Iterative platform for the synthesis of alpha functionalized products

Abstract

The use of microorganisms to make alpha-functionalized chemicals and fuels, (e.g. alpha-functionalized carboxylic acids, alcohols, hydrocarbons, amines, and their beta-, and omega-functionalized derivatives), by utilizing an iterative carbon chain elongation pathway that uses functionalized extender units. The core enzymes in the pathway include thiolase, dehydrogenase, dehydratase and reductase. Native or engineered thiolases catalyze the condensation of either unsubstituted or functionalized acyl-CoA primers with an alpha-functionalized acetyl-CoA as the extender unit to generate alpha-functionalized β-keto acyl-CoA. Dehydrogenase converts alpha-functionalized β-keto acyl-CoA to alpha-functionalized β-hydroxy acyl-CoA. Dehydratase converts alpha-functionalized β-hydroxy acyl-CoA to alpha-functionalized enoyl-CoA. Reductase converts alpha-functionalized enoyl-CoA to alpha-functionalized acyl-CoA. The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting alpha-functionalized acyl-CoA as primer and the aforementioned alpha-functionalized extender unit in subsequent turns of the cycle. Termination pathways acting on any of the four alpha-functionalized CoA thioester intermediates terminate the platform and generate various alpha-functionalized carboxylic acids, alcohols and amines with different β-reduction degree.

Claims

1. A composition comprising a genetically engineered bacteria in a media containing propionic acid or glycolic acid, said bacteria comprising a Megasphaera elsdenii pct gene, Ralstonia eutropha bktB and phaB1 genes; Aeromonas caviae phaJ gene; and Treponema denticola TdTer gene.

2. A composition comprising a genetically engineered bacteria in a media containing propionic acid or glycolic acid, and said bacteria comprising a Megasphaera elsdenii pct gene; Pseudomonas putida fadAx, fadB2x, fadB1x genes; and E. coli YdiI gene.

3. A composition comprising a genetically engineered bacteria in a media containing propionic acid or glycolic acid, and said bacteria comprising a Megasphaera elsdenii pct gene; Pseudomonas putida fadAx, fadB2x, fadB1x genes; and E. coli FabI gene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Platform for the synthesis of alpha-functionalized carboxylic acids, alcohols and amines. Acyl-CoA primer, which is either unsubstituted or functionalized, and alpha-functionalized extender unit are mainly activated from their acid form, which can be either supplemented in the media or derived from carbon sources. Primer and extender unit can also be derived from carbon sources without the need to generate their acid forms. The platform is composed of thiolases, dehydrogenases, dehydratases and reductases. Thiolases catalyze a condensation between acyl-CoA primer and alpha-functionalized acyl-CoA extender and generates alpha-functionalized β-keto acyl-CoA. Dehydrogenases convert alpha-functionalized β-keto acyl-CoA to alpha-functionalized β-hydroxy acyl-CoA. Dehydratases convert alpha-functionalized β-hydroxy acyl-CoA to alpha-functionalized enoyl-CoA. Reductases convert alpha-functionalized enoyl-CoA to alpha-functionalized acyl-CoA. Iterative operation can be realized by using alpha-functionalized acyl-CoA as primer and either acetyl-CoA or alpha-functionalized acetyl-CoA as extender unit in subsequent turns of the platform. Termination pathways starting from four alpha-functionalized CoA thioester intermediates terminate the platform and generate various alpha-functionalized carboxylic acids, alcohols and amines with different β-reduction degrees. There are three types of termination pathways: thioesterase/CoA-transferase/phosphotransacylase+kinase, which generates carboxylic acids; acyl-CoA reductase and alcohol dehydrogenase which generate alcohols; acyl-CoA reductase and transaminase which generate amine. R.sub.1 and R.sub.2 mean functionalized group from primer and extender unit respectively. Dashed line means multiple reaction steps or iteration.

(2) FIG. 2: Proposed platform depicted in FIG. 1 and its products utilizing propionyl-CoA as the extender unit (R.sub.2 in FIG. 1=—CH.sub.3).

(3) FIG. 3: Example pathway of synthesis of tiglic acid (trans-2-methyl-2-butenoic acid) and 2-methylbutyric acid through the proposed platform with acetyl-CoA as the primer and propionyl-CoA as the extender unit. Propionyl-CoA is activated by Pct from propionic acid (Step 1). The platform is composed of thiolase FadAx, which catalyzes the condensation between primer acetyl-CoA and extender unit propionyl-CoA to 2-methyl acetoacetyl-CoA (Step 2); dehydrogenase FadB2x, which converts 2-methyl acetoacetyl-CoA to 2-methyl-3-hydroxybutyryl-CoA (Step 3); dehydratase FadB1x, which converts 2-methyl-3-hydroxybutyryl-CoA to tiglyl-CoA (Step 4); reductase FabI, which reduces tiglyl-CoA to 2-methylbutyryl-CoA (Step 5). Termination reactions by endogenous thioesterases from tiglyl-CoA (Step 6) and 2-methylbutyryl-CoA (Step 7) finally generate products tiglic acid and 2-methylbutyric acid.

(4) FIG. 4: Example pathway of synthesis of trans-2-methyl-2-pentenoic acid and 2-methylvaleric acid through the proposed platform with propionyl-CoA as the primer and the extender unit. Propionyl-CoA is activated by Pct from propionic acid (Step 1). The platform is composed of thiolase FadAx, which catalyzes the condensation between two molecules of propionyl-CoA to 2-methyl-3-oxopentanoyl-CoA (Step 2); dehydrogenase FadB2x, which converts 2-methyl-3-oxopentanoyl-CoA to 2-methyl-3-hydroxypentanoyl-CoA (Step 3); dehydratase FadB1x, which converts 2-methyl-3-hydroxypentanoyl-CoA to 2-methyl pentenoyl-CoA (Step 4); reductase FabI, which reduces 2-methyl-2-pentenoyl-CoA to 2-methylvaleryl-CoA (Step 5). Termination reactions by endogenous thioesterases from 2-methyl-2-pentenoyl-CoA (Step 6) and 2-methylvaleryl-CoA (Step 7) finally generate products 2-methyl-2-pentenoic acid and 2-methylvaleric acid.

(5) FIG. 5: Titers of alpha-methylated products synthesized through the utilization of propionyl-CoA as the extender unit with either acetyl-CoA or propionyl-CoA priming. These products were produced from the E. coli strain overexpressing enzymes catalyzing Steps 1-5 depicted in FIG. 3-4. JC01(DE3), an E. coli strain deficient of mixed-acid fermentations, served as the host strain. The engineered strains were grown for 48 hours under 37° C. in 20 mL LB-like MOPS media supplemented with 20 g/L glycerol and 20 mM propionic acid.

(6) FIG. 6: Pathway for the improved production of tiglic acid through the proposed platform with acetyl-CoA as the primer and propionyl-CoA as the extender unit. Propionyl-CoA is activated by Pct from propionic acid (Step 1). Thiolase FadAx condenses acetyl-CoA and propionyl-CoA to 2-methyl acetoacetyl-CoA (Step 2). Dehydrogenase FadB2x converts 2-methyl acetoacetyl-CoA to 2-methyl-3-hydroxybutyryl-CoA (Step 3). Dehydratase FadB1x converts 2-methyl-3-hydroxybutyryl-CoA to tiglyl-CoA (Step 4). Finally, thioesterase YdiI can remove the CoA from tiglyl-CoA to generate the product tiglic acid (Step 5).

(7) FIG. 7: Results of improvement of tiglic acid production by removal of overexpression of FabI (ECR), addition of overexpression of YdiI (a thioesterase) and usage of JST06(DE3) as the host strain. JST06(DE3) is an E. coli strain deficient of mixed-acid fermentations, thioesterases. The engineered strains were grown for 48 h at 37° C. in 20 mL LB-like MOPS media supplemented with 20 g/L glycerol and 20 mM propionic acid.

(8) FIG. 8: Time course for tiglic acid production from JST06(DE3) strain overexpressing Pct, FadAx, FadB2x, FadB1x and YdiI in a fermentation conducted in a controlled bioreactor. The fermentation was performed under 37° C. in LB-like MOPS media supplemented with 30 g/L glycerol, and 20 mM propionic acid which was added at 0, 24, and 48 h.

(9) FIG. 9: Proposed platform depicted in FIG. 1 and its products utilizing glycolyl-CoA as the extender unit (R2 in FIG. 1=—OH).

(10) FIG. 10: Example pathway of synthesis of 2,3-dihydroxy-butyric acid through the proposed platform with acetyl-CoA as the primer and propionyl-CoA as the extender unit. Glycolyl-CoA is activated by Pct from glycolic acid (Step 1). Then, condensation by thiolase BktB converts glycolyl-CoA and acetyl-CoA to 2-hydroxy acetoacetyl-CoA (Step 2). Dehydrogenase PhaB converts 2-hydroxy acetoacetyl-CoA to 2,3-dihydroxy-butyryl-CoA (Step 3). CoA removal by endogenous thioesterases convert 2,3-dihydroxy-butyryl-CoA to the product 2,3-dihydroxy-butyric acid (Step 4).

(11) FIG. 11: Peak of product 2,3-dihydroxy-butyric acid in the GC-MS chromatogram of the fermentation sample from MG1655(DE3) ΔglcD (pET-P1-bktB-phaB-P2-phaJ) (pCDF-P1-pct-P2-tdTER). The strain was grown in 50 mL LB media supplemented with 10 g/L glucose and 40 mM glycolate for 96 hours under 30° C. in 250 mL flask.

(12) FIG. 12: Derivatization pathway of product 2-hydroxy acid and intermediate 2-hydroxyacyl-CoA of the proposed platform utilizing glycolyl-CoA as the extender unit depicted in FIG. 3, to a primary alcohol product. 2-hydroxyacyl-CoA can be degraded to primary aldehyde and formyl-CoA by 2-hydroxyacyl-CoA lyase. 2-hydroxy acid can be converted to α-keto acid by keto-dehydrogenase and α-keto acid can be decarboxylated to primary aldehyde by α-keto acid to primary aldehyde. Primary aldehyde is finally reduced to primary alcohol by alcohol dehydrogenase.

(13) FIG. 13: Vector map of pCDFDuet-1-P1-ntH6-HACL1 for overexpression and purification of codon-optimized 2-hydroxyacyl-CoA lyase HACL1 from Homo sapiens in E. coli.

(14) FIG. 14: SDS-PAGE analysis result of overexpression of Homo sapiens HACL1 in E. coli.

(15) FIG. 15: Vector map of pYES260-HACL1-SCopt for overexpression and purification of codon-optimized 2-hydroxyacyl-CoA lyase HACL1 from Homo sapiens in Saccharomyces cerevisiae.

(16) FIG. 16: SDS-PAGE analysis result of overexpression and purification of Homo sapiens HACL1 in S. cerevisiae.

(17) FIG. 17: GC-FID chromatograms of pentadecanal content in HACL1 degradative reaction (forward reaction) mixtures after extraction with hexane. HACL1 was expressed and purified from S. cerevisiae. Top: pentadecanal standard; Middle: HACL1 assay sampled; Bottom: no enzyme control. In samples containing HACL1, a pentadecanal peak is seen, while there is no peak in the sample in which enzyme was omitted.

(18) FIG. 18: GC-FID chromatograms of pentadecanal content demonstrating HACL1 activity in E. coli BL21(DE3) crude extract. The peak of pentadecanal is shown in the square.

(19) FIG. 19: Proposed platform depicted in FIG. 1 and its products utilizing phenylacetyl-CoA as the extender unit (R.sub.2 in FIG. 1=-Ph).

(20) FIG. 20: Proposed platform depicted in FIG. 1 and its products utilizing phenylacetyl-CoA as the extender unit (R.sub.2 in FIG. 1=—NH.sub.2).

(21) FIG. 21. A partial listing of embodiments of the invention, any one or more of which can be combined with any other.

DETAILED DESCRIPTION

(22) This disclosure generally relates to the use of microorganisms to make alpha-functionalized chemicals and fuels, (e.g. alpha-functionalized carboxylic acids, alcohols, hydrocarbons, amines, and their beta-, and omega-functionalized derivatives), by utilizing a novel iterative carbon chain elongation pathway that uses functionalized extender units to grow a carbon chain by two carbon units.

(23) The core enzymes in the pathway include thiolase, dehydrogenase, dehydratase and reductase. Native or engineered thiolases catalyze the condensation of either unsubstituted or functionalized acyl-CoA primers with an alpha-functionalized acetyl-CoA as the extender unit to generate alpha-functionalized p-keto acyl-CoA. Dehydrogenase converts alpha-functionalized β-keto acyl-CoA to alpha-functionalized β-hydroxy acyl-CoA. Dehydratase converts alpha-functionalized β-hydroxy acyl-CoA to alpha-functionalized enoyl-CoA. Reductase converts alpha-functionalized enoyl-CoA to alpha-functionalized acyl-CoA.

(24) The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting alpha-functionalized acyl-CoA as primer and the aforementioned omega-functionalized extender unit in subsequent turns of the cycle. Various termination pathways (FIG. 1 and Table 4) acting on any of the four alpha-functionalized CoA thioester intermediates terminate the platform and generate various alpha-functionalized carboxylic acids, alcohols and amines with different β-reduction degrees.

(25) Thioesterase or CoA transferase or phosphotransacylase+carboxylate kinase can terminate the platform by converting the alpha-functionalized acyl-CoAs to alpha-functionalized carboxylic acids. If alpha-functionalized carboxylic acids has keto group at the beta-site, it can then be converted to ketone through reactions by beta-keto acid decarboxylase. Acyl-CoA reductases can terminate the platform by converting the alpha-functionalized acyl-CoAs to alpha-functionalized aldehydes. Alpha-functionalized aldehydes can then be converted to alpha-functionalized alcohols and alpha-functionalized amines through reactions by alcohol dehydrogenase and transaminase respectively.

(26) This disclosure also relates to a novel primary alcohol synthesis incorporating the proposed iterative platform using glycolyl-CoA (alpha-hydroxy acetyl-CoA) as the extender unit. When the platform uses glycolyl-CoA as the extender unit, it generates alpha-hydroxyacyl-CoA, which can be converted to primary alcohol by termination pathways selected from: a) 2-hydroxyacyl-CoA lyase (HACL) that converts alpha-hydroxyacyl-CoA to primary aldehyde with one less carbon and formyl-CoA, and alcohol dehydrogenase subsequently converts the primary aldehyde to primary alcohol; b) acid-forming termination enzyme selected from thioesterase, CoA transferase and phosphotransacylase+carboxylate kinase that converts alpha-hydroxyacyl-CoA to alpha-hydroxy acid, keto-dehydrogenase that converts alpha-hydroxy acid to alpha-keto acid, alpha-keto acid decarboxylase that converts alpha-keto acid to primary aldehyde with one less carbon and alcohol dehydrogenase subsequently converts the primary aldehyde to primary alcohol.

(27) Many examples of thiolase enzymes which can potentially catalyze the non-decarboxylative condensation of an acyl-CoA primer and acetyl-CoA extender unit are provided herein and Table 1 provides several additional examples which can also serve as templates for engineered variants:

(28) TABLE-US-00002 TABLE 1 Example Thiolase Enzymes (EC Number 2.3.1.—) Source organism and gene name Protein Accession Numbers E. coli atoB NP_416728.1 E. coli yqeF NP_417321.2 E. coli fadA YP_026272.1 E. coli fadI NP_416844.1 Streptomyces collinus fadA Q93C88 Ralstonia eutropha bktB AAC38322.1 Pseudomonas sp. Strain B13 catF AAL02407.1 E coli paaJ NP_415915.1 Pseudomonas putida pcaF AAA85138.1 Rhodococcus opacus pcaF YP_002778248.1 Streptomyces sp. pcaF AAD22035.1 Ralstonia eutropha phaA AEI80291.1 Clostridium acetobutylicum thlA AAC26023.1 Clostridium acetobutylicum thlB AAC26026.1

(29) This technology takes the above thiolase initiated pathway one step further to make alpha functionalized products. The method entails developing a new pathway that is based on native or engineered thiolases capable of catalyzing the condensation of either unsubstituted or functionalized acyl-CoA primers with an omega-functionalized acetyl-CoA as the extender unit. This has been reported in neither the scientific, peer-reviewed literature nor the patent literature.

(30) Materials that can be used with the invention include those in Tables 2-5 below.

(31) TABLE-US-00003 TABLE 2 Activation enzymes Protein EC Enzyme Source organism Accession Reaction Illustration Numbers names and gene name Numbers Carboxylic acid .fwdarw. Acyl- CoA (including acyl-CoA 6.2.1.- Acyl-CoA synthetase E. coli paaK E. coli sucCD   E. coli fadK E. coli fadD NP_415916.1 NP_415256.1 NP_415257.1 NP_416216.4 NP_416319.1 primer, and E. coli prpE NP_414869.1 α- E. coli menE NP_416763.1 functionalized Penicillium CAJ15517.1 acetyl-CoA chrysogenum phl acting as the Salmonella AAL19325.1 extender typhimurium unit) LT2 prpE Bacillus subtilis bioW AAC00261.1 Cupriavidus basilensis ADE20402.1 hmfD Rhodopseudomonas CAJ18317.1 palustris badA R. palustris hbaA CAE26113.1 Pseudomonas NP_249687.1 aeruginosa PAO1 pqsA Arabidopsis thaliana Q42524.1 4cl 2.8.3- CoA E. coli atoD NP_416725.1 transferase E. coli atoA NP_416726.1 E. coli scpC NP_417395.1 Clostridium kluyveri AAA92346.1 cat1 Clostridium kluyveri AAA92344.1 cat2 Clostridium NP_149326.1, acetobutylicum ctfAB NP_149327.1 Pseudomonas putida NP_746081.1 pcalJ NP_746082.1 Megasphaera elsdenii WP_014015705.1 pct Acidaminococcus CAA57199.1 fermentans gctAB CAA57200.1 Acetobacter aceti AGG68319.1 aarC E. coli ydiF NP_416209.1 2.3.1.-; Phospho- Clostridium NP _349676.1 2.7.2.1; transacylase + acetobutylicum ptb 2.7.2.15 Carboxylate Enterococcus faecalis AAD55374.1 kinase ptb Salmonella enterica AAD39011.1 pduL Clostridium AAK81015.1 acetobutylicum buk Enterococcus faecalis AAD55375.1 buk Salmonella enterica AAD39021.1 pduW

(32) TABLE-US-00004 TABLE 3 Reactions of the platform Protein EC Enzyme Source organism Accession Reaction Illustration Numbers names and gene name Numbers Acyl-CoA + α- functionalized acetyl-CoA .fwdarw. α- functionalized β-ketoacyl- CoA 2.3.1.- Thiolase E. coli atoB E. coli yqeF E. coli fadA E. coli fadl Ralstonia eutropha bktB Pseudomonas sp. Strain B13 catF E coli paaJ Pseudomonas putida pcaF NP_416728.1 NP_417321.2 YP_026272.1 NP_4168441 AAC38322.1   AAL02407.1   NP_415915.1 AAA85138.1   Rhodococcus opacus pcaF Streptomyces sp. pcaF Ralstonia eutropha phaA Clostridium acetobutylicum thIA Clostridium YP_002778248.1   AAD22035.1   AEI80291.1   AAC26023.1   AAC26026.1 acetobutylicum thIB Pseudomonas AAK18168.1 putida fadA P. putida fadAx AAK18171.1 Acinetobacter sp. CAG68532.1 ADP1 dcaF E. coli paaJ NP_415915.1 α- functionalized β-ketoacyl- CoA .fwdarw. α- functionalized β- hydroxyacyl- CoA 1.1.1.35; 1.1.1.36 Hydroxyacyl- CoA dehydrogenase E. coli fadB E. coli fadJ E. coli paaH P. putida fadB P. putida fadB2x Acinetobacter sp. ADP1 dcaH Ralstonia eutrophus phaB NP_418288.1 NP_416843.1 NP_415913.1 AAK18167.2 AAK18170.1 CAG68533.1   P14697.1   3-oxoacyl- [acyl-carrier- protein] reductase Clostridium acetobutylicum hbd E. coli fabG AAA95971.1   NP_415611.1 α- functionalized β-hydroxyacyl- CoA .fwdarw. α- functionalized enoyl-CoA 4.2.1.17; 4.2.1.119 enoyl-CoA hydratase E. coli fadB E. coli fadJ E. coli paaF P. putida fadB P. putida fadBlx Acinetobacter sp. ADP1 dcaE Clostridium acetobutylicum crt NP_418288.1 NP_416843.1 NP_415911.1 AAK18167.2 AAK18173.1 CAG68535.1   AAA95967.1 3- hydroxyacyl- [acyl-carrier- protein] dehydratase Aeromonas caviae phaJ E. coli fabA E. coli fabZ 032472.1   NP_415474.1 NP_414722.1 α- functionalized enoyl-CoA .fwdarw. α- functionalized acyl-CoA 0 1.3.1.44 enoyl-CoA reductase Euglena gracilis TER Treponema denticola TER Clostridium acetobutylicum TER Q5EU90.1   4GGO_A   4EUH_A enoy-[acyl- carrier- protein] reductase E. coli fabl Enterococcus faecalis fabK Bacillus subtilis fabL Vibrio cholerae fabV NP_415804.1 NP_816503.1   KFK80655.1   ABX38717.1 acyl-CoA E. coli fadE NP_414756.2 dehydrogenase E. coli ydiO NP_416210.4

(33) TABLE-US-00005 TABLE 4 Termination Pathways Protein EC Enzyme Source organism Accession Reaction Illustration Numbers names and gene name Numbers Acyl-CoA.fwdarw. Carboxylic acid 3.1.2.- Thioesterase E. coli tesA E. coli tesB E. coli yciA E. coli fadM E. coli ydil NP_415027.1 NP_414986.1 NP_415769.1 NP_414977.1 NP_416201.1 E. coli ybgC NP_415264.1 E. coli paal NP_415914.1 Mus musculus P58137.1 acot8 Lycopersicon ADK38536.1 hirsutum f glabratum mks2 Alcanivorax YP_692749.1 borkumensis tesB2 Fibrobacter YP_005822012.1 succinogenes Fs2108 Prevotella YP_003574018.1 ruminicola Pr655 Prevotella YP_003574982.1 ruminicola Pr1687 2.8.3- CoA E. coli atoD NP_416725.1 transferase E. coli atoA NP_416726.1 E. coli scpC NP_417395.1 Clostridium kluyveri AAA92346.1 cat1 Clostridium kluyveri AAA92344.1 cat2 Clostridium NP_149326.1, acetobutylicum NP_149327.1 ctfAB Pseudomonas NP_746081.1 putida pcalJ NP_746082.1 Megasphaera WP _014015705.1 elsdenii pct Acidaminococcus CAA57199.1 fermentans gctAB CAA57200.1 Acetobacter aceti AGG68319.1 aarC E. coli ydiF NP_416209.1 2.3.1.-; Phospho- Clostridium NP _349676.1 2.7.2.1; transacylase + acetobutylicum ptb 2.7.2.15 Carboxylate Enterococcus AAD55374.1 kinase faecalis ptb Salmonella enterica AAD39011.1 pduL Clostridium AAK81015.1 acetobutylicum buk Enterococcus AAD55375.1 faecalis buk Salmonella enterica AAD39021.1 pduW Acyl-CoA.fwdarw. Aldehyde 1.2.1.10 Aldehyde forming CoA reductase Acinetobacter calcoaceticus acr1 Acinetobacter sp Strain M-1 acrM Clostridium AAC45217.1   BAB85476.1   AAT66436.1 beijerinckii ald E. coli eutE NP_416950.1 Salmonella enterica AAA80209.1 eutE Marinobacter YP_959769.1 aquaeolei VT8 maqu_2507 E. coli mhpF NP_414885.1 Clostridium kluyveri EDK35023.1 sucD Aldehyde.fwdarw. Alcohol 1.1.1.- Alcohol dehydrogenase E. coli betA E. coli dkgA E. coli eutG E. coli fucO E. coli ucpA NP_414845.1 NP_417485.4 NP_416948.4 NP_417279.2 NP_416921.4 E. coli yahK NP_414859.1 E. coli ybbO NP_415026.1 E. coli ybdH NP_415132.1 E. coli yiaY YP_026233.1 E. coli yjgB NP_418690.4 Marinobacter YP_959769.1 aquaeolei VT8 maqu_2507 Saccharomyces Q04894.1 cerevisiae ADH6 Clostridium kluyveri EDK35022.1 4hbD Acinetobacter sp. AAG10028.1 SE19 chnD Aldehyde.fwdarw. Amine 2.6.1.- Transaminase Arabidopsis thaliana At3g22200 Alcaligenes denitrificans AptA Bordetella NP _001189947.1   AAP92672.1   WP_015041039.1 bronchiseptica BB0869 Bordetella WP_010927683.1 parapertussis BPP0784 Brucella melitensis EEW88370.1 BAWG_0478 Burkholderia AFI65333.1 pseudomallei BP102613_I0669 Chromobacterium AAQ59697.1 violaceum CV2025 Oceanicola WP_007254984.1 granulosus OG2516_07293 Paracoccus ABL72050.1 denitrificans PD1222 Pden_3984 Pseudogulbenkiania WP_008952788.1 ferrooxidans ω- TA Pseudomonas P28269.1 putida ω-TA Ralstonia YP_002258353.1 solanacearum ω-TA Rhizobium meliloti NP_386510.1 SMc01534 Vibrio fluvialis ω- AEA39183.1 TA Mus musculus AAH58521.1 abaT Flavobacterium BAB13756.1 lutescens lat Streptomyces AAB39899.1 clavuligerus lat E. coli gabT YP_490877.1 E. coli puuE NP_415818.1 E. coli ygjG NP_417544.5 β-keto acid .fwdarw. ketone 4.1.1.56; β-keto acid decarboxylase Lycopersicon hirsutum f glabratum mks1 Clostridium acetobutylicum adc ADK38535.1     AAA63761.1

(34) TABLE-US-00006 TABLE 5 Enzymes for derivatization of 2-hydroxy acid to primary alcohol Protein EC Enzyme Source organism Accession Reaction Illustration Numbers names and gene name Numbers 2-hydroxy acid .fwdarw. α-keto acid 1.1.1- Keto- dehydrogenase Clostridium beijerinckii adh E. coli serA Gordonia sp. TY-5 adh1 Gordonia sp. TY-5 adh2 AAA23199.2   NP_417388.1 BAD03962.1   BAD03964.1 Gordonia sp. TY-5 BAD03961.1 adh3 Rhodococcus ruber WP_043801412.1 adh-A Acidaminococcus ADB47349.1 fermentans hgdH E. coli ldhA NP_415898.1 E. coli lldO NP_418062.1 E. coli leuB NP_414615.4 α-keto acid .fwdarw. primary aldehyde 4.1.1.1 α-keto acid decarboxylase Lactococcus lactis kivd Saccharomyces cerevisiae PDC1 S. cerevisiae PDC5 S. cerevisiae PDC6 S. cerevisiae AIS03677.1   CAA97573.1   CAA97705.1 CAA97089.1 NP_010668.3 ARO10 S. cerevisiae THI3 CAA98646.1 Zymomonas mobilis ADK13058.1 pdc Primary aldehyde.fwdarw. Primary alcohol 1.1.1.- Alcohol dehydrogenase E. coli betA E. coli dkgA E. coli eutG E. coli fucO E. coli ucpA E. coli yahK E. coli ybbO NP_414845.1 NP_417485.4 NP_416948.4 NP_417279.2 NP_416921.4 NP_414859.1 NP_415026.1 E. coli ybdH NP_415132.1 E. coli yiaY YP _026233.1 E. coli yjgB NP_418690.4 Saccharomyces Q04894.1 cerevisiae ADH6 Clostridium kluyveri EDK35022.1 4hbD Acinetobacter sp. AAG10028.1 SE19 chnD 2- hydroxyacyl- CoA .fwdarw. primary aldehyde + formyl-CoA 4.1.-.- 2-hydroxyacyl- CoA lyase Homo sapiens hac1 Rattus norvegicus hac1 Dictyostelium discoideum hac1 Mus musculus hac1 Q9UJ83   Q8CHM7   Q54DA9   Q9QXE0

(35) All strains used in this study are listed in Table 6. Gene deletions were performed using P1 phage transduction with single-gene knockout mutants from the National BioResource Project (NIG, Japan) as the specific deletion donor. The λDE3 prophage, carrying the T7 RNA polymerase gene and lacIq, was integrated into the chromosome through λDE3 lysogenization kit (Novagen, Darmstadt, Germany). All strains were stored in 32.5% glycerol stocks at −80° C. Plates were prepared using LB medium containing 1.5% agar, and appropriate antibiotics were included at the following concentrations: ampicillin (100 μg/mL), spectinomycin (50 μg/mL), kanamycin (50 μg/mL), and chloramphenicol (34 μg/mL).

(36) All plasmids used in this study and oligonucleotides used in their construction are listed in Tables 6 and 7. Plasmid based gene overexpression was achieved by cloning the desired gene(s) into either pETDuet-1 or pCDFDuet-1 (Novagen, Darmstadt, Germany) digested with appropriate restriction enzymes using In-Fusion PCR cloning technology (Clontech Laboratories, Inc., Mountain View, Calif.). Cloning inserts were created via PCR of ORFs of interest from their respective genomic or codon-optimized DNA with Phusion polymerase (Thermo Scientific, Waltham, Mass.). E. coli genes were obtained from genomic DNA, while heterologous genes were synthesized by GenScript (Piscataway, N.J.) or GeneArt (Life Technologies, Carlsbad, Calif.) with codon optimization except for bktB, phaB1, and pct, which were amplified from genomic DNA or cDNA of their source organisms. The resulting In-Fusion products were used to transform E. coli Stellar cells (Clontech Laboratories, Inc., Mountain View, Calif.) and PCR identified clones were confirmed by DNA sequencing.

(37) TABLE-US-00007 TABLE 6 Strains and plasmids used in this study. Strain/plasmid Genotype E. coli Strains MG1655 F-λ-ilvG-rfb-50 rph-1 JC01 MG1655 ΔldhA::FRT ΔpoxB::FRT Δpta::FRT ΔadhE::FRT ΔfrdA::FRT JC01(DE3) JC01 with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacl.sup.q JST06 JC01 ΔyciA:FRT ΔybgC::FRT Δydil::FRT ΔtesA::FRT ΔfadM::FRT ΔtesB::FRT JST06(DE3) JST06 with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacl.sup.q MG1655(DE3) MG1655 with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacl.sup.q MG1655(DE3) ΔglcD MG1655(DE3) ΔglcD::FRT BL21(DE3) F- ompT gal dcm lon hsdS.sub.B(r.sub.B.sup.−m.sub.B.sup.−) λ(DE3 [lacl lacUV5-T7 gene 1 ind1 sam7 nin5]) [malB.sup.+].sub.K-12(λ.sup.S) S. cerevisiae strains INVSc1 MATa his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52 Plasmids pETDuet ColE1(pBR322) ori, lacl, T7lac, pETDuet-P1-fadB2x-fadB1x ColE1 ori; Amp.sup.R; P.sub.T7lac-1: fadB2x-fadB1x pETDuet-P1-fadB2x-fadB1x- ColE1 ori; Amp.sup.R; P.sub.T7lac-1: fadB2x-fadB1x P.sub.T7lac-2: ydil P2-ydil pETDuet-P1- bktB-phaB1 ColE1 ori; Amp.sup.R; P.sub.T7lac-1: bktB-phaB1 pETDuet-P1- bktB-phaB1-P2- ColE1 ori; Amp.sup.R; P.sub.T7lac-1: bktB-phaB1 P.sub.T7lac-2: phaJ phaJ pCDFDuet-1 CloDF13 ori, lacl, T7lac, Strep.sup.R pCDFDuet-P1-pct-fadAx CloDF13 ori; Strep.sup.R; P.sub.T7lac-1: pct-fadAx pCDFDuet-P1-pct-fadAx-P2- CloDF13 ori; Strep.sup.R; P.sub.T7lac-1: pct-fadAx P.sub.T7lac-2: fabl fabI pCDFDuet-P1-pct-P2-tdTer CloDF13 ori; Strep.sup.R; P.sub.T7lac-1: pct P.sub.T7lac-2: tdTer pCDFDuet-1-P1-ntH6-HACL1 CloDF13 ori; Strep.sup.R; P.sub.T7lac-1: ntHis6-HACL1 pYE260-HACL1 ColE1 ori; Amp.sup.R; P.sub.GAL1: ntHis6-HACL1

(38) TABLE-US-00008 TABLE 7 Oligonucleotides used in this study for plasmid constructions Name Sequence pct-f1 5′-AGGAGATATACCATGAGAAAAGTAGAAATCATTAC-3′ pct-r1 5′-CGCCGAGCTCGAATTCTTATTTTTTCAGTCCCATGGGAC-3′ fabl-f1 5′-AAGGAGATATACATATGGGTTTTCTTTCCGGTAAG-3′ fabl-r1 5′-TTGAGATCTGCCATATGTTATTTCAGTTCGAGTTCGTTC-3′ fadAx-f1 5′-GAAAAAATAAGAATTTAAGGAGGAATAAACCATGACCCTGGCAAATGATCC-3′ fadAx-r1 5′-CGCCGAGCTCGAATTCTTAATACAGACATTCAACTGCC-3′ fadB2x-f1 5′-AGGAGATATACCATGCATATCGCCAACAAACAC-3′ fadB2x-r1 5′-CGCCGAGCTCGAATTCTTATTTTGCTGCCATGCGCAG-3′ fadB1x-f1 5′-AGCAAAATAAGAATTTAAGGAGGAATAAACCATGGCCTTTGAAACCATTCTG-3′ fadB1x-r1 5′-CGCCGAGCTCGAATTCTTAGCGATCTTTAAACTGTGC-3′ ydil-f1 5′-AAGGAGATATACATATGATATGGAAACGGAAAATCAC-3′ ydil-r1 5′-TTGAGATCTGCCATATGTCACAAAATGGCGGTCGTC-3′ bktB-f1 5′-AGGAGATATACCATGATGACGCGTGAAGTGGTAGT-3′ bktB-r1 5′-CGCCGAGCTCGAATTCTCAGATACGCTCGAAGATGG-3′ phaB1-f1 5′-GCGTATCTGAGAATTAGGAGGCTCTCTATGACTCAGCGCATTGCGTA phaB1-r1 5′-CGCCGAGCTCGAATTCTCAGCCCATGTGCAGGCC-3′ phaJ-f1 5′-AAGGAGATATACATATGTCGGCACAAAGCCTG-3′ phaJ-r1 5′-TTGAGATCTGCCATATGTTACGGCAGTTTCACCACC-3′ HACL1-f1 5′-GCCAGGATCCGAATTctATGCCGGACAGCAACTTC-3′ HACL1-r1 5′-CGCCGAGCTCGAATTcTTACATATTGCTACGGGTCAGC-3′

(39) Fermentation medium and conditions: The minimal medium designed by Neidhardt et al. with 125 mM MOPS and Na.sub.2HPO.sub.4 in place of K.sub.2HPO.sub.4 (1.48 mM for fermentations in flasks; 2.8 mM for fermentations in bioreactors), supplemented with 20 g/L glycerol, 10 g/L tryptone, 5 g/L yeast extract, 100 μM FeSO.sub.4, 5 mM calcium pantothenate, 5 mM (NH.sub.4).sub.2SO.sub.4, and 30 mM NH.sub.4Cl was used for all fermentations unless otherwise stated. Neutralized 20 mM glycolic acid or propionic acid was supplemented as needed. Antibiotics (50 μg/mL carbenicillin and 50 μg/mL spectinomycin) were included when appropriate. All chemicals were obtained from Fisher Scientific Co. (Pittsburgh, Pa.) and Sigma-Aldrich Co. (St. Louis, Mo.).

(40) Unless otherwise stated, fermentations were performed in 25 mL Pyrex Erlenmeyer flasks (narrow mouth/heavy duty rim, Corning Inc., Corning, N.Y.) filled with 20 mL fermentation medium and sealed with foam plugs filling the necks. A single colony of the desired strain was cultivated overnight (14-16 h) in LB medium with appropriate antibiotics and used as the inoculum (1%). After inoculation, flasks were incubated in a NBS 124 Benchtop Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, N.J.) at 200 rpm and 37° C., except fermentations supplemented with phenylacetic acid or isobutyric acid in which the temperature was 30° C. When optical density (550 nm, OD550) reached ˜0.3-0.5, 5 μM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added for plasmid based gene expression in all cases except the following: 1 μM IPTG was used for adipic acid production from glycerol without succinic acid supplementation and 10 μM IPTG was used during production of ω-phenylalkanoic acids. For induction of controlled chromosomal expression constructs, 0.1 mM cumate and 15 ng/mL anhydrotetracycline were also added when appropriate. Flasks were then incubated under the same conditions for 48 h post-induction unless otherwise stated.

(41) Additional fermentations were conducted in a SixFors multi-fermentation system (Infors HT, Bottmingen, Switzerland) with an air flow rate of 2 N L/hr, independent control of temperature (37° C.), pH (controlled at 7.0 with NaOH and H.sub.2SO.sub.4), and stirrer speed (720 rpm). Tiglic acid fermentations used the previously described fermentation media with 30 g/L glycerol, the inclusion of 5 μM sodium selenite, and 5 μM IPTG. Propionic acid (20 mM) was added at 0, 24, and 48 h. Pre-cultures were grown in 25 mL flasks as described above, incubated for 4 h post-induction, and used for inoculation as described above.

(42) Fermentations with glycolyl-CoA as a primer were conducted in 250 mL Erlenmeyer Flasks filled with 50 mL LB media supplemented with 10 g/L glucose and appropriate antibiotics. The cultivation of inoculum was same as above but 2% inoculation was used. After inoculation, cells were cultivated at 30° C. and 250 rpm in a NBS 124 Benchtop Incubator Shaker until an optical density of ˜0.8 was reached, at which point IPTG (0.1 mM) and neutralized glycolic acid (40 mM) were added. Flasks were then incubated under the same conditions for 96 h post induction.

(43) GC sample preparation: Sample preparation was conducted as follows: 2 mL culture supernatant samples were transferred to 5 mL glass vials (Fisher Scientific Co., Fair Lawn, N.J., USA) and 80 μL of 50% H.sub.2SO.sub.4 and 340 μL of 30% NaCl solution were added for pH and ionic strength adjustment, respectively. Tridecanoic acid (final concentration 50 mg/L) was added as internal standard and 2 mL of hexane-MTBE (1:1) added for extraction. The bottles were sealed with Teflonlined septa (Fisher Scientific Co., Fair Lawn, N.J., USA), secured with caps, and rotated at 60 rpm for 120 min. The samples were then centrifuged for 2 min at 2,375×g to separate the aqueous and organic layers. 1 mL of the dry organic layer was transferred into a 2 mL borosilicate glass vial, dried under N.sub.2, and re-suspended in 100 μL of pyridine. After vortexing, 100 μL of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) was added, the samples were heated at 70° C. for 30 min, dried under N.sub.2 and re-suspended in 1 mL hexane for analysis.

(44) GC-MS metabolite identification: Except identifications of 2,3-dihydroxybutyric acid, metabolite identification was conducted via GC-MS in an Agilent 7890A GC system (Agilent Technologies, Santa Clara, Calif.), equipped with a 5975C inert XL mass selective detector (Agilent) and Rxi-5Sil column (0.25 mm internal diameter, 0.10 μm film thickness, 30 m length; Restek, Bellefonte, Pa.). The sample injection amount was 2 μL with 40:1 split ratio. The injector and detector were maintained at 280° C. The column temperature was held initially at 35° C. for 1 min and increased to 200° C. at the rate of 6° C./min, then to 270° C. at the rate of 30° C./min. That final temperature was maintained for 1 min before cooling back to initial temperature. The carrier gas was helium (2.6 mL/min, Matheson Tri-Gas, Longmont, Colo.).

(45) Identification of 2,3-dihydroxybutyric acid was conducted by the Baylor College of Medicine Analyte Center (bcm.edu/research/centers/analyte, Houston, Tex.). An Agilent 6890 GC system (Agilent Technologies, Santa Clara, Calif.), equipped with a 5973 mass selective detector (Agilent Technologies) and HP-5 ms column (Agilent Technologies) was used. Sample extraction was conducted using Agilent Chem Elut liquid extraction columns (Agilent Technologies) according to manufacturer protocols.

(46) HPLC metabolite quantification: The concentration of products were determined via ion-exclusion HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, Md.) equipped with an HPX-87H organic acid column (Bio-Rad, Hercules, Calif.) with operating conditions to optimize peak separation (0.3 ml/min flow rate, 30 mM H.sub.2SO.sub.4 mobile phase, column temperature 42° C.).

(47) In vitro enzyme assay: Purified HACL1 was tested for its native catabolic activity by assessing its ability to cleave 2-hydroxyhexadecanoyl-CoA to pentadecanal and formyl-CoA. Enzyme assays were performed in 50 mM tris-HCl pH 7.5, 0.8 mM MgCl.sub.2, 0.02 mM TPP, 6.6 μM BSA, and 0.3 mM 2-hydroxyhexadecanoyl-CoA. The assay mixtures were incubated for one hour at 37° C., after which the presence of pentadecanal was assessed by extraction with hexane and analysis by GC-FID.

(48) 2-hydroxyhexadecanoyl-CoA was prepared by the n-hydroxysuccinimide method. In summary, the n-hydroxysuccinimide ester of 2-hydroxyhexadecanoic acid is prepared by reacting n-hydroxysuccinimide with the acid in the presence of dicyclohexylcarbodiimide. The product was filtered and purified by recrystallization from methanol to give pure n-hydroxysuccinimide ester of 2-hydroxyhexadecanoic acid. The ester was reacted with CoA-SH in presence of thioglycolic acid to give 2-hydroxyhexadecanoyl-CoA. The 2-hydroxyhexadecanoyl-CoA was purified precipitation using perchloric acid, filtration, and washing the filtrate with perchloric acid, diethyl ether, and acetone.

(49) For specific activity assays (reported in μmol substrate/mg protein/min) these supernatant fractions were utilized and protein concentration was established using the Bradford Reagent (Thermo Sci.) using BSA as the protein standard.

(50) Enzyme purification: A plasmid containing the codon optimized gene encoding human HIS-tagged HACL1 was constructed as described. The resulting construct was transformed into S. cerevisiae InvSC1 (Life Tech.). The resulting strain was cultured in 50 mL of SC-URA media containing 2% glucose at 30° C. for 24 hours. The cells were pelleted and the required amount of cells were used to inoculate a 250 mL culture volume of SC-URA media containing 0.2% galactose, 1 mM MgCl.sub.2, and 0.1 mM thiamine to 0.4 OD600. After 20 hours incubation with shaking at 30° C., the cells were pelleted and saved.

(51) When needed, the cell pellets were resuspended to an OD600 of approximately 100 in a buffer containing 50 mM potassium phosphate pH 7.4, 0.1 mM thiamine pyrophosphate, 1 mM MgCl.sub.2, 0.5 mM AEBSF, 10 mM imidazole, and 250 units of Benzonase nuclease. To the cell suspension, approximately equal volumes of 425-600 μm glass beads were added. Cells were broken in four cycles of 30 seconds of vortexing at 3000 rpm followed by 30 seconds on ice. The glass beads and cell debris were pelleted by centrifugation and supernatant containing the cell extract was collected. The HIS-tagged HACL1 was purified from the cell extract using Talon Metal Affinity Resin as described above, with the only modification being the resin bed volume and all subsequent washes were halved. The eluate was collected in two 500 μL fractions.

(52) Expression and purification of the desired protein can be confirmed by running cell pellet sample and eluate on SDS-PAGE.

(53) We demonstrated several cases of the iterative system can synthesize alpha-functionalized small molecules through the use of alpha-functionalized forms of acetyl-CoA as the extender unit. One case used of propionyl-CoA as the extender unit. To implement this, P. putida FadAx (thiolase), FadB2x (HACD), FadB1x (ECH), and E. coli FabI (ECR) were used with Pct for activation of exogenous propionic acid. Expression in JC01(DE3) resulted in the production of 2-methylbutyric acid (75 mg/L) and tiglic acid (573 mg/L) (FIG. 5), representing products of acid-forming endogenous termination enzymes at the acyl-CoA and enoyl-CoA pathway nodes.

(54) Interestingly, 2-methylpentanoic acid (49 mg/L) and (E)-2-methyl-2-pentenoic acid (84 mg/L) were also synthesized, as the result of propionyl-CoA serving as both the primer and the extender unit. Products resulting from non-functionalized extender units (acetyl-CoA) with acetyl-CoA or propionyl-CoA priming were also observed, demonstrating the nonspecific activity of the thiolase (and subsequent β-reduction enzymes). This represents a potential area for further improvement through the selection and engineering of a thiolase with maximal specificity for the desired condensation. Additional alpha-functionalization was demonstrated with glycolyl-CoA (i.e. α-hydroxylated acetyl-CoA) as the extender unit, which with acetyl-CoA priming supported the synthesis of 2,3-dihydroxybutyric acid (FIG. 11).

(55) The ability of the alpha-functionalization system to support high product titers was investigated by improving tiglic acid production. Omission of ECR and manipulation of the termination pathway through deletion of native thioesterases and controlled overexpression of YdiI, a thioesterase previously shown to act effectively on α,β-unsaturated enoyl-CoAs, resulted in further improvement, from 573 mg/L to 1.39 g/L (FIG. 7). When a controlled bioreactor with a higher initial glycerol concentration was used, tiglic acid production increased to 3.79 g/L (11.6% mol/mol glycerol) (FIG. 8).

(56) The host strains and plasmids used for production of above products are summarized in Table 8.

(57) TABLE-US-00009 TABLE 8 Host strains and plasmids enabling alpha-functionalized small molecule synthesis with listed primer/extender unit combinations Host strain Plasmid 1 Plasmid 2 Primer Extender unit Product JC01(DE3) pETDuet-P1- pCDFDuet-P1-pct- Acetyl-CoA Propionyl-CoA 2-methylbutyric fadB2x-fadB1x fadAx-P2-fabl acid Tiglic acid Propionyl-CoA Propionyl-CoA 2- methylpentanoic acid (E)-2-methyl-2- pentenoic acid JC01(DE3) pETDuet-P1- pCDFDuet-P1-pct- Acetyl-CoA Propionyl-CoA Tiglic acid fadB2x-fadB1x fadAx JST06(DE3) pETDuet-P1- pCDFDuet-P1-pct- Acetyl-CoA Propionyl-CoA N.A. fadB2x-fadB1x fadAx JST06(DE3) pETDuet-P1- pCDFDuet-P1-pct- Acetyl-CoA Propionyl-CoA Tiglic acid fadB2x-fadB1x- fadAx P2-ydil Acetyl-CoA Glycolyl-CoA 2,3- Acetyl-CoA Glycolyl-CoA 2,3- dihydroxybutyric dihydroxybutyric acid acid

(58) We also successfully expressed Homo sapiens 2-hydroxyacyl-CoA lyase HACL1 in Saccharomyces cerevisiae and Escherichia coli (FIGS. 14 and 16), and confirmed its activity of degradation of 2-hydroxyhexadecanoyl-CoA to pentadecanal (FIGS. 17-18). This provides the potential of combination of 2-hydroxyacyl-CoA lyase with proposed iterative platform using alpha-hydroxylated glycolyl-CoA as the extender unit for the synthesis of primary alcohols.

(59) We believe that, pathway and process optimization, in line with industrial biotechnology approaches, can further improve performance for a specific target product, as the underlying carbon and energy efficiency enables the feasibility of further advancing product titer, rate, and yield. Important areas include generating and balancing pools of priming and extender units and optimization of required pathway enzymes for a given target product. The former can exploit previously developed pathways for primers and extender units, whereas the latter includes identifying and engineering enzymes that may be flux limiting due to suboptimal enzyme specificity or activity. These approaches will be continually aided by developments in protein and metabolic engineering and synthetic and systems biology.

(60) The above experiments are repeated in Bacillus subtilis. The same genes can be used, especially since Bacillus has no significant codon bias. A protease-deficient strain like WB800N is preferably used for greater stability of heterologous protein. The E. coli-B. subtilis shuttle vector pMTLBS72 exhibiting full structural stability can be used to move the genes easily to a more suitable vector for Bacillus. Alternatively, two vectors pHT01 and pHT43 allow high-level expression of recombinant proteins within the cytoplasm. As yet another alternative, plasmids using the theta-mode of replication such as those derived from the natural plasmids pAMβ1 and pBS72 can be used. Several other suitable expression systems are available. Since the FAS genes are ubiquitous, the invention is predicted to function in Bacillus.

(61) The above experiments are repeated in yeast. The same genes can be used, but it may be preferred to accommodate codon bias. Several yeast E. coli shuttle vectors are available for ease of the experiments. Since the FAS genes are ubiquitous, the invention is predicted to function in yeast, especially since yeasts are already available with exogenous functional TE genes and the reverse beta oxidation pathway has also been made to run in yeast.

(62) Each of the following is incorporated by reference herein in its entirety for all purposes: US20130316413 Reverse beta oxidation pathway 62/140,628 BIOCONVERSION OF SHORT-CHAIN HYDROCARBONS TO FUELS AND CHEMICALS, Mar. 31, 2015 WO2015112988 TYPE II FATTY ACID SYNTHESIS ENZYMES IN REVERSE BETA-OXIDATION, Jan. 26, 2015 and 61/932,057, Jan. 27, 2014. 62/069,850 SYNTHETIC PATHWAY FOR BIOSYNTHESIS FROM 1-CARBON COMPOUNDS, Oct. 29, 2014 61/531/911, Sep. 7, 2011; 61/440,192, Feb. 7, 2011, US20140273110, WO2013036812 Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation Heath, R. J. & Rock, C. O. The Claisen condensation in biology. Nat. Prod. Rep. 19, 581-596 (2002). Haapalainen, A. M., et al., The thiolase superfamily: condensing enzymes with diverse reaction specificities. Trends in Biochemical Sciences 31, 64-71 (2006). Jiang, C., et al., Divergent evolution of the thiolase superfamily and chalcone synthase family. Molecular Phylogenetics and Evolution 49, 691-701 (2008). Choi, K. H., et al., β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis. J. Bacteriol. 182, 365-370 (2000). Pfleger, B. F., et al., Metabolic engineering strategies for microbial synthesis of oleochemicals. Metab. Eng. 29, 1-11 (2015). Dellomonaco, C., et al., Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355-359 (2011). Clomburg, J. M., et al., Synthetic Biology Approach to Engineer a Functional Reversal of the β-Oxidation Cycle. ACS Synthetic Biology 1, 541-554 (2012). 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). Cheong, S., Clomburg, J. M. and Gonzalez, R.* (2016). Energy- and carbon-efficient synthesis of functionalized small molecules in bacteria using non-decarboxylative Claisen condensation reactions. Nat. Biotechnol. 34 (5): doi:10.1038/nbt.3505.

(63) The following claims are provided to add additional clarity to this disclosure. Future applications claiming priority to this application may or may not include the following claims, and may include claims broader, narrower, or entirely different from the following claims. Further, any detail from any claim may be combined with any other detail from another claim, even if not yet so combined.