Nature Biotechnology: doi:10.1038/nbt · Strain MB263(DE3) ∆sucD overexpressing the engineered platform composed of PaaJ (thiolase), PaaH (HACDH), PaaF (ECH), TdTer (ECR), Acot8

Supplementary Figure 1

Combinatorial synthesis of functionalized small molecules through the proposed orthogonal, iterative carbon-chain elongation platform using functionalized primers and extender units in combination with various termination pathways.

Potential products are shown for 20 different combinations of ω or ω-1-functionalities from primers (R1) and α-functionalities from extender units (R2), listed in the first column, with varying β-functionalities from intermediate nodes with varying degrees of β-reductions (β-ketoacyl-CoA, β-hydroxyacyl-CoA, enoyl-CoA and acyl-CoA). In the structures of intermediate nodes and products, functional groupsfrom primers (R1) are displayed in red and functional groups from extender units (R2) are displayed in blue. Products shown are produced through either acid-forming termination reactions (catalyzed by ACT) or alcohol-forming termination reactions (catalyzed by ACR+ADH). n represents the number of iterations/cycles of the platform, which determines the length of CoA-thioester intermediates and products. Check marks indicate the combinations of primers and extender units evaluated in this study.

Nature Biotechnology: doi:10.1038/nbt.3505


Comparison of primers, extender units, pathway intermediates, enzymes, and products of (a) orthogonal iterative platform proposed and demonstrated in this study and (b) engineered reversal of β-oxidation1.

The ability of thiolases and β-reduction enzymes to function in an iterative fashion with functionalized substrates had not beendemonstrated to date. The generation of these functionalized intermediates dictates the operation of the proposed platform with non-physiological intermediates that are not part of the host anabolic network and enables this orthogonal, iterative platform to facilitate the combinatorial synthesis of functionally diverse small molecules previously inaccessible by other pathways based on non-decarboxylative Claisen condensations, such as the β-oxidation reversal. Products shown are those demonstrated from the acyl-CoA node in each case, with product synthesis from additional pathway nodes also valid (See text and Figure 1b for details of all productsproduced in this study). In the structures of intermediate nodes, functional groups from primers are displayed in red and functional groups from extender units are displayed in blue. n represents the chain length of products.



Decarboxylative and non-decarboxylative Claisen condensation reactions in biological systems.

Claisen condensations mediate the formation of carbon-carbon bonds (represented as a green, thick bond) between the α carbon of the extender unit and the carbonyl carbon of the primer. (a) The β-carboxyl group of the extender unit malonyl-CoA/ACP is released as CO2

during decarboxylative Claisen condensation with acetyl-CoA/ACP priming. Generation of malonyl-CoA/ACP requires the ATP-dependent carboxylation of acetyl-CoA. (b) Non-decarboxylative Claisen condensation directly utilizes acetyl-CoA for carbon elongation, thus circumventing ATP consumption.



Products synthesized through the use of glycolyl-CoA as ω-hydroxylated primer (a, c) or α-hydroxylated extender unit (b).

(a) Total ion GC-MS chromatogram showing peak of synthesized 4-hydroxybutyric acid. (b) Enlarged region of inset in (a) showing 2,3-dihydroxybutyric acid peak. 4-hydroxybutyric acid was produced through the platform utilizing glycolyl-CoA as the primer and acetyl-CoA as the extender unit, while 2,3-dihydroxybutyric acid was produced through the platform with same enzymatic components bututilizing acetyl-CoA as the primer and glycolyl-CoA as the extender unit with termination at β-hydroxyacyl-CoA node. The following enzymes provided the individual components of the pathway: BktB (thiolase) and PhaB1 (HACDH) from Ralstonia eutropha2,3, Aeromonas caviae PhaJ (ECH)4, Treponema denticola TdTer (ECR)5 with native enzymes catalyzing the acid-forming termination and Megasphaera elsdenii transferase Pct activating glycolic acid to glycolyl-CoA. MG1655 (DE3) ∆glcD served as the host strain. (c) Production of β-hydroxy-γ-butyrolactone through the engineered platform with enzymes Pct, BktB and PhaB1 utilizing the primerglycolyl-CoA and the extender unit acetyl-CoA with termination at β-hydroxyacyl-CoA node. β-hydroxy-γ-butyrolactone is the lactone of 3,4-dihydroxybutyric acid and is generated through spontaneous or endogenous enzyme-catalyzed lactonization of 3,4-dihydroxybutyric acid or β-hydroxy intermediate 3,4-dihydroxybutyryl-CoA. glcD encodes a subunit of glycolate oxidase, an enzyme involved in thedegradation of glycolic acid. Functional groups from primer and extender unit are marked in red and blue, respectively. Strains weregrown as described in supplementary methods.



Time course of adipic acid production, glycerol consumption, acetate production and cell growth for a fermentation conducted in acontrolled bioreactor.

Strain MB263(DE3) ∆sucD overexpressing the engineered platform composed of PaaJ (thiolase), PaaH (HACDH), PaaF (ECH), TdTer(ECR), Acot8 (ACT) and Cat1 (activation enzyme) grown with glycerol as the sole carbon source (i.e. no succinic acid supplementation)with fermentation conditions as described in the Methods section. This profile corresponds to one of three independent (i.e. biological replicates) bioreactor fermentations.


Supplementary Information

Supplementary Table 1. Comparison of attributes of different catabolic pathways, anabolic pathways and proposed platform of this study.

PATHWAY ATTRIBUTE

Lactate fermentation 13

Ethanol fermentation 14

Mixed acid fermentation 15

Acetone-Butanol-Ethanol fermentation 16, 17

Fatty acid biosynthesis 18-22

Polyketide biosynthesis 21, 23-25

α-keto acid elongation pathway26-29

Isoprenoid biosynthesis 19

β-oxidation reversal1

Proposed platform (this study)

Energy efficiency High High High High Low Low High Low High High

Carbon efficiency High High High High High High Low Low High High

Kinetics (Flux/rate) High High High High High Low Low Low High High

Product functionality Low Low Low Low High High High High Low High

Modularity N.A.b N.A. N.A. N.A. High High High Low High High

Iterative N.A. N.A. N.A. N.A. Yes Yes Yes Yes Yes Yes

C-elongation resolutiona N.A. N.A. N.A. N.A. High (+2) High (+2) High (+1) Low (+5) High (+2) High(+≥2)

Combinatorial nature N.A. N.A. N.A. N.A. High High High Low Low High

Metabolic orthogonality Low Low Low Low Low Low Low Low Low High

a. The number in the parenthesis means the number of carbons added in the carbon chain per cycle of elongation.

b. N.A.: “Not applicable”.


Supplementary Table 2. Host strains and plasmids enabling functionalized small molecule synthesis with listed primer/extender unit combinations. See Methods section for strain details.

Host strain Plasmid 1 Plasmid 2 Primer Extender unit Product

JC01(DE3) pETDuet-P1-ppfadA-ppfadB

pCDFDuet-P1-paaK-P2-fabI

Phenylacetyl-CoA Acetyl-CoA

4-phenylbutyric acid 6-

phenylhexanoic acid

JC01(DE3) pETDuet-P1-paaJ-paaH

pCDFDuet-P1-cat1-paaF-P2-

tdTer

Succinyl-CoA Acetyl-CoA Adipic acid

Glutaryl-CoA Acetyl-CoA Pimelic acid

JST06(DE3) pETDuet-P1-

paaJ-paaH-P2-cbjALD


tdTer

Succinyl-CoA Acetyl-CoA 6-

hydroxyhexanoic acid

Glutaryl-CoA Acetyl-CoA 7-

hydroxyheptanoic acid

JC01(DE3) bktBCT5fadBCT5 Δfa

dA egTerCT5ydiIA1 Δtes

B

pETDuet-P1-pct N.A. Isobutyryl-CoA Acetyl-CoA 4-

methylpentanoic acid

JST07 (DE3) bktBCT5fadBCT5 Δfa

dA egTerCT5 pETDuet-P1-pct pCDFDuet-P1-

maqu2507 Isobutyryl-CoA Acetyl-CoA 4-methylpentanol

JST06(DE3) ΔsdhB pETDuet-P1-

paaJ-paaH-P2-acot8


tdTer Succinyl-CoA Acetyl-CoA Adipic acid

JST06(DE3) ΔsdhB pETDuet-P1-

dcaF-dcaH-P2-acot8

pCDFDuet-P1-cat1-dcaE-P2-

tdTer

Succinyl-CoA Acetyl-CoA Adipic acid Succinyl-CoA Acetyl-CoA Suberic acid Succinyl-CoA Acetyl-CoA Sebacic acid

JST06(DE3) ΔsdhB pETDuet-P1-paaJ-P2-pcaIJ

pCDFDuet-P1-cat1 Succinyl-CoA Acetyl-CoA Levulinic acid


pCDFDuet-P1-cat1-P2-mks1 Succinyl-CoA Acetyl-CoA Levulinic acid


pCDFDuet-P1-cat1-P2-adc Succinyl-CoA Acetyl-CoA Levulinic acid

MB263 (DE3) pETDuet-P1-

paaJ-paaH-P2-acot8



MB263 (DE3) ΔsucD

pETDuet-P1-paaJ-paaH-P2-

acot8



JC01(DE3) pETDuet-P1-fadB2x-fadB1x

pCDFDuet-P1-pct-fadAx-P2-

fabI

Acetyl-CoA Propionyl-CoA 2-methylbutyric

acid Tiglic acid

Propionyl-CoA Propionyl-CoA

2-methylpentanoic

acid (E)-2-methyl-2-pentenoic acid

JC01(DE3) pETDuet-P1-fadB2x-fadB1x

pCDFDuet-P1-pct-fadAx Acetyl-CoA Propionyl-CoA Tiglic acid

JST06(DE3) pETDuet-P1-fadB2x-fadB1x

pCDFDuet-P1-pct-fadAx Acetyl-CoA Propionyl-CoA N.A.


JST06(DE3) pETDuet-P1-

fadB2x-fadB1x-P2-ydiI

pCDFDuet-P1-pct-fadAx Acetyl-CoA Propionyl-CoA Tiglic acid

MG1655(DE3) ΔglcD

pETDuet-P1-bktB-phaB1-P2-

phaJ

pCDFDuet-P1-pct-P2-tdTer

Glycolyl-CoA Acetyl-CoA 4-hydroxybutyric acid

Acetyl-CoA Glycolyl-CoA 2,3-

dihydroxybutyric acid

MG1655(DE3) pETDuet-P1-bktB-phaB1

pCDFDuet-P1-pct Glycolyl-CoA Acetyl-CoA β-hydroxy-γ-

butyrolactone MG1655(DE3)

ΔglcD pETDuet-P1-bktB-phaB1

pCDFDuet-P1-pct Glycolyl-CoA Acetyl-CoA β-hydroxy-γ-

butyrolactone


Supplementary Table 3. Full fermentation product profile from α-functionalization platform shown in Fig. 3a.a

Compound Concentration (g/L)

Glycerol consumed 10.5±1.6

Products from propionyl-CoA extension

2-methylbutyric acid 0.075±0.016

Tiglic acid 0.573±0.068

2-methylpentanoic acid 0.049±0.014

(E)-2-methyl-2-pentenoic acid 0.084±0.030

Products from acetyl-CoA extension

3-hydroxybutyric acid 0.559±0.114

Butyric acid 1.51±0.11

Pentanoic acid 0.268±0.082

Major by-products

Succinate 0.708±0.018

Pyruvate 1.23±0.22

Acetate 2.38±0.11

a. JC01 overexpressing of Pseudomonas putida FadAx (thiolase), FadB2x (HACDH), FadB1x (ECH), Escherichia coli FabI and Megasphaera elsdenii Pct (activation enzyme). Endogenous ACTs performed the acid-forming termination reaction. Strain was grown as described in the Methods section with glycerol as the carbon source and inclusion of propionyl-CoA precursor propionic acid (20 mM). Average and s.d. were calculated from at least 4 biological replicates.


Supplementary Table 4. Escherichia coli K12 strains and plasmids used in this study.

Strain/plasmid Genotype Source

Strains

MG1655 F-λ-ilvG-rfb-50 rph-1 6

JC01 MG1655 ΔldhA::FRT ΔpoxB::FRT Δpta::FRT ΔadhE::FRT ΔfrdA::FRT

7

JC01(DE3) JC01 with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIq

8

JC01(DE3) bktBCT5fadBCT5 ΔfadA egTerCT5ydiIA1 ΔtesB

JC01(DE3) FRT-cymR-PCT5-fadB ΔfadA::zeo FRT-cymR-PCT5-bktB ΔatoB FRT-cymR-PCT5-egTer @fabI chromosomal location FRT-kan-FRT-tetR-PA1-tetO2-tetO2-ydiI ΔtesB @ tesB chromosomal location

9

JST06 JC01 ΔyciA::FRT ΔybgC::FRT ΔydiI::FRT ΔtesA::FRT ΔfadM::FRT ΔtesB::FRT

10

JST07 JST06 ΔfadE::FRT 10

JST07(DE3) JST07 with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIq

10

JST07 (DE3) bktBCT5fadBCT5 ΔfadA egTerCT5

JST07(DE3) FRT-cymR-PCT5-fadB ΔfadA::zeo FRT-cymR-PCT5-bktB ΔatoB FRT-cymR-PCT5-egTer @fabI chromosomal location

10

MG1655(DE3) MG1655 with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIq

11

MB263 MG1655 ΔldhA::FRT ΔpoxB::FRT Δpta::FRT ΔadhE::FRT

12

JST06(DE3) JST06 with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIq

This study

JST06(DE3) ΔsdhB JST06(DE3) ΔsdhB::FRT This study

MG1655(DE3) ΔglcD MG1655(DE3) ΔglcD::FRT This study

MB263 (DE3) MB263 with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIq

This study

MB263 (DE3) ΔsucD MB263(DE3) ΔsucD::FRT This study

Plasmids

pETDuet ColE1(pBR322) ori, lacI, T7lac, Novagen (Darmstadt, Germany)

pETDuet-P1-pct ColE1 ori; AmpR; PT7lac-1: pct This study

pETDuet-P1-ppfadA-ppfadB ColE1 ori; AmpR; PT7lac-1: ppfadA-ppfadB This study

pETDuet-P1-paaJ-paaH ColE1 ori; AmpR; PT7lac-1: paaJ-paaH This study

pETDuet-P1-paaJ-paaH-P2-cbjALD

ColE1 ori; AmpR; PT7lac-1: paaJ-paaH PT7lac-2: cbjALD This study

pETDuet-P1-paaJ-paaH-P2-acot8

ColE1 ori; AmpR; PT7lac-1: paaJ-paaH PT7lac-2: acot8 This study

pETDuet-P1-paaJ-P2-pcaIJ ColE1 ori; AmpR; PT7lac-1: paaJ PT7lac-2: pcaI-pcaJ This study

pETDuet-P1-dcaF-dcaH-P2- ColE1 ori; AmpR; PT7lac-1: dcaF-dcaH PT7lac-2: acot8 This study


acot8

pETDuet-P1-fadB2x-fadB1x ColE1 ori; AmpR; PT7lac-1: fadB2x-fadB1x This study

pETDuet-P1-fadB2x-fadB1x-P2-ydiI

ColE1 ori; AmpR; PT7lac-1: fadB2x-fadB1x PT7lac-2: ydiI This study

pETDuet-P1- bktB-phaB1 ColE1 ori; AmpR; PT7lac-1: bktB-phaB1 This study

pETDuet-P1- bktB-phaB1-P2-phaJ

ColE1 ori; AmpR; PT7lac-1: bktB-phaB1 PT7lac-2: phaJ This study

pCDFDuet-1 CloDF13 ori, lacI, T7lac, StrepR Novagen (Darmstadt, Germany)

pCDFDuet-P1-pct CloDF13 ori; StrepR; PT7lac-1: pct 8

pCDFDuet-P1-maqu2507 CloDF13 ori; StrepR; PT7lac-1: maqu2507 10

pCDFDuet-P1-paaK-P2-fabI CloDF13 ori; StrepR; PT7lac-1: paaK PT7lac-2: fabI This study

pCDFDuet-P1-cat1 CloDF13 ori; StrepR; PT7lac-1: cat1 This study

pCDFDuet-P1-cat1-paaF-P2-tdTer

CloDF13 ori; StrepR; PT7lac-1: cat1-paaF PT7lac-2: tdTer This study

pCDFDuet-P1-cat1-dcaE-P2-tdTer

CloDF13 ori; StrepR; PT7lac-1: cat1-dcaE PT7lac-2: tdTer This study

pCDFDuet-P1-cat1-P2-mks1 CloDF13 ori; StrepR; PT7lac-1: cat1 PT7lac-2: mks1 This study

pCDFDuet-P1-cat1-P2-adc CloDF13 ori; StrepR; PT7lac-1: cat1 PT7lac-2: adc This study

pCDFDuet-P1-pct-fadAx CloDF13 ori; StrepR; PT7lac-1: pct-fadAx This study

pCDFDuet-P1-pct-fadAx-P2-fabI

CloDF13 ori; StrepR; PT7lac-1: pct-fadAx PT7lac-2: fabI This study

pCDFDuet-P1-pct-P2-tdTer CloDF13 ori; StrepR; PT7lac-1: pct PT7lac-2: tdTer This study


Supplementary Table 5. Oligonucleotides used in this study for plasmid constructions.

Name Sequence

tdTer-f1 5’-AAGGAGATATACATATGATTGTTAAGCCGATGGTCC-3’

tdTer-r1 5’-TTGAGATCTGCCATATGTTAGATGCGGTCAAAACGTTCA-3’

cat1-f1 5’-AGGAGATATACCATGAGCAAAGGCATTAAAAAC-3’

cat1-r1 5’-CGCCGAGCTCGAATTCTTATTTCATGGAGCCGGTTT-3’

pct-f1 5’-AGGAGATATACCATGAGAAAAGTAGAAATCATTAC-3’

pct-r1 5’-CGCCGAGCTCGAATTCTTATTTTTTCAGTCCCATGGGAC-3’

paaF-f1 5’-CATGAAATAAGAATTTAAGGAGGAATATGGCATGAGCGAA CTGAT-3’

paaF-r1 5’-CGCCGAGCTCGAATTCTTAGCGTCCTTTAAAGTCGGG-3’

paaJ-f1 5’-AGGAGATATACCATGCGTGAAGCCTTTATTTGT-3’

paaJ-r1 5’-CGCCGAGCTCGAATTCTCAAACACGCTCCAGAATCA-3’

paaH-f1 5’-GTGTTTGAGAATTCGAAGGAGGAATATACCATGATGATAA ATGTGCAAACTGTGG-3’

paaH-r1 5’-CCTGCAGGCGCGCCGAGCTCTCATGACTCATAACCGCTCT CCAG -3’

paaH-f2 5’-CCCAGGCAAGTGGGCCGTATGGATAATTCACCCCAAGACG-3’

paaH-r2 5’-CGTCTTGGGGTGAATTATCCATACGGCCCACTTGCCTGGG-3’

acot8-f1 5’-AAGGAGATATACATATGAGCGCCCCGGAAG-3’

acot8-r1 5’-TTGAGATCTGCCATATGTTACAGCTTCGATTCTGAGACTTGC-3’

cbjALD-f1 5’-AAGGAGATATACATATGAATAAAGACACACTAATACC-3’

cbjALD-r1 5’-TTGAGATCTGCCATATGTTAGCCGGCAAGTACACATC-3’

paaK-f1 5’- AGGAGATATACCATGATAACCAATACAAAGCTTG-3’

paaK-r1 5’- CGCCGAGCTCGAATTCTCAGGCACCAACAATATTGC-3’

fabI-f1 5’-AAGGAGATATACATATGGGTTTTCTTTCCGGTAAG-3’

fabI-r1 5’-TTGAGATCTGCCATATGTTATTTCAGTTCGAGTTCGTTC-3’

ppfadA-f1 5’-AGGAGATATACC ATGAGCCTGAATCCGCGTG-3’

ppfadA-r1 5’-CGCCGAGCTCGAATTCTTAAACACGTTCAAAAACGGTG-3’

ppfadB-f1 5’-ACGTGTTTAAGAATTTAAGGAGGAATAAACC ATGATCTATGAAGGCAAAGCC-3’

ppfadB-r1 5’-CGCCGAGCTCGAATTCTTAGTTAAAAAAGCGCTGACC-3’

dcaF-f1 5’-AGGAGATATACC ATGCTGAACGCCTATATCTATG-3’

dcaF-r1 5’-CGCCGAGCTCGAATTCTTAGCTCACATTTTCAATAACC-3’

dcaH-f1 5’-TGTGAGCTAAGAATTTAAGGAGGAATAAACC ATGACCCACCCGATCAAAAA-3’

dcaH-r1 5’-CGCCGAGCTCGAATTCTTAGGTGGTAAAGGTCAGCG-3’

dcaE-f1 5’-CATGAAATAAGAATTTAAGGAGGAATAAACC ATGATTCCGGATCAGGATAAC-3’

dcaE-r1 5’-CGCCGAGCTCGAATTCTTATTTGCCATGATAGCTCGG-3’

pcaJ-f1 5’-AAGGAGATATACAT ATGACCATCACCAAAAAACTG-3’


pcaJ-r1 5’-TTGAGATCTGCCATATGTTATTTGATCAGCGGAACACC-3’

pcaI-f1 5’-AAGGAGATATACATATGATCAACAAAACCTATGAGAG-3’

pcaI-r1 5’-TTGGTGATGGTCATAGTTTATTCCTCCTTATTTAATTAAACTGCT TTGGCAATGCTG-3’

mks1-f1 5’- AAGGAGATATACATATGGAGAAAAGCATGTCGCC-3’

mks-r1 5’- TTGAGATCTGCCATATGTTATTTATACTTGTTAGCGATGC-3’

adc-f1 5’-AAGGAGATATACAT ATGCTGAAAGACGAGGTGATC-3’

adc-r1 5’-TTGAGATCTGCCATATGTTATTTCAGGTAGTCATAAATAAC

fadAx-f1 5’-GAAAAAATAAGAATTTAAGGAGGAATAAACC ATGACCCTGGCAAATGATCC-3’

fadAx-r1 5’-CGCCGAGCTCGAATTCTTAATACAGACATTCAACTGCC-3’

fadB2x-f1 5’-AGGAGATATACCATGCATATCGCCAACAAACAC-3’

fadB2x-r1 5’-CGCCGAGCTCGAATTCTTATTTTGCTGCCATGCGCAG-3’

fadB1x-f1 5’-AGCAAAATAAGAATTTAAGGAGGAATAAACC ATGGCCTTTGAAACCATTCTG-3’

fadB1x-r1 5’-CGCCGAGCTCGAATTCTTAGCGATCTTTAAACTGTGC-3’

ydiI-f1 5’-AAGGAGATATACATATGATATGGAAACGGAAAATCAC-3’

ydiI-r1 5’-TTGAGATCTGCCATATGTCACAAAATGGCGGTCGTC-3’

bktB-f1 5’-AGGAGATATACCATGATGACGCGTGAAGTGGTAGT-3’

bktB-r1 5’-CGCCGAGCTCGAATTCTCAGATACGCTCGAAGATGG-3’

phaB1-f1 5’-GCGTATCTGAGAATTAGGAGGCTCTCT ATGACTCAGCGCATTGCGTA

phaB1-r1 5’-CGCCGAGCTCGAATTCTCAGCCCATGTGCAGGCC-3’

phaJ-f1 5’-AAGGAGATATACATATGTCGGCACAAAGCCTG-3’

phaJ-r1 5’-TTGAGATCTGCCATATGTTACGGCAGTTTCACCACC-3’


Supplementary References

1. Dellomonaco, C., Clomburg, J.M., Miller, E.N. & Gonzalez, R. Engineered reversal of

the b-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355-359

(2011).

2. Slater, S. et al. Multiple β-Ketothiolases Mediate Poly(β-Hydroxyalkanoate) Copolymer

Synthesis in Ralstonia eutropha. J. Bacteriol. 180, 1979-1987 (1998).

3. Peoples, O.P. & Sinskey, A.J. Poly-b-hydroxybutyrate (PHB) biosynthesis in Alcaligenes

eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). J.

Biol. Chem. 264, 15298-15303 (1989).

4. Fukui, T., Shiomi, N. & Doi, Y. Expression and characterization of (R)-specific enoyl

coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas

caviae. J. Bacteriol. 180, 667-673 (1998).

5. Tucci, S. & Martin, W. A novel prokaryotic trans-2-enoyl-CoA reductase from the

spirochete Treponema denticola. FEBS Lett. 581, 1561-1566 (2007).

6. Kang, Y.S. et al. Systematic mutagenesis of the Escherichia coli genome. J. Bacteriol.

186, 4921-4930 (2004).

7. Clomburg, J.M., Vick, J.E., Blankschien, M.D., Rodriguez-Moya, M. & Gonzalez, R. A

synthetic biology approach to engineer a functional reversal of the b-oxidation cycle. ACS

Synthetic Biology 1, 541-554 (2012).

8. Vick, J.E. et al. Escherichia coli enoyl-acyl carrier protein reductase (FabI) supports

efficient operation of a functional reversal of the β-oxidation cycle. Appl. Environ.

Microbiol. 81, 1406-1416 (2015).

9. Clomburg, J.M. et al. Integrated engineering of β-oxidation reversal and ω-oxidation

pathways for the synthesis of medium chain ω-functionalized carboxylic acids. Metab.


Eng. 28, 202-212 (2015).

10. Kim, S., Clomburg, J. & Gonzalez, R. Synthesis of medium-chain length (C6–C10) fuels

and chemicals via β-oxidation reversal in Escherichia coli. J. Ind. Microbiol. Biotechnol.

42, 465-475 (2015).

11. Tseng, H.-C., Martin, C.H., Nielsen, D.R. & Prather, K.L.J. Metabolic engineering of

Escherichia coli for enhanced production of (R)- and (S)-3-hydroxybutyrate. Appl.

Environ. Microbiol. 75, 3137-3145 (2009).

12. Blankschien, M.D., Clomburg, J. & Gonzalez, R. Metabolic engineering of Escherichia

coli for the production of succinate from glycerol. Metab. Eng. 12, 409-419 (2010).

13. Hofvendahl, K. & Hahn–Hägerdal, B. Factors affecting the fermentative lactic acid

production from renewable resources. Enzyme and Microbial Technology 26, 87-107

(2000).

14. Dombek, K.M. & Ingram, L.O. Ethanol production during batch fermentation with

Saccharomyces cerevisiae: changes in glycolytic enzymes and internal pH. Appl. Environ.

Microbiol. 53, 1286-1291 (1987).

15. Neidhardt, F.C. & Curtiss, R. Escherichia coli and Salmonella: cellular and molecular

biology. (ASM Press, Washington, D.C.; 1996).

16. Girbal, L., Croux, C., Vasconcelos, I. & Soucaille, P. Regulation of metabolic shifts in

Clostridium acetobutylicum ATCC 824. Fems Microbiol. Rev. 17, 287-297 (1995).

17. Qureshi, N. & Blaschek, H.P. Recent advances in ABE fermentation: hyper-butanol

producing Clostridium beijerinckii BA101. J Ind Microbiol Biotech 27, 287-291 (2001).

18. Steen, E.J. et al. Microbial production of fatty-acid-derived fuels and chemicals from

plant biomass. Nature 463, 559-U182 (2010).

19. d’Espaux, L., Mendez-Perez, D., Li, R. & Keasling, J.D. Synthetic biology for microbial

production of lipid-based biofuels. Current Opinion in Chemical Biology 29, 58-65


(2015).

20. Choi, K.-H., Heath, R.J. & Rock, C.O. β-ketoacyl-acyl carrier protein synthase III (FabH)

is a determining factor in branched-chain fatty acid biosynthesis. J. Bacteriol. 182, 365-

370 (2000).

21. Pfleger, B.F., Gossing, M. & Nielsen, J. Metabolic engineering strategies for microbial

synthesis of oleochemicals. Metab. Eng. 29, 1-11 (2015).

22. Howard, T.P. et al. Synthesis of customized petroleum-replica fuel molecules by targeted

modification of free fatty acid pools in Escherichia coli. Proceedings of the National

Academy of Sciences 110, 7636-7641 (2013).

23. Moore, B.S. & Hertweck, C. Biosynthesis and attachment of novel bacterial polyketide

synthase starter units. Nat. Prod. Rep. 19, 70-99 (2002).

24. Dunn, B.J. & Khosla, C. Engineering the acyltransferase substrate specificity of assembly

line polyketide synthases, Vol. 10. (2013).

25. Cummings, M., Breitling, R. & Takano, E. Steps towards the synthetic biology of

polyketide biosynthesis, Vol. 351. (2014).

26. Marcheschi, R.J. et al. A synthetic recursive "+1" pathway for carbon chain elongation.

ACS Chem. Biol. 7, 689-697 (2012).

27. Felnagle, E.A., Chaubey, A., Noey, E.L., Houk, K.N. & Liao, J.C. Engineering synthetic

recursive pathways to generate non-natural small molecules. Nat Chem Biol 8, 518-526

(2012).

28. Shen, C.R. & Liao, J.C. Metabolic engineering of Escherichia coli for 1-butanol and 1-

propanol production via the keto-acid pathways. Metab. Eng. 10, 312-320 (2008).

29. Cann, A.F. & Liao, J.C. Production of 2-methyl-1-butanol in engineered Escherichia coli.

Appl. Microbiol. Biotechnol. 81, 89-98 (2008).


Documents

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