Microorganisms and methods for increased diamine production

Abstract

The disclosure provides polypeptides and encoding nucleic acids of engineered diamine exporters. The disclosure also provides cells expressing engineered diamine exporter or exogenous diamine exporters. The disclosure further provides methods for diamine bioproduction.

Description

Attorney Docket No.199683-806002 / PCT MICROORGANISMS AND METHODS FOR INCREASED DIAMINE PRODUCTION CROSS-REFERENCE

[0001] This application claims benefit of U.S. Provisional Application No.63 / 503,854, filed on May 23, 2023, the entire contents of which are incorporated herein by reference. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted via Patent Center. The Sequence Listing titled 199683-806002_PCT_SL.xml, which was created on May 3, 2024 and is 59,147 bytes in size, is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION

[0003] The present disclosure relates generally to diamine exporter variants and methods of use thereof. The present disclosure also relates to non-naturally occurring microbial organisms that express diamine exporter variants, or exogenous diamine exporters, for bioproduction of a diamine. BACKGROUND OF THE INVENTION

[0004] Diamines represent key raw materials in the chemical industry. Diamines are often used as monomers to prepare polyamides, polyimides, polyureas, polyurethanes, as well as copolymers that include these materials. For instance, hexamethylenediamine (HMD) is used in the production of nylon-6,6, a polyamide common in the textile and plastic industries. HMD is also utilized to make hexamethylene diisocyanate, a monomer feedstock used in the production of polyurethane, as well as a cross-linking agent in epoxy resins. Other diamines, such as ethylenediamine, heptamethylenediamine, cadaverine and putrescine, are also widely used. Engineered microorganisms for fermentative production of these compounds and other diamines or their immediate precursors have been reported. However, processes for their fermentation and isolation of the diamine require acids and bases that generate salt by- products.

[0005] Thus, there remains a need for methods that facilitate production of diamines. The present invention satisfies this need and provides related advantages as well. 1 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT SUMMARY OF INVENTION

[0006] In one aspect, provided herein is an engineered diamine exporter comprising: a) a variant of amino acid sequence SEQ ID NO: 1 or a functional fragment thereof, wherein the engineered diamine exporter comprises one or more alterations at a position selected I24, V61, and I88; b) a variant of amino acid sequence SEQ ID NO: 46 or a functional fragment thereof, wherein the engineered diamine exporter comprises one or more alterations at a position selected from F2, G47, F102 and A133; or c) a variant of amino acid sequence SEQ ID NO: 48, or a functional fragment thereof, wherein the engineered diamine exporter comprises one or more alterations at a position selected from R148 and V286.

[0007] In some embodiments, the diamine has a carbon length of C4-C6. In some embodiments, the diamine is hexamethylenediamine (HMD).

[0008] In some embodiments, the diamine exporter does not export an HMD pathway intermediate. In some embodiments, the HMD pathway intermediate is 6-aminocaproic acid (6ACA).

[0009] In some embodiments, the engineered diamine exporter comprises an activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a diamine exporter consisting of the amino acid sequence of SEQ ID NO: 1. In other embodiments, the engineered diamine exporter comprises an activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a diamine exporter consisting of the amino acid sequence of SEQ ID NO: 46. In other embodiments, the engineered diamine exporter comprises an activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a diamine exporter consisting of the amino acid sequence of SEQ ID NO: 48. 2 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0010] In some embodiments, the one or more amino acid alterations are conservative amino acid substitutions. In some embodiments, the one or more amino acid alterations are non-conservative amino acid substitutions. In some embodiments, one or more amino acid alterations are insertions or deletions.

[0011] In some embodiments, the one or more amino acid alterations result in an engineered diamine exporter comprising: a) a M at a residue corresponding to position 61 in SEQ ID NO: 1; b) a T at a residue corresponding to position 88, and a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1; c) a F at a residue corresponding to position 24 in SEQ ID NO: 1; d) a stop codon at a residue corresponding to position 157, and an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1; e) a L at a residue corresponding to position 42 in SEQ ID NO: 46; f) a S at a residue corresponding to position 46 in SEQ ID NO: 46; g) a V at a residue corresponding to position 133 in SEQ ID NO: 46; h) a Y at a residue corresponding to position 102 in SEQ ID NO: 46; i) a C at a residue corresponding to position 148 in SEQ ID NO: 48; or j) a E at a residue corresponding to position 286 in SEQ ID NO: 48.

[0012] In some embodiments, the one or more amino acid alterations result in an engineered diamine exporter comprising: a) one or more of a M at a residue corresponding to position 61, a T at a reside corresponding to position 88, a F at a residue corresponding to position 24, a stop codon corresponding to position 157, a deletion of residues corresponding to positions 1 to 12, and an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1; b) one or more of a L at a residue corresponding to position 42, a S at a residue corresponding to position 46, a V at a residue corresponding to position 133, and a Y at a residue corresponding to position 102 in SEQ ID NO: 46; or c) one or more of a C at a residue corresponding to position 148, and a E at a residue corresponding to position 286 in SEQ ID NO: 48. In some embodiments, the one or more amino acid alterations result in an engineered diamine exporter having the sequence set forth in any one of SEQ ID NOS: 43- 45.

[0013] In some embodiments, the one or more amino acid alterations comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 alterations. In some embodiments, the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence 3 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT referenced in SEQ ID NO: 1. In some embodiments, the amino acid sequence, other than the one or more amino acid alterations, is identical to the amino acid sequence referenced in SEQ ID NO: 1. In other embodiments, the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence referenced in SEQ ID NO: 46. In some embodiments, the amino acid sequence, other than the one or more amino acid alterations, is identical to the amino acid sequence referenced in SEQ ID NO: 46. In other embodiments, the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence referenced in SEQ ID NO: 48. In some embodiments, the amino acid sequence, other than the one or more amino acid alterations, is identical to the amino acid sequence referenced in SEQ ID NO: 48.

[0014] In another aspect, provided herein is a recombinant nucleic acid encoding any of the engineered diamine exporter provided herein. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the engineered diamine exporter operatively linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a weak promoter. In other embodiments, the promoter is a strong promoter.

[0015] In some embodiments, provided herein is a vector comprising any of the recombinant nucleic acid provided herein.

[0016] In yet another aspect, provided herein is a non-naturally occurring microbial organism comprising a recombinant nucleic acid encoding any of the engineered diamine exporter provided herein.

[0017] In yet another aspect, also provided herein is a non-naturally occurring microbial organism comprising an exogenous nucleic acid encoding a diamine exporter having a sequence as set forth in any one of SEQ ID NOS: 1-42.

[0018] In yet another aspect, provided herein is a non-naturally occurring microbial organism comprising one or more disruptions in a gene encoding a diamine exporter having the sequence set forth in SEQ ID NO: 49. In some embodiments, the one or more disruptions 4 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT are in the 5’ regulatory region of the gene. In some embodiments, the one or more gene disruptions comprise one or more deletions.

[0019] In some embodiments, a non-naturally occurring microbial organism described herein is capable of producing at least 10% more diamine, is capable of at least 10% more diamine export, has a growth rate that is at least 10% higher, and / or has improved cell compared to a control microbial organism that does not comprise the recombinant nucleic acid or the exogenous nucleic acid. In some embodiments, the diamine export, or the production of diamine, is determined by measuring the levels of diamine in a culture media containing the microbial organism. In some embodiments, the diamine export, or the production of diamine, is determined by measuring the levels of diamine using a genetically encoded diamine reporter. In some embodiments, the growth rate, cell survival, diamine production, and / or diamine export are measured in the presence of exogenous diamine. In some embodiments, the diamine is HMD.

[0020] In some embodiments, a microbial organism described herein further includes a pathway that produces a diamine. In some embodiments, the pathway is for production of a diamine having a carbon length of C4 to C6. In some embodiments, the pathway is for production of HMD. In some embodiments, at least one enzyme of the pathway is encoded by an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid is heterologous to the microbial organism. In some embodiments, the exogenous nucleic acid is homologous to the microbial organism. In some embodiments, the exogenous nucleic acid encoding the at least one pathway enzyme is integrated into the genome of the microbial organism.

[0021] In some embodiments, a microbial organism described herein is in a micro- aerobic culture medium. In some embodiments, a microbial organism described herein is in a culture medium containing exogenous diamine.

[0022] In some embodiments, a microbial organism described herein is a species of bacteria, yeast, or fungus.

[0023] In some embodiments, a microbial organism described herein further includes a promoter operably linked to the recombinant nucleic acid, or to the exogenous nucleic acid. In some embodiments, the promoter is a constitutive promoter. The promoter may be a weak or a string promoter. In some embodiments, the promoter is a heterologous promoter. In 5 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT some embodiments, the recombinant nucleic acid, or the exogenous nucleic acid, is integrated into the genome of the microbial organism.

[0024] In yet another aspect, provided herein is a method of producing a diamine. In some embodiments, a method described herein includes culturing a non-naturally occurring microbial organisms described herein under conditions and for a sufficient period of time to produce the diamine.

[0025] In some embodiments, a method described herein includes separating the diamine from other components in the culture. In some embodiments, the separating includes extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.

[0026] In some embodiments, a method described herein includes culturing the microbial organisms in the absence of exogenous diamine. DETAILED DESCRIPTION OF THE INVENTION

[0027] The subject matter described herein relates to diamine exporters and microbial organisms comprising a diamine exporter that have desirable properties and are useful for producing desired products (e.g., a diamine). As used herein, a “diamine exporter” refers to a protein or enzyme that mediates transport of a diamine across a cellular membrane (e.g., a plasma membrane or an intracellular membrane). The diamine exporter may mediate passive transport based on the concentration difference of the diamine in each compartment, or may mediate ATP-dependent transport of the diamine. In some instances, the diamine exporter may mediate co-transport of diamine and a second substrate (e.g., an ion).

[0028] In some embodiments, the subject matter described herein relates to engineered diamine exporters, which are enzyme variants that have markedly different structural and / or functional characteristics compared to a wild-type diamine exporter that occurs in nature. Thus, the engineered diamine exporters provided herein are not naturally occurring enzymes. Such engineered diamine exporters provided are useful in an engineered cell, such as a microbial organism that has been engineered to produce a desired product (e.g., a diamine). In other embodiments, the subject matter described herein relates to non-naturally occurring 6 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT microbial organisms comprising a diamine exporter. The diamine exporter may be an engineered diamine exporter or an exogenous diamine exporter. The non-naturally occurring microbial organisms expressing a diamine exporter can be used to produce a desired product (e.g., a diamine). I. Definitions

[0029] As used herein the term “about” means ± 10% of the stated value. The term “about” can mean rounded to the nearest significant digit. Thus, about 5% means 4.5% to 5.5%. Additionally, about in reference to a specific number also includes that exact number. For example, about 5% also includes exact 5%.

[0030] As used herein, the term “alteration” or grammatical equivalents thereof when used in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refers to a change in structure of an amino acid residue or nucleic acid base relative to the starting or reference residue or base. An alteration of an amino acid residue includes, for example, deletions, insertions and substituting one amino acid residue for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a non-conservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein. An alteration of a nucleic acid base includes, for example, changing one naturally occurring base for a different naturally occurring base, such as changing an adenine to a thymine or a guanine to a cytosine or an adenine to a cytosine or a guanine to a thymine. An alteration of a nucleic acid base may result in an alteration of the encoding peptide, polypeptide or protein by changing the encoded amino acid residue or function of the peptide, polypeptide or protein. An alteration of a nucleic acid base may not result in an alteration of the amino acid sequence or function of encoded peptide, polypeptide or protein, also known as a silent mutation.

[0031] As used herein, the term “bioderived” means derived from or synthesized by a biological organism and can be considered a renewable resource since it can be generated by a biological organism. Such a biological organism, in particular the non-naturally occurring microbial organism disclosed herein, can utilize feedstock or biomass, such as, sugars (e.g., cellobiose, glucose, fructose, xylose, galactose (e.g., galactose from marine plant biomass), and sucrose), carbohydrates obtained from an agricultural, plant, bacterial, or animal source, and glycerol (e.g., crude glycerol by-product from biodiesel manufacturing) for synthesis of a desired bioderived compound. 7 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0032] As used herein, the term “conservative substitution” refers to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Alternatively, the term “non-conservative substitution” refers to the replacement of one amino acid residue for another such that the replaced residue is going from one family of amino acids to a different family of residues. Genetically encoded amino acids can be divided into four families: (1) acidic (negatively charged) = Asp (D), Glu (G); (2) basic (positively charged) = Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic) = Cys (C), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic = Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F); and (ii) moderately hydrophobic = Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar = Asn (N), Gln (Q), Ser (S), Thr (T). In alternative fashion, the amino acid repertoire can be grouped as (1) acidic (negatively charged) = Asp (D), Glu (G); (2) basic (positively charged) = Lys (K), Arg (R), His (H), and (3) aliphatic = Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic- hydroxyl; (4) aromatic = Phe (F), Tyr (Y), Trp (W); (5) amide = Asn (N), Glu (Q); and (6) sulfur-containing = Cys (C) and Met (M) (see, for example, Biochemistry, 4th ed., Ed. by L. Stryer, WH Freeman and Co., 1995, which is incorporated by reference herein in its entirety).

[0033] As used herein, the term “culture medium,” “medium,” “growth medium” or grammatical equivalents thereof refers to a liquid or solid (e.g., gelatinous) substance containing nutrients that support the growth of a cell, including a microbial organism, such as the microbial organism described herein. Nutrients that support growth include, but are not limited to, the following: a substrate that supplies carbon, such as, but are not limited to, cellobiose, galactose, glucose, xylose, ethanol, acetate, arabinose, arabitol, sorbitol and glycerol; salts that provide essential elements including magnesium, nitrogen, phosphorus, and sulfur; a source for amino acids, such as peptone or tryptone; and a source for vitamin content, such as yeast extract. Culture medium can be a defined medium, in which quantities of all ingredients are known, or an undefined medium, in which the quantities of all ingredients are not known. Culture medium can also include substances other than nutrients needed for growth, such as a substance that only allows select cells to grow (e.g., antibiotic or antifungal), which are generally found in selective medium, or a substance that allows for differentiation of one microbial organism over another when grown on the same medium, 8 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT which are generally found in differential or indicator medium. Such substances are well known to a person skilled in the art.

[0034] As used herein, the term “diamine” refers to an amine comprising two amino groups. Diamines may be linear diamines and may vary in the number of carbon atoms. For instance, a linear diamine can have carbon content of C2-C12, but preferably C4-C6. Hexamethylenediamine (HMD), also referred to as 1,6-diaminohexane or 1,6-hexanediamine, is a linear diamine with carbon length of C6. Other exemplary diamines include, but are not limited, to ethylenediamine (ethane-1,2-diamine), putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), and heptamethylenediamine (1,7-diaminoheptane), which are linear diamines having a carbon length of C2, C4, C5, and C7, respectively.

[0035] As used herein, the term “engineered” or “variant” when used in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refers to a sequence of amino acids or nucleic acids having at least one alteration at an amino acid residue or nucleic acid base as compared to a parent sequence. Such a sequence of amino acids or nucleic acids is not naturally occurring. The parent sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or a homolog thereof, or a modified variant of a wild-type sequence or homolog thereof.

[0036] “Exogenous” as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species whereas “homologous” refers to a molecule or activity derived from the 9 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid described herein can utilize either or both a heterologous or homologous encoding nucleic acid.

[0037] It is understood that, when more than one recombinant nucleic acid and / or exogenous nucleic acid is included into a microbial organism, the more than one recombinant nucleic acid and / or exogenous nucleic acid refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed herein. It is further understood, as disclosed herein, that such more than one recombinant nucleic acids or exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one recombinant nucleic acid and / or exogenous nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more recombinant and / or exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two recombinant and / or exogenous nucleic acids encoding an enzyme or protein having a desired activity are introduced into a host microbial organism, it is understood that the two recombinant and / or exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two recombinant and / or exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more recombinant or exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced recombinant or exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.

[0038] As used herein, the term “functional fragment” when used in reference to a peptide, polypeptide or protein is intended to refer to a portion of the peptide, polypeptide or protein that retains some or all of the activity (e.g., diamine export) of the original peptide, polypeptide or protein from which the fragment was derived. Such functional fragments include amino acid sequences that are about 200 to about 440, about 200 to about 430, about 200 to about 420, about 200 to about 410, about 200 to about 400, about 200 to about 390, 10 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT about 200 to about 380, about 200 to about 370, about 200 to about 360, about 200 to about 350, about 300 to about 440, about 300 to about 430, about 300 to about 420, about 300 to about 410, about 300 to about 400, about 300 to about 390, about 300 to about 380, about 300 to about 370, about 300 to about 350, about 300 to about 340, about 300 to about 330, about 300 to about 320, about 300 to about 310, about 400 to about 440, about 400 to about 430, about 400 to about 420, or about 400 to about 410 amino acids in length. These functional fragments can, for example, be truncations (e.g., C-terminal or N-terminal truncations) of a peptide, polypeptide, or protein. Functional fragments can also include one or more amino acid alterations, such as an amino acid alteration described herein.

[0039] As used herein, the term “isolated” when used in reference to a molecule (e.g., peptide, polypeptide, protein, nucleic acid, polynucleotide, vector) or a cell (e.g., a yeast cell) refers to a molecule or cell that is substantially free of at least one component with which the referenced molecule or cell is found in nature. The term includes a molecule or cell that is removed from some or all components with which it is found in its natural environment. Therefore, an isolated molecule or cell can be partly or completely separated from other substances with which it is found in nature or with which it is grown, stored or subsisted in non-naturally occurring environments.

[0040] As used herein, the terms “microbial,” “microbial organism” or “microorganism” are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

[0041] As used herein, the term “non-naturally occurring” when used in reference to a microbial organism described herein is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and / or other functional disruption of the microbial organism’s genetic material. Such modifications include, for example, genetic alterations within coding regions and functional fragments thereof. 11 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a biosynthesis pathway described herein.

[0042] As use herein, the term “operatively linked” when used in reference to a nucleic acid encoding an engineered aldehyde dehydrogenase refers to connection of a nucleotide sequence encoding an engineered aldehyde dehydrogenase described herein to another nucleotide sequence (e.g., a promoter) is such a way as to allow for the connected nucleotide sequences to function (e.g., express the engineered aldehyde dehydrogenase in the microbial organism).

[0043] As used herein, the term “pathway” when used in reference to production of a desired product (e.g., a diamine) refers to one or more polypeptides (e.g., proteins or enzymes) that catalyze the conversion of a substrate compound to a product compound and / or produce a co-substrate for the conversion of a substrate compound to a product compound. Such a product compound can be one of the bioderived compounds described herein, or an intermediate compound that can lead to the bioderived compound upon further conversion by other proteins or enzymes of the metabolic pathway. Accordingly, a metabolic pathway can be comprised of a series of metabolic polypeptides (e.g., two, three, four, five, six, seven, eight, nine, ten or more) that act upon a substrate compound to convert it to a given product compound through a series of intermediate compounds. The metabolic polypeptides of a metabolic pathway can be encoded by an exogenous nucleic acid as described herein or produced naturally by the host microbial organism.

[0044] As used herein, the term “recombinant” with respect to a nucleic acid, such as a nucleic acid comprising a gene that encodes a protein or polypeptide (e.g., an engineered diamine exporter described herein), refers to: a nucleic acid that has been artificially supplied to a biological system; a nucleic acid that has been modified within a biological system, or a nucleic acid whose expression or regulation has been manipulated within a biological system. The recombinant nucleic acid can be supplied to the biological system, for example, by introduction of the nucleic acid into genetic material of a microbial organism, such as by integration into a microbial organism chromosome, or as non-chromosomal genetic material such as a plasmid. A recombinant nucleic acid that is introduced into or expressed in a microbial organism may be a nucleic acid that comes from a different organism or species from the microbial organism, or may be a synthetic nucleic acid, or may be a nucleic acid that 12 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT is also endogenously expressed in the same organism or species as the microbial organism. A recombinant nucleic acid that is also endogenously expressed in the same organism or species as the microbial organism can be considered heterologous if: the sequence of the recombinant nucleic acid is modified relative to the endogenously expressed sequence, the sequence of a regulatory region such as a promoter that controls expression of the nucleic acid is modified relative to the regulatory region of the endogenously expressed sequence, the nucleic acid is expressed in an alternate location in the genome of the microbial organism relative to the endogenously expressed sequence, the nucleic acid is expressed in a different copy number in the microbial organism relative to the endogenously expressed sequence, and / or the nucleic acid is expressed as non-chromosomal genetic material such as a plasmid in the microbial organism.

[0045] As used herein, the term “promoter” when used in reference to a nucleic acid encoding an engineered aldehyde dehydrogenase refers to a nucleotide sequence where transcription of a linked open reading frame (e.g., a nucleotide sequence encoding an engineered aldehyde dehydrogenase) by an RNA polymerase begins. A promoter sequence can be located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to a promoter sequence and initiate transcription. Promoter sequences define the direction of transcription and indicate which DNA strand will be transcribed, e.g., the sense strand.

[0046] As used herein, the term “substantially anaerobic” when used in reference to a culture or growth condition is intended to mean that the amount of dissolved oxygen in a liquid medium is less than about 10% of saturation. The term also is intended to include sealed chambers maintained with an atmosphere of less than about 1% oxygen that include liquid or solid medium.

[0047] As used herein, the term “vector” refers to a compound and / or composition that transduces, transforms, or infects a microbial organism, thereby causing the microbial organism to express nucleic acids and / or proteins other than those native to the microbial organism, or in a manner not native to the cell. Vectors can be constructed to include one or more biosynthetic pathway enzyme or protein, such as a diamine exporter described herein, encoded by a nucleotide sequence operably linked to expression control sequences (e.g., promoter) that are functional in the microbial organism (“expression vector”). Expression vectors applicable for use in the microbial organisms described herein include, for example, 13 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more recombinant or exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of a recombinant or exogenous nucleic acid encoding an enzyme or protein involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid or its corresponding gene product (e.g., enzyme or protein). It is understood by those skilled in the art that the recombinant or exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

[0048] Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable microbial organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of 14 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT species homologs, in general, and in particular, orthologs, paralogs or non-orthologous gene displacements.

[0049] An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity that is less than 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

[0050] Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microbial organism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5’-3’ exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease and the polymerase from the second species and vice versa. 15 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0051] In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

[0052] A “non-orthologous gene displacement” is a non-orthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a non-orthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a non-orthologous gene product compared to a gene encoding the function sought to be substituted. Thus, a non- orthologous gene includes, for example, a paralog or an unrelated gene.

[0053] Therefore, in identifying and constructing the non-naturally occurring microbial organisms described herein having biosynthetic capability for a desired product, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and / or non-orthologous gene displacements are present in the referenced microbial organism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Similarly, for a gene disruption, evolutionally related genes can also be disrupted or deleted in a microbial organism to reduce or eliminate functional redundancy of enzymatic activities targeted for disruption. 16 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0054] Orthologs, paralogs and non-orthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is high enough to show the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, ClustalW and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well-known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

[0055] Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleotide sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch: - 2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences. 17 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT II. Diamine Exporters

[0056] In some aspects of the disclosure, provided herein are diamine exporters. In some embodiments, the diamine exporter has the sequence set forth in any one of SEQ ID NOS: 1- 42. In some embodiments, the diamine exporter has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 50% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 50% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 55% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 65% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 70% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 75% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 80% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 90% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 98% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter has at least 99% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the diamine exporter is identical to a sequence set forth in any one of SEQ ID NOS: 1-42.

[0057] In other embodiments, the diamine exporter has the sequence set forth in SEQ ID NO: 49. In some embodiments, the diamine exporter has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence set forth in SEQ ID NO: 49.

[0058] Also provided herein are engineered diamine exporters. In some embodiments, the engineered diamine exporter is a variant of a diamine exporter having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 46, or SEQ ID NO: 48. Such an engineered 18 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT diamine exporter is a variant of amino acid sequence SEQ ID NO: 1 and includes one or more alterations at a position selected from I24, V61, and I88; is a variant of amino acid sequence SEQ ID NO: 46 and includes one or more alterations at a position selected from F42, G47, F102 and A133; or is a variant of amino acid sequence SEQ ID NO: 48, and includes one or more alterations at a position selected from R148 and V286. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C2 to C12. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C2 to C7. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C4 to C6. In some embodiments, the engineered diamine exporter is selective for a diamine selected from ethylenediamine, putrescine, cadaverine, hexamethylenediamine (HMD), or heptamethylenediamine. In some embodiments, the diamine is hexamethylenediamine (HMD). In some embodiments, the engineered diamine exporter provided herein has higher activity for exporting HMD but low or no export activity for a HMD pathway intermediate, as compared to a diamine exporter having the sequence set forth in SEQ ID NO: 1. In some embodiments, the engineered diamine exporter provided herein has higher activity for exporting HMD but low or no export activity for a HMD pathway intermediate, as compared to a diamine exporter having the sequence set forth in SEQ ID NO: 46. In some embodiments, the engineered diamine exporter provided herein has higher activity for exporting HMD but low or no export activity for a HMD pathway intermediate, as compared to a diamine exporter having the sequence set forth in SEQ ID NO: 48. In some embodiments, the engineered diamine exporter has low or no export activity for a HMD pathway intermediate, for example 6-aminocaproic acid (6ACA).

[0059] It is understood that the engineered diamine exporters as described herein can carry out a similar activity as a wild-type diamine exporter, such as those having the sequence set forth in SEQ ID NOS: 1, 46 or 48. It is further understood that the variants of the diamine exporter enzyme can include alterations that provide a beneficial characteristic to the engineered diamine exporter, including but not limited to, increased activity (e.g., rate of diamine export, selectivity for a diamine of particular length, and / or selectivity of a diamine over a HMD pathway intermediate). In some embodiments, the engineered diamine exporter can exhibit one or more activities that are at least the same or higher than the activity of a diamine exporter having the sequence set forth in SEQ ID NO: 1 , that is, it has one or more activities that are the same or higher than an diamine exporter without the variant at the same 19 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT amino acid position(s). In some embodiments, the engineered diamine exporter can exhibit one or more activities that are at least the same or higher than the activity of a diamine exporter having the sequence set forth in SEQ ID NO: 46, that is, it has one or more activities that are the same or higher than an diamine exporter without the variant at the same amino acid position(s). In some embodiments, the engineered diamine exporter can exhibit one or more activities that are at least the same or higher than the activity of a diamine exporter having the sequence set forth in SEQ ID NO: 48, that is, it has one or more activities that are the same or higher than an diamine exporter without the variant at the same amino acid position(s). For example, the engineered diamine exporters provided here can one or more activities that are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher over a diamine exporter having the sequence set forth in SEQ ID NOS: 1, 46 or 48. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of an diamine exporter having the sequence set forth in SEQ ID NOS: 1, 46 or 48. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 10% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 20% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 30% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 40% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 50% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 60% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 70% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 80% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 90% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 100% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 110% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 120% higher. In some embodiments, 20 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT an engineered diamine exporter provided herein has an activity that is at least 130% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 140% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 150% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 160% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 170% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 180% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 190% higher. In some embodiments, an engineered diamine exporter provided herein has an activity that is at least 200% higher. It is understood that activity refers to the ability of an engineered diamine exporter described herein to export diamine relative to a diamine exporter having the sequence set forth in SEQ ID NOS: 1, 46 or 48 under the same assay conditions, such as those described herein.

[0060] In some embodiments, an engineered diamine exporter provided herein is a variant of amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes one or more alterations at a position selected from I24, V61, and I88 relative to SEQ ID NO: 1, wherein the one or more amino acid alterations are conservative amino acid substitutions. In some embodiments, an engineered diamine exporter provided herein includes one or more conservative amino acid substitutions relative to a diamine exporter comprising one or more alterations at a position selected from I24, V61, and I88 relative to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes one or more alterations at a position selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1, wherein the one or more amino acid alterations are non-conservative amino acid substitutions. In some embodiments, an engineered diamine exporter provided herein includes a conservative amino acid substitution and / or non-conservative amino acid substitution in 1 to 10 amino acid positions selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the engineered diamine exporter comprises one or more deletions or insertions relative to the sequence set forth in SEQ ID NO: 1. In some embodiments, the one or more insertions or deletions comprise an amino acid residue at a position selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the one or more insertions or 21 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT deletions comprise an amino acid residue in a position other than a position selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1.

[0061] In some embodiments, an engineered diamine exporter provided herein is a variant of amino acid sequence of SEQ ID NO: 46, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein includes one or more alterations at a position selected from F42, G47, F102 and A133 relative to SEQ ID NO: 46, wherein the one or more amino acid alterations are conservative amino acid substitutions. In some embodiments, an engineered diamine exporter provided herein includes one or more conservative amino acid substitutions relative to a diamine exporter comprising one or more alterations at a position selected from F42, G47, F102 and A133 relative to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein includes one or more alterations at a position selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46, wherein the one or more amino acid alterations are non-conservative amino acid substitutions. In some embodiments, an engineered diamine exporter provided herein includes a conservative amino acid substitution and / or non-conservative amino acid substitution in 1 to 10 amino acid positions selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46. In some embodiments, the engineered diamine exporter comprises one or more deletions or insertions relative to the sequence set forth in SEQ ID NO: 46. In some embodiments, the one or more insertions or deletions comprise an amino acid residue at a position selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46. In some embodiments, the one or more insertions or deletions comprise an amino acid residue in a position other than a position selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46.

[0062] In some embodiments, an engineered diamine exporter provided herein is a variant of amino acid sequence of SEQ ID NO: 48, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from R148 and V286, or a combination thereof, in SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein includes one or more alterations at a position selected from R148 and V286 relative to SEQ ID NO: 48, wherein the one or more amino acid alterations are conservative amino acid substitutions. In some embodiments, an engineered diamine 22 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT exporter provided herein includes one or more conservative amino acid substitutions relative to a diamine exporter comprising one or more alterations at a position selected from R148 and V286 relative to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein includes one or more alterations at a position selected from R148 and V286, or a combination thereof, in SEQ ID NO: 48, wherein the one or more amino acid alterations are non-conservative amino acid substitutions. In some embodiments, an engineered diamine exporter provided herein includes a conservative amino acid substitution and / or non- conservative amino acid substitution in 1 to 10 amino acid positions selected from R148 and V286, or a combination thereof, in SEQ ID NO: 48. In some embodiments, the engineered diamine exporter comprises one or more deletions or insertions relative to the sequence set forth in SEQ ID NO: 48. In some embodiments, the one or more insertions or deletions comprise an amino acid residue at a position selected from R148 and V286, or a combination thereof, in SEQ ID NO: 48. In some embodiments, the one or more insertions or deletions comprise an amino acid residue in a position other than a position selected from R148 and V286, or a combination thereof, in SEQ ID NO: 48.

[0063] In some embodiments, an engineered diamine exporter provided herein can further include a conservative amino acid substitution in from 1 to 50 amino acid positions, or alternatively from 2 to 50 amino acid positions, or alternatively from 3 to 50 amino acid positions, or alternatively from 4 to 50 amino acid positions, or alternatively from 5 to 50 amino acid positions, or alternatively from 6 to 50 amino acid positions, or alternatively from 7 to 50 amino acid positions, or alternatively from 8 to 50 amino acid positions, or alternatively from 9 to 50 amino acid positions, or alternatively from 10 to 50 amino acid positions, or alternatively from 15 to 50 amino acid positions, or alternatively from 20 to 50 amino acid positions, or alternatively from 30 to 50 amino acid positions, or alternatively from 40 to 50 amino acid positions, or alternatively from 45 to 50 amino acid positions, or any integer therein, wherein the positions are other than positions I24, V61, or I88, or a combination thereof, in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein can further include a conservative amino acid substitution in from 1 to 50 amino acid positions, or alternatively from 2 to 50 amino acid positions, or alternatively from 3 to 50 amino acid positions, or alternatively from 4 to 50 amino acid positions, or alternatively from 5 to 50 amino acid positions, or alternatively from 6 to 50 amino acid positions, or alternatively from 7 to 50 amino acid positions, or alternatively from 8 to 50 amino acid positions, or alternatively from 9 to 50 amino acid positions, or alternatively from 23 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT 10 to 50 amino acid positions, or alternatively from 15 to 50 amino acid positions, or alternatively from 20 to 50 amino acid positions, or alternatively from 30 to 50 amino acid positions, or alternatively from 40 to 50 amino acid positions, or alternatively from 45 to 50 amino acid positions, or any integer therein, wherein the positions are other than positions F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein can further include a conservative amino acid substitution in from 1 to 50 amino acid positions, or alternatively from 2 to 50 amino acid positions, or alternatively from 3 to 50 amino acid positions, or alternatively from 4 to 50 amino acid positions, or alternatively from 5 to 50 amino acid positions, or alternatively from 6 to 50 amino acid positions, or alternatively from 7 to 50 amino acid positions, or alternatively from 8 to 50 amino acid positions, or alternatively from 9 to 50 amino acid positions, or alternatively from 10 to 50 amino acid positions, or alternatively from 15 to 50 amino acid positions, or alternatively from 20 to 50 amino acid positions, or alternatively from 30 to 50 amino acid positions, or alternatively from 40 to 50 amino acid positions, or alternatively from 45 to 50 amino acid positions, or any integer therein, wherein the positions are other than positions R148 and V286, or a combination thereof, in SEQ ID NO: 48. Methods of identifying conservative amino acids are well known to one of skill in the art, any one of which can be used to generate the isolated engineered diamine exporters described herein.

[0064] An engineered diamine exporter provided herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 alterations relative to a diamine exporter having the sequence set for in SEQ ID NOS: 1, 46 or 48. An engineered diamine exporter provided herein may comprise at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, or at most 25 alterations relative to a diamine exporter having the sequence set for in SEQ ID NOS: 1, 46 or 48. In some embodiments, the one or more alterations may be located at one or more positions selected from I24, V61, and I88 in SEQ ID NO: 1. In other embodiments, the one or more alterations may be located at one or more positions corresponding to one or more positions selected from F42, G47, F102 and A133 in SEQ ID NO: 46. In other embodiments, the one or more alterations may be located at one or more positions corresponding to one or more positions selected from R148 and V286 in SEQ ID NO: 48. As used herein, the phrase “a residue corresponding to position X 24 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT in SEQ ID NO: Y” refers to a residue at a corresponding position following an alignment of two sequences. For example, the residue in SEQ ID NO: 1 corresponding to position I24 in SEQ ID NO: 1 is the isoleucine residue at position 24 in SEQ ID NO: 1.

[0065] An engineered diamine exporter provided herein can include any combination of the alterations at positions I24, V61, or I88 in SEQ ID NO: 1. One alteration alone, or in combination, can produce an engineered diamine exporter that retains or improves the activity as described herein relative to diamine exporter having the sequence set forth in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 alterations, including up to an alteration at all of positions I24, V61, and I88 in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes at least two alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes at least three alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes at least four alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes at least five alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes alterations at least six positions selected from I24, V61, and I88 in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein includes alterations at all of positions I24, V61, and I88 in SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein one or more alterations at positions other than I24, V61, and I88 in SEQ ID NO: 1, in addition to one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1.

[0066] An engineered diamine exporter provided herein can include any combination of the alterations at positions F42, G47, F102 and A133 in SEQ ID NO: 46. One alteration alone, or in combination, can produce an engineered diamine exporter that retains or improves the activity as described herein relative to diamine exporter having the sequence set forth in SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 alterations, including up to an alteration at all of positions F42, G47, F102 and 25 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT A133 in SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein includes at least two alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein includes at least three alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein includes at least four alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein includes alterations at all of positions F42, G47, F102 and A133 in SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein one or more alterations at positions other than F42, G47, F102 and A133 in SEQ ID NO: 46, in addition to one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46.

[0067] An engineered diamine exporter provided herein can include any combination of the alterations at positions R148 and V286 in SEQ ID NO: 48. One alteration alone, or in combination, can produce an engineered diamine exporter that retains or improves the activity as described herein relative to diamine exporter having the sequence set forth in SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 alterations, including up to an alteration at all of positions R148 and V286 in SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein includes at least two alterations at a position selected from R148 and V286 in SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein includes at least three alterations at a position selected from R148 and V286 in SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein includes at least four alterations at a position selected from R148 and V286 in SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein includes alterations at all of positions R148 and V286 in SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein one or more alterations at positions other than R148 and V286 in SEQ ID NO: 48, in addition to one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48.

[0068] In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having (a) a M at a residue corresponding to position 61 in SEQ ID NO: 1; (b) a T at a residue corresponding to position 88 in SEQ ID NO: 1; (c) a deletion 26 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT of residues corresponding to positions 1 to 12 in SEQ ID NO: 1; (d) a F at a residue corresponding to position 24 in SEQ ID NO: 1; (e) a stop codon at a residue corresponding to position 157 in SEQ ID NO: 1, (f) an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1; or (g) any combination of (a)-(f). In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having a M at a residue corresponding to position 61 in SEQ ID NO: 1. In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having a T at a residue corresponding to position 88, and a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1. In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having a F at a residue corresponding to position 24 in SEQ ID NO: 1. In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having a stop codon at a residue corresponding to position 157, and an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1. In some embodiments, the one or more amino acid alterations result in engineered exporter having the amino acid sequence set forth in any one of SEQ ID NOS: 43-45.

[0069] In other embodiments, the one or more amino acid alternations result in an engineered diamine exporter having (a) a L at a residue corresponding to position 42 in SEQ ID NO: 46; (b) a S at a residue corresponding to position 46 in SEQ ID NO: 46; (c) a V at a residue corresponding to position 133 in SEQ ID NO: 46; (d) a Y at a residue corresponding to position 102 in SEQ ID NO: 46; or (e) any combination of (a)-(d). In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having a L at a residue corresponding to position 42 in SEQ ID NO: 46. In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having a S at a residue corresponding to position 46 in SEQ ID NO: 46. In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having a V at a residue corresponding to position 133 in SEQ ID NO: 46. In some embodiment, the one or more amino acid alternations result in an engineered diamine exporter having a Y at a residue corresponding to position 102 in SEQ ID NO: 46.

[0070] In other embodiments, the one or more amino acid alternations result in an engineered diamine exporter having (a) a C at a residue corresponding to position 148 in SEQ ID NO: 48; b) a E at a residue corresponding to position 286 in SEQ ID NO: 48; or (c) any combination of (a)-(b). In some embodiments, the one or more amino acid alternations result 27 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT in an engineered diamine exporter having a C at a residue corresponding to position 148 in SEQ ID NO: 48. In some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having a E at a residue corresponding to position 286 in SEQ ID NO: 48.

[0071] In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one more alterations at a position selected from I24, V61, and I88, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 75% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one 28 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 1. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 1.

[0072] In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that is a variant of SEQ ID NO: 46 that includes one more alterations at a position selected from F42, G47, F102 and A133, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 46. Accordingly, in some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 75% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from F42, G47, F102 and 29 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 46.

[0073] In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that is a variant of SEQ ID NO: 48 that includes one more alterations at a position selected from R148 and V286, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 48. Accordingly, in some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other 30 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 75% identical to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 46. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 48. In some embodiments, an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the 31 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 48.

[0074] Sequence identity, homology or similarity refers to sequence similarity between two polypeptides or between two nucleic acid molecules. Identity can be determined by comparing a position in each sequence which may be aligned for comparison purposes. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polypeptide or polypeptide region (or a polynucleotide or polynucleotide region) has a certain percentage (for example, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of amino acids (or nucleotide bases) are the same in comparing the two sequences. The alignment of two sequences to determine their percent sequence identity can be done using software programs known in the art, such as, for example, those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Preferably, default parameters are used for the alignment. One alignment program well known in the art that can be used is BLAST set to default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the National Center for Biotechnology Information (see also Altschul et al., ” J. Mol. Biol.215:403-410 (1990)).

[0075] In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from I24, V61, and I88 in SEQ ID NO: 1, which results in increased production of a diamine when the engineered diamine exporter is expressed in an organism having a pathway for production of a diamine and assayed under conditions as described in Example II. In some embodiments, the diamine is HMD. In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from I24, V61, and I88 in SEQ ID NO: 1, which results in increased cell survival or growth when the engineered diamine exporter is expressed in an organism having a pathway for production of a diamine and assayed under conditions as described in 32 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT Example II. In some embodiments, the diamine is HMD. In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from I24, V61, and I88 in SEQ ID NO: 1, which results in low to no export activity for a diamine pathway intermediate. In some embodiments, the diamine pathway is an HMD pathway, and the intermediate is 6-aminocaproic acid (6ACA). For example, in some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having (a) a M at a residue corresponding to position 61 in SEQ ID NO: 1; (b) a T at a residue corresponding to position 88; (c) a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1; (d) a F at a residue corresponding to position 24 in SEQ ID NO: 1; (e) a stop codon at a residue corresponding to position 157 in SEQ ID NO: 1, (f) an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1; or (g) any combination of (a)- (f).

[0076] In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, which results in increased production of a diamine when the engineered diamine exporter is expressed in an organism having a pathway for production of a diamine and assayed under conditions as described in Example II. In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, which results in increased cell survival or growth when the engineered diamine exporter is expressed in an organism having a pathway for production of a diamine and assayed under conditions as described in Example II. In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, which results in low to no export activity for a diamine pathway intermediate. In some embodiments, the diamine has a carbon length of C2 to C12. In some embodiments, the diamine has a carbon length of C2 to C7. In some embodiments, the diamine has a carbon length of C4 to C6. In some embodiments, the diamine is selected from ethylenediamine, putrescine, cadaverine, hexamethylenediamine (HMD), or heptamethylenediamine. In some embodiments, the diamine pathway is an HMD pathway, and the intermediate is 6-aminocaproic acid (6ACA). For example, in some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having (a) a L at a residue corresponding to position 42 in SEQ ID NO: 46; (b) a S at a residue corresponding to position 46 in SEQ ID NO: 46; (c) a V at a residue corresponding to position 133 in SEQ ID NO: 46; (d) a Y at a residue corresponding to position 102 in SEQ 33 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT ID NO: 46; or (e) any combination of (a)-(d). In some embodiments, the one or more amino acid alterations result in engineered exporter having the amino acid sequence set forth in any one of SEQ ID NOS: 43-45.

[0077] In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from R148 and V286 in SEQ ID NO: 48, which results in increased production of a diamine when the engineered diamine exporter is expressed in an organism having a pathway for production of a diamine and assayed under conditions as described in Example VI. In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from R148 and V286 in SEQ ID NO: 48, which results in increased cell survival or growth when the engineered diamine exporter is expressed in an organism having a pathway for production of a diamine and assayed under conditions as described in Example VI. In some embodiments, the one or more amino acid alterations of the engineered diamine exporter are in a position selected from R148 and V286 in SEQ ID NO: 48, which results in low to no export activity for a diamine pathway intermediate. In some embodiments, the diamine has a carbon length of C2 to C12. In some embodiments, the diamine has a carbon length of C2 to C7. In some embodiments, the diamine has a carbon length of C4 to C6. In some embodiments, the diamine is selected from ethylenediamine, putrescine, cadaverine, hexamethylenediamine (HMD), or heptamethylenediamine. In some embodiments, the diamine pathway is an HMD pathway, and the intermediate is 6-aminocaproic acid (6ACA). For example, in some embodiments, the one or more amino acid alternations result in an engineered diamine exporter having (a) a C at a residue corresponding to position 148 in SEQ ID NO: 48; b) a E at a residue corresponding to position 286 in SEQ ID NO: 48; or (c) any combination of (a)- (b).

[0078] Methods of generating and assaying a diamine exporter (e.g., an engineered diamine exporter) as described herein are well known to one of skill in the art. Examples of such methods are described in the Examples provided herein. Any of a variety of methods can be used to generate a diamine exporter disclosed herein. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, 34 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2nd ed (2014)). One non-limiting example of a method for preparing a diamine exporter is to express recombinant nucleic acids encoding the engineered diamine exporter in a suitable microbial organism, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art.

[0079] In some embodiments, a diamine exporter provided herein is an isolated diamine exporter (e.g., an isolated engineered diamine exporter). An isolated diamine exporter provided herein can be isolated by a variety of methods well-known in the art, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol.182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay. III. Recombinant Nucleic Acids and Vectors

[0080] Provided herein are nucleic acids encoding a diamine exporter and vectors comprising a recombinant sequence encoding a diamine exporter.

[0081] In some embodiments, provided herein is a nucleic acid encoding a diamine exporter having the sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes a diamine exporter having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes an diamine exporter that has at least 50% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes an diamine exporter that has at least 50% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1- 42. In some embodiments, the nucleic acid encodes a diamine exporter that has at least 55% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes an diamine exporter that has at least 65% sequence 35 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes a diamine exporter that has at least 70% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes an diamine exporter that has at least 75% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes a diamine exporter that has at least 80% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1- 42. In some embodiments, the nucleic acid encodes an diamine exporter that has at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes a diamine exporter that has at least 90% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes a diamine exporter that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes a diamine exporter that has at least 98% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes a diamine exporter that has at least 99% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the nucleic acid encodes a diamine exporter that is identical to a sequence set forth in any one of SEQ ID NOS: 1-42.

[0082] In other embodiments, the nucleic acid encodes a diamine exporter that has the sequence set forth in SEQ ID NO: 49. In some embodiments, the nucleic acid encodes a diamine exporter that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence set forth in SEQ ID NO: 49.

[0083] Also provided herein is a recombinant nucleic acid that has a nucleotide sequence encoding an engineered diamine exporter described herein. Accordingly, in some embodiments, provided herein is a recombinant nucleic acid selected from (a) a nucleic acid molecule encoding an engineered diamine exporter that is a variant of a diamine exporter having the sequence set forth in SEQ ID NOS: 1, 46 or 48, such as an engineered diamine exporter that is a variant of amino acid sequence SEQ ID NO: 1 and includes one or more alterations at a position selected from I24, V61, and I88; is a variant of amino acid sequence SEQ ID NO: 46 and includes one or more alterations at a position selected from F42, G47, F102 and A133; or is a variant of amino acid sequence SEQ ID NO: 48 and includes one or more alterations at a position selected from R148 and V286; (b) a recombinant nucleic acid 36 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT that hybridizes to an isolated nucleic acid of (a) under highly stringent hybridization conditions; and (c) a recombinant nucleic acid that is complementary to (a) or (b).

[0084] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having (a) a M at a residue corresponding to position 61 in SEQ ID NO: 1; (b) a T at a residue corresponding to position 88; (c) a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1; (d) a F at a residue corresponding to position 24 in SEQ ID NO: 1; (e) a stop codon at a residue corresponding to position 157 in SEQ ID NO: 1, (f) an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1; or (g) any combination of (a)- (f). In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a M at a residue corresponding to position 61 in SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a M at a residue corresponding to position 61 in SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a T at a residue corresponding to position 88, and a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a F at a residue corresponding to position 24 in SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a stop codon at a residue corresponding to position 157, and an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered exporter having the amino acid sequence set forth in any one of SEQ ID NOS: 43-45.

[0085] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of amino acid sequence of SEQ ID NO: 46, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having (a) a L at a residue corresponding to position 42 in SEQ ID NO: 46; (b) a S at a residue corresponding to position 46 in SEQ ID NO: 46; (c) a V at a residue corresponding 37 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT to position 133 in SEQ ID NO: 46; (d) a Y at a residue corresponding to position 102 in SEQ ID NO: 46; or (e) any combination of (a)-(d). In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a L at a residue corresponding to position 42 in SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a S at a residue corresponding to position 46 in SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a V at a residue corresponding to position 133 in SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a Y at a residue corresponding to position 102 in SEQ ID NO: 46.

[0086] In other embodiments, provided herein is a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of amino acid sequence of SEQ ID NO: 48, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from R148 and V286. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having (a) a C at a residue corresponding to position 148 in SEQ ID NO: 48; b) a E at a residue corresponding to position 286 in SEQ ID NO: 48; or (c) any combination of (a)-(b). In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a C at a residue corresponding to position 148 in SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter having a E at a residue corresponding to position 286 in SEQ ID NO: 48.

[0087] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered diamine exporter provided herein has an amino acid sequence that is a variant of SEQ ID NO: 1 that includes one more alterations at a position selected from I24, V61, and I88, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a 38 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 75% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 1. 39 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0088] In some embodiments, provided herein is a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of SEQ ID NO: 46 that includes one more alterations at a position selected from F42, G47, F102 and A133, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 46. Accordingly, in some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 75% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 46. In some 40 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 46.

[0089] In some embodiments, provided herein is a recombinant nucleic acid encoding the recombinant nucleic acid encodes an engineered diamine exporter that is a variant of the recombinant nucleic acid encodes an engineered diamine exporter SEQ ID NO: 48 that includes one more alterations at a position selected from R148 and V286, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 48. Accordingly, in some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 75% identical to SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that 41 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT includes one more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encodes an engineered diamine exporter provided herein has an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 48.

[0090] In some embodiments, a recombinant nucleic acid provided herein encodes an engineered diamine exporter that is selective for a diamine having a carbon length of C2 to C12. In some embodiments, the diamine has a carbon length of C2 to C7. In some embodiments, the diamine has a carbon length of C4 to C6. In some embodiments, the 42 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT diamine is selected from ethylenediamine, putrescine, cadaverine, HMD, or heptamethylenediamine. In some embodiments, the diamine is HMD.

[0091] In some embodiments, provided herein is a recombinant nucleic acid that hybridizes under highly stringent hybridization conditions to an isolated nucleic acid encoding an engineered diamine exporter described herein. Accordingly, in some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered diamine exporter that is a variant of diamine exporter having the sequence set forth in SEQ ID NOS: 1, 46 or 48, such as an engineered diamine exporter that is a variant of amino acid sequence SEQ ID NO: 1 and includes one or more alterations at a position selected from I24, V61, and I88, a variant of amino acid sequence SEQ ID NO: 46 and includes one or more alterations at a position selected from F42, G47, F102 and A133; or a variant of amino acid sequence SEQ ID NO: 48 and includes one or more alterations at a position selected from R148 and V286. In some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered diamine exporter having one or more alterations at a position selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered diamine exporter having one or more alterations at a position selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid is an isolated nucleic acid that hybridizes under highly stringent hybridization conditions to a nucleic acid that encodes an engineered diamine exporter having one or more alterations at a position selected from R148 and V286, or a combination thereof, of SEQ ID NO: 48.

[0092] In some embodiments, provided herein is a recombinant nucleic acid that includes a nucleotide sequence encoding an engineered diamine exporter described herein that is operatively linked to a promoter. Such a promoter can express the engineered diamine exporter in a microbial organism as described herein. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a strong promoter. In some embodiments, the promoter is a weak promoter. 43 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0093] In some embodiments, provided herein is a vector containing a nucleic acid (e.g., a recombinant nucleic acid) described herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector comprises double stranded DNA.

[0094] A recombinant nucleic acid encoding an engineered diamine exporter described herein also includes a nucleic acid that hybridizes to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed. Hybridization conditions can include highly stringent, moderately stringent, or low stringency hybridization conditions that are well known to one of skill in the art such as those described herein. Similarly, a recombinant nucleic acid that can be used in the compositions and methods described herein can be described as having a certain percent sequence identity to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid molecule that encodes an amino acid sequence disclosed herein. For example, the nucleic acid can have at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or be identical, to a nucleotide described herein.

[0095] Stringent hybridization refers to conditions under which hybridized polynucleotides are stable. As known to those of skill in the art, the stability of hybridized polynucleotides is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of hybridized polynucleotides is a function of the salt concentration, for example, the sodium ion concentration, and temperature. A hybridization reaction can be performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Highly stringent hybridization includes conditions that permit hybridization of only those nucleotide sequences that form stable hybridized polynucleotides in 0.018M NaCl at 65°C, for example, if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.1X SSPE, and 0.1% SDS at 65°C. Hybridization conditions other than highly stringent hybridization conditions can also be used to describe the nucleotide sequences disclosed herein. For example, the phrase moderately stringent hybridization refers to conditions equivalent to hybridization in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing in 0.2X SSPE, 0.2% SDS, at 42°C. The phrase low stringency hybridization refers to conditions equivalent to 44 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT hybridization in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 22°C, followed by washing in 1X SSPE, 0.2% SDS, at 37°C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20X SSPE (sodium chloride, sodium phosphate, ethylene diamine tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable low, moderate and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

[0096] A recombinant nucleic acid encoding an engineered diamine exporter described herein can have at least a certain sequence identity to a nucleotide sequence disclosed herein. Accordingly, in some aspects described herein, a recombinant nucleic acid encoding an engineered diamine exporter has a nucleotide sequence of at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, or is identical, to a nucleic acid disclosed herein or a nucleic acid that hybridizes to a nucleic acid that encodes an amino acid sequence disclosed herein.

[0097] It is understood that a recombinant nucleic acid described herein or an engineered diamine exporter described here can exclude a parental sequence, for example an amino acid sequence set forth in SEQ ID NOS: 1 or 46. One skilled in the art will readily understand the meaning of a parental wild-type sequence based on what is well known in the art. It is further understood that such a recombinant nucleic acid described herein can exclude a nucleotide sequence encoding a naturally occurring amino acid sequence as found in nature. Similarly, an engineered diamine exporter described herein can exclude an amino acid sequence as found in nature. Thus, in some embodiments, the recombinant nucleic acid or engineered diamine exporter described herein is as set forth herein, with the proviso that the encoded amino acid sequence is not the wild-type parental sequence or a naturally occurring amino acid sequence and / or that the nucleotide sequence is not a wild-type or naturally occurring nucleotide sequence. A naturally occurring amino acid or nucleotide sequence is understood by those skilled in the art as relating to a sequence that is found in a naturally occurring organism as found in nature. Thus, a nucleotide or amino acid sequence that is not 45 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT found in the same state or having the same nucleotide or encoded amino acid sequence as in a naturally occurring organism is included within the meaning of a recombinant nucleotide and / or amino acid sequence described herein. For example, a nucleotide or amino acid sequence that has been altered at one or more nucleotide or amino acid positions from a parent sequence, including variants as described herein, are included within the meaning of a nucleotide or amino acid sequence described herein that is not naturally occurring. A recombinant nucleic acid described herein excludes a naturally occurring chromosome that contains the nucleotide sequence, and can further exclude other molecules, as found in a naturally occurring cell, such as DNA binding proteins, for example, proteins such as histones that bind to chromosomes within a eukaryotic cell.

[0098] Thus, a recombinant nucleic acid described here has physical and chemical differences compared to a naturally occurring nucleic acid. A recombinant or non-naturally occurring nucleic acid described herein does not contain or does not necessarily have some or all of the chemical bonds, either covalent or non-covalent bonds, of a naturally occurring nucleic acid as found in nature. A recombinant nucleic acid described herein thus differs from a naturally occurring nucleic acid, for example, by having a different chemical structure than a naturally occurring nucleic acid as found in a chromosome. A different chemical structure can occur, for example, by cleavage of phosphodiester bonds that release a recombinant nucleic acid from a naturally occurring chromosome. A recombinant nucleic acid described herein can also differ from a naturally occurring nucleic acid by isolating or separating the nucleic acid from proteins that bind to chromosomal DNA in either prokaryotic or eukaryotic cells, thereby differing from a naturally occurring nucleic acid by different non-covalent bonds. With respect to nucleic acids of prokaryotic origin, a non- naturally occurring nucleic acid described herein does not necessarily have some or all of the naturally occurring chemical bonds of a chromosome, for example, binding to DNA binding proteins such as polymerases or chromosome structural proteins, or is not in a higher order structure such as being supercoiled. With respect to nucleic acids of eukaryotic origin, a non- naturally occurring nucleic acid described herein also does not contain the same internal nucleic acid chemical bonds or chemical bonds with structural proteins as found in chromatin. For example, a non-naturally occurring nucleic acid described herein is not chemically bonded to histones or scaffold proteins and is not contained in a centromere or telomere. Thus, the non-naturally occurring nucleic acids described herein are chemically distinct from a naturally occurring nucleic acid because they either lack or contain different 46 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT van der Waals interactions, hydrogen bonds, ionic or electrostatic bonds, and / or covalent bonds from a nucleic acid as found in nature. Such differences in bonds can occur either internally within separate regions of the nucleic acid (that is cis) or such difference in bonds can occur in trans, for example, interactions with chromosomal proteins. In the case of a nucleic acid of eukaryotic origin, a cDNA is considered to be a recombinant or non-naturally occurring nucleic acid since the chemical bonds within a cDNA differ from the covalent bonds, that is the sequence, of a gene on chromosomal DNA. Thus, it is understood by those skilled in the art that recombinant or non-naturally occurring nucleic acid is distinct from a naturally occurring nucleic acid.

[0099] In some embodiments, provided herein is a method of constructing a host strain that can include, among other steps, introducing a vector disclosed herein into a microbial organism, for example, that is capable of expressing an amino acid sequence encoded by the vector and / or is capable of fermentation. Vectors described herein can be introduced stably or transiently into a microbial organism using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. Additional methods are disclosed herein, any one of which can be used in the method described herein. IV. Non-Naturally Occurring Microbial Organisms

[0100] In some aspects, provided herein are non-naturally occurring microbial organisms that express a diamine exporter.

[0101] In some embodiments, provided herein is a microbial organism, in particular a non-naturally occurring microbial organism, that expresses an exogenous diamine exporter. Thus, provided herein is a non-naturally occurring microbial organism having an exogenous nucleic acid encoding an exogenous diamine exporter. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has an exogenous nucleic acid encoding a diamine exporter having the sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C2 to C12. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C2 to C7. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C4 to C6. In some embodiments, the engineered diamine exporter is selective for a diamine selected from ethylenediamine, putrescine, cadaverine, hexamethylenediamine (HMD), or 47 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT heptamethylenediamine. In some embodiments, the engineered diamine export has little to no export activity for a HMD pathway intermediate (e.g., 6ACA).

[0102] In some embodiments, provided herein is a microbial organism (e.g., host microbial organism) that has an exogenous nucleic acid encoding a diamine exporter having the sequence set forth in any one of SEQ ID NOS: 1-42, or a functional fragment thereof. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1- 42. In some embodiments, the microbial organism has a exogenous nucleic acid that encodes an diamine exporter that has at least 50% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has a exogenous nucleic acid that encodes an diamine exporter that has at least 50% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter that has at least 55% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has a exogenous nucleic acid that encodes an diamine exporter that has at least 65% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter that has at least 70% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes an diamine exporter that has at least 75% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter that has at least 80% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes an diamine exporter that has at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter that has at least 90% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter that has at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter that has at least 98% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In 48 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter that has at least 99% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42. In some embodiments, the microbial organism has an exogenous nucleic acid that encodes a diamine exporter that is identical to a sequence set forth in any one of SEQ ID NOS: 1-42.

[0103] In some embodiments, provided herein is a microbial organism, in particular a non-naturally occurring microbial organism, that has one or more disruptions in a gene encoding a diamine exporter. Thus, provided herein is a non-naturally occurring microbial organism having one or more disruptions in a gene encoding a diamine exporter. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has one or more disruptions in a gene encoding a diamine exporter having the sequence set forth in any one of SEQ ID NO: 49. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C2 to C12. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C2 to C7. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C4 to C6. In some embodiments, the engineered diamine exporter is selective for a diamine selected from ethylenediamine, putrescine, cadaverine, hexamethylenediamine (HMD), or heptamethylenediamine. In some embodiments, the engineered diamine export has little to no export activity for a HMD pathway intermediate (e.g., 6ACA).

[0104] In some embodiments, provided herein is a microbial organism (e.g., host microbial organism) that has one or more disruptions in a gene encoding a diamine exporter having the set forth in SEQ ID NO: 49, or a functional fragment thereof. In some embodiments, the microbial organism has one or more disruptions in a gene that encodes a diamine exporter having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence set forth in of SEQ ID NO: 49. In some embodiments, the one or more disruptions are in a 5’ regulatory region of the gene. In some embodiments, the one or more disruptions include one or more deletions. In some embodiments, the one or more deletions are deletions of up to about 200 bp. In some embodiments, the one or more deletions are deletions of about 10 to 20 bp, about 20 to 50 bp, about 50 to 100 bp, about 100-150 bp, or about 150 to 200 bp. In some embodiments, the one or more deletions are a deletion of about 120 to about 160 bp. 49 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0105] Also provided herein is a microbial organism, in particular a non-naturally occurring microbial organism, that expresses an engineered diamine exporter described herein. Thus, provided herein is a non-naturally occurring microbial organism having a recombinant nucleic acid encoding an engineered diamine exporter described herein. Accordingly, in some embodiments, provided herein is microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of a diamine exporter having the sequence set forth in SEQ ID NOS: 1, 46 or 48, such as an engineered diamine exporter that is a variant of amino acid sequence SEQ ID NO: 1 and includes one or more alterations at a position selected from I24, V61, and I88, a variant of amino acid sequence SEQ ID NO: 46 and includes one or more alterations at a position selected from F42, G47, F102 and A133, or a variant of SEQ ID NO: 48 and includes one or more alterations at a position selected from R148 and V286. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C2 to C12. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C2 to C7. In some embodiments, the engineered diamine exporter is selective for a diamine having a carbon length of C4 to C6. In some embodiments, the engineered diamine exporter is selective for a diamine selected from ethylenediamine, putrescine, cadaverine, HMD, or heptamethylenediamine. In some embodiments, the diamine is HMD. In some embodiments, the engineered diamine export has little to no export activity for a HMD pathway intermediate (e.g., 6ACA).

[0106] In some embodiments, provided herein is a microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having (a) a M at a residue corresponding to position 61 in SEQ ID NO: 1; (b) a T at a residue corresponding to position 88; (c) a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1; (d) a F at a residue corresponding to position 24 in SEQ ID NO: 1; (e) a stop codon at a residue corresponding to position 157 in SEQ ID NO: 1, (f) an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1; or (g) any combination of (a)-(f). In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a M at a 50 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT residue corresponding to position 61 in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a M at a residue corresponding to position 61 in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a T at a residue corresponding to position 88, and a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a F at a residue corresponding to position 24 in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a stop codon at a residue corresponding to position 157, and an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1. In some embodiments, the microbial organisms has a recombinant nucleic acid that encodes an engineered exporter having the amino acid sequence set forth in any one of SEQ ID NOS: 43-45.

[0107] In some embodiments, provided herein is a microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of amino acid sequence of SEQ ID NO: 46, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having (a) a L at a residue corresponding to position 42 in SEQ ID NO: 46; (b) a S at a residue corresponding to position 46 in SEQ ID NO: 46; (c) a V at a residue corresponding to position 133 in SEQ ID NO: 46; (d) a Y at a residue corresponding to position 102 in SEQ ID NO: 46; or (e) any combination of (a)-(d). In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a L at a residue corresponding to position 42 in SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a S at a residue corresponding to position 46 in SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a V at a residue corresponding to position 133 in SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a Y at a residue corresponding to position 102 in SEQ ID NO: 46. 51 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0108] In other embodiments, provided herein is a microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of amino acid sequence of SEQ ID NO: 48, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from R148 and V286. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having(a) a C at a residue corresponding to position 148 in SEQ ID NO: 48; b) a E at a residue corresponding to position 286 in SEQ ID NO: 48; or (c) any combination of (a)-(b). In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a C at a residue corresponding to position 148 in SEQ ID NO: 48. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a E at a residue corresponding to position 286 in SEQ ID NO: 48.

[0109] In some embodiments, provided herein is a microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of SEQ ID NO: 1 that includes one more alterations at a position selected from I24, V61, and I88, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1. Accordingly, in some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 52 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT 75% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from I24, V61, and I88 in SEQ ID NO: 1, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 1.

[0110] In some embodiments, provided herein is a microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered diamine 53 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT exporter that is a variant of SEQ ID NO: 46 that includes one more alterations at a position selected from F42, G47, F102 and A133, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 46. Accordingly, in some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 75% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein 54 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter that has an amino acid sequence that includes one or more alterations at a position selected from F42, G47, F102 and A133 in SEQ ID NO: 46, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 46.

[0111] In some embodiments, provided herein is a microbial organism (e.g., host microbial organism) that has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of SEQ ID NO: 48 that includes one more alterations at a position selected from R148 and V286, wherein the engineered diamine exporter, other than the one or more amino acid alterations, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 48. Accordingly, in some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 65% identical to SEQ ID NO: 48. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 70% identical to SEQ ID NO: 48. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or 55 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT more amino acid alterations, is at least 75% identical to SEQ ID NO: 48. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 80% identical to SEQ ID NO: 46. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 85% identical to SEQ ID NO: 48. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 90% identical to SEQ ID NO: 48. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 95% identical to SEQ ID NO: 48. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 98% identical to SEQ ID NO: 48. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is at least 99% identical to SEQ ID NO: 48. In some embodiments, the microbial organism has an recombinant nucleic acid that encodes an engineered diamine having an amino acid sequence that includes one or more alterations at a position selected from R148 and V286 in SEQ ID NO: 48, wherein the engineered diamine exporter, other than the one or more amino acid alterations, is identical to SEQ ID NO: 48.

[0112] In some embodiments, provided herein are non-naturally occurring microbial organism provided that are capable of producing a diamine. In some embodiments, a 56 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT microbial organism that expresses a diamine exporter provided herein can be cultured for sufficient time and under sufficient conditions to produce a desired product (e.g., a diamine).

[0113] In some embodiments, provided herein is a non-naturally occurring microbial organism that is a capable of producing more diamine compared to a control microbial organism that does not having a recombinant nucleic acid that encodes a diamine exporter described herein. Such a microbial organism, in some embodiments, is capable of producing at least 10% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 20% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 30% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 40% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 50% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 60% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 70% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 80% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 90% more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1.1 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1.2 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1.3 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1.4 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1.5 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1.6 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1.7 fold more diamine compared to the control microbial organism. In some embodiments, the 57 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT microbial organism is capable of producing at least 1.8 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 1.9 fold more diamine compared to the control microbial organism. In some embodiments, the microbial organism is capable of producing at least 2 fold more diamine compared to the control microbial organism. In some embodiments, diamine production is determined by measuring the levels of diamine in a culture media comprising the microbial organism. In some embodiments, diamine production is determined by measuring the levels of diamine using a genetically encoded diamine reporter. In some embodiments, the diamine production is measured in the presence of exogenous diamine.

[0114] In some embodiments, provided herein is a non-naturally occurring microbial organism that is a capable of more diamine export compared to a control microbial organism that does not having a recombinant nucleic acid that encodes a diamine export exporter described herein. Such a microbial organism, in some embodiments, is capable of at least 10% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 20% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 30% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 40% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 50% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 60% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 70% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 80% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 90% more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1.1 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1.2 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1.3 fold more diamine export compared to the control microbial organism. In some embodiments, the 58 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT microbial organism is capable of at least 1.4 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1.5 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1.6 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1.7 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1.8 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 1.9 fold more diamine export compared to the control microbial organism. In some embodiments, the microbial organism is capable of at least 2 fold more diamine export compared to the control microbial organism. In some embodiments, diamine export is determined by measuring the levels of diamine in a culture media comprising the microbial organism. In some embodiments, diamine export is determined by measuring the levels of diamine using a genetically encoded diamine reporter. In some embodiments, the diamine export is measured in the presence of exogenous diamine.

[0115] In some embodiments, provided herein is a non-naturally occurring microbial organism that has a higher growth rate compared to a control microbial organism that does not having a recombinant nucleic acid that encodes a diamine export exporter described herein. Such a microbial organism, in some embodiments, has a growth rate that is at least 10% higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 20% higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 30% higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 40% higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 50% higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 60% higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 70% higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 80% higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 90% higher compared 59 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.1 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.2 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.3 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.4 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.5 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.6 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.7 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.8 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 1.9 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the microbial organism has a growth rate that is at least 2 fold higher compared to the growth rate of the control microbial organism. In some embodiments, the growth rate is measured in the presence of exogenous diamine.

[0116] The microbial organisms provided herein can be produced by introducing an expressible nucleic acid encoding a diamine exporter as provided herein. In some embodiments, an expressible nucleic acid encoding one or more of the enzymes or proteins of a pathway for diamine production (e.g., a HMD pathway). Depending on the host cell chosen, nucleic acids for some or all of a particular diamine pathway (e.g., a HMD pathway) can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for the diamine pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some diamine pathway genes, but is deficient in others, then an encoding nucleic acid is included for the deficient enzyme(s) or protein(s) to production of the diamine (e.g., HMD), or exogenous expression of endogenously expressed genes can be provided to increase expression of pathway enzymes, if 60 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT desired. Thus, a cell provided herein can be produced by introducing a diamine exporter provided herein, and optionally exogenous enzyme or protein activities to obtain a desired biosynthetic pathway, or by introducing one or more exogenous enzyme or protein activities, including an engineered aldehyde dehydrogenase provided herein that, together with one or more endogenous enzymes or proteins, produces a desired product such as a diamine (e.g., HMD), or a downstream product related thereto.

[0117] Although generally described herein as utilizing a cell that is a microbial organism as a host cell, particularly for producing a diamine (e.g., HMD), or a downstream product related thereto, it is understood that a host cell can be a cell line of a higher eukaryote, such as a mammalian cell line or insect cell line. Thus, it is understood that reference herein to a host cell that is a microbial organism can alternatively utilize a higher eukaryotic cell line to produce a desired product. Exemplary higher eukaryotic cell lines include, but are not limited to, Chinese hamster ovary (CHO), human (Hela, Human Embryonic Kidney (HEK) 293, Jurkat), mouse (3T3), primate (Vero), insect (Sf9), and the like. Such cell lines are commercially available (see, for example, the American Type Culture Collection (ATCC; Manassas VA); Life Technologies, Carlsbad CA). It is understood that any suitable host cell can be used to introduce an engineered aldehyde dehydrogenase provided herein, and optionally metabolic and / or genetic modifications to produce a desired product.

[0118] Depending on the diamine pathway constituents of a selected host cell, the non- naturally occurring cells provided herein will include at least one exogenously expressed diamine pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more diamine pathways, or a downstream product related thereto such as an ester or amide thereof, including an engineered aldehyde dehydrogenase provided herein. For example, diamine synthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid, including a diamine exporter provided herein. In a host deficient in all enzymes or proteins of a diamine pathway (e.g., a HMD pathway), or a downstream product related thereto, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins.

[0119] Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form 61 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT will, at least, parallel the diamine pathway deficiencies of the selected host cell if a diamine pathway (e.g., a HMD pathway), is to be included in the cell. Therefore, a non-naturally occurring cell provided herein can have one, two, three, four, five, six, seven, eight, and so forth, depending on the particular pathway, up to all nucleic acids encoding the enzymes or proteins constituting a diamine pathway disclosed herein. In some embodiments, the non- naturally occurring cells also can include other genetic modifications that facilitate or optimize diamine synthesis or that confer other useful functions onto the host cell. One such other functionality can include, for example, augmentation of the synthesis of one or more diamine pathway precursors.

[0120] Generally, a host cell is selected such that it can express a diamine exporter provided herein, and optionally produces the precursor of a pathway (e.g., a HMD pathway), in a cell containing such a pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host cell. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a cell that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a diamine pathway (e.g., a HMD pathway), or a downstream product related thereto, if desired.

[0121] In some embodiments, a non-naturally occurring cell provided herein is generated from a host that contains the enzymatic capability to produce a diamine (e.g., HMD), or a downstream product related thereto. In this specific embodiment it can be useful to increase the synthesis or reduce the accumulation of a diamine pathway product to, for example, drive diamine pathway reactions toward diamine production, or a downstream product related thereto. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described HMD pathway enzymes or proteins, including a diamine exporter provided herein. Overexpression of the enzyme or enzymes and / or protein or proteins of the diamine pathway (e.g., the HMD pathway) can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes, including exogenous expression of a diamine provided herein. Therefore, naturally occurring organisms can be readily converted to non-naturally occurring cells provided herein, for example, producing a diamine or a downstream product related thereto, through 62 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT overexpression of one, two, three, four, five, six, seven, eight, or more, depending on the diamine pathway, that is, up to all nucleic acids encoding diamine pathway enzymes or proteins, or enzymes that produce a downstream product related thereto. In addition, a non- naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the diamine pathway, or a downstream product related thereto.

[0122] It is understood that any of the pathways disclosed herein, as described in the Examples, including the pathways described herein, can be utilized to generate a cell that produces any pathway intermediate or product, as desired, in particular a pathway that utilizes a diamine exporter as described herein. As disclosed herein, a cell that produces a diamine pathway intermediate can be used in combination with another cell expressing one or more upstream or downstream diamine pathway enzymes to produce a desired product.

[0123] In some embodiments, provided herein is a non-naturally occurring microbial organism having a vector described herein comprising a nucleic acid described herein. Also provided a non-naturally occurring microbial organism having a nucleic acid described herein. In some embodiments, the nucleic acid is integrated into a chromosome of the organism. In some embodiments, the integration is site-specific. In an embodiment described herein, the nucleic acid is expressed. In some embodiments, provided herein is a non-naturally occurring microbial organism having a diamine exporter described herein.

[0124] The microbial organisms provided herein can be, for example, bacteria, yeast, fungus or any of a variety of microorganisms applicable or suitable to fermentation processes. In some embodiments, the microbial organism is a species of bacteria, yeast or fungus. In some embodiments, the microbial organism is a species of bacteria. In some embodiments, the microbial organism is a species of yeast. In some embodiments, the microbial organism is a species of fungus. Exemplary bacteria include any species selected from the order Enterobacteriales, family Enterobacteriaceae, including the genera Escherichia and Klebsiella; the order Aeromonadales, family Succinivibrionaceae, including the genus Anaerobiospirillum; the order Pasteurellales, family Pasteurellaceae, including the genera Actinobacillus and Mannheimia; the order Rhizobiales, family Bradyrhizobiaceae, including the genus Rhizobium; the order Bacillales, family Bacillaceae, including the genus Bacillus; the order Actinomycetales, families Corynebacteriaceae and Streptomycetaceae, including the genus Corynebacterium and the genus Streptomyces, respectively; order Rhodospirillales, 63 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT family Acetobacteraceae, including the genus Gluconobacter; the order Sphingomonadales, family Sphingomonadaceae, including the genus Zymomonas; the order Lactobacillales, families Lactobacillaceae and Streptococcaceae, including the genus Lactobacillus and the genus Lactococcus, respectively; the order Clostridiales, family Clostridiaceae, genus Clostridium; and the order Pseudomonadales, family Pseudomonadaceae, including the genus Pseudomonas. Non-limiting species of host bacteria include Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida. E. coli is a particularly useful host organism since it is a well characterized microbial organism suitable for genetic engineering.

[0125] Similarly, exemplary species of yeast or fungi species include any species selected from the order Saccharomycetales, family Saccaromycetaceae, including the genera Saccharomyces, Kluyveromyces and Pichia; the order Saccharomycetales, family Dipodascaceae, including the genus Yarrowia; the order Schizosaccharomycetales, family Schizosaccaromycetaceae, including the genus Schizosaccharomyces; the order Eurotiales, family Trichocomaceae, including the genus Aspergillus; and the order Mucorales, family Mucoraceae, including the genus Rhizopus. Non-limiting species of host yeast or fungi include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, Yarrowia lipolytica, and the like. A particularly useful host organism that is a yeast includes Saccharomyces cerevisiae.

[0126] In some embodiments, a non-naturally occurring microbial organism comprising a recombinant nucleic acid encoding a diamine exporter provided herein further comprises a diamine pathway (e.g., a diamine pathway or diamine biosynthesis pathway) . Accordingly, the non-naturally occurring microbial organisms occurring microbial organism comprising a recombinant nucleic acid encoding a diamine exporter further comprises a pathway to produce a diamine. Exemplary diamines pathways include, for example, pathway for production of hexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine or heptamethylenediamine. In some embodiments, the pathway for production of a diamine is a pathway for producing a linear diamine having a carbon length of C4-C6. In some 64 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT embodiments, the pathway is a pathway for production of HMD (e.g., a HMD pathway or HMD biosynthesis pathway).

[0127] In some embodiments, a non-naturally occurring microbial organism comprising a recombinant nucleic acid encoding a diamine exporter provided herein further comprises a diamine pathway, with at least one enzyme of the pathway encoded by a exogenous nucleic acid. The exogenous nucleic acid encoding the at least one diamine pathway enzymes may be heterologous or homologous. In some embodiments, the non-naturally occurring microbial organism comprising a recombinant nucleic acid encoding a diamine exporter and a diamine pathway, with at least two, three, four, five, six, seven, eight, nine, ten, or eleven exogenous nucleic acids encoding at least two, three, four, five, six, seven, eight, nine, ten or eleven enzymes of the diamine pathway. In some embodiments, the at least one enzyme of the diamine pathway is expressed in sufficient amount to produce HMD. In some embodiments, the diamine pathway is a pathway production of hexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine or heptamethylenediamine. In some embodiments, the diamine pathway is for producing a linear diamine having a carbon length of C4-C6.

[0128] In some embodiments, a non-naturally occurring microbial organism comprising a recombinant nucleic acid encoding a diamine exporter provided herein further comprises a HMD pathway, with at least one enzyme of the pathway encoded by a exogenous nucleic acid. The exogenous nucleic acid encoding the at least one HMD pathway enzyme may be heterologous or homologous. In some embodiments, the non-naturally occurring microbial organism comprising a recombinant nucleic acid encoding a diamine exporter and a HMD pathway with at least two, three, four, five, six, seven, eight, nine, ten, or eleven exogenous nucleic acids encoding at least two, three, four, five, six, seven, eight, nine, ten or eleven enzymes of the HMD pathway. In some embodiments, the at least one enzyme of the HMD pathway is expressed in sufficient amount to produce HMD. In some embodiments, the diamine exporter has the ability to export HMD.

[0129] In some embodiments, the HMD synthesis pathway comprises an intermediate compound selected from 3-oxoadipyl-CoA, adipate semialdehyde, 6-aminocaproate (6- ACA), 6-ACA semialdehyde, 2-aminopimelate, 3,6-dihydroxyhexanoyl-CoA or homolysine. In some embodiments, the HMD pathway comprises an 6-aminocaproate (6-ACA) as an intermediate. 65 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0130] In some embodiments, the HMD pathway comprises an enzyme selected from of 3-oxoadipyl-CoA thiolase, 6-ACA transaminase or dehydrogenase, 6-aminocaproyl-CoA reductase, 6-ACA reductase, adipyl-CoA reductase, adipate reductase, 6-hydroxy 3- oxohexanoyl-CoA dehydrogenase, 2-aminopimelate decarboxylase, or homolysine decarboxylase.

[0131] In some embodiments, the HMD pathway comprises an enzyme and substrate- product pair selected from 3-oxoadipyl-CoA thiolase that acts on succinyl-CoA and acetyl- CoA to make 3-oxoadipyl-CoA, 6-ACA transaminase that acts on adipyl-CoA to form 6- ACA, 6-aminocaproyl-CoA reductase that acts on 6-aminocaproayl-CoA to form 6-ACA semialdehyde, 6-ACA reductase that acts on 6-ACA and converts it directly to 6-ACA semialdehyde, adipyl-CoA reductase that acts on adipyl-CoA to form adipate semialdehyde, adipate reductase that acts on adipate and converts it directly to adipate semialdehyde, 6- hydroxy 3-oxohexanoyl-CoA dehydrogenase that reduces 6-hydroxy 3-oxohexanoyl-CoA to form 3,6-dihydroxy hexanoyl-CoA, 2-aminopimelate decarboxylase that decarboxylates 2- aminopimelate to form 6-ACA, or homolysine decarboxylase that decarboxylates homolysine to form HMD. In some embodiments, the HMD pathway is selected from the group of pathways (a) to (m): (a) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3- hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase and 6-ACA-CoA reductase, or 6-ACA reductase, HMDA transaminase or dehydrogenase; b) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3- hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (c) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3- hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA transferase, hydrolase or transferase, adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; 66 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT (d) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 3- hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA transferase, hydrolase or transferase, adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (e) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; (f) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (g) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase; (h) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase, HMDA transaminase or dehydrogenase; (i) an 4-hydroxy-2-oxoheptane-I,7-dioate (HODH aldolase); an 2-oxohept-4- ene-1,7-dioate (OHED) hydratase; an OHED formate-lyase and a pyruvate formate- lyase activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; or an adipate semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase (aminating); (j) a β-ketothiolase or an acetyl-CoA carboxylase and an acetoacetyl-CoA synthase, a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, and a trans-2-enoyl-CoA reductase for producing hexanoyl- CoA, one or more of a thioesterase, an aldehyde dehydrogenase, or a butanal 67 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT dehydrogenase, said host producing hexanal or hexanoates; one or more of a monooxygenase, an alcohol dehydrogenase, an aldehyde dehydrogenase, a 6- hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4- hydroxybutyrate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a 7- oxoheptanoate dehydrogenase, said host producing adipic acid or adipate semialdehyde; one or more of a monooxygenase, a transaminase, a 6- hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4- hydroxybutyrate dehydrogenase, and an alcohol dehydrogenase, said host producing 6-aminohexanoate; one or more of a carboxylate reductase, a w-transaminase, a deacetylase, a N-acetyl transferase, or an alcohol dehydrogenase, said host producing hexamethylenediamine; (k) acetyltransferase or thiolase to form 6-hydroxy-3-oxo-hexanoyl-CoA, 6- hydroxy-3-oxo-hexanoyl-CoA dehydrogenase, 3,4-dihydroxyhexanoyl-CoA dehydratase, 6-hydroxy-2-hexenoyl-CoA reductase, 6-hydroxyhexanoyl-CoA hydrolase to form 6-ACA, 6-hydroxycaproate dehydrogenase and transaminase to form HMDA; (l) homocitrate synthase, a homoaconitase and a homoisocitrate dehydrogenase to form 2-ketopimelate, 2-keto decarboxylase catalyzing the conversion of α-ketopimelate to adipate semialdehyde, 2-aminotransferase catalyzes the conversion of α-ketopimelate to 2-aminopimelate, 2-aminopimelate decarboxylase to decarboxylate 2-aminopimelate and form 6-ACA, aldehyde dehydrogenase catalyzes the conversion of 6-ACA to 6-aminohexanal and the aminotransferase catalyzes the conversion of 6-aminohexanal to 6-hexamethylenediamine; and (m) glutamyl-CoA transferase and / or ligase, beta-ketothiolase, 3-oxo-6- aminopimeloyl-CoA oxidoreductase, 3-hydroxy-6-aminopimeloyl-CoA dehydratase, 6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA reductase (aldehyde forming), 2-amino-7-oxoheptanoate aminotransferase and / or aminating oxidoreductase, homolysine decarboxylase, 6-aminopimeloyl-CoA hydrolase, transferase and / or ligase, 2-aminopimelate decarboxylase.

[0132] In any of the embodiments in the alternative pathways set out above, suitable enzymes may be selected from 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase, 68 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT 3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase, adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase, 3-oxoadipyl-CoA:acyl CoA transferase, 3- oxoadipate dehydrogenase, 3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase, adipyl-CoA transferase, lygase or hydrolase, 6-ACA transferase or synthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase, adipate reductase, 6-ACA transaminase or dehydrogenase, or 6-ACA reductase. Additional pathways and enzymes for HMD production are described in U.S. Pat. No.8,377,680, U.S. Pat. No.9,458,480, U.S. Pat. No. 10,711,289, and U.S. Pub. No.20120157717, all of which are hereby incorporated by reference.

[0133] The subject matter described herein includes general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well-known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction.

[0134] In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and / or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene’s promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible 69 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring cell.

[0135] It is understood that, in methods described herein, any of the one or more recombinant and / or exogenous nucleic acids can be introduced into a cell to produce a non- naturally occurring cell provided herein. The nucleic acids can be introduced so as to confer, for example, a diamine pathway (e.g., a HMD pathway) onto the cell, including introducing a nucleic acid encoding an engineered aldehyde dehydrogenase provided herein. Alternatively, encoding nucleic acids can be introduced to produce a cell having the biosynthetic capability to catalyze some of the required reactions to confer diamine biosynthetic capability to produce an intermediate. For example, a non-naturally occurring cell having a diamine pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, including a diamine exporter provided herein. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring cell provided herein, including an engineered aldehyde dehydrogenase provided herein. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring cell provided herein, as desired, so long as the combination of enzymes and / or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of five, six, seven, eight, nine, ten, eleven, twelve or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring cell provided herein, as desired, so long as the combination of enzymes and / or proteins of the desired biosynthetic pathway results in production of the corresponding desired product.

[0136] A recombinant nucleic acid encoding a diamine exporter provided herein, and optionally exogenous nucleic acid sequences involved in a pathway for production of a diamine (e.g., HMD), or a downstream product related thereto such as an ester or amide thereof, can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host 70 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

[0137] An expression vector or vectors can be constructed to include a recombinant nucleic acid encoding a diamine exporter provided herein, and / or optionally and / or an exogenous nucleic acid encoding a diamine production pathway (e.g., a HMD pathway), or nucleic acids encoding an enzyme that produces a downstream product related to a diamine, as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the host cells provided herein include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences encoding an engineered aldehyde dehydrogenase provided herein or encoding polypeptides involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or 71 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

[0138] A vector or expression vector can also be used to express an encoded nucleic acid to produce an encoded polypeptide by in vitro transcription and translation. Such a vector or expression vector will comprise at least a promoter, and includes the vectors described herein above. Such a vector for in vitro transcription and translation generally is double stranded DNA. Methods of in vitro transcription and translation are well known to those skilled in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). Kits for in vitro transcription and translation are also commercially available (see, for example, Promega, Madison, WI; New England Biolabs, Ipswich, MA; Thermo Fisher Scientific, Carlsbad, CA). V. Methods of Diamine Production

[0139] In yet another aspect of the disclosure, provided herein are methods for producing a diamine.

[0140] In some embodiments, the method for producing a diamine comprises comprising culturing a cell provided herein under conditions and for a sufficient period of time to produce the diamine. Such a cell expresses a diamine exporter provided herein, or is any of the microbial organisms that express a diamine exporter as provided herein. In some embodiments, the method is for producing a diamine having a carbon length of C2 to C12. In some embodiments, the method is for producing a diamine having a carbon length of C2 to C7. In some embodiments, the method is for producing a diamine having a carbon length of C4 to C6. In some embodiments, the method is for producing a diamine selected from ethylenediamine, putrescine, cadaverine, hexamethylenediamine (HMD), or heptamethylenediamine. In some embodiments, the cell is cultured in the absence of exogenous diamine. In a particular embodiment, the method further comprises separating the diamine from the other components in the culture, e.g., by distillation, chromatography, centrifugation, etc. 72 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0141] In some embodiments, the method comprises culturing a non-naturally occurring microbial organism that has an exogenous nucleic acid encoding a diamine exporter having the sequence set forth in any one of SEQ ID NOS: 1-42 under conditions and for a sufficient period of time to produce the diamine. In some embodiments, the method comprises culturing microbial organism has an exogenous nucleic acid that encodes a diamine exporter having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence set forth in any one of SEQ ID NOS: 1-42 under conditions and for a sufficient period of time to produce the diamine.

[0142] In some embodiments, the method comprises culturing a non-naturally occurring microbial organism that has one or more disruptions in a gene encoding a diamine exporter having the set forth in SEQ ID NO: 49 under conditions and for a sufficient period of time to produce the diamine. In some embodiments, the method comprises culturing microbial organism that has one or more disruptions in a gene that encodes a diamine exporter having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a sequence set forth in SEQ ID NO: 49 under conditions and for a sufficient period of time to produce the diamine. In some embodiments, the one or more disruptions are in a 5’ regulatory region of the gene. In some embodiments, the one or more disruptions include one or more deletions. In some embodiments, the one or more deletions are deletions of up to about 200 bp. In some embodiments, the one or more deletions are deletions of about 10 to 20 bp, about 20 to 50 bp, about 50 to 100 bp, about 100-150 bp, or about 150 to 200 bp. In some embodiments, the one or more deletions are a deletion of about 120 to about 160 bp.

[0143] In some embodiments, the method comprises culturing a non-naturally occurring microbial organism that has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from I24, V61, and I88, or a combination thereof, in SEQ ID NO: 1, under conditions and for a sufficient period of time to produce the diamine. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having (a) a M at a residue corresponding to position 61 in SEQ ID NO: 1; (b) a T at a residue corresponding to position 88; (c) a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1; (d) a F at a residue corresponding to position 24 in SEQ ID NO: 1; (e) a stop codon at a residue corresponding to position 157 in SEQ ID NO: 1, (f) an insertion 73 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT of up to ten amino acids at the C-terminus in SEQ ID NO: 1; or (g) any combination of (a)- (f). In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a M at a residue corresponding to position 61 in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a M at a residue corresponding to position 61 in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a T at a residue corresponding to position 88, and a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a F at a residue corresponding to position 24 in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a stop codon at a residue corresponding to position 157, and an insertion of up to ten amino acids at the C- terminus in SEQ ID NO: 1. In some embodiments, the microbial organism has a recombinant nucleic acid encodes an engineered exporter having the amino acid sequence set forth in any one of SEQ ID NOS: 43-45. In some embodiments, the microbial organism has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of SEQ ID NO: 1 that includes one more alterations at a position selected from I24, V61, and I88, wherein the engineered diamine exporter has, other than the one or more alterations, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 1.

[0144] In some embodiments, the method comprises culturing a non-naturally occurring microbial organism that has a recombinant nucleic acid encoding an engineered diamine exporter that isa variant of amino acid sequence of SEQ ID NO: 46, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from F42, G47, F102 and A133, or a combination thereof, in SEQ ID NO: 46, under conditions and for a sufficient period of time to produce the diamine. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having (a) a L at a residue corresponding to position 42 in SEQ ID NO: 46; (b) a S at a residue corresponding to position 46 in SEQ ID NO: 46; (c) a V at a residue corresponding to position 133 in SEQ ID NO: 46; (d) a Y at a residue corresponding to position 102 in SEQ ID NO: 46; or (e) any combination of (a)-(d). In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a L at a 74 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT residue corresponding to position 42 in SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a S at a residue corresponding to position 46 in SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a V at a residue corresponding to position 133 in SEQ ID NO: 46. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a Y at a residue corresponding to position 102 in SEQ ID NO: 46. In some embodiments, the microbial organisms has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of SEQ ID NO: 46 that includes one more alterations at a position selected from F42, G47, F102 and A133, wherein the engineered diamine exporter has, other than the one or more alterations, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 46.

[0145] In other embodiments the method comprises culturing a non-naturally occurring microbial organism that has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of amino acid sequence of SEQ ID NO: 48, or a functional fragment thereof, and the engineered diamine exporter has one or more alterations at a position selected from R148 and V286. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having(a) a C at a residue corresponding to position 148 in SEQ ID NO: 48; b) a E at a residue corresponding to position 286 in SEQ ID NO: 48; or (c) any combination of (a)-(b). In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a C at a residue corresponding to position 148 in SEQ ID NO: 48. In some embodiments, the microbial organism has a recombinant nucleic acid that encodes an engineered diamine exporter having a E at a residue corresponding to position 286 in SEQ ID NO: 48. In some embodiments, the microbial organism has a recombinant nucleic acid encoding an engineered diamine exporter that is a variant of SEQ ID NO: 48 that includes one more alterations at a position selected from R148 and V286, wherein the engineered diamine exporter has, other than the one or more alterations, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced as SEQ ID NO: 48. 75 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0146] Suitable purification and / or assays to test for the expression of a diamine exporter, or for production of a diamine (e.g., HMD), or a downstream product related thereto, can be performed using well known methods (see also Examples). Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art (see also Example).

[0147] The diamine (e.g., HMD), or other desired product, such as a downstream product related thereto, can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, or ultrafiltration. All of the above methods are well known in the art.

[0148] Any of the non-naturally occurring cells expressing a diamine exporter provided herein described herein can be cultured to produce and / or secrete the diamine (e.g., HMD) provided herein. For example, the cells that produce a diamine, or a downstream product related thereto, can be cultured for the biosynthetic production of the diamine, or a downstream product related f. Accordingly, in some embodiments, provided herein is a culture medium containing the diamine, or a downstream product related thereto, or a diamine pathway intermediate described herein. In some aspects, the culture medium can also be separated from the non-naturally occurring cells provided herein that produced the diamine (e.g., HMD), or a downstream product related thereto, or diamine pathway 76 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT intermediate. Methods for separating a cell from culture medium are well known in the art. Exemplary methods include filtration, flocculation, precipitation, centrifugation, sedimentation, and the like.

[0149] For the production of a diamine exporter provided herein, or of diamine, or a downstream product related thereto, in a cell expressing a diamine exporter provided herein, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United States publication 2009 / 0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. Fermentations can also be conducted in two phases, if desired. The first phase can be aerobic to allow for high growth and therefore high productivity, followed by an anaerobic phase of high yields of a desired product such as a diamine (e.g., HMD), or a downstream product related thereto.

[0150] If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

[0151] The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring cell. Such sources include, for example: sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch; or glycerol, and it is understood that a carbon source can be used alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. In one embodiment, the carbon source is a sugar. In one embodiment, the carbon source is a sugar-containing biomass. In some embodiments, the sugar is glucose. In one embodiment, the sugar is xylose. In another embodiment, the sugar is arabinose. In one embodiment, the sugar is galactose. In another embodiment, the sugar is fructose. In other embodiments, the sugar is sucrose. In one embodiment, the sugar is starch. In certain 77 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT embodiments, the carbon source is glycerol. In some embodiments, the carbon source is crude glycerol. In one embodiment, the carbon source is crude glycerol without treatment. In other embodiments, the carbon source is glycerol and glucose. In another embodiment, the carbon source is methanol and glycerol. In one embodiment, the carbon source is carbon dioxide. In one embodiment, the carbon source is formate. In one embodiment, the carbon source is methane. In one embodiment, the carbon source is methanol. In certain embodiments, methanol is used alone as the sole source of carbon or in combination with other carbon sources described herein or known in the art. In a specific embodiment, the methanol is the only (sole) carbon source. In one embodiment, the carbon source is chemoelectro-generated carbon (see, e.g., Liao et al. (2012) Science 335:1596). In one embodiment, the chemoelectro-generated carbon is methanol. In one embodiment, the chemoelectro-generated carbon is formate. In one embodiment, the chemoelectro-generated carbon is formate and methanol. In one embodiment, the carbon source is a carbohydrate and methanol. In one embodiment, the carbon source is a sugar and methanol. In another embodiment, the carbon source is a sugar and glycerol. In other embodiments, the carbon source is a sugar and crude glycerol. In yet other embodiments, the carbon source is a sugar and crude glycerol without treatment. In one embodiment, the carbon source is a sugar- containing biomass and methanol. In another embodiment, the carbon source is a sugar- containing biomass and glycerol. In other embodiments, the carbon source is a sugar- containing biomass and crude glycerol. In yet other embodiments, the carbon source is a sugar-containing biomass and crude glycerol without treatment. In some embodiments, the carbon source is a sugar-containing biomass, methanol and a carbohydrate. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods provided herein include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the cells provided herein for the expression of a diamine exporter provided herein, and optionally production of a diamine (e.g., HMD), or a downstream product thereof.

[0152] In addition to renewable feedstocks such as those exemplified above, the cells provided herein that a diamine (e.g., HMD), or a downstream product thereof, also can be 78 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes are expressed in the diamine-producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source.

[0153] The Wood-Ljungdahl pathway catalyzes the conversion of CO and H2 to acetyl- CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO2and CO2 / H2mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H2- dependent conversion of CO2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO2and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp.3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation: 2 CO2 + 4 H2 + n ADP + n Pi → CH3COOH + 2 H2O + n ATP Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO2and H2mixtures as well for the production of acetyl-CoA and other desired products.

[0154] The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolate:corrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC) (see WO2009 / 094485). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a diamine biosynthesis pathway, or a 79 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT downstream product related thereto, including a nucleic acid encoding a diamine exporter provided herein, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood- Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the cells provided herein such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.

[0155] Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and / or hydrogenase activities can also be used for the conversion of CO, CO2and / or H2to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha- ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and / or H2 by carbon monoxide dehydrogenase and hydrogenase are utilized to fix CO2via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase / phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can be converted to glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Acetyl-CoA can also be converted to acetoacetyl-CoA by, for example, acetoacetyl-CoA thiolase to funnel into a 1,3-BDO pathway, as disclosed herein. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a diamine (e.g., HMD), or a downstream product thereof, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the cells provided herein can be performed such that the modified organism contains a reductive TCA pathway.

[0156] Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring cell can be produced that produces and / or secretes the biosynthesized compounds provided herein when grown on a carbon source such as a carbohydrate. Such compounds include, for example, diamines (e.g., HMD), 80 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT or a downstream product thereof, and any of the intermediate metabolites in the diamine biosynthesis pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the biosynthetic pathways for the diamine, or a downstream product related thereto, including a diamine exporter provided herein. Accordingly, provided herein is a non-naturally occurring cell that produces and / or secretes a diamine (e.g., HMD), or a downstream product thereof, when grown on a carbohydrate or other carbon source and produces and / or secretes any of the intermediate metabolites in the diamine pathway when grown on a carbohydrate or other carbon source. The cells producing the diamine, or a downstream product related thereto, provided herein can initiate synthesis from an intermediate of a diamine biosynthesis pathway.

[0157] The non-naturally occurring cells provided herein are constructed using methods well known in the art as exemplified herein to exogenously express a diamine exporter provided herein, and optionally at least one nucleic acid encoding a diamine pathway enzyme or protein, or a downstream product related thereto. The enzymes or proteins can be expressed in sufficient amounts to produce the diamine, or a downstream product related thereto. It is understood that the cells provided herein are cultured under conditions sufficient to express a diamine exporter provided herein or produce a diamine (e.g., HMD), or a downstream product related thereto.

[0158] A microbial organism provided herein is cultured for production of a diamine (e.g., HMD) using well known methods. The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products provided herein can be obtained under anaerobic or substantially anaerobic culture conditions.

[0159] In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication 2009 / 0047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring cells as well as other anaerobic conditions well known in the art. 81 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0160] As described herein, one exemplary growth condition for achieving biosynthesis of a diamine (e.g., HMD), or a downstream product related thereto, includes micro-aerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring cells provided herein can be sustained, cultured or fermented under micro-aerobic or substantially micro-aerobic conditions.

[0161] The culture conditions described herein can be scaled up and grown continuously for manufacturing of a diamine (e.g., HMD), or a downstream product related thereto, by a microbial organism provided herein. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of diamine (e.g., HMD), or a downstream product related thereto. Generally, and as with non-continuous culture procedures, the continuous and / or near-continuous production of diamine, or a downstream product related thereto will include culturing a non-naturally occurring cell producing the diamine, or a downstream product related thereto provided herein in sufficient nutrients and medium to sustain and / or nearly sustain growth in an exponential phase. Continuous culture under such conditions can include, for example, growth or culturing for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms provided herein can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and / or near-continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the cell provided herein is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

[0162] Exemplary fermentation processes include, but are not limited to, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; and continuous fermentation and continuous separation. In an exemplary batch fermentation protocol, the production organism is grown in a suitably sized bioreactor sparged with an appropriate gas. As the cells grow and utilize the carbon source, additional carbon source(s) and / or other nutrients are fed into the bioreactor at a rate approximately balancing consumption of the carbon source and / or nutrients. The temperature of the bioreactor is 82 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT maintained at a desired temperature, generally in the range of 22-37 degrees C, but the temperature can be maintained at a higher or lower temperature depending on the growth characteristics of the production organism and / or desired conditions for the fermentation process. Growth continues for a desired period of time to achieve desired characteristics of the culture in the fermenter, for example, cell density, product concentration, and the like. In a batch fermentation process, the time period for the fermentation is generally in the range of several hours to several days, for example, 8 to 24 hours, or 1, 2, 3, 4 or 5 days, or up to a week, depending on the desired culture conditions. The pH can be controlled or not, as desired, in which case a culture in which pH is not controlled will typically decrease to pH 3- 6 by the end of the run. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit, for example, a centrifuge, filtration unit, and the like, to remove cells and cell debris. In the case where the desired product is expressed intracellularly, the cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. The fermentation broth can be transferred to a product separations unit. Isolation of product occurs by standard separations procedures employed in the art to separate a desired product from dilute aqueous solutions. Such methods include, but are not limited to, liquid- liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like) to provide an organic solution of the product, if appropriate, standard distillation methods, and the like, depending on the chemical characteristics of the product of the fermentation process.

[0163] In an exemplary fully continuous fermentation protocol, the production organism is generally first grown up in batch mode in order to achieve a desired cell density. When the carbon source and / or other nutrients are exhausted, feed medium of the same composition is supplied continuously at a desired rate, and fermentation liquid is withdrawn at the same rate. Under such conditions, the product concentration in the bioreactor generally remains constant, as well as the cell density. The temperature of the fermenter is maintained at a desired temperature, as discussed above. During the continuous fermentation phase, it is generally desirable to maintain a suitable pH range for optimized production. The pH can be monitored and maintained using routine methods, including the addition of suitable acids or 83 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT bases to maintain a desired pH range. The bioreactor is operated continuously for extended periods of time, generally at least one week to several weeks and up to one month, or longer, as appropriate and desired. The fermentation liquid and / or culture is monitored periodically, including sampling up to every day, as desired, to assure consistency of product concentration and / or cell density. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and product, are generally subjected to a continuous product separations procedure, with or without removing cells and cell debris, as desired. Continuous separations methods employed in the art can be used to separate the product from dilute aqueous solutions, including but not limited to continuous liquid-liquid extraction using a water immiscible organic solvent (e.g., toluene or other suitable solvents, including but not limited to diethyl ether, ethyl acetate, tetrahydrofuran (THF), methylene chloride, chloroform, benzene, pentane, hexane, heptane, petroleum ether, methyl tertiary butyl ether (MTBE), dioxane, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the like), standard continuous distillation methods, and the like, or other methods well known in the art.

[0164] Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of a diamine (e.g., HMD), or a downstream product related thereto, can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art and described herein.

[0165] In addition to the fermentation procedures described herein using the producers of a diamine (e.g., HMD), or a downstream product related thereto, provided herein for continuous production of substantial quantities of the diamine, or a downstream product related , producers also can be, for example, simultaneously subjected to chemical synthesis and / or enzymatic procedures to convert the product to other compounds, or the product can be separated from the fermentation culture and sequentially subjected to chemical and / or enzymatic conversion to convert the product to other compounds, if desired.

[0166] In some embodiments, the non-naturally occurring microbial organisms and methods provided herein can be assembled in a wide variety of sub-pathways to achieve biosynthesis of, for example, a diamine (e.g., HMD) or a downstream product related thereto such as an ester or amide thereof. In these embodiments, biosynthetic pathways for a desired 84 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT product provided herein can be segregated into different cells, and the different cells can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one cell is the substrate for a second cell until the final product is synthesized. For example, the biosynthesis of a diamine (e.g., HMD), or a downstream product related thereto, can be accomplished by constructing a cell that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, a diamine also can be biosynthetically produced from cells through co-culture or co- fermentation using two different cells in the same vessel, where the first cell produces a diamine intermediate and the second cell converts the intermediate to the diamine, or a downstream product related thereto.

[0167] Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring cells and methods provided herein together with other cells, with the co-culture of other non-naturally occurring cells having sub-pathways and with combinations of other chemical and / or biochemical procedures well known in the art to produce a diamine (e.g., HMD), or a downstream product related thereto.

[0168] Similarly, it is understood by those skilled in the art that a host organism can be selected based on desired characteristics for introduction of one or more gene disruptions to increase synthesis or production of a diamine (e.g., HMD), or a downstream product related thereto. Thus, it is understood that, if a genetic modification is to be introduced into a host organism to disrupt a gene, any homologs, orthologs or paralogs that catalyze similar, yet non-identical metabolic reactions can similarly be disrupted to ensure that a desired metabolic reaction is sufficiently disrupted. Because certain differences exist among metabolic networks between different organisms, those skilled in the art will understand that the actual genes disrupted in a given organism may differ between organisms. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the methods described herein can be applied to any suitable host microorganism to identify the cognate metabolic alterations needed to construct an organism in a species of interest that will increase biosynthesis of the diamine, or a downstream product related thereto.

[0169] Sources of encoding nucleic acids for a diamine biosynthesis pathway (e.g., a HMD biosynthesis pathway) enzyme or protein, or a downstream product related thereto, can include, for example, any species where the encoded gene product is capable of catalyzing 85 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacter species, including Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica, Thermotoga maritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcus fermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gamma proteobacterium, butyrate-producing bacterium, Nocardia iowensis, Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe, Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans, Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacter baumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, 86 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacterium smegmatis MC2155, Mycobacterium avium subsp. paratuberculosis K-10, Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, Cyanobium PCC7001, Dictyostelium discoideum AX4, Acidaminococcus fermentans, Acinetobacter baylyi, Acinetobacter calcoaceticus, Aquifex aeolicus, Arabidopsis thaliana, Archaeoglobus fulgidus, Aspergillus niger, Aspergillus terreus, Bacillus subtilis, Bos Taurus, Candida albicans, Candida tropicalis, Chlamydomonas reinhardtii, Chlorobium tepidum, Citrobacter koseri, Citrus junos, Clostridium acetobutylicum, Clostridium kluyveri, Clostridium saccharoperbutylacetonicum, Cyanobium PCC7001, Desulfatibacillum alkenivorans, Dictyostelium discoideum, Fusobacterium nucleatum, Haloarcula marismortui, Homo sapiens, Hydrogenobacter thermophilus, Klebsiella pneumoniae, Kluyveromyces lactis, Lactobacillus brevis, Leuconostoc mesenteroides, Metallosphaera sedula, Methanothermobacter thermautotrophicus, Mus musculus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium marinum, Mycobacterium smegmatis, Nicotiana tabacum, Nocardia iowensis, Oryctolagus cuniculus, Penicillium chrysogenum, Pichia pastoris, Porphyromonas gingivalis, Porphyromonas gingivalis, Pseudomonas aeruginos, Pseudomonas putida, Pyrobaculum aerophilum, Ralstonia eutropha, Rattus norvegicus, Rhodobacter sphaeroides, Saccharomyces cerevisiae, Salmonella enteric, Salmonella typhimurium, Schizosaccharomyces pombe, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoanaerobacter tengcongensis, Thermus thermophilus, Trypanosoma brucei, Tsukamurella paurometabola, Yarrowia lipolytica, Zoogloea ramigera and Zymomonas mobilis, Clostridum species, including but no limited to Clostridium saccharoperbutylacetonicum, Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium botulinum, Clostridium methylpentosum, Clostridium sticklandii, Clostridium phytofermentans, Clostridium saccharolyticum, Clostridium asparagiforme, Clostridium celatum, Clostridium carboxidivorans, Clostridium clostridioforme, Clostridium bolteae, Caldalkalibacillus thermarum, Clostridium botulinum, Pelosinus fermentans, Thermoanaerobacterium thermosaccharolyticum, Desulfosporosinus speices, Thermoanaerobacterium species, including but not limited to Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium xylanolyticum, Acetonema longum, Geobacillus species, including but not limited to Geobacillus thermoglucosidans, Bacillus azotoformans, Thermincola potens, Fusobacterium species, including but not limited to Fusobacterium nucleatum, Fusobacterium ulcerans, 87 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT Fusobacterium varium, Ruminococcus species, including but not limited to Ruminococcus gnavus, Ruminococcus obeum, Lachnospiraceae bacterium, Flavonifractor plautii, Roseburia inulinivorans, Acetobacterium woodii, Eubacterium species, including but not limited to Eubacterium plexicaudatum, Eubacterium hallii, Eubacterium limosum, Eubacterium yurii, Eubacteriaceae bacterium, Thermosediminibacter oceani, Ilyobacter polytropus, Shuttleworthia satelles, Halanaerobium saccharolyticum, Thermoanaerobacter ethanolicus, Rhodospirillum rubrum, Vibrio, Propionibacterium propionicum as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the diamine biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and non- orthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of the diamine (e.g., HMD), or a downstream product related thereto such as an ester or amide thereof, including expression of a diamine exporter provided herein, described herein with reference to a particular organism such as E. coli can be readily applied to other cells such as microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms.

[0170] In some instances, such as when an alternative diamine biosynthetic pathway exists in an unrelated species, diamine biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods provided herein can be applied to all cells using the cognate metabolic alterations to those exemplified herein to construct a cell in a species of interest that will synthesize the diamine (e.g., HMD), or a downstream product related thereto, if desired, including introducing a diamine exporter provided herein. 88 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0171] Methods for constructing and testing the expression levels of a non-naturally occurring host producing a diamine, or a downstream product related thereto, including a diamine exporter provided herein, can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). SEQUENCES

[0172] The sequences in the following TABLE 1 illustrate amino acid sequences that can be used to generate the diamine exporter sequences and perform the methods described herein. As needed, an RNA sequence can be readily deduced from the DNA sequence. TABLE 1: Sequences89 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT90 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT91 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT92 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT93 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT94 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT95 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT96 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT97 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT98 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT

[0173] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention. EXAMPLES Example 1: Screening and testing of HMD exporter candidate genes.

[0174] Genes encoding candidate hexamethylenediamine (HMD) exporters were identified bioinformatically from metagenomic libraries and public databases using a Basic Local Alignment Search Tool (BLAST). To evaluate the HMD exporter (HMD-Ex) candidate gene panel, plasmids carrying genes encoding the candidate transporters were 99 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT transformed into a DlysO strain of E. coli that also included genes encoding the following HMD pathway enzymes: (i) a thiolase (Thl), (ii) a 3-hydoxybutryl-CoA dehydrogenase (Hbd), (iii) a crotonase Crt), (iv) trans-enoyl-CoA reductase (Ter), (v) aldehyde dehydrogenase (Ald), (vi) transaminase (TA1), (vii) carboxylic acid reductase (CAR), and (viii) transaminase (TA2). Alternatively, the test HMD-ex gene was integrated onto the E. coli chromosome containing the HMD pathway genes.

[0175] The HMD-Ex strains produced by transformation of the HMD pathway production E. coli DlysO strain, and the HMD-Ex-chromosomal strains were tested for HMD production and export. The engineered E. coli cells were fed 5% glucose in minimal media, and the cells were harvested after a 16-24 h incubation at 35 °C. The level of HMD in the supernatant was then determined by standard LC / MS method or enzymatically using purified HMD transaminase. For the enzymatic detection of HMD levels, the absorbance at 450 nm was measured after incubation of purified HMD transaminase (3 mM), 50 U / mL bovine glutamate dehydrogenase (Sigma-Aldrich), 0.1 mM a-ketoglutarate, 0.1 mM NAD, 10 µM PMS (1-methoxy-5-methylphenazinium methyl sulfate and 2 mM XTT (2,3-Bis-(2-methoxy- 4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) in 0.1 M Tris-HCl, pH 7.4 buffer. A calibration curve containing known amounts of HMD was used to determine the level of HMD in the supernatant. For each HMD transporter gene evaluated, the relative HMD export was determined from the ratio of HMD in the supernatant of cells transformed with the plasmid containing the HMD-Ex gene to the HMD in the supernatant of cells transformed with empty vector (negative control) plasmid. Example 2: Improved HMD exporter variants of PACE family exporter. Identification of candidate HMD exporters

[0176] Two candidate transporters of the (Proteobacterial Antimicrobial Compound Efflux (PACE) family of transporters were identified as HMD exporters as described in Example 1. These HMD exporters, termed 11141G (SEQ ID NO: 1) and 11144B (SEQ ID NO: 46; also referred to as 11144A), conferred improved E.coli cell growth rate in the presence of exogenous HMD, and an increased capacity of HMD production from glucose. Directed Mutagenesis for Improved HMD exporter variants

[0177] Variants of 11141G and 11144B were generated through directed evolution to identify variants with improved function. Mutations were randomly inserted via error prone 100 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT PCR into the 11141G or 11144B gene sequence or the flanking DNA that controls gene expression using the Agilent GeneMorph II kit. The resulting gene variants were cloned onto low copy SC101 plasmid backbones with two different constitutive promoters. The plasmids were then transformed into engineered E.coli screening strain ECKh-14762. Each resulting cell library was plated on LB agar containing carbenicillin, and colonies were picked for colony PCR and sequencing of the amplicons. Library size for each cell library was estimated (Table 2), with the average mutation rate determined as 3 mutations per HMD exporter gene. Table 2: Library information for E.coli cells containing mutations in 11141G or 11144B.HMD export in HMD-ex variants

[0178] The four resulting cell libraries were then grown separately and plated on M9 agar containing glucose and 20 g / L HMD. Additionally, cells containing empty vector (“negative control” or “no exporter”) plasmid were also grown and tested for growth in media containing HMD, alongside cells transformed with plasmid containing the HMD exporter or HMD exporter variant.

[0179] Colonies expressing variants of 11141G or 11144B where then picked, grown in LB and used to prepare glycerol stocks. The variants were then tested for aerobic growth rates in M9 salts media containing 100 mM MOPS pH 7.5, 2% glucose, and 125 mM HMD. Strains that exhibited a higher growth rate were interpreted as having better export of HMD, and the HMD exporter variants from these cells were PCR amplified and sequenced. The growth rates and mutations for each of the tested HMD-ex variants are summarized in Tables 3 and 4. 101 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT Table 3: Comparison of growth rates of cells encoding variants of 11141G in the presence of 125 mM HMD.As shown in the table: “+” = lower than 0.2-fold growth rate constant value to negative control; “++” = greater than 0.2 to 1.0-fold growth rate constant value relative to negative control. Notes: Delta AA 1-12 in 11141Q indicates a deletion of codons 1-12 such that the new translation start site is at amino acid 13. The STOP157 mutation in 11141P indicates that the original stop codon mutated to encode amino acid glutamate (E, Glu) and also resulted in addition of 10 new amino acids on the C-terminus of the protein. Table 4: comparison of growth rates of cells encoding variants of 11144B in the presence of 125 mM HMD.As shown in the table: “-” = lower than 0.2-fold growth rate constant value relative to negative control; “+” = greater than 0.2 to 1.0-fold growth rate constant value relative to negative control.

[0180] In summary, E. coli strains expressing 11141G or 11144B exporter had higher growth rates in HMD containing media than E. coli strains lacking these exporters. Additionally, several variants of 11141G or 11144B further improved the ability of E. coli strains to grow in media containing HMD as compared to E. coli strains carrying the non- mutated 11141G or 11144B exporter. 102 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT Example 3: HMD production in cells with over expression of HMD exporters or HMD exporter variants.

[0181] Genes encoding candidate HMD exporters from E.coli or other organisms were codon optimized and cloned into expression plasmids, in frame with a constitutive promoter. If the gene corresponded to an E.coli gene, it was not further codon optimized for expression. These candidate HMD exporter plasmids were then transformed into an E.coli strain capable of producing HMD.

[0182] The resulting strains were tested for improved ability to produce HMD from glucose in a scaled-down experiment conducted for 24 hrs in a 384 multi-well plate as described in Example 1. Each strain and transporter combination were tested for HMD production in a total of eight replicate experiments. Table 5 shows the average HMD production titers for cells overexpressing the candidate HMD exporters. Table 5: Comparison of HMD production strains featuring different transporters overexpressed from plasmid.103 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCTAs shown in the table:= lower than 0.5-fold HMD production relative to no gene control; “+” = greater than 0.5 to 1.0-fold HMD production relative to no gene control;; “++” = greater than 1.0-fold HMD production relative to no gene control. The aceI variant corresponds to 11141P.

[0183] E.coli cells transformed with empty vector control, which lacked expression of any exporter gene, produced 16 mM HMD (Table 5), and cells that produced HMD at amounts ranging from 7 mM to 18 mM were considered to include exporters that had little to no HMD export activity (Table 5). Cells transformed with plasmids expressing Acinetobacter baumannii aceI 11141P variant produced higher amounts of HMD than cells expressing the A. baumannii aceI 11141G variant (45 mM and 42 mM HMD, respectively). Cells expressing a PACE family exporter from Acinetobacter nosocomialis produced the highest amount of HMD, reaching an average titer of 50 mM HMD. Additionally, cells expressing exporter E. coli ytfl produced 41 mM HMD, suggesting that this exporter is functional on HMD. Moreover, cells overexpressing E. coli emrE from a weak promoter (P115) produced about 27 mM HMD, while strains expressing E. coli emrE from a strong promoter (P108) produced around 40 mM HMD. 104 ACTIVE 697740502v1Attorney Docket No.199683-806002 / PCT Example 4: Comparative analysis of HMD production in cells expressing HMD exporter homologs.

[0184] Genes encoding HMD exporters homologs from different organisms were cloned into expression plasmids, transformed into an E. coli strain capable of HMD production, and evaluated for HMD production.

[0185] Table 6 shows the average HMD production titers for E. coli cells overexpressing each of the HMD exporter homologs.

[0186] Homologs from Acinetobacter larvae, Pseudomonas pohangensis, Acinetobacter pittii, Acinetobacter defluvii, Acinetobacter sp. KPC-SM-21, and Acinetobacter nosocomialis were identified as having the highest HMD exporter activity relative to the activity of the Acinetobacter baumanni ATCC 17978 HMD exporter (SEQ ID NO: 1) (Table 6). Exogenous expression of these transporters could be used to increase HMD transport in a cell. 105 ACTIVE 697740502v1TCP / 200608-386991.oNtekcoDyenrottAer 7 -p1uixe Crem03s rt 9i 16 muMsi1 uir 1snmirN s sueCvT ca 09 82t ea8v2 et_9eoA 3gui ietcmnue tdceslibe54KoLora 9cl 2ab_naretmaabi1renlael mesci nn aina ecH.Op Rs.preCLFshceL oase eaB tcgruetlama a s tc.plaUpF sba saec Cab53b craog u da 48 icr a cxa absano_ an ibeoaU g Ve1a0 et bi.Ob no 1- rel ro otllir 1Coortna BI ecca eicErreTtm oc o Oce vo ene bigiivY mo ciahPpadboifrfa ryC Ldic cAhSolX lOdu moe oc nit anretepsr beo hpA edFI sur ih on uSttrien oB R P rCmEPeatn icVmoitA oC cudorp9D942 023 39764 684 5M9F 41DIt 9 GTJE5E8T4EMoMPrV9V1G7UMvG QND2SK WI 05HPU2MA6J8Q2B2E3F2M5H1I M5AZ2R7GJ5P047:iD 6n00 0 0 1 1 1 1PA A A A A A A8A1A1AA2 2 27A A A96e U0 0 0lbA A A0 0 0A A A0A0 0 0A A A0 0 0E A A A VIaTTC ATCP / 200608-386991.oNtekcoDyenrottA4i iRivL- M1la uiulm Siii ire 42442a4n1it ..1.C CCfui-i MC BdrSttimotc7174 Cseteps 1C0T Te r P 1 aDp c ab- ENnas .AA msid etS zr o T .A i s m 36 iiinrc K..pi aanietso eaOps.p realuib0.iknoaetgcabpssllnicanrec C_ astcahtozT az 49 sur ab airraeitt eaxuladboettcaida01 h rciet aet c bioiih E.raak 8agrOotec rane a3 rtroCsefni c abre ittinibc aono 2 eA bhab ecibi se .F.re aioottSuBair cAtemH renaOcsote rcVME1tc hcanenimEni os ab irpiSchcAsu A oCcAeM Mon ehKor csCED4I90Z5H3E43L551F65 4R8 1R7S 1C1vtCoFTr2C1NRHHRJVJDY4ZE3YAJE8V ML2560205Pi S6T3U1V5A9 6G6G1DP9 0PR6ER0W44M03ML47n 2UA20A2A2A3DA3 3A3A3A4 4AA4A2A4M77B796A0A0A0 0AA A0 0 0 0 0 0 0 0A0A E A A A A A A A A A VITC ATCP / 200608-386991.oNtekcoDyenrottAA A Bmu3 Ci8-T a3 6tcH45-297C icaP -3SS0 1CoC reCt 6A 9ea niItN72 sMR98 C TniC A-1 C ATca 7 zs iimbCni esed ai ud scu uA1i 6 Ml ToAnnamsAA isnue2aac e ADulsFCi faniitr cncr it DS 21 CJ ci29 nain a 01oze ret au U- C.pihnamu txgi 2rna1c2ai4r5 r_eanr3mrearca q .pRsOeb et2xa33orcimarb b elretboarsa Aetreua r anc_elfpsetl etcateet iM cahcb etcibir a9a ba ul acabonicm caie SUbosrEet ambrec olrleiho b r bpdhuor cthcA orn h a ott e-oiertgbniho e ems niE Sei yshc n cya ybsot niC;c8V a PcPsMP enA7HA P ic9A71D6I6tJ 7 61v2o0J 1rS0JL4Q797J891K H4J3 59 98L 4T T 050PKG iBY83QEBV n3E5EA 0 3 X FA A F8 4NT Q3 EU U 0R0A D D477Q340P0 09U0 FNSU W XP P6A E VITC ATCP / 200608-386991.omNtt aht uaerore bkcp)toxDe de a ree c etc-rgabyeviutd =on” tenr aloei(+“ n;icttR)1 AoA:OtNevieDtIalvitetrQ rtal oepx- ErS( o8pxRe79e7D 1 M CHDIDICQ 1TdlAof-EQ:SE0SO89A / inN Nn.a1na%otmuahtbrreet90taer 1DcIaQO4 A / bgoEN4Nt=e ”+S nic+A“; o)t1e:viO CtCaNl DT)eI ertQA n rie Ennme gop S(oxe8ma t nsimN-(lD797n u 2aa 1orM tH1gCrbrΔ;nd8olo CTOetca7cf b97e -vi5.A0 inot1 ta n naengieah tm cnr uaA ewbolret=c”a-b“o:teelnDbic1Ivt9 atAorT eo 2UA ht 0te50P / i Dn vi47n0Ni tnal.)79UPweor1h t:6srO E V sopN ITAxeDIC ADocket No: 199683-806001 / PRO Example 5: Identification of additional HMD exporter candidates

[0187] E.coli strain containing a deletion of acrB was grown on plates of M9 agar with glucose and HMD. In these conditions, colonies arose at an estimated frequency of less than 1 in a million. The colonies were isolated from isocratic 10g / L HMD or 20 g / L HMD, as well as from plates containing a gradient of 5-45 g / L HMD. Selected colonies were tested for aerobic growth rate in the presence of HMD, and subjected to whole genome sequencing. Growth experiments were conducted in a minimal medium with glucose and 150 mM HMD. The genome mutations and growth rates of selected strains are summarized in Table 7. Genome mutations that resulted in higher growth rates in the presence of HMD are interpreted as improving net export of HMD. Table 7: Genome mutations and corresponding growth rates of selected colonies.As shown in the table: “-” = lower than 0.5-fold growth rate constant value relative to control; “+” = greater than 0.5 to 1.0-fold growth rate constant value relative to control; “++” = greater than 1.0-fold growth rate constant value relative to control.

[0188] As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present invention, it is intended that all subject matter contained in the above description, or defined in the appended claims, be interpreted as descriptive and illustrative of the present invention. Many modifications and variations of the present invention are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications and variances which fall within the scope of the appended claims.

[0189] Throughout this application various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in ACTIVE 697740502v1this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.111 ACTIVE 697740502v1

Claims

CLAIMS What is claimed is:

1. An engineered diamine exporter comprising: a. a variant of amino acid sequence SEQ ID NO: 1, or a functional fragment thereof, wherein the engineered diamine exporter comprises one or more alterations at a position selected from I24, V61, and I88; b. a variant of amino acid sequence SEQ ID NO: 46, or a functional fragment thereof, wherein the engineered diamine exporter comprises one or more alterations at a position selected from F42, G47, F102 and A133; or c. a variant of amino acid sequence SEQ ID NO: 48, or a functional fragment thereof, wherein the engineered diamine exporter comprises one or more alterations at a position selected from R148 and V286.

2. The engineered diamine exporter of claim 1, wherein the diamine has a carbon length of C4-C6.

3. The engineered diamine exporter of claim 1 or claim 2, wherein the diamine is hexamethylenediamine (HMD).

4. The engineered diamine exporter of any one of claims 1-3, wherein the diamine exporter does not export an HMD pathway intermediate.

5. The engineered diamine exporter of claim 4, wherein the HMD pathway intermediate is 6-aminocaproic acid (6ACA) .

6. The engineered diamine exporter of any one of claims 1 to 5, wherein a. the engineered diamine exporter comprises an activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a diamine exporter consisting of the amino acid sequence of SEQ ID NO: 1; ACTIVE 697740502v1b. the engineered diamine exporter comprises an activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a diamine exporter consisting of the amino acid sequence of SEQ ID NO: 46; or c. the engineered diamine exporter comprises an activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than the activity of a diamine exporter consisting of the amino acid sequence of SEQ ID NO:

48.

7. The engineered diamine exporter of any one of claims 1 to 6, wherein the one or more amino acid alterations are conservative amino acid substitutions.

8. The engineered diamine exporter of any one of claims 1 to 6, wherein the one or more amino acid alterations are non-conservative amino acid substitutions.

9. The engineered diamine exporter of any one of claims 1 to 6, wherein the one or more amino acid alterations are insertions or deletions.

10. The engineered diamine exporter of any one of claims 1 to 9, wherein the one or more amino acid alterations result in an engineered diamine exporter comprising: a. a M at a residue corresponding to position 61 in SEQ ID NO: 1; or b. a T at a residue corresponding to position 88, and a deletion of residues corresponding to positions 1 to 12 in SEQ ID NO: 1; or c. a F at a residue corresponding to position 24 in SEQ ID NO: 1; or d. a stop codon at a residue corresponding to position 157, and an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1; or e. a L at a residue corresponding to position 42 in SEQ ID NO: 46; or f. a S at a residue corresponding to position 46 in SEQ ID NO: 46; or g. a V at a residue corresponding to position 133 in SEQ ID NO: 46; or ACTIVE 697740502v1h. a Y at a residue corresponding to position 102 in SEQ ID NO: 46; or i. a C at a residue corresponding to position 148 in SEQ ID NO: 48; or j. a E at a residue corresponding to position 286 in SEQ ID NO:

48.

11. The engineered diamine exporter of any one of claims 1 to 10, wherein the one or more amino acid alterations result in an engineered diamine exporter comprising: a. one or more of a M at a residue corresponding to position 61, a T at a reside corresponding to position 88, a F at a residue corresponding to position 24, a stop codon corresponding to position 157, a deletion of residues corresponding to positions 1 to 12, and an insertion of up to ten amino acids at the C-terminus in SEQ ID NO: 1; or b. one or more of a L at a residue corresponding to position 42, a S at a residue corresponding to position 46, a V at a residue corresponding to position 133, and a Y at a residue corresponding to position 102 in SEQ ID NO: 46; or c. one or more of a C at a residue corresponding to position 148, and a E at a residue corresponding to position 286 in SEQ ID NO:

48.

12. The enginered diamine exporter of any one of claims 1 to 11, wherein the one or more amino acid alterations result in an engineered diamine exporter having the sequence set forth in any one of SEQ ID NOS: 43-45.

13. The engineered diamine exporter of any one of claims 1 to 12, wherein the one or more amino acid alterations comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 alterations.

14. The engineered diamine exporter of any one of claims 1 to 13, wherein: a. the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence referenced in SEQ ID NO: 1; b. the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at ACTIVE 697740502v1least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence referenced in SEQ ID NO: 46; or c. the amino acid sequence, other than the one or more amino acid alterations, has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence referenced in SEQ ID NO:

48.

15. The engineered diamine exporter of any one of claims 1 to 13, wherein: a. the amino acid sequence, other than the one or more amino acid alterations, is identical to the amino acid sequence referenced in SEQ ID NO: 1; b. the amino acid sequence, other than the one or more amino acid alterations, is identical to the amino acid sequence referenced in SEQ ID NO: 46; or c. the amino acid sequence, other than the one or more amino acid alterations, is identical to the amino acid sequence referenced in SEQ ID NO:

48.

16. A recombinant nucleic acid encoding the engineered diamine exporter of any one of claims 1 to 15.

17. The recombinant nucleic acid of claim 16, wherein the nucleic acid comprises a nucleotide sequence encoding the engineered diamine exporter operatively linked to a promoter.

18. The recombinant nucleic acid of claim 17, wherein the promoter is a constitutive promoter.

19. The recombinant nucleic acid of claim 17 or claim 18, wherein the promoter is a weak promoter.

20. The recombinant nucleic acid of claim 17 or claim 18, wherein the promoter is a strong promoter.

21. A vector comprising the recombinant nucleic acid of any one of claims 16 to 20. ACTIVE 697740502v122. A non-naturally occurring microbial organism comprising a recombinant nucleic acid encoding an engineered diamine exporter selected from any one of claims 1 to 15.

23. The non-naturally occurring microbial organism of claim 22, wherein the non- naturally occurring microbial organism further comprises a pathway that produces a diamine.

24. The non-naturally occuring microbial organisms of claim 23, wherein the diamine has a carbon length of C4 to C6.

25. The non-naturally occuring microbial organism of claim 23 or claim 24, wherein the diamine is HMD.

26. The non-naturally occurring microbial organism of any one of claims 23 to 25, wherein the non-naturally occurring microbial organism is capable of producing at least 10% more diamine compared to a control microbial organism that does not comprise the recombinant nucleic acid.

27. The non-naturally occurring microbial organism of any one of claims 23 to 26, wherein the non-naturally occurring microbial organism is capable of at least 10% more diamine export as compared to a control microbial organism that does not comprise the recombinant nucleic acid.

28. The non-naturally occurring microbial organism of any one of claims 23 to 27, wherein the non-naturally occurring microbial organism has a growth rate that is at least 10% higher compared to a control microbial organism that does not comprise the recombinant nucleic acid.

29. The non-naturally occurring microbial organism of any one of claims 23 to 28, wherein the non-naturally occurring microbial organism has improved cell survival compared to a control microbial organism that does not comprise the recombinant nucleic acid. ACTIVE 697740502v130. The non-naturally occurring organism of any one of claims 26 to 29, wherein the diamine export, or the production of diamine, is determined by measuring the levels of diamine in a culture media containing the microbial organism.

31. The non-naturally occurring organism of any one of claims 26 to 29, wherein the diamine export, or the production of diamine, is determined by measuring the levels of diamine using a genetically encoded diamine reporter.

32. The non-naturally occurring microbial organism of any one of claims 26 to 29, wherein the growth rate, cell survival, diamine production, and / or diamine export are measured in the presence of exogenous diamine.

33. The non-naturally occurring microbial organism of any one of claims 23 to 32, wherein at least one enzyme of the pathway is encoded by an exogenous nucleic acid.

34. The non-naturally occurring microbial organism of claim 33, wherein the exogenous nucleic acid is heterologous.

35. The non-naturally occurring microbial organism of claim 33, wherein the exogenous nucleic acid is homologous.

36. The non-naturally occurring microbial organism of any one of claims 22 to 35, wherein the non-naturally occurring microbial organism is in a micro-aerobic culture medium.

37. The non-naturally occurring microbial organism of any one of claims 22 to 36, wherein the non-naturally occurring microbial organism is in a culture medium containing exogenous diamine.

38. The non-naturally occurring microbial organism of any one of claims 22 to 37, wherein the microbial organism is a species of bacteria, yeast, or fungus.

39. The non-naturally occurring microbial organism of any of claims 22 to 38, wherein the recombinant nucleic acid is integrated into the genome of the microbial organism. ACTIVE 697740502v140. A non-naturally occurring microbial organism comprising an exogenous nucleic acid encoding a diamine exporter having a sequence as set forth in any one of SEQ ID NOS: 1-42.

41. A non-naturally occurring microbial organism comprising one or more disruptions in a gene encoding a diamine exporter having the sequence set forth in SEQ ID NO:

49.

42. The non-naturally occurring microbial organism of claim 41, wherein the one or more gene disruptions are in a 5’ regulatory region of the gene.

43. The non-naturally occurring microbial organism of claim 42, wherein the one or more gene disruptions comprise one or more deletions.

44. The non-naturally occurring microbial organism of any one of claims 40 to 43, wherein the non-naturally occurring microbial organism further comprises a pathway that produces a diamine.

45. The non-naturally occuring microbial organisms of any one of claims 40 to 44, wherein the diamine has a carbon length of C4 to C6.

46. The non-naturally occuring microbial organism of any one of claims 40 to 45, wherein the diamine is HMD.

47. The non-naturally occurring microbial organism of any one of claims 40 to 46, wherein the non-naturally occurring microbial organism is capable of producing at least 10% more diamine compared to a control microbial organism that does not comprise the exogenous nucleic acid.

48. The non-naturally occurring microbial organism of any one of claims 40 to 47, wherein the non-naturally occurring microbial organism is capable of at least 10% more diamine export as compared to a control microbial organism that does not comprise the exogenous nucleic acid encoding the diamine exporter.

49. The non-naturally occurring microbial organism of any one of claims 40 to 48, wherein the non-naturally occurring microbial organism a growth rate that is at least118 ACTIVE 697740502v110% higher compared to a control microbial organism that does not comprise the exogenous nucleic acid encoding the diamine exporter.

50. The non-naturally occurring microbial organism of any one of claims 40 to 49, wherein the non-naturally occurring microbial organism has improved cell survival compared to a control microbial organism that does not comprise the exogenous nucleic acid encoding the diamine exporter.

51. The non-naturally occurring organism of any one of claims 47 to 50, wherein the diamine export, or the production of diamine, is determined by measuring the levels of diamine in a culture media containing the microbial organism.

52. The non-naturally occurring organism of any one of claims 47 to 51, wherein the diamine export, or the production of diamine, is determined by measuring the levels of diamine using a genetically encoded diamine reporter.

53. The non-naturally occurring microbial organism of any one of claims 47 to 52, wherein the growth rate, cell survival, diamine production, and / or diamine export are measured in the presence of exogenous diamine.

54. The non-naturally occurring microbial organism of any one of claims 40 to 54, wherein at least one enzyme of the pathway is encoded by an exogenous nucleic acid.

55. The non-naturally occurring microbial organism of claim 54, wherein the exogenous nucleic acid is heterologous.

56. The non-naturally occurring microbial organism of claim 54, wherein the exogenous nucleic acid is homologous.

57. The non-naturally occurring microbial organism of any one of claims 40 to 56, wherein the non-naturally occurring microbial organism is in a micro-aerobic culture medium. ACTIVE 697740502v158. The non-naturally occurring microbial organism of any one of claims 40 to 57, wherein the non-naturally occurring microbial organism is in a culture medium containing exogenous diamine.

59. The non-naturally occurring microbial organism of any one of claims 40 to 58, wherein the microbial organism is a species of bacteria, yeast, or fungus.

60. The non-naturally occurring microbial organism of any of claims 40 to 59, wherein the exogenous nucleic acid is integrated into the genome of the microbial organism.

61. The non-naturally occurring microbial organism of any one of claims 40 to 60, wherein the microbial organism further comprises a promoter operably linked to the exogenous nucleic acid.

62. The non-naturally occurring microbial organism of 61, wherein the promoter is a constitutive promoter.

63. The non-naturally occurring microbial organism of claim 61 or claim 62, wherein the promoter is a weak promoter.

64. The non-naturally occurring microbial organism of claim 61 or claim 62, wherein the promoter is a strong promoter.

65. The non-naturally occurring microbial organism of any one of claims 61 to 64, wherein the promoter is a heterologous promoter.

66. The non-naturally occurring microbial organism of any one of claims 40 to 65, wherein the heterologous nucleic acid is integrated into the genome of the organism.

67. A method of producing a diamine, the method comprising culturing the microbial organism of any one of claims 22 to 39 under conditions and for a sufficient period of time to produce the diamine. ACTIVE 697740502v168. A method of producing a diamine, the method comprising culturing the microbial organism of any one of claims 40 to 66 under conditions and for a sufficient period of time to produce the diamine.

69. The method of claim 67 or claim 68, wherein the method further comprises separating the diamine from other components in the culture.

70. The method of claim 69, wherein the separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.

71. The method of any one of claims 67 to 70, wherein the culturing is performed in the absence of exogenous diamine. ACTIVE 697740502v1

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