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  • C12P7/44—Polycarboxylic acids

Definitions

• aspects of the invention relate to methods for the production of difunctional alkanes in microorganisms.
• aspects of the invention describe enzymes, and nucleic acids encoding such enzymes, associated with the difunctional alkane production from carbohydrates feedstocks in microorganisms.
• aspects of the invention describe recombinant microorganisms and metabolic pathways for the production of adipic acid, aminocaproic acid, hexamemylenediamine, 6- hydroxyhexanoate and 6-hydroxyhexanamine and 1,6-hexanediol, 5-arninopentanol, 5- aminopentanoate, 1,5-pentanediol, glutarate and 5-hydroxypentanoate.
• Crude oil is the number one starting material for the synthesis of key organic chemicals and polymers.
• biological processing of renewable raw materials in the production of chemicals using live microorganisms or their purified enzymes becomes increasingly interesting.
• Biological processing, in particular, fermentations have been used for centuries to make beverages.
• microorganisms have been used commercially to make compounds such as antibiotics, vitamins, and amino acids.
• the use of microorganisms for making industrial chemicals has been much less widespread. It has been realized only recently that microorganisms may be able to provide an economical route to certain compounds that are difficult or costly to make by conventional chemical means.
• the invention provides a microorganism producing 6- aminocaproic acid from lysine, and the microorganism includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the nucleic acid molecule is heterologous to the recombinant microorganism.
• the microorganism also includes a nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• 6-amino-3-hydroxyhexanoic acid to 6-amino-3-hydroxyhexanoyl-CoA
• iii) 6-aminohex-2-enoyl-CoA to 6-aminohex-2-enoic acid or iii) 6-aminohex-2-enoyl-CoA to 6-aminohex-2-enoic acid.
• the microorganism also includes a nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the invention provides in part a recombinant microorganism producing 6-aminocaproic acid from lysine, and the recombinant microorganism includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the at least one nucleic acid molecule is generally heterologous to the recombinant microorganism.
• the invention provides a recombinant microorganism producing 6-aminocaproic acid from lysine, the recombinant microorganism includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion that includes:
• a recombinant microorganism producing 6- aminocaproic acid from lysine where the recombinant microorganism includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the at least one nucleic acid molecule is heterologous to the recombinant microorganism.
• a recombinant microorganism producing 6- aminocaproic acid from lysine where the recombinant microorganism includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the recombinant microorganism also includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• 6-amino-2-hydroxyhexanoic to 6-amino-2-hydroxyhexanoyl-CoA
• 6-amino-2-hydroxyhexanoyl-CoA to 6-aminohex-2-enoyl-CoA
• the recombinant microorganism also includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the invention provides a recombinant microorganism producing 6-aminocaproic acid from L-2,3-dihydrodipicolinate, and the recombinant microorganism includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• 6-amino-2-oxohexanoic acid to 6-amino-2-hydroxyhexanoic acid
• 6-amino-2-hydroxyhexanoic acid to 6-aminohex-2-enoic acid
• 6-aminohex-2-enoic acid to 6-aminocaproic acid
• the at least one nucleic acid molecule is heterologous to the recombinant microorganism.
• a recombinant microorganism producing 6- aminocaproic acid from L-2,3-dihydrodipicolinate where the recombinant microorganism includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the at least one nucleic acid molecule is heterologous to the recombinant microorganism.
• a recombinant microorganism producing 6- aminocaproic acid from L-2,3-dihydrodipicolinate includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as: i) L-2,3-dihydrodipicolmate to A'-piperideine ⁇ e-dicarboxylate, ii) A 1 -piperideine-2,6-dicarboxylate to
• 6-amino-2-hydroxyhexanoic to 6-amino-2-hydroxyhexanoyl-CoA
• 6-amino-2-hydroxyhexanoyl-CoA to 6-aminohex-2-enoyl-CoA
• 6-aminohex-2-enoyl-CoA to 6-amiriohexanoyl-CoA
• a recombinant microorganism producing 6- aminocaproic acid from L-2,3-dihydrodipicolinate includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• a recombinant microorganism producing 6- ammocaproic acid from L-2,3-dihydrodipicolinate includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• L-2,3-dihydrodipicolinate to A 1 -piperideine-2,6-dicarboxylate ii) A 1 -piperideine-2,6-dicarboxylate to 2-amino-6-oxoheptanedioic acid, iii) 2-arnino-6-oxoheptanedioic acid to 6-amino-2-oxohexanoic acid, iv) 6-amino-2-oxohexanoic acid to 6-amino-hex-2-enoic acid, or v) 6-amino-hex-2-enoic acid to 6-aminocaproic acid.
• a recombinant microorganism producing 6- aminocaproic acid from L-2,3-dihydrodipicolinate includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• a recombinant microorganism producing 6- aminocaproic acid from L-lysine includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• a recombinant microorganism producing adipic acid from L-2,3-dihydrodipicolinate includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• a recombinant microorganism producing adipic acid from L-2,3-dihydrodipicolinate includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the recombinant microorganism includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• a recombinant microorganism producing 6- hydroxyhexanoate from L-2,3-dihydrodipicolinate includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• a recombinant microorganism producing 6- aminocaproic acid from lysine includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as: i) lysine to 6-amino-2-oxohexanoic acid,
• a recombinant microorganism producing 6- arninocaproic acid from lysine includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• a recombinant microorganism producing 6- arninocaproic acid from lysine includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• a recombinant microorganism producing hexamethylenediamine from lysine includes at least one nucleic acid encoding a polypeptide that catalyzes a substrate to product conversion such as:
• the recombinant microorganisms described herein are bacterial or yeast cells.
• one or more of the nucleic acids described herein encodes an enzyme.
• the enzyme exists in a natural biological system or, alternatively, the enzyme does not exist in a natural biological system.
• the nucleic acids encode a plurality of enzymes and the plurality of enzymes do not exist together in a natural biological system.
• nucleic acid preparations that encode at least one polypeptide that catalyzes a substrate to product conversion described herein.
• the nucleic acids include plasmids or other vector molecules.
• nucleic acid preparations containing one or more nucleic acid molecules that are engineered nucleic acids, and these engineered nucleic acids have less than 99%, less than 95%, less than 90%, less than 80%, less than 70%, or less than 60% identity with a natural nucleic acid.
• Nucleic acid preparations are provided that encode one or more proteins, which have less than 99%, less than 95%, less than 90%, less than 80%, less than 70%, or less than 60% identity with a natural protein.
• engineered metabolic pathways for the production of 6-aminocaproic acid in a recombinant microorganism which include a plurality of polypeptides that catalyze the substrate to product conversions described herein.
• the plurality of polypeptides does not exist in a natural biological system.
• an engineered metabolic pathway for the production of adipic acid in a recombinant microorganism which includes a plurality of polypeptides that catalyze the substrate to product conversions described herein.
• the plurality of polypeptides does not exist in a natural biological system.
• an engineered metabolic pathway for the production of 1,6-hexanediol in a recombinant microorganism includes a plurality of polypeptides that catalyze one or more of the substrate to product conversions described herein.
• the plurality of polypeptides does not exist in a natural biological system.
• an engineered metabolic pathway for the production of 6-hydroxyhexanoate in a recombinant microorganism that includes a plurality of polypeptides that catalyze a substrate to product conversion described herein.
• an engineered metabolic pathway for the production of hexamethylenediamine in a recombinant microorganism that includes a plurality of polypeptides that catalyze the substrate to product conversion described herein.
• Figure 1 shows the structure of L-lysine
• Figure 2 represents a flow diagram for the bioproduction of aminocaproic acid from lysine.
• Figure 3 A represents a flow diagram for the bioproduction of aminocaproic acid from L-2,3-dihydrodipicolinate.
• Figure 3B represents a flow diagram for the bioproduction of adipic acid
• Figure 4 represents a flow diagram of C6 difunctional hexanes from lysine via an N-heterocyclic ring intermediate.
• Figure 5 represents a flow diagram for the bioproduction of adipic acid, 6- hydroxyhexanoate, and 1,6-hexanediol from lysine.
• Figure 6 represents a flow diagram for the bioproduction of 6- hydroxyhexamine and hexamethylenediamine from lysine.
• Figure 7 represents a flow diagram for the bioproduction of C5 difunctional alkanes from lysine.
• Figure 8 represents a flow diagram for the bioproduction of C5 difunctional alkanes from L-pipecolate.
• Figure 9 represents a flow diagram for the bioproduction of C6 difunctional alkanes from lysine via a carbon extension process.
• organic molecule refers, for example, to any molecule that is made up predominantly of carbon and hydrogen, such as, for example, alkanes.
• Organic compounds of interest such as difunctional alkanes, diols, dicarboxylic acids, etc. can be used to synthesize plastic, nylons and other products usually derived from petroleum and hydrocarbons.
• aspects of the invention relate to the synthesis of difunctional n-alkanes with hydrocarbon chains derived from a hydrocarbon chain C n wherein n is a number of from about 1 to about 8, such as from about 2 to about 5, from about 3 to about 4, or preferably from 5 to 6.
• the difunctional n-alkanes are derived from lysine, lysine precursors and/or lysine degradation compounds.
• aspects of the invention relate to the production of difunctional alkanes of interest in a microorganism and provide methods for the production of difunctional alkanes from a carbohydrate source in a microorganism.
• difunctional alkanes refers to alkanes having two functional groups.
• the term "functional group” refers, for example, to a group of atoms arranged in a way that determines the chemical properties of the group and the molecule to which it is attached. Examples of functional groups include halogen atoms, hydroxyl groups (-OH), carboxylic acid groups (-COOH) and amine groups (-NH 2 ) and the like.
• Alcohol refers, for example, to an alkyl moiety in which one or more of the hydrogen atoms has been replaced by an -OH group.
• primary alcohol refers, for example to alcohols in which the -OH group is bonded to a terminal or chain-ending carbon atom, such as in 1-hexanol and the like.
• secondary alcohol refers, for example to alcohols in which the -OH group is bonded to a carbon atom that is bonded to one hydrogen atom and to two other carbon atoms, such as in 2-hexanol and the like.
• tertiary alcohol refers, for example to alcohols in which the -OH group is bonded to a carbon atom that is bonded to three other carbon atoms.
• “Amine” refers, for example, to an alkyl moiety in which one or more of the hydrogen atoms has been replaced by an -NH 2 group.
• the method incorporates microorganisms capable of producing one of the following C6-difunctional alkanes of interest, particularly, adipic acid, amino caproic acid, hexamethylenediamine (HMD), or 6-hydroxyhexanoate.
• Other difunctional alkanes of interest include 5-aminopentanol, 5-aminopentanoate, 1,5-pentanedioL glutarate, 5- hydroxypentanoate, cadaverine, etc.
• aspects of the invention provide engineered metabolic routes, methods to produce difunctional alkanes from sustainable feedstock, and materials associated therewith, including isolated nucleic acids or engineered nucleic acids, polypeptides or engineered polypeptides, and host cells or genetically engineered host cells.
• Carbon sources suitable as starting materials for such biosynthetic pathways include carbohydrates and synthetic intermediates.
• carbohydrates which cells are capable of metabolizing include sugars, dextroses, triglycerides and fatty acids. Intermediate products from metabolic pathway such as pyruvate, oxaloacetate, and 2-ketoglutatrate can also be used as starting materials.
• Aspects of the invention relate to engineered polypeptides, and polynucleotides encoding such polypeptides, having enzymatic activity or an improved activity for a natural or unnatural substrate or having broad substrate specificity (e.g., catalytic promiscuity such as substrate promiscuity).
• polypeptide refers to a polymer of amino acids, including, for example, gene products, naturally-occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the forgoing.
• polypeptide having enzymatic activity refers to any polypeptide that catalyzes a chemical reaction of other substances without itself being destroyed or altered upon completion of the reaction. Typically, a polypeptide having enzymatic activity catalyzes the formation of one or more products from one or more substrates.
• the catalytic promiscuity properties of some enzymes may be combined with protein engineering and may be exploited in novel metabolic pathways and biosynthesis applications.
• existing enzymes are modified for use in organic biosynthesis.
• the enzymes involved in the production of the Afunctional n- alkanes of interest include but are not limited to 2 amino-decarboxylases, 2- ketodecarboxylases, terminal-aminotransferases, 2-aminotransferases, 2 amino-aldehyde mutase, alcohol dehydrogenases, aldehyde dehydrogenases, amino-aldehyde dehydrogenases, dehydrogenases, dehydratases, CoA ligases, CoA-S transferases, deaminases and thioestrerases.
• the reaction mechanism of the reference enzyme(s) may be altered to catalyze new reactions, to change, expand or improve substrate specificity.
• enzyme structure e.g. crystal structure
• enzymes properties may be modified by rational redesign (see US patent application US20060160138, US20080064610 and US20080287320).
• Modification or improvement in enzyme properties may arise from the introduction of modifications into a polypeptide chain that may, in effect, perturb the structure-function of the enzyme and/or alter its interaction with another molecule (e.g., association with a natural substrate versus an unnatural substrate). It is well known in the art that certain regions of a protein may be critical for enzyme activity, for example amino acids involved in catalysis and substrate binding domains, such that small perturbations to these regions will have significant effects on enzyme function. Some amino acid residues may be at important positions for maintaining the secondary or tertiary structure of the enzyme, and thus also produce noticeable changes in enzyme properties when modified. In some embodiments, the potential pathway components are variants of any of the foregoing.
• the number of modifications to a reference parent enzyme that produces a variant enzyme having the desired property may comprise one or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, up to 30% of the total number of amino acids, up to 40% of the total number of amino acids making up the reference enzyme, or up to 50% of the total number of amino acids making up the reference enzyme.
• engineered pathways exemplified herein are described in relation to, but are not limited to, species specific genes and proteins and that the invention encompasses homologs and orthologs of such gene and protein sequences.
• Homolog and ortholog sequences possess a relatively high degree of sequence identity/similarity when aligned using methods known in the art.
• Such homologs or orthologs can suitably be obtained by means of any appropriate cloning strategy known to one skilled in the art.
• useful polypeptide sequences have at least 30%, at least 45%, at least 60%, at least 75%, at least 85%, or at least 95% identity to the amino acid sequence of the reference enzyme of interest.
• aspects of the invention relate to new microorganisms or "genetically modified” microorganisms or host cells that have been engineered to possess new metabolic capabilities or new metabolic pathways.
• host cell and “microorganism” are used interchangeably.
• genetically engineered microorganisms refers to microorganisms having at least one genetic alteration not normally found in the wild type strain of the reference species.
• genetically engineered microorganisms are engineered to express or overexpress at least one particular enzyme at critical points in a metabolic pathway, and/or to block the synthesis of other enzymes, to overcome or circumvent metabolic bottlenecks.
• metabolic pathway refers to a series of two or more enzymatic reactions in which the product of one enzymatic reaction becomes the substrate for the next enzymatic reaction.
• intermediate compounds are formed and utilized as substrates for a subsequent step. These compounds may be called “metabolic intermediates.”
• the products of each step are also called “metabolites.”
• aspects of the invention provide methods for designing and making engineered metabolic pathways.
• alternative pathways for making a product of interest from one or more available and sustainable substrates may be made in one or more host cells or microorganisms of interest.
• the engineered pathway for making difunctional alkanes of interest may involve multiple enzymes and therefore the flux through the pathway may not be optimum for the production of the product of interest. Consequently, in some aspects of the invention, the carbon flux is optimally balanced by modulating the activity level of the pathway enzymes relative to one another. Examples of such modulation are provided throughout the application.
• the term "carbon flux” refers to the number of feedstock molecules (e.g. glucose) which proceed down the engineered pathway relative to competitive paths.
• a host cell as used herein refers to an in vivo or in vitro eukaryotic cell, a prokaryotic cell or a cell from a multicellular organism (e.g. cell line) cultured as a unicellular entity.
• a host cell may be prokaryotic (e.g., a bacterial cell) or eukaryotic (e.g., a yeast, mammal or insect cell).
• host cells may be bacterial cells (e.g., Escherichia coli, Bacillus subtilis, Mycobacterium spp. , M.
• Eukaryotic or prokaryotic host cells can be, or have been, genetically modified (also referred as "recombinant”, “metabolically engineered” or “genetically engineered”) and used as recipients for a nucleic acid, such as an expression vector, that comprises a nucleotide sequence encoding one or more biosynthetic or engineered pathway gene products.
• Eukaryotic and prokaryotic host cells also denote the progeny of the original cell which has been genetically engineered by the nucleic acid.
• a host cell may be selected for its metabolic properties. For example, if a selection or screen is related to a particular metabolic pathway, it may be helpful to use a host cell that has a related pathway. Such a host cell may have certain physiological adaptations that allow it to process or import or export one or more intermediates or products of the pathway. However, in other embodiments, a host cell that expresses no enzymes associated with a particular pathway of interest may be selected in order to be able to identify all of the components required for that pathway using appropriate sets of genetic elements and not relying on the host cell to provide one or more missing steps.
• aerobic or anaerobic microorganisms are metabolically engineered.
• an anaerobic organism is any organism that does not require oxygen for growth (i.e. anaerobic conditions), such as certain bacterial cells.
• the bacterial cell can be an E. coli, C. glutanicum, B. flavum or B. lactofermentum cell; these strains are currently being employed industrially to make amino compounds using bacterial fermentation processes.
• C. glutanicum has been used extensively for amino acid production (e.g. L- glutamate, L-lysine, see Eggleging L et al., 2005, Handbook for Corynebacterium glutanicum. Boca Raton, USA: CRC Press).
• the metabolically engineered cell of the invention is made by transforming a host cell with at least one nucleotide sequence encoding enzymes involved in the engineered metabolic pathways.
• nucleotide sequence As used herein the terms “nucleotide sequence”, “nucleic acid sequence” and “genetic construct” are used interchangeably and mean a polymer of RNA or DNA, single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
• a nucleotide sequence may comprise one or more segments of cDN A, genomic DNA, synthetic DNA, or RNA.
• the nucleotide sequence is codon-optirnized to reflect the typical codon usage of the host cell without altering the polypeptide encoded by the nucleotide sequence.
• the term "codon optimization" or “codon-optimized” refers to modifying the codon content of a nucleic acid sequence without modifying the sequence of the polypeptide encoded by the nucleic acid to enhance expression in a particular host cell.
• the term is meant to encompass modifying the codon content of a nucleic acid sequence as a means to control the level of expression of a polypeptide (e.g., to either increase or decrease the level of expression).
• aspects of the invention include nucleic acid sequences encoding the enzymes involved in the engineered metabolic pathways.
• a metabolically engineered cell may express one or more polypeptides having an enzymatic activity necessary to perform the steps described throughout the description.
• a particular cell comprises one, two, three, four, five or more than five nucleic acid sequences with each one encoding the polypeptide(s) necessary to perform the conversion of lysine, lysine metabolite precursor and/or lysine degradation metabolites into difunctional alkane(s).
• a single nucleic acid molecule can encode one, or more than one, polypeptide.
• nucleic acid molecule can contain nucleic acid sequences that encode two, three, four or even five different polypeptides.
• Nucleic acid sequences useful for the invention described herein may be obtained from a variety of sources such as, for example, amplification of cDNA sequence, DNA libraries, de novo synthesis, excision of genomic segments, etc. The sequences obtained from such sources may then be modified using standard molecular biology and/or recombinant DNA technology to produce nucleic acid sequences having the desired modifications.
• nucleic acid sequences include for example, site directed mutagenesis, PCR mutagenesis, deletion, insertion, or substitution, or swapping portions of the sequence using restriction enzymes, optionally in combination with ligation, homologous recombination, site specific recombination or various combination thereof.
• the nucleic acid sequence may be a synthetic nucleic acid sequence. Synthetic polynucleotide sequences may be produce using a variety of methods described in U.S. patent 7,323,320, and in copending application having serial number 11/804,996 and in U.S. Patent Publication Nos. 2006/0160138, 2007/0269870, 2008/0064610, and 2008/0287320.
• a genetically modified host cell is genetically modified such that it produces, when cultured in vitro in a suitable medium, the product of interest or an intermediate at a level of at least 0.1 g/1, at least 1 g/1, at least lOg/l, at least 50 g 1, at least 100 g/1 or at least 150 g/1.
• the level of the metabolite of interest or its metabolic intermediates produced by a genetically modified host cell can be controlled in various ways.
• the level of expression is controlled by the number of copies of the nucleic acid sequences encoding one or more enzymes involved in the engineered pathway that are contained in the host cell (e.g. high copy expression vector versus medium or low copy expression vectors).
• the nucleic acid sequences are introduced into the cell using a vector.
• Low copy expression vectors generally provide fewer than 20 vector copies per cell (e.g. from 1 to about 5, from 5 to about 10, from 10 to about 15, from 15 to about 20 copies of the expression vector per cell).
• Suitable low copy expression vectors for prokaryotic cells include, but are not limited to pAYC184, pBeloBacl 1, pBR332, pBAD33, pBBRlMCS and its derivatives, pSClOl, SuperCos (cosmid) and pWE15 (cosmid).
• Medium copy number expression vectors generally provide from about 20 to about 50 expression vectors copies per cell or form about 20 to 80 expression vectors copies per cell.
• Suitable medium copy expression vectors for prokaryotic cells include, but are not limited to, pTrc99A, pBAD24 and vectors containing a ColEl origin of replication and its derivatives.
• High copy number expression vectors generally provide from about 80 to about 200 or more expression vector copies per cell.
• Suitable high copy expression vectors for prokaryotic cells include, but are not limited to, pUC, PCV1, pBluescript, pGEM and pTZ vectors.
• aspects of the invention provide expression cassettes comprising a nucleic acid or a subsequence thereof encoding a polypeptide involved in the engineered pathway.
• the expression cassette can comprise the nucleic acid operably linked to control sequences, such as a transcriptional element (e.g. promoter) and to a terminator.
• control sequences such as a transcriptional element (e.g. promoter) and to a terminator.
• cassette refers to a nucleotide sequence capable of expressing a particular gene if the gene is inserted so as to be operably linked to one or more regulatory sequences present in the nucleotide sequence.
• the expression cassette may comprise a heterologous gene which is desired to be expressed in the host cell.
• one or more expression cassettes may be introduced into a vector by known recombinant techniques.
• a promoter is a sequence of nucleotides that initiates and controls the transcription of a desired nucleic acid sequence by an RNA polymerase enzyme.
• the promoter may be inducible.
• promoters may be constitutive.
• suitable promoters for the use in prokaryotic host cells include a bacteriophage T7 RNA polymerase promoter, a trp promoter, a lac operon promoter and the like.
• suitable strong promoters for the use in prokaryotic cells include lacUV5 promoter, T5, T7, Trc, Tac and the like.
• Non limiting examples of suitable promoters for use in eukaryotic cells include a CMV immediate early promoter, a SV40 early or late promoter, a HSV thymidine kinase promoter and the like. Termination control regions may also be derived from various genes native to the preferred host.
• a first enzyme of the engineered pathway may be under the control of a first promoter and the second enzyme of the engineered pathway may be under the control of a second promoter, wherein the first and the second promoter have different strengths.
• the first promoter may be stronger than the second promoter or the second promoter may be stronger than the first promoter. Consequently, the level of a first enzyme may be increased relative to the level of a second enzyme in the engineered pathway by increasing the number of copies of the first enzyme and/or by increasing the promoter strength to which the first enzyme is operably linked to relative to the promoter strength to which the second enzyme is operably linked to.
• the plurality of enzymes of the engineered pathway may be under the control of the same promoter.
• altering the ribosomal binding site affects relative translation and expression of different enzymes in the pathway. Altering the ribosomal binding site can be used alone to control relative expression of enzymes in the pathway, or it can be used in concert with the aforementioned promoter modifications and codon optimization that also affects gene expression levels.
• expression of the potential pathway enzymes may be dependent upon the presence of a substrate that the pathway enzyme will act on in the reaction mixture.
• expression of an enzyme that catalyzes conversion of A to B may be induced in the presence of A in the media.
• Expression of such pathway enzymes may be induced either by adding the compound that causes induction or by the natural build-up of the compound during the process of the biosynthetic pathway (e.g., the inducer may be an intermediate produced during the biosynthetic process to yield a desired product).
• computer-implemented design techniques may be used to generate alternative pathways for generating an organic compound of interest.
• the databases contain genomic information and their link may be utilized for designing novel metabolic pathways.
• database are MetaCyc (a database of metabolic pathways and enzymes), the University of Minnesota biocatalysis biodegradation database (a database of microbial catalytic reactions and biodegradation pathways for organic chemical compounds), LGAND (a composite database that provides information about metabolites and other chemical compounds, substrate-product relations representing metabolic and other reactions and information about enzyme molecules).
• a database of pathway components may also contain components of predicted, putative, or unknown functions. It may also contain pseudo- components of defined function that may have an undefined composition.
• a program may design combinations of regulatory and/or functional elements that are in the public domain (e.g., that are not covered by patent rights and/or are not subject to a licensing fee).
• Databases of freely available genetic elements may be generated and/or used as a source of nucleic acid sequences that can be combined to produce altemative pathways.
• Alternative pathways containing different combinations of known functional and/or regulatory elements e.g., from different species
• Libraries including variations in enzymatic element regions may be used to ascertain the relative effects of different types of enzymes or of different variants of the same enzyme.
• Libraries including variations in regulatory element regions may be used to ascertain the optimal expression level or regulatory control among a set of genes.
• the functional properties of different engineered pathways may be tested in vivo by transforming host cells or organisms with the appropriate assembled nucleic acids, and assaying the properties of the engineered organisms.
• the functional properties of different engineered pathways may be tested in vitro by isolating components expressed from assembled nucleic acids and testing the appropriate combinations of components in an in vitro system.
• aspects of the invention relate to design and assembly of engineered pathways for the production of C6 difunctional alkanes of interest. Particularly, aspects of the invention relate to the production of adipic acid, amino caproic acid (a stable precursor of caprolactam acid), hexamethylene diamine, 6-amino hexanol, 1,6-hexanediol and 6-hydroxyhexanoate.
• Adipic acid consistently ranks as one of the top fifty chemicals produced in the US. Nearly 90% of domestic adipic acid is used to produce nylon-6,6. Other uses of adipic acid include production of lubricants resins, polyester polyols and plasticizers, and food acidulant.
• cyclohexane process There are three major commercial production processes: cyclohexane process, cyclohexanol process, butadiene carbonylation process.
• the dominant industrial process for synthesizing adipic acid employs initial air oxidation of cyclohexane to yield a mixture of cyclohexanone (ketone) and cyclohexanol (alcohol), which is designated KA (see for example U.S. Patent No. 5,221,800). Hydrogenation of phenol to yield KA is also used commercially, although this process accounts for just 2% of all adipic acid production.
• KA produced via both methods is oxidized with nitric acid to produce adipic acid.
• Reduced nitrogen oxides including NO2, NO, and N2O are produced as by-products and are recycled back to nitric acid at varying levels. It is becoming increasingly more interesting to industry and beneficial to the environment to engineer non-synthetic, biological routes to adipic acid. A number of microbiological routes have been described. Wild-type and mutant organisms have been shown to convert renewable feedstocks such as glucose and other hydrocarbons to adipic acid (see for example WO9507996, and U.S. Patent No 5,272,073, US 5,487,987 and US 5,616,496). Similarly, organisms possessing nitrilase activity have been shown to convert nitriles to carboxylic acids including adipic acid (see for example U.S. Patent No.
• cyclohexanol is converted in adipic acid, the enzymatic pathway comprising genes isolated from an Acinetobacter encoding hydroxylacylCoA dehydrogenase; enoylCoA hydratase, acylCoA dehydrogenase, ubiquinone oxidoreductase, monoxygenase, aldehyde dehydrogenase.
• Another enzymatic pathway for the conversion of cyclohexanol to adipic acid has been suggested as including the intermediates cyclohexanol, cyclohexanone, 2- hydroxycyclohexanone, ⁇ -caprolactone, 6-hydroxycaproic acid.
• Some specific enzyme activities in this pathway have been demonstrated, including cyclohexanol dehydrogenase, NADPH-linked cyclohexanone oxygenase, s-caprolactone hydrolase, and NAD (NADP)- linked 6-hydroxycaproic acid dehydrogenase (Tanaka et al., Hakko Kogaku Kaishi (1977), 55(2), 62-7).
• Aminocaproic acid (or e-aminocaproic acid, or IUPAC name 6- aminohexanoic acid) is a possible intermediate for the production of caprolactam.
• Caprolactam is primarily used in the manufacture of synthetic fibers, especially nylon 6 that is also used in bristle brushes, textile stiffeners, film coatings, synthetic leather, plastics, plasticizers, vehicles, cross linking for polyurethanes, and in the synthesis of lysine. About 2.5 billion tons of nylon 6 is produced annually on a worldwide basis. The production of nylon 6 is accomplished by the ring opening polymerization of the monomer ⁇ -caprolactam. The starting chemical compound for the production of ⁇ - caprolactam is benzene which is converted to either cyclohexane or phenol and either chemical is converted via cyclohexanone to cyclohexanone oxime and then this intermediate is heated in sulfuric acid.
• Nylon 6,6 and nylon 6,10 can be made into various kinds of nylon resins and nylon fiber.
• 6-hydroxyhexanoate (6HH) is a 6-carbon hydroxyalkanoate that can be circularized to caprolactone or directly polymerized to make polyester plastics (polyhydroxyalkanoate PHA).
• 1,6-hexanediol is a valuable intermediate for the chemical industry. It has applications in a variety of polymer syntheses such as the production of polyesters for polyurethane elastomers and polymeric plasticizers and is also used in gasoline refining.
• the problem to be solved therefore is to provide a synthesis route for difunctional alkanes which not only avoids reliance on environmentally sensitive starting materials such as petroleum but also makes efficient use of non-petrochemical inexpensive, renewable resources. It would further be desirable to provide a synthesis route for difunctional alkanes which avoids the need for significant energy inputs, optimizes carbon flux and minimizes the formation of toxic by-products.
• Lysine (Lys or K) is a 1,2,6-trifunctional hexane with the chemical formula NH 2 (Cffi) 4 CHN3 ⁇ 4COOH ( Figure 1).
• L-lysine is an important economic product obtained principally by industrial-scale fermentation utilizing the Gram positive Corynebacterium gl tamicum, Brevibacteriumflavum and Brevibacterium lactofermentum. A considerable amount is known regarding the biochemical pathway for L-lysine synthesis in Corynebacterium species (see for example WO04013341 and WO01665730).
• lysine is synthesized from aspartic acid, which is first converted to ⁇ -aspartyl-semialdehyde, cyclizated into dihydropicolinate, which is then reduced to A'-piperidine-l ⁇ -dicarboxylate. Ring-opening of this heterocycle gives a series of derivatives of pimelic acid, and ultimately leads to lysine.
• Enzymes involves in the lysine biosynthesis include (Lehninger, A. L., D. L. Nelson, and M. M. Cox. 2000. Lehninger Principles of Biochemistry, 3rd ed.
• aspartokinase ⁇ -aspartate semialdehyde dehydrogenase, dihydropicolinate synthase, Al-piperdine-2,6-dicarboxylate dehydrogenase, N-succinyl-2-amino-6- ketopimelate synthase, succinyl diaminopimelate aminotransferase, succinyl diaminopimelate desuccinylase, diaminopimelate epimerase, diaminopimelate decarboxylase.
• lysine is metabolized to give acetyl-CoA, via an initial transamination with a-ketoglutarate. The bacterial degradation of lysine yields to cadaverine by decarboxylation.
• lysine is an ideal precursor of difunctional alkanes as it can be produced in high yield and it contains an unbroken 6- carbon-chain with functionalized ends. Aspects of the invention provide potential pathways for the bioproduction of C6 difunctional alkanes. Detailed explanations of the relevant metabolic pathways are given hereinafter.
• aspects of the invention provide several metabolic pathways that can be used to produce organic compounds such as aminocaproic acid and its intermediates from lysine. These pathways are shown in Figures 2-3. Accordingly, aspects of the invention provide a recombinant microorganism having an engineered aminocaproic acid biosynthetic pathway.
• L-lysine production involves the use of molecular biology techniques to augment L-lysine production.
• recombinant microorganisms have at least one gene that is expressed at a level lower or higher than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated.
• Genes are selected from the group of genes which play a key role in the biosynthesis of lysine such as aspartokinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino- ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diaroinopimelate dehydrogenase, arginyl- t NA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucoses- phosphate dehydrogenase, transketolase, transaldolase, phosphogluconolactonase, fructose 1,6-biphosphatase,
• CoA are intermediates to several of the aminocaproic acid biosynthetic pathway.
• aspects of the invention provide a recombinant microorganism having an engineered 6-aminohex-2-enoic acid or 6-aminohex-2-enoyl-CoA biosynthetic pathway.
• the 6-aminohex-2-enoic acid biosynthetic pathway is the same as the aminocaproic biosynthetic pathway with omission of the last enzymatic step.
• the engineered microorganisms are used for the commercial production of aminocaproic acid or its intermediates.
• aminocaproic acid Nl3 ⁇ 4(CH2) 5 COOH is a derivative and analogue of the amino acid lysine H 2 (CH2) 4 CHNH 2 COOH and lysine is therefore an ideal biological precursor of the aminocaproic acid.
• aspects of the invention relate to a process for aminocaproic acid production and applying at least one enzymatic step A, B, B' and/or C as illustrated in the pathway shown in Figure 2. Accordingly, aspects of the invention provide a recombinant host cell or microorganism comprising at least one nucleic acid molecule encoding at least one polypeptide that catalyzes a substrate to product conversion as illustrated in Figure 2.
• Pathway I comprising enzymatic steps Al, B2, B3, B4, and A5;
• Pathway II comprising enzymatic steps Al, A2, A3, A4 and A5;
• Pathway III comprising enzymatic steps Al, A2, A3, A6, A7, B5 and B6;
• Pathway IV comprising enzymatic steps Al , A2, A3, A6, A7, B4 and A5;
• Pathway V comprising enzymatic steps Al, B2, B3, B5 and B6;
• Pathway VI comprising enzymatic steps Al, B' and A5;
• Pathway VII comprising enzymatic steps CI, C2, C3 and A5;
• Pathway VIII comprising enzymatic steps CI, C2, C4, C5, B4 and A5; and 9) Pathway EX comprising enzymatic steps CI, C2, C4, C5, B5 and B6.
• the aminocaproic biosynthetic pathway begins with the conversion of lysine to 3,6-diaminohexanoic acid by action of an aminomutase as shown in the substrate to product conversion step Al in Figure 2 (Pathways I through VI).
• the lysine aminomutase is a lysine 2,3-aminomutase (KAM or LAM) (EC 5.4.3.2) which facilitates the conversion of the amino acid lysine to 3,6- diaminohexanoic acid or beta-lysine as shown below:
• Lysine 2,3 aminomutase uses Pyridoxal phosphate, Zinc and a 4 Iron-4 Sulfur cluster as cofactors and a 5'-deoxyadenosyl radical formed in an S-Adenosyl methionine (SAM) activated radical reaction pathway.
• SAM S-Adenosyl methionine
• L-lysine 2,3-aminomutase examples include but are not limited, to EC
• enzymes having an aminotransferase or dehydrogenase activity are understood to be enzymes that catalyze the transfer of a 2-amino group from a lysine molecule to a recipient molecule, leaving behind a ⁇ -ketoacid (or 3-oxoacid) molecule containing a primary amino group.
• polypeptides useful for converting 3,6- aminohexanoic acid into 6-amino-3-oxohexanoic acid are exemplified but not limited to the enzymes listed in Table 1 and may be isolated from a variety of sources.
• enzymatic step A3 is further converted into 6-amino-3-hydroxyhexanoic acid (a3) by action of a reductase enzyme (enzymatic step A3, pathways II, III, and IV).
• a reductase enzyme enzymatic step A3, pathways II, III, and IV.
• enzymatic step A3 may be catalyzed by a dehydrogenase enzyme.
• the reductase / dehydrogenase enzyme catalyses the carbonyl reduction of the ⁇ -ketones to its corresponding hydroxyl-derivative (secondary alcohol).
• the oxidizing equivalent is supplied in the form of an oxidized nicotinamide cofactor, NAD(+) or NADP(+).
• reductase or dehydrogenase enzymes useful in the present invention are listed in Table 2 and may be available from different sources. Although there are no reported enzymes that catalyze the substrate to product conversion of a2 to a3, the substrate shows some similarity to those utilized in Table 2 and may be converted by action of an enzyme having a reductase or dehydrogenase activity as listed in Table 2.
• the reductase enzyme is L-carnitine dehydrogenase, 3- hydroxypropionate dehydrogenase, (S)-carnitine 3 -dehydrogenase, Hydroxyacid-oxoacid transhydrogenase, malonate semialdehyde reductase (NADPH) or 3-oxo-acyl-CoA reductase.
• enzymatic step A4 (pathway II, Figure 2), 6-amino-3-hydroxyhexanoic acid (a3) is converted into (E)-6-aminohex-2-enoic acid (a4) by a dehydratase or hydrolase which cleaves carbon-oxygen bonds by elimination of water.
• a dehydratase or hydrolase which cleaves carbon-oxygen bonds by elimination of water.
• the reductase enzyme is a trans-L-3-hydroxyproline dehydratase, a dimethyl- maleate hydratase, an L-carnitine dehydratase, a fumarate dehydratase, or a 2-oxohept-3- endioate hydroxylation.
• polypeptides having a reductase activity include, but are not limited to the ones listed below and may be isolated from a variety of sources. Table 3
• Crotonoyl-[acyl- carrier-protein] 11 II hydratase
• Pathways I, II, IV, VI, VII, and VIII conclude with the conversion of 6- aminohex-2-enoic acid into aminocaproic acid.
• the reduction of (E)-6-aminohex-2-enoic acid to aminocaproic acid (a4 ⁇ a5, enzymatic step A5) is catalyzed by a polypeptide having a reductase or a dehydrogenase activity.
• An example of an enzyme catalyzing this reaction is an enoate-reductase capable of hydrogenating the ⁇ , ⁇ -carbon-carbon double bond at the ⁇ , ⁇ -position next to a carboxylic acid group into a carbon-carbon single bond.
• Example of enzymes having a ⁇ , ⁇ enoate reductase activity can be found in US applications 20070117191 and 20070254341.
• Other exemplary polypeptides having an enoate reductase activity are listed in Table 4.
• the enoate reductase is a 2- enoate reductase, a NADH-dependent fumarate reductase, a succinate dehydrogenase, a coumarate reductase, a ⁇ -nitroacrylate reductase, a methylacetate reductase, or a ⁇ -butyrobetaine reductase.
• 6-amino-3-hydroxyhexanoic acid is converted to 6-amino-3-hydroxyhexanoyl-CoA.
• enzymes that catalyze the substrate to product conversion of 6-amino-3-hydroxyhexanoic acid into 6-amino-3- hydroxyhexanoyl-CoA have not been described, one skilled in the art will appreciate that the 6-amino-3-hydroxyhexanoic acid substrate is similar to substrates listed in table 5 and is likely to be an acceptable substrate for enzymes having a CoA transferase or a CoA ligase activity listed in Table 5.
• CoA-transferases catalyze reversible transfer reactions of Coenzyme A groups from CoA-thioesters to free carboxylic acids. Most CoA transferases operate with succinyl-CoA or acetyl-CoA as potential CoA donors and contain two different subunits.
• 6-amino-3-hydroxyhexanoyl-CoA is converted into
• 6-amino-hex-2-enoyl-CoA (enzymatic steps A7, pathways III and IV). Although enzymes that catalyze this conversion have not been described, one will appreciate that 6-amino-3- hydroxyhexanoyl-CoA may likely be an acceptable substrate for the dehydratase or hydro- lyase enzymes listed in Table 3.
• the reaction is catalyzed by enoyl-CoA hydratase (EC 4.2.1.17), a methyl-glutaconyl-CoA hydratase (EC 4.2.1.18), a crotonase (EC 4.2.1.55), a long-chain-enoylCoA hydratase (EC 4.2.1.74) or a 2-oxohept- 3-enedroate hydrolase (EC 4.2.1-).
• enoyl-CoA hydratase EC 4.2.1.17
• a methyl-glutaconyl-CoA hydratase EC 4.2.1.18
• a crotonase EC 4.2.1.55
• a long-chain-enoylCoA hydratase EC 4.2.1.74
• 2-oxohept- 3-enedroate hydrolase EC 4.2.1-).
• 6-aminohex-2-enoyl-CoA is converted to (E)-6- aminohex-2-enoic acid (b3 to b4 conversion, enzymatic step B4).
• enzymes that catalyze the substrate b3 to product b4 conversion of 6-aminohex-2-enoyl-CoA to (E)-6- aminohex-2-enoic acid have not been described, one skilled in the art will appreciate that the 6-aminohex-2-enoyl-CoA substrate is similar to glutaconyl CoA (see below) and the substrates listed in Table 5.
• 6-aminohex-2-enoyl-CoA is likely to be an acceptable substrate for enzymes having a CoA-transferase activity such as those listed below or in Table 5.
• Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition.
• the (E)-glutaconate:acetyl-CoA CoA- transferase (EC 2.8.3.12) which catalyzes the following reaction may catalyze the substrate to product conversion of 6-aminohex-2-enoylCoA to (E)-6-aminohex-2-enoic acid:
• the (E)-glutaconate:acetyl-CoA CoA-transferase enzyme is part of glutamate degradation V (via hydroxyglutarate) pathway and consists of two different subunits (Subunit A and B) encoded by the GctA and GctB genes (UniProt:Q59111 and UniProt:Q59112). i) Conversion of 6-aminohex-2-enoyl-CoA into 6-aminohexanoyl-CoA
• 6- aminohex-2-enoyl-CoA is converted into 6-aminohexanoyl-CoA (enzymatic step B5). Although this conversion has not been described, one will appreciate that the dehydrogenase enzymes listed in Table 4 may be able to convert 6-aminohex-2-enoyl- CoA to 6-aminohexanoyl-CoA.
• the enzymatic step B5 is catalyzed by a butyryl-CoA dehydrogenase (ECl.3.2.1, bed, Megasphaera elsdenii, Clostridium acetobutylicum), a Glutaryl-CoA dehydrogenase (EC 1.3.99.7), 2-methylacyl- CoA dehydrogenase (EC 1.3.99.10, acdH Streptomyces creermitilis), 2-methyl branched- chain acyl-CoA dehydrogenase (acdH (Streptomyces vermitilis), Acadsb (Rattus norvegicus), or a crotonobetaine reductase (EC 1.3.99.-, caiA Escherichia coli K12).
• a butyryl-CoA dehydrogenase ECl.3.2.1, bed, Megasphaera elsdenii, Clostridium acetobutylicum
• the dehydrogenase of enzymatic step B5 is an acyl-CoA dehydrogenase.
• Acyl-CoA dehydrogenases are a class of enzymes (EC 1.3.99.3, encoded by fadE in Escherichia coli K12, isovaleryl-CoA dehydrogenase encoded by aidB in Escherichia coli 12, encoded by acdB in Clostridium difficile) which catalyze the initial step in each cycle of fatty acid ⁇ -oxidation in the mitochondria of cells. Their action results in the introduction of a trans double-bond between C2 and C3 of the acyl-CoA thioester substrate.
• FAD is a required co-factor in the mechanism in order for the enzyme to bind to its appropriate substrate.
• l-amino-6-hexanoyl-CoA is converted to 6- aminohexanoic acid (Pathways ⁇ , V, and ⁇ ). Although enzymes that catalyze this substrate conversion have not been described, one skilled in the art will appreciate that the substrate l-amino-6-hexanoyl-CoA is similar to the substrates listed in Table 5.
• 1 -amino-6-hexanoyl-CoA is likely to be an acceptable substrate for enzymes having a CoA-transferase activity, such as those listed below and in Table 5.
• the CoA-transferase enzymes belong to the EC 2.8.1.x class.
• a preferable CoA transferase is a 2-hydroxyisocaproate CoA transferase.
• (S)-3,6-diaminohexanoic acid is converted to 3,6-diaminohexanoyl-CoA (enzymatic step B2).
• enzymes that catalyze the substrate to product conversion of (S)-3,6-diaminohexanoic acid to 3,6- diaminohexanoyl-CoA have not been described, one skilled in the art will appreciate that the (S)-3,6-diaminohexanoic acid substrate is similar to substrates listed in Table 5.
• (S)-3,6-diaminohexanoic acid is likely to be an acceptable substrate for enzymes having a CoA transferase or a CoA ligase activity, such as the enzymes listed in Table 5.
• CoA-transferases catalyze reversible transfer reactions of Coenzyme A groups from CoA-thioesters to free carboxylic acids. Most CoA transferases operate with succinyl-Co A or acetyl-CoA as potential CoA donors and contain two different subunits.
• step B3 (Pathways I and V), 3,6-diaminohexanoyl-CoA is converted to 6-aminohex-2-enoyl-CoA.
• enzymes that catalyze the substrate to product conversion of 3,6-diaminohexanoyl-CoA to 6-aminohex-2-enoyl-CoA have not been described, one skilled in the art will appreciate that the 3,6-diaminohexanoyl-CoA substrate is similar to substrates listed in Table 6. Therefore, 3,6-diaminohexanoyl-CoA is likely to be an acceptable substrate for enzymes having a deaminase activity, such as those listed in Table 6.
• (S)-3,6-diaminohexanoic acid is directly converted to 6-aminohex-2-enoic acid (enzymatic step B', Pathway VI) by a polypeptide having a deaminase activity.
• enzymes that catalyze the substrate to product conversion of 3,6-diaminohexanoate to 6-aminohex-2-enoyl have not been described, one skilled in the art will appreciate that the 3,6-diaminohexanoate substrate is similar to L-aspartate or (2S,3S)-3-methylaspartate.
• 3,6-diaminohexanoate is likely to be an acceptable substrate for Methylaspartate ammonia-lyase isolated for example from Citrobacter amalonaticus, Citrobacter freundii, Clostridium tetanomorphum, or Morganella morganiithat which catalyzes the following reaction:
• aspects of the invention provide metabolic pathways for the production of aminocaproic acid from lysine via a 6-amino-2-hydroxyhexanoic acid intermediate (Pathways VII, VIII and IX).
• lysine can be first converted to 6- amino-2-oxohexanoic acid (enzymatic step CI) and the resulting 6-amino-2-oxohexanoic acid is then converted to 6-amino-2-hydroxyhexanoic acid (enzymatic step C2).
• the resulting 6-amino-2-hydroxyhexanoic acid can be then converted to (E)-6-aminohex-2-enoic acid (enzymatic step C3) and finally the resulting ((E)-6- aminohex-2-enoic acid is converted to aminocaproic acid (as described in enzymatic step A5).
• 6-amino-2-hydroxyhexanoic acid is first converted to 6- amino-2-hydroxyhexanoyl-CoA (enzymatic step C4), then into 6-aminohex-2-enoyl-CoA (enzymatic step C5) and subsequently to 6-aminohexanoic acid as described above (enzymatic steps B5 followed by B6 or enzymatic steps B4 followed by A5).
• lysine is first converted to 6- amino-2-oxohexanoic acid (cl) by an aminotransferase enzyme (EC 2.6.1.x) or a dehydrogenase (EC 1.4.1.x or EC 1.4.3.x).
• the pathway involves a lysine racemase that converts L-lysine to D-lysine, which is then converted to cyclic ⁇ 1 - piperideine-2-carboxylate intermediate which is then spontaneously converted to 6-amino- 2-oxohexanoic acid in the presence of water (see Figure 4).
• Lysine racemase enzymes include enzymes of the EC 5.1.1.5 class.
• L-lysine is directly converted to cyclic A 1 -piperideine-2-carboxylate by a lysine dehydrogenase (EC 1.4.1.15, isolated from, for example, Agrobacterium tumefaciens or Candida albicans).
• L-lysine is converted to 6-amino-2-oxohexanoic acid by a lysine-a-oxidase (EC 1.4.3.14, Trichoderma viride i4).
• enzymes listed in Table 7 may be used or engineered to catalyze the conversion of L-lysine to 6-amino-2- oxohexanoic acid.
• 6-amino-2-oxohexanoic acid (cl) is converted to 6-amino-2-hydroxyhexanoic acid (c2, Pathways VII, VIII and EX).
• this reaction is catalyzed by a reductase or dehydrogenase enzyme.
• dehydrogenase enzymes capable of reducing the carboxylic group on c2 to a secondary alcohol may be considered. Examples of enzymes having, or which can be engineered to have, a 6- amino-2-oxohexanoic acid dehydrogenase activity can be isolated from a variety of sources and include, but are not limited to, the enzymes listed in Table 8.
• enzymatic step C3 (Pathway VIII) 6-amino-2-hydroxyhexanoic acid is converted to (E)-6-amino-hex-2-enoic acid by an enzyme having a dehydratase activity.
• Enzymatic reactions that produce enzymes having a dehydratase activity are part of the lyase class that catalyzes the removal of H 2 0, leaving a double bond as depicted in Figure 2.
• Polypeptides having dehydratase activity as well as nucleic acid encoding such polypeptides can be obtained from various species including, without limitation, Escherichia coli K12; Bacillus s btilis, Eubacterium barkeri, Glycine max, Anthracobia melaloma, Plicaria anthracina and Plicaria leiocarpa.
• Polypeptides having a dehydratase activity as well as nucleic acid encoding such polypeptides include, but are not limited to, enzymes listed in Table 3, and to enzymes EC 4.2.1.x, for example, EC 4.2.1.67 (D- fuconate hydratase), EC 4.2.1.68 (L-fuconate hydratase), EC 4.2.1.85 (dimethylmaleate hydratase), EC 4.2.1.- (crotonobetainyl-CoA hydratase) and EC 4.2.1.2 (Fumarate hydratase).
• q) Conversion of 6-amino-2-hydroxyhexanoic acid is first converted to 6-amino-2- hydroxyhexanoyl-CoA
• 6-amino-2-hydroxyhexanoic acid is first converted to 6-amino-2-hydroxyhexanoyl-CoA by a CoA transferase or a CoA ligase (enzymatic step C4, Pathways VIII and FX).
• Enzymes capable of catalyzing this reaction can be any suitable enzyme having a CoA transferase activity or a CoA ligase activity and include, but are not limited to, the enzymes listed in Table 5.
• polypeptides having a dehydratase activity as well as nucleic acids encoding such polypeptides include, but are not limited to, the enzymes listed in Table 3 and the following enzymes: EC 4.2.1.17 (enoyl-CoA hydratase or (3S)-3- hydroxyacyl-CoA hydro-lyase), EC 4.2.1.18 (methylglutaconyl-CoA hydratase or (S)-3- hydroxy-3-methylglutaryl-CoA hydro-lyase), EC 4.2.1.55 (Crotonase or 3- hydroxybutyryl-CoA dehydratase), EC 4.2.1.54 (lactyl-CoA dehydratase isolated in Clostridium propionicum), EC 4.2.1.56 (itaconyl-CoA hydratase isolated in
• HgdAB, CompD 2- Hydroxyglutaryl-CoA dehydratase
• a nitrogen-containing heterocyclic ring includes but is not limited to piperidine, pyridine, picolinate and pipecolate.
• a preferable nitrogen-containing heterocyclic ring is L-2,3-dihydropicolinate (also known as (S)-2,3-dihydropyridine-2,6-dicarboxylic acid), L-2,3,4,5,- terahydrodipicolinate (also know as A 1 -piperideine-2,6-dicarboxylate or (S)-2,3,4,5- terahydropyridin-2,6-dicarboxylic acid), A 1 -piperideine-2-dicarboxylate, A'-piperideine-6- dicarboxylate, 5,6-dihydropyridine-2-carboxylic acid, L-pipecolate and D-pipecolate.
• L-2,3-dihydropicolinate also known as (S)-2,3-dihydropyridine-2,6-dicarboxylic acid
• L-2,3,4,5,- terahydrodipicolinate also know as A 1 -piperideine-2,6-dicarboxylate or
• L-2,3-dihydropicolinate and ⁇ '-piperideine- 2,6-dicarboxylate are intermediate metabolites in the lysine biosynthesis pathways I, II, III, and VI.
• L-pipecolate, A 1 -piperideine-6-carboxylate, and A'-piperideine ⁇ -carboxylate are intermediate metabolites in the L-lysine or D-lysine degradation pathway.
• the bioconversion of lysine to A'-piperideine-2-carboxylate is catalyzed by a lysine dehydrogenase ( Figure 4, EC 1.4.1.15).
• Some aspects of the invention provide engineered metabolic pathways for the production of 6-amino-2-oxohexanoic acid from L-2-3-dihydrodipicolinate, an intermediate metabolite of the lysine biosynthetic pathway.
• Six lysine biosynthetic pathways have been described in bacteria, most algae, fungi and higher plants. These pathways are divided into the diaminopimelate pathways, and the a-aminoadipate pathways. In the pathways that belong to the diaminopimelate group, lysine is produced from aspartate (along with methionine, threonine and isoleucine).
• L- aspartate is first converted to L-aspartyl-4-phosphate by an aspartate kinase (or aspartokinase), and then to L-aspartate semialdehyde (aspartate semialdehyde dehydrogenase) and finally to L-2,3-dihydrodipicolinate
• Polypeptides having an aspartate kinase activity may be isolated from a variety of sources.
• suitable aspartate kinases and genes encoding for aspartate kinases are available from a variety of sources including, but not limited to, lysC ⁇ Escherichia coli KIT), thrA (Escherichia coli K12), metL (Escherichia coli KIT), AT3G02020 (Arabidopsis thaliana col), yclM (Bacillus subtilis), lysC , lysCa (Bacillus subtilis), lysC , lysCa (Corynebacterium glutamic m) and aspartokinase (Chromohalobacter salexigens).
• Polypeptides having an aspartate semialdehyde dehydrogenase activity, and genes encoding enzymes having an aspartate semialdehyde dehydrogenase activity may be isolated from a variety of sources such as asd from Escherichia coli K12.
• Polypeptides having a dihydropicolinate synthase activity, and genes encoding enzymes having a dihydropicolinate synthase activity may be isolated from a variety of sources.
• dihydrodipicolinate synthase and genes encoding for dihydrodipicolinate, are available from a variety of sources and include, but are not limited to, dapA (Escherichia coli KIT), dapA (Glycine max), dapA (Bacillus licheniformis) and dapA (Corynebacterium glutamicum).
• dapA Erichia coli KIT
• dapA Glycine max
• dapA Bacillus licheniformis
• dapA Corynebacterium glutamicum
• At least one of the genes for the lysine biosynthetic pathway or the lysine degradation pathway is functionally inactivated in the host cell.
• host cells may be microorganisms synthesizing less than 50%, less 40%, less than 30%, less than 20%, and preferably less than 10% of the amount of lysine naturally formed under the same conditions (e.g., in a host cell having an unmodified lysine biosynthetic and/or degradation pathway).
• C6 and C5 difunctional alkanes are produced from L-2,3-dihydropicolinate and 5,6-dihydropyridine-2-carboxylic acid.
• At least one of the following enzymes is inactivated at the genetic level: Dihydrodipicolinate reductase (EC 1.3.1.26, dapB gene), 2,3,4,5-tetrahydropyridine-2- carboxylate N-succinyltransferase (also known as Tetrahydrodipicolinate succinylase, EC 2.3.1.117, dapD gene), L,L-diaminopimelate aminotransferase (E.C. 2.6.1.83,
• AT4G33680 gene Hudson et al, 2006, Plant Physiol. 140(1):292-301; the enzyme was shown to be involved in lysine biosynthesis under physiological conditions by functional complementation of the E. coli lysine biosynthesis double mutant of dapD and dapE), or Tetrahydrodipicolinate N-acetyltransferase (EC 2.3.1.89, ykuQ (Bacillus subtilis)).
• C6 and C5 difunctional alkanes are produced from A'-piperideine- 2,6-dicarboxylate and preferably at least one of the following enzymes is inactivated at the genetic level: 2,3,4,5 etrahydropyridine-2-carboxylate N-succinyltransferase, L,L- diaminopimelate aminotransferase, Tetrahydrodipicolinate N-acetyltransferase, or Diaminopimelate dehydrogenase (EC 1.4.1.16, ddh (Corynebacterium glutamicum), dapdh (Lysinibacillus sphaericus)). Accordingly, aspects of the invention provide a recombinant microorganism having an engineered metabolic pathway for the production of C6 and C5 difunctional alkanes from L-2,3 dihydropicolinate.
• C5 and C6 afunctional alkanes are produced from A 1 -piperideine-2,6-dicarboxylate.
• 6- aminocaproate, adipic acid, 1,6-hexanediol and 6-hydroxyhexanoate are produced ( Figures 3A and 3B).
• L-2,3,dihydrodipicolinate is first converted to A'-piperidine- ⁇ -dicarboxylate (also known as L-2,3,4,5- tetrahydrodipicolinate, enzymatic step Dl), the resulting A'-piperidine- ⁇ e- dicarboxylate is converted to L-a-armno-s-ketopimelate (step D2), and the resulting L-a-amino-s-ketopimelate is converted to 6-amino-2-oxohexanoic acid (enzymatic step D3).
• 6-amino-2-oxohexanoic acid can be produced directly from lysine by a L-lysine a-oxidase ( Figure 4, EC 1.4.3.14, Trichoderma viride i4).
• L-2,3-dihydrodipicolinate is first converted to Al-piperidine-2,6-dicarboxylate (also known as L-2,3,4,5- tetrahydrodipicolinate, enzymatic step Dl), the resulting A ⁇ piperidine ⁇ - dicarboxylate is then converted to A'-piperidine ⁇ -carboxylate (enzymatic step D'2), and the resulting A 1 -piperidine-2-carboxylate is converted to 6-amino-2- oxohexanoic acid (step D'3).
• a ⁇ piperidine ⁇ - carboxylate may be first converted to L-pipecolate (enzymatic step D'4) and then to 6-amino-2-oxohexanoic acid (enzymatic step D'5).
• L-2,3-dihydrodipicolinate may be converted to ⁇ 1 piperidine-2,6-dicarboxylate (or tetrahydropicolinate) by action of an enzyme such as a dihydrodipicolinate reductase (belonging to EC 1.3.1.26, enzymatic step Dl) that catalyzes the following chemical reaction in the lysine biosynthesis pathway:
• Polypeptides having a dihydrodipicolinate reductase activity may be isolated from a variety of sources and include, but are not limited to, dapB from Escherichia coli K12, dapB from Bacillus subtilis, dapB from Corynebacterium glutamicum, dapB from Mycobacterium tuberculosis and dihydrodipicolinate reductase from Zea mays.
• a 1 -piperidine-2,6-dicarboxylate (or tetrahydropicolinate) is then converted to A'-piperidine ⁇ -carboxylate (or 3,4,5,6- tetrahydropyridine-2-carboxylic acid) by action of a decarboxylase.
• a decarboxylase Although there are no known enzymes having a A 1 -piperidine-2,6-dicarboxylate decarboxylase activity, polypeptides capable of removing carboxyl groups from aromatic rings may be used.
• the A'-piperidine ⁇ -carboxylate ring can then spontaneously open in the presence of water to produce 6-amino-2-oxohexanoic acid as depicted below in the lysine degradation pathways V and VII.
• Examples of enzymes having a decarboxylase activity include, but are not limited to, 3-hydroxy-2-methylpyridine-4,5-dicarboxylate 4-decarboxylase (EC 4.1.1.51), benzoylformate decarboxylase (EC 4.1.1.7) and 2,3-dihydrobenzoic acid decarboxylase (See US patent 6,440,704).
• 3-hydroxy-2-methylpyridine-4,5-dicarboxylate 4- decarboxylase has been identified in Mesorhizobium loti and in Pseudomonas sp. MA-1.
• Benzoylformate decarboxylase catalyzing the reaction as shown below
• the genes encoding such polypeptides may be found in a variety of species including, but not limited to, Pseudomonas fluorescens and Pseudomonas putida (gene mdlC, UniProt:P20906).
• a 1 -piperidine-2,6-dicarboxylate undergoes spontaneous dehydration to produce L- -amino-s-ketopimelate as in the lysine bios
• L-a-amino-s-ketopimelate is converted to 6-amino-2- hexanoic acid by action of a decarboxylase, for example, diaminopimelate decarboxylase (EC 4.1.1.20), an enzyme shown to catalyze the last reaction step in the lysine biosynthesis pathway:
• a decarboxylase for example, diaminopimelate decarboxylase (EC 4.1.1.20)
• EC 4.1.1.20 diaminopimelate decarboxylase
• gadA Erscherichia — * ⁇ C ⁇ 2 4 * ⁇ ⁇ a coli K12
• 6-amino-2-oxohexanoic acid is produced by decarboxylation of L-2,3-dihydropicolinate to 5,6-dihydropyridine-2-carboxylate (enzymatic step El), followed by a spontaneous hydration of 5,6-dihydropyridine-2- carboxylate to (E)-6-amino-2-oxohex-3-enoic acid (Step E2), and a subsequent dehydrogenation of (E)-6-amino-2-oxohex-3-enoic acid to 6-amino-2-oxohexanoic acid (enzymatic step E3) as depicted in Figure 3A.
• L-2,3-dihydropicolinate is first converted to 5,6- dihydrop
• decarboxylases include, but are not limited to, benzoylformate decarboxylase (EC 4.1.1.7), 3-hydroxy-2-methyl pyridine-4,5-dicarboxylate decarboxylase (EC 4.1.1.51), pyrrole-2-carboxylase (EC 4.1.1.-), gallate decarboxylase (4.1.1.59), Dopa decarboxylase (EC 4.1.1.28), Phenylpyruvate decarboxylase (EC 4.1.1.43), 4,5-dihydroxyphthalate decarboxylase (EC 4.1.1.55), 5-dihydroxybenzoate decarboxylase (EC 4.1.1.62), Uracil-5- carboxylate decarboxylase (EC 4.1.1.66), and Cis-l,2-dihydroxycyclohexa-3,5-diene-l- carboxylate dehydrogenase (EC 1.3.1.25).
• benzoylformate decarboxylase EC 4.1.1.7
• step (E3) the (E)-6-amino-2-oxohex-3-enoic acid is converted to 6-arnino-2-oxohexanoic acid.
• the enzymes listed in Table 4 may also have an activity towards (E)-6-arnino-2-oxohex-3-enoic acid or could be engineered to catalyze the reaction.
• Theses enzymes include, but are not limited to, 2-enoate reductase (EC 1.3.1.31), NADH-dependent fumarate reductase (EC 1.3.1.6), coumarate reductase (EC 1.3.1.11), ⁇ -nitroacrylate reductase (EC 1.3.1.16), Maleylacetate reductase (EC 1.3.1.32), N-ethylmaleimide reductase (EC 1.-.-.-) and EC 1.3.99.-.
• (E)-6-amino-2-oxohex-3-enoic acid could first be converted to (E)-6- amino-2-oxohex-3-enoyl-CoA, then to (E)-6-amino-2-oxohexanoyl-CoA, and then to (E)- 6-amino-2-hydroxyl-hexanoyl-CoA.
• the final steps may be identical to the ones detailed in Pathways VIII and EX.
• lysine may be directly converted to 6-amino-2- oxohexanoic acid by action of L-lysine oxidase as described in the lysine degradation pathway VII (EC 1.4.1.3.14) ( Figure 4).
• the L-lysine oxidase has been described in strains of the ascomycetes Trichoderma viride and Trichoderma harzianum.
• aspects of the invention relate to engineered metabolic pathways for the bioproduction of adipic acid, 1,6-hexanediol and 6-hydroxyhexanoate from nitrogen- containing heterocyclic rings.
• One aspect of the invention provides engineered metabolic pathways for the bioproduction of adipic acid, 6-hydroxyhexanoate (6HH) and 1,6- hexanediol from nitrogen-containing heterocyclic rings.
• engineered metabolic pathways for the production of 2-oxoadipate semialdehyde as a metabolite intermediate for the production of adipic acid, 6-hydroxyhexanoate and/or 1,6- hexanediol are provided.
• aspects of the invention provide recombinant microorganisms having an engineered pathway for the production of adipic acid.
• Other aspects of the invention provide a recombinant microorganism having an engineered pathway for the production of 6-hydroxyhexanoate.
• Yet other aspects of the invention provide a recombinant microorganism having an engineered pathway for the production of 1 ,6-hexanediol.
• the C6 difunctional alkanes are produced from
• L-pipecolate is an intermediate of the L-lysine and D-lysine degradation pathway. L-pipecolate may also be bioproduced according to Pathway D' as describe herein. According to some aspects of the invention, L-pipecolate is converted to ⁇ 1 - piperideine-6-L-carboxylate, and then A'-piperideine-e-L-carboxylate is converted to L-2- aminoadipate-6-semialdehyde; L-2-aminoadipate-6-semialdehyde is optionally converted to L-2-aminoadipate.
• A'-piperideine-e-L-carboxylate is converted to L-2- aminoadipate-6-semialdehyde
• L-2-aminoadipate-6-semialdehyde is optionally converted to L-2-aminoadipate.
• this set of reactions is part of the lysine degradation pathway.
• L-pipecolate is converted to A ⁇ piperideine-e-L-carboxylate by action of L-pipecolate dehydrogenase or L- pipecolate oxidase.
• L-pipecolate dehydrogenases and L-pipecolate oxidase are known in the art and are part of the lysine degradation pathway. Examples of L-pipecolate oxidases include, but are not limited to, EC 1.5.99.3 (Pseudomonas putida) and EC 1.5.3.7. (Schizosaccharomyces pombe, Arabidopsis thaliana, Homo sapiens).
• the enzyme is L-pipecolate dehydrogenase with EC number 1.5.99.3. [00123] The reactions catalyzed by L-pipecolate oxidase or dehydrogenase are shown below:
• L-pipecolate + 02 2,3,4,5-tetrahydropyridine-2-carboxylate + H202 reacts with water to form spontaneously L-2- aminoadipate-6-semialdehyde.
• A'-piperideine-e-L-carboxylate and L-2-aminoadipate-6- semialdehyde can then be converted to 2-aminoadipate by an L-aminoadipate- semialdehyde dehydrogenase or an oxido-reductase (EC 1.2.1.31), the oxidizing equivalent being supplied in the form of an oxidized nicotinamide cofactor NAD or NADH as detailed below:
• L-aminoadipate-semialdehyde dehydrogenase or oxido-reductase have been described in a variety of species including Pseudomonas putida (encoded by amaA), Streptomyces clavuligerus (encoded by pcd), Flavobacterium lutescens (encoded by pcd), Homo sapiens, Mus musculus, Rattus norvegicus, Schizosaccharomyces pombe, Acremonium chrysogenum, Penicillium chrysogenum and Candida albicans.
• 2-aminoadipate can be converted to adipic acid by removal of the a-amino group as described for the deamination of lysine in Pathways I through IX.
• 2-aminoadipate may be converted to oc-ketoadipate by a a- aminoadipate aminotransferase (EC 2.6.1.39, gene aadat, lysN) or a diaminopimelate dehydrogenase (EC 1.4.116, gene ddb or Dapdh) and subsequently to adipic acid as described in pathway VII through ⁇ .
• L-2-aminoadipate-6-semialdehyde is converted to adipate semialdehyde by removal of the a-amino group as described in Pathways I through ⁇ .
• Adipate semialdehyde can then finally be converted to 1,6- hexanediol or 6-hydroxyhexanoate by an aldehyde dehydrogenase and/or an alcohol dehydrogenase.
• Alcohol dehydrogenases catalyze the conversion of the aldehyde functional group into an alcohol functional group.
• Alcohol dehydrogenases (ADHs) (EC 1.1.1.1 and EC 1.1.1.2) catalyze the reversible reduction of ketones and aldehydes to alcohols with the reduction of NAD+ to NADH.
• the alcohol dehydrogenase includes, but is not limited, to adhA or adhB (from Z. mobilis), butanol dehydrogenase (from Clostridium acetobutylicum), propanediol oxidoreductase (from E.
• the hydro-carboxylic acid is subjected to dehydrogenation using an alcohol dehydrogenase or an aldehyde dehydrogenase to produce 1 ,6-hexane diol.
• Aldehyde NAD(+) dehydrogenase activity and alcohol NAD(+) dehydrogenase activities can be carried out by two different polypeptides, or carried out by a single polypeptide, such as a multi-functional aldehyde-alcohol dehydrogenase (EC 1.2.1.10) from E. coli (Goodlove et al.
• Polypeptides having aldehyde dehydrogenase (NAD(P)+) (EC 1.2.1.-) or aldehyde dehydrogenase (NAD(+)) (EC 1.2.1.3) activity can be used in combination with an alcohol dehydrogenase to reduce the remaining carboxylic acid to an alcohol, yielding a diol.
• Nucleic acids encoding such polypeptides can be obtained from various species including, without limitation, S. cerevisiae.
• Some aspects of the invention provide biosynthetic pathways for the production of adipic acid, 6-hydroxyhexanoate, 1,6-hexanediol from lysine and from 2- oxoadipate semialdehyde. All these pathways share the first enzymatic step in which lysine is first converted to 2-amino-adipate-6-semialdehyde (see Figure 5).
• lysine in enzymatic step 5a or 5m, lysine is first converted to a-aminoadipate semialdehyde (also known as 2-aminoadipate-6-semialdehyde), the resulting a-aminoadipate semialdehyde is then converted to 2-oxoadipate semialdehyde (enzymatic step 5b).
• a-aminoadipate semialdehyde also known as 2-aminoadipate-6-semialdehyde
• 2-oxoadipate semialdehyde enzymatic step 5b
• lysine is converted to a-aminoadipate semialdehyde by action of an enzyme having a lysine amino-transferase activity.
• the first enzyme is a lysine amino-transferase (enzymatic step 5a, EC 2.6.1.36).
• Enzymes catalyzing this substrate to product conversion are well known and participate in the lysine degradation pathway VI and employ pyridoxal phosphate as a cofactor.
• L-lysine 6-aminotransferase has been demonstrated in yeasts and bacteria. Examples of suitable L-aminotransferase enzymes are available from a variety of sources, for example, Nocardia lactamdurans (UniProt accession number Q05174),
• Flavobacterium lutescens (gene lat, Fujii et al., 2000 , J. Biochem. ,128(3);391-7), Candida utilis, Streptomyces clavuligerus (UniProt: Q01767, gene lat), Rhodococcus sp., Mycobacterium marinum, Mycobacterium ulcerans, Saccharopolyspora erythraea, Frankia alni and Frankia sp.
• the second known enzyme capable of catalyzing the substrate to product conversion of lysine to a-aminoadipate semialdehyde is L-lysine- ⁇ -dehydrogenase (enzymatic step 5m).
• L-lysine ⁇ -dehydrogenase catalyzes the oxidative deamination of the lysine epsilon amino group in an NAD+-dependent reaction (EC 1.4.18; Lysine degradation pathway VIII):
• L-lysine ⁇ -dehydrogenase enzymes are available from a variety of sources, for example, Agrobacterium tumefaciens and Geobacillus stearothermophilus (gene: lysDH)
• 2-aminoadipate-6-semialdehyde can then be converted to adipate semialdehyde (enzymatic step 5b followed by enzymatic step 5g or enzymatic step 5f) for the production of adipic acid, 6HH, or 1,6-hexanediol.
• adipate semialdehyde is produced via an engineered pathway comprising enzymatic steps 5b and 5g.
• 2- arriinoadipate-6-semialdehyde is converted to 2-oxoadipate semialdehyde by action of an enzyme having a 2-aminoadipate activity.
• an enzyme having a 2-aminoadipate activity There are two known types of biochemical reactions that could affect the substrate to product conversion, namely the 2-aminoadipate aminotransferase (EC 2.6.1.39) and the Kynurenine aminotransferase II (EC 2.6.1.7) as depicted below.
• Enzymes of the EC 2.6.1.39 class catalyze this substrate to product conversion and participate in the lysine degradation II, lysine degradation V, lysine biosynthesis IV and lysine biosynthesis V pathways.
• suitable a-aminoadipate aminotransferase enzymes are available from a variety of sources, for example, Thermits thermophilus (gene LysN, UniProt: Q5SL82, Q7X567, Q72LL6, Miyazaki et al.
• step 5g 2-ketopadipate semialdehyde is converted to adipate semialdehyde by first action of a dehydrogenase (enzymatic step 5gl) to convert the ketone group to a secondary alcohol group (2-hydroxyadipate semialdehyde), followed by action of a dehydratase that catalyzes the conversion of the secondary alcohol group to an alkene (enzymatic step 5g2), and finally followed by the action of a dehydrogenase (enzymatic step 5g3) that catalyzes the conversion of the alkene to an alkane.
• a dehydrogenase enzyme to convert the ketone group to a secondary alcohol group (2-hydroxyadipate semialdehyde
• a dehydratase that catalyzes the conversion of the secondary alcohol group to an alkene
• step 5g3 a dehydrogenase
• ketoadipate semialdehyde is first converted to the thiol ester by CoA transferase and then subjected to enzymatic steps 5gl to 5g3.
• Enzymes capable of catalyzing enzymatic steps 5g include, but are not limited to, enzymes listed in enzymatic steps C2, C4, C5, C3, B4, B5, B6 and A5.
• 2-ketopadipate semialdehyde is first converted to the corresponding acp-compound.
• the dehydrogenase is a 3- oxoacyl-[acyl-carrier protein] reductase (EC 1.1.1.100) shown to catalyze the following reaction in the fatty acid biosynthesis superpathway:
• the dehydrogenase is a ⁇ -ketoacyl-facyl-camer- protein] reductase (FabG gene from E. Coli), the fatty acid synthase (Fas2 gene from Saccharomyces cerevisiae) or the fatty acid synthase (FASN gene from Homo sapiens).
• the dehydratase is a 3-hydroxyoctanoyl-[acyl-carrier-protein] which catalyses the following reaction:
• 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratases include, but are not limited to, 3-hydroxyacyl-ACP dehydrase from Spinacia oleracea, ⁇ - hydroxyacyl-ACP dehydrase (FabA gene from E. Coli), and ⁇ -hydroxyacyl-ACP dehydratase (i3 ⁇ 4i>Zgene from E. Coli).
• the second dehydrogenase is an enoyl acyl carrier protein reductase (EC 1.3.1.9) such as enoyl-ACP reductase ( aWgene form E. Coli) shown to catalyze the following reaction:
• the first and the second dehydrogenase are identical fatty acid synthases (Fas2 gene from Saccharomyces cerevisiae or FASN gene from Homo sapiens).
• adipate semialdehyde can be used to produce 1,6- hexanediol, 6HH, adipic acid, aminocaproic acid, hexamemylenediamine or 6- aminohexanol.
• adipate semialdehyde is converted to 6HH by simple hydrogenation and the reaction is catalyzed by an alcohol dehydrogenase (EC 1.1.1.1).
• This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donors with NAD + or NADP + as acceptors.
• a 6-hydroxyhexanoate dehydrogenase (EC 1.1.1.258) that catalyzes the following chemical reaction is used:
• alcohol dehydrogenases include but are not limited to adhA or adhB
• butanol dehydrogenase from Clostridium acetobutylicwn
• propanediol oxidoreductase from E. coli
• ADHIV alcohol dehydrogenase from Saccharomyces
• adipate semialdehyde is converted to 6- hydroxyhexanoate by an alcohol dehydrogenase and then to 1,6-hexanediol by action of an alcohol dehydrogenase or an aldehyde dehydrogenase (enzymatic step 5i).
• Alcohol dehydrogenases (ADHs) (EC 1.1.1.1 and EC 1.1.1.2) catalyze the reversible reduction of ketones and aldehydes to alcohols with the reduction of NAD + to NADH.
• examples of alcohol dehydrogenases include but are not limited to adhA or adhB (from Z.
• Aldehyde NAD(+) dehydrogenase activity and alcohol NAD(+) dehydrogenase activities can be carried out by two different polypeptides, or carried out by a single polypeptide, such as a multi-functional aldehyde- alcohol dehydrogenase (EC 1.2.1.10) from E. coli (Goodlove et al. Gene 85:209-14, 1989; GenBank Accession No. M33504).
• EC 1.2.1.10 multi-functional aldehyde- alcohol dehydrogenase
• Polypeptides having aldehyde dehydrogenase (NAD(P)+) (EC 1.2.1.-) or aldehyde dehydrogenase (NAD(+)) (EC 1.2.1.3) activity, as well as nucleic acids encoding such polypeptides, can be obtained from various species including, without limitation, S. cerevisiae.
• 2-aminoadipate-6-semialdehyde is first converted to 2-aminoadipate (enzymatic step 5c), the resulting 2-aminoadipate is converted to 2-ketoadipate (enzymatic step 5d), and finally to adipic acid (enzymatic step 5h).
• 2-aminoadipate-6-semialdehyde is converted to 2-aminoadipate by action of a L-aminoadipate-semialdehyde dehydrogenase (EC 1.2.1.31).
• L-aminoadipate-semialdehyde dehydrogenase enzymes are available form a number of sources, for example, Streptomyces clavuligerus (pcd), Flavobacterium lutescens (pcd), Acremonium chrysogenum, Candida albicans, Candida maltosa, Homo sapiens, Kluyveromyces lactis, Mus museums, Penicillium chrysogenum, Pichia guilliermondii, Pseudomonas putida, Pseudomonas sp., Rattus norvegicus, Saccharomyces cerevisiae and Schizosaccharomyces pombe.
• enzymatic step 5d the resulting 2-aminoadipate is converted to 2- ketoadipate by action of a 2-aminoadipate aminotransferase (EC 2.6.1.3 ) which participates in the amino-acid degradation pathway; L-lysine degradation via saccharopine pathway and in the glutaryl-CoA from L-lysine pathway.
• 2-ketoadipate is then converted to adipic acid (enzymatic steps 5h) by successive enzymatic steps described for the conversion of 2-ketoadipate semialdehyde to adipate semialdehyde.
• 2-amino adipate is converted to adipic acid by successive enzymatic steps as described above for the conversion of l-aminoadipate-6- semialdehyde to adipate semialdehyde (enzymatic step 5f, see Pathways I through ⁇ ).
• aspects of the invention relates to the production of 6- hydroxyhexanamine and/or hexamethylenediamine. Accordingly, aspects of the invention provide a recombinant microorganism having an engineered pathway for the production of 6-hydroxyhexanamine and hexamethylenediamine.
• lysine is converted to 2,6-diaminohexanal by action of an amino aldehyde dehydrogenase (enzymatic step 6a).
• 2,6-diaminohexanal can then be converted to 6-aminohexanal by deamination (enzymatic step 6b) and subsequently to 6-aminohexanol by an alcohol dehydrogenase (enzymatic step 6d).
• 2,6-diaminohexanal is converted to 2-oxo-l,6-diaminohexane (enzymatic step 6c) by a semialdehyde aminomutase.
• Glutamate-1 -semialdehyde 2,1-aminomutase may catalyze this reaction.
• Glutamate-1 - semialdehyde 2,1-aminomutase has been isolated in a variety of species including, but not limited to, Escherichia coli, Synechococcus sp, Xanthomonas campestris, and
• Glutamate-1 -semialdehyde 2,1-aminomutase genes include, but are not limited to, hemL ⁇ Escherichia coli K12, Salmonella typhimurium) and GSAl (Arabidopsis thaliana col).
• 2-oxo-l,6-diaminohexane is then converted to 1,6-diaminohexane by removal of the ketone group (enzymatic step 6h).
• 2-oxo-l,6-diaminohexane is first reduced to 2-hydroxy- 1,6- diaminohexane (enzymatic step 6e) by an alcohol dehydrogenase, and then further reduced to 1,6-diamino-hexene (enzymatic step 6f) which can then be converted to hexamethylenediamine (enzymatic step 6e).
• aspects of the invention relate to the bioproduction of C5 difunctional alkanes.
• C5 difunctional alkanes of interest include 5-hydroxypentanoate, ghitarate, 1,5- pentanediol, 5-aminopentanoate, 5-aminopentanol and cadaverine.
• aspects of the invention provide a recombinant microorganism having an engineered pathway for the production 5-hydroxypentanoate, ghitarate, 1 ,5-pentanediol, 5-aminopentanoate, 5- aminopentanol and cadaverine.
• C5 difunctional alkanes are produced from lysine as shown in Figure 7.
• Methods for producing cadaverine by introducing a lysine decarboxylation gene and or a lysine-cadaverine antiporter gene into a lysine producing microorganism have been described (see for example JP 2002223770 and WO2008/092720).
• Lysine decarboxylase catalyzes the decarboxylation of L-lysine into cadaverine.
• the enzyme has been classified as EC 4.1.1.18.
• the enzymes isolated from Escherichia coli having lysine decarboxylase activity are the cadA gene product and the ldc gene product.
• the C5 difunctional alkanes engineered pathway begins with the conversion of lysine to 2-amino-5-oxo-hexanoate (or a-aminoadipate semialdehyde) by a L-lysine-6-aminotransferase (classified as EC 2.6.1.36 and EC 1.4.1.18) as described in the bioproduction of C6 difunctional alkanes (enzymatic step 5a).
• a-arninoadipate semialdehyde is converted to 5-aminopentanal by a decarboxylase (enzymatic step 7b).
• decarboxylase examples include, but are not limited to, lysine decarboxylase, L-Orm1bine carboxylase (EC 4.1.1.17, ode gene product and spec speF gene product), Aspartate 4-decarboxylase (EC 4.1.1.12), and glutamate decarboxylase (gadA ⁇ Escherichia coli K12), gadB (Escherichia coli KIT), GAD1 (Saccharomyces cerevisiae S288Q, GAD2 and GAD 1 (Homo sapiens, Arabidopsis thaliana col).
• 5-aminopentanal can be converted to 5-aminopentanol by an alcohol dehydrogenase or to 5-aminopentanoate by an aldehyde dehydrogenase.
• a-aminoadipate semialdehyde is converted to oc- ketoadipate semialdehyde by a a-aminoadipate aminotransferase (enzymatic step 5b, EC 2.6.1.39, lysine degradation pathway).
• the glutarate and the 1,5-pentanediol pathways involve a ketodecarboxylase to convert a-ketoadipate semialdehyde to 5-oxopentanal (enzymatic step 7e) and an alcohol dehydrogenase to convert 5-oxopentanal to 1,5- pentanediol (enzymatic step 7f) or an aldehyde dehydrogenase to convert 2-oxopentanal to glutarate (enzymatic step 7g).
• ketodecarboxylases Although there are no known enzymes shown to catalyze the decarboxylation of a-ketoadipate semialdehyde to 5-oxopentanal, a list of ketodecarboxylases was generated based on the following criteria: (i) demonstrated activity on a 2-ketocarboxylate, and (ii) availability of protein sequence information.
• Alcohol dehydrogenases (EC 1.1.1.1 and 1.1.1.2) catalyze the reversible reduction of ketones and aldehydes to alcohols with the reduction of NAD+ to NADH.
• examples of alcohol dehydrogenases include, but are not limited to, adhA or adhB (from Z. mobilis), butanol dehydrogenase (from Clostridium acetobutylicum), propanediol oxidoreductase (from E. coli), ADHIV alcohol dehydrogenase (from Saccharomyces), and ADH6 (from S. cerevisiae).
• Aldehyde NAD(+) dehydrogenase activity and alcohol NAD(+) dehydrogenase activities can be carried out by two different polypeptides, or carried out by a single polypeptide, such as a multi-functional aldehyde-alcohol dehydrogenase (EC 1.2.1.10) from E. coli (Goodlove et al. Gene 85:209-14, 1989; GenBank Accession No. M33504).
• EC 1.2.1.10 multi-functional aldehyde-alcohol dehydrogenase from E. coli
• Polypeptides having aldehyde dehydrogenase (NAD(P)+) (EC 1.2.1.-) or aldehyde dehydrogenase (NAD(+)) (EC 1.2.1.3) activity can be used in combination with an alcohol dehydrogenase to reduce the remaining carboxylic acid to an alcohol, yielding a diol.
• Nucleic acids encoding such polypeptides can be obtained from various species including, without limitation, S. cerevisiae.
• a-ketoadipate semialdehyde is converted to ketoadipate by an aldehyde dehydrogenase including, but not limited to, acetaldehyde dehydrogenase (EC 1.2.1.4 encoded by aldB ⁇ Escherichia coli K12), ALD6
• enzymatic step 7h 2-ketoadipate is then converted to glutarate semialdehyde by a keto-decarboxylase listed in Table 10 and then to 5-hydroxypentanoic acid by an alcohol dehydrogenase.
• aspects of the invention relate to the bioproduction of C5 difunctional alkanes from nitrogen-containing heterocyclic ring ( Figure 8).
• 2-aminoadipate semialdehyde can be generated from, for example, L-pipecolate as discussed in the bioproduction of C6 difunctional alkanes.
• 2-aminoadipate semialdehyde can then be converted to C5 difunctional alkane as disclosed above.
• enzymes catalyzing enzymatic steps 5b may also catalyze enzymatic step 7a; and enzymes catalyzing enzymatic step 5e may also catalyze enzymatic step 7j.
• the carbon chain of lysine is elongated to form a
• the carbon chain of lysine is elongated following a pathway similar to the a-keto-elongation pathway.
• the a-keto-elongation pathway comprises three enzymes: an homocitrate synthase, an homoaconitase and a homo-isocitrate dehydrogenase.
• the lysine biosynthesis pathway IV provides enzymes for the elongation of 2-ketoglutarate to a-ketoadipate.
• 2-ketoglutarate is converted to homocitrate by a homocitrate synthase (EC 2.3.3.14, LYS20 and LYS21 (Sacharomyces cerevisiae), nifV (Klebsiella pneumoniae), hcs (Thermus thermophilusj).
• a homocitrate synthase EC 2.3.3.14, LYS20 and LYS21 (Sacharomyces cerevisiae), nifV (Klebsiella pneumoniae), hcs (Thermus thermophilusj).
• Homocitrate is then converted to cis-homoaconitate and then to homoisocitrate by a homoaconitase (EC 4.2.1.36, LYS4 (Sacharomyces cerevisiae), lysU, lysT (Thermus thermophilusj) and homoisocitrate is converted to a- ketoadipate by a homo-isocitrate dehydrogenase (EC 1.1.1.87, LYS12 (Sacharomyces cerevisiae), hicdh (Thermus thermophilusj).
• a homoaconitase EC 4.2.1.36, LYS4 (Sacharomyces cerevisiae), lysU, lysT (Thermus thermophilusj)
• homoisocitrate is converted to a- ketoadipate by a homo-isocitrate dehydrogenase (EC 1.1.1.87, LYS12 (Sacharomyces cerevisi
• 2,7-diaminoheptanoic acid is converted to hexamethylenediamine in a single enzymatic step catalyzed by a decarboxylase.
• the decarboxylase is lysine decarboxylase. Lysine decarboxylases (EC 4.1 .1 .18) isolated from Escherichia coli having lysine decarboxylase activity are the cadA gene product and the ldc gene product. Other decarboxylases capable of catalyzing the substrate to product reaction are listed in Table 9.
• a 2,7-diaminoheptanoic acid is converted to 7- amino-2-oxoheptanoic acid by an aminotransferase enzyme (EC 2.6.1.x) or a dehydrogenase (EC 1.4.1.x or EC 1.4.3.x).
• aminotransferase enzyme EC 2.6.1.x
• dehydrogenase EC 1.4.1.x or EC 1.4.3.x
• Preferred enzymes are listed in Table 7 and may be engineered to catalyze the desired substrate to product reaction.
• lysine is first converted to 6-amino-2- oxohexanoic acid by an aminotransferase enzyme (EC 2.6.1.x) or a dehydrogenase (EC 1.4.1.x or EC 1.4.3.x).
• aminotransferase enzyme EC 2.6.1.x
• dehydrogenase EC 1.4.1.x or EC 1.4.3.x
• Preferred enzymes are listed in Table 7 and may be engineered to catalyze the desired substrate to product reaction.
• 6-amino-2- oxohexanoic acid is subjected to a carbon elongation enzymatic step to produce 7-amino- 2-oxoheptanoic acid.
• 7-amino-2-oxoheptanoic acid is converted to 6- aminohexanal by a decarboxylase.
• Preferred decarboxylases are lysine decarboxylases (EC 4.1.1.18).
• the 7-amino-2-oxoheptanoic acid to 6- arninohexanal conversion is catalyzed by an enzyme listed in Table 9.
• 6-aminohexanal is subsequently converted to 6-aminocaproic acid by an aldehyde dehydrogenase (EC 1.2.1.3) or aldehyde oxidase (EC 1.2.3.1, AA02 (Arabidopsis thaliana col), AAOl (Arabidopsis thaliana col), AOX1 (Homo sapiens)).
• aldehyde dehydrogenase EC 1.2.1.3
• aldehyde oxidase EC 1.2.3.1, AA02 (Arabidopsis thaliana col), AAOl (Arabidopsis thaliana col), AOX1 (Homo sapiens)
• 2,7-diaminoheptanoic acid is converted to 6- aminohexamide by a monooxygenase.
• Preferred monooxygenase is a L-lysine monooxygenase (EC 1.13.12.12, davB in Pseudomonas fluorescens) that catalyses the conversion of L-lysine to 5-aminopentamide in the lysine degradation IV pathway.
• 6- aminohexamide is converted to 6-aminocaproic acid by an amidase.
• the amidase is a ⁇ -aminovaleramidase (EC 3.5.1.30, davA
• Microorganisms may be cultivated continuously or discontinuously in a batch process (batch cultivation) or in a fed-batch process (feed process) or repeated fed- batch process (repetitive feed process) for the purposes of difunctional alkanes.
• the culture medium to be used must satisfy in a suitable manner the requirements of the respective strains. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). Media must contain suitable carbon sources such as monosaccharides (e.g. glucose and fructose), oligosaccharides (e.g. sucrose, lactose), polysaccharides (e.g. starch and cellulose), oils and fats or mixture thereof.
• monosaccharides e.g. glucose and fructose
• oligosaccharides e.g. sucrose, lactose
• polysaccharides e.g. starch and cellulose
• Media must contain a nitrogen source such as organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soy bean flour and urea, or inorganic compounds such as ammomum sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammomum nitrate.
• a nitrogen source such as organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soy bean flour and urea, or inorganic compounds such as ammomum sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammomum nitrate.
• the nitrogen sources may be used individually or as a mixture.
• media In addition to the carbon sources and nitrogen sources, media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for growth of the culture and promotion of the enzymatic pathways for C5 and C6 difunctional alkanes.
• Suitable growth media includes common commercially available media such as Luria Bertani (LB) broth, Yeast medium (YM) or any synthetic or defined media.
• Suitable pH ranges are between pH 5.0 to pH 9.0.
• basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid are used as appropriate.
• Culture may be performed under aerobic or anaerobic conditions.
• reaction mixtures for pathway development may be carried out in any vessel that permits cell growth and/or incubation.
• a reaction mixture may be a bioreactor, a cell culture flask or plate, a multiwell plate (e.g., a 96, 384, 1056 well microliter plates, etc.), a culture flask, a fermentor, or other vessel for cell growth or incubation.
• Screening may be carried out by detection of expression of a selectable marker, which, in some genetic circumstances, allows cells expressing the marker to survive while other cells die (or vice versa). Efficient screening techniques are needed to provide efficient development of novel pathways using the methods described herein.
• suitable screening techniques for compounds produced by the enzymatic pathways allow for a rapid and sensitive screen for the properties of interest.
• Visual (colorimetric) assays are optimal in this regard, and are easily applied for compounds with suitable light absorption properties.
• More sophisticated screening technologies include, for instance, high-throughput HPLC-MS analysis, SPME (Solid Phase Microextraction) and GC-MS (Gas chromatography-mass spectrometry) (see Handbook of analytical derivatization reaction, D. R. Knapp; John Wiley & Sons, 1979).
• screening robots are connected to HPLC-MS systems for automated injection and rapid sample analysis. These techniques allow for high-throughput detection and quantification of virtually any desired compound.
• Bioproducts of interest may be isolated from the fermentation medium or cell extract using methods known in the art. For example, solids or cell debris may be removed by centrifugation, filtration, decantation and the like. Bioproducts of interest may be isolated by distillation, liquid-liquid extraction, membrane evaporation, adsorption, or using any methods known in the art.
• identification of the product of interest may be performed using an UPLC.
• the standard samples are prepared with known amounts of the organic product in the medium (e.g. adipic acid, amino caproic acid).
• the retention time of the adipic acid produced can then be compared to that of the authentic standard.
• identification of the product of interest may be performed using a GC-MS.
• the resolved samples are then analyzed by a mass selective detector and compared to previous mass spectra and retention time of authentic standards.
• cellular extracts may be screened for enzyme activity.
• oxohexanoate dehydrogenase activity may be detected by measuring the rate of increase of absorbance at 340 nm as described in Donoghue and Trudgill (Eur. J. Biochem., 1975, 60:1-7).
• the present invention provides among other things compositions and methods for metabolic engineering. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

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