Fluoride Control of Secondary Metabolism
Regulates Cellular Organo fluorine Biosynthesis
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Expanding the Fluorine Chemistry of Living Systems
Using Engineered Polyketide Synthase Pathways
Experimental purposes:
• Constructed pathways involving two polyketide synthase systems,
fluoroacetate can be used to incorporate fluorine into the polyketide
backbone in vitro.
• Fluorine can be inserted site-selectively and introduced into polyketide
products in vivo.
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Synthetic biology of fluorine
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SDS-PAGE gels of purified proteins
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Assembly PCR
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Structural alignment of NphT7 and the DEBS
Mod5 ketosynthase (KS) domain
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Enzymatic production of a ctivated extender
units f or C–C bond-formation reactions
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Formation of malonyl-CoA Formation of fluoromalonyl-CoA
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HPLC chromatograms monitoring
fluoroacetyl-CoA formation by A260 nm
Plot of the conversion of free CoA to
fluoroacetyl-CoA
Kinetic parameters for AckA and Pta
Enzymatic production of a ctivated extender
units f or C–C bond-formation reactions
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Kinetic parameters for malonate activation
Enzymatic production of a ctivated extender
units f or C–C bond-formation reactions
Steady state kinetic analysis of MatB
Malonate Methylmalonate Fluoromalonate
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NMR spectra of enzymatically synthesized fluoromalonyl-CoA
1H NMR 13C NMR
19F NMR
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A chain-extension and ketoreduction cycle with a fluorinated extender
using a simple polyketide synthase, NphT7
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Reactions catalyzed by NphT7 and PhaB
Steady-state kinetic parameters for NphT7 -catalyzed C–C bond formation measured using a
coupled assay with PhaB.
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Characterization of enzymatically synthesized 2-fluoro-3-
hydroxybutyryl-CoA
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19F NMR
LC/MS 1H-19F HMBC
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Production of fluorinated polyketides
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Reaction catalyzed by DEBS Mod6+TE using the NDK-SNAC substrate
Triketide lactones monitored by LC-MS
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Amplification of TKL formation using MatB
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Dependence of TKL formation on
methylmalonyl-CoA
Comparison of TKL yield with and without
MatB regeneration
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LC/MS traces monitoring TKL formation Plot of NDK-SNAC and TKL concentrations
LC/MS traces monitoring F-TKL formation Plot of NDK-SNAC and F-TKL concentrations
Time-course for TKL and F-TKL formation by
DEBS Mod6+TE with substrate regeneration
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1D-NMR spectra of synthetic F-TKL standard in CDCl3
2D-NMR spectra of synthetic F-TKL standard in CDCl3
Stereochemical analysis for F-TKL
Molecular modeling results for F-TKL
Analysis for F-TKL
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Hydrolysis and regeneration reactions for F-TKL
production by DEBS Mod6+TE
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Reaction scheme
showing enzymes
present in F-TKL
forming reactions
including observed
non-productive
hydrolysis reactions
(red) and the ATP
regenerating system
(blue).
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Selectivity of DEBS Mod6+TE and DEBS Mod3+TE for the
methylmalonyl-CoA vrsus fluoromalonyl-CoA extender unit
Production of fluorinated polyketides in vivo
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LC-MS traces showing regio selective tetraketidelactone formation using the DEBS mini-PKS
Production of fluorinated polyketides in vivo
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ESI-MS/MS data for tetraketide lactones
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F-TKL production in vivo
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LC/MS traces show ing F-TKL formation In vivo selectivity data showing F-TKL
production compared to H-TKL and TKL
LC/MS traces showing F-TKL formation (m/z 173) by E.coli cell culture upon feeding with NDK-SNAC
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Thank you
Wangxiaoying
2013/9/28