WO2016007258A9

WO2016007258A9 - Omega-aminated carboxylic acids

Info

Publication number: WO2016007258A9
Application number: PCT/US2015/035664
Authority: WO
Inventors: Ramon Gonzalez; James M. CLOMBURG
Original assignee: William March Rice University
Priority date: 2014-06-12
Filing date: 2015-06-12
Publication date: 2016-03-10
Also published as: WO2015191972A2; WO2015191972A3; WO2016007258A1; WO2015191422A1

Abstract

Omega functionalized chemicals are made using a reverse beta oxidation cycle either by beginning with particular primers or by omega functionalization of β-oxidation intermediate(s)/product(s). Bacteria and methods for same are provided.

Description

OMEGA- AMINATED CARBOXYLIC ACIDS

PRIOR RELATED APPLICATIONS

[0001] This application claims priority to 62/011,474, Omega-Carboxylated

Carboxylic Acids, filed June 12, 2014, 62/012,113, Omega-Animated Carboxylic Acids, filed June 13, 2014 and 62/011,465, Omega-Hydroxylated Carboxylic Acids, filed June 12, 2014, as well as to 61/531,911, Synthesis Of Alpha- And Omega-Functionalized Carboxylic Acids And Alcohols, filed Sept. 7, 2011, WO2013036812, Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation, filed Sept 7, 2012, and US20140273110 (14/199,528), Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation, filed 3/6/2014. Each is expressly incorporated by reference herein in its entirety for all purposes.

FEDERALLY SPONSORED RESEARCH STATEMENT [0002] Not applicable.

FIELD OF THE DISCLSOURE

[0003] The disclosure generally relates to the biological synthesis of various chemicals through a reverse beta oxidation cycle.

BACKGROUND OF THE DISCLOSURE

[0004] We have already demonstrated that an engineered reversal of the beta- oxidation cycle can be used to generate straight-chain aliphatic carboxylic acids and n- alcohols with side chains of different lengths and functionalities (WO2012109176, filed 2/7/2012 based on 61/440,192, filed 2/7/2011, both incorporated by reference herein in their entireties). In all cases the synthesized molecules were primary n-alcohols or carboxylic acids with a methyl group at the omega end.

[0005] The initial reverse beta oxidation work employed primer acetyl-CoA (for even length products) or propionyl-CoA (for odd chain length products) and corresponding termination pathways that then lead to the synthesis of carboxylic acids and alcohols as products. [0006] However, a later filed case (WO2013036812, filed 9/7/2012, based on

61/531/911, filed 9/7/2011) used one of 14 primers, none of them being acetyl-CoA or propionyl-CoA (although acetyl-coA does condense with the primer, acting as an extender unit, to add two carbon units thereto). These, in combination with different termination pathways, allowed the synthesis of diols, dicarboxylic acids, hydroxy acids, carboxylated alcohols, amines, amino acids, hydroxylated amines, diamines, amides, carboxylated amides, hydroxylated amides, diamides, hydroxamic acids and their beta substituted derivatives thereof.

[0007] This invention takes the development of the reverse beta-oxidation cycle even further, elaborating significantly on the production of omega-aminated carboxylic acids.

SUMMARY OF THE DISCLOSURE

[0008] We have demonstrated that an engineered reversal of the β-oxidation cycle can be used to generate straight-chain aliphatic carboxylic acids and n-alcohols with side chains of different lengths and functionalities (Nature 476, 355-359, 2011). More recently, we have utilized a synthetic approach in which the core modules required for a functional β- oxidation reversal (thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3- hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase) were characterized and assembled to enable the synthesis of carboxylic acids of different chain lengths (ACS Synthetic Biology 1, 541-554, 2012). While in all cases the synthesized molecules were n-alcohols or carboxylic acids with a methyl group at the omega (ω) end, the modular nature of the engineered pathway can be exploited for the synthesis of product families with a wide range of functionalities. This can be achieved by manipulating any of the modules composing the β-oxidation reversal, namely priming, core or termination modules.

[0009] Here, we sought to establish further diversification of product families generated via a functional reversal of the β-oxidation cycle. Specifically, the omega carbon of n-alcohols and carboxylic acids generated by the β-oxidation reversal can be functionalized by introducing carboxylic, alcohol, or amine groups.

[0010] Examples of potential products to be generated include co-hydroxylated carboxylic acids, ω-carboxylated n-alcohols, dicarboxylic acids, diols, omega amino acids and omega amino n-alcohols. In all cases, products of different chain lengths can be obtained: i.e. products with an internal/spacer chain between the alpha and omega ends of different lengths, depending on the number of turns of the cycle, and containing different functionalities, depending on the β-oxidation intermediate used as precursor for their synthesis. The latter can include a hydroxy or keto group in the beta carbon or an α, β unsaturation.

[0011] Two general approaches were employed to functionalize the omega carbons.

In the first approach, the priming step is engineered to use a primer or starter with a functionalized (hydroxylated, carboxylated, or aminated) omega carbon (examples illustrated in FIG. 2). Omega-functionalized intermediates of varying chain length are generated from one or multiple turns of a beta-oxidation reversal, which can be converted to various products through the use of different terminations pathways (examples illustrated in FIG. 1). Specific combinations of priming molecules and termination pathway leading to the synthesis of omega-amino carboxylic acids (sometimes referred to as amino acids herein) are illustrated in FIGS. 3-5.

[0012] In the second approach, alternate termination pathways are engineered to functionalize (hydroxylate, carboxylate, or aminate) the omega carbon of an intermediate or a product of the engineered reversal of the β-oxidation cycle (illustrated by the omega- oxidation and amination of carboxylic acids in FIG. 6). The latter could take place before or after the intermediates of the engineered reversal of the β-oxidation cycle have been converted to carboxylic acids and n-alcohols by the appropriate termination enzymes. These two approaches are briefly described below.

[0013] Use of primers with a functionalized omega carbon— The

"normal/standard" starter used in the engineered reversal of the β-oxidation cycle is acetyl- CoA, which leads to the synthesis of even-chain n-alcohols and carboxylic acids. Propionyl- CoA can also be used as starter unit/primer by thiolase(s), thus enabling the synthesis of odd- chain carboxylic acids and n-alcohols.

[0014] A methyl group is always found at the omega end of both of the aforementioned starter/primer molecules. The use of starter/primer molecules with an omega hydroxylated, omega carboxylated, or omega-aminated carbon (i.e. a functionalized omega end) will then lead to the synthesis of carboxylic acids and n-alcohols through the β-oxidation reversal that will contain an omega hydroxylated/carboxylated/aminated end: e.g. ω- hydroxylated carboxylic acids, ω-carboxylated n-alcohols, dicarboxylic acids, diols, ω-amino carboxylic acids, ω-amino alcohols. FIG. 2 illustrates the first reaction of the β-oxidation reversal (i.e. non-decarboxylative condensation catalyzed by thiolases) for the use of representative ω-functionalized primers with carboxylated, hydroxylated, and aminated omega carbons.

[0015] The functionalized priming molecule can be generated either internally or for the purposes of proof of concept studies can be exogenously supplied as the acid form. In the latter case, and in certain instances through internal generation, the activation of the acid form of the functionalized primer to a CoA intermediate is required before subsequent condensation with acetyl-CoA can take place (FIG. 2). This approach requires: 1) identification/engineering of appropriate activation enzymes for the conversion of the ω- functionalized acid to its CoA intermediate, 2) a thiolase enzyme(s) capable of condensing an ω-functionalized acyl-CoA with acetyl-CoA, 3) enzymes for the dehydrogenation, dehydration, and reduction steps of the core β-oxidation reversal that are active on corresponding ω-functionalized substrates, 4) appropriate termination pathways leading to product synthesis (FIG. 1).

[0016] Omega functionalization of β -oxidation intermediate(s)/product(s)— This second approach entails the engineering of appropriate termination pathways that act on intermediate(s)/product(s) of the β-oxidation reversal. Two primary strategies can be employed. First, ω-hydroxylation and further oxidation to the carboxylic acid group will be achieved by using the ω-oxidation pathway. This pathway is used by industrially important yeasts and bacteria during the degradation of alkanes and long chain fatty acids. The methyl group at the omega carbon is first oxidized to a hydroxyl group, then to an oxo group, and finally to a carboxyl group.

[0017] These enzymes can be used to functionalize the omega carbon of carboxylic acids and n-alcohols generated by the action of thioesterases and aldehyde-forming acyl-CoA reductases and alcohol dehydrogenases, respectively, on the different intermediates of the β- oxidation reversal. Thus, this ω-oxidation pathway can be used in conjunction with a functional reversal of the β-oxidation pathway to generate carboxylic acids and n-alcohols with hydroxylated or carboxylated omega carbons (producing dicarboxylic acids, ω- hydroxyacids, or diols depending on the starting product and the extent of omega-oxidation). This approach is illustrated in FIG. 6 with carboxylic acids as the starting point/product generated via the β-oxidation reversal. [0018] In many cases, the use of transaminases or omega-amino acid/alcohol dehydrogenases can be used to synthesize ω-amino acids or ω-amino alcohols from the corresponding acid/alcohol semialdehydes (FIGS. 4-6). The required acid/alcohol semialdehydes can be generated through the oxidation of ω-hydroxyacids produced via functionalized priming or via the ω-oxidation pathways described above. It is important to note that the required acid/alcohol semialdehydes can also be generated through the reduction of omega-hydroxylated and omega-carboxylated acyl-CoAs, which are intermediates of the β-oxidation reversal when ω-hydroxylated and ω-carboxylated primers are used. All potential priming molecule and termination pathway combinations enabling omega-amino acid synthesis are illustrated in FIGS. 3-6.

[0019] Our initial cloning experiments proceeded in E. coli for convenience since the needed genes were already available in plasmids suitable for expression in E. coli, and some of the tested strains may already have been available, but the addition of genes to bacteria and other microorganisms is of nearly universal applicability, so it will be possible to use a wide variety of organisms with the selection of suitable vectors for same.

[0020] Bacteria from a wide range of species have been successfully modified, and may be the easiest to transform and culture, since the methods were invented in the 70 's and are now so commonplace, that even school children perform genetic engineering experiments using bacteria. Such species include e.g., Bacillus, Streptomyces, Azotobacter, Trichoderma, Rhizobium, Pseudomonas, Micrococcus, Nitrobacter, Proteus, Lactobacillus, Pediococcus, Lactococcus, Salmonella, and Streptococcus, or any of the completely sequenced bacterial species. Indeed, hundreds of bacterial genomes have been completely sequenced, and this information greatly simplifies both the generation of vectors encoding the needed genes, as well as the planning of a recombinant engineering protocol. Such species are listed along with links at http://en.wikipedia.org/wiki/List_of_sequenced_bacterial_genomes.

[0021] Additionally, yeasts are a common species used for microbial manufacturing, and many species can be successfully transformed. In fact, rat acyl ACP thioesterase has already been successfully expressed in yeast Saccharomyces and functional reversal of the beta oxidation cycle has also been achieved in Saccharomyces, demonstrating that this method has wide applicability to microbes, as expected since the beta oxidation pathway is ubiquitous (Lian 2015). Other species include but are not limited to Candida, Aspergillus, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha (Pichia angusta), Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae and Yarrowia lipolytica, to name a few.

[0022] It is also possible to genetically modify many species of algae, including e.g.,

Spirulina, Apergillus, Chlamydomonas, Laminaria japonica, Undaria pinnatifida, Porphyra, Eucheuma, Kappaphycus, Gracilaria, Monostroma, Enteromorpha, Arthrospira, Chlorella, Dunaliella, Aphanizomenon, Isochrysis, Pavlova, Phaeodactylum, Ulkenia, Haematococcus, Chaetoceros, Nannochloropsis, Skeletonema, Thalassiosira, and Laminaria japonica, and the like. Indeed, the microalga Pavlova lutheri is already being used as a source of economically valuable docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), and Crypthecodinium cohnii is the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas.

[0023] Furthermore, a number of databases include vector information and/or a repository of vectors that can be selected for use in these various microbes. See e.g., Addgene.org, which provides both a repository and a searchable database allowing vectors to be easily located and obtained from colleagues. See also Plasmid Information Database (PlasmID) and DNASU having over 191,000 plasmids. A collection of cloning vectors of E. coli is also kept at the National Institute of Genetics as a resource for the biological research community. Furthermore, vectors (including particular ORFS therein) are usually available from colleagues.

[0024] As used herein, "fatty acids" means any saturated or unsaturated aliphatic acids having the common formulae of CnH2n±xCOOH, wherein x<n, which contains a single carboxyl group. "Odd chain" fatty acids have an odd number of carbons in the chain (n=even), whereas "even chain" have an even number (n=odd). Acid and base names are used interchangeably herein, e.g., succinic acid and succinate.

[0025] As used herein, reference to the "omega" carbon of an intermediate or product refers to the last carbon, opposite the CoA group, which is on the "alpha" or second carbon (see figures). This nomenclature is retained throughout the entire pathway, even if certain intermediates become more oxidized at the omega end, and thus should take nomenclature priority. Thus, the omega carbon will be retained throughout the pathway, regardless of oxidation state. The alpha and beta carbons will of course advance by two with every turn of the cycle, retaining the second and third carbon position, respectively. The first carbon is 1. [0026] As used herein, "reduced activity" is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species. Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, by knock-out, by adding stop codons, by frame shift mutation, and the like. Reduction in activity is indicated by a negative superscript, e.g., FadD^".

[0027] By "knockout" or "null" mutant what is meant is that the mutation produces almost undetectable amounts of protein activity. A gene can be completely (100%) reduced by knockout or removal of part of all of the gene sequence. Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can also completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein. All knockout mutants herein are signified by Agene.

[0028] As used herein, "overexpression" or "overexpressed" is defined herein to be at least 150% of protein activity as compared with an appropriate control species, or any expression in a species that otherwise lacks the activity. Preferably, the activity is increased 200-500%. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like. Overexpressed genes or proteins can be signified herein by "+".

[0029] As used herein, all accession numbers are to GenBank or UniProt unless indicated otherwise.

[0030] Exemplary gene or protein species are provided herein. However, gene and enzyme nomenclature varies widely (esp. in bacteria), thus any protein (or gene encoding same) that catalyzes the same reaction can be substituted for a named protein herein. Further, while exemplary protein sequence accession numbers are provided herein, each is linked to the corresponding DNA sequence, and to related sequences. Further, related sequences can be identified easily by homology search and requisite activities confirmed as by enzyme assay, as is known in the art.

[0031] E. coli gene and protein names (where they have been assigned) can be ascertained through ecoliwiki.net/ and enzymes can be searched through brenda- enzymes. info/, ecoliwiki.net/ in particular provides a list of alternate nomenclature for each enzyme/gene. Many similar databases are available including UNIPROTKB, PROSITE; 5 EC2PDB; ExplorEnz; PRIAM; KEGG Ligand; IUBMB Enzyme Nomenclature; IntEnz; MEDLINE; and MetaCyc, to name a few.

[0032] By convention, genes are written in italic, and corresponding proteins in regular font. E.g., fadD is the gene encoding FadD or acyl-CoA synthetase.

[0033] Generally speaking, we may use the gene name and protein names interchangeably herein, based on the protein name as provided in ecoliwiki.net. The use of a protein name as an overexpressed protein (e.g., FabH⁺) signifies that protein expression can occur in ways other than by adding a vector encoding same, since the protein can be upregulated in other ways. The use of FadD^" signifies that the protein has been downregulated in some way, whereas the use of AfadD means that the gene has been directly downregulated to a null or knockout mutant.

[0034] The use of the word "a" or "an" when used in conjunction with the term

"comprising" in the claims or the specification means one or more than one, unless the context dictates otherwise.

[0035] The term "about" means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.

[0036] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[0037] The terms "comprise", "have", "include" and "contain" (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.

[0038] The phrase "consisting of is closed, and excludes all additional elements.

[0039] The phrase "consisting essentially of excludes additional material elements, but allows the inclusions of non-material elements that do not substantially change the nature of the invention, such as instructions for use, buffers, background mutations that do not effect the reverse beta oxidation pathways, additional purification steps, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 : Reverse β-oxidation for the synthesis of functionalized products.

Initial condensation of priming molecule with acetyl-CoA (1) is followed by subsequent dehydrogenation (2), dehydration (3), and reduction (4) with product chain length dependent on the number of elongation cycles (5). Product synthesis achieved through termination pathways functioning on CoA intermediates at each pathway step. Carboxylic acid production through thioesterase termination (6) and alcohol production through alcohol forming CoA reductase termination (7) depicted as examples. Omega-functionalization achieved through use of omega-functionalized CoA primer or omega-functionalization of intermediates/products generated from a β-oxidation reversal.

[0041] FIG. 2: Priming the β-oxidation reversal with functionalized primers, ω-

Amino carboxylic acids can be produced through the condensation of acetyl-CoA with ω- aminated CoA (A), ω-carboxylated CoA (B), or co-hydroxylated CoA (C) priming molecules and subsequent steps of a β-oxidation reversal and appropriate termination enzymes.

[0042] FIG. 3: Synthesis of omega-amino carboxylic acids and their α,β functionalized derivatives through omega-amino priming. Initial priming of a functional β- oxidation reversal with an omega-aminated primer and n elongation cycles generates omega- functionalized CoA intermediates that can be converted to omega-amino carboxylic acids through the termination pathways depicted.

[0043] FIG. 4: Synthesis of omega-amino acids and their α,β functionalized derivatives through omega-hydroxylated priming. Initial priming of a functional β-oxidation reversal with an omega-hydroxylated primer and n elongation cycles generates omega- functionalized CoA intermediates that can be converted to omega-amino acids through the termination pathways depicted.

[0044] FIG. 5: Synthesis of omega-amino carboxylic acids and their α,β functionalized derivatives with omega-carboxylated priming molecules. Initial priming of a functional β-oxidation reversal with an omega-carboxylated primer and n elongation cycles generates omega-functionalized CoA intermediates that can be converted to omega-amino acids through the termination pathways depicted. Note that the a and β carbons are named according to the initial CoA intermediate (and not the final product) resulting in different products for the case of α,β functionalized derivatives compared to those illustrated in FIG. 3 and 4.

[0045] FIG. 6: Synthesis omega-amino carboxylic acids and their α,β functionalized derivatives through omega-functionalization termination. Initial priming of a functional β- oxidation reversal with acetyl-CoA and n elongation cycles generates CoA intermediates that can be converted to omega-amino carboxylic acids through the termination pathways depicted.

[0046] FIG. 7: Activation of omega-aminated priming molecules by C. propionicum

Actl . CoA transferase activities (nmol/min/mg) from crude cell extract containing Actl shown for omega-aminated acids of various chain length. CoA activation of these compounds results in the formation of an omega-aminated molecule that can serve as the priming molecule for the synthesis of longer chain length omega-aminated acids through a beta-oxidation reversal.

[0047] FIG. 8: ω-hydroxyacid production through omega-hydroxylated priming.

Total ion chromatogram showing GC-MS identified 4-hydroxybutyrate production from a one-turn beta-oxidation reversal with glycolyl-CoA priming. 6-Hydroxyhexanoic acid production from overexpression of genes encoding thiolase (BktB), 3-hydroxyacyl-CoA dehydrogenase (PhaBl), Aeromonas caviae enoyl-CoA hydratase PhaJ (acPhaJ), and trans- enoyl-CoA reductase T. denticola TER (taTTER) components, along with activation enzyme for glycolate to glycolyl-CoA conversion— propionyl-CoA transferase from Megasphaera ekdenii (mePCT). MG1655 (DE3) AglcD (pET-Pl-bktB-phaBl-P2-acPhaJ) (pCDF-Pl- mePCT-P2-taTTER) grown at 30°C in LB media with 10 g/L Glucose and 40 mM Glycolate. co-hydroxyacids produced through this route can be converted to omega-aminated acids through the action of an alcohol dehydrogenase and a transminase (FIG. 11-13).

[0048] FIG. 9: ω-hydroxyacid production through omega-carboxylated priming. 6-

Hydroxyhexanoic acid production from succinyl-CoA priming with overexpression of genes encoding thiolase (PaaJ), 3-hydroxyacyl-CoA dehydrogenase (PaaH), enoyl-CoA hydratase (PaaF), and trans-enoyl-CoA reductase (taTTER) components, along with activation enzyme for succinate to succinyl-CoA conversion (Catl). Data shown for strain expressing above enzymes with or without the additional overexpression of an acyl-CoA reductase termination pathway (acyl-CoA reductase Aid from Clostridium beijerinckii-cbjA D). co-hydroxyacids produced through this route can be converted to omega-aminated acids through the action of an alcohol dehydrogenase and a transminase (FIG. 11-13).

[0049] FIG. 10: co-hydroxyacid production through omega-carboxylated priming. 7-

Hydroxyheptanoic acid production from glutaryl-CoA priming with overexpression of genes encoding thiolase (PaaJ), 3-hydroxyacyl-CoA dehydrogenase (PaaH), enoyl-CoA hydratase (PaaF), and trans-enoyl-CoA reductase (taTTER) components, along with activation enzyme for glutarate to glutaryl-CoA conversion (Catl). Data shown for strain expression above enzymes with or without the additional overexpression of an acyl-CoA reductase termination pathway (ctyALD). ω-hydroxyacids produced through this route can be converted to omega- aminated acids through the action of an alcohol dehydrogenase and a transminase (FIG. 11- 13).

[0050] FIG. 11 : Synthesis of C6-C10 ω-hydroxyacids through the ω-oxidation of carboxylic acids generated from a β-oxidation reversal. 6-Hydroxyhexanoic acid, 8- hydroxyoctanoic acid, and 10-hydroxydecanoic acid production shown from 72 hr fermentations with JCOl (DE3) bktB^CT5 fadB^CT5 AfadA egTER^CT5 ydiI^M AtesB containing pETDuet-l-Pl-P2-a/ 5Gr using rich (LB) medium with glycerol as the carbon source, co- hydroxyacids produced through this route can be converted to omega-aminated acids through the action of an alcohol dehydrogenase and a transminase (FIG 11-13).

[0051] FIG. 12: NAD(P)+-dependent co-hydroxyacid oxidation activity (μιηοΐ/mg protein/min) from various alcohol dehydrogenases. Activity measured from crude extract of BL21(DE3) cells expressing indicated enzyme in pCDFDuet-1, no NAD(P)+-dependent activity detected with blank pCDFDuet-1 vector. For ChnD, the NAD+-dependent activity shown, no NADP+-dependent activity detected. For YahK and YjgB, NADP+-dependent activity shown, no NAD+-dependent activity detected. Oxidation of omega-hydroxyacids to omega-oxo-carboxylic acids is a key step in multiple routes to omega-amino acid production (See FIG. 3-5).

[0052] FIG. 13: Purification of Chromobacterium violaceum CV2025 and aminotransferase activity of CV2025. FIG. 13A SDS/PAGE gel showing expression and purification of histidine tagged CV2025. FIG. 13B Thin-layer chromatogram showing the appearance of L-alanine when CV2025 (cell lysate or purified form) is incubated with indicated amino-acid substrate and pyruvate (amino group donor and acceptor). L-alanine synthesis demonstrates functional activity in the reverse direction through the transfer of the amine group from the substrate to pyruvate, forming L-alanine.

[0053] FIG. 14: Demonstration of CV2025 transaminase activity in vector constructs expressing a carboxylic acid omega-hydroxylase (AlkBGT) and/or an alcohol dehydroganse (ChnD). Thin-layer chromatogram showing the appearance of L-alanine in cell lysates of cells containing the indicated vectors expressing CV2025, incubated with methylbenzylamine (MBA) and pyruvate (amino group donor and acceptor). Controls showing lack of L-alanine synthesis without pyruvate addition also shown. Vectors can be utilized for the synthesis of omega-amino acids with either functionalized priming to form an omega-hydroxyacid (pETDuet-Pl-chnD-CV2025) or the synthesis of carboxylic acids (pETDuet-Pl-chnD- CV2025-P2-alkBGT) through a beta-oxidation reversal.

[0054] FIG. 15: Relevant genes for activation, priming, core/elongation, termination, ω-oxidation, and amination modules of a functional reversal of the β-oxidation cycle for ω- amino carboxylic acid synthesis (See FIG. 1 for pathway details).

DETAILED DESCRIPTION

[0055] The production of ω-amino carboxylic acids from a functional reversal of the β-oxidation requires either the initial condensation of an ω-functionalized priming molecule with acetyl-CoA or the omega-functionalization of carboxylic acids generated from a β- oxidation reversal at the omega carbon (FIGS. 3-6) or combinations thereof. For the former, various omega-functional priming can be exploited for synthesis of ω-amino carboxylic acids through either direct use of ω-amino priming molecules (FIG. 3) or the use of omega- functionalized primers such as omega-hydroxylated and omega-carboxylated molecules that can be paired with appropriate termination pathways to generate precursor molecules (omega-oxo carboxylic acids) to which amino-functionality can be introduced (FIGS. 4 and 5). Alternatively, these same precursor molecules can be generated through the synthesis of carboxylic acids through a beta-oxidation reversal, followed by omega-hydroxylation and subsequent oxidation of the omega-hydroxyl group (FIG. 6). In the case of the generation of omega-oxo carboxylic acids, these compounds are converted to omega-aminated carboxylic acids through amination reactions, while the use of an omega-aminated primer inserts amino functionality during priming of a beta-oxidation reversal.

OMEGA-AMINO CARBOXYLIC ACIDS VIA OMEGA-AMINATED PRIMERS

[0056] For the production of omega-amino carboxylic acids through omega-aminated priming, either internal generation or external addition of a functionalized acid molecule followed by its activation to a CoA intermediate is required to provide the priming molecule that can be condensed with acetyl-CoA. While several potential metabolic pathways are available for the synthesis of amino acids, the determination of enzymes that can activate these potential priming molecules is a prerequisite to determine the best potential priming molecules. [0057] One potential candidate for this activation enzyme is the Co A transferase Actl from Clostridium propionicum (cpActl). C. propionicum is capable of fermenting amino acids, such as L- and beta-alanine, to ammonium, acetate, C0₂, and propionate, and increased induction of cpActl is observed when this organism is grown on beta-alanine (FEBS Journal 272, 813-821, 2005). In order to determine the potential of cpActl as an activation enzyme for a beta-oxidation reversal with omega-aminated priming, the gene encoding this CoA transferase was cloned into pCDFDuet, and crude protein extract from E. coli cells expressing this enzyme were tested for CoA transferase activity on a variety of omega- aminated substrates. Utilizing acetyl-CoA as the CoA group donor, transferase activity was observed with beta-alanine and 4-aminobutyrate as substrates, while no activity was detected with glycine or 5 -amino valerate (FIG. 7).

[0058] With the establishment of cpActl as an enzyme for activation of potential amino-functionalized priming molecules, the use of core/elongation modules of the β- oxidation reversal capable of working on ω-aminated intermediates can be exploited for the synthesis of ω-amino carboxylic acids. For this, our knowledge base on thiolase, 3- hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase enzymes that have been demonstrated to function in a beta-oxidation reversal with intermediates of varying omega- functionality can be employed as potential components to operate a beta-oxidation reversal with omega-aminated priming (FIG.15).

OMEGA-AMINO CARBOXYLIC ACID PRECURSORS

[0059] As described above, one approach for the synthesis of omega-amino acids utilizes an aminated priming molecule which is subsequently lengthened by 2 carbon units in each turn of a beta-oxidation reversal before carboxylic acid termination. An alternative approach for introducing amino functionality is the generation of amino acid precursor intermediates through a beta-oxidation reversal, which can then be converted to omega amino acids through amination reactions (FIGS. 4-6). Key to this amination reaction is the generation of omega-oxo (aldehyde) carboxylic acids, which can then be converted to omega-amino acids through the action of transaminase enzymes. While the in vivo toxicity and relative instability of aldehyde intermediates makes their uses as primers/intermediates of a beta oxidation reversal difficult, the synthesis of omega-hydroxyacids also provides a route to these key precursors in which the alcohol group of omega-hydroxyacids can be further oxidized to an aldehyde. This in turn minimizes the number of intermediates in the pathway possessing inherent toxicity and instability.

[0060] The synthesis of ω-hydroxyacids from a functional reversal of the β-oxidation can take place through either the initial condensation of an ω-functionalized priming molecule with acetyl-CoA or the hydroxylation of carboxylic acids generated from a β- oxidation reversal at the omega carbon. For the former, the use of either omega-hydroxylated or omega-carboxylated priming molecules can be utilized with appropriate termination pathways (carboxylic acid forming and alcohol forming, respectively) to generate the required omega-hydroxy acids. For this approach, an omega-hydroxylated primer, such as glycolate/glycolyl-CoA, represents a molecule that when utilized as the primer for a beta- oxidation reversal will generate intermediates of varying chain length with omega- hydroxylation. As such, when this type of priming is paired with carboxylic acid forming termination, the synthesis of omega-hydroxyacids can be achieved (FIG. 4).

[0061] For this, the identification and characterization of enzymes that can enable a functional beta-oxidation reversal with glycolate/glycolyl-CoA priming was required. Through the use of in vitro characterization and in vivo functional testing, Ralstonia eutropha BktB (reBktB), Ralstonia eutropha PhaBl (rePhaBl), Aeromonas caviae PhaJ (acPhaJ) or Pseudomonas aeruginosa PhaJ4) (paPhaJ4), and T. denticola or Methylobacillus flagellates TER (taTTER or m TER) were established as functional enzymes for the thiolase, 3- hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase steps for beta-oxidation reversal with omega-hydroxylated intermediates. The expression of these genes encoding enzymes for the 4 steps of a beta-oxidation reversal in combination with the expression of the propionyl-CoA transferase from Megasphaera elsdenii (mePCT), an enzyme demonstrated to convert glycolate to glycolyl-CoA (Journal of Biotechnology 156, 214-217, 2011; Nature Communications 4, 1414, 2013), resulted in the synthesis of 4-hydroxybutyrate when cultured with glucose as the carbon source with addition of 40 mM glycolate (FIG. 8).

[0062] In addition to the use of omega-hydroxylated primers with carboxylic acid forming termination pathways, omega-carboxylated primers can be utilized in a beta- oxidation reversal to form omega-hydroxyacids when combined with alcohol forming termination pathways (FIG. 5). It should also be noted that this approach can directly synthesize the omega-oxo carboxylic acids required as substrates for the transamination reaction when an aldehyde forming acyl-CoA reductase is used as the termination enzyme (FIG. 5). For this approach, succinate/succinyl-CoA represents one potential primer that can result in omega-hydroxyacids when combined with appropriate termination pathways and, as with glycolate, the possibility to generate succinate internally (e.g. from glucose or glycerol) could allow production from a single carbon source.

[0063] A similar approach to that for omega-hydroxylated priming was used for the identification and characterization of enzymes that can enable a functional beta-oxidation reversal with succinate/succinyl-CoA priming. This investigation established E. coli PaaJ, PaaH, PaaF (beta-ketoadipyl-CoA thiolase, 3-hydroxyadipyl-CoA dehydrogenase PaaH, and 2,3-dehydroadipyl-CoA hydratase, respectively; European Journal of Biochemistry 270, 3047-3054, 2003), and the trans-2-enoyl-CoA reductase from T. denticola {ΐάϊΈ ; FEBS Letters 581, 1561-1566, 2007) as functional enzymes for the thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase steps a beta-oxidation reversal with omega- carboxylated intermediates. The expression of these enzymes in combination with the acetyl- CoA:succinate CoA transferase Catl from Clostridium kluyveri (Journal of Bacteriology 178, 871-880, 1996) resulted in the synthesis of the 6-carbon dicarboxylic acid, apidic acid (data not shown).

[0064] In order to utilize this approach for the synthesis of omega-hydroxyacids, the expression of a termination enzyme(s) capable of reducing the CoA group of the omega- carboxylated acyl-CoA intermediate to an alcohol is required. For this, the acyl-CoA reductase Aid from Clostridium beijerinckii (cbjA D; Applied and Environmental Microbiology 65, 4973-4980, 1999) was selected as a potential termination enzyme given its role in the production of butanol in C beijerinckii. In order to demonstrate the potential for this combination of enzymes to result in omega-hydroxyacid production through omega- carboxylated priming of a beta-oxidation reversal, a background strain devoid in both native fermentation pathways ( ldhA, Δρία, ΔροχΒ, adhE, bfrdA) and several endogenous thioesterases ( tesA, tesB, yciA, Δ/adM, ydil, and ybgC) was selected to maximize acetyl-CoA/succinyl-CoA generation, as well as reduce the activity of endogenous acid generating termination pathways.

[0065] As seen in FIG. 9, when this combination of beta-oxidation reversal and activiation enzymes (PaaJ, PaaH, PaaF, tdTER, Catl, cbjALD) was expressed in this strain (JST06; Journal of Industrial Microbiology and Biotechnology 42, 465-475, 2015), the production of 6-hydroxyhexanoic acid was observed. Furthermore, when glutarate, the five carbon dicarboxylic acid, was used in place of succinate with the above strain, JST06 (DE3) (pET-Pl-paaJ-paaH-P2-cty^'ALD) (pCDF-Pl-catl-paaF-P2-tdTER), the synthesis of 7- hydroxyheptanoic acid was observed (FIG. 10). This seven carbon product represents the final product of a one turn beta-oxidation reversal with glutaryl-CoA/acetyl-CoA condensation with an alcohol forming termination pathway.

[0066] It should be noted that with the above results, the expression of an acyl-CoA reductase (cbjALD) was required, as no omega-hydroxyacids were observed in the control strain without cbjALD expression. These results demonstrate the potential for the use of omega-carboxylated primers in combination with alcohol forming termination pathways to produce omega-hydroxyacids through a beta-oxidation reversal, which as with the use of omega-hydroxylated primers, provide a route to precursors that can be further oxidized to an omega-oxo carboxylic acid and aminated through the action of a transaminase for omega- amino acid synthesis.

[0067] An alternative route to these precursor molecules is the use of omega- oxidation pathways to convert carboxylic acids generated through a β-oxidation reversal to ω-hydroxyacids (FIG. 6). The omega-functionalization of carboxylic acids to ω- hydroxyacids requires the identification of enzymes capable of adding a hydroxyl group to the aliphatic chain of the carboxylic acid. While several enzymes have been characterized for the hydroxylation of various compounds at sub-terminal positions (Biotechnology Advances 31, 1473-1485, 2013; Chemical Society Reviews 41, 1218-1260, 2012), the higher energy associated with the omega methyl group makes omega-hydroxylation a more challenging reaction.

[0068] Despite the challenges associated with this reaction, several industrially important yeasts and bacteria possess enzymes capable of this ω-hydroxylation as part of a pathway for the degradation of alkanes and long chain fatty acids (Applied Microbiology and Biotechnology 74, 13-21, 2007; WIREs System Biology and Medicine 5, 575-585, 2013). One promising candidate is the alkane hydroxylase system of P. putida (Microbiology 147, 1621-1630, 2001), encoded by alkBGT, that part of the pathway that enables growth on linear alkanes C6-C16 and has been recently shown to ω-hydroxylate medium chain length fatty acid methyl esters (Advanced Synthesis & Catalysis 353, 3485-3495, 2011). [0069] Our ability to investigate ω-hydroxyacid synthesis via this route required development of reliable expression systems for the independent control of the core/elongation and termination modules leading to the synthesis of carboxylic acids. The construction of strains with controlled chromosomal expression of the thiolase {bktB), dehydrogenase (fadB), dehydratase (fadB), and reductase (E. gracilis TER, egTER) modules along with independent chromosomal expression of thioestarase (ydil) termination resulted in the ability to produce C6, C8, and CIO chain length carboxylic acids, providing products generated from a β-oxidation reversal that through ω-functionalization will enable the synthesis of our target products.

[0070] The construction of a background strain with chromosomal expression of all required modules for carboxylic acid production (JC01(DE3) bktB fadB AfadA egTER^CT5 ydiI^Ai, see Clomburg et al, 2015 for additional details) enabled us to directly test ω-hydroxylase expression with independent control from Duet system vectors. As seen in FIG. 11, when expressed individually from Duet vectors in our background strain producing C6-C10 carboxylic acids, the production of ω-hydroxyacids was observed. The expression of alkBGT resulted in the synthesis of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10- hydroxydecanoic acid (FIG. 11), demonstrating the ability of this approach to synthesize co- hydroxyacids, a key precursor to ω-amino acids.

[0071] Regardless of the route taken to co-hydroxyacids, the production of ω-amino acids through a β-oxidation reversal with amination reactions requires an additional enzyme (alcohol dehydrogenases) with activity toward the alcohol group of the synthesized ω- hydroxyacid. This oxidizes the alcohol group to an aldehyde (FIG. 12), the functional group required for the transamination reaction. For this, a group of enzymes involved in the degradation of cyclic alcohols in certain microorganisms have potential to function with the chain length ω-hydroxyacids of interest. The alcohol dehydrogenase ChnD and aldehyde dehydrogenase ChnE of Acinetobacter sp. Strain SE19 have been shown to be involved in the degradation of cyclohexanol, with indirect evidence pointing to their roles in the oxidation of 6-hydroxyhexanoic acid to adipic acid (Journal of Bacteriology 182, 4744-4751, 2000).

[0072] Oxidation activity on co-hydroxyacids of varying chain length was measured from crude extracts of cells expressing ChnD. While no activity was detected in crude extracts from cells with the control pCDFDuet vector, when ChnD was expressed from this vector, crude extracts displayed NAD⁺ dependent oxidation activity for C5-C 10 chain length substrates (FIG. 12). The high specific activities (greater than 10 μηιοΐ/mg protein/min) for C₆, C₈, and C₁₀ ω-hydroxyacid oxidation make ChnD an ideal candidate for the alcohol dehydrogenase step in the conversion of ω-hydroxyacids the corresponding omega-oxo carboxylic acids.

[0073] In addition to ChnD, potential native E. coli enzymes for the oxidation of ω- hydroxyacids were identified through a BLAST search using ChnD as the template. While numerous alcohol dehydrogenases showed sequence similarity to ChnD, only 2 E. coli enzymes tested possessed target chain length ω-hydroxyacid oxidation activity (data not shown). These enzymes, YahK and YjgB, resulted in the NADP⁺-dependent oxidation of C₆- Cio omega-hydroxyacids when crude extract of cells expressing the enzymes were tested (FIG. 12), but did not possess NAD⁺-dependent oxidation activity with the same chain ω- hydroxyacids (data not shown), in concurrence to their co-factor specificity on other substrates (Applied Microbiology and Biotechnology 97, 5815-5824, 2013).

[0074] Based on the ability of these characterized enzymes to function with ω- hydroxyacids, the expression of an alcohol dehydrogenase such as those described above, in combination with any of the routes to ω-hydroxyacids (e.g. omega-hydroxylated primers, omega-carboxylated primers, or hydroxylation of carboxylic acids) provides a functional route to generate omega-oxo carboxylic acids. Omega-amino functionality can then be introduced into these compounds through the action of an omega-transaminase, replacing the oxo group with an amine group as described in the next section.

AMINATION OF OMEGA-OXO CARBOXYLIC ACIDS

[0075] The introduction of omega-amino functionality to generated omega-oxo carboxylic acids can be accomplished through the action of omega-transaminases, enzymes that catalyze reversible amino group transfer from a donor to an acceptor, whereby one of the two compounds can be a non-activated aldehyde, ketone, or amine (ACS Catalysis 4, 129- 143, 2014).

[0076] A wide range of omega-transaminases from various microorganisms have been characterized with amination activity toward a variety of substrates (Applied Microbiology and Biotechnology 94, 1163-1171, 2012; Proteins 81, 774-787, 2013; Trends in Biotechnology 28, 324-332, 2010). Examples of potential omega-transaminases that can be exploited for the conversion of generated omega-oxo carboxylic acids to omega-amino acids include those from Alcaligenes denitrificans aptA (AAP92672.1), Bordetella bronchiseptica (WP_015041039.1), Bordetella parapertussis BPP0784 (WP O 10927683.1), Brucella melitensis (EEW88370.1), Burkholderia pseudomallei BMEI1757 (AFI65333.1), Oceanicola granulosus OG2516 07293 (WP_007254984.1), Paracoccus denitrificans PD1222 (ABL72050.1), Pseudogulbenkiania ferrooxidans (WP_008952788.1), Pseudomonas putida (P28269.1), Ralstonia solanacearum RALTA_B1822 (YP_002258353.1), and Vibrio fluvialis (AEA39183.1), among others.

[0077] Considering the current chain length of omega-hydroxyacids demonstrated through a beta-oxidation reversal with omega-functionalized priming or omega- functionalization termination, as well as the characterized specificity of potential omega-oxo carboxylic acid generating enzymes, the omega-transaminase CV2025 (AAQ59697.1) from Chromobacterium violaceum is of special interest. This enzyme has been demonstrated to aminate a number of various chain length aldehyde substrates, as well utilize L-alanine as the amino group donor with high specificity (Enzyme and Microbial Technology 41, 628-637, 2007). The latter is an important characteristic giving the relative ease at which L-alanine can be synthesized and regenerated in vivo for continued availability of the amino donor (Applied Microbiology and Biotechnology 77, 355-366, 2007). Furthermore, CV2025 has recently been utilized for the omega-amino functionalization of 12-oxododecanoic acid methyl ester (Advanced Synthesis and Catalysis 355, 1693-1697, 2013).

[0078] Given these characteristics, we sought to characterize CV2025 for the potential omega-amino functionalization of omega-oxo carboxylic acids generated through a beta-oxidation reversal. The gene encoding CV2025 was cloned with an N-terminal HIS-tag, expressed in E. coli, and purified to enable further characterization (FIG. 13 A). In order to ensure functional activity, the purified enzyme was incubated with methylbenzylamine (MBA) (amino donor) with and without the addition of pyruvate as the amino group acceptor in a standard assay mixture (Enzyme and Microbial Technology 41, 628-637, 2007). Following incubation, thin layer chromatography was utilized to detect the presence of L- alanine, the compound resulting from amino group transfer from the donor to pyruvate. As seen in FIG. 13B, the presence of L-alanine was only detected with the enzyme was incubated with both MBA and pyruvate, indicating the functional activity of CV2025 purified from E. coli.

[0079] Following confirmation of functional activity, purified CV2025 was then tested on various omega-amino carboxylic acids to determine its potential in the context of amino-functionilzation of omega-oxo carboxylic acids. As seen in FIG. 13B, when CV2025 was incubated with pyruvate and 4-aminobutyric acid, 6-aminohexanoic acid, or 8- aminooctanoic acid, the synthesis of L-alanine was observed. These results demonstrate the functional activity of CV2025 with omega-amino carboxylic acids in the reverse direction through the transfer of the amine group from the substrate to pyruvate. Therefore in the context of a beta-oxidation reversal for the synthesis of omega-amino carboxylic acids, CV2025 represents an amino functionalization enzyme that can be utilized to aminate the omega-oxo carboxylic acids generated.

[0080] Based on this functionality, the gene encoding CV2025 was cloned into various constructs than can be utilized for the production of omega-amino carboxylic acids through the amination of omega-oxo carboxylic acids generated through either omega- functionalized priming or omega-functionalization termination. Those constructed included CV2025 both individually and with an co-hydroxyacid oxidation enzyme (e.g. pETDuet-Pl- CV2025 and pKTDuQt-Vl-chnD-CV2025) for use with functionalized priming modules, or in combination with omega-hydroxylation and ω-hydroxyacid oxidation enzymes (e.g. p TDuQt-Pl-chnD-CV2025-V2-alkBGT) for the complete omega-amination of carboxylic acids generated from a beta-oxidation reversal.

[0081] Functional activity of CV2025 was verified in these constructs with MBA and pyruvate as described above. The appearance of L-alanine when crude protein extracts of cells containing these vectors were incubated with both MBA and pyruvate confirms the functional activity of CV2025 in these constructs (FIG. 14), thus providing genetic constructs that can be combined with the above described strains generating precursors for a complete pathway for the synthesis of omega-amino carboxylic acids from a beta-oxidation reversal.

[0082] The following references are incorporated by reference in their entirety for all purposes.

[0083] 61/440,192, Reverse beta oxidation pathway, filed 2/7/2011.

[0084] WO2012109176, Reverse beta oxidation pathway, filed 2/7/2012.

[0085] 61,531,911, Synthesis Of Alpha- And Omega-Functionalized Carboxylic

Acids And Alcohols, filed Sept. 7, 2011

[0086] WO2013036812, Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation, filed Sept 7, 2012. [0087] US20140273110, Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation, filed 3/6/2014.

[0088] Dellomonaco C. et al., Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals, Nature 476, 355-359, 2011.

[0089] Clomburg, J.M., et al, A synthetic biology approach to engineer a functional reversal of the beta-oxidation cycle, ACS Synthetic Biology 1, 541-554, 2012.

[0090] Clomburg, J.M. et al, Integrated engineering of β-oxidation reversal and co- oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids Metabolic Engineering 28: 202-212, 2015.

[0091] Lian J. & Zhao H., 2015. Reversal of the β-oxidation cycle in Saccharomyces cerevisiae for production of fuels and chemicals, ACS Synth Biol. 4(3):332-41.

[0092] What is claimed is :

Claims

A genetically engineered microorganism comprising: a) one or more overexpressed enzymes that allow production of an omega-aminated Co A thioester primer selected from 2-aminoacetyl-CoA, 3-aminopropionoyl-CoA, or 4-aminobutyryl-CoA; b) an overexpressed thiolase that catalyzes condensation of said omega-aminated acyl-CoA primer with acetyl-CoA to produce an omega-aminated β-ketoacyl-CoA; c) an overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-[acyl-carrier- protein] reductase that catalyzes reduction of said omega-aminated β-ketoacyl- CoA to an omega-aminated β-hydroxyacyl-CoA; d) an overexpressed enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydratase, or 3- hydroxyacyl-[acyl-carrier-protein] dehydratase that catalyzes dehydration of said omega-aminated β-hydroxyacyl-CoA to an omega-aminated trans-enoyl-CoA; e) an overexpressed acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl- [acyl-carrier-protein] reductase that catalyzes reduction of said omega-aminated trans-enoyl-CoA to an omega-aminated acyl-CoA; f) an overexpressed termination enzyme(s) selected from the group consisting of a thioesterase, an acyl-CoA:acetyl-CoA transferase, and a phosphotransacylase and a carboxylate kinase catalyzing conversion of a product of steps b, c, d, or e above to omega-amino carboxylic acid; g) reduced expression of fermentation enzymes leading to reduced production of lactate, acetate, ethanol and succinate; wherein said microorganism has a reverse beta oxidation pathway beginning with said omega-aminated CoA thioester primer and running in a biosynthetic direction.

The genetically engineered microorganism of claim 1 , wherein said genetically engineered microorganism produces a product selected from the group consisting of omega-amino carboxylic acids, β-hydroxy omega-amino carboxylic acids, β-keto omega- amino carboxylic acids, and α,β-unsaturated omega-amino carboxylic acids.

The microroganism of claim 1 , wherein said overexpressed thiolase is encoded by a gene selected from the group consisting of E. coli atoB (NP_416728.1), E. coliyqeF

(NP_417321.2), E. colifadA (YP_026272.1), E. colifadl (NP_416844.1), Ralstonia eutropha bktB (AAC38322.1), Pseudomonas sp. Strain B13 catF (AAL02407.1), E. coli paaJ (NP_415915.1), Pseudomonas putida pcaF (AAA85138.1), Acinetobacter baylyi dcaF (Q6FBN0), 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), and homologs.

4) The microorganism of claim 1, wherein said overexpressed 3-hydroxyacyl-CoA

dehydrogenase or 3-oxoacyl-[acyl-carrier-protein] reductase is encoded by a gene selected from the group consisting of E. colifadB (NP_418288.1), E. colifadJ

(NP_416843.1), Ralstonia eutropha phaBl (YP_725942.1), Ralstonia eutropha phaB2 (YP_726470.1), Ralstonia eutropha phaB3 (YP_726636.1), Acinetobacter baylyi dcaH (Q937T5), E. colipaaH (P76083), E. colifabG (NP 415611.1), and homologs.

5) 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 selected from the group consisting of E. colifadB (NP_418288.1), E. colifadJ (NP_416843.1), Aeromonas caviae phaJ (032472.1), Pseudomonas aeruginosa phaJl (BAA92740.1), Pseudomonas aeruginosa phaJ2 (BAA92741.1), Pseudomonas aeruginosa phaJ3 (BAC44834.1), Pseudomonas aeruginosa phaJ4 (BAC44835.1), Acinetobacter baylyi dcaE (Q937T3), E. colipaaF (P76082), E. colifabA

(NP_415474.1), E. colifabZ (NP_414722.1), and homologs.

6) 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 selected from the group consisting of E. coliydiO (P0A9U8), Euglena gracilis TER (Q5EU90.1), Treponema denticola TER (NP_971211.1), Methylobacillus flagellatus TER (Q1H0P3), E. colifabl (NP_415804.1), Enterococcus faecalis fabK 16503.1), Bacillus subtilis fabL (KFK80655.1), Vibrio choleraefabV (ΑΒΧ38ΊΠ A), and homologs.

7) The microorganism of claim 1 , wherein said overexpressed thioesterase is encoded by a gene selected from the group consisting of E. coli tesA (NP_415027.1), E. coli tesB (NP_414986.1), E. coliyciA (NP_415769.1), E. colifadM (NP_414977 A), E. coliydil (NP_416201.1), E. coliybgC (NP_415264.1), Alcanivorax borkumensis tesB2

(YP_692749.1), Fibrobacter succinogenes Fs2108 (YP 005822012.1), Prevotella ruminicola Pr655 (YP_003574018.1), Prevotella ruminicola Prl687 (YP_003574982.1), Mus musculus ACOT8 (P58137), and homologs. 8) The microorganism of claim 1, wherein said overexpressed acyl-CoA:acetyl-CoA transferase is encoded by a gene selected from the group consisting of E. coli atoD (NP_416725.1), Clostridium kluyveri cat2 (AAA92344.1), Clostridium acetobutylicum ctfAB (NPJ49326.1, NPJ49327.1), E. coliydiF (NP_416209.1), and homologs.

9) The microorganism of claim 1 , wherein said overexpressed phosphotransacylase is

encoded by a gene selected from the group consisting of Clostridium acetobutylicum ptb (NP_349676.1), Enterococcus faecalis ptb (AAD55374.1), Salmonella enterica pduL (AAD39011.1), and homologs.

10) The microorganism of claim 1, wherein said overexpressed carboxylate kinase is encoded by a gene selected from the group consisting of Clostridium acetobutylicum buk

(AAK81015.1), Enterococcus faecalis buk (AAD55375.1), Salmonella enterica pduW (AAD39021.1), and homologs.

11) The microorganism of any of claims 1-10, wherein said reduced expression of

fermentation enzymes are AadhE, (Apta or AackA or AackApta), ApoxB, AldhA, and AfrdA and less acetate, lactate, ethanol and succinate are thereby produced.

12) A genetically engineered microorganism comprising: a) one or more overexpressed enzymes that allow production of an omega- hydroxylated Co A thioester primer selected from 2-hydroxyacetyl-CoA, 3- hydroxypropionyl-CoA, or 4-hydroxybutyryl-CoA; b) an overexpressed thiolase that catalyzes condensation of said omega-hydroxylated acyl-CoA primer with 2-carbon donor acetyl-CoA to produce an omega- hydroxylated β-ketoacyl-CoA; c) an overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-[acyl-carrier- protein] reductase that catalyzes reduction of said omega-hydroxylated β-ketoacyl- CoA to an omega-hydroxylated β-hydroxyacyl-CoA; d) an overexpressed enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydratase, or 3- hydroxyacyl-[acyl-carrier-protein] dehydratase that catalyzes dehydration of said omega-hydroxylated β-hydroxyacyl-CoA to an omega-hydroxylated trans-enoyl- CoA; e) an overexpressed acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl- [acyl-carrier-protein] reductase that catalyzes reduction of said omega- hydroxylated trans-enoyl-CoA to an omega-hydroxylated acyl-CoA; f) an overexpressed termination enzyme(s) selected from the group consisting of a thioesterase, an acyl-CoA:acetyl-CoA transferase, and a phosphotransacylase and a carboxylate kinase catalyzing conversion of a product of steps b, c, d, or e above to an omega-hydroxy carboxylic acid; g) an overexpressed alcohol oxidase/dehydrogenase that catalyzes oxidation of the omega-hydroxy carboxylic acid of step f to an omega-oxo carboxylic acid; h) an overexpressed omega-transaminase that catalyzes conversion of the omega-oxo carboxylic acid of step g to an omega-amino carboxylic acid; i) reduced expression of fermentation enzymes leading to reduced production of lactate, acetate, ethanol and succinate; wherein said microorganism has a reverse beta oxidation pathway beginning with said omega-hydroxylated acyl-CoA primer and running in a biosynthetic direction.

13) The genetically engineered microorganism of claim 12, wherein said genetically

engineered microorganism produces a product selected from the group consisting of omega-amino carboxylic acids, β-hydroxy omega-amino carboxylic acids, β-keto omega- amino carboxylic acids, and α,β-unsaturated omega-amino carboxylic acids.

14) The microorganism of claim 12, wherein said overexpressed thiolase is encoded by a gene selected from the group consisting of E. coli atoB (NP_416728.1), E. coliyqeF (NP_417321.2), E. colifadA (YP_026272.1), E. colifadl (NP_416844.1), Ralstonia eutropha bktB (AAC38322.1), Pseudomonas sp. Strain B13 catF (AAL02407.1), E. coli paaJ (NP_415915.1), Pseudomonas putida pcaF (AAA85138.1), Acinetobacter baylyi dcaF (Q6FBN0), 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), and homologs.

15) The microorganism of claim 12, wherein said overexpressed 3-hydroxyacyl-CoA

16) The microorganism of claim 12, wherein said overexpressed enoyl-CoA hydratase, 3- hydroxyacyl-CoA dehydratase, or 3-hydroxyacyl-[acyl-carrier-protein] dehydratase is encoded by a gene selected from the group consisting of E. colifadB (NP_418288.1), E. colifadJ (NP_416843.1), Aeromonas caviae phaJ (032472.1), Pseudomonas aeruginosa phaJl (BAA92740.1), Pseudomonas aeruginosa phaJ2 (BAA92741.1), Pseudomonas aeruginosa phaJ3 (BAC44834.1), Pseudomonas aeruginosa phaJ4 (BAC44835.1), Acinetobacter baylyi dcaE (Q937T3), E. colipaaF (P76082), E. colifabA

(NP_415474.1), E. colifabZ (NP_414722.1), and homologs.

17) The microorganism of claim 12, wherein said acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl-[acyl-carrier-protein] reductase is encoded by a gene selected from the group consisting of E. coliydiO (P0A9U8), Euglena gracilis TER (Q5EU90.1), Treponema denticola TER (NP_971211.1), Methylobacillus flagellatus TER (Q1H0P3), E. colifabl (NP_415804.1), Enterococcus faecalis fabK 16503.1), Bacillus subtilis fabL (KFK80655.1), Vibrio choleraefabV (ΑΒΧ38ΊΠ A), and homologs.

18) The microorganism of claim 12, wherein said overexpressed thioesterase is encoded by a gene selected from the group consisting of E. coli tesA (NP_415027.1), E. coli tesB (NP_414986.1), E. coliyciA (NP_415769.1), E. colifadM (NP_414977 A), E. coliydil (NP_416201.1), E. coliybgC (NP_415264.1), Alcanivorax borkumensis tesB2

(YP_692749.1), Fibrobacter succinogenes Fs2108 (YP 005822012.1), Prevotella ruminicola Pr655 (YP_003574018.1), Prevotella ruminicola Prl687 (YP_003574982.1), Mus musculus ACOT8 (P58137), and homologs.

19) The microorganism of claim 12, wherein said overexpressed acyl-CoA:acetyl-CoA

transferase is encoded by a gene selected from the group consisting of E. coli atoD (NP_416725.1), Clostridium kluyveri cat2 (AAA92344.1), Clostridium acetobutylicum ctfAB (NPJ49326.1, NPJ49327.1), E. coliydiF (NP_416209.1), and homologs.

20) The microorganism of claim 12, wherein said overexpressed phosphotransacylase is encoded by a gene selected from the group consisting of Clostridium acetobutylicum ptb (NP_349676.1), Enterococcus faecalis ptb (AAD55374.1), Salmonella enterica pduL (AAD39011.1), and homologs. 21) The microorganism of claim 12, wherein said overexpressed carboxylate kinase is encoded by a gene selected from the group consisting of Clostridium acetobutylicum buk (AAK81015.1), Enterococcus faecalis buk (AAD55375.1), Salmonella enterica pduW (AAD39021.1), and homologs.

22) The microorganism of claim 12, wherein said overexpressed alcohol

oxidase/dehydrogenase is encoded by a gene selected from the group consisting of Rhodococcus ruber SCI cddC (AAL14237.1), Acinetobacter sp. SE19 chnD

(AAG10028.1), E. coliyahK (NP_414859.1), E. coliyjgB (NP_418690.4), and homologs.

23) The microorganism of claim 12, wherein said overexpressed omega-transaminase is an enzyme selected from the group consisting of Arabidopsis thaliana At3g22200

(NP 001189947.1), Alcaligenes denitrificans AptA (AAP92672.1), Bordetella

bronchiseptica BB0869 (WP 015041039.1), Bordetella parapertussis BPP0784

(WP O 10927683.1), Brucella melitensis BAWG_0478 (EEW88370.1), Burkholderia pseudomallei BP 1026B I0669 (AFI65333.1), Chromobacterium violaceum CV2025 (AAQ59697.1), Oceanicola granulosus OG2516_07293 (WP_007254984.1), Paracoccus denitrificans PD 1222 Pden_3984 (ABL72050.1), Pseudogulbenkiania ferrooxidans ω- TA (WP_008952788.1), Pseudomonas putida ω-ΤΑ (P28269.1), Ralstonia solanacearum ω-ΤΑ (YP_002258353.1), Rhizobium meliloti SMc01534 (NP_386510.1), and Vibrio fluvialis ω-ΤΑ (AEA39183.1), Mus musculus AbaT (AAH58521.1), E. coli GabT

(YP 490877.1), and homologs.

24) The microorganism of any of claims 12-23, wherein said reduced expression of

fermentation enzymes are AadhE, (Apta or AackA or AackApta), ApoxB, AldhA, and AfirdA and less acetate, lactate, ethanol and succinate are thereby produced.

25) A genetically engineered microorganism comprising: a) one or more overexpressed enzymes that allow production of an omega- carboxylated acyl-CoA primer selected from oxalyl-CoA, malonyl-CoA, or succinyl-CoA; b) an overexpressed thiolase that catalyzes condensation of said omega-carboxylated acyl-CoA primer with acetyl-CoA to produce an omega-carboxylated β-ketoacyl- CoA; c) an overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-[acyl-carrier- protein] reductase that catalyzes reduction of said omega-carboxylated β-ketoacyl- CoA to an omega-carboxylated β-hydroxyacyl-CoA; d) an overexpressed enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydratase, or 3- hydroxyacyl-[acyl-carrier-protein] dehydratase that catalyzes dehydration of said omega-carboxylated β-hydroxyacyl-CoA to an omega-carboxylated trans-enoyl- CoA; e) an overexpressed acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl- [acyl-carrier-protein] reductase that catalyzes reduction of said omega- carboxylated trans-enoyl-CoA to an omega-carboxylated acyl-CoA; f) an overexpressed termination enzyme(s) selected from: i) the group consisting of an alcohol-forming coenzyme-A thioester reductase, and alcohol oxidase/dehydrogenase catalyzing conversion of a product of steps b, c, d, or e to a 1-oxo omega-carboxylic acid; or ii) an aldehyde-forming CoA thioester reductase catalyzing the conversion of a

product of steps b, c, d, or e to a 1-oxo omega-carboxylic acid; g) an overexpressed omega-transaminase that catalyzes conversion of the 1-oxo

omega-carboxylic acid of step f to a 1 -amino omega-carboxylic acid; h) reduced expression of fermentation enzymes leading to reduced production of lactate, acetate, ethanol and succinate; wherein said microorganism has a reverse beta oxidation pathway beginning with said omega-carboxylated CoA thioester primer and running in a biosynthetic direction.

26) The genetically engineered microorganism of claim 25, wherein said genetically

engineered microorganism produces a product selected from the group consisting of 1- amino omega-carboxylic acids, β-hydroxy 1 -amino omega-carboxylic acids, β-keto 1- amino omega-carboxylic acids, and α,β-unsaturated 1 -amino omega-carboxylic acids.

27) The microorganism of claim 25, wherein said overexpressed thiolase is encoded by a gene selected from the group consisting of E. coli atoB (NP_416728.1), E. coliyqeF (NP_417321.2), E. colifadA (YP_026272.1), E. colifadl (NP_416844.1), Ralstonia eutropha bktB (AAC38322.1), Pseudomonas sp. Strain B13 catF (AAL02407.1), E. coli paaJ (NP_415915.1), Pseudomonas putida pcaF (AAA85138.1), Acinetobacter baylyi dcaF (Q6FBN0), Rhodococcus opacus pcaF (YP_002778248.1), Streptomyces sp. pcaF (AAD22035.1), Ralstonia eutropha phaA (AEI80291.1), Clostridium acetobutylicum thlA (AAC26023.1), or Clostridium acetobutylicum MB (AAC26026.1), and homologs.

28) The microorganism of claim 25, wherein said overexpressed 3-hydroxyacyl-CoA

29) The microorganism of claim 25, wherein said overexpressed enoyl-CoA hydratase, 3- hydroxyacyl-CoA dehydratase, or 3-hydroxyacyl-[acyl-carrier-protein] dehydratase is encoded by a gene selected from the group consisting of E. colifadB (NP_418288.1), E. colifadJ (NP_416843.1), Aeromonas caviae phaJ (032472.1), Pseudomonas aeruginosa phaJl (BAA92740.1), Pseudomonas aeruginosa phaJ2 (BAA92741.1), Pseudomonas aeruginosa phaJ3 (BAC44834.1), Pseudomonas aeruginosa phaJ4 (BAC44835.1), Acinetobacter baylyi dcaE (Q937T3), E. colipaaF (P76082), E. colifabA

(NP_415474.1), E. colifabZ (NP_414722.1), and homologs.

30) The microorganism of claim 25, wherein said acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl-[acyl-carrier-protein] reductase is encoded by a gene selected from the group consisting of E. coliydiO (P0A9U8), Euglena gracilis TER (Q5EU90.1), Treponema denticola TER (NP_971211.1), Methylobacillus flagellatus TER (Q1H0P3), E. colifabl (NP_415804.1), Enterococcus faecalis fabK 16503.1), Bacillus subtilis fabL (KFK80655.1), Vibrio choleraefabV (ΑΒΧ38ΊΠ A), and homologs.

31) The microorganism of claim 25, wherein said overexpressed alcohol-forming coenzyme - A thioester reductase is encoded by a gene selected from the group consisting of Clostridium acetobutylicum adhE2 (YP_009076789.1), Arabidopsis thaliana At3gll980 (AEE75132.1), Arabidopsis thaliana At3g44560 (AEE77915.1), Arabidopsis thaliana At3g56700 (AEE79553.1), Arabidopsis thaliana At5g22500 (AED93034.1), Arabidopsis thaliana CER4 (AEE86278.1), Marinobacter aquaeolei VT8 maqu_2220

(YP_959486.1), Marinobacter aquaeolei VT8 maqu_2507 (YP 959769.1), and homologs. 32) The microorganism of claim 25, wherein said overexpressed aldehyde-forming CoA thioester reductase is encoded by a gene selected from the group consisting of

Acinetobacter calcoaceticus acrl (AAC45217.1), Acinetobacter sp Strain M-l acrM (BAB85476.1), Clostridium beijerinckii aid (AAT66436.1), E. coli eutE (NP 416950.1), Salmonella enterica eutE (AAA80209.1), E. coli mhpF (NP_414885.1), and homologs.

33) The microorganism of claim 25, wherein said overexpressed alcohol

34) The microorganism of claim 25, wherein said overexpressed omega-transaminase is an enzyme selected from the group consisting of Arabidopsis thaliana At3g22200

(NP 001189947.1), Alcaligenes denitrificans AptA (AAP92672.1), Bordetella

bronchiseptica BB0869 (WP 015041039.1), Bordetella parapertussis BPP0784

(WP 010927683.1), Brucella melitensis BAWG_0478 (EEW88370.1), Burkholderia pseudomallei BP 1026B I0669 (AFI65333.1), Chromobacterium violaceum CV2025 (AAQ59697.1), Oceanicola granulosus OG2516_07293 (WP_007254984.1), Paracoccus denitrificans PD1222 Pden_3984 (ABL72050.1), Pseudogulbenkiania ferrooxidans ω- TA (WP_008952788.1), Pseudomonas putida ω-ΤΑ (P28269.1), Ralstonia solanacearum ω-ΤΑ (YP_002258353.1), Rhizobium meliloti SMc01534 (NP_386510.1), Vibrio fluvialis ω-ΤΑ (AEA39183.1), Mus musculus AbaT (AAH58521.1), E. coli GabT (YP_490877.1) and homologs.

35) The microorganism of any of claims 25-34, wherein said reduced expression of

36) A genetically engineered microorganism comprising: a) an overexpressed thiolase that catalyzes condensation of an acyl-CoA primer with acetyl-CoA to produce a β-ketoacyl-CoA; b) an overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-[acyl-carrier- protein] reductase that catalyzes reduction of said β-ketoacyl-CoA to a β- hydroxyacyl-CoA; c) an overexpressed enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydratase, or 3- hydroxyacyl-[acyl-carrier-protein] dehydratase that catalyzes dehydration of said β-hydroxyacyl-CoA to a trans-enoyl-CoA; d) an overexpressed acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl- [acyl-carrier-protein] reductase that catalyzes reduction of said trans-enoyl-CoA to an acyl-CoA; e) an overexpressed termination enzyme(s) selected from the group consisting of a thioesterase, an acyl-CoA:acetyl-CoA transferase, a phosphotransacylase and a carboxylate kinase catalyzing the conversion of a product of steps b, c, d, or e to a carboxylic acid; f) an overexpressed omega-hydroxylase that catalyzes omega-hydroxylation of the carboxylic acid of step e to an omega-hydroxy carboxylic acid; g) an overexpressed alcohol oxidase/dehydrogenase that catalyzes oxidation the

omega-hydroxy carboxylic acid of step f to an omega-oxo carboxylic acid; h) an overexpressed omega-transaminase that catalyzes conversion of the omega-oxo carboxylic acid of step g to an omega-amino carboxylic acid; i) reduced expression of fermentation enzymes leading to reduced production of lactate, acetate, ethanol and succinate; wherein said microorganism has a reverse beta oxidation pathway beginning with acetyl- CoA and running in a biosynthetic direction.

37) The genetically engineered microorganism of claim 36, wherein said genetically

38) The microorganism of claim 36, wherein said overexpressed thiolase is encoded by a gene selected from the group consisting of E. coli atoB (NP_416728.1), E. coliyqeF (NP_417321.2), E. colifadA (YP_026272.1), E. colifadl (NP_416844.1), Ralstonia eutropha bktB (AAC38322.1), Pseudomonas sp. Strain B13 catF (AAL02407.1), E. coli paaJ (NP_415915.1), Pseudomonas putida pcaF (AAA85138.1), Acinetobacter baylyi dcaF (Q6FBN0), Rhodococcus opacus pcaF (YP_002778248.1), Streptomyces sp. pcaF (AAD22035.1), Ralstonia eutropha phaA (AEI80291.1), Clostridium acetobutylicum thlA (AAC26023.1), Clostridium acetobutylicum MB (AAC26026.1), and homologs.

39) The microorganism of claim 36, wherein said overexpressed 3-hydroxyacyl-CoA

40) The microorganism of claim 36, wherein said overexpressed enoyl-CoA hydratase, 3- hydroxyacyl-CoA dehydratase, or 3-hydroxyacyl-[acyl-carrier-protein] dehydratase is encoded by a gene selected from the group consisting of E. colifadB (NP_418288.1), E. colifadJ (NP_416843.1), Aeromonas caviae phaJ (032472.1), Pseudomonas aeruginosa phaJl (BAA92740.1), Pseudomonas aeruginosa phaJ2 (BAA92741.1), Pseudomonas aeruginosa phaJ3 (BAC44834.1), Pseudomonas aeruginosa phaJ4 (BAC44835.1), Acinetobacter baylyi dcaE (Q937T3), E. colipaaF (P76082), E. colifabA

(NP_415474.1), E. colifabZ (NP_414722.1), and homologs.

41) The microorganism of claim 36, wherein said acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl-[acyl-carrier-protein] reductase is encoded by a gene selected from the group consisting of E. coliydiO (P0A9U8), Euglena gracilis TER (Q5EU90.1), Treponema denticola TER (NP_971211.1), Methylobacillus flagellatus TER (Q1H0P3), E. colifabl (NP_415804.1), Enterococcus faecalis fabK 16503.1), Bacillus subtilis fabL (KFK80655.1), Vibrio choleraefabV (ΑΒΧ38ΊΠ A), and homologs.

42) The microorganism of claim 36, wherein said overexpressed thioesterase is encoded by a gene selected from the group consisting of E. coli tesA (NP_415027.1), E. coli tesB (NP_414986.1), E. coliyciA (NP_415769.1), E. colifadM (NP_414977 A), E. coliydil (NP_416201.1), E. coliybgC (NP_415264.1), Alcanivorax borkumensis tesB2

43) The microorganism of claim 36, wherein said overexpressed acyl-CoA:acetyl-CoA

44) The microorganism of claim 36, wherein said overexpressed phosphotransacylase is

45) The microorganism of claim 36, wherein said overexpressed carboxylate kinase is

encoded by a gene selected from the group consisting of Clostridium acetobutylicum buk (AAK81015.1), Enterococcus faecalis buk (AAD55375.1), Salmonella enterica pduW (AAD39021.1), and homologs.

46) The microorganism of claim 36, wherein said overexpressed carboxylic acid omega

hydroxylase is selected from the group consisting of the enzymes Pseudomonas putida AlkBGT (YP 009076004.1, Q9WWW4.1, Q9L4M8.1), Marinobacter aquaeolei

CYP153A (ABM17701 \ Mycobacterium marinum CYP153A16 (YP 001851443.1), Polaromonas sp. CYP153A (YP_548418.1), Nicotiana tabacum CYP94A5

(AAL54887.1), Vicia sativa CYP94A1 (AAD10204.1), Vicia sativa CYP94A2

(AAG33645.1), Arabidopsis thaliana CYP94B1 (BAB08810.1), Arabidopsis thaliana CYP86A8 (CAC67445.1), Candida tropicalis CYP52A1 (AAA63568.1, AAA34354.1, AAA34334.1), Candida tropicalis CYP52A2 (AAA34353.2, CAA35593.1), Homo sapiens CYP4A11 (AAQ56847.1), and homologs.

47) The microorganism of claim 36, wherein said overexpressed alcohol

48) The microorganism of claim 36, wherein said overexpressed omega-transaminase is an enzyme selected from the group consisting of Arabidopsis thaliana At3g22200

(NP 001189947.1), Alcaligenes denitrificans AptA (AAP92672.1), Bordetella

bronchiseptica BB0869 (WP 015041039.1), Bordetella parapertussis BPP0784

(WP O 10927683.1), Brucella melitensis BAWG_0478 (EEW88370.1), Burkholderia pseudomallei BP 1026B I0669 (AFI65333.1), Chromobacterium violaceum CV2025 (AAQ59697.1), Oceanicola granulosus OG2516_07293 (WP_007254984.1), Paracoccus denitrificans PD 1222 Pden_3984 (ABL72050.1), Pseudogulbenkiania ferrooxidans ω- TA (WP_008952788.1), Pseudomonas putida ω-ΤΑ (P28269.1), Ralstonia solanacearum ω-ΤΑ (YP_002258353.1), Rhizobium meliloti SMc01534 (NP_386510.1), and Vibrio fluvialis ω-ΤΑ (AEA39183.1), Mus musculus AbaT (AAH58521.1), E. coli GabT (YP 490877.1), and homologs.

49) The microorganism of any of claims 36-48 wherein said reduced expression of

50) A method of a producing a product selected from the group consisting of omega-amino carboxylic acids, β-hydroxy omega-amino carboxylic acids, β-keto omega-amino carboxylic acids, α,β-unsaturated omega-amino carboxylic acids, 1 -amino omega- carboxylic acids, β-hydroxy 1 -amino omega-carboxylic acids, β-keto 1 -amino omega- carboxylic acids, or α,β-unsaturated 1 -amino omega-carboxylic acids, comprising growing a genetically engineered microorganism according to any of claims 1-49 in a culture broth containing glycerol or a sugar, extending a CoA thioester primer by using a reverse beta oxidation pathway to produce a product at least two carbons longer than said primer, and isolating said product.