WO2015191972A2 - Omega-carboxylated carboxylic acids and derivities - Google Patents

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- CARBOXYLATED CARBOXYLIC ACIDS AND DERIVITIVES

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] That 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-carboxylated carboxylic acids (dicarboxylic 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 or alcohol groups.

[0010] Examples of potential products to be generated include ω- hydroxylated carboxylic acids, ω-carboxylated n-alcohols, dicarboxylic acids, and diols. 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 or carboxylated) 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-carboxylated carboxylic acids are illustrated in FIGS. 3-6.

[0012] In the second approach, alternate termination pathways are engineered to functionalize (hydroxylate or carboxylate) the omega carbon of an intermediate or a product of the engineered reversal of the β-oxidation cycle (illustrated by the omega-oxidation of carboxylic acids in FIGS. 7 and 8). 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 or omega carboxylated 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 hydroxy lated/carboxylated end: e.g. ω-hydroxylated carboxylic acids, ω- carboxylated n-alcohols, dicarboxylic acids, and diols. 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 and hydroxylated 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).

[0016] 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).

[0017] Omega functionalization of β -oxidation intermediate(s)/product(s)— The 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 can 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. The long chain dicarboxylates derived from omega-oxidation then enter the β-oxidation cycle for further degradation (WIREs System Biology and Medicine 5, 575-585, 2013). 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-hydroxy acids, or diols depending on the starting product and the extent of omega-oxidation). This approach for the synthesis of dicarboxylic acids is illustrated in FIGS. 7 and 8 with termination from a beta-oxidation reversal leading to carboxylic acids or alcohols followed by omega-oxidation resulting in the desired product. [0018] 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.

[0019] 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.

[0020] 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.

[0021] 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. [0022] 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.

[0023] 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.

[0024] 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.

[0025] 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^".

[0026] 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.

[0027] 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 "+".

[0028] As used herein, all accession numbers are to GenBank or UniProt unless indicated otherwise.

[0029] 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 shown in the art.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The phrase "consisting of is closed, and excludes all additional elements.

[0038] 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

[0039] FIG. 1. Reverse beta-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 any 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 either an omega-functionalized CoA primer or by the omega-functionalization of intermediates/products generated from a beta-oxidation reversal.

[0040] FIG. 2. Priming the β-oxidation reversal with functionalized primers.

Dicarboxylic acids can be produced through the condensation of acetyl-CoA with ω- carboxylated CoA (A) or co-hydroxylated CoA (B) priming molecules and subsequent steps of a β-oxidation reversal and appropriate termination enzymes. [0041] FIG. 3. Synthesis of dicarboxylic acids and their alpha, beta functionalized derivatives through omega-carboxylated priming. Initial priming of a functional beta- oxidation reversal with an omega-carboxylated primer and n elongation cycles generates omega-functionalized CoA intermediates that can be converted to di-carboxylic acids through the termination pathways depicted.

[0042] FIG. 4. Synthesis of dicarboxylic acids and their alpha, beta functionalized derivatives with omega-carboxylated priming molecules. Initial priming of a functional beta- oxidation reversal with an omega-carboxylated primer and n elongation cycles generates omega-functionalized CoA intermediates that can be converted to di-carboxylic acids through the termination pathways depicted.

[0043] FIG. 5. Synthesis of dicarboxylic acids and their alpha, beta functionalized derivatives through omega-hydroxylated priming. Initial priming of a functional beta- oxidation reversal with an omega-hydroxylated primer and n elongation cycles generates omega-functionalized CoA intermediates that can be converted to di-carboxylic acids through the termination pathways depicted.

[0044] FIG. 6. Synthesis of dicarboxylic acids and their alpha, beta functionalized derivatives with omega-hydroxylated priming molecules. Initial priming of a functional beta- oxidation reversal with an omega-hydroxylated primer and n elongation cycles generates omega-functionalized CoA intermediates that can be converted to di-carboxylic acids through the termination pathways depicted.

[0045] FIG. 7. Synthesis of dicarboxylic acids and their alpha, beta functionalized derivatives through carboxylic acid/omega-functionalization termination. Initial priming of a functional beta-oxidation reversal with acetyl-CoA and n elongation cycles generates CoA intermediates that can be converted to dicarboxylic acids through the termination pathways depicted.

[0046] FIG. 8. Synthesis of dicarboxylic acids and their alpha, beta functionalized derivatives through alcohol/omega-functionalization termination. Initial priming of a functional beta-oxidation reversal with acetyl-CoA (or propionyl-coA) and n elongation cycles generates CoA intermediates that can be converted to dicarboxylic acids through the termination pathways depicted.

[0047] FIG. 9. Dicarboxylic acid production through omega-carboxylated priming.

Adipic 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 (tdTER) components, along with activation enzyme for succinate to succinyl-CoA conversion (catl) and thioesterase termination (ACOT8). Data shown for pathway expression in strain background with deletion of fermentative product pathways, JC01(DE3, and the further deletion of numerous native thioesterases, JST06(DE3), cultivated for 48 hr using rich (LB) medium with glycerol as the carbon source and addition of 20 mM succinate.

[0048] FIG. 10. Dicarboxylic acid production through omega-carboxylated priming.

Pimelic 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 (TER from T. denticola, tdTER) components, along with activation enzyme for glutarate to glutaryl-CoA conversion (Catl) and thioesterase termination (ACOT8). Data shown for pathway expression with varying IPTG concentrations in strain background with deletion of fermentative product pathways and numerous native thioesterases, JST06(DE3), cultivated for 48 hr using rich (LB) medium with glycerol as the carbon source and addition of 20 mM glutarate.

[0049] FIG. 11. Dicarboxylic acid production through omega-carboxylated priming with multiple beta-oxidation reversal turns. Adipic acid (C6) and suberic acid (C8) production from succinyl-CoA priming with overexpression of genes encoding thiolase (DcaF), 3-hydroxyacyl-CoA dehydrogenase (DcaH), enoyl-CoA hydratase (DcaE), and trans- enoyl-CoA reductase (tdTER) components, along with activation enzyme for succinate to succinyl-CoA conversion (Catl) and thioesterase termination (ACOT8). Data shown for pathway expression in JST06(DE3) sdhB cultivated for 48 hr using rich (LB) medium with glycerol as the carbon source and addition of 20 mM succinate.

[0050] FIG. 12. co-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), enoyl-CoA hydratase (acPhaJ), and trans-enoyl-CoA reductase (tdTER) components, along with activation enzyme for glycolate to glycolyl-CoA conversion (mePCT). MG1655 (DE3) AglcD (pET-Pl-bktB-phaBl-P2-acPhaJ) (pCDF-Pl-mePCT-P2- tdTER) grown at 30 °C in LB media with 10 g/L Glucose and 40 mM Glycolate. The omega alcohol group of ω-hydroxyacids produced through this route can be further oxidized to an omega-carboxylic group to produce dicarboxylic acids.

[0051] FIG. 13. Synthesis of C6-C10 co-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. Note that egTEPv refers to the trans-2-enoyl-CoA reductase from Euglena gracilis. The omega alcohol group of co-hydroxyacids produced through this route can be further oxidized to an omega-carboxylic group to produce dicarboxylic acids (FIGS. 14 and 15).

[0052] FIG. 14. Production of C₆-Cio dicarboxylic acids through the omega-oxidation of carboxylic acids produced from a beta-oxidation reversal. C₆, C₈, and C₁₀ co-hydroxyacid (black bars) and dicarboxylic acid (white bars) production shown from 72 hr fermentations with JCOl (DE3) bktB^CT5 fadB^CT5 AfadA egTER^CT5 ydiI^M AtesB expressing AlkBGT using rich (LB) medium with glycerol as the carbon source with either FIG. 14A ChnD and ChnE (pKTOuQt-l-Pl-chnD-chnE-P2-alkBGT) or FIG. 14B YjgB and ChnE (pETDuet-l-Pl-jygff- chnE-?2-alkBGT). Glycerol consumption shown for each strain.

[0053] FIG. 15. C₆-Ci₀ dicarboxylic acid production with JCOl (DE3) bktB^CJ5 fadB^CT5 AfadA egTER^CT5 ydiI^M AtesB pETOuQt-l-Pl-chnD-chnE-P2-alkBGT) in minimal media. FIG. 15A. Cell growth (squares), glycerol consumption (circles), and total C₆-Cio dicarboxylic acids (diamonds) in shake flask fermentations run for various time points. FIG. 15B. Distribution of carboxylic acid, co-hydroxyacid, and dicarboxylic acid chain length after 96 hr fermentations. FIG. 15C. Total ion chromatogram of 96 hr sample showing GC-MS identified co-hydroxyacids and dicarboxylic acids. Positive identification of 8- hydroxyoctanoic acid (C₈-OH), 10-hydroxydecanoic acid (Cio-OH), adipic acid (C₆-DCA), suberic acid (C₈-DCA), and sebacic acid (C₁₀-DCA) confirmed through comparison of fragmentation patterns of peaks to that of analytical standards.

[0054] FIG. 16. Relevant genes for activation, priming, core/elongation, termination, and ω-oxidation modules of a functional reversal of the β-oxidation cycle for dicarboxylic acid synthesis (See FIG. 1 for pathway details).

[0055] FIG. 17. Genotypes of strains resulting in dicarboxylic acid synthesis from the use of co-carboxylated primers in combination with carboxylic acid forming termination pathways through a reversal of the β-oxidation cycle (See FIG. 16 for details/source of genes).

[0056] FIG. 18. Genotypes of strains resulting in dicarboxylic acid synthesis from the omega-oxidation of carboxylic acids generated from a functional reversal of the β- oxidation cycle (See FIG. 16 for details/source of genes).

DETAILED DESCRIPTION

[0057] Considering the two potential routes to omega-functionalized products discussed above and our knowledge base on the beta-oxidation reversal platform, the synthesis of dicarboxylic acids through both functionalized priming and omega-oxidation of carboxylic acids was investigated. With the selection of dicarboxylic as our defined target products, it is important to evaluate the potential for either route to these products. In order to determine the overall potential for this product class to be produced within the context of a functional beta-oxidation reversal, the theoretical and maximum yields were calculated with the use of glucose as the carbon and energy source (Table 1).

[0058] Table 1. Dicarboxylic theoretical and maximum yields from either omega- oxidation of carboxylic acids generated from a beta-oxidation reversal or using a beta- oxidation reversal with functionalized priming (See FIG. 1 for pathway details). Flux Balance Analysis and Flux Variability Analysis were used to identify the solution space for the synthesis of products of different chain lengths through the β-oxidation reversal and omega oxidation pathways (Metabolic Engineering 23, 100-115, 2014). "Maximum yield" refers to a non-growing culture satisfying constraints on redox balance and generation of ATP for maintenance while "yield" refers to the optimal solution where a coupling between cell growth and product synthesis is observed.

Dicarboxylic Acid Production Through Dicarboxylic Acids

o-Oxidation C6 CIO C14

Yield (w/w glucose) 0.49 0.39 0.36

Maximum Yield (w/w glucose) 0.54 0.45 0.41

Dicarboxylic acid Production Through Dicarboxylic Acids

Functionalized Priming C6 CIO C14

Yield (w/w glucose) 0.51 0.40 0.36

Maximum Yield (w/w glucose) 0.65 0.50 0.44 [0059] Significant production of dicarboxylic acids can theoretically be achieved through either potential route. The only case in which major differences in yields between the use of functionalized primers and the omega-functionalization of beta-oxidation intermediates/products were observed was for C6 products (for CIO and C 14 the differences are 10% or less).

[0060] It is also important to note that while these calculated yields are representative of the use of glucose as the sole carbon and energy source, the use of glycerol as the carbon and energy source would further increase the yield. However, this increase in product yield is significant only in the case of C6 products.

[0061] Considering the potential for target product synthesis through either route, the use functionalized priming or omega-functionalization of beta-oxidation intermediates/products for the production of dicarboxylic acids such as suberic acid (octanedioic acid) and sebacic acid (decanedioic acid) (C8 and CIO chain length dicarboxylic acids) was further explored. It is also important to note that to date, no production of these target products from "unrelated", single carbon sources has been reported, and thus their production through either route (functionalized primers or omega-functionalization) provides an opportunity to establish proof of concept.

DICARBOXYLIC ACIDS VIA OMEGA-CARBOXYLATED PRIMERS

[0062] The production of dicarboxylic 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 oxidation of carboxylic acids generated from a β-oxidation reversal at the omega carbon. For the former, 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.

[0063] Given the comprehensive knowledge base on the production of succinate from a number of industrially relevant carbon sources (Metabolic Engineering 7, 229-239, 2005; Metabolic Engineering 12, 409-419, 2010), succinate/succinyl-CoA represents an attractive potential primer that can result in dicarboxylic acids when combined with appropriate termination pathways (FIGS. 3 and 4). In order to determine the potential of succinate as a functionalized primer for the β-oxidation reversal, the identification and characterization of enzymes capable of functioning with omega-carboxylated intermediates was required. [0064] In the context of β-oxidation reversal capable of working on ω-carboxylated intermediates, the thiolase enzyme represents perhaps the most important enzyme as its selectivity for condensation of a functionalized primer with acetyl-CoA compared to the condensation of two acetyl-CoA molecules is a significant determining factor in the control of product synthesis.

[0065] For this step, the 3-ketoadipyl-CoA thiolase encoded by paaJ from E. coli is a promising candidate owning to its reported function in the phenylacetate catabolism pathway functioning with omega-carboxylated molecules (European Journal of Biochemistry 270, 3047-3054, 2003). In order to determine the ability for this enzyme to condense succinyl- CoA and acetyl-CoA, HPLC-MS analysis was conducted. For this, PaaH, the 3- hydroxyadipyl-CoA dehydrogenase from E. coli was also included, as the reduction of the 3- oxo-acyl-CoA with the consumption of NADH makes the overall reaction more thermodynamically favored.

[0066] Upon incubation of purified PaaJ and PaaH with succinyl-CoA, acetyl-CoA, and NADH, HPLC-MS analysis showed the appearance of a peak at an m/z ratio corresponding to 3-hydroxyadipyl-CoA (data not shown). These results indicate not only that these 2 enzymes could perform the thiolase and dehydrogenase modules with omega- carboxylated intermediates, but also underlie a potential thermodynamic limitation within the pathway, as a highly active dehydrogenase module may be required to ensure sufficient reduction of the 3-oxo-acyl intermediate and avoid thio lytic cleavage back to the 2 corresponding acyl-CoA intermediates.

[0067] With the establishment of functional enzymes for the thiolase and 3- hydroxyacyl-CoA dehydrogenase enzymes, the next steps for achieving a functional β- oxidation reversal with carboxylated priming required the identification of enzymes for the dehydratase and reductase modules capable of acting on ω-carboxylated intermediates. Given the identified function of PaaJ and PaaH with ω-carboxylated intermediates, a third member of the phenylacetate catabolism pathway, PaaF, the predicted 2,3-dehydroadipyl- CoA hydratase, was selected as the enoyl-CoA hydratase component.

[0068] These 3 enzymes, along with the Co A transferase Catl from Clostridium kluyveri (Journal of Bacteriology 178, 871-880, 1996) for the activation of succinate to succinyl-CoA, were then cloned into appropriate vectors with the trans-2-enoyl-CoA reductase from T. denticola {ΐάΊΈ ; FEBS Letters 581, 1561-1566, 2007). When this combination of enzymes was overexpressed in a strain devoid of native fermentation pathways ( ldhA, Δρία, ΔροχΒ, adhE, Δ/rdA), adipic acid production was observed from glycerol as the carbon source and addition of 20 mM succinate (data not shown).

[0069] While this indicates that endogenous termination pathways (thioesterases) are present for the conversion of adipyl-CoA to adipate, a more precise control of the termination pathway represents a potential area to improve target product synthesis. For this, the expression of the acyl-CoA thioesterase ACOT8 from Mus musculus, an enzyme shown to hydrolyze various dicarboxylyl-CoA esters including adipyl-CoA (Journal of Biological Chemistry 280, 38125-38132, 2005), was tested as a means of improving production. Furthermore, ACOT8 expression in combination with the deletion of endogenous thioesterases ( tesA, tesB, yciA, Δ/adM, ydil, and ybgC) was also evaluated to potentially provide more precise control of the available termination enzymes.

[0070] While the expression of ACOT8 with PaaJ, PaaH, PaaF, tdTER, and Catl resulted in slight increases to adipic acid production, a near 40% increase in production was observed when the overexpression of these enzymes was done in the JST06 background (Journal of Industrial Microbiology and Biotechnology 42, 465-475, 2015) lacking numerous endogenous thioesterases (FIG. 9). These results thus not only establish a functional set of enzymes for the production of dicarboxyic acids through a beta-oxidation reversal, but also demonstrate of the manipulation of termination pathways can have a profound impact on the level of product synthesis.

[0071] To provide further evidence for the potential of this route to dicarboxylic acids, the strain described above was tested with externally added glutarate. When this five carbon dicarboxylic acid was used in place of succinate with the above strain, JST06 (DE3) (pET-Pl-paaJ-paaH-P2-ACOT8) (pCDF-Pl-catl-paaF-P2-tdTER), the synthesis of pimelic acid was observed (FIG. 10). This seven carbon dicarboxylic acid product represents the final product of a one turn beta-oxidation reversal with glutaryl-CoA/acetyl-CoA condensation with a carboxylic acid forming termination pathway.

[0072] While the production of apidic and pimelic acids demonstrates the potential for a beta-oxidation reversal to function with omega-carboxylated intermediates six and seven carbons in length, the lack of longer chain dicarboxylic acids indicates that only a single turn beta-oxidation reversal was taking place. Given the role of PaaJ, PaaH, and PaaF in phenylacetate catabolism, the absence of products from a multiple turn beta-oxidation reversal could indicate the inability for these enzymes to function with longer chain length intermediates. As such, in order to demonstrate an important aspect of this pathway, i.e. the ability to produce omega-functionalized products of various chain lengths, potential other enzymes were explored.

[0073] Of the many thiolases available, the thiolase (DcaF), 3-hydroxyacyl-CoA dehydrogenase (DcaH), and enoyl-CoA hydratase (DcaE) from Acinetobacter baylyi are promising candidates due to their role in the beta-oxidation of dicarboxylic acids and the ability for A. baylyi to grow on dicarboxylic acids in the C6-C10 range (Applied and Environmental Microbiology 67, 4817-4827, 2001).

[0074] These 3 genes were cloned into appropriate vectors, along with catl, tdTER, and ACOT8 to produce pET-Pl-dcaF-dcaH-P2-ACOT8 and pCDF-Pl-catl-dcaE-P2-tdTER. The resulting vectors were utilized for pathway expression in JST06(DE3) sdhB, with the deletion in sdhB designed to maximize succinate/succinyl-CoA availability through the disruption of the succinate succinate :quinone oxidoreductase.

[0075] As seen in FIG. 11, the use of DcaF, DcaH, and DcaE enabled the synthesis of not only adipic acid, but also resulted in the production of suberic acid. This eight carbon dicarboxylic acid, represents the product of a 2-turn beta-oxidation reversal with succinyl- CoA priming, demonstrating the potential for this pathway to be exploited for the synthesis of longer chain length dicarboxylic acids. It should also be noted that even when PaaJ, PaaH, and PaaF were utilized with the additional pathway enzymes in JST06(DE3) sdhB, adipic acid was still the only longer chain dicarboxylic acid, indicating the importance of identifying/utilizing enzymes with various chain specificity in dictating product formation.

DICARBOXYLIC ACIDS VIA OMEGA-HYDROXYLATED PRIMERS

[0076] In addition to the synthesis of dicarboxylic acids through the combination of omega-carboxylated primers with carboxylic acid forming termination pathways (FIGS. 3 and 4), an additional approach for the synthesis of this class of product involves the use of omega-hydroxylated primers in combination with carboxylic acid or alcohol forming primary termination pathways, followed by further oxidation of the alcohol group(s) (FIGS. 5 and 6). Carboxylic acid forming termination pathways will result in omega-hydroxyacids as an intermediate in the pathway (FIG. 5), while the use of alcohol forming termination pathways enables the production of Ι,ω-diols with varying a and β functionalities (FIG. 6). For the latter, the terminal alcohol groups (i.e. alcohol groups at the 1 and ω positions) can in turn be oxidized to carboxylic acid groups resulting in dicarboxylic acids. For this approach, glycolate/glycolyl-CoA represents one potential primer that can be used in this approach when combined with appropriate termination pathways and, as with succinate, the possibility to generate glycolate internally (e.g. from glucose or glycerol) could allow production from a single carbon source.

[0077] A similar approach to that described above for succinate/succinyl-CoA was used for the identification and characterization of enzymes that can enable a functional beta- oxidation reversal with glycolate/glycolyl-CoA priming. This investigation established Ralstonia eutropha BktB, Ralstonia eutropha PhaBl, Aeromonas caviae PhaJ (acPhaJ) or Pseudomonas aeruginosa PhaJ4) (paPhaJ4), and T. denticola or Methylobacillus flagellates TER (taTTER or m TER) 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.

[0078] 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. 12).

[0079] The use of omega-hydroxylated primers in a beta-oxidation reversal such as that detailed above, results in the synthesis of products (omega-hydroxyacids or Ι,ω-diols depending on the termination pathway) which can be converted to dicarboxylic acids through the further oxidation of the alcohol group(s) (FIGS. 5 and 6). For this conversion, enzymes (alcohol and aldehyde dehydrogenases) with activity toward the alcohol group of the synthesized products are required, and are described in the next section.

DICARBOXYLIC ACIDS VIA OMEGA-OXIDATION

[0080] In order to further demonstrate synthesis of dicarboxylic acids, we also looked to exploit the other potential route to product formation through the use of omega-oxidation pathways to convert carboxylic acids or n-alcohols generated through a β-oxidation reversal to dicarboxylic acids (FIG. 7 and 8). The omega-functionalization of these products to dicarboxylic acids requires enzymes capable of adding a hydroxyl group to the aliphatic chain.

[0081] 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. Despite the challenges, 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). Furthermore, once ω-hydroxylation of carboxylic acid or n- alcohol takes place, further oxidation of this alcohol group(s) is required in order to produce a dicarboxylic acid (FIG. 7 and 8) becomes possible. Alcohol and aldehyde dehydrogenases represent potential enzymes that can be exploited for this oxidation of ω-hydroxyacids to dicarboxylic acids.

[0082] Our ability to investigate dicarboxylic acid 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.

[0083] 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. It was determined that the expression of the alkane monooxygenase system of Pseudomonas putida, encoded by alkBGT, enabled the synthesis of C6-C10 omega-hydroxyacids (FIG. 13), which can serve as a platform to demonstrate the potential for dicarboxylic acid synthesis.

[0084] The production of dicarboxylic acids through a combination of a β-oxidation reversal and ω-oxidation pathways requires additional enzymes (alcohol and aldehyde dehydrogenases) with activity toward the alcohol group of the synthesized ω-hydroxyacid. For this, a group of enzymes involved in the degradation of cyclic alcohols in certain microorganisms holds potential to function with the chain length co-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).

[0085] Oxidation activity on ω-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-C10 chain length substrates (Table 2). The high specific activities (greater than 10 μιηοΐ/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 to target chain length dicarboxylic acids.

[0086] Table 2. NAD(P)⁺-dependent co-hydroxyacid oxidation activity (μιηοΐ/mg protein/min) from various alcohol dehydrogenases ω-Hydroxyacid Enzyme³

Chain Length ChnD^b YahK^c YjgB^c

C₅ 5.0 ± 0.1 0.009 ± 0.001 0.050 ± 0.001

C₆ 35.4 ± 0.2 0.036 ± 0.001 0.29 ± 0.01

C₈ 26.4 ± 0.7 0.135 ± 0.003 1.79 ± 0.04

Cio 11.2 ± 0.4 0.194 ± 0.001 2.9 ± 0.1

a: 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 ^b: NAD⁺-dependent activity shown, no NADP⁺-dependent activity detected

c: NADP⁺-dependent activity shown, no NAD⁺-dependent activity detected

[0087] In addition to ChnD, potential native E. coli enzymes for the oxidation of co- 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 (Table 2), 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).

[0088] Given their in vitro characteristics, both ChnD and YjgB were selected for potential in vivo production of dicarboxylic acids. Due to the lack of commercially available substrates, in vitro characterization of potential enzymes for the second oxidation step (conversion of aldo-acids to dicarboxylic acids) was not yet investigated. However, the functionality of ChnD along with its previously reported function indicates that ChnE could provide the needed activity and was therefore selected as the aldehyde dehydrogenase component.

[0089] When chnD and chnE were co-expressed with alkBGT in JCOl (DE3) bktB^CT5 fadB AfadA egTER ydil AtesB, the production of adipic acid, suberic acid, and sebacic acid (C₆, C₈, and C₁₀ dicarboxylic acids, respectively) was observed with glycerol as the sole carbon source (FIG. 14A). The inclusion of ChnD and ChnE resulted in the complete conversion of all ω-hydroxyacids to dicarboxylic acids under these conditions (-400 mg/L of total C₆-Cio dicarboxylic acids after 72 hours). The inclusion of YjgB and ChnE with AlkBGT also resulted in the production of adipic, suberic, and sebacic acids, but in this case not all ω-hydroxyacids were converted to the corresponding dicarboxylic acids (FIG. 14B). This residual ω-hydroxyacid production appears to result from the lower specific activity of YjgB, compared to ChnD, for the target chain length ω-hydroxyacids (Table 2).

[0090] With the establishment of the set of required enzymes for the synthesis of omega-hydroxyacids through the omega-oxidation route, the further potential of this pathway was demonstrated through the production of omega-hydroxyacids in minimal media. Utilizing the same background strain (JCOl (DE3) bktB^CJ5 fadB^CJ5 AfadA egTER^CT5 ydiI^Ai AtesB) containing an appropriate vector for dicarboxylic acid production (pET-Pl-c/mD- chnE-?2-alkBGT), product synthesis profiles from shake flask fermentations were determined at various time points in minimal media with glycerol as the sole carbon and energy source. Under these conditions, when chnD and chnE were expressed in combination with alkBGT, the synthesis of nearly 500 mg/L total C₆-Cio dicarboxylic acids was observed (FIG. 15 A). This included 170 ± 7 mg/L adipic acid, 254 ± 13 mg/L suberic acid, and 61 ± 4 mg/L sebacic acid (FIG. 15B). The identity of these compounds was confirmed through GC- MS (FIG. 15C), using analytical standards for comparison of peak fragmentation patterns (data not shown).

[0091] The following references are incorporated by reference in their entirety for all purposes.

[0092] 61/440, 192, Reverse beta oxidation pathway, filed 2/7/2011.

[0093] WO2012109176, Reverse beta oxidation pathway, filed 2/7/2012.

[0094] 61,531,911, Synthesis Of Alpha- And Omega-Functionalized Carboxylic

Acids And Alcohols, filed Sept. 7, 2011

[0095] WO2013036812, Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation, filed Sept 7, 2012.

[0096] US20140273110, Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation, filed 3/6/2014.

[0097] Dellomonaco C. et al., Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals, Nature 476, 355-359, 2011.

[0098] 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.

[0099] 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.

[00100] 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.

[00101] What is claimed is:

Claims

1) 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, and succinyl-CoA; b) an overexpressed thiolase that catalyzes condensation of said omega-carboxylated acyl-CoA primer with acetyl-CoA to an omega-carboxylated B-ketoacyl-CoA; c) an overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-[acyl-carrier- protein] reductase that catalyzes reduction of said omega-carboxylated B-ketoacyl- CoA to produce an omega-carboxylated B-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 B-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 a thioesterase, an acyl-CoA:acetyl-CoA transferase, a phosphotransacylase and a carboxylate kinase catalyzing conversion of a product of steps b, c, d, or e to a dicarboxylic acid, ii) the group consisting of an alcohol-forming coenzyme-A thioester reductase, an alcohol oxidase/dehydrogenase, and an aldehyde dehydrogenase catalyzing conversion of a product of steps b, c, d, or e to a dicarboxylic acid, iii) or the group consisting of an aldehyde-forming CoA thioester reductase and an aldehyde dehydrogenase catalyzing conversion of a product of steps b, c, d, or e to a dicarboxylic acid; g) reduced expression of fermentation enzymes leading to reduced production of lactate, acetate, ethanol and succinate; and wherein said microorganism has a reverse beta oxidation pathway beginning with said omega-carboxylated acyl-CoA primer and running in a biosynthetic direction.

2) The genetically engineered microorganism of claim 1 , wherein said genetically

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

3) The microorganism 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 homo logs.

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), or homologs.

11) The microorganism of claim 1, 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.

12) The microorganism of claim 1, wherein said overexpressed aldehyde-forming Co A

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.

13) The microorganism of claim 1, 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.

14) The microorganism of claim 1, wherein said overexpressed aldehyde dehydrogenase is encoded by a gene selected from the group consisting of Rhodococcus ruber SC 1 cddD (AAL14238.1), Acinetobacter sp. SE19 chnE (AAG10022.1), and homologs.

15) The microorganism of any of claims 1-14, 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.

16) A genetically engineered microorganism comprising: a) one or more overexpressed enzymes that allow production of an omega-hydroxylated acyl-CoA 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 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: i) the group consisting of a thioesterase, an acyl-CoA:acetyl-CoA transferase, a phosphotransacylase and a carboxylate kinase catalyzing conversion of a product of steps b, c, d, or e to an omega-hydroxy carboxylic acid, ii) the group consisting of an alcohol-forming coenzyme-A thioester reductase catalyzing conversion of a product of steps b, c, d, or e to a 1, omega diol, iii) the group consisting of an aldehyde-forming CoA thioester reductase and an

alcohol dehydrogenase catalyzing conversion of a product of steps b, c, d, or e to a 1 , omega diol; g) an overexpressed alcohol oxidase/dehydrogenase and aldehyde dehydrogenase that catalyzes oxidation of a product of step f to a dicarboxylic 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-hydroxylated CoA thioester primer and running in a biosynthetic direction.

17) The genetically engineered microorganism of claim 16, wherein said genetically

engineered microorganism produces a product selected from the group consisting of dicarboxylic acids, β-hydroxy dicarboxylic acids, β-keto dicarboxylic acids, α,β- unsaturated dicarboxylic acids, Ι,ω-diols, β-hydroxy Ι,ω-diols, β-keto Ι,ω-diols, and α,β- unsaturated Ι,ω-diols.

18) The microorganism of claim 16, 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.

19) The microorganism of claim 16, 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. 20) The microorganism of claim 16, 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. coli fabZ (Ν?_4\4Ί 22 A), and homologs.

21) The microorganism of claim 16, 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 (ABX38717.1), and homologs.

22) The microorganism of claim 16, 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 ), 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.

23) The microorganism of claim 16, 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.

24) The microorganism of claim 16, 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.

25) The microorganism of claim 16, 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.

26) The microorganism of claim 16, 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.

27) The microorganism of claim 16, 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.

28) The microorganism of claim 16, 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.

29) The microorganism of claim 16, wherein said overexpressed aldehyde dehydrogenase is encoded by a gene selected from the group comprising Rhodococcus ruber SC 1 cddD (AAL14238.1), Acinetobacter sp. SE19 chnE (AAG10022.1), and homologs.

30) The microorganism of any of claims 16-29, 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.

31) 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 conversion of a product of steps a, b, c, or d 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 and aldehyde dehydrogenase that catalyzes oxidation of the omega-hydroxy carboxylic acid of step f to a dicarboxylic 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 acetyl- CoA and running in a biosynthetic direction.

32) The genetically engineered microorganism of claim 31 , wherein said genetically

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

33) The microorganism of claim 31 , 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. 34) The microorganism of claim 31, 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.

35) The microorganism of claim 31, 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.

36) The microorganism of claim 31, 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.

37) The microorganism of claim 31 , 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.

38) The microorganism of claim 31, 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. 39) The microorganism of claim 31 , 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.

40) The microorganism of claim 31 , 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.

41) The microorganism of claim 31, wherein said overexpressed omega hydroxylase is

selected from the group consisting of enzymes Pseudomonas putida AlkBGT

(YP 009076004.1, Q9WWW4.1, Q9L4M8.1), Marinobacter aquaeolei CYP153A (ABM17701.1), 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.

42) The microorganism of claim 31 , 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.

43) The microorganism of claim 31, wherein said overexpressed aldehyde dehydrogenase is encoded by a gene selected from the group consisting of Rhodococcus ruber SC 1 cddD (AAL14238.1), Acinetobacter sp. SE19 chnE (AAG10022.1), and homologs.

44) The microorganism of any of claims 31-43, 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.

45) 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) is selected from: i) the group consisting of an alcohol-forming coenzyme-A thioester reductase

catalyzing conversion of a product of steps a, b, c, or d to a 1 -alcohol, ii) the group consisting of an aldehyde-forming CoA thioester reductase and an

alcohol dehydrogenase catalyzing conversion of a product of steps a, b, c, or d to a 1 -alcohol, f) an overexpressed omega-hydroxylase that catalyzes omega-hydroxylation of the 1- alcohol of step e resulting in an 1 , omega-diol; g) an overexpressed alcohol oxidase/dehydrogenase and aldehyde dehydrogenase that catalyzes oxidation of an 1 , omega-diol of step f to a dicarboxylic 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 acetyl- CoA and running in a biosynthetic direction.

46) The genetically engineered microorganism of claim 45, wherein said genetically

engineered microorganism produces a product selected from the group consisting of dicarboxylic acids, β-hydroxy dicarboxylic acids, β-keto dicarboxylic acids, α,β- unsaturated dicarboxylic acids, Ι,ω-diols, β-hydroxy Ι,ω-diols, β-keto Ι,ω-diols, and α,β- unsaturated Ι,ω-diols.

47) The microorganism of claim 45, 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.

48) The microorganism of claim 45, 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.

49) The microorganism of claim 45, 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.

50) The microorganism of claim 45, 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.

51) The microorganism of claim 45, 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.

52) The microorganism of claim 45, 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.

53) The microorganism of claim 45, wherein said overexpressed alcohol forming alcohol dehydrogenase is encoded by a gene selected from the group consisting of E. coli betA (NP_414845.1), E. coli dkgA (NP_417485.4), E. coli eutG (NP_416948.4), E. colifucO (NP_417279.2), E. coli ucpA (NP_416921.4), E. coli yahK (NP_414859.1), E. coliybbO (NP_415026.1), E. coli ybdH (NP_415132.1), E. coliyia Y (YP_026233.1), E. coliyjgB (NP 418690.4), and homologs.

54) The microorganism of claim 45, wherein said overexpressed omega hydroxylase is

selected from the group consisting of enzymes Pseudomonas putida alkBGT

(YP 009076004.1, Q9WWW4.1, Q9L4M8.1), Marinobacter aquaeolei CYP153A (ΑΒΜΠ701 A),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.

55) The microorganism of claim 45, 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. coli yahK (NP_414859.1), E. coliyjgB (NP_418690.4), and homologs.

56) The microorganism of claim 45, wherein said overexpressed aldehyde dehydrogenase is encoded by a gene selected from the group consisting of Rhodococcus ruber SC 1 cddD (AAL14238.1), Acinetobacter sp. SE19 chnE (AAG10022.1), and homologs. 57) The microorganism of any of claims 45-56, 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.

58) The microorganisms of any of claims 1-57 comprising fadR, atoC(c), AarcA, Acrp, crp*.

59) A method of a producing a product selected from the group consisting of dicarboxylic acids, β-hydroxy dicarboxylic acids, β-keto dicarboxylic acids, α,β-unsaturated dicarboxylic acids, Ι,ω-diols, β-hydroxy Ι,ω-diols, β-keto Ι,ω-diols, and α,β-unsaturated Ι,ω-diols, comprising growing a genetically engineered microorganism according to any of claims 1-58 in a culture broth containing glycerol or a sugar, extending a Co A 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.