SYNTHESIS OF OMEGA FUNCTIONALIZED PRODUCTS

Abstract

The use of microorganisms to make omega- and/or omega-1-functionalized products through an iterative carbon chain elongation pathway that we call a reverse beta oxidation pathway. The pathway uses omega-functionalized CoA thioesters as primers and acetyl-CoA as the extender unit in a non-decarboxylative Claisen condensation, and then uses beta oxidation or fatty acid synthesis enzymes to complete the cycle, via reductase, dehydratase and reductase reactions. Various termination enzymes that act on the functionalized beta-keto acyl-CoA intermediates of the pathway and produce omega or omega-1 functionalized products. The action of termination enzymes on such intermediates yield a large variety of products.

Claims

1) A genetically engineered microorganism, comprising: a) an overexpressed activation enzyme(s) able to produce an omega-1-(-1) functionalized CoA thioester primer, wherein said activation enzyme is selected from: i) an acyl-CoA synthase that generates an -1-functionalized CoA thioester primer from an -1-functionalized acid; ii) an acyl-CoA transferase that generates the -1-functionalized CoA thioester primer from an -1-functionalized acid; iii) a phosphotransacylase and a carboxylate kinase that generates the -1-functionalized CoA thioester primer from an -1-functionalized acid; iv) one or more enzymes that generates the -1-functionalized CoA thioester primer from a carbon source without proceeding via an -1-functionalized acid; b) an overexpressed thiolase that catalyzes the condensation of said -1-functionalized acyl-CoA primer with acetyl-CoA to form an -1-functionalized beta-ketoacyl-CoA; c) an overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-[ACP] reductase that catalyzes the reduction of said -1-functionalized beta-ketoacyl-CoA to produce an -1-functionalized beta-hydroxyacyl-CoA; d) an overexpressed enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydratase, or 3-hydroxyacyl-[ACP] dehydratase that catalyzes the dehydration of said -1-functionalized beta-hydroxyacyl-CoA to an -1-functionalized trans-enoyl-CoA; e) an overexpressed acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl-[ACP] reductase that catalyzes the reduction of said -1-functionalized trans-enoyl-CoA to an -1-functionalized acyl-CoA; f) an overexpressed termination enzyme(s) able to remove -1-intermediates from steps b-e and produce an -1-functionalized product; g) optionally reduced expression of fermentation genes leading to reduced production of lactate, acetate, ethanol and succinate; and h) wherein said microorganism has a reverse beta-oxidation pathway beginning with said -1-functionalized CoA thioester primer and running in a biosynthetic direction.

2) The microorganism of claim 1, wherein said -1-functionalized CoA thioester primer is functionalized with a group selected from alkyl group, hydroxyl group, carboxyl group, aryl group, halogen, amino group, hydroxyacyl group, carboxyacyl group, aminoacyl group, ketoacyl group, halogenated acyl group, and a functionalized acyl group.

3) The microorganism of claim 1, wherein said termination pathway comprises one or more of: a) a thioesterase, or an acyl-CoA transferase, or a phosphotransacylase and a carboxylate kinase catalyzing a conversion of an omega-1-functionalized thioester intermediate of steps b, c, d, or e to a carboxylic acid; b) an alcohol-forming acyl-CoA reductase catalyzing a conversion of said omega-1-functionalized intermediates of steps b, c, d, or e to an alcohol; c) an aldehyde-forming acyl-CoA reductase catalyzing a conversion of said omega-1-functionalized thioester intermediates of steps b, c, d, or e to an aldehyde, and an alcohol dehydrogenase catalyzing a conversion of said aldehyde to an alcohol; d) an aldehyde-forming acyl-CoA reductase catalyzing a conversion of said omega-1-functionalized thioester intermediates of steps b, c, d, or e to an aldehyde, and an aldehyde decarbonylase catalyzing a conversion of said aldehyde to an alkane; or e) an aldehyde-forming acyl-CoA reductase catalyzing a conversion of said omega-1-functionalized thioester intermediates of steps b, c, d, or e to an aldehyde, and a transaminase catalyzing a conversion of said aldehyde to an amine; f) an overexpressed -keto acid decarboxylase catalyzing a conversion of an omega-1-functionalized -keto-acid to an omega-1-functionalized methyl ketone; g) an overexpressed amidohydrolase catalyzing a conversion of an omega-1 amino acid to a lactam; or h) an overexpressed lactonase catalyzing the conversion of an omega-1 hydroxy acid to a lactone.

4) The microorganism of claim 1, wherein said activation enzyme is encoded by a gene(s) selected from E. coli paaK; E. coli sucCD; E. coli fadK; E. coli fadD; E. coli prpE; E. coli menE; Penicillium chrysogenum phl; Salmonella typhimurium LT2 prpE; Bacillus subtilis bioW; Cupriavidus basilensis hmfD; Rhodopseudomonas palustris badA; R. palustris hbaA; Pseudomonas aeruginosa PAO1 pqsA; Arabidopsis thaliana 4cl; E. coli atoD; E. coli atoA; E. coli scpC; Clostridium kluyveri cat1; Clostridium kluyveri cat2; Clostridium acetobutylicum ctfAB; Pseudomonas putida pcaIJ; Megasphaera elsdenii pct; Acidaminococcus fermentans gctAB; Acetobacter aceti aarC; E. coli ydiF; Clostridium acetobutylicum ptb; Enterococcus faecalis ptb; Salmonella enterica pduL; Clostridium acetobutylicum buk, Enterococcus faecalis buk and Salmonella enterica pduW.

5) The microorganism of claim 1, wherein said overexpressed thiolase is encoded by a gene(s) selected from the group consisting of E. coli atoB, E. coli yqeF, E. coli fadA, E. coli fadI, Ralstonia eutropha bktB, Pseudomonas sp. B13 catF, E coli paaJ, Rhodococcus opacus pcaF, Pseudomonas putida pcaF, Streptomyces sp. pcaF, P. putida fadAx, P. putida fadA, Ralstonia eutropha phaA, Acinetobacter sp. ADP1 dcaF, Clostridium acetobutylicum thlA, and Clostridium acetobutylicum thlB.

6) The microorganism of claim 1, wherein said overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-[acyl-carrier-protein] reductase is encoded by a gene(s) selected from the group consisting of E. coli fabG, E. coli fadB, E. coli fadB, E. coli paaH, P. putida fadB, P. putida fadB2x, Acinetobacter sp. ADP1 dcaH, Ralstonia eutrophus phaB, and Clostridium acetobutylicum hbd.

7) The microorganism of claim 1, wherein said overexpressed enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydratase, or 3-hydroxyacyl-[acyl-carrier-protein] dehydratase is encoded by a gene(s) selected from the group consisting of E. coli fabA, E. coli fabZ, E. coli fadB, E. coli fadJ, E. coli paaF, P. putida fadB, P. putida fadBlx, Acinetobacter sp. ADP1 dcaE, Clostridium acetobutylicum crt, and Aeromonas caviae phaJ.

8) The microorganism of claim 1, wherein said acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl-[acyl-carrier-protein] reductase is encoded by a gene(s) selected from the group consisting of E. coli fadE, E. coli ydiO, Euglena gracilis TER, Treponema denticola TER, Clostridium acetobutylicum TER, E. coli fabI, Enterococcus faecalis fabK, Bacillus subtilis fabL, and Vibrio cholerea fabV.

9) The microorganism of claim 1, wherein said termination enzymes are selected from one or more enzymes encoded by a gene(s) selected from E. coli tesA; E. coli tesB; E. coli yciA; E. coli fadM; E. coli ydiI; E. coli ybgC; E. coli paal; Mus musculus acot8; Lycopersicon hirsutum f glabratum mks2; Alcanivorax borkumensis tesB2; Fibrobacter succinogenes Fs2108; Prevotella ruminicola Pr655; Prevotella ruminicola Pr1687; E. coli atoD; E. coli atoA; E. coli scpC; Clostridium kluyveri cat1; Clostridium kluyveri cat2; Clostridium acetobutylicum ctfAB; Pseudomonas putida pcaIJ; Megasphaera elsdenii pct; Acidaminococcus fermentans gctAB; Acetobacter aceti aarC; E. coli ydiF; Clostridium acetobutylicum ptb; Enterococcus faecalis ptb; Salmonella enterica pduL; Clostridium acetobutylicum buk, Enterococcus faecalis buk, Salmonella enterica pduW; Lycopersicon hirsutum f glabratum mks1; Clostridium acetobutylicum adc; Arabidopsis thaliana At3g22200; Alcaligenes denitrificans AptA; Bordetella bronchiseptica BB0869; Bordetella parapertussis BPP0784; Brucella melitensis BAWG_0478; Burkholderia pseudomallei BP1026B_I0669; Chromobacterium violaceum CV2025; Oceanicola granulosus OG2516_07293; Paracoccus denitrificans PD1222 Pden_3984; Pseudogulbenkiania ferrooxidans -TA; Pseudomonas putida -TA; Ralstonia solanacearum -TA; Rhizobium meliloti SMc01534; Vibrio fluvialis -TA; Mus musculus abaT; Flavobacterium lutescens lat; Streptomyces clavuligerus lat; E. coli gabT; E. coli puuE; E. coli ygjG; Clostridium beijerinckii adh; E. coli sera; Gordonia sp. TY-5 adh1; Gordonia sp. TY-5 adh2; Gordonia sp. TY-5 adh3; Rhodococcus ruber adh-A; Acidaminococcus fermentans hgdH; Comamonas testosteroni pmdD; Xanthomonas campestris XCC1745; Homo sapiens PON1; Mesorhizobium loti Mlr6805; Pseudomonas sp. P51 tcbE; Flavobacterium sp. KI72 nylB; Arthrobacter sp. KI72 nylA; Homo sapiens DPYS; Brevibacillus agri pydB; E. coli pyrC; Pseudomonas putida crnA; Pseudomonas fluorescens puuE; Acinetobacter calcoaceticus acr1; Acinetobacter sp Strain M-1 acrM; Clostridium beijerinckii ald; E. coli eutE; Salmonella enterica eutE; E. coli mhpF; Clostridium kluyveri sucD; E. coli betA; E. coli dkgA; E. coli eutG; E. coli fucO; E. coli ucpA; E. coli yahK; E. coli ybbO; E. coli ybdH; E. coli yiaY; E. coli yjgB; Saccharomyces cerevisiae ADH6; Clostridium kluyveri 4hbD; Acinetobacter sp. SE19 chnD; Arabidopsis thaliana At3g22200; Alcaligenes denitrificans AptA; Bordetella bronchiseptica BB0869; Bordetella parapertussis BPP0784; Brucella melitensis BAWG_0478; Burkholderia pseudomallei BP1026B_I0669; Chromobacterium violaceum CV2025; Oceanicola granulosus OG2516_07293; Paracoccus denitrificans PD1222 Pden_3984; Pseudogulbenkiania ferrooxidans -TA; Pseudomonas putida -TA; Ralstonia solanacearum -TA; Rhizobium meliloti SMc01534; Vibrio fluvialis -TA; Mus musculus abaT; Flavobacterium lutescens lat; Streptomyces clavuligerus lat; E. coli gabT; E. coli ygjG; and E. coli puuE.

10) The microorganism of claim 1, wherein said reduced expression of fermentation enzymes are adhE, (pta or ackA or ackApta), poxB, ldhA, and frdA.

11) The microorganism of claim 1, comprising the following mutations: fadR, atoC(c), arcA, crp, crp*.

12) A recombinant microorganism, said microorganism having a reverse beta oxidation pathway running in a biosynthetic direction and comprising overexpressed enzymes including 1) a thiolase catalyzing a non-decarboxylative Claisen condensation between an -1-functionalized primer and acetyl-CoA, 2) a hydroxyacyl-CoA dehydrogenase, 3) an enoyl-CoA hydratase, 4) an enoyl-CoA reductase and 5) a termination enzyme(s) catalyzing conversion of intermediates of said reverse beta oxidation pathway to one or more -1-functionalized product(s).

13) The recombinant microorganism of claim 12, said microorganism being a bacteria.

14) The recombinant microorganism of claim 12, said microorganism being E. coli.

15) A method of making -1-functionalized products, comprising growing a microorganism of claim 1 in a nutrient broth under conditions such that said enzymes are overexpressed, said microorganism producing -functionalized product using said overexpressed enzymes, and isolating said -functionalized product or a derivative of said -functionalized product.

16) The method of claim 15, said nutrient broth being supplemented with an -1-functionalized acid.

17) The method of claim 15, wherein said microorganism is a microorganism of claim 3.

18) The method of claim 15, wherein said microorganism a microorganism of claim 4.

19) The method of claim 16, wherein said microorganism a microorganism of claim 4.

20) The method of claim 16, wherein said microorganism a microorganism of claim 9.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG. 1A-B. (81): Platform for the synthesis of /-1-functionalized products, where the /-1-functionalized primer is mainly activated from its acid form, which can be either supplemented in the media or derived from carbon sources, catalyzed by CoA-synthase, CoA transferase or phosphotransacylase+kinase. Primer can also be derived from carbon sources without this step. Condensation between /-1-functionalized primer and acetyl-CoA catalyzed by thiolase forms /-1-functionalized -keto acyl-CoA. Further carbon chain elongation is achieved by subsequent reactions by dehydrogenase, dehydratase and reductase and iterations of the cycle.

[0076] Termination pathways are quite diverse, but exemplary termination pathways include CoA removal by thioesterase or CoA transferase and phosphotransacylase+kinase and decarboxylation by decarboxylase generate /-1-functionalized methyl ketone from /-1-functionalized -keto acyl-CoA. Subsequent dehydrogenation by keto-dehydrogenase and amino group transfer by transaminase convert /-1-functionalized methyl ketone into /-1-functionalized 2-alcohol and 2-amine respectively.

[0077] R means functional group and n means length of primers, intermediates and products. Dashed line means multiple reaction steps or iteration. Here, we showed only the functional groups, but it is understood that the figure applies to both and -1 functionalized primers, intermediates and products.

[0078] FIG. 2A-B (81): Synthesis of -1-carboxylated methyl ketones, 2-alcohols and 2-amines, namely -1 ketoacids, hydroxyacids and amino acids, through the platform depicted in FIG. 1 (R in FIG. 1=COOH). Omega-carboxylated acyl-CoA, which is activated from ,-diacid, serves as the primer.

[0079] FIG. 3A-B (81): Derivatives of -1 ketoacids, hydroxyacids and amino acids, which could be synthesized through additional enzymatic and metabolic reactions. Products shown include omega-functionalized methyl ketones, 2-alcohols, 2-amines, and their derivatives, including lactams, lactones, ,-1-diamines, -1-amino-1-alcohols, -amino methyl ketones, -hydroxy methyl ketones, -amino-2-alcohols, ,-1-diols.

[0080] FIG. 4 (81): Example of synthesis of levulinic acid (4-oxopentanoic acid) through the proposed platform with succinyl-CoA as the primer. Succinyl-CoA is activated from succinate by Cat1. Levulinic acid is produced after subsequent condensation between succinyl-CoA and acetyl-CoA catalyzed by PaaJ, CoA removal catalyzed by PcaIJ and decarboxylation by Mks1/Adc.

[0081] FIG. 5 (81): Titers of levulinic acid synthesized through the platform depicted in FIG. 4 with different enzymes catalyzing the first four steps. JST06(DE3) sdhB, an E. coli strain deficient of mixed-acid fermentations, thioesterases and TCA cycle, served as the host strain. The engineered strains were grown for 48 hours at 37 C. in 20 mL LB-like MOPS media supplemented with 20 g/L glycerol and 20 mM succinate.

[0082] FIG. 6A-B (81): The pathway to validate and demonstrate the iterative carbon elongation platform utilizing thiolase-catalyzed non-decarboxylative Claisen condensation which accepts omega-functionalized acyl-CoA primers. The validation is through analyzing whether omega-functionalized carboxylic acids or omega-functionalized alcohols are produced after adding termination pathways acyl-CoA thioesterase/transferase (ACT) or acyl-CoA reductase+alcohol dehydrogenase (ACR+ADH) respectively at the acyl-CoA node of the platform. Omega-functionalization was demonstrated (see FIG. 7-11): omega-phenylation (R=-Ph); omega-carboxylation (R=COOH); omega-hydroxylation (R=OH) and omega-1-methylation (RCH(CH.sub.3).sub.2).

[0083] FIG. 7 (81): Titers of omega-phenylalkanoic acids produced with phenylacetyl-CoA (R=Phenyl) as the primer. Utilized host strain and enzymatic components are listed in the bottom part. Endogenous refers to native enzymes without overexpression. The engineered strain was grown for 48 hours at 30 C. in 20 mL LB-like MOPS media supplemented with 20 g/L glycerol and 5 mM phenylacetic acid.

[0084] FIG. 8 (81): Titers of dicarboxylic acids and omega-hydroxy acids produced with succinyl-CoA and glutaryl-CoA (R=COOH) as the primer, and omega-1-methyl fatty acid and omega-1-methyl alcohol with isobutyryl-CoA (R=CH(CH.sub.3).sub.2) as the primer. Utilized host strain and enzymatic components are listed in the bottom part. Endogenous herein refers to native enzymes without overexpression. The engineered strains were grown for 48 hours at 37 C. (when using succinyl-CoA or glutaryl-CoA as the primer) or 30 C. (when using isobutyryl-CoA as the primer) in 20 mL LB-like MOPS media supplemented with 20 g/L glycerol and 20 mM succinate or glutaric acid or isobutyric acid.

[0085] FIG. 9 (81): Total ion GC-MS chromatogram showing peak of 4-hydroxybutyric acid synthesized with glycolyl-CoA (R=OH) as the primer. The following enzymes provided the individual components of the pathway: BktB (thiolase) and PhaB1 (HACDH) from Ralstonia eutropha, Aeromonas caviae PhaJ (ECH), Treponema denticola TdTer (ECR) 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. The engineered strain was grown for 96 hours at 30 C. in 50 mL LB media supplemented with 10 g/L glucose and 40 mM glycolic acid.

[0086] FIG. 10 (81): Improvement of adipic acid synthesis and synthesis of dicarboxylic acids of different chain lengths through the iterative system depicted in FIG. 6 (81) with succinyl-CoA priming and specified pathway enzymes listed in the bottom part. The engineered strains were grown for 48 hours at 37 C. in 20 mL LB-like MOPS media supplemented with 20 g/L glycerol and 20 mM succinate.

[0087] FIG. 11 (81): Adipic acid production from glycerol through the pathway depicted in FIG. 6 priming from succinyl-CoA without the addition of primer precursor succinic acid in either shake flasks or controlled bioreactors.

[0088] FIG. 12A-B (81): Synthesis of omega-phenyl methyl ketones, 2-alcohols and 2-amines, through the platform depicted in FIG. 1(R=-Phenyl). Omega-phenylacyl-CoA, which is activated from omega-phenylalkanoic acid, serves as the primer.

[0089] FIG. 13A-B (81): Synthesis of omega-1-methyl methyl ketones, 2-alcohols and 2-amines, through the platform depicted in FIG. 1 (R=CH(CH.sub.3).sub.2). Omega-1-methyl acyl-CoA, which is activated from omega-1-methylated carboxylic acid, serves as the primer.

[0090] FIG. 14A-B (81): Synthesis of -hydroxy methyl ketones, ,-1-diols and -1-amino-1-alcohols, through the platform depicted in FIG. 1 (R=OH). Omega-hydroxyacyl-CoA, which is activated from omega-hydroxyacid, serves as the primer.

[0091] FIG. 15A-B (81): Synthesis of -amino methyl ketones, -amino-2-alcohols and ,-1-diamines, through the platform depicted in FIG. 1 (R=NH.sub.2). Omega-amino acyl-CoA, which is activated from omega-amino acid, serves as the primer.

[0092] FIG. 16A-B (81): Synthesis of -halogenated methyl ketones, -halogenated 2-alcohols and -halogenated 2-amines, through the platform depicted in FIG. 1 (R=X). Omega-halogenated acyl-CoA, which is activated from omega-halogenated carboxylic acid, serves as the primer.

[0093] FIG. 17 A-C (81): A partial listing of embodiments, any one or more or which can be combined with any other, even if not yet so combined.

[0094] FIG. 18A-B (1-84): Platform for the synthesis of omega-1-functionalized carboxylic acids, alcohols, amines, hydrocarbons, and methyl ketones. The platform is composed of thiolase, dehydrogenase, dehydratase and reductase. Thiolase(s) catalyzes the condensation between omega-1-functionalized primer and extender unit acetyl-CoA and generates omega-1-functionalized -keto acyl-CoA. Dehydrogenase converts omega-1-functionalized -keto acyl-CoA to omega-1-functionalized -hydroxy acyl-CoA. Dehydratase converts omega-1-functionalized -hydroxy acyl-CoA to omega-1-functionalized enoyl-CoA. Reductase converts omega-1-functionalized enoyl-CoA to omega-1-functionalized acyl-CoA. The platform can be iterated by using synthesized omega-1-functionalized acyl-CoA as the primer for the next turn of the platform.

[0095] Termination pathways starting from four omega-1-functionalized CoA thioester intermediates terminate the platform and generate various omega-1-functionalized carboxylic acids, alcohols and amines with different -reduction degrees. There are four types of termination pathways: 1) thioesterase/CoA-transferase/phosphotransacylase+kinase which generates carboxylic acids; 2) alcohol-forming acyl-CoA reductase or aldehyde-forming acyl-CoA reductase and alcohol dehydrogenase which generates alcohols; 3) aldehyde-forming acyl-CoA reductase and aldehyde decarbonylase which generates hydrocarbons (not pictured); and 4) aldehyde-forming acyl-CoA reductase and transaminase which generates amines.

[0096] Secondary termination pathways are also possible. For example, omega-1-functionalized methyl ketone can be generated by subsequent decarboxylation of omega-1-functionalized -keto acid. Omega-1-functionalized acyl-CoA thioester primers be generated from their acid form, which can be either supplemented in the media or derived from other carbon sources, or directly synthesized through additional cellular pathways.

[0097] R means functionalized group of primers, intermediates and products. n means length of primers, intermediates and products. Dashed line means multiple reaction steps or iteration.

[0098] FIG. 19A-B (2-84): Proposed platform and its products utilizing omega-1-methyl acyl-CoA as the primer (R=CH3).

[0099] FIG. 20A-B (3-84): Example pathway of synthesis of 4-methylpentanoic acid and 4-methylpentanol through the proposed platform with isobutyryl-CoA as the primer and acetyl-CoA as the extender unit. Isobutyryl-CoA is activated by Pct from isobutyric acid. The platform is composed of thiolase BktB, which catalyzes the condensation between primer isobutyryl-CoA and extender unit acetyl-CoA to form 4-methyl-3-oxopentanoyl-CoA; dehydrogenase and dehydratase FadB, which catalyzes the conversion of 4-methyl-3-oxopentanoyl-CoA to 4-methyl-3-hydroxypentanoyl-CoA and the subsequent dehydration of 4-methyl-3-hydroxypentanoyl-CoA to 4-methyl-2-pentenoyl-CoA; reductase Fab1, which reduces 4-methyl-2-pentenoyl-CoA to 4-methylpentanoyl-CoA. Termination reaction by endogenous thioesterases or overexpressed YdiI converts 4-methylpentanoyl-CoA to the product 4-methylpentanoic acid. Acyl-CoA reductase and alcohol dehydrogenase Maqu_2507 terminates the platform and catalyzes the termination reaction of reduction of 4-methylpentanoyl-CoA to 4-methylpentanal and the subsequent reduction of 4-methylpentanal to the product 4-methylpentanol.

[0100] FIG. 21A-B (6-84): Proposed platform and its products utilizing omega-1-amino acyl-CoA as the primer (R=NH2).

[0101] FIG. 22A-B (7-84): Proposed platform and its products utilizing omega-1-hydroxy acyl-CoA as the primer (R=OH).

[0102] FIG. 23A (8a-84) Derivatization reaction of omega-1 amino acid, one of the products of the platform depicted in FIG. 21A-B, to lactam, catalyzed by amidohydrolase.

[0103] FIG. 23B (8b-84) Derivatization reaction of omega-1 hydroxy acid, one of the products of the platform depicted in FIG. 22A-B, to lactone, catalyzed by lactonase.

[0104] FIG. 24 A-G (9-84) A partial listing of preferred embodiments, and one or more of which can be combined with any other one or more shown here.

[0105] FIG. 25A-B (2-85): Example pathways for the generation of omega-phenyl acyl-CoA thioester primers benzoyl-CoA, phenylacetyl-CoA and phenylpropionyl-CoA from carbon sources such as glucose or glycerol via chorismate, the intermediate of biosynthesis of aromatic amino acids phenylalanine and tryptophan.

[0106] FIG. 26A-B (3-85): Example pathway of synthesis of 4-phenylbutyric acid and 6-phenylhexanoic acid through the proposed platform with phenylacetyl-CoA as the primer and acetyl-CoA as the extender unit. Phenylacetyl-CoA is activated by E. coli enzyme PaaK from phenylacetic acid. The platform is composed of thiolase FadA from Pseudomonas putida, which catalyzes the condensation between primer phenylacetyl-CoA and extender unit acetyl-CoA to 4-phenylacetoacetyl-CoA; dehydrogenase and reductase FadB from P. putida, which catalyzes the conversion of 4-phenylacetoacetyl-CoA to 4-phenyl-3-hydroxybutyryl-CoA and the subsequent dehydration of 4-phenyl-3-hydroxybutyryl-CoA to 4-phenylcrotonyl-CoA; reductase Fab1 from E. coli or Ter from Treponema denticola (tdTER), which reduces 4-phenylcrotonyl-CoA to 4-phenylbutyryl-CoA. Termination by an acid forming reaction, such as those catalyzed by thioesterases, can convert the intermediate of one-turn of the pathway, 4-phenylbutyryl-CoA, to the product 4-phenylbutyric acid. Pathway iteration using the generated 4-phenylbutyryl-CoA as a primer with similar thiolase, dehydrogenase, dehydratase and reductase steps results in 6-phenylhexonyl-CoA, which can be converted to 6-phenylhexanoic acid through acid forming termination pathways.

[0107] FIG. 27A-C(6-85): Maps of vectors overexpressing required enzymes for the production of even chain omega-phenyl products, such as 4-phenylbutyric acid and 6-phenylhexanoic acid, through the proposed platform depicted in FIG. 26A-B with phenylacetyl-CoA as the primer.

[0108] FIG. 28A-B (7-85): Example pathway of synthesis of 5-phenylpentanoic acid through the proposed platform with phenylpropionyl-CoA as the primer and acetyl-CoA as the extender unit. Phenylpropionyl-CoA is activated by Penicillium chrysogenum enzyme Phl from phenylpropionic acid. The platform is composed of thiolase FadA from Pseudomonas putida, which catalyzes the condensation between primer phenylpropionyl-CoA and extender unit acetyl-CoA to 5-phenyl-3-oxopentanoyl-CoA; dehydrogenase and reductase FadB from P. putida, which catalyzes the conversion of 5-phenyl-3-oxopentanoyl-CoA to 5-phenyl-3-hydroxypentanoyl-CoA and the subsequent dehydration of 5-phenyl-3-hydroxypentanoyl-CoA to 5-phenyl-2-pentenoyl-CoA; reductase Fab1 from E. coli or TdTer, which reduces 5-phenyl-2-pentenoyl-CoA to 5-phenylpentanoyl-CoA. Termination by an acid forming reaction, such as those catalyzed by thioesterases, converts 5-phenylpentanoyl-CoA to the product 5-phenylpentanoic acid.

[0109] FIG. 29A-C (8-85): Maps of vectors overexpressing enzymes for the production of odd chain omega-phenyl products, such as 5-phenylpentanoic acid, through the proposed platform depicted in FIG. 28A-B with phenylpropionyl-CoA as the primer.

[0110] FIG. 30A-E (9-85): A partial listing of preferred embodiments, and one or more of which can be combined with any other one or more.

[0111]

[0112] TABLE 1 ACTIVATION ENZYMES

[0113] TABLE 2 REACTIONS OF THE PLATFORM

[0114] TABLE 3 PRIMARY & SECONDARY TERMINATION ENZYMES

[0115] TABLE 4 STRAINS & PLASMIDS

[0116] TABLE 5 OLIGONUCLEOTIDES

[0117] TABLE 6 HOST STRAINS AND PLASMIDS ENABLING OMEGA-FUNCTIONALIZED SMALL MOLECULE SYNTHESIS WITH LISTED PRIMER/EXTENDER UNIT COMBINATIONS

DETAILED DESCRIPTION

[0118] The disclosure generally relates to the use of microorganisms to make omega- and omega-1-functionalized products. The method entails developing a new pathway that is based on native or engineered thiolases capable of catalyzing the condensation of omega-functionalized acyl-CoA primers with an acetyl-CoA as the extender unit. This has been reported in neither the scientific, peer-reviewed literature nor the patent literature.

[0119] The first enzyme needed in the new pathway are activation enzymes. TABLE 1 lists several activation enzymes. Once the functionalized initiating primer is ready, it must be condensed with another Acetyl-CoA by a thiolase. Thiolases that will work with these functionalized primers are listed in TABLE 2. The remaining reactions in the platform tend to be less fussy about substrates, so many known enzymes will work with functionalized intermediates. These are also listed in TABLE 2. TABLE 3 shows various termination pathways, including both primary pathways and secondary pathways, and exemplary enzymes that can be used therein.

[0120] The following description provides additional details, any one of which can be subject to patenting in combination with any other. The specification in its entirety is to be treated as providing a variety of details that can be used interchangeably with other details, as the specification would be of inordinate length if one were to list every possible combination of genes/vectors/enzymes/hosts that can be made to enable carbon source conversion into omega- or omega-1-functionalized products.

Methods

[0121] Initial demonstration of the engineered pathway was conducted in E. coli for convenience. Enzymes of interest where expressed from vectors such as pETDuet-1 or pCDFDuet-1 (MERCK, Germany), which makes use of the DE3 expression system. Genes can be codon optimized according to the codon usage frequencies of the host organism and synthesized by a commercial vendor or in-house. However, thousands of expression vectors and hosts are available, and this is a matter of convenience.

[0122] Pathway enzymes can also be inserted into the host chromosome, allowing for the maintenance of the pathway without requiring antibiotics to ensure the continued upkeep of plasmids. A large number of genes that can be placed on the chromosome, as chromosomal expression does not require separate origins of replication as is the case with plasmid expression.

[0123] Engineered strains expressing pathway components can be cultured under the following or similar conditions. Overnight cultures started from a single colony can be used to inoculate flasks containing appropriate media. Cultures are grown for a set period of time, and the culture media analyzed. The conditions will be highly dependent on the specifications of the actual pathway and what exactly is to be tested. For example, the ability for the pathway to be used for omega-1-functionalized product synthesis can be tested by the glycerol or sugars as a substrate in MOPS minimal media, as described by Neidhardt et al (1974), supplemented with appropriate antibiotics, and inducers. Depending on the strain chosen, primers or precursors for primers can be added to the medium, or they can be internally generated.

[0124] Wild-type K12 Escherichia coli strain MG1655 was used as the host for all genetic modifications. All resulting strains used in this study are listed in TABLE 4. 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 DE3 prophage, carrying the T7 RNA polymerase gene and lacIq, was integrated into the chromosome through DE3 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 m/mL), spectinomycin (50 g/mL), kanamycin (50 g/mL), and chloramphenicol (34 m/mL).

[0125] All plasmids used in this study and oligonucleotides used in their construction are listed in TABLE 5 and TABLE 6. 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, phaB 1, pct, cbjALD and mks1, which were amplified from genomic DNA or cDNA of their source organisms. The recognition site of NdeI in the paaH sequence was eliminated via overlap PCR. 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.

[0126] 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 M 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.

[0127] Neutralized 5 mM phenylacetic acid or 20 mM succinic acid, glutaric acid, isobutyric acid, glycolic acid, or propionic acid was supplemented as needed. Antibiotics (50 g/mL carbenicillin and 50 g/mL spectinomycin) were included when appropriate. All chemicals were obtained from Fisher Scientific Co. (Pittsburgh, Pa.) and Sigma-Aldrich Co. (St. Louis, Mo.).

[0128] 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, OD.sub.550) reached 0.3-0.5, 5 M isopropyl -D-1-thiogalactopyranoside (IPTG) was added for plasmid based gene expression in all cases except the following: 1 M IPTG was used for adipic acid production from glycerol without succinic acid supplementation and 10 M IPTG was used during production of -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.

[0129] 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 (660 rpm for adipic acid production and 720 rpm for tiglic acid production). Fermentations for adipic acid production used the above fermentation media with 45 g/L glycerol, the inclusion of 5 M sodium selenite, and 1 M IPTG. Pre-cultures were grown in 25 mL Pyrex Erlenmeyer flasks as described above and incubated for 24 h post-induction. An appropriate amount of this pre-culture was centrifuged, washed twice with fresh media, and used for inoculation (400 mL initial volume).

[0130] 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 for production of 4-hydroxybutyric acid.

[0131] For analysis of dicarboxylic acids and -hydroxy acids, extractions were performed as previously described (Clomburg et al. 2015), with 12-hydroxydodecanoic acid as the internal standard and diethyl ether as the organic solvent. With the exception of 4-methylpentanol analysis, extraction of all other analysis samples was conducted as previously described (Kim et al. 2015), with tridecanoic acid as the internal standard and hexane:MTBE (1:1) as the organic solvent.

[0132] Extracted products were then derivatized by BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) as previously described (Clomburg et al. 2015) for GC-MS or GC-FID analysis. For GC-FID analysis of 4-methylpentanol, extraction was performed with hexane:MTBE as described above with tridecanol as the additional internal standard. Acetylation was then conducted by adding a 1:1 pyridine:acetic anhydride mixture, following the previously described method (Kim et al. 2015). For GC-MS analysis of 4-methylpentanol, samples were extracted with hexane, with 1-heptanol as the internal standard, with subsequent BSTFA derivatization

[0133] GC-MS metabolite identification: Except for identifications of 4-hydroxybutyric acid, metabolite identification was conducted via GC-MS as previously described 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 m film thickness, 30 m length; Restek, Bellefonte, Pa.). The sample injection amount was 2 L 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.).

[0134] Identification of 4-hydroxbutyric acid was conducted by the Baylor College of Medicine Analyte Center (www.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.

[0135] Product quantification was conducted using previously reported gas chromatography methods. Quantification was performed in Varian CP-3800 gas chromatograph (Varian Associates, Inc., Palo Alto, Calif.), equipped with a flame ionization detector (GC-FID) and an Agilent HP-5 capillary column (0.32 mm internal diameter, 0.50 m film thickness, 30 m length. Agilent). The temperature was initially 50 C., held for 3 min, then increased to 250 C. at 10 C./min, and finally 250 C. was held for 10 min. Helium (1.8 mL/min, Matheson Tri-Gas) was used as the carrier gas. The injector and detector temperatures were 220 and 275 C., respectively. The sample was injected at 1 L without splits.

[0136] The concentration of glycerol, adipic acid, 6-hydroxyhexanoic acid, 7-hydroxyheptanoic acid and 4-methylpentanoic acid 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.).

Omega Functionalized Products

[0137] We first validated the iterative operation of the proposed carbon chain elongation platform consisting of thiolase accepting various -functionalized primers, along with HACD, ECH and ECR, and achieved synthesis of various -functionalized carboxylic acids and alcohols after termination at the acyl-CoA node by ACT and ACR+ADH respectively (FIG. 6A-B).

[0138] The aromatic primer phenylacetyl-CoA, with acetyl-CoA as the extender unit, was used to achieve iterative pathway operation and synthesis of corresponding aromatic products. Pseudomonas putida thiolase FadA (ppFadA) was used, with P. putida FadB (ppFadB) providing HACD and ECH activities, Escherichia coli Fab1 as the ECR, and E. coli acyl-CoA synthetase PaaK to activate externally supplied phenylacetic acid.

[0139] These and subsequent enzymes for all of the pathways described herein were selected on the basis of literature reports of the specific enzymes' and organisms' ability to function with the required intermediates. When expressed in mixed-acid fermentation-deficient E. coli MG1655 ldhApoxBptaadhEfrdA (JC01), these enzymes enabled the synthesis of 4-phenylbutyric acid (177 mg/L) and 6-phenylhexanoic acid (49 mg/L) (FIG. 7). These products result from the action of endogenous termination pathways, possibly native ACTs, on acyl-CoA's that are generated by one and two turns of the pathway, respectively.

[0140] Omega-carboxylated primers can support the synthesis of products such as -hydroxyacids and dicarboxylic acids. In this context, we selected succinyl-CoA and glutaryl-CoA, which can be generated from corresponding acids by the Clostridium kluyveri CoA transferase Cat1. Overexpression of E. coli PaaJ (thiolase), PaaH (HACD), and PaaF (ECH), with Treponema denticola trans-enoyl-CoA reductase (TdTer) as the ECR in JC01 led to production of C6 (adipic, 170 mg/L) and C7 (pimelic, 25 mg/L) dicarboxylic acids from endogenous acid-producing termination enzymes following succinic or glutaric acid supplementation, respectively (FIG. 8).

[0141] The system's modularity was exploited to achieve the synthesis of -hydroxyacids by manipulation of termination pathways. Minimizing activity of endogenous acid-producing termination reactions (by deletion of native thioesterases) and using Clostridium beijerinckii ACR cbjALD (with native ADH enzymes) in combination with the other pathway components enabled the synthesis of 6-hydroxyhexanoic acid (34 mg/L) and 7-hydroxyheptanoic acid (87 mg/L) following supplementation with exogenous succinic or glutaric acid, respectively (FIG. 8). This strategy used the thioesterase-deficient strain JST06 (JC01 yciAybgCydiItesAfadMtesB), as -hydroxyacids were not observed when JC01 was used as the host strain. This demonstrates the importance of engineering the termination pathway(s) for product selectivity, and it represents an area in which further optimization could improve target product synthesis and reduce byproduct formation via nonspecific and/or endogenous enzymes.

[0142] Usage of -hydroxylated primer glycolyl-CoA can lead to the synthesis of -hydroxyacid 4-hydroxybutyric acid through the proposed pathway (FIG. 6A-B, FIG. 9). The following enzymes provided the individual components of the pathway: BktB (thiolase) and PhaB1 (HACD) from Ralstonia eutropha, Aeromonas caviae PhaJ (ECH), Treponema denticola TdTer (ECR) 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.

[0143] The use of functionalized primers and termination pathways enables the synthesis of a wide range of products, albeit at relatively low titers. One potential cause of low product titers is the intracellular concentrations of primers and/or extender units available for condensation. To determine whether low primer concentrations affected product synthesis, we attempted to maximize succinyl-CoA availability by deleting sdhB (encoding a subunit of succinate dehydrogenase), thereby reducing succinate consumption through the tricarboxylic acid (TCA) cycle. This deletion was introduced into JST06 to reduce undesirable hydrolysis of priming (succinyl-CoA) and extending units (acetyl-CoA) by native thioesterases, with Mus musculus dicarboxylic ACT Acot8 then overexpressed as the termination enzyme. This re-engineered strain produced a higher adipic acid titer (334 mg/L compared to 170 mg/L in the JC01 background) in the presence of succinic acid (FIG. 10).

[0144] Further product diversification from the use of succinyl-CoA can be achieved through iterative pathway operation. Replacement of the thiolase (PaaJ), HACD (PaaH) and ECH (PaaF) pathway components with the Acinetobacter sp. ADP1 enzymes DcaF, DcaH, and DcaE resulted in the production of suberic (34 mg/L) and sebacic (13 mg/L) acids in addition to adipic acid (95 mg/L) (FIG. 10). These C8 and C10 diacids, products of two and three turns of the pathway, respectively, were not observed when using PaaJHF, demonstrating how selecting individual pathway components with desired specificity can control product synthesis. This type of approach could be used to further increase product diversity, as well as overall performance, through the selection and engineering of enzymes with required specificity and efficiency for desired functionalization.

[0145] Although our system can synthesize functionalized products, primer precursor supplementation and low overall titers need to be overcome to achieve industrial scale viability. To show the potential for higher product titer from a single carbon source, improvement in adipic acid production was targeted, given the industrial importance of this compound. The intracellular generation of succinic acid/succinyl-CoA was accomplished using strain MG1655 ldhApoxBptaadhE (MB263), which retains the reductive branch of the TCA cycle, along with the overexpression of PaaJ, PaaH, PaaF, TdTer, Cat1, and Acot8, resulting in 0.24 g/L adipic acid from a single carbon source (glycerol, FIG. 11). Maximization of primer availability through deletion of sucD, which encodes a subunit of succinyl-CoA synthetase, part of the TCA cycle, was again used to improve product titer (0.35 g/L, FIG. 11). When grown in a controlled bioreactor with a higher initial glycerol concentration, this strain produced 2.5 g/L adipic acid (4.1% mol/mol glycerol) (FIG. 11). Further improvement is envisioned through minimizing acetate formed directly through the transferase for primer activation. Acetate recycling (to acetyl-CoA) or use of an acetyl-CoA-independent activation enzyme offers a potential solution to improve adipic acid titer, a strategy that can also be applied to other combinations of primer and extenders.

[0146] Once we demonstrated the iterative operation of the proposed platform and its acceptance of various -functionalized primers, we then utilized this platform to demonstrate the synthesis of -functionalized methyl ketone. We chose -carboxylated succinyl-CoA as the primer, and -carboxylated methyl ketone levulinic acid, the product from first cycle of -ketoacyl-CoA node and a key building block for the chemical industry. Levulinic acid production was observed in JST06 sdhB strain overexpressing PaaJ, and Cat1 along with P. putida CoA transferase PcaIJ which generates 3-oxoadipic acid from 3-oxoadipyl-CoA, the product of condensation between succinyl-CoA and acetyl-CoA (48 mg/L) (FIG. 5). 3-oxoadipic acid was believed to be spontaneously decarboxylated to levulinic acid in this strain. Additional overexpression of the decarboxylases Solanum habrochaites Mks1 or Clostridium acetobutylicum Adc increased levulinic acid titers to 71 mg/L and 159 mg/L, respectively (FIG. 5). All the strains were grown with glycerol and succinic acid for the synthesis of levulinic acid.

Omega-1 Products

[0147] For these experiments, isobutyrate (precursor for the initiating omega-1 methyl (CH.sub.3) primer) was added to a concentration of 20 mM in the medium. Isobutyryl-CoA priming (FIG. 20A-B) was assessed with the following individual pathway components: Megasphaera elsdenii Pct (transferase for isobutyric acid activation), Ralstonia eutropha BktB (thiolase), E. coli FadB (HADCH and ECH), and Euglena gracilis EgTer (ECR). Overexpression of these enzymes in mixed-acid fermentation-deficient E. coli MG1655 ldhA poxB pta adhE frdA (JC01), enabled the synthesis of 4-methylpentanoic acid (FIG. 8). This product, representing a one-turn reversal with isobutyryl-CoA priming, resulted from endogenous termination pathways. Overexpression of E. coli YdiI (thioesterase) resulted in slight increases to 4-methylpentanoic acid titer (FIG. 8), indicting the value of overexpressing termination enzymes.

[0148] The use of different termination pathways enables the production of products with varying functionality, even when exploiting the same initial omega-1-functionalzied primer. For example, engineering termination pathways through replacing YdiI with the Marinobacter aquaeolei alcohol-forming acyl-CoA reductase Maqu2507, along with the use of host strain with deletion to native thioesterases (JC01 yciA ybgC ydiI tesA fadM tesB fadE), enabled production of 4-methylpentanol (FIG. 8). Whereas the production of 4-methylpentanoic acid results from an acid forming termination pathways from 4-methylpentanoyl-CoA, 4-methylpentanol is the result of the 2-step reduction of this omega-1-methylated intermediate to form the corresponding alcohol. As such, the modular nature of the engineered pathway provides the opportunity to produce a wide range of products through the combinatorial engineering of the primers and termination pathway utilized.

[0149] Iterative pathway operation using primers such as omega-1-methyl- (FIG. 19A-B), omega-1-amino- (FIG. 21A-B)), and omega-1-hydroxyl-acyl-CoA thioesters (FIG. 22A-B) as the initiating primer, in combination with various termination pathways) enables the engineered pathway to synthesize various omega-1-functionalized carboxylic acids, alcohols, hydrocarbons, and amines with different degrees of -reduction and carbon chain length as described herein.

Omega Phenyl Products

[0150] Generation of the required omega-phenyl acyl-CoA thioester primer can make use of externally supplied phenylalkanoic acids or -CoA form thereof or can be accomplished from a carbon source such as glycerol or sugars through the pathways depicted in FIG. 25A-B. Exploiting components of pathways for the biosynthesis of aromatic amino acids phenylalanine and tryptophan, the generation of omega-phenyl acyl-CoA thioester primers benzoyl-CoA, phenylacetyl-CoA and phenylpropionyl-CoA can be accomplished via chorismate, enabling the synthesis of required omega-phenyl primers, and subsequent omega-phenyl products, from industrially relevant single carbon sources such as sugars or glycerol.

[0151] Combining the core engineered pathway with enzymes/pathways for the generation of the initial omega-phenyl acyl-CoA thioester primer provides a route for the generation of omega-phenyl acyl-CoA intermediates with varying beta-functionality. These intermediates can then be converted to numerous products of interest through action of various termination pathways. For example, the use of acid forming termination pathways, such as thioesterases, enables the synthesis of omega-phenyl carboxylic acids, while alcohol forming termination pathways, such as acyl-CoA reductases/alcohol dehydrogenases, provides a route to various omega-phenyl alcohols. The combinatorial expression of core pathway components with termination pathways allows the synthesis of omega-phenyl products, including omega-phenyl carboxylic acids, alcohols, hydrocarbons, amines, methyl ketones and their beta-functionalized derivatives.

[0152] Initial demonstration of the engineered pathway was conducted in E. coli for convenience, and focused on the synthesis of omega-phenyl carboxylic acids. Enzymes of interest where expressed from vectors such as pETDuet-1 or pCDFDuet-1 (MERCK, Germany), which makes use of the DE3 expression system. Genes can be codon optimized according to the codon usage frequencies of the host organism and synthesized by a commercial vendor or in-house. However, thousands of expression vectors and hosts are available, and this is a matter of convenience. The vectors used in initial demonstration of the engineered pathway are shown in FIG. 27A-C and FIG. 29A-C.

[0153] For these experiments, phenylacetate or phenylpropionate was added to the growth medium at a concentration of 5 mM to provide the starting substrate. The aromatic primer phenylacetyl-CoA, with acetyl-CoA as the extender unit, was used to achieve pathway operation and demonstrate the synthesis of phenylalkanoic acids.

[0154] Pseudomonas putida thiolase FadA, P. putida FadB (providing both HACD and ECH activities) was tested with either E. coli Fab1 or T. denticola TER as the ECR, and E. coli acyl-CoA synthetase PaaK to activate externally supplied phenylacetic acid. Overexpression of either combination of enzymes in mixed-acid fermentation-deficient E. coli MG1655 ldhA poxB pta adhE frdA (JC01), enabled the synthesis of 4-phenylbutyric acid (FIG. 7). This product resulted from the action of endogenous termination pathways, possibly native thioesterases, on phenylbutyryl-CoA generated by one turn of the pathway. In addition to demonstrating overall pathway functionality, the use of either T. denticola TER or E. coli Fab1 with FadA and FadB for 4-phenylbutyric acid synthesis also demonstrates how both -oxidation enzymes and fatty acid biosynthesis enzymes acting on the required CoA intermediates can be used in this context.

[0155] Iterative pathway operation (e.g. the use of the omega-phenyl acyl-CoA generated from a turn of the pathway as a primer for the next round) was also demonstrated through the use of P. putida thiolase FadA, P. putida FadB (providing HACD and ECH activities), E. coli Fab1 (ECR), and E. coli acyl-CoA synthetase PaaK in the JC01 strain background. Varying induction levels by altering IPTG concentration (10 M) as well as incubation at 30 C., resulted in the synthesis of 6-phenylhexanoic acid in addition to higher levels of 4-phenylbutyric acid, compared to the above results with the same set of enzymes (FIG. 7). This demonstrates the ability to synthesize omega-phenyl products of various chain length through the iterative addition of 2 carbon units (via acetyl-CoA as the donor) to the growing omega-phenyl acyl-CoA primer.

[0156] Combination of iterative pathway operation using any of benzoyl-CoA (FIG. 25A-B, phenylacetyl-CoA (FIG. 25A-B and FIG. 26A-B) and phenylpropionyl-CoA (FIG. 25A-B) and FIG. 28A-B) as the initial primer with various termination pathways enables the engineered pathway to synthesize various omega-phenyl carboxylic acids, alcohols, hydrocarbons, and amines with different degrees of -reduction and carbon chain length as described herein.

[0157] In addition, pathway and process optimization, in line with industrial biotechnology approaches, can 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 of optimization 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.

Other Species

[0158] 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 pAM1 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.

[0159] 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.

[0160] Each of the following is incorporated by reference herein in its entirety for all purposes: [0161] 61/440,192, filed Feb. 7, 2011, WO2012109176, filed Feb. 7, 2012, and US20130316413 Reverse beta-oxidation pathway [0162] 62/140,628, Mar. 31, 2015, WO2017020043 Biosynthesis of polyketides [0163] 61/932,057, filed Jan. 27, 2014, WO2015112988, US20160340699, TYPE II FATTY ACID SYNTHESIS ENZYMES IN REVERSE beta-OXIDATION. [0164] 62/069,850, filed Oct. 29, 2014, WO2016069929, SYNTHETIC PATHWAY FOR BIOSYNTHESIS FROM 1-CARBON COMPOUNDS [0165] 61/531,911, filed Sep. 7, 2011; 61/440,192, filed Feb. 7, 2011; WO2013036812, US20140273110 Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation [0166] 62/011,465, Filed Jun. 12, 2014; WO2015191972, WO2015191972, Omega-hydroxylated carboxylic acids [0167] 62/012,113, filed Jun. 13, 2014; WO2015191422A1, WO2015191972, WO2016007258, Omega-aminated carboxylic acids [0168] 62/011,474, filed Jun. 12, 2014; WO2015191422A1, WO2015191972 Omega-carboxylated carboxylic acids and derivatives [0169] 62/154,397, filed Apr. 29, 2015, WO2016176347, SYNTHESIS OF OMEGA-1 FUNCTIONALIZED PRODUCTS AND DERIVATIVES THEREOF (herein referred to as 81); [0170] 62/148,248, filed Apr. 16, 2015, WO2016168708, SYNTHESIS OF OMEGA FUNCTIONALIZED METHYLKETONES, 2-ALCOHOLS, 2-AMINES, AND DERIVATIVES THEREOF (herein referred to as 84); and [0171] 62/154,010, filed Apr. 28, 2015, WO2016176339, SYNTHESIS OF OMEGA-PHENYL PRODUCTS AND DERIVATIVES THEREOF (herein referred to as 85) [0172] Cheong, S., et al., Energy- and carbon-efficient synthesis of functionalized small molecules in bacteria using non-decarboxylative Claisen condensation reactions. Nat. Biotechnol. 34 (5) (2016). [0173] Choi, K. H., et al., -Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis. J. Bacteriol. 182, 365-370 (2000). [0174] Clomburg, J. M. et al. Integrated engineering of -oxidation reversal and -oxidation pathways for the synthesis of medium chain -functionalized carboxylic acids. Metab. Eng. 28, 202-212 (2015). [0175] Clomburg, J. M., et al., A Synthetic Biology Approach to Engineer a Functional Reversal of the -Oxidation Cycle. ACS Synthetic Biology 1, 541-554 (2012). [0176] Dellomonaco, C., et al., Engineered reversal of the -oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355-359 (2011). [0177] Haapalainen, A. M., et al., The thiolase superfamily: condensing enzymes with diverse reaction specificities. Trends in Biochemical Sciences 31, 64-71 (2006). [0178] Heath, R. J. & Rock, C. O. The Claisen condensation in biology. Nat. Prod. Rep. 19, 581-596 (2002). [0179] Jiang, C., et al., Divergent evolution of the thiolase superfamily and chalcone synthase family. Molecular Phylogenetics and Evolution 49, 691-701 (2008). [0180] Kim, S., Clomburg, J. M. & Gonzalez, R. Synthesis of medium-chain length (C6-C10) fuels and chemicals via -oxidation reversal in Escherichia coli. J. Ind. Microbiol. Biotechnol. 42, 465-475 (2015). [0181] Lan, E. I., et al., Metabolic engineering of 2-pentanone synthesis in Escherichia coli. Aiche J. 59, 3167-3175 (2013). [0182] Lian J. & Zhao, H., Reversal of the -Oxidation Cycle in Saccharomyces cerevisiae for Production of Fuels and Chemicals, ACS SYN. BIOL. 4, 332-341 (2015). [0183] Neidhardt, F. C., et al., Culture medium for enterobacteria. J. Bacteriol. 119, 736-747 (1974). [0184] Pfleger, B. F., et al., Metabolic engineering strategies for microbial synthesis of oleochemicals. Metab. Eng. 29, 1-11 (2015). [0185] Vick, J. E. et al. Escherichia coli enoyl-acyl carrier protein reductase (Fab1) supports efficient operation of a functional reversal of the -oxidation cycle. Appl. Environ. Microbiol. 81, 1406-1416 (2015).

[0186] 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. Furthermore, any detail from any claim may be combined with any other detail from another claim, even if not yet so combined.