BIOSYNTHESIS

FIELD OF THE INVENTION

Aspects of the present disclosure relate to the general field of biotechnology and, more particularly, to the biosynthesis of compounds.

BACKGROUND OF THE INVENTION

Nonribosomal peptides (NRP) are a class of peptide secondary metabolites, often produced by microorganisms such as bacteria and fungi. Nonribosomal peptides are synthesized by nonribosomal peptide synthetases, which, unlike ribosomal peptides, are independent of messenger RNA. Each nonribosomal peptide synthetase can typically synthesize only one type of peptide, although the synthesis of most nonribosomal peptides requires more than one nonribosomal peptide synthetase. Nonribosomal peptides often have a cyclic and/or branched structures, can contain non-proteinogenic amino acids including D- amino acids, carry modifications such as N-methyl and N-formyl groups, and/or are glycosylated, acylated, halogenated or hydroxylated. Nonribosomal peptides are a diverse family of products with a broad range of biological activities and pharmacological properties. Examples of nonribosomal peptides include siderophores, and certain toxins, pigments, antibiotics, cytostatics and immunosuppressants.

SUMMARY OF THE INVENTION

Dihydroxybenzoate, synthesized from chorismate, is the precursor in the biosynthesis of several nonribosomal peptides, such as siderophores, including enterobactin {Escherichia coli) and vibriobactin {Vibrio cholera). The present disclosure is based, at least in part, on surprising results showing that a library of functionally and structurally diverse molecules (e.g., nonribosomal, iron-chelating proteins) can be produced by combining in a single bacterial cell a compressed pathway comprising select biosynthetic genes obtained from the E. coli. enterobactin gene cluster and select biosynthetic genes obtained from the V. cholera vibriobactin gene cluster, and then feeding the cells various amine or polyamine (e.g., diamine) linker precursors.

Peptides synthesized independently of the ribosome in plants, fungi and bacteria are clinically relevant molecules. They display anticancer, anti- hemochromatosis, and anti-viral activity, among many others. Despite their natural origin, there is an increased difficulty in finding new molecules, as many niches, species and genomes get tapped. Thus, expanding the chemical diversity of libraries to include new entities can be challenging.

Provided herein are compressed synthetic pathways from Escherichia coli and Vibrio cholerae genes, capable of being programmed for the production of new, synthetic nonribosomal peptides. These molecules are analogs of the iron chelators, serratiochelins. While initially unable to be produced using the native biosynthetic genes, these molecules were successfully produced using ancestral homologs obtained from Escherichia coli and Vibrio cholerae. By expressing the ancestral homologs in E. coli and feeding the organism with different precursors, more than 30 molecules were produced, more than 20 of which are new and display high degrees of drug-likeness.

This new approach to the engineering of biosynthetic pathways, where ancestral genes from different pathways enable heterologous expression of nonribosomal peptides, allows for the bioproduction of many intractable molecules.

Some aspects of the present disclosure are directed to modified bacterial cells comprising a compressed biosynthetic pathway that comprises (a) biosynthetic genes obtained from one species encoding enzymes active in the bioassembly of a nonribosomal molecule, (b) biosynthetic genes obtained from another species encoding enzymes active in the bioassembly of a nonribosomal molecule that is different from the nonribosomal molecule of (a), and (c) a gene encoding an amide synthase.

Some aspects of the present disclosure are directed to modified bacterial cells comprising a compressed biosynthetic pathway that comprises (a) biosynthetic genes obtained from one species encoding enzymes active in the bioassembly of a nonribosomal molecule, (b) biosynthetic genes obtained from at least one other (e.g., at least two other) species encoding enzymes active in the bioassembly of at least one nonribosomal molecule that is different from the nonribosomal molecule of (a), and (c) a gene encoding an amide synthase.

In some embodiments, the biosynthetic genes of (a) are Escherichia coli biosynthetic genes. For example, the Escherichia coli biosynthetic genes may include entD gene, an entC gene, an entE gene, an entB gene and an entA gene.

In some embodiments, the biosynthetic genes of (b) are Vibrio cholera biosynthetic genes. For example, the Vibrio cholera biosynthetic genes may include a vibH gene and a vibF gene.

In some embodiments, the amide synthase is a vibH gene. In some embodiments, the modified bacterial cell is a modified Escherichia coli cell. In some embodiments, endogenous entD, entC, entE, entB, entA and entF genes are deleted from the cell.

In some embodiments, the nonribosomal molecule is a nonribosomal peptide.

Some aspects of the present disclosure are directed to methods of producing a nonribosomal molecule, the method comprising culturing at least one of the modified bacterial cell provided herein, in the presence of an exogenous diamine linker precursor, under conditions that result in the production of a nonribosomal molecule that is different from the nonribosomal molecules of (a) and (b).

Some aspects of the present disclosure are directed to engineered vectors comprising a promoter operably linked to nucleic acid comprising an entD gene, an entC gene, an entE gene, an entB gene, an entA gene, a vibH gene and a vibF gene.

In some embodiments, the promoter is inducible.

The present disclosure also provides bacterial cells comprising the engineered vector as described herein.

Some aspects of the present disclosure are directed to methods of producing a nonribosomal molecule, the methods comprising culturing, in the presence of a diamine linker precursor at least one bacterial cell as described herein under conditions that result in the production of a nonribosomal molecule.

The present disclosure also provides nonribosomal molecules produced by the method as described herein.

Also provided herein are compounds of any one of formula (I)-(XXXVI) or

(XXXVII)-(LV), or chemical analogs thereof (see Table 2).

Further provided herein are modified Escherichia coli (E. coli) cells that comprise an entA gene, an entB gene, an entC gene, an entD gene, a vibF gene, a vibH gene, and a deletion in an entF gene.

Some aspects provide methods comprising culturing a modified E. coli cell that comprises an entA gene, an entB gene, an entC gene, an entD gene, a vibF gene, a vibH gene, and a deletion in an entF gene in the presence of a polyamine linker precursor to produce a nonribosomal molecule.

Some aspects provide methods comprising culturing a modified E. coli cell that comprises an entA gene, an entB gene, an entC gene, an entD gene, a vibF gene, a vibH gene, and a deletion in an entF gene in the presence of an amine linker precursor to produce a nonribosomal molecule precuror. Further provided herein are modified Escherichia coli (E. coli) cells that comprise an entB gene, an entD gene and an entE gene, a vibF gene, a vibH gene, a deletion in an entA gene, a deletion in an entC gene and a deletion in an entF gene.

The present disclosure also provides methods of culturing a modified E. coli cell that comprises a deletion in an entA gene, an entC gene and an entF gene, an e«tS gene, an entD gene and an entE gene, and a vibF gene and a vibH gene in the presence of a polyamine linker precursor and a polyhydroxybenzoate to produce a nonribosomal molecule.

In some embodiments, the polyhydroxybenzoate is 2,5-Dihydroxybenzoic acid (DHB). In some embodiments, the polyhydroxybenzoate is vanillic acid, gallic acid, caffeic acid, 5-Bromo-2,4-Dihydroxybenzoic acid or 3,4-Dihydroxy-5-methoxybenzoic acid.

In some embodiments, the modified E. coli cell is cultured in iron-deficient media (media that is free of iron, or media that contains less than 10% iron).

In some embodiments, the polyamine linker precursor is selected from the group consisting of: 1,3-Diaminopropane, N-(3-Aminopropyl)-l,4-diaminobutane, N,N'-Bis(3- aminopropyl)-l,4-diaminobutane, 1,5-Diaminopentane, 1 ,4-Butanediamine dihydrochloride, Bis(3-aminopropyl)amine, m-Xylylenediamine, N,N'-Bis(2-aminoethyl)-l ,3-propanediamine, N-Benzylethylenediamine, 4-Aminobenzylamine, 4-(2-Aminoethyl)aniline, 4,4'- Oxydianiline, 4,4'-Diaminodiphenylmethane, 1,5-Diaminonaphthalene, 2,2'- Thiobisacetamide, Sulfaguanidine, p-Aminobenzenesulfonamide, Urea, N-Phenylthiourea, 3,3'-Diamino-N-methyldipropylamine, and 1, 8-Diaminooctane.

In some embodiments, the nonribosomal molecule is selected from the group consisting of: N-(4-(2,3-dihydroxybenzamido)butyl)-2-(2,3-dihydroxyphenyl)- 5-methyl-4,5- dihydrooxazole-4-carboxamide; (2R,3S)-3-amino-4-((4-(2,3- dihydroxybenzamido)butyl)amino)-4-oxobutan-2-yl 2,3-dihydroxybenzoate; N-(5-(2,3- dihydroxybenzamido)pentyl)-2-(2,3-dihydroxyphenyl)-5-methyl- 4,5-dihydrooxazole-4- carboxamide; (2R,3S)-3-amino-4-((5-(2,3-dihydroxybenzamido)pentyl)amino)- 4-oxobutan- 2-yl 2,3-dihydroxybenzoate; (4S)-N-(4-((3-(2,3-dihydroxybenzamido)propyl)amino)butyl)-2- (2,3-dihydroxyphenyl)-5-methyl-4,5-dihydrooxazole-4-carboxam ide; N-(3-((4-((2S,3R)-2- (2,3-dihydroxybenzamido)-3-hydroxybutanamido)butyl)amino)pro pyl)-2,3- dihydroxybenzamide; N-(3-(2,3-dihydroxybenzamido)propyl)-2-(2,3-dihydroxyphenyl) -5- methyl-4,5 -dihydrooxazole-4-carboxamide; (2R,3 S)-3-amino-4-((3 -(2,3 - dihydroxybenzamido)propyl)amino)-4-oxobutan-2-yl 2,3-dihydroxybenzoate; N-(3 -((2,3 dihydroxybenzamido)methyl)benzyl)-2-(2,3-dihydroxyphenyl)-5- methyl-4,5-dihydrooxazole- 4-carboxamide; (2R,3S)-3-amino-4-((3-((2,3-dihydroxybenzamido)methyl)benzyl )amino)-4- oxobutan-2-yl 2,3-dihydroxybenzoate; N-(2-((3-((2-(2,3- dihydroxybenzamido)ethyl)amino)propyl)amino)em^

4,5-dihydrooxazole-4-carboxamide; (14S,15R)-14-amino-l-(2,3-dihydroxyphenyl)-l,13- dioxo-2,5,9,12-tetraazahexadecan-15-yl 2,3-; dihydroxybenzoate; N,N'-Bis(2-aminoethyl)- l,3-propanediamineN-(2-(N-benzyl-2,3-dihydroxybenzamido)ethy l)-2-(2,3- dihydroxyphenyl)-5 -methyl-4,5 -dihydrooxazole-4-carboxamide; (2R,3 S)-3 -amino-4- (benzyl(2-(2,3-dihydroxybenzamido)ethyl)amino)-4-oxobutan-2- yl 2,3-dihydroxybenzoate; N-(4-(2,3-dihydroxybenzamido)phenethyl)-2-(2,3-dihydroxyphen yl)-5-methyl-4,5- dihydrooxazole-4-carboxamide; (2R,3S)-3-amino-4-((4-(2-(2,3- dihydroxybenzamido)ethyl)phenyl)amino)-4-oxobutan-2-yl 2,3-dihydroxybenzoate; N-(4-(4- (2,3-dihydroxybenzamido)phenoxy)phenyl)-2-(2,3-dihydroxyphen yl)-5-methyl-4,5- dihydrooxazole-4-carboxamide; (2R,3S)-3-amino-4-((4-(4-(2,3- dihydroxybenzamido)phenoxy)phenyl)amino)-4-oxobutan-2-yl 2,3-dihydroxybenzoate; N-(4- (4-(2,3-dihydroxybenzamido)benzyl)phenyl)-2-(2,3-dihydroxyph enyl)-5-methyl-4,5- dihydrooxazole-4-carboxamide; (2R,3S)-3-amino-4-((4-(4-(2,3- dihydroxybenzamido)benzyl)phenyl)amino)-4-oxobutan-2-yl 2,3-dihydroxybenzoate; (4S)- N-(3 -((4-((2-(2,3 -dihydroxybenzamido)ethyl)amino)butyl)amino)propyl)-2-(2,3 - dihydroxyphenyl)-5 -methyl-4,5 -dihydrooxazole-4-carboxamide; N-((l 6S, 17R)- 1 -(2,3- dihydroxyphenyl)- 17-hydroxy- 1 , 15-dioxo-2,6, 11,14-tetraazaoctadecan- 16-yl)-2,3 - dihydroxybenzamide; N-(5-(2,3-dihydroxybenzamido)naphthalen-l-yl)-2-(2,3- dihydroxyphenyl)-5 -methyl-4,5 -dihydrooxazole-4-carboxamide; (2R,3 S)-3 -amino-4-((5 -(2,3- dihydroxybenzamido)naphthalen- 1 -yl)amino)-4-oxobutan-2-yl 2,3-dihydroxybenzoate; N-(2- ((2-(2,3-dihydroxybenzamido)-2-oxoethyl)thio)acetyl)-2-(2,3- dihydroxyphenyl)-5-methyl- 4,5-dihydrooxazole-4-carboxamide; (2R,3S)-3-amino-4-(2-((2-(2,3-dihydroxybenzamido)-2- oxoethyl)thio)acetamido)-4-oxobutan-2-yl 2,3-dihydroxybenzoate; N-(N-((4-(2,3- dihydroxybenzamido)phenyl)sulfonyl)carbamimidoyl)-2- 2,3-dihydroxyphenyl)-5-methyl- 4,5- dihydrooxazole-4-carboxamide; (2R,3S)-3-amino-4-((4-(N-((Z)-N'-(2,3- dihydroxybenzoyl)carbamimidoyl)sulfamoyl)phenyl); amino)-4-oxobutan-2-yl 2,3- dihydroxybenzoate; N-((4-(2,3 -dihydroxybenzamido)phenyl)sulfonyl)-2-(2,3 - dihydroxyphenyl)-5 -methyl-4,5 -dihydrooxazole-4-carboxamide; (2R,3S)-3-amino-4-((4-(N- (2,3-dihydroxybenzoyl)sulfamoyl)phenyl)amino)-4-oxobutan-2-y l 2,3-dihydroxybenzoate; N-((2,3-dihydroxybenzoyl)carbamoyl)-2-(2,3-dihydroxyphenyl)- 5-methyl-4,5- dihydrooxazole-4-carboxamide; (2R,3S)-3-amino-4-(3-(2,3-dihydroxybenzoyl)ureido)-4- oxobutan-2-yl 2,3 -dihydroxybenzoate; N-((2,3 -dihydroxybenzoyl)(phenyl)carbamothioyl)-2- (2,3-dihydroxyphenyl)-5-methyl-4,5-dihydrooxazole-4-carboxam ide; (2R,3S)-3-amino-4-(3- (2,3-dihydroxybenzoyl)-l-phenylthioureido)-4-oxobutan-2-yl 2,3-dihydroxybenzoate; N-(4- (2,3-dihydroxybenzamido)butyl)-2-(2,3-dihydroxyphenyl)-5-met liyl-4,5-diliydrooxazole-4- carboxamide; (2R,3S)-3-amino-4-((4-(2,3-dihydroxybenzamido)butyl)amino)-4 -oxobutan-2- yl 2,3-dihydroxybenzoate; (2R)-2-(2,3-dihydroxybenzamido)-3-(((2R)-2-(2,3- dihydroxybenzamido)-3-(((2R)-2-(2,3-dihydroxybenzamido)-3- hydroxybutanoyl)oxy)butanoyl)oxy)butanoic acid; (2R)-2-(2,3-dihydroxybenzamido)-3- (((2R)-2-(2,3-dihydroxybenzamido)-3-hydroxybutanoyl)oxy)buta noic acid; (2R)-2-(2,3- dihydroxybenzamido)-3-hydroxybutanoic acid; N-(3-aminopropyl)-2,3-dihydroxybenzamide; N-(3-((4-aminobutyl)amino)propyl)-2,3 -dihydroxybenzamide; (S)-N-(3 -((4-(2-(2,3 - dihydroxybenzamido)-3 -hydroxypropanamido)butyl)amino)propyl)-2,3 - dihydroxybenzamide; (S)-N-(2-((4-((3-(2,3- dihydroxybenzamido)propyl)amino)butyl)amino)ethyl)-2-(2,3-di hydroxyphenyl)-4,5- dihydrooxazole-4-carboxamide; (S)-N-( 1 -(2,3-dihydroxyphenyl)- 17-hydroxy- 1,15 -dioxo- 2,6,11,14-tetraazaheptadecan-16-yl)-2,3 -dihydroxybenzamide; N-(5-aminopentyl)-2,3- dihydroxybenzamide; N-(4-aminobutyl)-2,3-dihydroxybenzamide (Aminochelin); N-(3-((3- aminopropyl)amino)propyl)-2,3 -dihydroxybenzamide; N-(3 -(aminomethyl)benzyl)-2,3 - dihydroxybenzamide; N-(2-(benzylamino)ethyl)-2,3-dihydroxybenzamide; (S)-N-benzyl-N- (2-(2,3-dihydroxybenzamido)ethyl)-2-(2,3-dihydroxyphenyl)-4, 5-dihydrooxazole-4- carboxamide; N-(4-(aminomethyl)phenyl)-2,3-dihydroxybenzamide; N-(4-(2- aminoethyl)phenyl)-2,3-dihydroxybenzamide; N-(4-(4-aminophenoxy)phenyl)-2,3- dihydroxybenzamide; and N-(8-aminooctyl)-2,3 -dihydroxybenzamide (or any one of the molecules listed in Table II or depicted in Figs. 9A-9W).

Also provided herein are any of the foregoing nonribosomal molecules

The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Each of the above embodiments and aspects may be linked to any other embodiment or aspect. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. For purposes of clarity, not every component may be labeled in every drawing.

Fig. 1A shows the closed-ring chemical structure of a nonribosomal siderophore, serratiochelin, produced by Serratia plymuthica. The diamine linker is circled (dotted circle). Fig. IB shows the open-ring chemical structure of serratiochelin.

Fig. 2A depicts compressed pathways Ec/Vc-entF (top) and Ec/Vc-vibF (bottom). The Ec/Vc-entF pathway includes the following genes: entD, entF, entC, entE, entB, entA and vibH. The Ec/Vc-vibF pathway includes the following genes: entD, entC, entE, entB, entA, vibH and vibF. Fig. 2B depicts compressed pathways Sp-schFO (top) and Sp- schFlF2F3 (bottom). The Sp-schFlF2F3 pathway includes the following genes: schG, schFO, schC, schE, schB, schA and schH. The Sp-schFO pathway includes the following genes: schG, schC, schE, schB, schA, schH, schFl, schF2, and schF3.

Fig. 3 depicts a plasmid map of the compressed pathway containing the following genes: entD, entC, entE, entB, entA, vibH and vibF, which was used to produce a library of functionally and structurally diverse nonribosomal peptides.

Figs. 4A-4B show examples of compressed synthetic pathways for heterologous expression of natural and unnatural nonribosomal peptides. Heterologous expression of serratiochelms in E. coli Ent- cells was initially attempted by cloning their biosynthetic genes into a single operon, driven by pDSW204 promoter (Fig. 4A). Genes from E. coli MG1655 and V. cholerae A1552, which are homologous to those involved in the biosynthesis of serratiochelms, were then cloned into a single operon (Fig. 4B). The enzymes responsible for activating an amino acid and incorporating it into the nascent molecule differ slightly in their core motif: VibF DMFVAGLI (SEQ ID NO: 8) Ser/Thr; SchF2 DMFCAGLI (SEQ ID NO: 9) Ser/Thr; EntF DVWHFSLV (SEQ ID NO: 10) Ser; and MbtB DMLNAGLV (SEQ ID NO: 11) Ser/Thr. Based on its core motif, VibF, similar to serratiochelin SchF2 and mycobactin MbtB, should activate L-serine and L-threonine.

Fig. 5 shows structures for the DHB-polyamine intermediates assembled by the compressed pathway. By adding other polyamines to the growth medium, VibH was found to catalyze the reaction between foreign free polyamines and the tethered DHB. Each molecule is identified by the letter "M" and a number, corresponding to the polyamine added to the medium.

Fig. 6 shows structures for new serratiochelin analogs. The acylation of the remaining primary amine by VibF occurs with a 2-(2,3- dihydroxyphenyl)-5- methyloxazolinyl and a 2-(2,3-dihydroxyphenyl)-oxazolinyl. In some samples, the amino acid incorporated in the intermediate did not go through an additional cyclization, thus remaining in the open conformation as dihydroxybenzoyl-L-threonine and as

dihydroxybenzoyl-L-serine. The second acylation of a primary amine occurred only for the intermediates depicted with up to four alternative radicals. Each molecule is identified by the letter "M" and a number, corresponding to the polyamine number, as well as the amino acid incorporated and its conformation. R _n indicates the alternative radicals for the structures proposed and detected in the samples.

Figs. 7A-7D show the calculated and observed mass (left) and structure and observed versus calculated fragmentation pattern (right) of indicated molecules produced using the methods of the present disclosure.

Figs. 8A-8C show the calculated and observed mass (left) and structure and fragmentation pattern (right) of indicated molecules produced using the methods of the present disclosure.

Figs.9A-9W show the calculated and observed mass (left) and structure and fragmentation pattern (right) of indicated molecules produced using the methods of the present disclosure. So: serine incorporation, open configuration; Sc: serine incorporation, closed configuration; To: threonine incorporation, open configuration; Tc: threonine incorporation, closed configuration.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein, in some aspects, is a platform for producing structurally and functionally diverse nonribosomal molecules (e.g., nonribosomal peptides, for example, iron- chelating nonribosomal peptides, such as siderophores). This is achieved, in some embodiments, by expressing in modified bacterial cells "compressed" biosynthetic pathways that include clusters of biosynthetic genes obtained from heterologous organisms (e.g., two different species of bacteria, such as Escherichia coli and Vibrio cholera), that encode enzymes active in the bioassembly of nonribosomal molecules. Generally, by combining genes obtained from two different organisms, each organism capable of producing a single specific nonribosomal molecule, the platform provided herein can be used to produce non- naturally-occurring nonribosomal molecules, which differ structurally and functionally from the nonribosomal molecules produced naturally by each of the two organisms from which the biosynthetic genes were obtained. It should be understood that the platform provided herein, in some embodiments, may also be used to produce naturally-occurring NRPs (e.g., serratiochelin).

A prolific collection of metabolites, which have extended life expectancy and bettered quality of life, are naturally produced by plants, fungi and bacteria. Such metabolites are used in therapeutics capable of treating some of the most daunting pathologies, such as cancer and bacterial infections. These natural products have the particularity of not being a direct result of gene translation. Instead, they are assembled by very large enzymes.

Depending on whether these enzymes catalyze reactions where an amino acid or a a- carboxyacyl-CoA is activated and condensed into nascent molecules, they are classified as nonribosomal peptides (NRP) or polyketides (PK), respectively. Less commonly, mixed- gene operons or hybrid genes can also generate hybrid molecules, with both NRP and PK character. The biosynthetic genes are composed of units (modules), each of which can be further divided into catalytic domains.

Described herein are combinations of precursor-directed biosynthesis, combinatorial genetics and heterologous expression of biosynthetic genes towards the assembly of new, unnatural molecules (e.g., nonribosomal peptides), in a programmable fashion and on demand. As an example, to demonstrate the capability of the methods of the present disclosure, iron-chelating nonribosomal peptides referred to as siderophores were produced. These molecules are key for cell survival under low-soluble iron availability. By linking the production of new molecules to survival, the organism used herein was driven to produce new molecules or otherwise perish. As discussed in the Examples, the Serratia plymuthica serratiochelin biosynthetic pathway was deconstructed and a simple and reduced pathway, incorporating only biosynthetic genes, was reconstructed. An equivalent pathway was also constructed, using homologous genes from E. coli and V. cholerae, which are responsible for the biosynthesis of enterobactin and vibriobactin, respectively. This homologous pathway was capable of generating both natural (e.g., serratiochelin and enterobactin) and non-natural molecules on demand, using exogenous supplementation of precursors, which were incorporated into the molecule. A "natural" or "naturally-occurring" molecule is one that is normally produced by an unmodified organism (in nature). It should be understood, however, that the modified organism and biosynthetic pathways described herein may be used to synthetically produce molecules that are found in nature. Serratiochelin and enterobactin are examples of molecules that are produced synthetically using the methods provided herein and are also produced naturally by unmodified (e.g., not genetically modified or manipulated) organisms. The new, synthetic molecules assembled by the multi-enzymatic pathway were subsequently analyzed in silico to determine their usefulness at the clinical level.

Thus, the present disclosure provides, in some embodiments, for the use of ancestral, homologous genes, coupled with a lethal selective pressure, for successful heterologous expression of an assortment of new and unnatural/synthetic molecules {e.g., nonribosomal peptides).

Nonribosomal Molecules

Nonribosomal molecules— secondary metabolites generally produced by

microorganism— are a diverse family of compounds with a broad range of biological activities and pharmacological properties. Nonribosomal peptides, for example, are synthesized independently of mRNA by nonribosomal peptide-synthetases (NRPS).

Generally, NRPS genes for specific peptides are contained in a single operon in bacteria and contain an initiation or starting module, elongation or extending modules, and a termination or releasing module. The initiation module may contain the following domains: F-domain (formylation), NMT-domain (N-methylation), A-domain (adenylation), and PCP-domain (thiolation and peptide carrier protein with attached 4'-phosphopantetheine). The elongation module may contain the following domains: condensation domain (forms amide bonds), cyclization domain (into thiazolines or oxazolines to thizolidines or oxazolidines), NMT- domain (N-methylation), A-domain (adenylation), PCP-domain (thiolation and peptide carrier protein), and E-domain (epimerization into D-amino acids). The termination module may contain the following domains: TE-domain (termination by a thioesterase) and R- domain (reduction to terminal aldehyde or alcohol).

Biosynthesis of nonribosomal peptides typically commences with a loading stage, during which the first amino acid is activated with ATP as a mixed acyl-phosphoric acid anhydride with AMP by the A-domain and is loaded onto the serine-attached 4'- phosphopantethine sidechain of the PCP-domain, resulting in thiolation. The newly-bound amino group may be formylated by an F-domain or methylated by an NMT-domain at this stage. During elongation, each module loads its specific amino acid onto its respective PCP- domain, and the C-domain catalyzes amide bond formation between the thioester group of the growing chain with the amino group of the current module, which attaches to the current PCP-domain. The C-domain may be replaced by the Cy-domain, which catalyzes the reaction of a serine, threonine, or cysteine sidechain with amides, forming oxazolidines and thiazolidine, respectively, in addition to the amide bond. An E-domain may be present, which epimerizes the innermost amino acid of the peptide chain to its D-configuration. The elongation cycle repeats for each elongation module present. During the termination stage, the TE-domain hydrolyzes the completed polypeptide chain from the ACP-domain of the final elongation module, often forming cyclic amides (lactams) or cyclic esters (lactones). Alternatively, the polypeptide may be released by an R-domain, which reduces the terminal aldehyde or alcohol's thioester bond. The released polypeptide may be further modified via glycosylation, acylation, halogenation, or hydroxylation due to the actions of enzymes usually associated with the synthetase complex. The polypeptide becomes functional after priming (attachment of the 4'-phosphopantetheine sidechain of acyl-CoA to the PCP -domain by 4'PP transferases) and deblocking (removal of the S-attached acyl group by specialized associated thioesterases).

There are several classes of nonribosomal molecules, any of which may be produced by the methods of the present disclosure, including, without limitation, pigments, antibiotics, such as actinomycin, bacitracin, calcium-dependent antibiotic, daptomycin, vancomycin, teixobactin, tyrocidine, gramicidin, zwittermicin A, antibiotic precursors, such as ACV- tripeptide, toxins, such as microcystins and nodularins, phytotoxins, such as HC -toxin, AM- toxin, and victorin, and immunosuppressants, such as ciclosporin. Cytostatics, which inhibit cell growth and multiplication, including epothilone and bleomycin, are also contemplated herein.

Some siderophores, which are small, high-affinity iron-chelating compounds, are also a result of nonribosomal peptide synthetases. Siderophores are secreted by microorganisms, including bacteria, fungi, and grasses, in response to environmental iron deficiencies. The molecules are excreted into the extracellular environment where they generally form a stable, hexadentate, octahedral complex preferentially with the Fe3 ⁺ ion. The siderophores are then recognized by cell-specific receptors on the outer membrane and are transported across the cell membrane. Microbes usually reduce the ion to Fe2 ⁺ internally, releasing it from the siderophore which has low affinity for the reduced ion.

There are three major groups of siderophores encompassing over 250 different structures: catecholates (including, enterobactin, from is. coli, bacillibactin, from Bacillus subtillis and 5. anthracis, vibriobactin, from Vibrio cholera, and serratiochelin from Serratia sp.), hydroxamates (including ferrichrome, from Ustilago sphaerogena, Desferoxamine B, from Streptomyces pilosus, Desferoxamine E, from Streptomyces coelicolor, fusarinine C, from Fusarium roseum, ornibactin, from Burkholderia cepacia, and rhodotorulic acid, from Rhodotorula pillmanae), and carboxylates (derivatives of citric acid). Compressed Biosynthetic Pathways

A "compressed biosynthetic pathway," as used herein, refers to a biosynthetic pathway (e.g., genes located on a single vector or on multiple vectors) that contains primarily (e.g., all) biosynthetic genes, which are genes actively involved in the bioassembly of a nonribosomal molecule (e.g., enterobactin, vibriobactin or serratiochelin). A biosynthetic pathway is considered to contain "primarily" biosynthetic genes if at least 80%, at least 85%, at least 95%, at least 98%, or at least 100% of the genes in the biosynthetic pathway are biosynthetic genes (as opposed to regulatory genes). For example, Fig. 3 depicts an example of a plasmid containing a compressed biosynthetic pathway of the present disclosure, whereby the genes of the pathway are primarily biosynthetic genes, including entD, entC, entE, entB, entA, vibH, and vibF (see Table 1).

Table 1.

Thus, examples of bioactive genes include, without limitation, entD, entF, entC, entE, entB, and entA of the Escherichia coli enterobactin gene cluster; vz ^' ?H and vibF of the Vibrio cholera vibriobactin gene cluster; and schG, schFO, schC, schE, schB, schA, schH, schF2, schFl and schF3 of the Serratia plymuthica V4 serratiochelin gene cluster. Other bioactive genes actively involved in the biosynthesis of nonribosomal proteins are contemplated herein.

Compressed biosynthetic pathways of the present disclosure typically contain genes obtained from at least two (e.g., 2, 3, 4 or more) different gene clusters obtained from at least two different organisms. A "cluster" of biosynthetic genes, as used herein, refers to a group of two or more biosynthetic genes found within an organism's genome that encode for similar molecules (e.g., polypeptides, or proteins), which collectively share a generalized function and are often located within a few thousand base pairs of each other. For example, entD, entF, entC, entE, entB, and entA are components of the Escherichia coli enterobactin gene cluster. As another example, vibH and vibF are components of the Vibrio cholera vibriobactin gene cluster. As yet another example schG, schFO, schC, schE, schB, schA, schH, schF2, schFl and schF3 are components of the S. plymuthica V4 serratiochelin gene cluster. Thus, a compressed biosynthetic pathway may contain at least one (e.g., 1, 2, 3, 4, 5, or more) gene from the Escherichia coli enterobactin gene cluster and at least one gene from the Vibrio cholera vibriobactin gene cluster.

Nonribosomal molecules, such as nonribosomal peptides, are typically

"bioassembled" from two or more compounds. For example, for the production of serratiochelin, an amide synthase (SchH) condenses diaminopropane with an acylated dihydroxybenzoyl intermediate, and SchF3 completes the synthesis of serratiochelin. Thus, serratiochelin is considered "bioassembled" from diaminopropane and acylated

dihydroxybenzoyl. "Enzymes active in the bioassembly of a nonribosomal molecule" are enzymes that catalyze the bioassembly of a nonribosomal molecule. Such enzymes, in some embodiments, may be referred to as nonribosomal peptide synthetases (e.g., Strieker et al. Current Opinion in Structural Biology, 2010, 20, 2, 234-240, incorporated by reference herein).

An "amide synthase," as used herein, refers to an enzyme that catalyze the joining of either ammonia or an amide with another molecule, in which the linkage is in the form of a carbon-nitrogen bond (e.g., EC 6.3.1). Examples of amide synthases for use as provided herein include, without limitation, VibHmd SchH.

Enterobactin Pathway

In some embodiments, genes of a compressed biosynthetic pathway are obtained from the E. coli enterobactin gene cluster (e.g., Escherichia coli MG1655). Enterobactin, Ν,Ν',Ν"- ((3S,7S,11S)-2,6,10- trioxo-l,5,9-trioxacyclododecane- 3,7,1 l-triyl)tris(2,3- dihydroxybenzamide), is a high affinity siderophore mainly found in Gram-negative bacteria, including Escherichia coli and Salmonella typhimurium. It is secreted from bacterial cells in response to iron deficiency, resulting in the formation of FeEnt, a coordination complex consisting of a ferric ion chelated to the conjugate base of enterobactin. In E. coli, FepA in the bacterial outer membrane permits entrance of FeEnt to the bacterial periplasm. Using an ATP-binding cassette transporter, FepB, C, D, and G all participate in the transportation of FeEnt through the inner membrane. Ferrienterobactin esterase then cleaves FeEnt to remove the iron, yielding three 2,3-dihydroxybenzoyl-L-serine units. Enterobactin is created from chorismic acid, an aromatic amino acid precursor.

Chorismic acid is converted to 2,3-dihydroxybenzoic acid (DHB) by a series of enzymes, EntA, EntB, and EntC. DHB forms an amide link to L-serine through reactions catalyzed by EntD, EntE, EntF, and EntB. Three molecules of DHB-Ser undergo intermolecular cyclization, resulting in the formation of enterobactin.

Several protein-coding genes are found in E. coli and are necessary for the formation of enterobactin. EntA, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, is a protein- coding gene found in E. coli. It catalyzes the formation of DHB. EntB, 2,3-dihydro-2,3- dihydroxybenzoate synthase, is an aryl carrier protein that is converted into its holo-form by EntD and then activates DHB. EntC, isochorismate synthase, catalyzes the reversible conversion of chorismate to isochorismate during the formation of enterobactin. EntD encodes an Sfp-type phosphopantetheinyl transferase (PPTase) and catalyzes the transfer of the 4'-phosphopantetheine (Ppant) moiety from coenzyme A to the apo-domains of EntB and EntF, resulting in their respective holo-forms. The holo-forms of EntB and EntF then activate DHB and L-serine, respectively. EntE, 2,3-dihydroxybenzoate-AMP ligase, catalyzes the formation of an amide link between DHB and L-serine. EntF, a four domain

(condensation-adenylation-peptidyl carrier protein-thioesterase) nonribosomal peptide synthase, cyclotrimerizes lactone synthase and catalyzes an elongation to permit the ester- bond formation between covalently tethered DHB-serine moieties.

Vibriobactin Pathway

In some embodiments, genes of a compressed biosynthetic pathway are obtained from the V. cholera vibriobactin gene cluster {e.g., Vibrio cholera El Tor A1552). Vibriobactin, N(l)-(2,3-dihydroxybenzoyl)-N(5),N(9)-bis[2-(2, 3-dihydroxyphenyl)-5-methyloxazolinyl-4- carboxamidojnorspermidine, is a siderophore synthesized in Vibrio cholerae. It is biosynthezied from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L- threonine, and one molecule of norspermidine. The reactions leading to functional mature vibriobactin require several nonribosomal peptide synthases, in a process analogous to that described for enterobactin above. VibE and VibB are homologous to EntE and EntB, respectively, from Escherichia coli enterobactin synthetase; VibE, a 2,3-dihydroxybenzoate- adenosyl monophosphate ligase, activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's aryl carrier protein domain. VibB is a bifunctional isochorismate lyase-aryl carrier protein (ArCP). VibH, a novel amide synthase that acts as a free-standing condensation (C) domain, condenses the resulting DHB thioester (the donor) with the small molecule norspermidine (the acceptor), forming N(l)-(2, 3- dihydroxybenzoyl)norspermidine (DHB-NSPD). VibH acts upon an upstream carrier- protein-bound donor and a downstream amine, resulting in a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carrier protein thioesters. VibF contains a rare NRPS domain organization: cyclization-cyclization- adenylation-condensation-peptidyl carrier protein-condensation. It activates and covalently loads its PCP with L-threonine, and together with VibE and VibB, it condenses and heterocyclizes 2, 3-dihydroxybenzoyl-VibB with L-Thr to 2-dihydroxyphenyl-5- methyloxazolinyl-4-carboxy-VibF, an oxazoline. The enzyme-bound aryl oxazoline can be transferred by VibF to various amine acceptors, but it is transferred most efficiently to N(l)- (2, 3-dihydroxybenzoyl)norspermidine), the product of 2,3 -dihydroxybenzoyl -VibB, norspermidine, and VibH. The diacylated product then undergoes a second aryl oxazoline acylation on its remaining secondary amine, also catalyzed by VibF, and yields vibriobactin. Serratiochelin Pathway

In some embodiments, genes of a compressed biosynthetic pathway are obtained from the Serrati asp. or S. plymuthica V4 serratiochelin gene clusters (which include two clusters located at different loci). Serratiochelins, bis-catecholate siderophores, may be terra- or hexadentate in structure. They contain a propane- 1,3-diamine linker and are found in

Serratia species. One of the serratiochelin gene clusters (ca. 21 kb) contains genes

schCEBAGFO and the other (ca. 15 kb) contains genes schFlF2F3H. Some of the serratiochelin genes are involved in siderophore export or ferric-siderophore uptake and utilization. The remaining six genes are involved in serratiochelin synthesis. SchG, homologous to acetolactate synthase, catalyzes the first step in branched-chain amino acid synthesis. The remaining five genes show high sequence identity with the corresponding enterobactin-synthesizing genes. SchC, similar to EntC, is a putative isochorismate synthase, and catalyzes the reaction of chorismate to isochorismate. SchB, similar to EntB, is a bifunctional isochorismate lyase-aryl carrier protein (ArCP), which generates the next intermediate. SchA, similar to EntA, is a dehydrogenase, which converts the intermediate to 2,3-dihydroxybenzoate. SchE, like EntE, adenylates 2,3-dyhydroxybenzoate and transfers it to the ArCP domain of SchB. SchFO may incorporate L-threonine into serratiochelin. SchH, with a 45% sequence similarity to VibH, is also required for the biosynthesis of

serratiochelin. It is a free-standing amide synthase and condenses a diamine with the acylated dihydroxybenzoyl intermediate. SchFl, SchF2, and SchF3 match the domain architecture of VibF completely; SchFl is a putative free-standing NRPS cyclization domain, SchF2 is a putative Cy-A domain, where the A domain codes for L-threonine, and SchF3 matches the C-terminal domain of VibF, containing a C-T-C domain arrangement and finishing the biosynthesis of the serratiochelin.

Nucleic Acids

A "nucleic acid" is at least two nucleotides covalently linked together, and in some instances, may contain phosphodiester bonds {e.g., a phosphodiester "backbone"). An "engineered nucleic acid" is a nucleic acid that does not occur in nature. It should be understood, however, that while an engineered nucleic acid as a whole is not naturally- occurring, it may include nucleotide sequences that occur in nature. In some embodiments, an engineered nucleic acid comprises nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered nucleic acid includes a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, and/or a viral nucleotide sequence. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. A "recombinant nucleic acid" is a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) and, in some embodiments, can replicate in a living cell. A "synthetic nucleic acid" is a molecule that is amplified or chemically, or by other means, synthesized. A synthetic nucleic acid includes those that are chemically modified, or otherwise modified, but can base pair with naturally-occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.

In some embodiments, a nucleic acid of the present disclosure is considered to be a nucleic acid analog, which may contain, at least in part, other backbones comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages and/or peptide nucleic acids. A nucleic acid may be single-stranded (ss) or double- stranded (ds), as specified, or may contain portions of both single-stranded and double- stranded sequence. In some embodiments, a nucleic acid may contain portions of triple- stranded sequence. A nucleic acid may be DNA, both genomic and/or cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Nucleic acids of the present disclosure may include one or more genetic elements. A "genetic element" refers to a particular nucleotide sequence that has a role in nucleic acid expression (e.g., promoter, enhancer, terminator) or encodes a discrete product of an engineered nucleic acid (e.g., a nucleotide sequence encoding a guide RNA, a protein and/or an RNA interference molecule).

Nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press).

In some embodiments, nucleic acids are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5 ^' exonuclease, the 3 ^' extension activity of a DNA polymerase and DNA ligase activity. The 5 ^' exonuclease activity chews back the 5 ^' end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. Vector and Associated Genetic Elements

In some embodiments, a compressed biosynthetic pathway is delivered to a cell on a vector. A "vector" refers to a nucleic acid (e.g., DNA) used as a vehicle to artificially carry genetic material (e.g., an engineered nucleic acid construct) into a cell where, for example, it can be replicated and/or expressed. In some embodiments, a vector is an episomal vector (see, e.g., Van Craenenbroeck K. et al. Eur. J. Biochem. 267, 5665, 2000, incorporated by reference herein). A non-limiting example of a vector is a plasmid (e.g., Fig. 3). Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors typically contain an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert. Plasmids may have more features, including, for example, a "multiple cloning site," which includes nucleotide overhangs for insertion of a nucleic acid insert, and multiple restriction enzyme consensus sites to either side of the insert. Another non-limiting example of a vector is a viral vector. Expression of compressed biosynthetic pathway is driven by a promoter operably linked to a nucleic acid containing the genes of the pathway. A "promoter" refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. A promoter may also contain sub- regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof.

Herein, a promoter is considered to be "operably linked" when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control ("drive") transcriptional initiation and/or expression of that sequence.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as "endogenous."

In some embodiments, a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring" such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906).

In some embodiments, a promoter is an "inducible promoter," which refer to a promoter that is characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by an inducer signal. An inducer signal may be endogenous or a normally exogenous condition (e.g., light), compound (e.g., chemical or non-chemical compound) or protein that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter. Thus, a "signal that regulates transcription" of a nucleic acid refers to an inducer signal that acts on an inducible promoter. A signal that regulates transcription may activate or inactivate transcription, depending on the regulatory system used. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor that is preventing the promoter from driving transcription.

Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.

The administration or removal of an inducer signal results in a switch between activation and inactivation of the transcription of the operably linked nucleic acid sequence. Thus, the active state of a promoter operably linked to a nucleic acid sequence refers to the state when the promoter is actively regulating transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is expressed). Conversely, the inactive state of a promoter operably linked to a nucleic acid sequence refers to the state when the promoter is not actively regulating transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is not expressed).

An inducible promoter of the present disclosure may be induced by (or repressed by) one or more physiological condition(s), such as changes in light, pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agent(s). An extrinsic inducer signal or inducing agent may comprise, without limitation, amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or combinations thereof.

Inducible promoters of the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically -regulated and physically- regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid- regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

In some embodiments, an inducer signal of the present disclosure is an N-acyl homoserine lactone (AHL), which is a class of signaling molecules involved in bacterial quorum sensing. Quorum sensing is a method of communication between bacteria that enables the coordination of group based behavior based on population density. AHL can diffuse across cell membranes and is stable in growth media over a range of pH values. AHL can bind to transcriptional activators such as LuxR and stimulate transcription from cognate promoters.

In some embodiments, an inducer signal of the present disclosure is

anhydrotetracycline (aTc), which is a derivative of tetracycline that exhibits no antibiotic activity and is designed for use with tetracycline-controlled gene expression systems, for example, in bacteria.

Other inducible promoter systems are known in the art and may be used in accordance with the present disclosure.

In some embodiments, inducible promoters of the present disclosure function in prokaryotic cells (e.g., bacterial cells). Examples of inducible promoters for use prokaryotic cells include, without limitation, bacteriophage promoters (e.g. Plslcon, T3, T7, SP6, PL) and bacterial promoters (e.g., Pbad, PmgrB, Ptrc2, Plac/ara, Ptac, Pm), or hybrids thereof (e.g. PLlacO, PLtetO). Examples of bacterial promoters for use in accordance with the present disclosure include, without limitation, positively regulated E. coli promoters such as positively regulated σ70 promoters (e.g., inducible pBad/araC promoter, Lux cassette right promoter, modified lamdba Prm promote, plac Or2-62 (positive), pBad/AraC with extra REN sites, pBad, P(Las) TetO, P(Las) CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), aS promoters (e.g., Pdps), σ32 promoters (e.g., heat shock) and σ54 promoters (e.g., glnAp2); negatively regulated E. coli promoters such as negatively regulated σ70 promoters (e.g., Promoter (PRM+), modified lamdba Prm promoter, TetR - TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ, RecA DlexO DLacOl, dapAp, FecA, Pspac-hy, pel, plux-cl, plux-lac, CinR, CinL, glucose controlled, modified Pr, modified Prm+, FecA, Pcya, rec A (SOS), Rec A (SOS), EmrR regulated, Betl regulated, pLac lux, pTet Lac, pLac/Mnt, pTet/Mnt,

LsrA/cI, pLux/cI, Lacl, LacIQ, pLacIQl, pLas/cI, pLas/Lux, pLux/Las, pRecA with LexA binding site, reverse BBa ROOl 1, pLacI/ara-1, pLacIq, rrnB PI, cadC, hns, PfhuA, pBad/araC, nhaA, OmpF, RcnR), aS promoters (e.g., Lutz-Bujard LacO with alternative sigma factor σ38), σ32 promoters (e.g., Lutz-Bujard LacO with alternative sigma factor σ32), and σ54 promoters (e.g., glnAp2); negatively regulated B. subtilis promoters such as repressible B. subtilis σΑ promoters (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank) and σΒ promoters. Other inducible microbial promoters may be used in accordance with the present disclosure.

Cells and Cell Expression

Nucleic acids of the present disclosure may be expressed in a broad range of host cell types. In some embodiments, engineered nucleic acids are expressed in bacterial cells, yeast cells, insect cells, mammalian cells or other types of cells.

Bacterial cells of the present disclosure include bacterial subdivisions of Eubacteria and Archaebacteria. Eubacteria can be further subdivided into gram-positive and gram- negative Eubacteria, which depend upon a difference in cell wall structure. Also included herein are those classified based on gross morphology alone {e.g., cocci, bacilli). In some embodiments, the bacterial cells are Gram-negative cells, and in some embodiments, the bacterial cells are Gram-positive cells. Examples of bacterial cells of the present disclosure include, without limitation, cells from Escherichia spp., Yersinia spp., Klebsiella spp., Acinetobacter spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp.,

Streptomyces spp., Bacteroides spp., Prevotella spp., Clostridium spp., Bifidobacterium spp., or Lactobacillus spp. In some embodiments, the bacterial cells are from Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae,

Lactococcus lactis, Leuconostoc lactis, Actinobacillus actinobycetemcomitans,

cyanobacteria, Escherichia coli, Helicobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphlococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Streptococcus spp., Enterococcus faecalis, Bacillus coagulans, Bacillus ceretus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Zymomonas mobilis, Streptomyces phaechromogenes, or Streptomyces ghanaenis. "Endogenous" bacterial cells refer to non-pathogenic bacteria that are part of a normal internal ecosystem such as bacterial flora.

In some embodiments, bacterial cells of the invention are anaerobic bacterial cells {e.g., cells that do not require oxygen for growth). Anaerobic bacterial cells include facultative anaerobic cells such as, for example, Escherichia coli, Shewanella oneidensis and Listeria monocytogenes. Anaerobic bacterial cells also include obligate anaerobic cells such as, for example, Bacteroides and Clostridium species.

In some embodiments, the bacterial cells are Escherichia coli (E. coli) cells. E. coli is a Gram-negative, anaerobic, rod-shaped bacterium commonly found in the large intestine of endotherms. Frequently used as prokaryotic model organism, E. coli contains a circular DNA molecule of 4288 annotated protein-coding genes, seven ribosomal RNA operons, and 86 transfer RNA genes. The genome contains a number of transposable genetic elements, repeat elements, cryptic prophages, and bacteriophage remnants as well. As a host cell, E. coli is versatile, allowing for the production of heterologous proteins and molecular cloning into its vector plasmids.

Cells of the present disclosure, in some embodiments, are modified. A modified cell is a cell that contains an exogenous nucleic acid or a nucleic acid that does not occur in nature (e.g., an engineered nucleic acid). In some embodiments, a modified cell contains a mutation in a genomic nucleic acid. In some embodiments, a modified cell contains an exogenous independently replicating nucleic acid (e.g., an engineered nucleic acid present on an episomal vector). In some embodiments, a modified cell is produced by introducing a foreign or exogenous nucleic acid into a cell. A nucleic acid may be introduced into a cell by conventional methods, such as, for example, electroporation (see, e.g., Heiser W.C.

Transcription Factor Protocols: Methods in Molecular Biology™ 2000; 130: 117-134), chemical (e.g., calcium phosphate or lipid) transfection (see, e.g., Lewis W.H., et ah, Somatic Cell Genet. 1980 May; 6(3): 333-47; Chen C, et al., Mol Cell Biol. 1987 August; 7(8): 2745- 2752), fusion with bacterial protoplasts containing recombinant plasmids (see, e.g., Schaffher W. Proc Natl Acad Sci USA. 1980 Apr; 77(4): 2163-7), transduction, conjugation, or microinjection of purified DNA directly into the nucleus of the cell (see, e.g., Capecchi M.R. Cell. 1980 Nov; 22(2 Pt 2): 479-88).

In some embodiments, a cell is modified to express a reporter molecule. In some embodiments, a cell is modified to express an inducible promoter operably linked to a reporter molecule (e.g., a fluorescent protein such as green fluorescent protein (GFP) or other reporter molecule).

In some embodiments, a cell is modified to overexpress an endogenous protein of interest (e.g., via introducing or modifying a promoter or other regulatory element near the endogenous gene that encodes the protein of interest to increase its expression level). In some embodiments, a cell is modified by mutagenesis. In some embodiments, a cell is modified by introducing an engineered nucleic acid into the cell in order to produce a genetic change of interest (e.g., via insertion or homologous recombination).

In some embodiments, a modified cell contains a gene deletion. That is, a cell may be modified to remove a gene normally expressed in nature. In some embodiments, a cell is an Escherichia coli cell containing one or more of the following gene deletions: AentD, A entC, AentE, AentB, AentA and AentF.

Methods of Producing Nonribosomal Molecules

Some aspects of the present disclosure are directed to methods of producing a nonribosomal molecule, the methods comprising culturing at least one modified cell comprising a compressed biosynthetic pathway, in the presence of an exogenous polyamine (e.g., diamine) linker precursor, under conditions that result in the production of a nonribosomal molecule. Other aspects of the present disclosure are directed to methods of producing a nonribosomal molecule precursor, the methods comprising culturing at least one modified cell comprising a compressed biosynthetic pathway, in the presence of an exogenous amine linker precursor, under conditions that result in the production of a nonribosomal molecule precursor.

"Conditions that result in the production of a nonribosomal molecules" may be vary and may be based on any one or more of the following conditions: type of cell used for gene expression, volume of cell culture, composition of cell culture media, length of cell culture period, and temperature at which cells are cultured.

In some embodiments, cells are cultured in minimal medium, which, in some embodiments, is optimized for production of a particular nonribosomal molecule of interest.

Minimal medium may comprise, in some embodiments, Na ₂ HP0 ₄ (e.g., 1 to 10 g/L, such as 4 to 6 g/L (e.g., 5.96 g/L)), K ₂ HP0 ₄ (e.g., 1 to 10 g/L, such as 1 to 4 g/L (e.g., 3.0 g/L)), NH ₄ C1 (e.g., 1 to 10 g/L, such as 1 to 3 g/L (e.g., 1.0 g/L)), NaCl (e.g., 1 to 10 g/L, such as 1 to 2 g/L (e.g., 0.5 g/L)), MgS0 ₄ (0.05 to 1 g/L, such as 0.05 to 1.0 g/L (e.g., 0.058 g/L)), C ₆ H ₁₂ 0 ₆ (e.g., 1 to 10 g/L, such as 4 to 6 g/L (e.g., 5.0 g/L)) and IPTG (e.g., 1 to 5 nM, such as 2 to 3 mM (e.g., 1 mM)).

In some embodiments, the cells are cultured at a pH of 4 to 8, or 4 to 10. For example, the cells may be cultured at a pH of 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the cells are cultured at (e.g., the minimal medium has) a pH value of 7.0.

In some embodiments, cells (e.g., bacterial cells) are cultured in the presence of an exogenous polyamine linker precursor. A "polyamine linker precursor," as used herein, refers to an amine that has at least two amine groups with one or two hydrogen atoms. Non- limiting examples of polyamine linker precursor are shown in Table 2 and include norspermidine, cadaverine, spermidine, diaminopropane, m-xylylenediamine, N,N'-bis(2- aminoethyl)-l,3-propanediamine, N-benzylethylenediamine, 4-(2-Aminoethyl)aniline, 4,4'- oxydianiline, 4,4'-diaminodiphenylmethane, spermine, 1,5-diaminonaphthalene, 2,2'- thiobisacetamide, sulfaguanidine, p-aminobenzenesulfonamide, urea, N-phenylthiourea and putrescine. An "amine linker precursor" refers to an amine group with at least one hydrogen atom.

In some embodiments, the precursors are added to a cell culture or other reaction medium at a final concentration of 0.1 μΜ to 0.05 mM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μΜ), or 0.05 to 20 mM (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM).

In some embodiments, an iron chelator is added to a cell culture or reaction medium.

For example, 2,2'-bipyridyl may be added to a cell culture or reaction medium. An iron chelator, in some embodiments, may be added to a final concentration of 0.05 to 1 mM (e.g., 0.05, 0.1, 0.15 mM).

In some embodiments, the cells are grown (e.g., cultured) for 1 to 20 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days), or more, with or without shaking (e.g., 200 to 250 rpm). For example, cell may be cultured for 1 to 5, 1 to 10, or 1 to 15. In some embodiments, cells are cultured for 5 days.

Compositions and Molecules

Also provided herein are compositions comprising nonribosomal molecules (e.g., nonribosomal peptide) produced by the methods of the present disclosure. A composition may comprise any one or more of the nonribosomal molecules listed in Table 2, or an analog thereof. Table 2. Nonribosomal molecules

Exact Mass: 555.16

Exact Mass: 569.12

Exact Mass: 492.1380

Microorganisms display an extraordinary ability to synthesize molecules that can target cancer cells, parasites, iron overload and bacterial infections. For this, they have evolved sets of very large enzymes that harmoniously interact to assemble acyl-CoA or peptide-based molecules, polyketides or nonribosomal peptides, respectively. The genes encoding these enzymes are modular and each module is responsible for the incorporation of one unit.

Tapping this proficiency has nonetheless posed a great challenge to researchers. Besides the difficulties in cultivating some of the producer organisms, it can be also challenging to find the conditions that lead to molecule production, or to engineer the pathway for inducible or constitutive expression. More frequently than not, attempts to alter these pathways for production of new molecules, or even a mere attempt at their heterologous expression, results in a complete shutdown of molecule production.

Provided herein are methods for effective production and structural diversification of nonribosomal molecules, for example. Using these methods, biosynthetic pathways containing ancestral biosynthetic genes were successfully constructed and used in a heterologous and programmable fashion to produce serratiochelins, for example, and their new analog molecules, in demand.

The enterobactin and vibriobactin biosynthetic pathways were constructed to create a single hybrid pathway, comprised of genes involved only in the biosynthesis of each of the molecules. More specifically, entABCDE and vibFH were cloned in a single operon, driven by a lower-expression version of the IPTG-inducible ptrc99a, pDSW204, as discussed below. Expression occurred from an enterobactin-deficient E.coli strain (E. coli Ent ^" ) that was generated to lack entABCDEF.

An assortment of structurally diverse nonribosomal peptides were produced by supplementing the iron-deprived growth medium with different small molecule precursors, the substrate of VibH. These molecules were analogs of serratiochelin and its intermediate. Additional structural diversity was generated due to the capacity of VibF to activate not only L-threonine, but L-serine as well, for incorporation into the nascent molecule. In vivo VibF activation of L-serine has never been reported before. Nonetheless, not all precursors could serve as a substrate to VibF or VibH, and even when they could, there seemed to be a slight preference for L-threonine over L-serine activation as well.

The new molecules generated result, in part, from the precursor added to the medium. Thus, the structure of the resulting molecule can be predicted. Nonetheless, if there is a specific moiety that one desires to include in a nascent molecule, the corresponding precursor can be supplied to the medium for incorporation, if it contains at least one amine group. Although, the enzymes active sites can further limit molecule diversity.

Nearly half of the precursors tested herein were incorporated into the nascent molecule and over half of these led to additional new structures, as detected by LC-MS/MS. An algorithm-based analysis of molecular bioactivity on clinically relevant targets also revealed that several of these molecules (particularly the smaller ones) showed promising scores and can potentially be developed into useful drugs.

Besides producing molecules in demand, the synthetic pathways of the present disclosure were used to assembled the cyclic and linear versions of enterobactin, as well as its monomer and its dimer. The synthetic pathways also assembled a new version of linear enterobactin, and its dimer and monomers, containing not L-serine but L-threonine.

Such observation sheds insight onto the evolution of gene collectives, which are thought to be sets of genes that co-evolved quickly to lead to new molecules with the least effort. The following Examples show that the combination of genes from independent pathway can produce new molecules, and known molecules, assembled by different enzymes.

The methods described herein may be expanded to other nonribosomal pathways for which the heterologous expression has posed a problem or when diversification of the structure of the molecules being produced is desired. Using these methods, molecular diversification can be achieved and altered by means of heterologous expression and precursor supplementation, for example. In silico molecule design and its in vivo assembly may also be implemented using microfluidics systems for consistent, streamlined and on demand production of programmed molecules

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references

(including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teachings that are referenced herein. EXAMPLES

Example 1

The present study directed to the production of new molecules based on, but structurally and functionally distinct from, dihydroxybenzoate. This was achieved, ultimately, by providing VibH with different substrates (e.g., polyamine linkers) to condense dihydroxybenzoate (Fig. 1, circled). The polyamine linkers were selected based on the availability of at least two amines groups with one or two hydrogen atoms (see examples in Table 2, "precursors").

Four different constructs, each containing a "compressed pathway," were prepared for the initial biosynthesis of dihydroxybenzoate (Figs. 2A-2B). These biosynthetic genes were amplified by PCR using 5' - 3' primers that added the E. coli ribosomal binding site

GAGGAGA to the region upstream of the translation initiation codon, when required. Genes schCEBA were amplified together having a ribosomal binding site (RBS) been only added upstream of schC. The native RBSs, when existent, were maintained for schEBA (in some cases the genes {e.g., in operons) overlapped {e.g., the 3' of the upstream gene with the 5' of the downstream gene) to promote polycistronic expression).

The first compressed pathway included the following genes: entD, entF, entC, entE, entB, entA and vibH (Fig. 2A, top). The second compressed pathway included the following genes: entC, entD, entE, entB, entA, vibH md vibF (Fig. 2A, bottom). The third compressed pathway included the following genes: schG, schFO, schC, schE, schB, schA and schH (Fig. 2B, top). The fourth compressed pathway included the following genes: schG, schC, schE, schB, schA, schH, schFl, schF2, and schF3 (Fig. 2B, bottom). Serratia plymuthica V4 was the donor for the serratiochelin {sch) genes. Escherichia coli MG1655 was the donor for the enterobactin {ent) genes entCEBA, entD and entF, while Vibrio cholera El Tor A1552 was the donor for the vibriobactin {vib) genes vibF and vibH. Unexpectedly, the only compressed pathway capable of producing the iron chelating molecules required for survival of the strain in the growth conditions used was the second compressed pathway (Fig. 2A, bottom, and Fig. 3), which contained both vz ^' ?H and vibF. This pathway was used to generate molecules with structural and functional diversity relative to dihydroxybenzoate.

This compressed pathway was cloned into a medium copy plasmid pDSW204 (E. coli) (Fig. 3). The biosynthetic genes (e.g., entD, entC, entE, entB, entA, vibH and vibF) were cloned into a single operon, using their native or standard E. coli. ribosomal binding site (RBS). The isopropyl β-D-l-thiogalactopyranoside (IPTG)-inducible trc promoter was used to drive expression of the compressed pathway. Given the size of the compressed pathway, a cos site was cloned in for stability.

E. coli MG1655 was selected as a host, given its robustness. In order to avoid the potential noise created by the enterobactin pathway in the chromosome of E. coli,

endogenous genes entD, entF and entCEBA were deleted from its chromosome (via Lambda Red Recombination). entD was replaced with a chloramphenicol -resistance cassette, entCEBA was replaced with kanamycin-resistance cassette, and entF was replaced with gentamicin-resistance cassette. The removal of the enterobactin biosynthetic genes disabled this organism's capacity to assemble this siderophore. The assembly of the genes in a single pathway was assessed in yeast by amplifying by PCR the junctions (e.g., forward primer on one region, reverse primer on the next region). With such method, a correct product size is indicative of proper assembly. Thus, the genes entF, entD, entCEBA, vibF, vibH, schFO, schFl, schF2F3, schH, schG and schCEBA were PCR-amplified with overhangs homologous to the genes located up and downstream in the compressed pathway. The genes at the beginning and end of each pathway were amplified with 5' or 3' primers, respectively, which added a Spel restriction site and homology to the YAC pYES-lL (Life Technologies). The amplicons were transformed into, and assembled into full pathways, by S. cerevisiae using the Geneart® High Order Genetic Assembly kit (Life Technologies). The compressed pathways were released from pYes-lL by digestion with the restriction enzyme Spel. The expression vector derived from pDSW205 was digested with the same enzyme, downstream of the trc promoter, and the compressed pathway cloned in.

All strains were maintained on Lysogeny Broth (Miller) medium, supplemented with 15% agar. Seeking to activate all iron uptake native mechanisms, the E. coli MG1655

AentBAentCEBAAentF carrying the compressed pathway was grown in iron-deprived medium: minimal medium (e.g., 3.0 Κ ₂ ΗΡ0 ₄ , 5.96 Na ₂ HP0 ₄ , 5.0 g/L glucose, 1.0 g/L NH4CI, 0.5 g/L NaCl, and 0.058 g/L MgS0 ₄ , at pH 7.0) supplemented with lmM IPTG, 100 μg/L ampicillin and 0.1 mM bipyridyl. The growth medium was supplemented with precursors (also referred to as polyamine linkers) to a final concentration of 0.05 μΜ to 10 mM. The cells were grown in a 250 mL of medium in a 1 L Erlenmeyer flask, shaking at 250 rpm, for 5 days (ODeiOnm ^¾ 0.500) at 30 °C (surprisingly, the growth rate was slower, and survival poorer, at 37 °C). After incubation for 5 days the cultures were spun-down (5000 rpm, 5 min, 4 °C), the supernatant was filter sterilized, the sterile supernatant was loaded into C18 Reversed-phase Sep-Pac columns, and the molecules of interest were eluted with 100% acetonitrile. The panoply of precursors was selected based on the presence of at least two amine groups having one or two free hydrogen atoms. The precursors were also selected based on their potential to endow the molecule with alternative or additional functionalities.

The various precursors used and molecules (in closed chain form and open chain form) produced are shown in Table 2. Example 2

Serratiochelins are catechol siderophores produced by Serratia plymuthica V4. These siderophores utilize catechol moieties for iron coordination, obtaining them from the conversion of endogenous chorismate to dihydroxybenzoate (DHB). This pathway appears to be extremely conserved among catechol siderophores. Additional enzymes can then use this precursor to form a wide diversity of catechol-based molecules, such as enterobactin, fluvibactin, vibriobactin, photobactin, petrobactin and vulnibactin.

The experiments described in this example address whether E. coli is capable of producing enterobactin as well as serratiochelins by testing (1) whether the machinery responsible for the import and export of siderophores in E. coli would recognize

serratiochelins and its catechol moieties; (2) whether E. coli can uptake polyamines (Table 3), (3) whether S. plymuthica genes would be functional in E. coli (Table 4), and (4) whether expressing the DHB pathway proteins in a different organism and supplementing the media in which the organisms with a desired precursor would result in the generation of new analogs. Table 3. List of precursors, reference number and final working concentration.

Table 4. Levels of similarity between homologous proteins involved in the assembly of siderophores in E. coli, V. cholerae and S. plymuthica.

Max Total Query

Comparison E value Identity

Score Score Coverage

EntE vs. SchE 781 781 99% 0.0 70%

EntF vs. SchFO 1452 1452 98% 0.0 58%

VibF vs.

899 2131 96% 0.0 51%

SchFlF2F3

EntD vs. SchG 19.6 52.0 58% 0.11 24%

VibH vs. SchH 207 222 95% 2xl0 ^"66 32%

The precursor 1,3-diaminopropane (diaminopropane) is required for the assembly of serratiochelins and is naturally produced by S. plymuthica but not by E. coli. In order to produce serratiochelins using E. coli, diaminopropane was added to the growth medium. The S. plymuthica genes involved in the biosynthesis of serratiochelins were cloned in a single operon and were driven by an inducible promoter pDSW204 (pSP S), a weaker version of ptrc99A. This synthetic operon is a compressed version of the 2-cluster pathway responsible for biosynthesizing serratiochelins (Fig. 4A). It contains only (1) genes schABCEG, which are homologous to the genes entABCDE involved in the biosynthesis of enterobactin, (2) genes schFlF2F3, which together are homologous to vibF, and (3) schH, a vz ^' ?Hhomolog. Genes vibF and vibH are involved in the biosynthesis of vibriobactin, a siderophore obtained from V. cholerae.

The constructs were transformed into a strain of E. coli Ent ^" from which genes entABCDEF (homologous to schABCEG) had been removed. The E. coli strain Ent ^" , carrying either pSP S or empty vector, were grown in the presence or absence of diaminopropane under iron-deprived conditions at 30 °C with agitation. Growth was not observed in either case. Without being bound by theory, the lack of growth may reflect the incapacity of the E. coli transcriptional and translational machinery to operate on S.

plymuthica genes, or the resulting enzymes might not have found in E. coli conditions favorable to their activation and processing. The enzymes responsible for assembling nonribosomal peptides function as assembly lines. This suggests that if one single enzyme is not present or is non- functional, the target molecule might not be made.

Example 3

As noted above, the biosynthetic pathway in Example 1 includes genes homologous to E. coli and V. cholerae genes. In E. coli and V. cholerae, these homologs form part of the biosynthetic pathways that produce enterobactin and vibriobactin, respectively. The following experiments tested whether these genes could replace the S. plymuthica homologs to produce serratiochelins. Thus, an additional pathway was constructed for the biosynthesis of serratiochelins and new analogs.

E. coli genes entABCDE and V. cholerae gene vibFH, ancestral homologs to S.

plymuthica V4 genes, were cloned into the same empty plasmid backbone, the plasmid was introduced into E. coli Ent ^" , and the resulting construct was designated pEV S (Fig. 4B, bottom). Holo-EntB, acylated with DHB by EntE, serves as a substrate for the activity of VibH, similarly to VibB. pEV S enabled the growth of E. coli Ent ^" under iron-limited conditions in the presence of diaminopropane. The production of both a serratiochelin precursor (Fig. 5, Ml) and serratiochelin (Fig. 6, Ml) was confirmed upon analysis of the tC 18-purified supernatant.

Unexpectedly, growth for E. coli Ent ^" carrying pEV S was also observed in the absence of diaminopropane, indicating that another siderophore, independent of the polyamine, could be assembled by enzymes encoded by the biosynthetic pathway. This unexpected observation was investigated, and upon analysis of the tCl 8-purified supernatant, the production of enterobactin was detected (Fig. 7A) as well as linear enterobactin (Fig. 7B) and its dimers (Fig. 7C) and monomers (Fig. 7D). These results shows that VibF can replace EntF to assemble this enterobactin, independent of any precursors. Enterobactin was detected in all samples analyzed, except those to which norspermidine or sulfaguanidine had been added. Linear enterobactin and its dimers and monomers were also found in most samples. E. coli Ent-, which is the empty strain, could not grow under the same conditions.

Based on the results above, experiments were designed to investigate whether this pathway could produce thr-enterobactin analogs. Only linear thr-enterobactin (Fig. 8A), its dimers (Fig. 8B) and monomers (Fig. 8C) were detected, and only in a reduced number of samples. These results, however, do not exclude the possibility that thr-enterobactin was produced at levels too low to be detected.

This study demonstrates in vivo VibF activation of L-serine to produce enterobactin, and L-threonine activation to produce a new linear thr-enterobactin. The alternative pathway to enterobactin removed the selective pressure exerted on the compressed pathway to condense polyamines and generate serratiochelin analogs.

Example 4

Having established a hybrid pathway capable of producing molecules, as a function of the precursor added, experiments were then designed to determine the extent of the hybrid pathway's programming capabilities. Polyamines with a varying number of carbons and amine groups, with and without other moieties, as well as 4 dipeptides, were added to the growth medium {see Table 3). The concentration used was determined as the highest concentration that would not inhibit the growth of the producer strain, in the absence of iron.

The biosynthetic and programmable pathway was capable of generating, on demand, several of the predicted intermediate nonribosomal peptides, where a polyamine was condensed with DHB (Fig. 5). VibH condense linear polyamines as well as aromatic polyamines, such as aminobenzylamine (Fig. 5, M10 and Fig. 9U) and oxydianiline (Fig. 5, M21 and Fig. 9W). The annotated MS/MS spectra for the structures shown in Fig. 9 were also obtained.

The capacity of the pathway to generate fully-assembled serratiochelin mutasynthons seemed to be mostly restricted to linear polyamines, containing up to 4 amine groups and 10 carbons (Table 3, polyamine 3, Fig. 6, M3R _n ). Nonetheless, VibF incorporated L-serine or L- threonine in the molecules and, for some molecules, depending on the precursor

supplemented, cyclized the molecule {see Figs. 9A-9W). VibH was flexible in the substrate it can act upon, transferring the activated DHB from EntB to a diversity of acceptors amines (Fig. 5 and Figs. 9A-9W).

While vibriobactin was not assembled in the presence of norspermidine in the medium (Table 3, polyamine 6), its intermediate with only the primary amines acylated (Fig. 9N, M6Tc) and an additional analog (Fig. 90, M6To) was detected.

A factor limiting the diversity of molecules generated appeared to be VibF. Given that several of the intermediates were detected in the supernatant, VibF seems incapable of condensing dihydroxyphenyl-5-methoxyxazoline (and L-serine-containing derivative) with the polyamine-containing intermediate. Several approaches could potentially extend the performance of this synthetic pathway in terms of assembly full-sized molecules. For example, VibF may be subjected to directed evolution and other VibF/SchFlF2F3 homologs tested. It is also possible that the molecules were indeed assembled but could not be exported to the external milieu.

Results also showed a preferential orientation for condensing asymmetrical polyamines using the e«tS-tethered DHB. For molecules M9 (Fig. 5 and Fig. 9Q), M3Sc (Fig. 9G), M3To (Fig. 9H), M3So (Fig. 91) and M9Tc (Fig. 9R), a single fragmentation pattern, corresponding to that of a single orientation, was found. Nonetheless, the alternative conformation could exist at lowers levels.

M5 corresponds to aminochelin (Fig. 5), a molecule that can act as a siderophore and can be incorporated into azotochelin, both produced by Azotobacter vinelandii. Thus, novel intermediated generated via this programmable pathway may possess metal chelating abilities similar to their larger counterparts.

To investigate whether the engineered non-natural siderophores could serve other therapeutically-relevant purposes, the Simplified Molecular-Input Line-Entry System

(SMILES) specifications were run using three online tools that compute the likelihood of the submitted structure having particular activities of biological interest. The SMILES of the new molecules assembled on demand by the programmable pathway provided herein were run using these algorithms, and the scores were compiled in Table 5. Bioactivity prediction was calculated using the web-based platform Molinspiration. Target prediction was calculated with the Swiss Target Prediction web-based tool. Drug-likeness was calculated using MolSoft, a web-based algorithm.

Table 5. Algorithm-based bioactivity, ligand target and drug-likeness prediction.

Three of the 21 intermediate, and all but three full-sized molecules, were predicted to bind GPRCs (Score>0.20) (Table 5). GPRCs are the largest class of Eukaryotic cell-surface receptors involved over 30 human diseases, such as retinitis pigmentosa and nephrogenic diabetes insipidus. More than 60% of all prescribed drugs target these receptors. The molecules with a score over the threshold were those that contained linear polyamines.

Six intermediates containing linear polyamines (Table 5, Ml -6, M21), but none of the full molecules, were predicted to target ion channel modulators. These modulators are membrane proteins that control de passage of ions (e.g., Ca ²⁺ , K ⁺ , H ⁺ and CI ^" ) across the cell membrane. They are involved in the treatment of multiple human diseases, such as epilepsy, coronary heart disease and chronic pain. A single molecule (Table 5, Ml 2) was predicted to inhibit kinases. These enzymes phosphorylate proteins altering their activity and are important to restore aberrant phosphorylation associated with disease.

Seven full-sized molecules (Table 5, M2Tc, M2So, M3To, M3So, M5Sc, M6To and M9To) were predicted to inhibit proteases, a feature typical of anti-viral molecules, such as antiretrovirals.

A total of seven molecules were predicted to act as enzyme inhibitors (Table 5, M2, M6, M21, Ml Sc, M2Sc, M3Sc and M9Sc), such as anti-cancer molecules that inhibit telomerases.

The Swiss Target Prediction algorithm returns instead a target and the associated 2D similarity score, for each molecule submitted. 2D-based similarity is based on structural similarity of fragments of the molecules. The SMILES of several molecules submitted returned similar hits, though with different scores as shown on Table 5. Those with the highest score were predicted to target the catechol O-methyltransferase (Table 5, Ml, M6, S=0.86). This enzyme degrades catecholamines and its impaired activity is connected to psychiatric disorders. Other molecules were determined to potentially target: telomerase reverse transcriptase (Table 5, M10, S=0.89), which is responsible for telomere maintenance and genome stability; microtubule-associated tau protein (Table 5, Ml 2, S=0.82), which stabilizes microtubules and is thought to be associated with

neurodegenerative diseases; arachidonate 5 -lipoxygenase (Table 5, Ml 1, S=0.80), whose polymorphism is thought to be connected with Alzheimer's disease; D(2) dopamine receptor (Table 5, M9, S=0.80), associated with addiction; melanin-concentrating hormone receptor 1 (Table 5, M2, M21, S=0.77), associated with obesity; muscle blind-like protein 1 (Table 5, M5, S=0.77; M4, S=0.75), involved in mRNA maturation in mammals and associated with myotonic dystrophy; and opioid receptors (Table 5, M9Tc, S=0.72), involved in addiction. Despite sharing some of these predicted targets, all full-sized molecules had lower scores than their smaller counterparts (S<70), though their values are still significant (S>0.5).

The overall non target-specific drug-likeness of each molecule (Table 5), was significant for all structures (S>0), varying between 0.50 (Table 5, M5Sc) and 1.63 (Table 5, M6 and M9). These results highlight the potential clinical relevance of the molecules generated.

The Lipinski Rule of Five aims to be a straightforward method for prediction of the solubility, absorption and permeation of any molecule, in the human body (Lipinski CA et al. Adv. Drug Deliv. Rev. 64, 4-17, 2012). At most, 10% of the drugs in the dataset did not respect these rules, when combined in any pairs. If two parameters do not respect the rules, "poor absorption or permeability" is possible, and in the many thousand drugs tested, only a minute number falls outside these parameters. Based on this calculation (Table 6), none of the intermediate molecules of the present disclosure, and only 8 out of the 17 full-sized molecules synthesized on demand, could potentially be poorly absorbed (Table 6). The most common types of violation, for the latter molecules, were the Topological Polar Surface Area (TPSA>140A), their molecular weight (MW>450) and the number of H-bond donors (OHNF 5).

Table 6. Lipinski's Rule of Five, number of Rule violations, rotatable bond number and molecular volume of all molecules produced.

MLogP, Moriguchi's logP, octanol/water partition coefficient, under 4.15

TPSA, Molecular Polar Surface Area, prediction of passive transport of drugs in vivo, no higher than 140 A Atoms, number of atoms between 20 and 70 (Ghose-Viswanadhan-Wendoloski CMC drug-like index at 80%)

MW, molecular weight, between 200 and 450

ON, number of H-bond acceptors (N+O), up to 10

OHNH, number of H-bond donors, up to 5

Violations, calculated number of unmet criteria

RBN, rotatable bond number, is a measure of molecular flexibility, which is related with membrane permeability, from 1 to 9

Volume, molecular volume, impacts the ability of molecules to be transported Example 5

This examples addresses whether use of a condensed pathway would result in further diversity by supplying polyhydroxybenzoates exogenously, permitting control over two parts of the nascent molecules.

To achieve control over the selection of (di)hydroxybenzoates to be tethered to the thiol group of EntE, a new biosynthetic pathway was built. This pathway contained solely the biosynthetic genes entB, entE, vibF and vibH, in addition a to the PPTase-encoding entD. By supplementing exogenously dihydroxybenzoates, genes entCA are no longer necessary, as they convert chorismate to 2,3-dihydroxybenzoate. entB is still required, as it is a bifunctional protein. Its N-terminus contains the isochorismate lyase, which converts isochorismate to (2S,3S)-2,3-dihydroxy-2,3-dihydrobenzoate, whereas its C-terminus contains the aryl-carrier protein domain. This domain is phosphopantetheinylated by EntD.

By removing entC and entA, the autonomy of E. coli for the production of 2,3 - Dihydroxybenzoate was removed. The growth medium was supplemented with 2,3 - Dihydroxybenzoate and similar molecules, to be taken up by the cell. Vanillic acid, gallic acid, caffeic acid, 5-Bromo-2,4-Dihydroxybenzoic acid and 3,4-Dihydroxy-5- methoxybenzoic acid as alternatives to 2,3 -Dihydroxybenzoate were also used as supplements used. Methods

Strains, plasmids and general growth media

Serratia plymuthica V4, the original producer of serratiochelins donated the serratiochelin biosynthetic genes (schCEBA, schFlF2F3, schG and sch ) and clusters.

Escherichia coli MG1655 (ID NC 000913.3) was the donor of the enterobactin genes entCEBA (IDs 945511, 947426, 946178 and 945284) and entD (ID 945194), while Vibrio cholerae El Tor A1552 (ID N16961) donated genes vibF (ID 2614958) and vibH (ID

2615318) (Table 7).

Table 7. List of bacterial strains and their genotype and/or phenotype, and respective source and of plasmids, their characteristics and source.

All E. coli strains were maintained on Lysogeny Broth (Miller, Lab Express) medium, supplemented with 15% agar and antibiotic when required. V. cholerae was maintained on agar plates prepared with Marine Broth 2216 (BD Diagnostics). Saccharomyces cerevisiae was maintained on Complete Supplement Mixture medium (CSM, Sunrise Science Products) or CSM-tryptophan dropout, for selection and maintenance of the yeast artificial chromosome (YAC, pYES-lL) carrying the assembled pathways.

Construction of an E. coli strain for heterologous expression of serratiochelins

The serratiochelins biosynthetic pathway shares homology with that of enterobactin. In order to guarantee that the synthetic pathway genes were indeed the ones involved in molecule biosynthesis - versus the homologous in the chromosome - genes entD, entF and entCEBA were removed from the chromosome of E. coli MG1655.

This was achieved using the Lambda Red Recombination system, having entD, entCEBA and entF been replaced with chloramphenicol, kanamycin and gentamicin- resistance cassettes, respectively.

The removal of the enterobactin biosynthetic genes disabled this organism's capacity to assemble this siderophore and grow in iron-limited conditions.

Construction of compressed synthetic pathways for the assembly of serratiochelin analogs From both clusters, only the genes actively involved in the bioassembly of serratiochelins were used (Figs. 4A and 4B). These genes were amplified by PCR using 5' - 3' primers that added the E. coli ribosomal binding site GAGGAGA to the region upstream of the translation initiation codon. In the particular case of genes schCEBA and schFlF2, these were amplified together having an RBS been only added upstream of schC and schFl . The native RBSs were maintained for schEBA and schF2. In the serratiochelins producer, S. plymuthica V4, the condensation-domain containing SchFO is not involved in the assembly of these molecules, despite its high similarity to EntF. Instead, the V. cholerae VibF homologs SchFl F2F3 are indeed involved.

Given that the serratiochelins biosynthetic pathway seems to descend from the enterobactin and vibriobactin pathways, whether the ancestral genes could assemble the molecule was also investigated as well. Thus, besides building the sch-based compressed synthetic pathway, their ancestral genes, from E. coli and V. cholera, were also used to assemble a homologous pathway.

The genes entD, entCEBA, vibF, vibH, schFl, schF2F3, schH, schG and schCEBA were PCR-amplified from E. coli MG1655 (ent genes), S. plymuthica V4 (sch genes) or V. cholerae El Tor A1552 (vib genes) with overhangs homologous to the genes to be located up and downstream in the compressed pathway. The genes at the beginning and end of each pathway were amplified with 5' primers that added a Spel restriction site and homology to the YAC pYES-lL (Life Technologies). The genes at the end of the pathways was amplified with 3' overhangs to a cos site (for large construct stability), which was amplified with a 3' primer with homology to the YAC backbone and also contained a Spel site

The amplicons were transformed, and assembled into full pathways, by S. cerevisiae using the Geneart ^® High Order Genetic Assembly kit (Life Technologies). The compressed pathways (Figs. 4A and 4B) were released from pYes-lL by digestion with the restriction enzyme Spel and each of the inserts was cloned into the same restriction site, added to pDSW204 by PCR.

The level of similarity between the homologous proteins was assessed utilizing the BLAST® blastp suite from the National Center for Biotechnology Information (NCBI).

Criteria for selection of exogenously-supplied precursors

The substrate limits for VibH to use several amine-containing small molecules as DHB acceptors were tested. The selection of precursors aimed to generate a wide diversity of molecules with a range of chemical properties, as a proof of principle. All polyamine precursors were purchased from Sigma- Aldrich, whereas the dipeptides were synthesized by Biomatik. All precursors selected contained at least two amine groups with at least one hydrogen atom. The product references, names and concentrations used are listed on Table 3.

Putrescine and spermidine are naturally occurring polyamines in E. coli. Though their molecular functions are yet to be fully understood, it has been found that they facilitate mRNA translation. Despite being synthesized endogenously, these compounds were supplied exogenously as well to enable the strain to uptake them and use them for incorporation into the unnatural NRPs. The endogenous levels of production were predicted to be too low and unavailable for the assembly of molecules, besides their natural physiological function.

In order to test whether the compressed and hybrid pathway could assemble serratiochelin and vibriobactin, 1,3 - diaminopropane (diaminopropane) and Bis(3- aminopropyl)amine (norspermidine) were added to the medium, respectively. To test whether other analogs could be generated, several different polyamines to be added to the growth medium were selected, e.g., molecules with up to 12 carbons and 4 amine groups (1,5- Diaminopentane, 1 ,4-Butanediamine dihydrochloride, N,N-Bis(3-aminopropyl)-l,4- diaminobutane, N-(3-Aminopropyl)-l,4-diaminobutane and N,N'-Bis(2-aminoethyl)-l,3- propanediamine), as they were the most similar to diaminopropane and norspermidine. Next, 2,2'-Thiobisacetamide was selected, as the two amides could potentially contribute for metal chelation, similarly to EDTA, as well as provide the amine groups necessary for the condensing reaction catalyzed by VibH. Siderophores are uptaken by cells through specialized transporters. Due to this easy access to the intracellular milieu, some antibiotic molecules have evolved to structurally resemble siderophores. Given the structure of the synthetic antibiotics sulfonamides, two were selected for tentatively generating sideromycins. The two sulfonamides selected, sulfaguanidine and p-aminobenzenesulfonamide, contained two amine groups that in theory VibH could use for condensing with the dihydroxybenzoyl and the threonine-containing intermediate. Next, efforts were made to enhance the fluorescence property of the analogs. In order to achieve this, we selected precursors that contained two benzene rings, in addition to the required amine groups (4,4'-Oxydianiline, 4,4'-Diaminodiphenylmethane and 1,5-Diaminonaphthalene). By providing it with fluorescent properties, it was hypothesized that these molecules could be, e.g., tracked during their export and import process across the membrane and inside the cell. They could also, for

2_ _| _

example, be used as a Fe sensor in the medium, as bacteria will only secrete the iron chelator in low soluble iron conditions.

Despite being a nonribosomal peptide, serratiochelin and other NRP siderophores incorporate natural amino acids in their structure.

Antimicrobial peptides as small as 12 amino acids long, such as KR-12, display strong activity against some bacteria. Thus, whether this pathway would be able to incorporate dipeptides in its structure was tested. Four dipeptides were selected, based on their polarity, hydrophobicity and structural conformation. It is well established how the most efficient antimicrobial peptides are positively charged, for interaction with cellular structures.

Thus the incorporation of dipeptides lysine-lysine (KK), lysine-arginine (KR), lysine- glutamine (KQ) and glutamine-asparagine (QN) were tested.

Production and purification of hybrid unnatural nonribosomal peptides

Minimal medium optimized for the production of serratiochelins was used for molecule production. It was composed of Na ₂ HP0 ₄ (5.96 g/L), K ₂ HP0 ₄ (3.0 g/L), NH ₄ C1 (1.0 g/L), NaCl (0.5 g/L), MgS0 ₄ (0.058 g/L ), C ₆ H ₁₂ 0 ₆ (5.0 g/L) and IPTG (1 mM), at pH 7.0.

The precursors were added to final concentrations of 0.05 to 10 mM (Table 3). The siderophore production and related machinery was further induced by adding the iron chelator 2,2'-bipyridyl (Sigma- Aldrich D216305) to a final concentration of 0.1 mM to the growth medium.

The cultures were grown for up to 7 days at 30 C with 250 rpm shaking, to an optical density (600 nm) of »0.500. After growth cells were spun down and the supernatant filter- sterilized. The cell-free supernatant was loaded onto Sep-Pak tC18 (5 g) Reversed-Phase columns (Waters ^® ). The columns were washed with water and the molecules eluted with 100% acetonitrile. Liquid chromatography and tandem mass-spectrometry (LC -MS/MS) sample analysis was performed at the Small Molecule Mass Spectrometry core facilities at Harvard

University. Two-hundred and fifty microliter-aliquots of each sample were injected into a high-resolution, accurate mass Q Exactive Plus Orbitrap, with positive ionization and mass scan ranging from 66.7 to 1000 m/z (resolution 70,000 FWHM) and operated over the course of 30 minutes at a flow rate of 3 mL/min, with a gradient of 10 ACN in H ₂ 0 to 100% ACN. Molecules displaying masses matching the expected one were fragmented (35,000 FWHM) and the respective fragmentation patterns were compared against those of the predicted structures.

The predicted structures for the natural and unnatural molecules potentially produced in-demand were drawn using ChemDraw ^® Professional 10 (Perkin Elmer). The prediction of the structure was performed based on previous knowledge on the NRPS-based assembly of serratiochelins. As the molecule assembly process had already been elucidated, it was possible to determine the mass and possible configurations of the new, unnatural

nonribosomal molecules.

In-silico prediction of biological activity

In silico tools for prediction of small molecule activity against an array of targets have matured over the course of the last few years. At the current state-of-the art, some of these can be used and reliable indicators of activity and as a first sieve through libraries containing thousands of molecules and pick out those most likely to bind to specific targets.

In order to get an insight into the potential activity of the molecules generated by our programmable compressed pathway, the corresponding SMILES in the online tools

Bioactivity Score Calculator (BSC) by Molinspiration, MolSoft and the Swiss Target Prediction (STP) were run.

BSC utilizes Bayesian statistics to compare the structure of active and inactive molecules, on a particular target, to identify new possibly active molecules. Instead of computing an overall value of drug-likeness, BSC focuses on 6 drug classes: GPRC ligands, ion channel blockers, nuclear receptor ligands and protease, kinase and enzyme inhibitors.

MolSoft is an algorithm used to predict the drug-likeness score of molecules, using a set of 5000 active molecules and 10000 inactive molecules.

STP was developed by the Swiss Institute of Bioinformatics and combines 2D and 3D measures of similarity to predict bioactivity against over 2000 targets in humans, horses, mice, rat and cows. The smaller, intermediate molecules were further analyzed for compliance with Lipinski's Rule of Five. This rule was developed in 1997 by Christopher Lipinski and colleagues, from Pfizer. It considers that an orally active drug has no more than a single violation of the following criteria: (1) 5 or less hydrogen bond donors, (2) 10 or less hydrogen bond acceptors, (3) less than 500 Da and (4) an octanol -water partition (logP) of 5 or less.

SEQUENCES

entA (SEQ ID NO: 1)

ATGGATTTCAGCGGTAAAAATGTCTGGGTAACCGGCGCAGGTAAAGGTATCGGCTAC GCCACGGC GCTGGCGTTTGTTGAGGCGGGAGCGAAAGTTACAGGTTTTGATCAAGCGTTCACTCAGGA GCAAT ATCCCTTTGCGACCGAAGTGATGGATGTTGCCGACGCTGCGCAGGTCGCGCAAGTGTGTC AGCGAC TGTTAGCTGAAACGGAGCGACTGGACGCGCTGGTCAATGCGGCGGGAATTTTACGCATGG GCGCG ACCGATCAGCTCAGTAAAGAGGACTGGCAGCAGACTTTTGCGGTTAACGTCGGCGGTGCG TTTAA CCTGTTCCAGCAAACCATGAACCAGTTTCGCCGTCAGCGGGGCGGGGCGATTGTCACTGT GGCGTC CGACGCCGCGCACACGCCGCGTATTGGCATGAGTGCTTATGGCGCATCGAAAGCGGCGCT GAAAA GCCTGGCGTTGAGCGTCGGGCTGGAACTGGCGGGTAGCGGCGTGCGCTGTAATGTGGTTT CGCCTG GCTCCACCGACACCGATATGCAACGCACGCTGTGGGTGAGCGATGACGCCGAAGAACAGC GTATT CGCGGCTTTGGCGAGCAGTTTAAACTCGGCATTCCGCTGGGGAAAATCGCCCGTCCACAA GAGAT CGCCAACACGATTTTGTTCCTCGCCTCTGACCTCGCCAGCCATATTACCCTACAGGATAT TGTGGTC GATGGCGGCTCAACGCTGGGGGCATAA entB (SEQ ID NO: 2)

ATGGCTATTCCAAAATTACAGGCTTACGCACTGCCGGAGTCTCACGATATTCCGCAG AATAAAGTT GACTGGGCCTTTGAACCGCAACGTGCCGCGTTGTTAATCCATGATATGCAGGACTATTTT GTCAGC TTCTGGGGCGAGAACTGCCCGATGATGGAGCAGGTGATCGCGAATATTGCTGCGCTGCGC GACTA CTGCAAACAGCACAATATCCCGGTTTATTACACCGCCCAGCCGAAAGAGCAGAGCGATGA AGATC GGGCGCTGTTGAATGATATGTGGGGGCCGGGCCTGACCCGCTCGCCGGAACAGCAAAAGG TGGTG GATCGCCTGACGCCAGATGCCGACGACACGGTGCTGGTGAAGTGGCGCTACAGCGCGTTT CATCG TTCTCCGCTGGAGCAAATGCTGAAAGAGAGTGGACGTAACCAGCTGATTATTACCGGGGT ATATG CCCACATTGGCTGTATGACCACCGCAACCGACGCATTTATGCGCGATATTAAACCGTTTA TGGTGG CGGATGCGCTGGCCGATTTCAGCCGTGACGAGCATTTGATGTCGCTGAAATATGTGGCCG GACGTT CTGGCCGGGTGGTGATGACTGAAGAATTACTGCCAGCACCTATCCCCGCCAGCAAAGCGG CGCTG CGTGAGGTGATCCTGCCGTTGCTGGACGAGTCCGATGAACCGTTCGATGACGACAACCTG ATCGAC TACGGTCTGGATTCGGTGCGCATGATGGCGCTGGCGGCGCGCTGGCGCAAAGTGCATGGT GATAT CGACTTTGTCATGCTGGCGAAAAACCCGACCATCGACGCCTGGTGGAAGCTACTCTCCCG CGAGGT GAAATAA

e«iC (SEQ ID NO: 3)

ATGGATACGTCACTGGCTGAGGAAGTACAGCAGACCATGGCAACACTTGCGCCCAATCGC TTTTTC TTTATGTCGCCGTACCGCAGTTTTACGACGTCAGGATGTTTCGCCCGCTTCGATGAACCG GCTGTG AACGGGGATTCGCCCGACAGTCCCTTCCAGCAAAAACTCGCCGCGCTGTTTGCCGATGCC AAAGC GCAGGGCATCAAAAATCCGGTGATGGTCGGGGCGATTCCCTTCGATCCACGTCAGCCTTC GTCGCT GTATATTCCTGAATCCTGGCAGTCGTTCTCCCGTCAGGAAAAACAAGCTTCCGCACGCCG TTTCAC CCGCAGCCAGTCGCTGAATGTGGTGGAACGCCAGGCAATTCCGGAGCAAACCACGTTTGA ACAGA TGGTTGCCCGCGCCGCCGCACTTACCGCCACGCCGCAGGTCGACAAAGTGGTGTTGTCAC GGTTGA TTGATATCACCACTGACGCCGCCATTGATAGTGGCGTATTGCTGGAACGGTTGATTGCGC AAAACC CGGTTAGTTACAACTTCCATGTTCCGCTGGCTGATGGTGGCGTCCTGCTGGGGGCCAGCC CGGAAC TGCTGCTACGTAAAGACGGCGAGCGTTTTAGCTCCATTCCGTTAGCCGGTTCCGCGCGTC GTCAGC CGGATGAAGTGCTCGATCGCGAAGCAGGTAATCGTCTGCTGGCGTCAGAAAAAGATCGCC ATGAA CATGAACTGGTGACTCAGGCGATGAAAGAGGTACTGCGCGAACGCAGTAGTGAGTTACAC GTTCC TTCTTCTCCACAGCTGATCACCACGCCGACGCTGTGGCATCTCGCAACTCCCTTTGAAGG TAAAGC GAATTCGCAAGAAAACGCACTGACTCTGGCCTGTCTGCTGCATCCGACCCCCGCGCTGAG CGGTTT CCCGCATCAGGCCGCGACCCAGGTTATTGCTGAACTGGAACCGTTCGACCGCGAACTGTT TGGCGG CATTGTGGGTTGGTGTGACAGCGAAGGTAACGGCGAATGGGTGGTGACCATCCGCTGCGC GAAGC TGCGGGAAAATCAGGTGCGTCTGTTTGCCGGAGCGGGGATTGTGCCTGCGTCGTCACCGT TGGGTG AGTGGCGCGAAACAGGCGTCAAACTTTCTACCATGTTGAACGTTTTTGGATTGCATTAA eni£> (SEQ ID NO: 4)

ATGAAAACTACGCATACCTCCCTCCCCTTTGCCGGACATACGCTGCATTTTGTTGAGTTC GATCCGG CGAATTTTTGTGAGCAGGATTTACTCTGGCTGCCGCACTACGCACAACTGCAACACGCTG GACGTA AACGTAAAACAGAGCATTTAGCCGGACGGATCGCTGCTGTTTATGCTTTGCGGGAATATG GCTATA AATGTGTGCCCGCAATCGGCGAGCTACGCCAACCTGTCTGGCCTGCGGAGGTATACGGCA GTATTA GCCACTGTGGGACTACGGCATTAGCCGTGGTATCTCGTCAACCGATTGGCATTGATATAG AAGAAA TTTTTTCTGTACAAACCGCAAGAGAATTGACAGACAACATTATTACACCAGCGGAACACG AGCGA CTCGCAGACTGCGGTTTAGCCTTTTCTCTGGCGCTGACACTGGCATTTTCCGCCAAAGAG AGCGCA TTTAAGGCAAGTGAGATCCAAACTGATGCAGGTTTTCTGGACTATCAGATAATTAGCTGG AATAAA CAGC AGGTC ATCATTCATCGTGAGAATGAGATGTTTGCTGTGCACTGGC AGATAAAAGAAAAGAT AGTCATAACGCTGTGCCAACACGATTAA

entE (SEQ ID NO: 5)

ATGAGCATTCCATTCACCCGCTGGCCGGAAGAGTTTGCCCGTCGCTATCGGGAAAAAGGC TACTGG CAGGATTTGCCGCTGACCGACATTCTGACGCGACATGCTGCGAGTGACAGCATCGCGGTT ATCGAC GGCGAGCGACAGTTGAGTTATCGGGAGCTGAATCAGGCGGCGGATAACCTCGCGTGTAGT TTACG CCGTCAGGGCATTAAACCTGGTGAAACCGCGCTGGTACAACTGGGTAACGTCGCTGAATT GTATAT TACCTTTTTCGCGCTGCTGAAACTGGGCGTTGCGCCGGTGCTGGCGTTGTTCAGCCATCA GCGTAG TGAACTGAACGCCTATGCCAGCCAGATTGAACCCGCATTGCTGATTGCCGATCGCCAACA TGCGCT GTTTAGCGGGGATGATTTCCTCAATACTTTCGTCACAGAACATTCCTCCATTCGCGTGGT GCAACTG CTCAACGACAGCGGTGAGCATAACTTGCAGGATGCGATTAACCATCCGGCTGAGGATTTT ACTGCC ACGCCATCACCTGCTGATGAAGTGGCCTATTTCCAGCTTTCCGGTGGCACCACCGGCACA CCGAAA CTGATCCCGCGCACTCATAACGACTACTACTACAGCGTGCGTCGTAGCGTCGAGATTTGT CAGTTC ACACAACAGACACGCTACCTGTGCGCGATCCCGGCGGCTCATAACTACGCCATGAGTTCG CCAGG ATCGCTGGGCGTCTTTCTTGCCGGAGGAACGGTTGTTCTGGCGGCCGATCCCAGCGCCAC GCTCTG TTTCCCATTGATTGAAAAACATCAGGTTAACGTTACCGCGCTGGTGCCACCCGCAGTCAG CCTGTG GTTGCAGGCGCTGATCGAAGGCGAAAGCCGGGCGCAGCTTGCCTCGCTGAAACTGTTACA GGTCG GCGGCGCACGTCTTTCTGCCACCCTTGCGGCGCGTATTCCCGCTGAGATTGGCTGTCAGT TGCAGC AGGTGTTTGGCATGGCGGAAGGGCTGGTGAACTACACCCGACTTGATGATAGCGCGGAGA AAATT ATCCATACCCAGGGTTACCCAATGTGTCCGGATGACGAAGTATGGGTTGCCGATGCCGAA GGAAA TCCACTGCCGCAAGGGGAAGTCGGACGCCTGATGACGCGCGGGCCGTACACCTTCCGCGG CTATT ACAAAAGTCCACAGCACAATGCCAGCGCCTTTGATGCCAACGGTTTTTACTGTTCCGGCG ATCTGA TCTCTATTGATCCAGAGGGTTACATCACCGTGCAGGGGCGCGAGAAAGATCAGATTAACC GTGGC GGCGAGAAGATCGCTGCCGAAGAGATCGAAAACCTGCTGCTGCGCCACCCGGCGGTGATC TACGC CGCACTGGTGAGCATGGAAGATGAGCTGATGGGCGAAAAAAGCTGCGCTTATCTGGTGGT AAAAG AGCCGCTGCGCGCGGTGCAGGTGCGTCGTTTCCTGCGTGAACAGGGTATTGCCGAATTTA AATTAC CGGATCGCGTGGAGTGTGTGGATTCACTTCCGCTGACGGCGGTCGGGAAAGTCGATAAAA AACAA TTACGTCAGTGGCTGGCGTCACGCGCATCAGCCTGA

v¾ (SEQ ID NO: 6)

ATGAAAGAAATGACAGCAATGCAAGCGGCTTATTGGCTAGGGCGTCAACACGACTGCTTG CTCGA TGGTGTGGCCGCGCATCTCTACGCCGAATTTGATGGTCAAGCATTAAATCGACAGGCGCT AACGGA AGCGGTACGTGCGCTGTACGCGAAACATCCCATGTTACGTTTAGCGATCACCAAGGATGG ACAGC AGAAGATCTTGCCGCTCTCAACTTTCCATCAGCTTAAAGTGGATGACCTATCACAATGGA AGCCAG ATGAGGTGGAGTCTTTTGTGCATACTAAGCGCCAGCGAATGACCCACCAGATGCTCGATT TAACTC AGGGAAACCCGATTGAGATCAGCTTGACTCTGTTGCCTGAGGGCAAACACCGATTACACA TTGAT GCGGATATGATTGCGTGTGATGCACAAAGTTTTCGGCTGTTGGTGGACGATCTCACCTCA CTCTAT TTAGAGGCCATAGAGCATCGATTAGAGATCATTGAGTCTGATGTGGTGACTTTTTTCCAA TATCTT GATGCTCAGCAAGCCGATCGTGCTTTGGCAAAACGCAAAGAGGTGGATAAGAAATGGTGG CAAGA GCGTCTTGCGACGATTCCCGCCGAGCCAAGTTTGCCTTATCAACCCGTACCAACCGACGC CGTTAG CGCAAACAGCCAAAGGTTTGCACACTGGTTCACTCCAGTGGAGCGCAAAGGGTTGGCGGA GGTTG CGCGGCAACATCATCTCACACTGACCCAACTGACATTGGCACTCTTTTCACAAGTGATTG CCAATG CTTGCCAAGAGAGACAATTTCGACTCAATGTACCTACATTCCATCGCGGCAATCGCTCTT TGGATA TTGAACACACGATCGGCGATTTTTCCAATTTGCTGATTTTTAGCGCCGATGTGGGAACGA CTCAGA CCCTGTTAAGCCTGTGTCAGCAAACGGCTAACCAACTGCATCAATTACTTCGCCATGAAA G

CTATTCAGGGGTTAGCGTGATGCGCGATTTATCGCGGAAACAGGGTGGGGTGCAGCG TTCACCGA TAGTCTTTACCTCAGGCATGGAAATGCGAGACGAGGAGATCTTTTCCGATCATGTCACTC AGCATT TAGGGCGTATGAATTGGGTGATTTCGCAGGGAGCGCAGGTGACTCTCGATGCGCAAATTG CTCCG GCTTATGAAGGGATTTTGCTCAATTGGGATGTGCGAATGGAAAACTTCGCTGATAAGGAC ATCACA GCGCTTTTCGCGCATTACGTTGATTTGATCCGCTGTGTGGCGCTGCATCCTGAGATGATG CAGCAA AGCGTACAACAGATTGACGCGCAACTCGGTTATGCTCGCCGCGAGTCGATTCAGGAGATG CCACT GACCCCTTTACAACAAGCTTACTTGCTAGGTCGCTCAACCCAAATTGCCTTAGGCGGCGT CGCGAT GCATGAGTTTCGTGAGTATCGAGGTCACATCGACACTCAGTCACTGCATAGCCGTCTGCT CTATCT CGTTGAGCATATTCCTGCCTTGAGAACTCGCATTGATCAAGAGAAATGGATTCAGTGGGT CTCTCC TTGCATAGCGTTAAATTGGCAAGCCATCGATTTACAGCATCTTTCCCGCGAACAAGCCTT ATTAGC GGTTGAGCCAGTCAGGCAGCAATATCAGCAGCGGATGCATGATTTAACGCGCTCGCCCTG GCAAA TCTGTGTGGTGCAACTGCCAATAGAAGAGCAAGAGGAGTTCAGCTCCATCGTATTGACCA GCTTTG ATGCTTTGATTGTCGATGGTCGCACCCATGCACTCATTCTTGCTGCACTACTAGGAAGCG AAGAAC CCGATATAACTCAGGTGGTGCAAAATGCTCGTGATACTCAGTCCATCTCGCCGCAATTCG CCTCCA AAAAAGCGCAAGATGAAGCGTATTGGAAAAGCAAACTTCATCCTGATTGCCCGCCGCCAG CACTG CCTTGGAAACAAGCATTAGAGACGATCACTACGTCTCGTTATGCTCGTGAAAGTTTGCAA ATACCC AAAGAGAGTGTCGGTAAGCTCAATCGATGCGGCATTGAAAATGGCTTATTTCTCAATTCC TTGCTG ACAGCAACCATCTTGGACGTGCTCTCCTATTGGACAACCGAAATCGGGATGCGAGTGGGT TTTCCG GTATTAATTCCAAGCAGTAATGCGATTGATGGCAATGAGTCATCCTTTGTGATCTTAGAG CATGAA AAATCCACCCTGAGTTTGCTGAGCCAAGCGAGCAAGCTACAGCGTGAGATGCTAGAGGCT CTTGA GCATCTTGCTTTTTCTGGCGTCGATCTTAACCGTCTGTTGATGAATCAAGCGCCGCAAGC TCTGGTG CTGCCGGTTGTGTTAACCAATGGTTTGTCTTGGAAAACCCTGAATCCAGAGGACGCTGTC ACTCTG TTCGATGGGGTAACGCAAACTCCGCAGTGGCTTTAGATATTCGCTTAACTTACGACGAGC AGAAAA ACCTCATCATCAGCTTTGATTATGCTTTAGCGGTTTTAGAGACTGAGCTTATCCGCGAGA TGTTAAG TGCCTTACATCATCGCTTGAGTCAGATCACTTCGTCAGCATCATTGGCCGCGCCGCTTGA GCCATGT ATCGACCTTTCACACTACCGTTTTAATAGCGATGAGTCGGCCAGTCACGATTGCGATTTT CTTGCCA AGTTAGCGCAGCAATTGTTTGTCCGCACAGAGGATAAAACCGCTGTGATCTGCGGTGAAC AAACC CTTTCTTATGCTCAATTGGGTGAGCAGGTTCAGCGCGTGATGTGGCAGCTCAAAGCGCGA GGGTTG ACCACAGGAAATGTGCTCGCCATTTGCTTGCCACGCAGTGTTGAACATATCGTCATCAGT TTGGCT TCTGCACTTTCGGGGATTATCTGGGTACCTATTGATGCTGCGTCTCCCAAAGAGCGTCTG AATTACC TTTTAGAAAATTGTCACGCCGATCTGGTGGTGATGGATAAGCCCTGTGAGTTTGGCAATG TGATCG CTTTTGATGCCTTGATTGAGCCTGTCTTATTCGCCGATGGAGTACCTGACGTTACACCAC TCGATCA ATTAAGCCGATTAAGCCAAAGCCAACAAACGGCTTATTATCTTTATACCTCTGGCACTAC GGGCAA ACCTAAGTGCGTGGTGGTCAATAATCAGGCGACCTCGAATGTGATTGGTCAAACTGGCCA AGCGT GGCATCTCACCAGTGAGGATGTGGTGATGTCAGTGACGCCATTGCATCATGATATGTCGG TGTTTG ATCTCTTTGCCACCTTGAGCTTCGGAGCGACGTTAGTTCTTCCCGCTGGGCATGAAGAAA AAGATG CGTTGCAATGGAATCGCTTAATTGAGCGGCATCAAGTGACGATTTGGGTCTCCGTCCCCG CTATCT TGGAGATGTTGCTCTCTTGCACTCAAGCAGGGCAATTACACTCACTTCGTCTGATTGCCC AAGGGG GGGATTACATCAAACCTGCCACCATCGCCCAATTACGCGCTGGTTCAAATCCGCCGCGGT TGATCT CTTTGGGCGGTCCTACCGAGACGACGATTTGGAGCATCTGGCATGAGCTGACGGCAGATG ATGTC AGCGTGATCCCATATGGTCGGCCGTTGGCGGGAAACCGTTATTTCATTATGGATGAGATA CAACGC CATGTACCGCAAGGTGTGGTCGGCCGTATCTTCACCTCTGGGGTCAATTTAGCGCAAGGT TATCTT GAAGACGGCGAACTGAAACAAACCGATTTTGTCACCGTATTGGATGAGCATGGCCACCCT GTGCG CGCGTTTCGAACGGGCGATCAAGGCTACTACCGCGCGGACGGCAATATCATTTTTGCTAG CCGCAT CAATGGTTATGTGAAAGTTCGTGGGGTACGAGTGTCACTGCCGGATATCGAAAAGCAGCT GCAAA CTCATCCGGCGCTCGCGAGCGTGGTGGTGGTCGATTATGCGGATACTAATGGAGATACCG CTTTAG CCGCATTATTTAGCGTTAAACCTCAGCAATCAGCATCGAGTCAGGCGTTACGAGAGTTTG CCAAAC AATCGCTACCGTCTTCGCATATTCCGAGTCGGTTTATTGCTTTAGAGGCTTTACCGCTTT CTGCGAA TGGTAAAGTGGATCGCAAACAGTGCCAAGCGCATGTTCAGCGACAATCGATCTCTGTTGA ACCAG TAGGTCAACCGAATCAAAGTCAACCTCTCTCACCACCTACCTTGTCATCGGCTTCCTTGA GTGAGTT TGAACAGCGAGTCTGGCGACAGCATCAACGACATGGTGACGGACGCAATCAATATGCGAC CGCTT ATCGATTGAGCGGAAAAGTGAATATCGCGCGCTTAATCACAGCGCTCAGTCAGTTGCCCA ATCATT TTCCAGTGTTAAACCGACGCTATGTGCTTGATGAGGCATCCGGCTTAACCTTGTACTCGG CGAAGC CAACACCGCTTGAGATCCATTTTGAGCATGTTGAGTCAATGGATGAGGCGTTTGAACATC TTGTGC GATGGCAAAATCAGCCGATGGATTTAGCCAAGCAAGCCACACTAAGTTTTTGCTTGTTGA GTTTGG GGAGCGAAGAGCGAGTGCTTGGGGTGATAAGCCATCAAATTATCAGCGAGCAATGGGATT GGCGG CGCGTATTTGAGTGCGTAACCAATGGGTATAACCAAATCGCCTGTGACATCGATCCTGTG ATGGAG GGGGAGGATCTTTCCCTCGGCTTTACCCCTGTGATGCCGCAAACGCCATTATCCCAAGCG TTGTTG CCGTGGTTACAGCCTGCGAGCCACGCTACATGGATTGAGCAGAGCTTGCCGAGCGCAAAT AGCGC CATCAATACGCTTTGCAGTCGTAAATATCGGATTGCCCTACACAGCAGCGTACTGGCGGA GCATGG TTTAGCGCAGCCGGATAAGCAGGAAGTGCTTGCACTGATCGCCGCGTTATTTGCTCGCTA CCTTGC CGCAAGTGCGAAACTTGGCCAATTTGAGCTTTACGTTCCGCATGATGTGGAGCGCAAAAC GCGTG AGTTGAATGGTTCTATGATTGAAAGCCAGTTGGTGCATATCGCCCTTAAAGGCTTTGAGC GTCCGC TTTCGCAACTGAGCCAAGAGATCTTAGACGCTATGCGCCAGCCGTTAACGGGGGGAGTCG CTAGC GAAACTCACGCCTCTGCCGCAGCCTTGGTGACTTGGTTAGTGGATCCTTCAGTGCATCTA CAATTG GAGGGCTTGCGCTGCGAAAAATTACTTTTTGCAGCCATGCACCCCAAATTTGAAGTCGCC TTGGGG GTTGGGCTTAATTGCCAAGGTGCCCTAGTACTTGAGCTGGCGTTAGATGCCACGGTTTCC CCCCAT GTCGGAGCCTACTTGCTTGAGCAATTTGTCGCTGCGATAGGTGGGCGAACAATGCCTTCT TCGACA ACCTCATCCTCTCATGTCTCAGCCATTGAGTTGACGTCAAACAATCCGCCGACCTCAAAC AATGCG CATGCGGCGTTGGCAGAGTCTGAGCTTGAATTGAGCGCTGTAAATTCCTCTGCGGTTGAA GAGTGC ATTTTGGCTGAGTTTCGCTCAGCACTTGGTGTTGCCGAGATGACGGCAGAGGATGATTTC TTTGATT TTGGCGGACATTCCCTGATCGCAACGCGAGTCATTGGTCGCTTGTTGAGCGAGCAAGGTA TTGAAC TGCACATCAATGATATGTTTAGTTTCCCTAATGCCAAGCAACTGGCGCAGCAAGCCGTAC TTCACC GTAAACCCACCAGCACATCATTCGCAGTGAGTGAGGTGGTCGAGTCGTCCAAAGCGCCTT TGTCGT TAGCACAAGCCTCTTTATGGAAGGCGATGAGCAAATACGCCAAGTTTGGTTTGACCCACA TCTTCA ATCTTCCGTTTGCGCTTAAATTTTTAGATGAGGTCAATGAGCAAGCATTTGGTGAGGCTT TTCACTG GCTTTTACTGCGTCATGCTGGGTTACGAACCCACTTTGGCCTAGAAGATGGGCAGCCTTA CCAGCA CGTGATCGCAGCCAGCAACATAGAACATTACCAGTGGTTCTGGACGTCTAAAGATAACGC CGCCC AATCCGTGGCCAGACTACTGGCGCAAGAGGCCGAGCACACCTTTGACCTGAGCCAAGAAT TGCCA CTTCGACTCAATTTCGTACGTGATGAACAGACGGGGACGCAATATCTTTCACTTCTGTTC CACCAT ATTGTGTTGGATGAGTGGTCGATCAATATTTTGATGGATGAATTAGCACAGGTGTATCAA CACAGT GTGCAAGGTACTCGTCCACAGTGGCAAACGGAACCCTTACCTTTCCATGAGTTTGCTCGC AAGCAG CGTTCTTCGGCATTCAATCAAACGCATCTTAACTATTGGTTAACCAAGTTTGCCGGTGTG CCTTGGG CACAGCCTCTGTTTGCTGCGGATCATCCTTTAAGTAACAGCACTGGCGTCGATCTTGGAG AAGGCG GCTGGGTCGAAATAAAATTGCCGAAATCAACCATGGTATCGCTGTATCAATTGGCTAAAG CACGC CATGCTTCGTTGTTTAACGTGATGTATGCGGCGATTTCTGCGTCGGTACATTGTCTCGGC GCACCAG AAAAGCTGCTGGTTGGCACACCTGCTTCCGGTCGGCTGGATGCCGAGTTTTTCGACACAG TCGGTT ATTTCACTACCATGGGCGTTCAGTTGGTGGATTTCACGAAAGTACAAACGGTGTGGCAAC TCATCG AGCAAGTGAAAAACAGCATTAACCAGTCGATGCCATACACAGATATTCCGATTGACCTGA TTGAA GAAGGGCTCAAAGGTGTTGAGCATGAGACGGAAGGTCACATGTTTGAGGTCTTTATACAA CTGCA TGCAAAAAATAAGCTGCATGGAGAGCTACTGTTGACAGAAGGGCATGCGATCCGCTTCCA ACAAG TCGATCCCGATAAAAGTGAATCCGGCTTAGGTTTACAGTTTGAAATTTTAGAAGAGAGGA TCGAGC AAAAGCAGACGTTGAGAGTCATGATGAGTTATATGTCGAAACACTATAGCCCAGCTCAAG TCGCT CTGTTGACCAAGGTAACCAGTGGCATGTTTGAGCGGTTTTCCGATTGTATTGCACAAGAT ATTGCA CTGCCGACGCTGAAAAAGCAGGTGAGGCAGCTTGAAGATGAAGCTTGTCGCTCTCCGTCC ATGGG GTAG

v¾# (SEQ ID NO: 7)

GTGTCTATGTTATTGGCTCAAAAGCCTTTTTGGCAGCGCCATCTGGCTTATCCTCACATT AATCTCG

ACACCGTTGCCCATTCGCTACGCCTGACAGGGCCTTTAGATACCACACTTTTGCTGC GCGCATTGC

ATTTAACCGTCAGTGAGATAGATCTGTTCCGTGCTCGCTTTTCTGCGCAAGGTGAGC TGTATTGGC ACCCATTTTCTCCGCCTATCGACTATCAAGACCTCAGCATCCACCTTGAAGCAGAACCTT TAGCTTG GCGGCAAATAGAGCAGGATTTACAGCGCTCAAGCACACTGATTGACGCGCCAATAACGAG CCATC AAGTGTATCGTTTGTCACACAGTGAGCACCTTATCTATACCCGTGCGCACCACATTGTGC TGGACG GTTATGGCATGATGCTGTTTGAGCAGCGCCTCAGCCAACATTACCAATCCTTGCTCAGTG GGCAGA CGCCAACTGCCGCGTTTAAACCTTATCAATCCTATCTGGAAGAAGAGGCGGCTTATCTTA CCAGCC ATCGCTACTGGCAAGATAAGCAGTTTTGGCAAGGCTATTTACGCGAAGCTCCCGACTTAA CGCTCA CCTCTGCAACCTATGATCCTCAACTTAGTCATGCCGTGAGCCTCTCTTACACACTCAATA GCCAACT CAACCATTTGTTACTCAAGCTCGCTAACGCGAATCAGATTGGCTGGCCTGATGCCTTAGT CGCGCT GTGCGCGCTCTATTTGGAATCGGCTGAACCTGATGCGCCTTGGCTGTGGCTGCCCTTTAT GAACCG ATGGGGCAGCGTGGCCGCTAATGTGCCCGGTTTAATGGTTAACTCGCTGCCCTTGCTGCG TCTTTCT GCGCAGCAAACCTCGCTCGGCAACTACCTTAAACAGTCAGGGCAAGCGATACGCTCACTC TATTTG CATGGCCGCTATCGAATTGAACAAATCGAACAAGATCAAGGGCTAAATGCTGAGCAAAGT TATTT TATGAGCCCATTTATCAATATTTTGCCTTTTGAATCCCCACACTTTGCTGATTGCCAGAC TGAGCTC AAGGTGCTGGCTTCAGGCTCTGCAGAAGGGATTAATTTTACCTTTCGTGGCTCACCCCAG CATGAA CTCTGTCTAGACATCACCGCAGATTTAGCCAGCTACCCGCAATCCCACTGGCAAAGTCAT TGCGAG CGGTTTCCACGTTTTTTCGAGCAGTTACTCGCCCGTTTCCAACAGGTGGAGCAAGATGTT GCACGC CTACTCGCAGAGCCTGCGGCATTGGCAGCCACCACCTCCACACGGGCTATCGCCAGTTGA

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While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as

"comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.