WO2015191422A1

WO2015191422A1 - Omega-hydroxylated carboxylic acids

Info

Publication number: WO2015191422A1
Application number: PCT/US2015/034629
Authority: WO
Inventors: Ramon Gonzalez; James M. CLOMBURG
Original assignee: William Marsh Rice University
Priority date: 2014-06-12
Filing date: 2015-06-08
Publication date: 2015-12-17
Also published as: WO2015191972A2; WO2015191972A3; WO2016007258A9; WO2016007258A1

Abstract

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

Description

OMEGA-HYDROXYLATED CARBOXYLIC ACIDS

PRIOR RELATED APPLICATIONS

[0001] This application claims priority to the following: U.S. App. Ser. No.

62/011,465, Omega-Hydroxylated Carboxylic Acids, Filed June 12, 2014; U.S. App. Ser. No. 62/012,113, Omega-aminated Carboxylic Acids, filed June 13, 2014; U.S. App. Ser. No. 62/011,474, Omega-carboxylated carboxylic acids, filed June 12, 2014; U.S. App. Ser. No. 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 U.S. App. Ser. No. 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 relates to biological synthesis of various chemicals through a reverse beta oxidation pathway.

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-hydroxylated 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 -hydroxy acyl- 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 (to) 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-hydroxy carboxylic acids are illustrated in FIGS. 3-5.

[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 made with a traditional primer (illustrated by the omega-oxidation 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 or primer 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 therefore 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). 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 and essentially modifies the beta oxidation intermediate(s) as they leave the cycle.

[0017] 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 hydroxy 1 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-hydroxyacids, or diols depending on the starting product and the extent of omega-oxidation). This approach for the synthesis of omega-hydroxy carboxylic acids is illustrated in FIG. 6 with termination from a beta-oxidation reversal leading to carboxylic acids 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, plus any of the algal species named above. 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, "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^".

[0025] 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.

[0026] 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 "+".

[0027] As used herein, all accession numbers are to GenBank or UniProt unless indicated otherwise.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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. [0033] 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.

[0034] 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.

[0035] 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.

[0036] The phrase "consisting of is closed, and excludes all additional elements.

[0037] 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.

DESCRIPTION OF FIGURES

[0038] 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 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 beta-oxidation reversal.

[0039] FIG. 2. Priming the β-oxidation reversal with functionalized primers. Omega- hydroxyacids can be produced through the condensation of acetyl-CoA with co-carboxylated CoA (A) or co-hydroxylated CoA (B) priming molecules and subsequent steps of a β- oxidation reversal and appropriate termination enzymes.

[0040] FIG. 3. Synthesis of omega-hydroxy carboxylic 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 omega- hydroxy carboxylic acids through the termination pathways depicted. [0041] FIG. 4. Synthesis of omega-hydroxy carboxylic 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 with n elongation cycles generates omega-functionalized CoA intermediates that can be converted to omega- hydroxy carboxylic acids through the termination pathways depicted. Note that the alpha and beta carbons are named according to the initial CoA intermediate (and not the final product) resulting in different products for the case of alpha, beta functionalized derivatives compared to those illustrated in Figure 3.

[0042] FIG. 5. Synthesis of omega-hydroxy carboxylic 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 omega- hydroxy carboxylic acids through the termination pathways depicted. Note that the alpha and beta carbons are named according to the initial CoA intermediate (and not the final product) resulting in different products for the case of alpha, beta functionalized derivatives depending on the termination pathway selected.

[0043] FIG. 6. Synthesis of omega-hydroxy carboxylic acids and their alpha, beta functionalized derivatives through 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 omega-hydroxy carboxylic acids through the termination pathways depicted.

[0044] FIG. 7. 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.

[0045] FIG. 8. 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 (tdTER) components, along with activation enzyme for succinate to succinyl-CoA conversion {catl). Data shown for JST06(DE3) strain expressing above enzymes with or without the additional overexpression of an acyl-CoA reductase termination pathway (cbjALD) and cultivated for 48 hr using rich (LB) medium with glycerol as the carbon source and addition of 20 mM succinate.

[0046] FIG. 9. 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 (tdTER) 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 (cbjALD) and cultivated for 48 hr using rich (LB) medium with glycerol as the carbon source and addition of 20 mM glutarate.

[0047] FIG. 10. Synthesis of 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^CJ5fadB^CJ5 AfadA egTER^CT5 ydiI^M AtesB expressing either alkBGT or CPR2 from pETDuet vector using rich (LB) medium with glycerol as the carbon source.

[0048] FIG. 1 1A-C. co-hydroxyacid production with JCOl (DE3) bktB^CT5 fadB^CT5

AfadA egTER^CT5 ydiI^M AtesB (pETDuet-l-Pl-P2-a/£5G7) in minimal media. A. Cell growth (squares), glycerol consumption (circles), and total C₆-Cio co-hydroxyacid (triangles) in shake flask fermentations run for various time points. B. Distribution of carboxylic acid and co- hydroxyacid chain length after 96 hr fermentations. C. Total ion chromatogram of 96 hr sample showing GC-MS identified co-hydroxyacids. Positive identification of 6- hydroxyhexanoic acid (C₆-OH), 8-hydroxyoctanoic acid (Cs-OH), and 10-hydroxydecanoic acid (Cio-OH) confirmed through comparison of fragmentation patterns of peaks to that of analytical standards.

[0049] FIG. 12. Relevant genes for activation, priming, core/elongation, termination, and ω-oxidation modules of a functional reversal of the β-oxidation cycle for co-hydroxyacid synthesis (See FIG. 1 for pathway details).

[0050] FIG. 13. Genotypes of strains resulting in co-hydroxyacid synthesis from the use of co-hydroxylated primers in combination with carboxylic acid forming termination pathways through a reversal of the β-oxidation cycle (See FIG. 12 for details/source of genes).

[0051] FIG. 14. Genotypes of strains resulting in co-hydroxyacid synthesis from the use of ω-carboxylated primers in combination with alcohol forming termination pathways through a reversal of the β-oxidation cycle (See FIG. 12 for details/source of genes).

[0052] FIG. 15: Genotypes of strains resulting in ω-hydroxyacid synthesis from the omega-oxidation of carboxylic acids generated from a functional reversal of the β-oxidation cycle (See FIG. 12 for details/source of genes).

DETAILED DESCRIPTION

[0053] Considering the two potential routes to omega-functionalized products discussed above and our knowledge base on the beta-oxidation reversal platform, the synthesis of co-hydroxylated carboxylic acids through both functionalized priming and omega-oxidation of carboxylic acids was investigated. With the selection of co-hydroxyacids 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).

[0054] Table 1. Omega-hydroxyacid 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.

Functionalized Priming C6 CIO C14

Yield (w/w glucose) 0.44 0.36 0.33

Maximum Yield (w/w glucose) 0.59 0.46 0.39

[0055] Significant production of co-hydroxyacids 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).

[0056] 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.

[0057] Considering the potential for target product synthesis through either route, the use of functionalized priming or omega-functionalization of beta-oxidation intermediates/products for the production of omega-hydroxyacids such as 6-hydroxyhexanoic acid, 7-hydroxyheptanoic acid, 8-hydroxyoctanoic acid, 9-hydroxynonanoic acid, and 10- hydroxydecanoic acid 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.

PRODUCTION OF OMEGA-HYDROXYACIDS THROUGH OMEGA- HYDROXYLATED PRIMERS

[0058] The production of ω-hydroxyacids from a functional reversal of the β- oxidation requires 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, 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. Glycolic acid/glycolyl-CoA represents one potential primer that can result in omega-hydroxyacids when combined with appropriate termination pathways (FIGS. 3 and 5), and the possibility to generate glycolate internally (e.g. from glucose or glycerol) could allow production from a single carbon source. In order to determine the potential of glycolate as a functionalize primer for the β-oxidation reversal, we first identified and characterized enzymes capable of converting glycolate acid to glycolyl-CoA.

[0059] For this, the propionyl-CoA transferase from Megasphaera elsdenii (mePCT) was selected due to its reported activity with a variety of hydroxylated short chain carboxylic acids (Journal or Bacteriology 124, 1462-1474, 1975) as well as for the conversion of glycolate to glycolyl-CoA (Journal of Biotechnology 156, 214-217, 2011; Nature Communications 4, 1414, 2013). mePCT utilizes acetyl-CoA as a donor for the transfer of CoA to glycolate resulting in the conversion of acetyl-CoA and glycolate to acetate and glycolyl-CoA. To confirm this reported activity, mePCT was purified and characterized through HPLC-MS analysis of reaction substrates/products. When incubated with glycolate and acetyl-CoA, mePCT resulted in the formation of glycolyl-CoA with an associated decrease in acetyl-CoA, confirming its ability to activate glycolate to glycolyl-CoA (data not shown).

[0060] With the establishment of mePCT as an enzyme for activation of the functionalized priming molecule, the identification of core/elongation modules of the β- oxidation reversal capable of working on ω-hydroxylated intermediates was then required. Of these core modules, the thiolase enzyme represents perhaps the most critical 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.

[0061] For this step, the 3-ketoacyl-CoA thiolase encoded by bktB from Ralstonia eutropha was a promising candidate owning to its reported ability to function with hydroxylated molecules (Nature Communications 4, 1414, 2013) as well as its potential for condensing longer chain acyl-CoA compounds (JACS 133, 11399-11401, 2011). For HPLC- MS analysis of the potential for bktB, the 3-hydroxybutyryl-CoA dehydrogenase PhaBl from Ralstonia eutropha was included, as the reduction of the 3-oxo-acyl-CoA with the consumption of NADH makes the overall reaction more thermodynamically favored. Upon inclusion of PhaB 1 with BktB and mePCT, in addition to acetyl-CoA, glycolate, and NADH, HPLC-MS analysis showed the appearance of a peak at an m/z ratio corresponding to 3,4- dihydroxybutyryl-CoA (data not shown). These results confirm not only that these 2 enzymes could perform the thiolase and dehydrogenase modules with omega-hydroxylated 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.

[0062] Based on the in vitro characterization of these enzymes, we next explored the expression of the activation (mePCT), thiolase {bktB), and dehydrogenase (phaBI) modules for the in vivo production of 3,4-dihydroxybutyrate with exogenous glycolate. This approach ensured that appropriate conditions could be determined enabling in vivo pathway functionality and allow the further testing of dehydratase and reductase modules. The latter was of great importance owing to the fact that substrates for testing potential enzymes for these steps with hydroxylated CoA intermediates were not commercially available.

[0063] For the in vivo testing of these enzymes, bktB, phaBI, and mePCT were cloned into vectors of the Novagen Duet system for expression in an E. coli K-12 MG1655 background harboring a λϋΕ3 Lysogen. Fermentations were then conducted with glucose and exogenous ly added glycolate. While 3,4-dihydroxybutyrate was not seen directly in fermentation supernatants, 0.295 g/L of β-hydroxy-Y-butyrolactone (HBL), its corresponding lactone, was observed upon expression of bktB, phaBI, and mePCT, confirming the ability for these enzymes to enable hydroxylated product synthesis in vivo (data not shown). Further improvement of HBL production to 0.34 g/L was achieved through the deletion of glcD, encoding glycolate oxidase, which converts glycolate to glyoxylate as part of the native pathway for glycolate utilization in E. coli.

[0064] Once in vivo hydroxylated product synthesis was confirmed, the next steps for achieving a functional β-oxidation reversal with hydroxylated priming required the identification of enzymes for the dehydratase and reductase modules capable of acting on ω- hydroxylated intermediates. This approach entailed cloning of numerous prospective enzymes for each step into the Duet vector framework for their expression with bktB, phaBI, and mePCT. For the dehydratase module, several R-specific dehydratases, required due to the stereospecificity of PhaBI, including P. aeruginosa phaJ4, P. aeruginosa phaJl, A. caviae phaJ, and E. coli paaZ were selected for testing.

[0065] Enzymes for potential use as the reductase module focused on NADH- dependent trans-2-enoyl-CoA reductases (TER), a class of enzymes that has been extensively studied in recent years for the reduction of the 2,3 double bond of various chain length enoyl- CoA molecules. TER enzymes from Idiomarina loihiensis, Cytophaga hutchinsonii, Methylobacillus flagellates, and Treponema denticola were selected and their associated genes cloned into the Duet vector framework for testing.

[0066] Various combinations of dehydratase and reductase enzymes were then tested in vivo through their expression along with bktB, phaBl, and mePCT in an MG1655 (DE3) Ag/cD background under the same conditions utilized for HBL production. While the potential product of a full one-turn β-oxidation reversal with glycolate priming, 4- hydroxybutyate (4-HB), is not commercially available and hence difficult to quantify, fermentation samples were analyzed via GC-MS to identify enzyme combinations that resulted in product formation.

[0067] After analysis, it was determined that the combination of either P. aeruginosa phaJ4 or A. caviae phaJ with the TER from either Methylobacillus flagellates or Treponema denticola resulted in 4-HB production in the mg/L range when expressed with bktB, phaBl, and mePCT. The total ion chromatogram showing GC-MS identified 4-hydroxybutyrate production from a one -turn beta-oxidation reversal with glycolyl-CoA priming with the use of mePCT, bktB, phaBl, phaJ from A. caviae , and TER from T denticola is shown in FIG. 7.

PRODUCTION OF OMEGA-HYDROXYACIDS THROUGH OMEGA- CARBOXYLATED PRIMERS

[0068] In addition to the synthesis of omega-hydroxyacids through the combination of omega-hydroxylated primers with carboxylic acid forming termination pathways (FIGS. 3 and 5), an additional approach for the synthesis of this class of product involves the use of omega-carboxylated primers in combination with alcohol forming termination pathways (FIG. 4). 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.

[0069] A similar approach to that described above for glycolate/glycolyl-CoA 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 (tdlL ; 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).

[0070] In order to utilize this approach for the synthesis of omega-hydroxy acids, 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 (cbjALD; 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, Δ/rdA) 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. As seen in FIG. 8, when this combination of beta- oxidation reversal and activiation enzymes (PaaJ, PaaH, PaaF, taTTER, 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.

[0071] To provide further evidence for the potential of this route to omega- hydroxyacids, 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-cbjALD) (pCDF-Pl-catl-paaF-P2-tdTER), the synthesis of 7- hydroxyheptanoic acid was observed (FIG. 9). 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. It should be noted that was 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. As such, 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.

PRODUCTION OF OMEGA-HYDROXYACIDS THROUGH OMEGA-OXIDATION

OF CARBOXYLIC ACIDS

[0072] In order to further demonstrate synthesis of ω-hydroxyacids, we also looked to exploit the other potential route to product formation through 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.

[0073] 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). These include the alkane hydroxylase system of P. putida (Microbiology 147, 1621-1630, 2001) as well as the soluble fusion protein CPR2, containing the monooxygenase CYP153A from Marinobacter aquaeolei and the reductase domain from Bacillus megaterium P450_BM3 (Microbial Biotechnology 6, 694-707, 2013). The alkane hydroxylase system of P. putida, encoded by alkBGT, is 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).

[0074] 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.

[0075] 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. 10, 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, while CPR2 expression enabled the synthesis of 10-hydroxydecanoic acid as the sole omega-hydroxyacid produced (FIG. 10).

[0076] 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 co-hydroxyacid production (pET-Pl-P2-a/ 5G7), 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, the expression of alkBGT resulted in the synthesis of more than 800 mg/L of C₆- Cio co-hydroxyacids, including 271 ± 20 mg/L 6-hydroxyhexanoic acid, 403 ± 24 mg/L 8- hydroxyoctanoic acid, and 150 ± 8 mg/L 10-hydroxydecanoic acid after 96 hours (Fig. 11). The production of these compounds was verified via GC-MS (Fig. 11), with comparison of fragmentation patterns of peaks to that of analytical standards confirming their identity (data not shown).

[0077] The following references are incorporated by reference in their entirety for all purposes.

[0078] 61/440,192, Reverse beta oxidation pathway, filed 2/7/2011.

[0079] WO2012109176, Reverse beta oxidation pathway, filed 2/7/2012.

[0080] 62/011,465, Omega-Hydroxylated Carboxylic Acids, Filed June 12, 2014. [0081] 61,531,911, Synthesis Of Alpha- And Omega-Functionalized Carboxylic

Acids And Alcohols, filed Sept. 7, 2011

[0082] WO2013036812, Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation, filed Sept 7, 2012.

[0083] US20140273110, Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation, filed 3/6/2014.

[0084] Dellomonaco C. et al., Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals, Nature 476, 355-359, 2011.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] What is claimed is:

Claims

A genetically engineered microorganism comprising: a) one or more overexpressed enzymes that allow the production of an omega- hydroxylated CoA thioester primer selected from hydroxyacetyl-CoA, 3- hydroxypropionyl-CoA, or 4-hydroxybutyryl-CoA; b) an overexpressed thiolase that catalyzes the condensation of an omega-hydroxylated acyl-CoA primer with 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 the 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 the 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 the reduction of said omega- hydroxylated trans-enoyl-CoA to an omega-hydroxylated acyl-CoA; f) an overexpressed termination enzyme(s) able to act on an omega-hydroxylated CoA- thioester substrate of steps b, c, d, or e, wherein said termination pathway is selected from: i) the group consisting of a thioesterase, an acyl-CoA:acetyl-CoA transferase, a phosphotransacylase and a carboxylate kinase catalyzing the conversion of the CoA moiety of the reaction products of steps b, c, d, or e above to a carboxylic acid group, ii) the group consisting of an alcohol-forming coenzyme-A thioester reductase, an alcohol oxidase/dehydrogenase, and an aldehyde dehydrogenase catalyzing the conversion of the CoA moiety of the reaction products of steps b, c, d, or e above to a carboxylic acid group through hydroxyl and aldehyde intermediates, iii) or the group consisting of an aldehyde-forming CoA thioester reductase and an aldehyde dehydrogenase catalyzing the conversion of the CoA moiety of the reaction products of steps b, c, d, or e above to a carboxylic acid group through an aldehyde intermediate; 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-hydroxylated CoA thioester 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 omega-hydroxylated carboxylic acids, β-, omega-dihydroxy carboxylic acids, β-keto, omega-hydroxy carboxylic acids, and α,β-unsaturated omega-hydroxylated carboxylic acids.

3) The microorganism of claim 1, wherein said overexpressed thiolase is encoded by a gene from the group consisting ofE. coli atoB (NP_416728.1), E. coli yqeF (NP_417321.2), E. colifadA (YP_026272.1), E. coli fadl (NP_4\6844 A), 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 homologues.

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 homologues.

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 homologues. 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 egTER (Q5EU90.1), Treponema denticola tdTER (NP_971211.1), Methylobacillus flagellatus m TER

(Q1H0P3), E. colifabl (NP_415804.1), Enterococcus faecalis fabK (NP_816503.1), Bacillus subtilis fabL (KFK80655.1), Vibrio cholerae ^α¾Γ(ΑΒΧ38717.1), and homologues.

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 homologues.

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 homologues.

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 homologues.

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 homologues.

11) The microorganism of claim 1, wherein said overexpressed alcohol-forming coenzyme -A thioester reductase is selected from the group consisting of the gene or enzyme 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 homologues.

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 homologues.

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 homologues.

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 homologues.

15) The microorganism 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 the production of an omega- carboxylated CoA thioester primer selected from oxalyl-CoA, malonyl-CoA, or succinyl-CoA; b) an overexpressed thiolase that catalyzes the condensation of an 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 the 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 the 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 the reduction of said omega- carboxylated trans-enoyl-CoA to an omega-carboxylated acyl-CoA; f) an overexpressed termination enzyme(s) able to act on said omega-carboxylated CoA- thioester substrate of steps b, c, d, or e, wherein said termination pathway is selected from: i) the group consisting of an alcohol-forming coenzyme-A thioester reductase

catalyzing the conversion of the CoA moiety of the reaction products of steps b, c, d, or e above to a hydroxy 1 group, ii) the group consisting of an aldehyde-forming CoA thioester reductase and an

alcohol dehydrogenase catalyzing the conversion of the CoA moiety of the reaction products of steps b, c, d, or e above to a hydroxyl group through an aldehyde intermediate, 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-carboxylated CoA thioester primer and running in a biosynthetic direction.

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

18) The microorganism of claim 16, wherein said overexpressed thiolase is encoded by a gene 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 homologues. 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

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. colifabZ (NP_414722.1), and homologues.

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 egTER (Q5EU90.1), Treponema denticola tdTER (NP_971211.1), Methylobacillus flagellatus m TER

22) The microorganism of claim 16, wherein said overexpressed alcohol-forming coenzyme - A thioester reductase is selected from the group consisting the gene or enzyme 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 homologues.

23) The microorganism of claim 16, wherein said overexpressed aldehyde-forming CoA thioester reductase is encoded by a gene selected from the group consisting of

24) The microorganism of claim 16, wherein said overexpressed alcohol dehydrogenase is encoded by a gene selected from the group consisting of E. coli bet A (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. coliybdH (NP_415132.1), E. coli yia Y (YP_026233.1), E. coliyjgB (NP_418690.4), and homologues.

25) The microorganism of claims 16-24, 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.

26) A genetically engineered microorganism comprising: a) an overexpressed thiolase that catalyzes the 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 the 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 the 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 the reduction of said trans-enoyl-CoA to an acyl-CoA; e) an overexpressed termination enzyme(s) able to act on a CoA-thioester substrate or its beta-functionalized derivative, wherein said termination pathway is selected from the group consisting of a thioesterase, or an acyl-CoA:acetyl-CoA transferase, or a phosphotransacylase and a carboxylate kinase catalyzing the conversion of the CoA moiety of the reaction products of steps b, c, d, or e above to a carboxylic acid group; f) an overexpressed omega-hydroxylase that catalyzes the omega-hydroxylation of the reaction product of step e resulting in a omega-hydroxy 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 acetyl- CoA and running in a biosynthetic direction.

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

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

28) The microorganism of claim 26, 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 homologues.

29) The microorganism of claim 26, wherein said overexpressed 3-hydroxyacyl-CoA

30) The microorganism of claims claim 26, 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. coli fadJ (N? 16843.1), 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. coli paaF (P76082), E. colifabA (NP_415474.1), E. colifabZ (NP_414722.1), and homologues. 31) The microorganism of claim 26, 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 egTER (Q5EU90.1), Treponema denticola tdTER (NP_971211.1), Methylobacillus flagellatus m TER

32) The microorganism of claim 26, 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

33) The microorganism of claim 26, 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) or E. coliydiF (NP_416209.1), and homologues.

34) The microorganism of claim 26, 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 homologues.

35) The microorganism of claim 26, 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 homologues.

36) The microorganism of claim 26, wherein said overexpressed carboxylic acid omega

hydroxylase is encoded by a gene(s) selected from the group consisting of Pseudomonas putida alkBGT (YP 009076004.1, Q9WWW4.1, Q9L4M8.1), Marinobacter aquaeolei CYP153A (ABM 17701.1 ^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 homologues.

37) The microorganism of claims 26-36, wherein said reduced expression of fermentation enzymes are AadhE, (Apia or AackA or AackApta), ApoxB, AldhA, and AfrdA and less acetate, lactate, ethanol and succinate are thereby produced.

38) A method of a producing a product selected from the group consisting of omega- hydroxylated carboxylic acids, β-, omega-dihydroxy carboxylic acids, β-keto, omega- hydroxy carboxylic acids, and α,β-unsaturated omega-hydroxylated carboxylic acids comprising growing a genetically engineered microorganism according to any of claims 1-37 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.