1

Literature Report--- Synthetic Biology and

Enzymatic Fluorinations

Wangxiaoying

2013/9/28

Michelle C. Chang

2

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)

Michelle C. Chang

3

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

4

Constructing de Novo Biosynthetic Pathways for

Chemical Synthesis inside Living Cells

5

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

Biosynthetic Pathways for Chemical Synthesis

6

Pipeline for construction of a de novo metabolic pathway

Biosynthetic Pathways for Chemical Synthesis

7

Chemical phenotypes of interest for de novo

metabolic pathway construction

8

Specialized structural motifs and unusual functional

groups in natural products

Biosynthetic Pathways for Chemical Synthesis

Methods for functional gene annotation

9

limit the scope of possible reactions

limit the size of libraries to determine

enzyme function

Functional genomic approaches

Engineering new or altered enzyme

Neutral drift mechanism of enzyme evolution

10

Engineering pathway balance

11

(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

12

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

13

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.

Synthetic biology of fluorine

14

SDS-PAGE gels of purified proteins

15

Assembly PCR

16

Structural alignment of NphT7 and the DEBS

Mod5 ketosynthase (KS) domain

Enzymatic production of a ctivated extender

units f or C–C bond-formation reactions

17

Formation of malonyl-CoA Formation of fluoromalonyl-CoA

18

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

19

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

20

NMR spectra of enzymatically synthesized fluoromalonyl-CoA

1H NMR 13C NMR

19F NMR

A chain-extension and ketoreduction cycle with a fluorinated extender

using a simple polyketide synthase, NphT7

21

Reactions catalyzed by NphT7 and PhaB

Steady-state kinetic parameters for NphT7 -catalyzed C–C bond formation measured using a

coupled assay with PhaB.

Characterization of enzymatically synthesized 2-fluoro-3-

hydroxybutyryl-CoA

22

19F NMR

LC/MS 1H-19F HMBC

Production of fluorinated polyketides

23

Reaction catalyzed by DEBS Mod6+TE using the NDK-SNAC substrate

Triketide lactones monitored by LC-MS

Amplification of TKL formation using MatB

24

Dependence of TKL formation on

methylmalonyl-CoA

Comparison of TKL yield with and without

MatB regeneration

25

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

26

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

Hydrolysis and regeneration reactions for F-TKL

production by DEBS Mod6+TE

27

Reaction scheme

showing enzymes

present in F-TKL

forming reactions

including observed

non-productive

hydrolysis reactions

(red) and the ATP

regenerating system

(blue).

28

Selectivity of DEBS Mod6+TE and DEBS Mod3+TE for the

methylmalonyl-CoA vrsus fluoromalonyl-CoA extender unit

Production of fluorinated polyketides in vivo

29

LC-MS traces showing regio selective tetraketidelactone formation using the DEBS mini-PKS

Production of fluorinated polyketides in vivo

ESI-MS/MS data for tetraketide lactones

30

F-TKL production in vivo

31

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

32

Thank you

Wangxiaoying

2013/9/28