WO2016176339A1 - Synthesis of omega-phenyl products and derivatives thereof - Google Patents

Abstract

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

Description

SYNTHESIS OF OMEGA-PHENYL PRODUCTS AND DERIVATIVES THEREOF

PRIOR RELATED APPLICATIONS

[0001] This application claims priority to US Serial No. 62/154,010, filed April 28,

2015 and incorporated by reference herein in its entirety for all purposes.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] This invention was made with government support under Grant Nos. EEC-

0813570, CBET-1134541, and CBET-1067565 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

[0003] This disclosure generally relates to the use of microorganisms to make omega- phenyl products, including omega-phenyl carboxylic acids, alcohols, amines, hydrocarbons, methyl ketones, and their beta-functionalized derivatives. This is achieved by utilizing an iterative carbon chain elongation pathway that uses omega-phenyl CoA thioester primers.

BACKGROUND OF THE DISCLOSURE

[0004] Reactions that catalyze the iterative formation of carbon-carbon bonds are instrumental for many metabolic pathways, such as the biosynthesis of fatty acids, polyketides, and many other molecules with applications ranging from biofuels and green chemicals to therapeutic agents. These pathways typically start with small precursor metabolites that serve as building blocks, which are subsequently condensed and modified in an iterative fashion until the desired chain length and functionality are achieved.

[0005] Most iterative carbon-carbon bond forming reactions in nature take place through a Claisen condensation mechanism in which the nucleophilic a-anion of an acyl- thioester, serving as the extender unit, attacks the electrophilic carbonyl carbon of another acyl-thioester, serving as the primer. Depending on how the nucleophilic a-anion is generated, the Claisen condensation reaction can be classified as decarboxylative or non- decarboxylative.

[0006] Many natural iterative carbon chain elongation pathways, like fatty acid and polyketide biosynthesis pathways, utilize decarboxylative Claisen condensation reactions with malonyl thioesters as extender units. Products synthesized through these pathways include fatty acids, alcohols, polyketides, esters, alkanes and alkenes with diverse chain lengths, structures and functionalities. This diversity is achieved by using functionalized primers, a-functionalized malonyl thioesters as extender units, and an array of pathways for termination of carbon chain elongation and subsequent product modification. [0007] Despite the structural and functional diversity of these products, the use of malonyl thioester as a C2 extender unit requires the ATP-dependent activation of acetyl-CoA to malonyl-CoA, which in turn limits the energy efficiency of these pathways. Furthermore, owing to the decarboxylative nature of the condensation reaction, the β site of the extender units of the decarboxylative Claisen condensation must be a carboxylic group, restricting the range of extender units and potentially limiting the diversity of products that can be generated through these carbon chain elongation pathways.

[0008] In order to overcome the inherently low ATP efficiency of the aforementioned pathways, we have recently implemented a novel approach by driving beta-oxidation in reverse to make carboxylic/fatty acids (and other products) instead of degrading them (see US20130316413, WO2013036812, each incorporated by reference in its entirety for all purposes).

[0009] Unlike the fatty acid biosynthesis pathway, the reversal of the β-oxidation cycle operates with coenzyme-A (CoA) thioester intermediates and uses acetyl-CoA directly for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl- CoA). In the reversal of the β-oxidation cycle, thiolases catalyze a non-decarboxylative Claisen condensation in which acetyl-CoA, instead of malonyl thioesters, serves as the extender unit, and subsequent β-reduction reactions by hydroxyacyl-CoA dehydrogenases (HACDs), enoyl-CoA hydratases (ECHs) and enoyl-CoA reductases (ECRs) enable iteration. [0010] Compared to pathways utilizing decarboxylative Claisen condensation, thiolase-based pathways like the beta-oxidation reversal are more energy efficient due to less ATP consumption for the supply of extender unit acetyl-CoA than with malonyl thioesters. However, these thiolases only utilize acetyl-CoA or propionyl-CoA as the primer units, thus limiting the functionality of synthesized products. A novel non-decarboxylative Claisen condensation reaction able to accept wider range of primer units and proceed in an iterative manner is required to diversify the product range of carbon-chain elongation.

[0011] What is needed in the art is a novel biological pathway for synthesizing omega functionalized products, in particular omega-phenyl products. SUMMARY OF THE DISCLOSURE

[0012] This disclosure generally relates to the use of microorganisms to make omega- phenyl products, including omega-phenyl carboxylic acids, alcohols, amines, hydrocarbons, methyl ketones and their beta-functionalized derivatives. This is achieved by utilizing an iterative carbon chain elongation pathway that uses omega-phenyl CoA thioester primers. [0013] A general CoA-dependent carbon elongation platform based on the use of thiolase-catalyzed non-decarboxylative Claisen condensation, which accepts omega-phenyl acyl-CoA primer units is described. Together with suitable hydroxyacyl-CoA dehydrogenases (HACDs, also referred to as 3-ketoacyl-CoA reductases), enoyl-CoA hydratases (ECHs, also referred to as 3-hydroxyacyl-CoA dehydratases) and enoyl-CoA reductases (ECRs) (FIG. 1), an iterative cycle is developed that grows an omega-phenyl CoA thioester primer by two carbons with each turn of the cycle.

[0014] In more detail, the pathway includes native or engineered thiolases to catalyze the condensation of omega-phenyl CoA thioester primers with acetyl-CoA as the donor or extender unit to generate omega-phenyl β-keto acyl-CoA. Dehydrogenase converts omega- phenyl β-keto acyl-CoA to omega-phenyl β-hydroxy acyl-CoA. Dehydratase converts omega-phenyl β-hydroxy acyl-CoA to omega-phenyl enoyl-CoA. Reductase converts omega- phenyl enoyl-CoA to omega-phenyl acyl-CoA. The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting omega-phenyl acyl-CoA as primer and acetyl-CoA as extender unit in subsequent turns of the cycle. Termination pathways acting on any of the four omega-phenyl CoA thioester intermediates terminate the platform enabling product synthesis. A wide-range of omega-phenyl product families (e.g. omega-phenyl products, including omega-phenyl carboxylic acids, alcohols, amines, hydrocarbons, methyl ketones and their beta-functionalized derivatives) can be obtained through this iterative platform.

[0015] The process involves performing traditional fermentations using industrial organisms (E. coli, S. cerevisiae, B. subtillis, and the like) that convert different feedstocks into desired products (e.g. omega-phenyl-functionalized carboxylic acids, alcohols, amines, hydrocarbons, and their beta-functionalized derivatives). These organisms are considered workhorses of modern biotechnology. Media preparation, sterilization, inoculum preparation, and fermentation are the main steps of the process.

[0016] As used herein, a "primer" is a starting molecule for the iterative cycle to add two carbon donor units to a growing acyl-CoA thioester. The initial primer is typically either acetyl-CoA or propionyl-CoA, but as the chain grows by adding donor units in each cycle, the primer will accordingly increase in size. In some cases, the bacteria can also be provided with larger primers, e.g., C4 primers, etc. added to the media or obtained from other cellular pathways. In this invention, non-traditional primers are used in which the terminal omega carbon has been functionalized (i.e., omega-phenyl). The omega-phenyl initiating primers are either provided to the cell in the media, or made in the cell by the addition of appropriate enzymes, or combinations thereof (e.g., adding a phenyl alkanoic acid substrate that can be converted to a -CoA form in the cell).

[0017] As used herein, the "donor" of the 2 carbon units is acetyl-CoA.

[0018] As used herein, the "omega" position is the last carbon in a straight chain, wherein position 1 is determined by the -CoA activator. We do not change the nomenclature even after the -CoA is removed, and the nomenclature might otherwise change. Thus, an "omega-phenyl" group refers to a phenyl on the last carbon in the straight chain, as referenced as the end opposite from where the -CoA is or was.

[0019] As used herein, a reverse beta-oxidation (BOX-R) pathway uses -CoA activated intermediates to grow a carbon chain by two units per turn of the cycle. The cycle can include beta-oxidation enzymes, or type II fatty acid synthesis enzyme (FAS II) enzymes that act on -CoA activated intermediates (in addition to the usual ACP-carried intermediates).

[0020] As used herein "type II fatty acid synthesis enzymes" refer to those enzymes that function independently, e.g., are discrete, monofunctional enzymes, used in fatty acid synthesis. Type II enzymes are found in archaea and bacteria. Type I systems, in contrast, utilize a single large, multifunctional polypeptide.

[0021] Thiolases are ubiquitous enzymes that have key roles in many vital biochemical pathways, including the beta-oxidation pathway of fatty acid degradation and various biosynthetic pathways. Members of the thiolase family can be divided into two broad categories: degradative thiolases (EC 2.3.1.16), and biosynthetic thiolases (EC 2.3.1.9). The forward and reverse reactions are shown below:

[0022] These two different types of thiolase are found both in eukaryotes and in prokaryotes: acetoacetyl-CoA thiolase (EC:2.3.1.9) and 3-ketoacyl-CoA thiolase (EC:2.3.1.16). 3-ketoacyl-CoA thiolase (also called thiolase I) has a broad, chain-length specificity for its substrates and is involved in degradative pathways such as fatty acid beta- oxidation. Acetoacetyl-CoA thiolase (also called thiolase II) is specific for the thiolysis of acetoacetyl-CoA and involved in biosynthetic pathways such as poly beta-hydroxybutyric acid synthesis or steroid biogenesis. [0023] Furthermore, the degradative thiolases can be made to run in the forward direction by building up the level of left hand side reactants (primer and donor), thus driving the equilibrium in the forward direction and/or by overexpressing same or by expressing a mutant of same.

[0024] As used herein, a "thiolase able to use omega-phenyl-functionalized primer" is an enzyme that catalyzes the condensation of an omega-phenyl acyl-CoA thioester with acetyl-CoA as the 2-carbon donor for chain elongation to produce an omega-phenyl β- ketoacyl-CoA (ω -phenyl is omega-phenyl; β-ketoacyl is beta-ketoacyl) in a non- decarboxylative condensation reaction:

[0025] As used herein a "hydroxyacyl-CoA dehydrogenase" or "HACD", is an enzyme that catalyzes the reduction of an omega-phenyl β-ketoacyl-CoA to an omega-phenyl β-hydroxyacyl-CoA:

[0026] As used herein, "enoyl-CoA hydratase" or "ECH" is an enzyme that catalyzes the dehydration of an omega-phenyl β-hydroxyacyl-CoA to an omega-phenyl enoyl-CoA:

[0027] As used herein, an "enoyl-CoA reductase" or "ECR" is an enzyme that catalyzes the reduction of an omega-phenyl enoyl-CoA to an omega-phenyl acyl-CoA:

[0028] As used herein, "termination pathway" refers to one or more enzymes (or genes encoding same) that will pull reaction CoA thioester intermediates out the iterative cycle and produce the desired end product.

[0029] By "termination pathway" what is meant is a CoA thioester intermediate from the iterative cycle is pulled out of the iterative cycle by one (which can have more than one activity) or more termination enzymes and results in i) carboxylic acids, ii) primary alcohols, iii) hydrocarbons, iv) primary amines, or v) derivatives thereof from CoA thioesters intermediates as described in FIG. 1.

[0030] Many examples of termination pathways are provided herein and the following

Table 1 provides several examples:

[0031] Many microbes do not make significant amounts of free fatty acids (FFAs), but can be made to do so by adding a gene coding for an acyl-ACP thioesterase (called a "TE" gene herein), which are promiscuous enzymes that also work on -CoA activated intermediates, as well as ACP-carried intermediates in many cases. It is also known to change the chain length of the FFAs by changing the TE: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl- ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and, 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs.

[0032] For example, most thioesterases exhibit the highest specificities in the C16-C18 range, including thaliana FatA (18: 1 Δ9), Madhuca longifolia FatB (16:0, 16: 1, 18:0, 18: 1), Coriandrum sativum FatA (18: 1Δ9), A. thaliana FatB (16:0, 18: 1, 18:0, 16: 1), Helianthus annuus FatA (18: 1, 16: 1), and Brassica juncea FatB2 (16:0, 18:0), among numerous others. Medium-chain acyl-ACP thioesterases include Cuphea palustris FatBl and C. hookeriana FatB2 (8:0, 10:0), C palustris FatB2 (14:0, 16:0); and Umbellularia californica FatB (12:0, 12: 1, 14:0, 14: 1). Arecaceae (palm family) and Cuphea accumulate large quantities of fatty acids that are shorter (between 8 and 12 carbon atoms), and several enzymes are also available in bacteria. Exemplary thioesterase families and common names of their members are shown in Table 2. Thousands of such sequences are available.

[0033] As used herein, the expressions "microorganism," "microbe," "strain" "cell" and the like may be used interchangeably and all such designations include their progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0034] As used herein, reference to a "cell," "microbe," etc. is generally understood to include a culture of such cells, as the work described herein is done in cultures having 10^9"15 cells.

[0035] As used herein, "growing" cells used its art accepted manner, referring to exponential growth of a culture of cells, not the few cells that may not have completed their cell cycle at stationary phase or have not yet died in the death phase or after harvesting.

[0036] As used in the claims, "homolog" means an enzyme with at least 40% identity to one of the listed sequences and also having the same general catalytic activity, although of course Km, Kcat, and the like can vary. While higher identity (60%, 70%, 80%) and the like may be preferred, it is typical for bacterial sequences to diverge significantly (40-60%), yet still be identifiable as homologs, while mammalian species tend to diverge less (80-90%).

[0037] Reference to proteins herein can be understood to include reference to the gene encoding such protein. Thus, a claimed "permease" protein can include the related gene encoding that permease. However, it is preferred herein to refer to the protein by standard name per ecoliwiki or HUGO since both enzymatic and gene names have varied widely, especially in the prokaryotic arts.

[0038] Once an exemplary protein is obtained, many additional examples of proteins with similar activity can be identified by BLAST search. Further, every protein record is linked to a gene record, making it easy to design overexpression vectors. Many of the needed enzymes are already available in vectors, and can often be obtained from cell depositories or from the researchers who cloned them. But, if necessary, new clones can be prepared based from the researchers who cloned them. But, if necessary, new clones can be prepared based on available sequence information using RT-PCR techniques. Thus, it should be easily possible to obtain all of the needed enzymes/genes for overexpression.

[0039] Another way of finding suitable enzymes/genes for use in the invention is to consider other enzymes with the same EC number, since these numbers are assigned based on the reactions performed by a given enzyme. An enzyme that thus be obtained, e.g., from AddGene or from the author of the work describing that enzyme, and tested for functionality as described herein. In addition, many sites provide lists of proteins that all catalyze the same reaction. [0040] Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides that encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species. Using such databases, a gene or cDNA may be "optimized" for expression in E. coli, yeast, algal or other species using the codon bias for the species in which the gene will be expressed.

[0041] Initial cloning experiments have proceeded in E. coli for convenience since most of the required genes are already available in plasmids suitable for bacterial expression, but the addition of genes to bacteria is of nearly universal applicability. Indeed, since recombinant methods were invented in the 70' s and are now so commonplace, 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, Streptococcus, Paracoccus, Methanosarcina, and Methylococcus, 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.

[0042] Additionally, yeasts, such as Saccharomyces, are a common species used for microbial manufacturing, and many species can be successfully transformed. Indeed, yeasts are already available that express recombinant thioesterases— one of the termination enzymes described herein— and the reverse beta-oxidation pathway has also been achieved in yeast. Other species include but are not limited to Candida, Aspergillus, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha (Pichia angusta), Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica, to name a few. [0043] 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.

[0044] Furthermore, a number of databases include vector information and/or a repository of vectors and can be used to choose vectors suitable for the chosen host species. 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.

[0045] The enzymes can be added to the genome or via expression vectors, as desired.

Preferably, multiple enzymes are expressed in one vector or multiple enzymes can be combined into one operon by adding the needed signals between coding regions. Further improvements can be had by overexpressing one or more, or even all of the enzymes, e.g., by adding extra copies to the cell via plasmid or other vector. Initial experiments may employ expression plasmids hosting 3 or more ORFs for convenience, but it may be preferred to insert operons or individual genes into the genome for long-term stability.

[0046] Still further improvements in yield can be had by reducing competing pathways, such as those pathways for making e.g., acetate, formate, ethanol, and lactate, and it is already well known in the art how to reduce or knockout these pathways. See e.g., the Rice patent portfolio by Ka-Yiu San and George Bennett (US7569380, US7262046, US8962272, US8795991) and patents by these inventors (US8129157 and US8691552) (each incorporated by reference herein in its entirety for all purposes). Many others have worked in this area as well.

[0047] In calculating "% identity" the unaligned terminal portions of the query sequence are not included in the calculation. The identity is calculated over the entire length of the reference sequence, thus short local alignments with a query sequence are not relevant (e.g., % identity = number of aligned residues in the query sequence/length of reference sequence). Alignments are performed using BLAST homology alignment as described by Tatusova TA & Madden TL (1999) FEMS Microbiol. Lett. 174:247-250, and available through the NCBI website. The default parameters were used, except the filters were turned OFF.

[0048] "Operably associated" or "operably linked", as used herein, refer to functionally coupled nucleic acid or amino acid sequences.

[0049] "Recombinant" is relating to, derived from, or containing genetically engineered material. In other words, the genetics of an organism was intentionally manipulated by the hand of man in some way.

[0050] "Reduced activity" is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species (e.g., the wild type gene in the same host 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. All reduced activity genes or proteins are signified herein by

[0051] By "null" or "knockout" what is meant is that the mutation produces undetectable active protein. 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 null mutants herein are signified by "Δ."

[0052] "Overexpression" or "overexpressed" is defined herein to be at least 150% of protein activity as compared with an appropriate control species, or any detectable expression in a species that lacks the activity altogether. Preferably, the activity is increased 100-500%) or even ten-fold. 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. All overexpressed genes or proteins are signified herein by "+".

[0053] In certain species it is possible to genetically engineer the endogenous protein to be overexpressed by changing the regulatory sequences or removing repressors. However, overexpressing the gene by inclusion on selectable plasmids or other vectors that exist in hundreds of copies in the cell may be preferred due to its simplicity and ease of exerting externals controls, although permanent modifications to the genome may be preferred in the long term for stability reasons.

[0054] The term "endogenous" or "native" means that a gene originated from the species in question, without regard to subspecies or strain, although that gene may be naturally or intentionally mutated, or placed under the control of a promoter that results in overexpression or controlled expression of said gene. Thus, genes from Clostridia would not be endogenous to Escherichia, but a plasmid expressing a gene from E. coli or would be considered to be endogenous to any genus of Escherichia, even though it may now be overexpressed.

[0055] "Expression vectors" are used in accordance with the art accepted definition of a plasmid, virus or other propagatable sequence designed for protein expression in cells. There are thousands of such vectors commercially available, and typically each has an origin of replication (ori); a multiple cloning site; a selectable marker; ribosome binding sites; a promoter and often enhancers; and the needed termination sequences. Most expression vectors are inducible, although constitutive expression vectors also exist. [0056] As used herein, "inducible" means that gene expression can be controlled by the hand of man by adding e.g., a ligand to induce expression from an inducible promoter. Exemplary inducible promoters include the lac operon inducible by IPTG, the yeast AOX1 promoter inducible with methanol, the strong LAC4 promoter inducible with lactate, and the like. Low level of constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters.

[0057] As used herein, an "integrated sequence" means the sequence has been integrated into the host genome, as opposed to being maintained on an expression vector. It will still be expressible, and preferably is inducible as well. [0058] 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.

[0059] 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. [0060] 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.

[0061] 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.

[0062] The phrase "consisting of is closed, and excludes all additional elements. [0063] 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 invention, and the like.

[0064] The following abbreviations are used herein:

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1A-B: Platform for the synthesis of omega-phenyl carboxylic acids, alcohols, amines, hydrocarbons, and methyl ketones. The platform is composed of thiolase, dehydrogenase, dehydratase and reductase enzymes. Thiolase(s) catalyze the condensation between omega-phenyl acyl-CoA thioester primer and extender unit acetyl-CoA and generates omega-phenyl β-ketoacyl-CoA. Dehydrogenase converts omega-phenyl β-keto acyl-CoA to omega-phenyl β-hydroxy acyl-CoA. Dehydratase converts omega-phenyl β-hydroxy acyl-CoA to omega-phenyl enoyl-CoA. Reductase converts omega-phenyl enoyl-CoA to omega-phenyl acyl-CoA, which can then function as primer+2 for the next turn of the cycle.

[0066] Termination pathways starting from four omega-phenyl CoA thioester intermediates terminate the platform and generate various omega-phenyl carboxylic acids, alcohols, hydrocarbons, and amines with different β-reduction degrees. There are four types of termination pathways: 1) thioesterase/ CoA-transf erase/ phosphotransacylase+kinase which generates carboxylic acids; 2) alcohol-forming acyl-CoA reductase or aldehyde-forming acyl- CoA reductase and alcohol dehydrogenase which generates alcohols; 3) aldehyde-forming acyl- CoA reductase and aldehyde decarbonylase which generates hydrocarbons; and 4) aldehyde- forming acyl-CoA reductase and transaminase which generates amines. Secondary termination pathways are also possible: modifying any of the above products further. For example, omega- phenyl methyl ketone can be generated by subsequent decarboxylation of omega-phenyl β-keto acid. Omega-phenyl acyl-CoA thioester primers be generated from their acid form, which can be either supplemented in the media or derived from other carbon sources or directly synthesized through additional pathways, such as those outlined in FIG. 2A-B. n means length of primers, intermediates and products. Dashed line means multiple reaction steps or iteration.

[0067] FIG. 2A-B: Example pathways for the generation of omega-phenyl acyl-CoA thioester primers benzoyl-CoA, phenylacetyl-CoA and phenylpropionyl-CoA from carbon sources such as glucose or glycerol via chorismate, the intermediate of biosynthesis of aromatic amino acids phenylalanine and tryptophan.

[0068] FIG. 3A-B: Example pathway of synthesis of 4-phenylbutyric acid and 6- phenylhexanoic acid through the proposed platform depicted in FIG. 1A-B with phenylacetyl- CoA as the primer and acetyl-CoA as the extender unit. Phenylacetyl-CoA is activated by E. coli enzyme PaaK from phenylacetic acid. The platform is composed of thiolase FadA from Pseudomonas putida, which catalyzes the condensation between primer phenylacetyl-CoA and extender unit acetyl-CoA to 4-phenylacet ogenase and reductase FadB from P. putida, which catalyzes the conversion of 4-phenylacetoacetyl-CoA to 4-phenyl-3- hydroxybutyryl-CoA and the subsequent dehydration of 4-phenyl-3-hydroxybutyryl-CoA to 4- phenylcrotonyl-CoA; reductase Fabl from E. coli or Ter from Treponema denticola (tdTER), which reduces 4-phenylcrotonyl-CoA to 4-phenylbutyryl-CoA. Termination by an acid forming reaction, such as those catalyzed by thioesterases, can convert the intermediate of one-turn of the pathway, 4-phenylbutyryl-CoA, to the product 4-phenylbutyric acid. Pathway iteration using the generated 4-phenylbutyryl-CoA as a primer with similar thiolase, dehydrogenase, dehydratase and reductase steps results in 6-phenylhexonyl-CoA, which can be converted to 6- phenylhexanoic acid through acid forming termination pathways.

[0069] FIG. 4: Titers of 4-phenylbutyric acid synthesized through the platform depicted in FIG. 3A-B with overexpression of different reductases Fabl and TdTer catalyzing the reductase step. JC01(DE3), an E. coli strain deficient of mixed-acid fermentations, served as the host strain. The strain also overexpressed PaaK, FadA and FadB. The engineered strains were grown for 48 hours under 37°C in 20 mL LB-like MOPS media supplemented with 20 g/L glycerol and 5 mM phenylacetic acid and induced with 5 μΜ IPTG.

[0070] FIG. 5: Titers of 4-phenylbutyric acid and 6-phenylhexanoic acid synthesized through the platform depicted in FIG. 3A-B. JC01(DE3), an E. coli strain deficient of mixed- acid fermentations, served as the host strain containing vectors for the IPTG inducible expression of PaaK, FadA, FadB, and Fabl. Strains were grown for 48 hours under 30°C in 20 mL LB-like MOPS media supplemented with 20 g/L glycerol and 5 mM phenylacetic acid and induced with 10 μΜ IPTG.

[0071] FIG. 6: Maps of vectors overexpressing required enzymes for the production of even chain omega-phenyl products, such as 4-phenylbutyric acid and 6-phenylhexanoic acid, through the proposed platform depicted in FIG. 3A-B with phenylacetyl-CoA as the primer.

[0072] FIG. 7A-B: Example pathway of synthesis of 5-phenylpentanoic acid through the proposed platform depicted in FIG. 1A-B with phenylpropionyl-CoA as the primer and acetyl-

CoA as the extender unit. Phenylpropionyl-CoA is activated by Penicillium chrysogenum enzyme Phi from phenylpropionic acid. The platform is composed of thiolase FadA from

Pseudomonas putida, which catalyzes the condensation between primer phenylpropionyl-CoA and extender unit acetyl-CoA to 5-phenyl-3-oxopentanoyl-CoA; dehydrogenase and reductase

FadB from P. putida, which catalyzes the conversion of 5-phenyl-3-oxopentanoyl-CoA to 5- phenyl-3-hydroxypentanoyl-CoA and the subsequent dehydration of 5-phenyl-3- hydroxypentanoyl-CoA to 5-phenyl-2-pentenoyl-CoA; reductase Fabl from E. coli or TdTer, which reduces 5-phenyl-2-pentenoyl-Co yl-CoA. Termination by an acid forming reaction, such as those catalyzed by thioesterases, converts 5-phenylpentanoyl-CoA to the product 5-phenylpentanoic acid.

[0073] FIG. 8: Maps of vectors overexpressing enzymes for the production of odd chain omega-phenyl products, such as 5-phenylpentanoic acid, through the proposed platform depicted in FIG. 7A-B with phenylpropionyl-CoA as the primer.

[0074] FIG. 9: A partial listing of preferred embodiments, and one or more of which can be combined with any other one or more.

DETAILED DESCRIPTION

[0075] This disclosure generally relates to the use of microorganisms to make omega- phenyl products, including omega-phenyl carboxylic acids, alcohols, amines, hydrocarbons, methyl ketones and their beta-functionalized derivatives.

[0076] The engineered pathway starts with thiolase-catalyzed condensation reactions that accept omega-phenyl CoA thioesters as primers (FIG. 1). Native or engineered thiolases catalyze the condensation of omega-phenyl CoA thioester primers with acetyl-CoA as the extender unit to generate omega-phenyl β-keto acyl-CoA. Subsequent β-reduction reactions

catalyzed by hydroxyacyl-CoA dehydrogenases (HACDHs), enoyl-CoA hydratases (ECHs) and enoyl-CoA reductases (ECRs) create an iterative elongation cycle in which the dehydrogenase converts omega-phenyl β-keto acyl-CoA to omega-phenyl β-hydroxy acyl-CoA, the dehydratase converts omega-phenyl β-hydroxy acyl-CoA to omega-phenyl enoyl-CoA, and the reductase converts omega-phenyl enoyl-CoA to omega-phenyl acyl-CoA (FIG. 1).

[0077] The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting omega-phenyl acyl-CoA as primer (now two carbons longer) and acetyl-CoA as extender unit in subsequent turns of the cycle. Termination pathways acting on any of the four omega-phenyl CoA thioester intermediates terminate the platform and generate various omega- phenyl carboxylic acids, alcohols, hydrocarbons, and amines with different degrees of β- reduction (FIG. 1 and Table 1).

[0078] Many examples of thiolase enzymes that can potentially catalyze the non- decarboxylative condensation of an acyl-CoA primer and acetyl-CoA extender unit are provided herein and Table 3 provides several additional examples, which can also serve as templates for engineered variants.

[0079] The ubiquitous nature of β-oxidation provides numerous enzymes for each of the

3 β-reduction steps that convert the omega-phenyl β-keto acyl-CoA generated from the thiolase reaction into an omega-phenyl acyl-CoA. Furthermore, the ability of enzymes from the type II fatty acid biosynthesis pathway to function with CoA intermediates as substrates (PCT/US15/12932) provides another large set of potential enzymes that can be exploited for this purpose. Many examples of enzymes for each of the 3 β-reduction steps are provided herein and Table 3 provides several additional examples:

[0080] In addition to the required thiolase and β-reduction enzymes, additional enzymes enabling the generation of the omega-phenyl acyl-CoA can be exploited to ensure sufficient primer generation. Key enzyme(s) for primer generation include activation enzymes such as acyl-CoA synthetases or CoA transferases, which catalyze the conversion of endogenous or exogenous phenylalkanoic acids to the corresponding omega-phenyl acyl-CoA thioester primer. Many examples of these enzymes are provided herein and Table 4 provides several additional examples:

[0081] Generation of the required omega-phenyl acyl-CoA thioester primer can make use of externally supplied phenylalkanoic acids or -CoA form thereof or can be accomplished from a carbon source such as glycerol or sugars through the pathways depicted in FIG. 2. Exploiting components of pathways for the biosynthesis of aromatic amino acids phenylalanine and tryptophan, the generation of omega-phenyl acyl-CoA thioester primers benzoyl-CoA, phenyl acetyl -CoA and phenylpropionyl-CoA can be accomplished via

[0082] Combining the core engineered pathway with enzymes/pathways for the generation of the initial omega-phenyl acyl-CoA thioester primer provides a route for the

generation of omega-phenyl acyl-CoA intermediates with varying beta-functionality (FIG. 1). These intermediates can then be converted to numerous products of interest through action of various termination pathways. For example, the use of acid forming termination pathways, such as thioesterases, enables the synthesis of omega-phenyl carboxylic acids, while alcohol forming termination pathways, such as acyl-CoA reductases/alcohol dehydrogenases, provides a route to various omega-phenyl alcohols (FIG. 1). The combinatorial expression of core pathway components with termination pathways, such as those in Table 1, facilitates the synthesis of omega-phenyl products, including omega-phenyl carboxylic acids, alcohols, hydrocarbons, amines, methyl ketones and their beta- functionalized derivatives.

[0083] The following description provides additional details, any one of which can be subject to patenting in combination with any other. The specification in its entirety is to be treated as providing a variety of details that can be used interchangeably with other details, as the specification would be of inordinate length if one were to list every possible combination of genes/vectors/enzymes/hosts that can be made to enable carbon source conversion into omega-phenyl products.

[0084] Initial demonstration of the engineered pathway was conducted in E. coli for convenience, and focused on the synthesis of omega-phenyl carboxylic acids. Enzymes of interest where expressed from vectors such as pETDuet-1 or pCDFDuet-1 (MERCK, Germany), which makes use of the DE3 expression system. Genes can be codon optimized according to the codon usage frequencies of the host organism and synthesized by a commercial vendor or in-house. However, thousands of expression vectors and hosts are available, and this is a matter of convenience. The vectors used in initial demonstration of the engineered pathway are shown in FIG. 6 and FIG. 8.

[0085] Pathway enzymes can also be inserted into the host chromosome, allowing for the maintenance of the pathway without requiring antibiotics to ensure the continued upkeep of plasmids. A large number of genes that can be placed on the chromosome, as chromosomal expression does not require separate origins of replication as is the case with plasmid expression. [0086] Engineered strains expressing pathway components can be cultured under the following or similar conditions. Overnight cultures started from a single colony can be used to inoculate flasks containing appropriate media. Cultures are grown for a set period of time, and the culture media analyzed. The conditions will be highly dependent on the specifications of the actual pathway and what exactly is to be tested. For example, the ability for the pathway to be used for omega-phenyl product synthesis can be tested by the glycerol or sugars as a substrate in MOPS minimal media, as described by Neidhardt et al (1974), supplemented with appropriate antibiotics and inducers.

[0087] The minimal medium designed by Neidhardt et al. (1974), with 125 mM MOPS and Na₂HP0₄ in place of K₂HP0₄, supplemented with 20 g/L glycerol, 10 g/L tryptone, 5 g/L yeast extract, 100 μΜ FeS0₄, 5 mM calcium pantothenate, 1.48 mM Na₂HP0₄, 5 mM (NH₄)₂S0₄, and 30 mM NH₄C1 was used for the below described fermentations. Phenylacetate or phenylpropionate was added to a concentration of 5 mM when appropriate. Fermentations were conducted in 25 mL Pyrex Erlenmeyer flasks (Corning Inc., Corning, NY) filled with 20 mL of the above culture medium and sealed with foam plugs filling the necks. A single colony of the desired strain was cultivated overnight (14-16 hrs) in LB medium with appropriate antibiotics and used as the inoculum (1%). After inoculation, flasks were incubated at 37°C or 30°C and 200 rpm in an NBS Benchtop Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, NJ) until an optical density of -0.3-0.5 was reached, at which point IPTG (5 μΜ unless otherwise stated) was added when appropriate. Flasks were then incubated under the same conditions for 48 hrs post-induction.

[0088] The aromatic primer phenyl acetyl -Co A, with acetyl-CoA as the extender unit, was used to achieve pathway operation and demonstrate the synthesis of phenylalkanoic acids. Pseudomonas putida thiolase FadA, P. putida FadB (providing both HACDH and ECH activities) was tested with either E. coli Fabl or T. denticola TER as the ECR, and E. coli acyl-CoA synthetase PaaK to activate externally supplied phenylacetic acid. Overexpression of either combination of enzymes in mixed-acid fermentation-deficient E. coli MG1655 AldhA ApoxB Apia AadhE AfrdA (JCOl), enabled the synthesis of 4- phenylbutyric acid (FIG. 4). This product resulted from the action of endogenous termination pathways, possibly native thioesterases, on phenylbutyryl-CoA generated by one turn of the pathway. In addition to demonstrating overall pathway functionality, the use of either T. denticola TER or E. coli Fabl with FadA and FadB for 4-phenylbutyric acid synthesis also demonstrates how both β-oxidation enzymes and fatty acid biosynthesis enzymes acting on the required CoA intermediates can be used in this context.

[0089] Iterative pathway operation (e.g. the use of the omega-phenyl acyl-CoA generated from a turn of the pathway as a primer for the next round) was also demonstrated through the use of P. putida thiolase FadA, P. putida FadB (providing HACDH and ECH activities), E. coli Fabl (ECR), and E. coli acyl-CoA synthetase PaaK in the JCOl strain background. Varying induction levels by altering IPTG concentration (10 μΜ) as well as incubation at 30°C, resulted in the synthesis of 6-phenylhexanoic acid in addition to higher levels of 4-phenylbutyric acid, compared to the above results with the same set of enzymes (FIG. 5). This demonstrates the ability to synthesize omega-phenyl products of various chain length through the iterative addition of 2 carbon units (via acetyl-CoA as the donor) to the growing omega-phenyl acyl-CoA primer.

[0090] Combination of iterative pathway operation using any of benzoyl-CoA (FIG.

2), phenylacetyl-CoA (FIG. 2 and FIG. 3) and phenylpropionyl-CoA (FIG. 2 and FIG. 7) as the initial primer with various termination pathways (FIG. 1 and Table 1) enables the engineered pathway to synthesize various omega-phenyl carboxylic acids, alcohols, hydrocarbons, and amines with different degrees of β-reduction and carbon chain length as described herein.

[0091] The above experiments are repeated in Bacillus subtilis. The same genes can be used, especially since Bacillus has no significant codon bias. A protease-deficient strain like WB800N is preferably used for greater stability of heterologous protein. The E. coli - B. subtilis shuttle vector pMTLBS72 exhibiting full structural stability can be used to move the genes easily to a more suitable vector for Bacillus. Alternatively, two vectors pHTOl and pHT43 allow high-level expression of recombinant proteins within the cytoplasm. As yet another alternative, plasmids using the theta-mode of replication such as those derived from the natural plasmids ρΑΜβΙ and pBS72 can be used. Several other suitable expression systems are available. Since the FAS and BOX-R enzymes are ubiquitous, the invention is predicted to function in Bacillus. [0092] The above experiments are repeated in yeast. The same genes can be used, but it may be preferred to accommodate codon bias. Several yeast E. co\\ shuttle vectors are available for ease of the experiments. Since the FAS and BOX enzymes are ubiquitous, the invention is predicted to function in yeast, especially since yeast are already available with exogenous functional TE genes and the reverse beta oxidation pathway has also been made to run in yeast.

[0093] The following are incorporated by reference herein in its entirety for all purposes:

[0094] US20130316413 Reverse beta oxidation pathway [0095] 62/140,628 BIOCONVERSION OF SHORT-CHAIN HYDROCARBONS

TO FUELS AND CHEMICALS, March 31, 2015

[0096] PCT/US 15/12932 TYPE II FATTY ACID SYNTHESIS ENZYMES IN

REVERSE BETA-OXIDATION, January 26, 2015 and 61/932,057, January 27, 2014.

[0097] 62/069,850 SYNTHETIC PATHWAY FOR BIOSYNTHESIS FROM 1- CARBON COMPOUNDS, October 29, 2014

[0098] WO2013036812 Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation

[0099] All accession numbers (generally in brackets after a gene or protein) are expressly incorporated by reference for all purposes herein. Inclusion of the information at each accession entry, would render the patent of inordinate length, and thus, incorporation of all sequences (and other information found therein) by reference is preferred. A person of ordinary skill in the art will recognize the accession numbers and be able to access them from a variety of databases.

[00100] Choi, K.-H, Heath, R.J. & Rock, CO. β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis. J. Bacteriol. 182, 365-370 (2000). [00101] Clomburg, J.M., Vick, J.E., Blankschien, M.D., Rodriguez-Moya, M. &

Gonzalez, R. A Synthetic Biology Approach to Engineer a Functional Reversal of the β- Oxidation Cycle. ACS Synthetic Biology 1, 541-554 (2012).

[00102] Dellomonaco, C, Clomburg, J.M., Miller, E.N. & Gonzalez, R. Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355- 359 (2011).

[00103] Heath, R.J. & Rock, CO. The Claisen condensation in biology. Nat. Prod. Rep. 19, 581-596 (2002).

[00104] Haapalainen, A.M., et al., The thiolase superfamily: condensing enzymes with diverse reaction specificities. Trends in Biochemical Sciences 31, 64-71 (2006).

[00105] Jiang, C, et al., Divergent evolution of the thiolase superfamily and chalcone synthase family. Molecular Phylogenetics and Evolution 49, 691-701 (2008).

[00106] Neidhardt F.C., Bloch P.L & Smith D.F. Culture Medium for Enter obacteria.

J Bacterid 119(3), 736-747 (1974). [00107] Pfleger, B.F., et al., Metabolic engineering strategies for microbial synthesis of oleochemicals. Metab. Eng. 29, 1-11 (2015).

[00108] Lian J. & Zhao H., Reversal of the β-oxidation cycle in Saccharomyces cerevisiae for production of fuels and chemicals., ACS Synth Biol. 4(3):332-41 (2015).

[00109] Vick, J.E. et al. Escherichia coli enoyl-acyl carrier protein reductase (Fabl) supports efficient operation of a functional reversal of the β-oxidation cycle. Appl. Environ. Microbiol. 81, 1406-1416 (2015).

Claims

A genetically engineered microorganism comprising: a) one or more overexpressed activation enzyme(s) able to produce an omega-phenyl acyl-CoA thioester primer, wherein said activation enzyme(s) is selected from: i) an acyl-CoA synthase, an acyl-CoA transferase, or a phosphotransacylase and a carboxylate kinase which catalyze the conversion of an exogenously added omega-phenyl acid to a omega-phenyl acyl-CoA thioester primer; ii) one or more enzymes as depicted in FIG. 2 that allows the production of said omega-phenyl acyl-CoA thioester primer from a carbon source such as glycerol or sugars without the exogenous addition of said omega-phenyl acid; b) an overexpressed thiolase that catalyzes the condensation of said omega-phenyl acyl- CoA thioester primer and acetyl-CoA to form an omega-phenyl B-ketoacyl-CoA; c) an overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-[acyl-carrier- protein] reductase that catalyzes the reduction of said omega-phenyl B-ketoacyl-CoA to produce an omega-phenyl B-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-phenyl β-hydroxyacyl-CoA to an omega-phenyl 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-phenyl trans-enoyl-CoA to an omega-phenyl acyl-CoA; f) iterations of steps b to e, wherein said iteration is achieved by utilizing an omega- phenyl acyl-CoA-thioester product generated in step e of the last turn as the primer unit of step b in the next turn of iteration; g) an overexpressed termination enzyme(s) able to act on said omega-phenyl thioester intermediates of steps b, c, d, or e, wherein said termination pathway is selected from: i) the group consisting of a thioesterase, or an acyl-CoA transferase, or a

phosphotransacylase and a carboxylate kinase catalyzing the conversion of omega-phenyl thioester intermediates of steps b, c, d, or e to a carboxylic acid; ii) an alcohol-forming acyl-CoA reductase catalyzing the conversion of omega- phenyl thioester intermediates of steps b, c, d, or e to an alcohol; iii) an aldehyde-forming acyl-CoA reductase catalyzing the conversion of omega- phenyl thioester intermediates of steps b, c, d, or e to an aldehyde and an alcohol dehydrogenase catalyzing the conversion of said aldehyde to an alcohol; iv) an aldehyde-forming acyl-CoA reductase catalyzing the conversion of omega- phenyl thioester intermediates of steps b, c, d, or e to an aldehyde and an aldehyde decarbonylase catalyzing the conversion of said aldehyde to an alkane; v) an aldehyde-forming acyl-CoA reductase catalyzing the conversion of omega- phenyl thioester intermediates of steps b, c, d, or e to an aldehyde and a transaminase catalyzing the conversion of said aldehyde to an amine; and, h) optionally reduced expressions 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-phenyl acyl-CoA thioester primer and running in the biosynthetic direction.

The microorganism of claim 1, wherein said an omega-phenyl CoA thioester primer is an acyl CoA thioester whose omega group is a phenyl group.

The microorganism of claim 1, wherein said omega-phenyl acid is the acid form of omega-phenyl acyl-CoA thioester primer whose omega group is a phenyl group.

The microorganism of claim 1, wherein said omega-phenyl acid is supplemented in the media or generated intracellularly from a given carbon source such as sugars or glycerol.

The genetically engineered microorganism of claim 1, wherein said genetically engineered microorganism produces a product selected from the group consisting of 3- keto acids, 3-keto alcohols, 3-keto amines, 3-hydroxy acids, 1,3-diols, 3-hydroxy amines, A²-fatty acids, A²-fatty alcohols, A²-amines, fatty acids, alcohols, alkanes, alkene, and amines whose omega group is a phenyl group.

6) The genetically engineered microorganism of claim 1, wherein said termination pathway acts on omega phenyl β-ketoacyl-CoA-thioester products generated in step b forming an omega-phenyl β-keto-acid, further comprising an overexpressed β-keto acid

decarboxylase catalyzing the conversion of said omega-phenyl β-keto-acid to an omega- phenyl methyl ketone.

7) The genetically engineered microorganism of claim 6, wherein said genetically

engineered microorganism produces an omega-phenyl methyl ketone.

8) The microorganism of claim 1, wherein said overexpressed acyl-CoA synthase is encoded by a gene(s) selected from the group consisting of E. coli sucC, E. coli sucD, E. coli paaK, E. coliprpE, E. coli menE, E. colifadK, E. colifadD, Penicillium chrysogenum phi, Salmonella typhimurium LT2 prpE, Bacillus subtilis bioW, Cupriavidus basilensis hmfD, Rhodopseudomonas palustris badA, R. palustris hbaA, Pseudomonas aeruginosa PAOl pqsA, Arabidopsis thaliana 4cl, or homologues.

9) The microorganism of claim 1, wherein said overexpressed acyl-CoA transferase is

encoded by a gene(s) selected from the group consisting of E. coli atoD, E. coli scpC, E. coli ydiF, E. coli atoA, E. coli atoD, Clostridium acetobutylicum ctfA, C. acetobutylicum ctfB, Clostridium kluyveri cat2, C kluyveri catl, P. putida peal, P. putida pcaJ,

Megasphaera elsdenii pet, Acidaminococcus fermentans gctA, Acidaminococcus fermentans gctB, Acetobacter aceti aarC, or homologues.

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

encoded by a gene(s) selected from the group consisting of Clostridium acetobutylicum ptb, Enterococcus faecalis ptb, Salmonella enterica pduL, or homologues.

11) The microorganism of claim 1, wherein said overexpressed carboxylate kinase is encoded by a gene(s) selected from the group consisting of Clostridium acetobutylicum buk, Enterococcus faecalis buk, Salmonella enterica pduW, or homologues. 12) The microorganism of claim 1, wherein said overexpressed thiolase is encoded by a gene(s) selected from the group consisting of E. coli atoB, E. coli yqeF, E. colifadA, E. colifadl, Ralstonia eutropha bktB, Pseudomonas sp. B13 catF, E colipaaJ, Rhodococcus opacus pcaF, Pseudomonas putida pcaF, Streptomyces sp. pcaF, P. putida fadAx, P. putida fadA, Ralstonia eutropha phaA, Acinetobacter sp. ADP1 dcaF, Clostridium acetobutylicum thlA, Clostridium acetobutylicum MB, or homologues.

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

dehydrogenase or 3-oxoacyl-[acyl-carrier-protein] reductase is encoded by a gene(s) selected from the group consisting of E. colifabG, E. colifadB, E. colifadJ, E. coli paaH, P. putida fadB, P. putida fadB2x, Acinetobacter sp. ADP1 dcaH, Ralstonia eutrophus phaB, Clostridium acetobutylicum hbd, or homologues.

14) The microorganism of claim 1, wherein said overexpressed enoyl-CoA hydratase, 3- hydroxyacyl-CoA dehydratase, or 3-hydroxyacyl-[acyl-carrier-protein] dehydratase is encoded by a gene(s) selected from the group consisting of E. colifabA, E. colifabZ, E. colifadB, E. colifadJ, E. colipaaF, P. putida fadB, P. putida fadBlx, Acinetobacter sp.

ADP1 dcaE, Clostridium acetobutylicum crt, Aeromonas caviae phaJ, or homologues.

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

16) The microorganism of claim 1, wherein said overexpressed thioesterase is encoded by a gene(s) selected from the group consisting of E. coli tesA, E. coli tesB, E. coli yciA, E. colifadM, E. coliydil, E. coliybgC, E. coli paal, Mus musculus acot8, Alcanivorax borkumensis tesB2, Fibrobacter succinogenes Fs2108, Prevotella ruminicola Pr655,

Prevotella ruminicola Prl687, Lycopersicon hirsutum f glabratum mks2, or homologues.

17) The microorganism of claim 1, wherein said overexpressed aldehyde-forming acyl-CoA reductase is encoded by a gene(s) selected from the group consisting Acinetobacter calcoaceticus acrl, Acinetobacter sp Strain M-l acrM, Clostridium beijerinckii aid, E. coli eutE, Salmonella enter ica eutE, Marinobacter aquaeolei VT8 maqu 2507, E. coli mhpF, Clostridium kluyveri sucD, or homologues.

18) The microorganism of claims 1, wherein said overexpressed alcohol dehydrogenase is encoded by a gene(s) selected from the group consisting E. coli betA, E. coli dkgA, E. coli eutG, E. colifucO, E. coli ucpA, E. coliyahK, E. coliybbO, E. coliybdH, E. coliyiaY, E. coliyjgB, Saccharomyces cerevisiae ADH6, Marinobacter aquaeolei VT8 maqu 2507, Clostridium kluyveri 4hbD, Acinetobacter sp. SE19 chnD, or homologues.

19) The microorganism of claim 1, wherein said overexpressed aldehyde decarbonylase is encoded by a gene(s) selected from the group consisting of Synechococcus elongatus PCC7942 orfl593, Nostoc punctiforme PCC73102 npun Rl 711, Prochlorococcus marinus MIT9313 pmtl231, or homologues.

20) The microorganism of claim 1, wherein said overexpressed transaminase is encoded by a gene(s) selected from the group consisting of Arabidopsis thaliana At3g22200,

Alcaligenes denitrificans aptA, Bordetella bronchiseptica BB0869, Bordetella

parapertussis BPP0784, Brucella melitensis BAWG 0478, Burkholderia pseudomallei

BP1026B I0669, Chromobacterium violaceum CV2025, Oceanicola granulosus

OG2516 07293, Paracoccus denitrificans PD1222 Pden_3984, Caulobacter crescentus CC 3143, Pseudogulbenkiania ferrooxidans co-TA, Pseudomonas putida ω -TA,

Ralstonia solanacearum ω -TA, Rhizobium meliloti SMc01534, Vibrio fluvialis ω -TA, Bacillus megaterium SC6394 ω -TA, Mus musculus abaT, Flavobacterium lutescens lat,

Streptomyces clavuligerus lat, E. coli gabT, E. colipuuE, E. coliygjG, or homologues.

21) The microorganism of claim 6, wherein said overexpressed β-keto acid decarboxylase is encoded by a gene(s) selected from the group consisting of Clostridium acetobutylicum adc, Solanum habrochaites mksl, or homologues. 22) The microorganism of claims 1 and 6, wherein said reduced expressions of fermentation enzymes are AadhE, (Apta or AackA or AackApta), ApoxB, AldhA, and AfrdA and less acetate, lactate, ethanol and succinate are thereby produced. 23) The microorganism of claims 1 and 6, comprising one or more of the following mutations: fadR, atoC(c), AarcA, Acrp, crp*.

24) The microorganism of claim 1, comprising one or more termination enzymes from Table 1.

25) A method of producing a product comprising growing a genetically engineered

microorganism according to any of claims 1-24 in a culture broth containing glycerol or a sugar, and extending a generated omega-phenyl acyl-CoA thioester primer using non- decarboxylative condensation and beta-reduction reactions to produce an omega-phenyl product at least two carbons longer than said primer, and isolating said omega-phenyl product.

26) A genetically engineered microorganism comprising a reverse beta oxidation pathway using an omega-phenyl primer, said microorganism comprising: a) an overexpressed thiolase that catalyzes the condensation of a omega-phenyl acyl- CoA thioester primer and acetyl-CoA to form an omega-phenyl B-ketoacyl-CoA; b) an overexpressed 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-ACP reductase that catalyzes the reduction of said omega-phenyl β-ketoacyl-CoA to produce an omega-phenyl B-hydroxyacyl-CoA; c) an overexpressed enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydratase, or 3- hydroxyacyl-ACP dehydratase that catalyzes the dehydration of said omega-phenyl β- hydroxyacyl-CoA to an omega-phenyl trans-enoyl-CoA; d) an overexpressed acyl-CoA dehydrogenase, trans-enoyl-CoA reductase, or enoyl- ACP reductase that catalyzes the reduction of said omega-phenyl trans-enoyl-CoA to an omega-phenyl acyl-CoA; and, e) an overexpressed termination enzyme(s) able to act on said omega-phenyl thioester intermediates of steps a, b, c, or d to produce an omega-phenyl product.

27) A method of producing an omega-phenyl product, comprising growing the

microorganism of claim 26 in a culture broth containing an omega-phenyl alkanoic acid or a -CoA activated for thereof, and extending an omega-phenyl acyl-CoA thioester primer using non-decarboxylative condensation and beta-reduction reactions to produce an omega-phenyl product at least two carbons longer than said primer, and isolating said omega-phenyl product.