US20190002936A1 - Transgenic microorganisms and synthesis of piperazic acid, piperazic acid containing products, and derivatives thereof - Google Patents

Transgenic microorganisms and synthesis of piperazic acid, piperazic acid containing products, and derivatives thereof Download PDF

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US20190002936A1
US20190002936A1 US16/024,077 US201816024077A US2019002936A1 US 20190002936 A1 US20190002936 A1 US 20190002936A1 US 201816024077 A US201816024077 A US 201816024077A US 2019002936 A1 US2019002936 A1 US 2019002936A1
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streptomyces
actinomadura
piz
ornithine
seq
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Joshua Van Dyke-Blodgett
Yifei Hu
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Washington University in St Louis WUSTL
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/1381—L-Ornithine N5-monooxygenase (1.14.13.B10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01012—Ornithine cyclodeaminase (4.3.1.12)

Definitions

  • Sequence Listing which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention.
  • the subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
  • the present disclosure generally relates to the synthesis of piperazic acid.
  • Piperazic acid is a nonproteinogenic amino acid that contains a characteristic and biochemically unusual N—N bond. Piz is a proline structural mimic, and Piz-containing compounds are of significant interest for drug discovery. Piz itself is not bioactive, but peptidic compounds incorporating Piz as a building block include antibacterial, antiviral, immunomodulatory, and anticancer drug leads. Intriguingly, all naturally-occurring Piz containing compounds discovered thus far have been bioactive.
  • the present disclosure provides for a transgenic microorganism for the synthesis of L-piperazic acid and derivatives thereof and additional biosynthetic processes for the production of L-piperazic acid and derivatives thereof.
  • the present disclosure is directed to methods of producing piperazic acid, especially L-piperazic acid and derivatives thereof.
  • Synthesis of enantiopure L-Piz has been elusive and expensive.
  • the methods and transgenic organisms as described herein have overcome many of the challenges currently faced regarding the synthesis of enantiopure L-Piz.
  • L-Piz and derivatives thereof can be used as a starting material for a large range of bioactive molecules, including many currently known therapeutics and can be isotopically labeled for use in drug discovery analyses and imaging modalities.
  • the new synthetic routes can give access to isotope (e.g., 15 N, 13 C, 2 H) or radioisotopically-labeled piperazic acid for which no synthetic pathways are currently reported.
  • One aspect of the present disclosure includes transgenic microorganisms (e.g., bacteria) engineered to accumulate piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.
  • transgenic microorganisms e.g., bacteria
  • Piperazic acid (Piz)-containing product e.g., a piperazic acid (Piz)-containing product.
  • Another aspect of the present disclosure includes biochemical and biological methods for producing piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.
  • compositions and methods of using isotopically labeled piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product includes compositions and methods of using isotopically labeled piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.
  • Another aspect of the present disclosure provides for a method for preparing a piperazic acid (Piz)-containing product.
  • the method comprises: (i) providing N 5 —OH-Ornithine or derivative thereof; (ii) providing a suitable enzyme comprising a N 5 —OH Ornithine cyclase/dehydratase; or (iii) optionally, buffer salts, a NADPH cofactor, Fe +2 salts, and a catalytic Flavin Adenine Dinucleotide (FAD) cofactor.
  • the method further comprises: (i) providing an ornithine or a derivative thereof; or (ii) providing a suitable enzyme comprising an ornithine N 5 hydroxylase.
  • the (i) the N 5 —OH-Ornithine or derivative thereof is an enantiopure L-Ornithine or derivative thereof;
  • the enzyme comprising N 5 —OH Ornithine cyclase/dehydratase is a L-N 5 —OH Ornithine cyclase/dehydratase or a PzbB enzyme; or
  • the enzyme comprising ornithine N 5 hydroxylase is an L-ornithine N 5 —OHase or a PzbA enzyme.
  • the method is carried out in the absence of O 2 , substantially no O 2 , or in the presence of low O 2 .
  • the method comprises a coupled enzyme assay.
  • the piperazic acid (Piz)-containing product comprises a compound of formula:
  • R 5 is a hydrogen, an alkyl, a piperazic acid, an acetyl, or a carboxyl protecting group
  • each R 1 and R 2 are independently selected from hydrogen or an amino protecting group, wherein R 1 and R 2 may be taken together to form a fused bicyclic or tricyclic amino protecting group
  • each R 3 and R 4 are independently selected from a hydrogen, a halo (e.g., a chloro, a fluoro, a bromo, a iodo), or a hydroxyl.
  • R 1 and R 2 are not simultaneously hydrogen.
  • the piperazic acid (Piz)-containing product is used as a starting material in a synthetic method of making a bioactive Piz-containing composition selected from the group consisting of: (i) an antibacterial agent, an antibiotic agent, an antitumor agent, an antiviral agent, an immunomodulatory agent, or an anti-inflammatory agent; (ii) a molecular probe, anticancer drug, or drug lead; (ii) a metalloprotease inhibitor, a caspase inhibitor, an angiotensin converting enzyme (ACE) inhibitor, an inflammatory peptide C5a antagonist, an oxytocin receptor antagonist, or a matylastin type-IV collagenase inhibitor; (iii) a dehydropiperazic acid; a chloropiperazic acid; a hydroxypiperazic acid; a monamycin, an aurantimycin, an antrimycin, an azinothricin, a luzopeptin, a kettapeptin, a qui
  • the transgenic microorganism comprises, as operably associated components in the 5′ to 3′ direction of transcription: (I)(a) a promoter functional in the microorganism; (b)(i) a first polynucleotide comprising a nucleotide sequence encoding a first polypeptide having a L-Ornithine N 5 hydroxylase activity; (ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide having a L-Ornithine N 5 cyclase activity or L-Ornithine N 5 dehydratase activity; or (iii) a third polynucleotide comprising a nucleotide sequence encoding a third polypeptide having a L-Ornithine N 5 hydroxylase activity and a L-Ornithine N 5
  • the microorganism comprises (a)(i) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 1-SEQ ID NO: 81 or SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N 5 hydroxylase activity; or (ii) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 82-SEQ ID NO: 166 or SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N 5 cyclase activity and L-Ornithine N 5 dehydratase activity; or (b) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N 5 hydroxylase activity, L-Ornithine
  • the microorganism comprises: (i) a PzbA ortholog with at least about 25% identity to SEQ ID NO: 1-SEQ ID NO: 81 or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbA activity to produce a piperazic acid (Piz)-containing product; (ii) a PzbB ortholog with at least about 25% identity to SEQ ID NO: 82-SEQ ID NO: 166 or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbB activity to produce a piperazic acid (Piz)-containing product; or (iii) a PzbAB ortholog with at least about 25% identity to or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbA and PzbB activity to produce a piperazic acid (Piz)-containing product.
  • the microorganism is an Actinobacteria selected from the group consisting of Streptomyces, Corynebacterium, Kutzneria , and Actinomadura ; is a heterologous population of microorganisms; is an Actinobacteria (optionally, an actinomycete); or is selected from the group consisting of Streptomyces lividans or Corynebacterium glutamicum , optionally carrying one or more copies of a native or non-native pzbA and optionally carrying one or more copies of pzbB.
  • the transgenic microorganism overproduces L-Ornithine; the pzbA or the pzbB are cloned from a sanglifehrin biosynthetic locus of Streptomyces flaveolus ; or a piperazic acid (Piz)-containing product accumulates within the microorganism.
  • Another aspect of the present disclosure provides for a method for producing a piperazic acid (Piz)-containing product.
  • the method comprises: (i) providing a transgenic microorganism capable of accumulating a piperazic acid (Piz)-containing product; (ii) cultivating the microorganism; or (iii) isolating accumulated piperazic acid (Piz)-containing product.
  • the method comprises providing a transgenic microorganism and providing a feedstock, wherein the transgenic microorganism comprises at least one copy of pzbA and at least one copy of pzbB under a constitutive promoter; or the at least one pzbA is optionally a native copy.
  • the transgenic microorganism is (i) a heterologous population of microorganisms; (ii) an Actinobacteria (optionally, an actinomycete); or (ii) selected from the group consisting of Streptomyces lividans or Corynebacterium glutamicum , optionally carrying one or more copies of a native or non-native pzbA and optionally carrying one or more copies of pzbB.
  • the pzbA or pzbB are cloned from a sanglifehrin biosynthetic locus of Streptomyces flaveolus ; or a piperazic acid (Piz)-containing product accumulates within the microorganism.
  • the method is carried out in the absence of O 2 , substantially no O 2 , or in the presence of low O 2 .
  • the piperazic acid (Piz)-containing product comprises a compound of formula:
  • R 5 is a hydrogen, an alkyl, a piperazic acid, an acetyl, or a carboxyl protecting group; each R 1 and R 2 are independently selected from hydrogen or an amino protecting group, wherein R 1 and R 2 may be taken together to form a fused bicyclic or tricyclic amino protecting group; or each R 3 and R 4 are independently selected from a hydrogen, a halo (e.g., a chloro, a fluoro, a bromo, a iodo), or hydroxyl. In some embodiments, R 1 and R 2 are not simultaneously hydrogen.
  • the piperazic acid (Piz)-containing product is used as a starting material in the synthesis of a bioactive Piz-containing composition selected from the group consisting of: (i) an antibacterial agent, an antibiotic agent, an antitumor agent, an antiviral agent, an immunomodulatory agent, or an anti-inflammatory agent; (ii) a molecular probe, anticancer drug, or drug lead; (iii) a metalloprotease inhibitor, a caspase inhibitor, an angiotensin converting enzyme (ACE) inhibitor, an inflammatory peptide C5a antagonist, an oxytocin receptor antagonist, or a matylastin type-IV collagenase inhibitor; (iv) a dehydropiperazic acid; a chloropiperazic acid; a hydroxypiperazic acid; a monamycin, an aurantimycin, an antrimycin, an azinothricin, a luzopeptin, a kettapeptin, a quinoxa
  • compositions comprising a radiolabeled piperazic acid-containing product or a pharmaceutically acceptable salt, solvate, or polymorph thereof, including all tautomers and stereoisomers thereof, optionally in combination with one or more pharmaceutically acceptable excipients.
  • Another aspect of the present disclosure provides for a method comprising a process for preparation of a radiolabeled piperazic acid-containing product comprising: (i) providing a radiolabeled N 5 —OH-Ornithine or derivative thereof; (ii) providing a suitable N 5 —OH Ornithine cyclase/dehydratase; or (iii) optionally, buffer salts, a NADPH cofactor, Fe +2 salts, and a catalytic Flavin Adenine Dinucleotide (FAD) cofactor.
  • FAD Flavin Adenine Dinucleotide
  • the method comprises: (i) providing a radiolabeled ornithine or a derivative thereof; or (ii) providing a suitable ornithine N 5 hydroxylase.
  • the radiolabeled N 5 —OH-Ornithine or derivative thereof is an enantiopure radiolabeled L-Ornithine or derivative thereof;
  • the enzyme comprising N 5 —OH Ornithine cyclase/dehydratase is L-N 5 —OH Ornithine cyclase/dehydratase or the enzyme PzbB, or
  • the enzyme comprising ornithine N 5 hydroxylase is a L-ornithine N 5 —OHase or the enzyme PzbA.
  • the method comprises a coupled enzyme assay.
  • Another aspect of the present disclosure provides for a method of detecting radiolabeled piperazic acid-containing product.
  • the method comprises: (i) providing a microorganism; (ii) contacting the microorganism with a radiolabeled piperazic acid-containing product; or (iii) detecting a radiolabeled natural product, a radiolabeled biocatalysis product, or a radiolabeled metabolite.
  • radiolabeled piperazic acid-containing product is: (i) labeled for use as a biologically active molecular probe as a drug discovery agent; or (ii) labeled for use in detecting a natural product drug lead compound.
  • a piperazic acid (Piz)-containing product comprises: (i) a single radiolabel; (ii) a radiolabel selected from the group consisting of 2 H (D or deuterium), 3 H (T or tritium), 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, and 131 I; (iii) a radiolabel selected from the group consisting of 15 N, 13 C, and 2 H; or (iv) a radiolabeled L-Piz or L-Piz derivative.
  • the composition can be used in mass spectrometry, gamma imaging, magnetic resonance imaging, magnetic resonance spectroscopy, or fluorescence spectroscopy.
  • FIG. 1 is a series of chemical structures showing examples of piperazic acid (Piz) family natural products.
  • Piz and modified Piz dehydropiperazic, chloropiperazic and hydroxypiperazic acid
  • red All of these molecules are bioactive, with sanglifehrin (top left) under consideration as an immunosuppressant and Hepatitis-C antiviral.
  • the small molecule in the center (Sch 382583) is a member of an emerging group of Piz containing metalloprotease inhibitors with clinical relevance as metastatic cancer and antibacterial antibiotic leads. All of these molecules are currently thought to be exclusively produced by actinobacteria.
  • Piz and modified Piz dehydropiperazic, chloropiperazic and hydroxypiperazic acid
  • FIG. 2 shows orthologs of both PzbA (yellow) and PzbB (red) are found within biosynthetic gene clusters for known Piz-containing antibiotics. As these clusters encode molecules that are structurally dissimilar except for the incorporation of Piz, parsimony suggests both pzbA and pzbB (previously unrecognized) are involved in Piz biosynthesis.
  • FIG. 3 shows HPLC-ESI-MS detection of products and substrates with assay time points at time 0 min, 15 min, and 30 min showing the consumption of L-Orn, accumulation of the known intermediate N 5 —OH-Orn, and the concomitant formation of Piz.
  • a coupled enzymatic reaction containing purified PzbA, PzbB buffer salts, NADPH cofactor, Fe +2 salts, and catalytic FAD (Flavin Adenine Dinucleotide) cofactor according to Scheme 2.
  • the enzyme product is N 5 —OH-Orn and no Piz is formed.
  • FIG. 4 is a series of LC/MS spectra of biosynthetic Piz compared against an authentic L-Piz standard (top row) showing in vivo production of L-Piz in a heterologous bacterial host, Streptomyces lividans.
  • S. lividans WT parent, no Piz production
  • S. lividans harboring a single copy of pzbA (sfaB) alone, pzbB (sfaC) alone, or co-expressing pzbA and pzbB (sfaBC) cloned from the sanglifehrin biosynthetic locus of Streptomyces flaveolus .
  • LC/MS detection of biosynthetic Piz was compared against an authentic L-Piz standard (top row).
  • pzbA is dispensable in the heterologous system because S. lividans encodes a native copy of the gene as part of a siderophore biosynthetic pathway unrelated to Piz production.
  • pzbA remains required for Piz production, but its role in bacteria is not limited to Piz anabolism.
  • pzbB is only found associated with Piz production.
  • FIG. 5 is a series of LC/MS spectra showing the detection of sanglifehrin, a Piz-containing compound produced by Streptomyces flaveolus .
  • sfaC pzbB
  • FIG. 6 is a Marfey's derivatization analysis of the product of PzbB in an assay with L-N5 hydroxy Ornithine substrate (the product of PzbA) showing that the synthesized compound is enantiopure L-Piz.
  • FIG. 7 is a graph showing L-Piz production from various Streptomyces strains. Randomly selected environmental Streptomyces isolates were transformed with pYH015 via intergeneric conjugation as described for S. lividans.
  • the present disclosure is based, at least in part, on the discovery of a complete biosynthetic pathway to L-Piz from the central metabolite L-Orn (the complete biosynthetic pathway not previously known).
  • the present disclosure provides for biological and biochemical production of enantiopure L-piperazic acid.
  • the present disclosure provides for in vitro coupled enzyme assay furnished L-Piz or d 7 -L-Piz.
  • the present disclosure provides for in vivo L-Piz production using genetically engineered S. lividans (natively containing pzbA-gene, pzbB engineered), and data indicating incorporation of L-Piz in L-Piz containing sanglifehrin.
  • Advantages of the methods as described herein include a more cost-effective method of producing L-Piz; the methods as described herein avoid the multi-step synthetic processes currently known in the art; the enzyme catalysts are typically stereospecific providing enantiopure products.
  • One aspect of the present disclosure provides for green biocatalysis of L-Piz in vitro, where no organic solvents and fewer reagents are used (see e.g., Example 2). Another aspect of the present disclosure provides an enzymatic route to heavy isotope-labelled Piz (see e.g., Example 3). Another aspect of the present disclosure provides green biocatalysis of L-Piz in vivo (see e.g., Example 4). Another aspect of the present disclosure provides Directed discovery of drugs and drug-like compounds using heavy isotope L-Piz (see e.g., Example 5). The processes as described herein enable a more efficient and less expensive means to produce L-Piz or isotopically labeled L-Piz. Also provided herein are genes or enzymes encoding Piz production.
  • piperazic acid (Piz)-containing products can be produced using a biochemical or biological approach.
  • a piperazic acid (Piz)-containing product can be piperazic acid or a derivative thereof (e.g., L-piperazic acid (L-Piz)).
  • Piperazic acid (Piz) (aka hexahydropyridazine-3-carboxylic acid) is a nonproteinogenic amino acid that contains a characteristic and biochemically unusual N—N bond.
  • Piz is a proline structural mimic, and Piz-containing compounds are of significant interest for drug discovery.
  • Piz itself is not bioactive, but peptidic compounds incorporating Piz as a building block include antibacterial, antiviral, immunomodulatory, and anticancer drug leads (see e.g., Oelke et al. 2011 Nat. Prod. Rep. (28) 1445-1471.
  • peptidic compounds incorporating Piz as a building block include antibacterial, antiviral, immunomodulatory, and anticancer drug leads (see e.g., Oelke et al. 2011 Nat. Prod. Rep. (28) 1445-1471.
  • Piz-containing metalloprotease inhibitors for drugging bacterial N-formylpeptidases, validated targets for antibiotic development. Intriguingly, all known naturally-occurring Piz containing compounds discovered thus far are bioactive.
  • Piz natural products i.e., naturally occurring compounds produced by live organisms
  • synthetic chemists are attracted to Piz as a synthetic building block for incorporation into drug-like compounds, molecular probes, and the like.
  • bioactive piperazic acid-containing products there are many bioactive piperazic acid-containing products.
  • a piperazic acid-containing product can be any product comprising a piperazic acid, piperazic acid moiety, a piperazic add dipeptide fragment, or a derivative thereof.
  • a piperazic acid-containing product can be Piz, L-Piz, a Piz derivative, a modified Piz, or a Piz-containing compound.
  • a Piz-containing compound or Piz derivative-containing compound can be:
  • a Piz derivative can be a dehydropiperazic acid, a chloropiperazic acid, or a hydroxypiperazic acid.
  • a Piz derivative can be sanglifehrin or Sch 382583.
  • a Piz derivative can be:
  • a starting material comprising Piz or a Pi-z derivative can be a useful reagent for expanding chemical space in small molecule library, molecular analog construction, and molecular probes.
  • a Piz-containing product can be a monamycin.
  • exemplary monomycins are shown below.
  • a Piz-containing product can be an antrimycin.
  • exemplary antrimycins are shown below.
  • a Piz-containing product can be an azinothricin.
  • exemplary azinothricins are shown below.
  • a Piz-containing product can be chloptosin or himastatin.
  • a Piz-containing product can be a luzopeptin or a quinoxapeptin.
  • a luzopeptin or a quinoxapeptin Exemplary luzopeptins and quinoxapeptins are shown below.
  • a Piz-containing product can be a lydiamycin.
  • exemplary lydiamycins are shown below.
  • a Piz-containing product can be a piperazimycin.
  • exemplary piperazimycins are shown below.
  • a Piz-containing product can be a sanglifehrin.
  • Exemplary sanglifehrins are shown below.
  • Piperazic acid-containing products can be antibacterial, antiviral, immunomodulatory, or anticancer drug leads.
  • Piperazic acid-containing products can be caspase (apoptosis, cytokine activation) inhibitors, angiotensin converting enzyme (ACE) inhibitors, anti-inflammatory agents (e.g., sanglifehrin), antitumor antibiotics (e.g., azinothricin, verucopeptin, himastatin, luzopeptin A, immunosuppressants (e.g., L-156,602 an inflammatory peptide C5a antagonist), antibiotics (e.g., Aurantimycin A (inhibits Gram-positive bacteria growth), monamycins), oxytocin receptor antagonist (e.g., L-156,373) (modulate behaviors), or Matylastin type-IV collagenase inhibitors.
  • Piperazic acid-containing products can be antivirals (e.g., sangamides NVP018, NVP019
  • R 5 is a hydrogen, alkyl, a piperazic acid, acetyl, or carboxyl protecting group; and each R 1 and R 2 are independently selected from hydrogen or an amino protecting group, wherein R 1 and R 2 may be taken together to form a fused bicyclic or tricyclic amino protecting group; and each R 3 and R 4 are independently selected from hydrogen, halo (e.g., chloro, fluoro, etc.), or hydroxyl.
  • halo e.g., chloro, fluoro, etc.
  • R groups e.g., R 1 , R 2 , R 3 , R 4 , R 5
  • formula (I) can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; hydroxyl; amine; C 1-10 carboxylic acid; C 1-10 carboxyl, straight chain or branched C 1-10 alkyl, optionally containing unsaturation; a C 2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C 1-10 alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with
  • amino compound refers to a compound that includes an “imine” or an “imino” group as defined herein.
  • hydroxyl as used herein, unless otherwise indicated, includes —OH.
  • halogen and “halo”, as used herein, unless otherwise indicated, include a chlorine, chloro, Cl; fluorine, fluoro, F; bromine, bromo, Br; or iodine, iodo, or I.
  • aryl as used herein, unless otherwise indicated, include a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, or anthracenyl.
  • amine and “amino”, as used herein, unless otherwise indicated, include a functional group that contains a nitrogen atom with a lone pair of electrons and wherein one or more hydrogen atoms have been replaced by a substituent such as, but not limited to, an alkyl group or an aryl group.
  • alkyl as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight or branched moieties, such as but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl groups, etc.
  • Representative straight-chain lower alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; while branched lower alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-di
  • carboxyl as used herein, unless otherwise indicated, includes a functional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group (—COOH).
  • alkenyl as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety.
  • An alkenyl can be partially saturated or unsaturated.
  • alkynyl as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above.
  • An alkynyl can be partially saturated or unsaturated.
  • acyl as used herein, unless otherwise indicated, includes a functional group derived from an aliphatic carboxylic acid, by removal of the hydroxyl (—OH) group.
  • alkoxyl includes O-alkyl groups wherein alkyl is as defined above and O represents oxygen.
  • Representative alkoxyl groups include, but are not limited to, —O-methyl, —O-ethyl, —O-n-propyl, —O-n-butyl, —O-n-pentyl, —O-n-hexyl, —O-n-heptyl, —O-n-octyl, —O-isopropyl, —O-sec-butyl, —O-isobutyl, —O-tert-butyl, —O-isopentyl, —O-2-methylbutyl, —O-2-methylpentyl, —O-3-methylpentyl, —O-2,2-dimethylbutyl, —O-2,3-dimethylbutyl, —O-2,2-dimethylpentyl, —O-2,2-dimethyl
  • cycloalkyl includes a non-aromatic, saturated, partially saturated, or unsaturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 3 to 10 carbon atoms, preferably 3 to 8 ring carbon atoms.
  • cycloalkyls include, but are not limited to, C 3 -C 8 cycloalkyl groups include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl.
  • cycloalkyl also includes -lower alkyl-cycloalkyl, wherein lower alkyl and cycloalkyl are as defined herein.
  • -lower alkyl-cycloalkyl groups include, but are not limited to, —CH 2 -cyclopropyl, —CH 2 -cyclobutyl, —CH 2 -cyclopentyl, —CH 2 -cyclopentadienyl, —CH 2 -cyclohexyl, —CH 2 -cycloheptyl, or —CH 2 -cyclooctyl.
  • heterocyclic includes an aromatic or non-aromatic cycloalkyl in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N.
  • heterocycle examples include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1,4)-dioxane, (1,3)-dioxolane, 4,5-dihydro-1H-imidazolyl, or tetrazolyl.
  • Heterocycles can be substituted or unsubstituted. Heterocycles can also be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heterocyclic ring). A heterocyclic can be saturated, partially saturated, or unsaturated.
  • cyano as used herein, unless otherwise indicated, includes a —CN group.
  • alcohol as used herein, unless otherwise indicated, includes a compound in which the hydroxyl functional group (—OH) is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms.
  • solvate is intended to mean a solvate form of a specified compound that retains the effectiveness of such compound.
  • examples of solvates include compounds of the invention in combination with, for example: water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.
  • DMSO dimethylsulfoxide
  • the term “mmol”, as used herein, is intended to mean millimole.
  • the term “equiv”, as used herein, is intended to mean equivalent.
  • the term “mL”, as used herein, is intended to mean milliliter.
  • the term “g”, as used herein, is intended to mean gram.
  • the term “kg”, as used herein, is intended to mean kilogram.
  • the term “ ⁇ g”, as used herein, is intended to mean micrograms.
  • the term “h”, as used herein, is intended to mean hour.
  • the term “min”, as used herein, is intended to mean minute.
  • the term “M”, as used herein, is intended to mean molar.
  • the term “ ⁇ L”, as used herein, is intended to mean microliter.
  • ⁇ M is intended to mean micromolar.
  • nM is intended to mean nanomolar.
  • N is intended to mean normal.
  • amu is intended to mean atomic mass unit.
  • ° C. is intended to mean degree Celsius.
  • wt/wt is intended to mean weight/weight.
  • v/v is intended to mean volume/volume.
  • MS mass spectroscopy.
  • HPLC is intended to mean high performance liquid chromatograph.
  • RT as used herein, is intended to mean room temperature.
  • e.g. is intended to mean example.
  • N/A is intended to mean not tested.
  • salts refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention.
  • Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-n
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
  • the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
  • the expression “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the invention.
  • solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
  • pharmaceutically acceptable hydrate refers to a compound of the invention, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
  • the host genetically engineered to accumulate a Piz compound can be any microorganism.
  • One aspect of the present disclosure is directed to a transgenic microorganism engineered to accumulate L-piperazic acid (L-Piz).
  • L-Piz L-piperazic acid
  • a microorganism can be used in the biosynthesis of piperazic acid and piperazic acid derivatives.
  • Exemplary microorganisms that can be engineered to accumulate Piz or Piz containing compounds include, but are not limited to, bacteria (e.g., actinobacteria, proteobacteria) or fungi (e.g., yeast).
  • the microorganism can be a bacterium.
  • the microorganism can be in the Phylum, Actinobacteria or Proteobacteria. Any actinobacteria or proteiobacteria with native pzBA or pzbB genes can be suitable for use as a heterologous host.
  • Exemplary Proteobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be Collimonas (a divergent member of the gram negative Burkholderiales).
  • the Collimonas can be of the species Collimonas arenas; Collimonas fungivorans +; Collimonas pratensis; Collimonas sp. 16.2.3 ; Collimonas sp. 16.2.7 ; Collimonas sp. 16.3.1 ; Collimonas sp. 5.15 ; Collimonas sp. 8.2.7 ; Collimonas sp. A6AGF; Collimonas sp.
  • A6ATD5 Collimonas sp. A9 1b-26a; Collimonas sp. AA5ATF; Collimonas sp. AD101 ; Collimonas sp. AD102 ; Collimonas sp. AD103 ; Collimonas sp. AD137 ; Collimonas sp. AD19 ; Collimonas sp. AD23 ; Collimonas sp. AD33 ; Collimonas sp. AD58 ; Collimonas sp. AD59 ; Collimonas sp. AD60 ; Collimonas sp. AD61 ; Collimonas sp.
  • Collimonas sp. MPS11E8 Collimonas sp. NAR2(8); Collimonas sp. NAR7(1); Collimonas sp. NAR7(12); Collimonas sp. NAR7(15); Collimonas sp. NAS7(14); Collimonas sp. NAS9(14); Collimonas sp. NBRC 3740 ; Collimonas sp. NCCB 100027 ; Collimonas sp. RE1 ; Collimonas sp. RX265 ; Collimonas sp. S2U21 ; Collimonas sp.
  • Collimonas sp. S3.TSA.015 Collimonas sp. S5.ACT.019 ; Collimonas sp. S5.CEL.014 ; Collimonas sp. S5.TSA.011 ; Collimonas sp. S5.TSA.20 ; Collimonas sp. UR 9-06 ; Collimonas sp. wged101 ; Collimonas sp. wged148 ; Collimonas sp. wged41 ; Collimonas sp. wged45 ; Collimonas sp. wged84 ; Collimonas sp. wged96; or Collimonas sp. ZL261.
  • Exemplary Actinomycetes that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be Actinoalloteichus, Actinomadura, Actinosynnema, Amycolatopsis, Frankia, Kibdelosporangium, Kutzneria, Lentzea, Mycobacterium, Pseudonocardia, Rhodococcus, Salinispora, Streptacidiphilus , or Streptomyces .
  • These exemplary Actinomycetes are known to have strains with native pzbB, which would indicate that they can be heterologous hosts for Piz or Piz derivative production.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Actinoalloteichus .
  • the Actinoalloteichus can be of the species Actinoalloteichus alkalophilus; Actinoalloteichus cyanogriseus +, Actinoalloteichus hymeniacidonis; Actinoalloteichus nanshanensis; Actinoalloteichus sp. 10-82 ; Actinoalloteichus sp. 2216-6 ; Actinoalloteichus sp. 3BG8 ; Actinoalloteichus sp.
  • Actinoalloteichus sp. CA Actinoalloteichus sp. CA1 , Actinoalloteichus sp. FXJ7.260 ; Actinoalloteichus sp. JAJ70 , Actinoalloteichus sp. JAJ71 ; Actinoalloteichus sp. L2004 ; Actinoalloteichus sp. MA-32 ; Actinoalloteichus sp. MHA15 , Actinoalloteichus sp. NPS-702 ; Actinoalloteichus sp. QAII6 ; Actinoalloteichus sp.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Actinomadura .
  • the Actinomadura can be of the species Actinomadura alba; Actinomadura apis; Actinomadura atramentaria+; Actinomadura bangladeshensis; Actinomadura catellatispora; Actinomadura chibensis+; Actinomadura chokoriensis; Actinomadura citrea; Actinomadura coerulea; Actinomadura cremea+; Actinomadura echinospora; Actinomadura fibrosa; Actinomadura flavalba+; Actinomadura formosensis+; Actinomadura fulvescens; Actinomadura geliboluensis; Actinomadura glauciflava+; Actinomadura hallen
  • Actinomadura sp. 10-44 Actinomadura sp. 13670A; Actinomadura sp. 13679C; Actinomadura sp. 171712 ; Actinomadura sp. 171810 ; Actinomadura sp. 171812 ; Actinomadura sp. 171817 ; Actinomadura sp. 171824 ; Actinomadura sp. 171828 ; Actinomadura sp. 171839 ; Actinomadura sp. 171848 ; Actinomadura sp. 171849 ; Actinomadura sp.
  • Actinomadura sp. 172301 Actinomadura sp. 172301y; Actinomadura sp. 172302a; Actinomadura sp. 172315 ; Actinomadura sp. 172320 ; Actinomadura sp. 172512 ; Actinomadura sp. 1A01698 ; Actinomadura sp. 1g12710 ; Actinomadura sp. 21G792 ; Actinomadura sp. 2602GPT1-42 ; Actinomadura sp. 28a-59-3 ; Actinomadura sp. 28a-77-2 ; Actinomadura sp. 2EPS; Actinomadura sp.
  • Actinomadura sp. AC104 Actinomadura sp. AF-555 ; Actinomadura sp. AML286 ; Actinomadura sp. AML34 ; Actinomadura sp. AML691 ; Actinomadura sp. AMS667 ; Actinomadura sp. ANSum10 ; Actinomadura sp. ART34 ; Actinomadura sp. ART64 ; Actinomadura sp. AV1 ; Actinomadura sp. AW310 ; Actinomadura sp. BK148 ; Actinomadura sp.
  • Actinomadura sp. new-30-5s-4-2 Actinomadura sp. new-30-5s-4-5 ; Actinomadura sp. NN236 ; Actinomadura sp. NN242 ; Actinomadura sp. NTRHn4 ; Actinomadura sp. OS1-43 ; Actinomadura sp. OS3-82 ; Actinomadura sp. OS3-83 ; Actinomadura sp. OS3-87 ; Actinomadura sp. OS3-89 ; Actinomadura sp. P3829 ; Actinomadura sp. P3842 ; Actinomadura sp.
  • R-Ac152 Actinomadura sp. R10-32 ; Actinomadura sp. R16-14 ; Actinomadura sp. R17-27 ; Actinomadura sp. R39 ; Actinomadura sp. RD001933 ; Actinomadura sp. RK2_75 ; Actinomadura sp. RK59 ; Actinomadura sp. RK75 ; Actinomadura sp. RK79 ; Actinomadura sp. RS-52 ; Actinomadura sp. RtII23 ; Actinomadura sp.
  • TFS 1200 Actinomadura sp. TFS 455 ; Actinomadura sp. TP-A0878 ; Actinomadura sp. UKMCC_L29 ; Actinomadura sp. VAN305 ; Actinomadura sp. WMMB 441 ; Actinomadura sp. WMMB 499 ; Actinomadura sp. WMMB 616 ; Actinomadura sp. XM-11-5 ; Actinomadura sp. XM-17-1 ; Actinomadura sp. XM-17-10 ; Actinomadura sp. XM-17-11 ; Actinomadura sp.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Actinosynnema .
  • the Actinosynnema can be of the species Actinosynnema mirum or Actinosynnema pretiosum.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Amycolatopsis .
  • the Amycolatopsis can be of the species Amycolatopsis alba; Amycolatopsis albidoflavus; Amycolatopsis azurea; Amycolatopsis balhimycina; Amycolatopsis coloradensis; Amycolatopsis decaplanina; Amycolatopsis eurytherma; Amycolatopsis fastidiosa; Amycolatopsis japonica; Amycolatopsis kentuckyensis; Amycolatopsis keratiniphila; Amycolatopsis lexingtonensis; Amycolatopsis lurida; Amycolatopsis mediterranei; Amycolatopsis methanolica; Amycolatopsis orientalis; Amycolatopsis palatopharyngis; Amycolatopsis pretoriens
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Frankia .
  • the Frankia can be of the species Frankia brunchorstii or Frankia subtilis.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Kibdelosporangium .
  • the Kibdelosporangium can be of the species Kibdelosporangium albatum; Kibdelosporangium aridum ; or Kibdelosporangium philippinense.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Lentzea .
  • the Lentzea can be of the species Lentzea albida; Lentzea albidocapillata; Lentzea californiensis; Lentzea flaviverrucosa; Lentzea jiangxiensis; Lentzea kentuckyensis; Lentzea sp. 132 ; Lentzea sp. 173316 ; Lentzea sp. 173591 ; Lentzea sp. 173892 ; Lentzea sp.
  • KLBMP 1096 Lentzea sp. LM 058 ; Lentzea sp. LM 121 ; Lentzea sp. mCFU23 ; Lentzea sp. ML457-mF8 ; Lentzea sp. MS-15 ; Lentzea sp. MS-20 ; Lentzea sp. MS-5 ; Lentzea sp. MS6 ; Lentzea sp. SAUK6214 ; Lentzea sp. YIM 48827 ; Lentzea sp. YIM 48828 ; Lentzea sp. YIM 65117 ; Lentzea sp.
  • YIM 75756 Lentzea sp. YIM 75760 ; Lentzea sp. YIM 75761 ; Lentzea sp. YIM 75778 ; Lentzea sp. YIM 75796 ; Lentzea sp. YM-11 ; Lentzea sp. YN-8-6 ; Lentzea violacea ; or Lentzea waywayandensis.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Mycobacterium .
  • the Mycobacterium can be of the species Mycobacterium abscessus; Mycobacterium africanum; Mycobacterium agri; Mycobacterium aichiense; Mycobacterium alvei; Mycobacterium arupense; Mycobacterium asiaticum; Mycobacterium aubagnense; Mycobacterium aurum; Mycobacterium austroafricanum; Mycobacterium avium+; Mycobacterium boenickei; Mycobacterium bohemicum; Mycobacterium bolletii; Mycobacterium botniense; Mycobacterium bovis +; Mycobacterium branderi; Mycobacterium brisbanense; Mycobacterium brumae; Mycobacterium canariasense; Mycobacterium caprae; Mycobacterium celatum;
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Pseudonocardia .
  • the Pseudonocardia can be of the species Pseudonocardia alaniniphila; Pseudonocardia alni; Pseudonocardia asaccharolytica; Pseudonocardia aurantiaca; Pseudonocardia autotrophica; Pseudonocardia azurea; Pseudonocardia benzenivorans; Pseudonocardia chloroethenivorans; Pseudonocardia compacta; Pseudonocardia halophobica; Pseudonocardia hydrocarbonoxydans; Pseudonocardia kongjuensis; Pseudonocardia nitrificans; Pseudon
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Rhodococcus .
  • the Rhodococcus can be of the species Rhodococcus luberonensis; Rhodococcus marchali; Rhodococcus perornatus; Rhodococcus rosaeluteae; Rhodococcus sariuoni; Rhodococcus spiraeae ; or Rhodococcus turanicus.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Salinispora .
  • the Salinispora can be of the species Actinocatenispora; Actinoplanes; Amorphosporangium; Ampullariella; Asanoa; Catellatospora; Catenuloplanes; Couchioplanes; Dactylosporangium; Krasilnikovia; Longispora; Luedemannella; Micromonospora; Myceliochytrium; Pilimelia; Planopolyspora; Planosporangium; Polymorphospora; Salinispora; Spirilliplanes; Verrucosispora; Virgisporangium corrig.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Streptacidiphilus .
  • the Streptacidiphilus can be of the species Streptacidiphilus albus, Streptacidiphilus carbonis, Streptacidiphilus neutrinimicus, Streptacidiphilus anmyonensis, Streptacidiphilus durhamensis, Streptacidiphilus hamsterleyensis, Streptacidiphilus jiangxiensis, Streptacidiphilus melanogenes, Streptacidiphilus oryzae , or Streptacidiphilus rugosus.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Streptomyces .
  • the Streptomyces can be of the species Streptomyces coelicolor, S. lividans, S. albicans, S. griseus , or S. plicatosporus .
  • the Streptomyces can be of the species Streptomyces abietis; Streptomyces abikoensis; Streptomyces aburaviensis; Streptomyces achromogenes; Streptomyces acidiscabies; Streptomyces actinomycinicus; Streptomyces acrimycini; Streptomyces actuosus; Streptomyces aculeolatus; Streptomyces abyssalis; Streptomyces afghaniensis; Streptomyces aidingensis; Streptomyces marinus; Streptomyces alanosinicus; Streptomyces albaduncus; Streptomyces albiaxialis; Streptomyces albidochromogenes; Streptomyces albiflavescens; Streptomyces albiflaviniger Streptomyces albidoflavus; Streptomyces albofaciens; Strepteptomy
  • the microorganism can be a streptomyces species with azinothricin as the founding member, Steptomyces flaveolus DSM 9954, Streptomyces MK498-98F14 strain, Steptomyces sp. RJA2928, Streptomyces hygroscopicus strain ATCC 53653, Streptomyces lycidus (strain HKI0343), Streptomyces strain CNQ-593, Streptomyces sp. (A92-308110), or Streptomyces himastatinicus ATCC 53653.
  • the microorganism can be a Streptomyces strain BB10EC, ES09EC, LM04EC, CS08EC, CM04EC, PF8EC, MRY08EC, LM08EC, JMO5EC, BB04EC, PF1EC, PF5EC, JV594, or JV596.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus, Corynebacterium .
  • the Corynebacterium can be of the species Corynebacterium glutamicum .
  • the Corynebacterium can be of the species Corynebacterium efficiens, Corynebacterium diphtheriae group, Corynebacterium xerosis, Corynebacterium striatum, Corynebacterium minutissimum, Corynebacterium amycolatum, Corynebacterium glucuronolyticum, Corynebacterium argentoratense, Corynebacterium matruchotii, Corynebacterium glutamicum, Corynebacterium sp., Nonfermentative corynebacteria, Corynebacterium afermentans subsp.
  • Afermentans Corynebacterium auris, Corynebacterium pseudodiphtheriticum, Corynebacterium propinquum, Corynebacterium uropygiale, Corynebacterium jeikeium, Corynebacterium urealyticum, Corynebacterium afermentans subsp. lipophilum, Corynebacterium accolens, Corynebacterium macginleyi , CDC coryneform groups F-1 and G, Corynebacterium bovis , or Corynebacterium kroppenstedtii.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus, Kutzneria .
  • the Kutzneria can be of the species Kutzneria spp. 744 , Kutzneria albida, Kutzneria kofuensis, Kutzneria viridogrisea ), (see e.g., Neuman et al. 2012 13(7) 972-976).
  • Kutzneria were previously known to be in the family of Streptosporangiaceae (suborder Streptosporangineae) and were known as Streptosporangium albidum, Streptosporangium viridogriseum (subspecies kofuense ), or Streptosporangium viridogriseum.
  • an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Actinomadura .
  • the Actinomadura can be of the species Actinomadura luzonensis, Actinomadura dessertvillei, Actinomadura madurae, Actinomadura pelletieri, Actinomadura sputi, Actinomadura meyerae, Actinomadura hibisca, Actinomadura pusilla, A. fastidiosa, A. ferruoinea, A. helvata, A. kijaniata, A. libanotica, A. roseola, A. roseoviolacea, A. rubra., A. salmonea , or A. spiralis.
  • the microorganism can be a fungi.
  • the gene can be refactored and insterted into eukaryal vectors for yeast or fungal expression.
  • some fungi also encode functionally orthologous PzbA enzymes (SidA).
  • the microorganism can be in the Phylum, Ascomycota or the genus, Aspergillus .
  • the species can be Aspergillus caesiellus, Aspergillus candidus, Aspergillus carneus, Aspergillus clavatus, Aspergillus deflectus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus israelii, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae, Aspergillus sydowii, Aspergillus tamari, Aspergillus terreus, Aspergillus ustus , or Aspergillus versicolor.
  • transformed microorganisms can accumulate at least about 1 ⁇ M to at least about 1 M L-Piz.
  • transformed microorganisms can accumulate about 1 ⁇ M; about 10 ⁇ M; about 20 ⁇ M; about 30 ⁇ M; about 40 ⁇ M; about 50 ⁇ M; about 60 ⁇ M; about 70 ⁇ M; about 80 ⁇ M; about 90 ⁇ M; about 100 ⁇ M; about 110 ⁇ M; about 120 ⁇ M; about 130 ⁇ M; about 140 ⁇ M; about 150 ⁇ M; about 160 ⁇ M; about 170 ⁇ M; about 180 ⁇ M; about 190 ⁇ M; about 200 ⁇ M; about 210 ⁇ M; about 220 ⁇ M; about 230 ⁇ M; about 240 ⁇ M; about 250 ⁇ M; about 260 ⁇ M; about 270 ⁇ M; about 280 ⁇ M; about 290 ⁇ M; about 300 ⁇ M; about 310 ⁇
  • transformed microorganisms can accumulate between at least about 1 mg and at least about 3 mg of Piz or Piz derivatives (e.g., L-Piz, see e.g., Examples 4 or 14) per liter in about 3 days (or at least about 14 ⁇ g/L per hour or at least about 0.2 ⁇ g/L per minute). In some embodiments, transformed microorganisms can accumulate at least about 0.1 ⁇ g up to about 10 ⁇ g of a Piz or Piz derivatives (e.g., L-Piz) per minute per L.
  • Piz or Piz derivatives e.g., L-Piz
  • transformed microorganisms can accumulate at least about 0.1 ⁇ g, at least about 0.2 ⁇ g, at least about 0.3 ⁇ g, at least about 0.4 ⁇ g, at least about 0.5 ⁇ g, at least about 0.6 ⁇ g, at least about 0.7 ⁇ g, at least about 0.8 ⁇ g, at least about 0.9 ⁇ g, or at least about 1 ⁇ g of Piz or Piz derivatives (e.g., L-Piz) per minute per L.
  • various transformed microorganisms accumulate similar amounts of Piz or Piz derivatives (e.g., L-Piz). Recitation of each of these discrete values is understood to include ranges between each value. Recitation of each of a range is understood to include discrete values within the range.
  • a microorganism e.g., the bacteria, Streptomyces lividans
  • Hydroxylase (e.g., L-Ornithine N 5 -hydroxylase) activity can be engineered into a microorganism by way of one or more individual genes encoding a polypeptide having hydroxylase (e.g., L-Ornithine N 5 -hydroxylase) activity. It is contemplated these activities can likewise be engineered in other microorganisms.
  • Cyclase e.g., L-Ornithine N 5 -cyclase activity or dehydratase (e.g., L-Ornithine N 5 -dehydratase) activity
  • dehydratase e.g., L-Ornithine N 5 -dehydratase
  • activity can be engineered into a microorganism by way of one or more of the individual genes.
  • cyclase e.g., L-Ornithine N 5 -cyclase activity or dehydratase (e.g., L-Ornithine N 5 -dehydratase) activity
  • dehydratase e.g., L-Ornithine N 5 -dehydratase
  • a microorganism by way of one or more genes encoding a polypeptide having cyclase (e.g., L-Ornithine N 5 -cyclase) activity or encoding a polypeptide having dehydratase (e.g., L-Ornithine N 5 -dehydratase) activity; or by one gene encoding both cyclase (e.g., L-Ornithine N 5 -cyclase) and dehydratase (e.g., L-Ornithine N 5 -dehydratase).
  • cyclase e.g.
  • L-Ornithine N 5 -cyclase activity and L-Ornithine N 5 -dehydratase activity can be present in a polypeptide or a fusion polypeptide. It is contemplated these activities can likewise be engineered in other microorganisms.
  • the Piz (e.g., L-Piz) can be endogenous or exogenous to the microorganism. Where the Piz is endogenous, the microorganism can be engineered to produce increased levels of Piz. Where Piz is exogenous, the microorganism can be engineered to produce such exogenous Piz.
  • the microorganism can be engineered to synthesize and accumulate the desired Piz continuously, after some developmental state, or upon being induced to do so.
  • Induction of Piz synthesis can be according to the actions of an inducible promoter associated with the encoded hydroxylase, cyclase, or dehydratase and an inducing agent, as discussed in further detail herein.
  • the promoters as recited herein are only as examples of useful promoters. It is contemplated to adjust copy number (e.g., plasmid as self replicating high copy, low copy, or chromosomally insertional), in conjunction with promoters driving high, medium, or low expression of pzbA and pzbB combinations.
  • the composition can be Piz, a Piz derivative, or a Piz-containing compound.
  • the radiolabeled compound can be for use as a drug discovery agent or an imaging agent.
  • references herein to “radiolabeled” include a compound where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring).
  • One non-limiting exception is 19 F, which allows detection of a molecule which contains this element without enrichment to a higher degree than what is naturally occurring.
  • Compounds carrying the substituent 19 F may thus also be referred to as “labelled” or the like.
  • the term radiolabeled may be interchangeably used with “isotopically-labelled”, “labelled”, “isotopic tracer group”, “isotopic marker”, “isotopic label”, “detectable isotope”, or “radioligand”.
  • the compound comprises a single radiolabeled group.
  • radiolabel groups can include: 2 H (D or deuterium), 3 H (T or tritium), 11 C, 13 C, 14 C 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, or 131 I.
  • an isotopically labeled compound needs only to be enriched with a detectable isotope to, or above, the degree which allows detection with a technique suitable for the particular application, e.g., in a detectable compound labeled with 11 C, the carbon-atom of the labeled group of the labeled compound may be constituted by 12 C or other carbon-isotopes in a fraction of the molecules.
  • the radionuclide that is incorporated in the radiolabeled compounds will depend on the specific application of that radiolabeled compound.
  • “heavy” isotope-labeled compounds e.g., compounds containing deuterons/heavy hydrogen, heavy nitrogen, heavy oxygen, heavy carbon
  • compounds that incorporate 3 H, 14 C, or 125 I can be useful for in vitro labelling or in competition assays.
  • 11 C, 13 C, 18 F, 19 F, 120 I, 123 I, 131 I, 75 Br, or 76 Br can generally be useful.
  • the radiolabel is 11 C.
  • the radiolabel is 14 C.
  • the radiolabel is 13 C.
  • a gene of particular interest for engineering a microorganism to accumulate Piz or Piz derivative is the active pzbB gene from Streptomyces flaveolus (see e.g., Example 3).
  • Another gene of interest for engineering a microorganism to accumulate Piz is the active pzbA gene.
  • pzbA is natively encoded on the S. lividans chromosome.
  • pzbA or pzbB can be expressed in another host that does not natively express the pzbA or pzbB gene or the host can be engineered to carry more than one copy of the a non-natively expressed pzbA or pzbB gene.
  • an pzbA- or pzbB-encoding nucleotide sequence is cloned from its native source (e.g., Streptomyces flaveolus, S. lividans ) and inserted into a host microorganism (see e.g., Example 3).
  • a transformed host microorganism comprises a pzbA or pzbB polynucleotide of SEQ ID NO: 177-SEQ ID NO: 178 (pzbA) or SEQ ID NO: 179-SEQ ID NO: 181 (pzbB).
  • a microorganism is transformed with a nucleotide sequence encoding pzbA or pzbB polypeptide of SEQ ID NO: 1-SEQ ID NO: 81 or SEQ ID NO: 82-SEQ ID NO: 166.
  • a transformed host microorganism comprises a pzbA and pzbB polynucleotides of SEQ ID NO: 167-SEQ ID NO: 176.
  • a transformed host microorganism comprises a nucleotide sequence having at least about 25% sequence identity to SEQ ID NO: 177-SEQ ID NO: 178 or a nucleotide sequence encoding a polypeptide having L-Ornithine N 5 hydroxylase activity and at least about 80% sequence identity to SEQ ID NO: 1-SEQ ID NO: 81.
  • a transformed host microorganism such as a bacterium
  • a transformed host microorganism can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 1-SEQ ID NO: 81, wherein the transformed host exhibits L-Ornithine N 5 hydroxylase activity, pzbA activity and/or accumulation of Piz.
  • a transformed host microorganism can comprise a nucleotide sequence that hybridizes under stringent conditions to SEQ ID NO: 177-SEQ ID NO: 178 over the entire length of SEQ ID NO: 177-SEQ ID NO: 178, and which encodes an active pzbA polypeptide.
  • a transformed host microorganism can comprise the complement to any of the above sequences.
  • a transformed host microorganism comprises a nucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 179-SEQ ID NO: 181 or a nucleotide sequence encoding a polypeptide having L-Ornithine N 5 cyclase activity or L-Ornithine N 5 dehydratase activity and at least about 80% sequence identity to SEQ ID NO: 82-SEQ ID NO: 166.
  • a transformed host microorganism such as a bacterium
  • a transformed host microorganism can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 82-SEQ ID NO: 166, wherein the transformed host exhibits L-Ornithine N 5 cyclase activity or L-Ornithine N 5 dehydratase activity, or pzbB activity and/or accumulation of Piz.
  • a transformed host microorganism can comprise a nucleotide sequence that hybridizes under stringent conditions to SEQ ID NO: 179-SEQ ID NO: 181 over the entire length of SEQ ID NO: 179-SEQ ID NO: 181, and which encodes an active pzbB polypeptide.
  • a transformed host microorganism can comprise the complement to any of the above sequences.
  • L-Ornithine N 5 hydroxylase (see e.g., SEQ ID NO: 177-SEQ ID NO: 178 encoding pzbA gene and SEQ ID NO: 1-SEQ ID NO: 81 encoding pzbA polypeptide), or homologue thereof, is engineered to be expressed or overexpressed in a transformed microorganism.
  • a microorganism can be transformed with a nucleotide having a sequence of 1SEQ ID NO: 177-SEQ ID NO: 178 so as to express L-Ornithine N 5 hydroxylase.
  • a microorganism can be transformed with a nucleotide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to SEQ ID NO: 177-SEQ ID NO: 178 encoding a polypeptide having L-Ornithine N 5 hydroxylase activity.
  • a transformed host microorganism can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 1-SEQ ID NO: 81, wherein the transformed host exhibits L-Ornithine N 5 hydroxylase activity, pzbA activity, and/or accumulation of Piz.
  • L-Ornithine N 5 cyclase or L-Ornithine N 5 dehydratase is engineered to be expressed or overexpressed in a transformed microorganism.
  • a microorganism can be transformed with a nucleotide having a sequence of SEQ ID NO: 179-SEQ ID NO: 181 so as to express L-Ornithine N 5 cyclase or L-Ornithine N 5 dehydratase.
  • a microorganism can be transformed with a nucleotide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to SEQ ID NO: 179-SEQ ID NO: 181 encoding a polypeptide having L-Ornithine N 5 hydroxylase activity.
  • a transformed host microorganism can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 82-SEQ ID NO: 166, wherein the transformed host exhibits L-Ornithine N 5 cyclase activity, L-Ornithine N 5 dehydratase activity, pzbB activity, and/or accumulation of Piz.
  • a microorganism e.g., a bacterium
  • a microorganism is engineered to express one or more of pzbA, pzbB, L-Ornithine N 5 hydroxylase, L-Ornithine N 5 cyclase, or L-Ornithine N 5 dehydratase.
  • heterologous DNA sequence each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • a “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
  • Expression vector expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
  • a “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid.
  • An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus.
  • the promoter is iducible by an agent selected from the group consisting of temperature, pH, a metabolite, light, an osmotic agent, a heavy metal, and an antibiotic.
  • the promoter is selected from the group consisting of a constitutive promoter to produce L-Piz.
  • a promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest.
  • compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
  • transcription start site or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative.
  • “Operably-linked” or “functionally linked” refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
  • the two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent.
  • a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
  • a “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
  • a constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3′ transcription termination nucleic acid molecule.
  • constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3′-untranslated region (3′ UTR).
  • constructs can include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct.
  • 5′ UTR 5′ untranslated regions
  • These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
  • transgenic refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.
  • Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
  • Transformed refers to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999).
  • Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.
  • the term “untransformed” refers to normal cells that have not been through the transformation process.
  • Wild-type refers to a virus or organism found in nature without any known mutation.
  • pzbA, pzbB and/or polypeptide (e.g., pzbA, pzbB) variants having, for example, at least 95%-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
  • Nucleotide and/or amino acid sequence identity percent is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • conservative substitutions can be made at any position so long as the required activity is retained.
  • conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gin by Asn, Val by lie, Leu by lie, and Ser by Thr.
  • amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine).
  • Aliphatic amino acids e.g., Glycine, Alanine, Valine, Leucine, Isoleucine
  • Hydroxyl or sulfur/selenium-containing amino acids e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine
  • Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids.
  • Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
  • “Highly stringent hybridization conditions” are defined as hybridization at 65° C. in a 6 ⁇ SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (T m ) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65° C. in the salt conditions of a 6 ⁇ SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65° C. in the same salt conditions, then the sequences will hybridize.
  • T m melting temperature
  • Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
  • transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods.
  • exogenous is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express.
  • exogenous gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell.
  • the type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
  • Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
  • RNA interference e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA)
  • siRNA small interfering RNAs
  • shRNA short hairpin RNA
  • miRNA micro RNAs
  • RNAi molecules are commercially available from a variety of sources (e.g., Ambion, Tex.; Sigma Aldrich, Mo.; Invitrogen).
  • siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iTTM RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing).
  • Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3′ overhangs.
  • numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.”
  • the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
  • the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.
  • the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise.
  • the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
  • FIG. 2 Select examples of piperazic acid (Piz) family of natural products are shown in FIG. 2 .
  • Piz and modified Piz (e.g., dehydropiperazic, chloropiperazic, hydroxypiperazic acid) molecular components are shown in red in FIG. 2 . All of these molecules are bioactive, with sanglifehrin (top left of FIG. 2 ) under consideration as an immunosuppressant and Hepatitis-C antiviral.
  • the small molecule in the center of FIG. 2 (Sch 382583) is a member of an emerging group of Piz containing metalloprotease inhibitors with clinical relevance as metastatic cancer and antibacterial antibiotic leads. All of these molecules are exclusively produced by actinobacteria.
  • FIG. 2 shows the HPLC-ESI-MS detection of products and substrates with assay time points at time 0 min, 15 min, and 30 min showing the consumption of L-Orn, accumulation of the known intermediate N 5 —OH-Orn, and the concomitant formation of Piz. Data (not shown) in the same assay lacking PzbB, the enzyme product is N 5 —OH-Orn and no Piz is formed.
  • L-Piz can be synthesized chemically, but to date a fermentative pathway to the amino acid has eluded researchers. Enantiopure synthetic L-Piz is expensive: ($2800/gram, 95% pure). DL-Piz synthesized as a mix of isomers, which is significantly less chemically desirable, is less expensive ($800/gram, 95% pure), but still of significant cost.
  • enantiopure (as currently understood) L-Piz can be made from the inexpensive feedstock enantiopure L-Ornithine ($1.40/gram, >99% pure, Sigma-Aldrich), buffer salts, NADPH cofactor, Fe +2 salts, and catalytic FAD (Flavin Adenine Dinucleotide) cofactor (see e.g., FIG. 3 ).
  • Heavy isotope-labeled compounds e.g., compounds containing deuterons/heavy hydrogen, heavy nitrogen, heavy oxygen, heavy carbon
  • d 7 -L-Orn the feasible production of d 7 L-Piz using the reaction described in Example 2 above has been demonstrated.
  • any heavy isotope labeled L-Orn could yield similarly labeled L-Piz.
  • Coupled PzbA/PzbB enzymatic reactions could be scaled to produce and market variously heavy isotopically labeled or radioisotopically labeled versions of L-Piz, for which there are current no known synthetic paths.
  • This example shows a greener production of L-Piz (no organic solvents and fewer reagents than conventional methods).
  • Micro-organisms such as bacteria and fungi are preferred producers of amino acids in the biotechnology industry. This is because the cellular enzyme catalysts of life are typically stereospecific, giving enantiopure products. Enantiopurity can be more difficult to achieve in synthetic chemistry. Also, inexpensive feedstocks are provided for growth, significantly reducing the cost of amino acid production in contrast to fine chemical starting points often required for synthetic chemistry.
  • L-Piz fermentation in a heterologous, genetically engineered host ( Streptomyces lividans ) grown on standard lab media, and with no investment in yield optimization (see e.g., FIG. 4 ) has been demonstrated.
  • S. lividans (WT parent, no Piz production) is compared against S. lividans harboring a single copy of pzbA (sfaB) alone, pzbB (sfaC) alone, or co-expressing pzbA and pzbB (sfaBC) cloned from the sanglifehrin biosynthetic locus of Streptomyces flaveolus in FIG. 4 .
  • LC/MS detection of biosynthetic Piz was compared against an authentic L-Piz standard (top row, FIG. 4 ). In contrast with the in vitro data in FIG. 4 , pzbA is dispensable in the heterologous system because S.
  • lividans encodes a native copy of the gene as part of a siderophore biosynthetic pathway unrelated to Piz production.
  • pzbA remains required for Piz production, but its role in bacteria is not limited to Piz anabolism.
  • pzbB is only found associated with Piz production.
  • S. lividans is carrying at minimum a single copy of a suitable pzbB gene (one or more native pzbA's are natively encoded on the S. lividans chromosome, and therefore is not absolutely required for heterologous expression) under a constitutive promoter to produce micromolar L-Piz.
  • a suitable pzbB gene one or more native pzbA's are natively encoded on the S. lividans chromosome, and therefore is not absolutely required for heterologous expression
  • Measurably higher ( ⁇ 1 mM) L-Piz titers can be achieved using a heterologous S. lividans producer carrying one or more copies of a non-native pzbA in conjunction with heterologous pzbB.
  • lividans serves as a proof of concept host, not necessarily an industrial endpoint.
  • Much higher L-Piz production can likely be achieved by expressing suitable pzbA and pzbB genes in a heterologous host that overproduces the critical feedstock L-Ornithine.
  • One such candidate host is the actinobacterial industrial producer of L-Orn, Corynebacterium glutamicum (20.8-51.5 grams/liter).
  • at least one such industrial L-Orn producing strain is publicly available through the American Type Culture Collection (ATCC), making strain engineering from a high producer feasible.
  • ATCC American Type Culture Collection
  • Piz-derived small molecules e.g., isotopically labeled Piz compound
  • FIG. 5 shows LC/MS detection of sanglifehrin, a Piz-containing compound produced by Streptomyces flaveolus .
  • sanglifehrin a Piz-containing compound produced by Streptomyces flaveolus .
  • pzbB sfaC
  • FIG. 6 shows the Marfey's derivatization analysis of the product of PzbB in an assay with L-N5 hydroxy Ornithine substrate (the product of PzbA). This conclusively shows the product of PzbB has the same stereochemistry (L) and mass as the same derivative produced using L-Piz authentic standard (see e.g., FIG. 6 ).
  • a PzbB ortholog can have as little as around 25% sequence identity to another PzbB ortholog and still produce L-Piz or retain PzbB activity.
  • Bioinformatic data showed PzbB orthologs that can be used to produce L-Piz have an estimated protein identity (functional cutoff) to be around 25% (some predicted PzbB orthologs have identity scores in the 30% range and most have 45% or above.
  • Example 8 SfaBC (Co-Expressing pzbA and pzbB) Combined Ornithine In Vitro Assay Method
  • Example 9 SfaBC (Co-Expressing pzbA and pzbB) Combined D 7 -Ornithine In Vitro Assay Method
  • Example 11 SfaC (Expressing pzbB) N 5 —OH-L-Ornithine In Vitro Assay
  • concentrations in time were plotted, and fitted to a line. The slope of the line was used as the rate of the reaction. Hemin increased the slope by 14.4 times.
  • This example describes L-Piz production from various Streptomyces strains (see e.g., FIG. 7 ) (methods are as described above unless stated otherwise). Randomly selected environmental Streptomyces isolates were transformed with pYH015 via intergeneric conjugation as described for S. lividans . L-Piz production was quantified via SRM LC/MS from triplicate growths essentially as noted for the S. lividans transformants. Resulting strain to strain Piz production is variable, ranging from very low to nearly the same as S. lividans carrying pYH015 (JV594). Note S.
  • Micromonospora _ pattaloongensis _DSM_45245 MSETDSATVRQVVGVGFGPANLALAIAAGEVAGPDGRTLLDECVFLERQP SFGWHRGMLLDGATMQVSFLKDLATLRSPSSRYTFTSYLHDVGRLTDFIN SKTLYPYRTDFHTYLEWAADRLPADVRYGTEVVSVTPERTDDVVRELLVR TGDGRTFRTRNLVIGTGMTPCFPDGVQRGPRVWHSAELLTRLAAPAPTRP RTFAVVGAGQSAAEVVEHLHATHPEADVHAIFGRFGYSMSDDSPFANQIF DPDSVDEFYHAPGEVRDALMGYHANTNYSVVDLDLIRSLHGTAYREHIAG RRRLHFHHASRITRQTVTGEGVHLDVEFLPTGTIRQIDADAIVYATGYRP SDPRQLLGDLADECKTDDRGRLALARDYRVITSDGVRCGIYVHGAAAERT HGLSAGLLSNVAVRAGEILAAIRSL 54.
  • Rhodococcus _ fascians _02-815 MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPSESEDLEGSTL FGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDSVAPTWNYVSVHLRGKLQPVA DFEETLKVVQLTVSTYEQKFGSGWEMDSSLDHYRRIGPAVGAFSFEVESADGMFKLSQEQ NLETRRRVADHFSANHAGRGKELASFMREYSHGDYNNF 93.
  • Rhodococcus _ fascians _A3b MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG KELASFMREYSHGDYNNF 148.
  • Rhodococcus _ fascians _A73a MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG KELASFMREYSHGDYNNF 149.
  • Rhodococcus _ fascians _02-816c MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG KELASFMREYSHGDYNNF 153.
  • Rhodococcus _ fascians _05-339-1 MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS VAPTWNYVSVHLRGKLQPVADFEETLKVVQLIVSTYEQKFGSGWEMDSSL DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG KELASFMREYSHGDYNNF 154.
  • Rhodococcus _ fascians _LMG_3605 MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG KELASFMREYSHGDYNNF 155.
  • Rhodococcus _ fascians _LMG_3616 MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG KELASFMREYSHGDYNNF 156.
  • Rhodococcus _ fascians _LMG_3623 MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG KELASFMREYSHGDYNNF 157.
  • Rhodococcus _ fascians _A22b MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG KELASFMREYSHGDYNNF 158.

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Abstract

Among the various aspects of the present disclosure is the provision of a biological and biochemical production of piperazic acid derived from the newly discovered production pathway for L-piperazic acid. One aspect of the present disclosure includes a transgenic microorganism (e.g., bacteria) engineered to accumulate piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product. Another aspect of the present disclosure includes biochemical and biological methods for producing piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product. Another aspect of the present disclosure includes compositions and methods of using isotopically labeled piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application claims priority from U.S. Provisional Application Ser. No. 62/527,586 filed on 30 Jun. 2017, which is incorporated herein by reference in its entirety.
    • STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
  • Not Applicable.
    • MATERIAL INCORPORATED-BY-REFERENCE
  • The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
    • FIELD OF THE INVENTION
  • The present disclosure generally relates to the synthesis of piperazic acid.
    • BACKGROUND OF THE INVENTION
  • Piperazic acid (Piz) is a nonproteinogenic amino acid that contains a characteristic and biochemically unusual N—N bond. Piz is a proline structural mimic, and Piz-containing compounds are of significant interest for drug discovery. Piz itself is not bioactive, but peptidic compounds incorporating Piz as a building block include antibacterial, antiviral, immunomodulatory, and anticancer drug leads. Intriguingly, all naturally-occurring Piz containing compounds discovered thus far have been bioactive.
    • SUMMARY OF THE INVENTION
  • Among the various aspects of the present disclosure is the provision of a biological and biochemical production of enantiopure piperazic acid derived from the newly discovered production pathway for L-piperazic acid. For example, the present disclosure provides for a transgenic microorganism for the synthesis of L-piperazic acid and derivatives thereof and additional biosynthetic processes for the production of L-piperazic acid and derivatives thereof.
  • Briefly, therefore, the present disclosure is directed to methods of producing piperazic acid, especially L-piperazic acid and derivatives thereof. Synthesis of enantiopure L-Piz has been elusive and expensive. The methods and transgenic organisms as described herein have overcome many of the challenges currently faced regarding the synthesis of enantiopure L-Piz. L-Piz and derivatives thereof can be used as a starting material for a large range of bioactive molecules, including many currently known therapeutics and can be isotopically labeled for use in drug discovery analyses and imaging modalities. The new synthetic routes can give access to isotope (e.g., 15N, 13C, 2H) or radioisotopically-labeled piperazic acid for which no synthetic pathways are currently reported.
  • One aspect of the present disclosure includes transgenic microorganisms (e.g., bacteria) engineered to accumulate piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.
  • Another aspect of the present disclosure includes biochemical and biological methods for producing piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.
  • Another aspect of the present disclosure includes compositions and methods of using isotopically labeled piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.
  • Another aspect of the present disclosure provides for a method for preparing a piperazic acid (Piz)-containing product. In some embodiments, the method comprises: (i) providing N5—OH-Ornithine or derivative thereof; (ii) providing a suitable enzyme comprising a N5—OH Ornithine cyclase/dehydratase; or (iii) optionally, buffer salts, a NADPH cofactor, Fe+2 salts, and a catalytic Flavin Adenine Dinucleotide (FAD) cofactor.
  • In some embodiments, the method further comprises: (i) providing an ornithine or a derivative thereof; or (ii) providing a suitable enzyme comprising an ornithine N5 hydroxylase.
  • In some embodiments, the (i) the N5—OH-Ornithine or derivative thereof is an enantiopure L-Ornithine or derivative thereof; (ii) the enzyme comprising N5—OH Ornithine cyclase/dehydratase is a L-N5—OH Ornithine cyclase/dehydratase or a PzbB enzyme; or (iii) the enzyme comprising ornithine N5 hydroxylase is an L-ornithine N5—OHase or a PzbA enzyme.
  • In some embodiments, the method is carried out in the absence of O2, substantially no O2, or in the presence of low O2.
  • In some embodiments, the method comprises a coupled enzyme assay.
  • In some embodiments, the piperazic acid (Piz)-containing product comprises a compound of formula:
  • Figure US20190002936A1-20190103-C00001
  • where R5 is a hydrogen, an alkyl, a piperazic acid, an acetyl, or a carboxyl protecting group; each R1 and R2 are independently selected from hydrogen or an amino protecting group, wherein R1 and R2 may be taken together to form a fused bicyclic or tricyclic amino protecting group; or each R3 and R4 are independently selected from a hydrogen, a halo (e.g., a chloro, a fluoro, a bromo, a iodo), or a hydroxyl. In some embodiments, R1 and R2 are not simultaneously hydrogen.
  • In some embodiments, the piperazic acid (Piz)-containing product is used as a starting material in a synthetic method of making a bioactive Piz-containing composition selected from the group consisting of: (i) an antibacterial agent, an antibiotic agent, an antitumor agent, an antiviral agent, an immunomodulatory agent, or an anti-inflammatory agent; (ii) a molecular probe, anticancer drug, or drug lead; (ii) a metalloprotease inhibitor, a caspase inhibitor, an angiotensin converting enzyme (ACE) inhibitor, an inflammatory peptide C5a antagonist, an oxytocin receptor antagonist, or a matylastin type-IV collagenase inhibitor; (iii) a dehydropiperazic acid; a chloropiperazic acid; a hydroxypiperazic acid; a monamycin, an aurantimycin, an antrimycin, an azinothricin, a luzopeptin, a kettapeptin, a quinoxapeptin, a lydiamycin, a piperazimycin, or a sangamide; or (iv) sanglifehrin A, pandanamide A, azinothricin, Sch392583, luzopeptin A, kutzernide 2, piperazic acid, L-piperazic acid, antrimycin, kettapeptin, GE3, A83586C, chloptosin, himastatin, luzopeptin, quinoxapeptin, lydiamycin, piperazimycin, sanglifehrin, sangamide NVP018, sangamide NVP019, sanglifehrin, Sch 382583; chloptosin, himastatin, verucopeptin, luzopeptin A, L-156,602, aurantimycin A, or L-156,373.
  • Another aspect of the present disclosure provides for a transgenic microorganism comprising an artificial DNA construct. In some embodiments, the transgenic microorganism comprises, as operably associated components in the 5′ to 3′ direction of transcription: (I)(a) a promoter functional in the microorganism; (b)(i) a first polynucleotide comprising a nucleotide sequence encoding a first polypeptide having a L-Ornithine N5 hydroxylase activity; (ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide having a L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity; or (iii) a third polynucleotide comprising a nucleotide sequence encoding a third polypeptide having a L-Ornithine N5 hydroxylase activity and a L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity; or (c) a transcriptional termination sequence; or (II)(a) a promoter functional in the microorganism; (b)(i) a first polynucleotide comprising a nucleotide sequence encoding a first polypeptide having PzbA activity; (ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide having PzbB activity; or (iii) a third polynucleotide comprising a nucleotide sequence encoding a first polypeptide having PzbA activity and PzbB activity; or (c) a transcriptional termination sequence. In some embodiments, the transgenic microorganism accumulates increased levels of a piperazic acid (Piz)-containing product, optionally L-Piz, compared to a microorganism not comprising the DNA construct.
  • In some embodiments, the microorganism comprises (a)(i) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 1-SEQ ID NO: 81 or SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N5 hydroxylase activity; or (ii) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 82-SEQ ID NO: 166 or SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N5 cyclase activity and L-Ornithine N5 dehydratase activity; or (b) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N5 hydroxylase activity, L-Ornithine N5 cyclase activity, and L-Ornithine N5 dehydratase activity.
  • In some embodiments, the microorganism comprises: (i) a PzbA ortholog with at least about 25% identity to SEQ ID NO: 1-SEQ ID NO: 81 or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbA activity to produce a piperazic acid (Piz)-containing product; (ii) a PzbB ortholog with at least about 25% identity to SEQ ID NO: 82-SEQ ID NO: 166 or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbB activity to produce a piperazic acid (Piz)-containing product; or (iii) a PzbAB ortholog with at least about 25% identity to or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbA and PzbB activity to produce a piperazic acid (Piz)-containing product.
  • In some embodiments, the microorganism is an Actinobacteria selected from the group consisting of Streptomyces, Corynebacterium, Kutzneria, and Actinomadura; is a heterologous population of microorganisms; is an Actinobacteria (optionally, an actinomycete); or is selected from the group consisting of Streptomyces lividans or Corynebacterium glutamicum, optionally carrying one or more copies of a native or non-native pzbA and optionally carrying one or more copies of pzbB.
  • In some embodiments, the transgenic microorganism overproduces L-Ornithine; the pzbA or the pzbB are cloned from a sanglifehrin biosynthetic locus of Streptomyces flaveolus; or a piperazic acid (Piz)-containing product accumulates within the microorganism.
  • Another aspect of the present disclosure provides for a method for producing a piperazic acid (Piz)-containing product. In some embodiments, the method comprises: (i) providing a transgenic microorganism capable of accumulating a piperazic acid (Piz)-containing product; (ii) cultivating the microorganism; or (iii) isolating accumulated piperazic acid (Piz)-containing product.
  • In some embodiments, the method comprises providing a transgenic microorganism and providing a feedstock, wherein the transgenic microorganism comprises at least one copy of pzbA and at least one copy of pzbB under a constitutive promoter; or the at least one pzbA is optionally a native copy.
  • In some embodiments, the transgenic microorganism is (i) a heterologous population of microorganisms; (ii) an Actinobacteria (optionally, an actinomycete); or (ii) selected from the group consisting of Streptomyces lividans or Corynebacterium glutamicum, optionally carrying one or more copies of a native or non-native pzbA and optionally carrying one or more copies of pzbB.
  • In some embodiments, the pzbA or pzbB are cloned from a sanglifehrin biosynthetic locus of Streptomyces flaveolus; or a piperazic acid (Piz)-containing product accumulates within the microorganism.
  • In some embodiments, the method is carried out in the absence of O2, substantially no O2, or in the presence of low O2.
  • In some embodiments, the piperazic acid (Piz)-containing product comprises a compound of formula:
  • Figure US20190002936A1-20190103-C00002
  • where: R5 is a hydrogen, an alkyl, a piperazic acid, an acetyl, or a carboxyl protecting group; each R1 and R2 are independently selected from hydrogen or an amino protecting group, wherein R1 and R2 may be taken together to form a fused bicyclic or tricyclic amino protecting group; or each R3 and R4 are independently selected from a hydrogen, a halo (e.g., a chloro, a fluoro, a bromo, a iodo), or hydroxyl. In some embodiments, R1 and R2 are not simultaneously hydrogen.
  • In some embodiments, the piperazic acid (Piz)-containing product is used as a starting material in the synthesis of a bioactive Piz-containing composition selected from the group consisting of: (i) an antibacterial agent, an antibiotic agent, an antitumor agent, an antiviral agent, an immunomodulatory agent, or an anti-inflammatory agent; (ii) a molecular probe, anticancer drug, or drug lead; (iii) a metalloprotease inhibitor, a caspase inhibitor, an angiotensin converting enzyme (ACE) inhibitor, an inflammatory peptide C5a antagonist, an oxytocin receptor antagonist, or a matylastin type-IV collagenase inhibitor; (iv) a dehydropiperazic acid; a chloropiperazic acid; a hydroxypiperazic acid; a monamycin, an aurantimycin, an antrimycin, an azinothricin, a luzopeptin, a kettapeptin, a quinoxapeptin, a lydiamycin, a piperazimycin, or a sangamide; or (v) sanglifehrin A, pandanamide A, azinothricin, Sch392583, luzopeptin A, kutzernide 2, piperazic acid, L-piperazic acid, antrimycin, kettapeptin, GE3, A83586C, chloptosin, himastatin, luzopeptin, quinoxapeptin, lydiamycin, piperazimycin, sanglifehrin, sangamide NVP018, sangamide NVP019, sanglifehrin, Sch 382583; chloptosin, himastatin, verucopeptin, luzopeptin A, L-156,602, aurantimycin A, or L-156,373.
  • Another aspect of the present disclosure provides for a composition comprising a radiolabeled piperazic acid-containing product or a pharmaceutically acceptable salt, solvate, or polymorph thereof, including all tautomers and stereoisomers thereof, optionally in combination with one or more pharmaceutically acceptable excipients.
  • Another aspect of the present disclosure provides for a method comprising a process for preparation of a radiolabeled piperazic acid-containing product comprising: (i) providing a radiolabeled N5—OH-Ornithine or derivative thereof; (ii) providing a suitable N5—OH Ornithine cyclase/dehydratase; or (iii) optionally, buffer salts, a NADPH cofactor, Fe+2 salts, and a catalytic Flavin Adenine Dinucleotide (FAD) cofactor.
  • In some embodiments, the method comprises: (i) providing a radiolabeled ornithine or a derivative thereof; or (ii) providing a suitable ornithine N5 hydroxylase.
  • In some embodiments, (i) the radiolabeled N5—OH-Ornithine or derivative thereof is an enantiopure radiolabeled L-Ornithine or derivative thereof; (ii) the enzyme comprising N5—OH Ornithine cyclase/dehydratase is L-N5—OH Ornithine cyclase/dehydratase or the enzyme PzbB, or (iii) the enzyme comprising ornithine N5 hydroxylase is a L-ornithine N5—OHase or the enzyme PzbA.
  • In some embodiments, the method comprises a coupled enzyme assay.
  • Another aspect of the present disclosure provides for a method of detecting radiolabeled piperazic acid-containing product. In some embodiments, the method comprises: (i) providing a microorganism; (ii) contacting the microorganism with a radiolabeled piperazic acid-containing product; or (iii) detecting a radiolabeled natural product, a radiolabeled biocatalysis product, or a radiolabeled metabolite.
  • Another aspect of the present disclosure provides for a the radiolabeled piperazic acid-containing product is: (i) labeled for use as a biologically active molecular probe as a drug discovery agent; or (ii) labeled for use in detecting a natural product drug lead compound.
  • Another aspect of the present disclosure provides for a piperazic acid (Piz)-containing product comprises: (i) a single radiolabel; (ii) a radiolabel selected from the group consisting of 2H (D or deuterium), 3H (T or tritium), 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl, 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I, and 131I; (iii) a radiolabel selected from the group consisting of 15N, 13C, and 2H; or (iv) a radiolabeled L-Piz or L-Piz derivative.
  • In some embodiments, the composition can be used in mass spectrometry, gamma imaging, magnetic resonance imaging, magnetic resonance spectroscopy, or fluorescence spectroscopy.
  • Other objects and features will be in part apparent and in part pointed out hereinafter.
    • DESCRIPTION OF THE DRAWINGS
  • Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
  • FIG. 1 is a series of chemical structures showing examples of piperazic acid (Piz) family natural products. Piz and modified Piz (dehydropiperazic, chloropiperazic and hydroxypiperazic acid) molecular components are shown in red. All of these molecules are bioactive, with sanglifehrin (top left) under consideration as an immunosuppressant and Hepatitis-C antiviral. The small molecule in the center (Sch 382583) is a member of an emerging group of Piz containing metalloprotease inhibitors with clinical relevance as metastatic cancer and antibacterial antibiotic leads. All of these molecules are currently thought to be exclusively produced by actinobacteria. Piz and modified Piz (dehydropiperazic, chloropiperazic and hydroxypiperazic acid) molecular components are shown in red.
  • FIG. 2 shows orthologs of both PzbA (yellow) and PzbB (red) are found within biosynthetic gene clusters for known Piz-containing antibiotics. As these clusters encode molecules that are structurally dissimilar except for the incorporation of Piz, parsimony suggests both pzbA and pzbB (previously unrecognized) are involved in Piz biosynthesis.
  • FIG. 3 shows HPLC-ESI-MS detection of products and substrates with assay time points at time 0 min, 15 min, and 30 min showing the consumption of L-Orn, accumulation of the known intermediate N5—OH-Orn, and the concomitant formation of Piz. In vitro reconstitution of L-Piz production from L-Orn in a coupled enzymatic reaction containing purified PzbA, PzbB buffer salts, NADPH cofactor, Fe+2 salts, and catalytic FAD (Flavin Adenine Dinucleotide) cofactor according to Scheme 2. Not shown: In the same assay lacking PzbB, the enzyme product is N5—OH-Orn and no Piz is formed.
  • FIG. 4 is a series of LC/MS spectra of biosynthetic Piz compared against an authentic L-Piz standard (top row) showing in vivo production of L-Piz in a heterologous bacterial host, Streptomyces lividans. S. lividans (WT parent, no Piz production) is compared against S. lividans harboring a single copy of pzbA (sfaB) alone, pzbB (sfaC) alone, or co-expressing pzbA and pzbB (sfaBC) cloned from the sanglifehrin biosynthetic locus of Streptomyces flaveolus. LC/MS detection of biosynthetic Piz was compared against an authentic L-Piz standard (top row). In contrast with the in vitro data above, pzbA is dispensable in the heterologous system because S. lividans encodes a native copy of the gene as part of a siderophore biosynthetic pathway unrelated to Piz production. Thus, pzbA remains required for Piz production, but its role in bacteria is not limited to Piz anabolism. In contrast, it is currently thought that pzbB is only found associated with Piz production.
  • FIG. 5 is a series of LC/MS spectra showing the detection of sanglifehrin, a Piz-containing compound produced by Streptomyces flaveolus. Four major isobaric isomers of sanglifehrin A detected in WT S. flaveolus fermentation extracts. As expected from the results above, an unmarked gene deletion of pzbB (sfaC) from S. flaveolus abrogates sanglifehrin production. Genetic complementation of this mutant with an additional copy of pzbB, or exogenously supplied 50 μM authentic L-Piz (top), restored the production of the four sanglifehrin A isobars. L-Piz is therefore cell penetrant and qualitatively nontoxic. These data additionally link pzbB function with Piz production in vivo, which agrees with the in vitro assay data.
  • FIG. 6 is a Marfey's derivatization analysis of the product of PzbB in an assay with L-N5 hydroxy Ornithine substrate (the product of PzbA) showing that the synthesized compound is enantiopure L-Piz.
  • FIG. 7 is a graph showing L-Piz production from various Streptomyces strains. Randomly selected environmental Streptomyces isolates were transformed with pYH015 via intergeneric conjugation as described for S. lividans.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present disclosure is based, at least in part, on the discovery of a complete biosynthetic pathway to L-Piz from the central metabolite L-Orn (the complete biosynthetic pathway not previously known). As shown herein, the present disclosure provides for biological and biochemical production of enantiopure L-piperazic acid. For example, the present disclosure provides for in vitro coupled enzyme assay furnished L-Piz or d7-L-Piz. As another example, the present disclosure provides for in vivo L-Piz production using genetically engineered S. lividans (natively containing pzbA-gene, pzbB engineered), and data indicating incorporation of L-Piz in L-Piz containing sanglifehrin.
  • Advantages of the methods as described herein include a more cost-effective method of producing L-Piz; the methods as described herein avoid the multi-step synthetic processes currently known in the art; the enzyme catalysts are typically stereospecific providing enantiopure products.
  • One aspect of the present disclosure provides for green biocatalysis of L-Piz in vitro, where no organic solvents and fewer reagents are used (see e.g., Example 2). Another aspect of the present disclosure provides an enzymatic route to heavy isotope-labelled Piz (see e.g., Example 3). Another aspect of the present disclosure provides green biocatalysis of L-Piz in vivo (see e.g., Example 4). Another aspect of the present disclosure provides Directed discovery of drugs and drug-like compounds using heavy isotope L-Piz (see e.g., Example 5). The processes as described herein enable a more efficient and less expensive means to produce L-Piz or isotopically labeled L-Piz. Also provided herein are genes or enzymes encoding Piz production.
  • Piperazic Acid-Containing Products
  • As described herein, piperazic acid (Piz)-containing products can be produced using a biochemical or biological approach.
  • A piperazic acid (Piz)-containing product can be piperazic acid or a derivative thereof (e.g., L-piperazic acid (L-Piz)).
  • Piperazic acid (Piz) (aka hexahydropyridazine-3-carboxylic acid) is a nonproteinogenic amino acid that contains a characteristic and biochemically unusual N—N bond.
  • Figure US20190002936A1-20190103-C00003
  • Piz is a proline structural mimic, and Piz-containing compounds are of significant interest for drug discovery. Piz itself is not bioactive, but peptidic compounds incorporating Piz as a building block include antibacterial, antiviral, immunomodulatory, and anticancer drug leads (see e.g., Oelke et al. 2011 Nat. Prod. Rep. (28) 1445-1471. Especially therapeutically interesting are Piz-containing metalloprotease inhibitors for drugging bacterial N-formylpeptidases, validated targets for antibiotic development. Intriguingly, all known naturally-occurring Piz containing compounds discovered thus far are bioactive. Beyond Piz natural products (i.e., naturally occurring compounds produced by live organisms), synthetic chemists are attracted to Piz as a synthetic building block for incorporation into drug-like compounds, molecular probes, and the like. As described herein, there are many bioactive piperazic acid-containing products.
  • For example, a piperazic acid-containing product can be any product comprising a piperazic acid, piperazic acid moiety, a piperazic add dipeptide fragment, or a derivative thereof.
  • In some embodiments, a piperazic acid-containing product can be Piz, L-Piz, a Piz derivative, a modified Piz, or a Piz-containing compound. For example, a Piz-containing compound or Piz derivative-containing compound can be:
  • Figure US20190002936A1-20190103-C00004
    Figure US20190002936A1-20190103-C00005
  • As another example, a Piz derivative can be a dehydropiperazic acid, a chloropiperazic acid, or a hydroxypiperazic acid. As another example, a Piz derivative can be sanglifehrin or Sch 382583.
  • As another example, a Piz derivative can be:
  • Figure US20190002936A1-20190103-C00006
  • A starting material comprising Piz or a Pi-z derivative (e.g., L-Piz) can be a useful reagent for expanding chemical space in small molecule library, molecular analog construction, and molecular probes.
  • Previous synthetic routes (see e.g., U.S. Pat. No. 6,632,942, incorporated herein by reference) have a lower yield (˜80%) than the processes as described herein (˜100%). Furthermore, the previous methods require multi-step synthetic procedures (6 steps).
  • As an example, a Piz-containing product can be a monamycin. Exemplary monomycins are shown below.
  • Figure US20190002936A1-20190103-C00007
    Compound R1 R2 R3 R4
    Monamycin A H H Me H
    Monamycin B1 H H Me H
    Monamycin B2 H Me H H
    Monemycin B3 Me H H H
    Monamycin C Me H Me H
    Monamycin D1 Me H Me H
    Monamycin D2 H Me Me H
    Monamycin E Me Me Me H
    Monamycin F Me Me Me H
    Monamycin G1 H H Me Cl
    Monamycin G2 H Me H Cl
    Monamycin G3 Me H H Cl
    Monamycin H1 Me H Me Cl
    Monamycin H2 H Me Me Cl
    Monamycin I Me Me Me Cl
  • As another example, a Piz-containing product can be an antrimycin. Exemplary antrimycins are shown below.
  • Figure US20190002936A1-20190103-C00008
    Compound R1 R2
    Antrimycin A Me Et
    Antrimycin B Et Et
    Antrimycin C n-Pr Et
    Antrimycin D i-Bu Et
    Antrimycin Av Me Me
    Antrimycin Bv Et Me
    Antrimycin Cv n-Pr Me
    Antrimycin Dv i-Bu Me
  • As another example, a Piz-containing product can be an azinothricin. Exemplary azinothricins are shown below.
  •
    Figure US20190002936A1-20190103-C00009
    Compound R1 R2 R3 R4
    Azinothricin OMe Me H Me
    Kettapeptin OMe H H Me
    A38586C H H H Me
    GE3 H H i-Pr H
  • As another example, a Piz-containing product can be chloptosin or himastatin.
  • Figure US20190002936A1-20190103-C00010
  • As another example, a Piz-containing product can be a luzopeptin or a quinoxapeptin. Exemplary luzopeptins and quinoxapeptins are shown below.
  •
    Figure US20190002936A1-20190103-C00011
    Compound R1 R2
    Luzopeptin A Ac Ac
    Luzopeptin B H Ac
    Luzopeptin C H H
    Figure US20190002936A1-20190103-C00012
    Figure US20190002936A1-20190103-C00013
    Compound R1 R2
    Quinoxapeptin A
    Figure US20190002936A1-20190103-C00014
    Figure US20190002936A1-20190103-C00015
    Quinoxapeptin B Ac
    Figure US20190002936A1-20190103-C00016
    Quinoxapeptin C H H
  • As another example, a Piz-containing product can be a lydiamycin. Exemplary lydiamycins are shown below.
  • Figure US20190002936A1-20190103-C00017
    Compound R1 R2 X—Y
    Lydiamycin A H H CH2—NH
    Lydiamycin B OH H CH2—NH
    Lydiamycin C H H CH═N
    Lydiamycin D OH OH CH2—NH
  • As another example, a Piz-containing product can be a piperazimycin. Exemplary piperazimycins are shown below.
  •
    Figure US20190002936A1-20190103-C00018
    Compound R1 R2
    Piperazimycin A OH Me
    Piperazimycin B H Me
    Piperazimycin C OH Et
  • As another example, a Piz-containing product can be a sanglifehrin. Exemplary sanglifehrins are shown below.
  • Figure US20190002936A1-20190103-C00019
  • Piperazic acid-containing products can be antibacterial, antiviral, immunomodulatory, or anticancer drug leads. Piperazic acid-containing products can be caspase (apoptosis, cytokine activation) inhibitors, angiotensin converting enzyme (ACE) inhibitors, anti-inflammatory agents (e.g., sanglifehrin), antitumor antibiotics (e.g., azinothricin, verucopeptin, himastatin, luzopeptin A, immunosuppressants (e.g., L-156,602 an inflammatory peptide C5a antagonist), antibiotics (e.g., Aurantimycin A (inhibits Gram-positive bacteria growth), monamycins), oxytocin receptor antagonist (e.g., L-156,373) (modulate behaviors), or Matylastin type-IV collagenase inhibitors. Piperazic acid-containing products can be antivirals (e.g., sangamides NVP018, NVP019 against chronic Hepatitis B).
  • In some embodiments the Piz-containing product can have the formula:
  • Figure US20190002936A1-20190103-C00020
  • wherein: R5 is a hydrogen, alkyl, a piperazic acid, acetyl, or carboxyl protecting group; and each R1 and R2 are independently selected from hydrogen or an amino protecting group, wherein R1 and R2 may be taken together to form a fused bicyclic or tricyclic amino protecting group; and each R3 and R4 are independently selected from hydrogen, halo (e.g., chloro, fluoro, etc.), or hydroxyl.
  • R groups (e.g., R1, R2, R3, R4, R5) or formula (I) can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; hydroxyl; amine; C1-10carboxylic acid; C1-10carboxyl, straight chain or branched C1-10alkyl, optionally containing unsaturation; a C2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C1-10alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein the unsubstituted phenyl ring or substituted phenyl ring can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; hydroxyl; amine; C1-10carboxylic acid; C1-10carboxyl, straight chain or branched C1-10alkyl, optionally containing unsaturation; straight chain or branched C1-10alkyl amine, optionally containing unsaturation; a C2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C1-10alkyl amine; heterocyclyl; heterocyclic amine; aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms; and the unsubstituted heterocyclyl or substituted heterocyclyl can be optionally substituted with one or more groups independently selected from the group consisting of hydroxyl; hydroxyl; amine; C1-10carboxylic acid; C1-10carboxyl; straight chain or branched C1-10alkyl, optionally containing unsaturation; straight chain or branched C1-10alkyl amine, optionally containing unsaturation; a C2-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; heterocyclyl; straight chain or branched C1-10alkyl amine; heterocyclic amine; and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms.
  • The term “imine” or “imino”, as used herein, unless otherwise indicated, includes a functional group or chemical compound containing a carbon-nitrogen double bond. The expression “imino compound”, as used herein, unless otherwise indicated, refers to a compound that includes an “imine” or an “imino” group as defined herein.
  • The term “hydroxyl”, as used herein, unless otherwise indicated, includes —OH.
  • The terms “halogen” and “halo”, as used herein, unless otherwise indicated, include a chlorine, chloro, Cl; fluorine, fluoro, F; bromine, bromo, Br; or iodine, iodo, or I.
  • The term “aryl”, as used herein, unless otherwise indicated, include a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, or anthracenyl.
  • The terms “amine” and “amino”, as used herein, unless otherwise indicated, include a functional group that contains a nitrogen atom with a lone pair of electrons and wherein one or more hydrogen atoms have been replaced by a substituent such as, but not limited to, an alkyl group or an aryl group.
  • The term “alkyl”, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight or branched moieties, such as but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl groups, etc. Representative straight-chain lower alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; while branched lower alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, unsaturated C1-C8 alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, or -3-methyl-1 butynyl. An alkyl can be saturated, partially saturated, or unsaturated.
  • The term “carboxyl”, as used herein, unless otherwise indicated, includes a functional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group (—COOH).
  • The term “alkenyl”, as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety. An alkenyl can be partially saturated or unsaturated.
  • The term “alkynyl”, as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above. An alkynyl can be partially saturated or unsaturated.
  • The term “acyl”, as used herein, unless otherwise indicated, includes a functional group derived from an aliphatic carboxylic acid, by removal of the hydroxyl (—OH) group.
  • The term “alkoxyl”, as used herein, unless otherwise indicated, includes O-alkyl groups wherein alkyl is as defined above and O represents oxygen. Representative alkoxyl groups include, but are not limited to, —O-methyl, —O-ethyl, —O-n-propyl, —O-n-butyl, —O-n-pentyl, —O-n-hexyl, —O-n-heptyl, —O-n-octyl, —O-isopropyl, —O-sec-butyl, —O-isobutyl, —O-tert-butyl, —O-isopentyl, —O-2-methylbutyl, —O-2-methylpentyl, —O-3-methylpentyl, —O-2,2-dimethylbutyl, —O-2,3-dimethylbutyl, —O-2,2-dimethylpentyl, —O-2,3-dimethylpentyl, —O-3,3-dimethylpentyl, —O-2,3,4-trimethylpentyl, —O-3-methylhexyl, —O-2,2-dimethylhexyl, —O-2,4-dimethylhexyl, —O-2,5-dimethylhexyl, —O-3,5-dimethylhexyl, —O-2,4dimethylpentyl, —O-2-methylheptyl, —O-3-methylheptyl, —O-vinyl, —O-allyl, —O-1-butenyl, —O-2-butenyl, —O-isobutylenyl, —O-1-pentenyl, —O-2-pentenyl, —O-3-methyl-1-butenyl, —O-2-methyl-2-butenyl, —O-2,3-dimethyl-2-butenyl, —O-1-hexyl, —O-2-hexyl, —O-3-hexyl, —O-acetylenyl, —O-propynyl, —O-1-butynyl, —O-2-butynyl, —O-1-pentynyl, —O-2-pentynyl and —O-3-methyl-1-butynyl, —O-cyclopropyl, —O-cyclobutyl, —O-cyclopentyl, —O-cyclohexyl, —O-cycloheptyl, —O-cyclooctyl, —O-cyclononyl and —O-cyclodecyl, —O—CH2-cyclopropyl, —O—CH2-cyclobutyl, —O—CH2-cyclopentyl, —O—CH2-cyclohexyl, —O—CH2-cycloheptyl, —O—CH2-cyclooctyl, —O—CH2-cyclononyl, —O—CH2-cyclodecyl, —O—(CH2)2-cyclopropyl, —O—(CH2)2-cyclobutyl, —O—(CH2)2-cyclopentyl, —O—(CH2)2-cyclohexyl, —O—(CH2)2-cycloheptyl, —O—(CH2)2-cyclooctyl, —O—(CH2)2-cyclononyl, or —O—(CH2)2-cyclodecyl. An alkoxyl can be saturated, partially saturated, or unsaturated.
  • The term “cycloalkyl”, as used herein, unless otherwise indicated, includes a non-aromatic, saturated, partially saturated, or unsaturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 3 to 10 carbon atoms, preferably 3 to 8 ring carbon atoms. Examples of cycloalkyls include, but are not limited to, C3-C8 cycloalkyl groups include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl.
  • The term “cycloalkyl” also includes -lower alkyl-cycloalkyl, wherein lower alkyl and cycloalkyl are as defined herein. Examples of -lower alkyl-cycloalkyl groups include, but are not limited to, —CH2-cyclopropyl, —CH2-cyclobutyl, —CH2-cyclopentyl, —CH2-cyclopentadienyl, —CH2-cyclohexyl, —CH2-cycloheptyl, or —CH2-cyclooctyl.
  • The term “heterocyclic”, as used herein, unless otherwise indicated, includes an aromatic or non-aromatic cycloalkyl in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N. Representative examples of a heterocycle include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1,4)-dioxane, (1,3)-dioxolane, 4,5-dihydro-1H-imidazolyl, or tetrazolyl. Heterocycles can be substituted or unsubstituted. Heterocycles can also be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heterocyclic ring). A heterocyclic can be saturated, partially saturated, or unsaturated.
  • The term “cyano”, as used herein, unless otherwise indicated, includes a —CN group.
  • The term “alcohol”, as used herein, unless otherwise indicated, includes a compound in which the hydroxyl functional group (—OH) is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms.
  • The term “solvate” is intended to mean a solvate form of a specified compound that retains the effectiveness of such compound. Examples of solvates include compounds of the invention in combination with, for example: water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.
  • The term “mmol”, as used herein, is intended to mean millimole. The term “equiv”, as used herein, is intended to mean equivalent. The term “mL”, as used herein, is intended to mean milliliter. The term “g”, as used herein, is intended to mean gram. The term “kg”, as used herein, is intended to mean kilogram. The term “μg”, as used herein, is intended to mean micrograms. The term “h”, as used herein, is intended to mean hour. The term “min”, as used herein, is intended to mean minute. The term “M”, as used herein, is intended to mean molar. The term “μL”, as used herein, is intended to mean microliter. The term “μM”, as used herein, is intended to mean micromolar. The term “nM”, as used herein, is intended to mean nanomolar. The term “N”, as used herein, is intended to mean normal. The term “amu”, as used herein, is intended to mean atomic mass unit. The term “° C.”, as used herein, is intended to mean degree Celsius. The term “wt/wt”, as used herein, is intended to mean weight/weight. The term “v/v”, as used herein, is intended to mean volume/volume. The term “MS”, as used herein, is intended to mean mass spectroscopy. The term “HPLC”, as used herein, is intended to mean high performance liquid chromatograph. The term “RT”, as used herein, is intended to mean room temperature. The term “e.g.”, as used herein, is intended to mean example. The term “N/A”, as used herein, is intended to mean not tested.
  • As used herein, the expression “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion. As used herein, the expression “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. As used herein, the expression “pharmaceutically acceptable hydrate” refers to a compound of the invention, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
  • Host
  • The host genetically engineered to accumulate a Piz compound can be any microorganism. One aspect of the present disclosure is directed to a transgenic microorganism engineered to accumulate L-piperazic acid (L-Piz). As described herein, a microorganism can be used in the biosynthesis of piperazic acid and piperazic acid derivatives. Exemplary microorganisms that can be engineered to accumulate Piz or Piz containing compounds include, but are not limited to, bacteria (e.g., actinobacteria, proteobacteria) or fungi (e.g., yeast).
  • As described herein, the microorganism can be a bacterium. In some embodiments, the microorganism can be in the Phylum, Actinobacteria or Proteobacteria. Any actinobacteria or proteiobacteria with native pzBA or pzbB genes can be suitable for use as a heterologous host.
  • Exemplary Proteobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be Collimonas (a divergent member of the gram negative Burkholderiales). As an example, the Collimonas can be of the species Collimonas arenas; Collimonas fungivorans +; Collimonas pratensis; Collimonas sp. 16.2.3; Collimonas sp. 16.2.7; Collimonas sp. 16.3.1; Collimonas sp. 5.15; Collimonas sp. 8.2.7; Collimonas sp. A6AGF; Collimonas sp. A6ATD5; Collimonas sp. A9 1b-26a; Collimonas sp. AA5ATF; Collimonas sp. AD101; Collimonas sp. AD102; Collimonas sp. AD103; Collimonas sp. AD137; Collimonas sp. AD19; Collimonas sp. AD23; Collimonas sp. AD33; Collimonas sp. AD58; Collimonas sp. AD59; Collimonas sp. AD60; Collimonas sp. AD61; Collimonas sp. AD62; Collimonas sp. AD63; Collimonas sp. AD64; Collimonas sp. AD65; Collimonas sp. AD66; Collimonas sp. AD67; Collimonas sp. AD68; Collimonas sp. AD69; Collimonas sp. AD70; Collimonas sp. AD71; Collimonas sp. AD76; Collimonas sp. AD77; Collimonas sp. AD88; Collimonas sp. AD89; Collimonas sp. AD95; Collimonas sp. AD97; Collimonas sp. AD98; Collimonas sp. AD99; Collimonas sp. AR5(10); Collimonas sp. AR5(11); Collimonas sp. AR5(6); Collimonas sp. AS3(2); Collimonas sp. AS3(5); Collimonas sp. BJC15-A11; Collimonas sp. BJC15-A32; Collimonas sp. BPN72; Collimonas sp. BPN73; Collimonas sp. C2PN21; Collimonas sp. CB13; Collimonas sp. CB20; Collimonas sp. CT; Collimonas sp. CT_MP11E6; Collimonas sp. CT_MP11E8; Collimonas sp. CTO 113 b214; Collimonas sp. DEC-B5; Collimonas sp. ES3-61; Collimonas sp. F11; Collimonas sp. F14; Collimonas sp. GCM11; Collimonas sp. HPML71; Collimonas sp. HPN72; Collimonas sp. HPN73; Collimonas sp. III-15; Collimonas sp. III-27; Collimonas sp. III-32; Collimonas sp. III-35; Collimonas sp. III-47; Collimonas sp. III-48; Collimonas sp. III-5; Collimonas sp. III-9; Collimonas sp. IS343; Collimonas sp. IS0468_OTU1303; Collimonas sp. IS0613_OTU1303; Collimonas sp. ISO615_OTU1303; Collimonas sp. IS0616_OTU1303; Collimonas sp. ISO644_OTU1303; Collimonas sp. ISO648_OTU1303; Collimonas sp. KN-1; Collimonas sp. KW19; Collimonas sp. M1Ju29; Collimonas sp. M1U16; Collimonas sp. M1U8; Collimonas sp. M1U9; Collimonas sp. MF3_1; Collimonas sp. MH6; Collimonas sp. MPS11E8; Collimonas sp. NAR2(8); Collimonas sp. NAR7(1); Collimonas sp. NAR7(12); Collimonas sp. NAR7(15); Collimonas sp. NAS7(14); Collimonas sp. NAS9(14); Collimonas sp. NBRC 3740; Collimonas sp. NCCB 100027; Collimonas sp. RE1; Collimonas sp. RX265; Collimonas sp. S2U21; Collimonas sp. S2U31; Collimonas sp. S3.TSA.015; Collimonas sp. S5.ACT.019; Collimonas sp. S5.CEL.014; Collimonas sp. S5.TSA.011; Collimonas sp. S5.TSA.20; Collimonas sp. UR 9-06; Collimonas sp. wged101; Collimonas sp. wged148; Collimonas sp. wged41; Collimonas sp. wged45; Collimonas sp. wged84; Collimonas sp. wged96; or Collimonas sp. ZL261.
  • Exemplary Actinomycetes that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be Actinoalloteichus, Actinomadura, Actinosynnema, Amycolatopsis, Frankia, Kibdelosporangium, Kutzneria, Lentzea, Mycobacterium, Pseudonocardia, Rhodococcus, Salinispora, Streptacidiphilus, or Streptomyces. These exemplary Actinomycetes are known to have strains with native pzbB, which would indicate that they can be heterologous hosts for Piz or Piz derivative production.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Actinoalloteichus. As an example, the Actinoalloteichus can be of the species Actinoalloteichus alkalophilus; Actinoalloteichus cyanogriseus +, Actinoalloteichus hymeniacidonis; Actinoalloteichus nanshanensis; Actinoalloteichus sp. 10-82; Actinoalloteichus sp. 2216-6; Actinoalloteichus sp. 3BG8; Actinoalloteichus sp. AH97; Actinoalloteichus sp. CA; Actinoalloteichus sp. CA1, Actinoalloteichus sp. FXJ7.260; Actinoalloteichus sp. JAJ70, Actinoalloteichus sp. JAJ71; Actinoalloteichus sp. L2004; Actinoalloteichus sp. MA-32; Actinoalloteichus sp. MHA15, Actinoalloteichus sp. NPS-702; Actinoalloteichus sp. QAII6; Actinoalloteichus sp. SH18(2011); Actinoalloteichus sp. SHA6; Actinoalloteichus sp. TRM46408; Actinoalloteichus sp. TSI127-17, Actinoalloteichus sp. WH1-2216-6; or Actinoalloteichus spitiensis+.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Actinomadura. As an example, the Actinomadura can be of the species Actinomadura alba; Actinomadura apis; Actinomadura atramentaria+; Actinomadura bangladeshensis; Actinomadura catellatispora; Actinomadura chibensis+; Actinomadura chokoriensis; Actinomadura citrea; Actinomadura coerulea; Actinomadura cremea+; Actinomadura echinospora; Actinomadura fibrosa; Actinomadura flavalba+; Actinomadura formosensis+; Actinomadura fulvescens; Actinomadura geliboluensis; Actinomadura glauciflava+; Actinomadura hallensis; Actinomadura hibisca+; Actinomadura keratinilytica; Actinomadura kijaniata+; Actinomadura latina+; Actinomadura livida; Actinomadura luteofluorescens; Actinomadura macra+; Actinomadura madurae+; Actinomadura maheshkhaliensis; Actinomadura melliaura; Actinomadura meridiana; Actinomadura mexicana; Actinomadura meyerae; Actinomadura miaoliensis; Actinomadura namibiensis; Actinomadura napierensis; Actinomadura nitritigenes; Actinomadura ochracea; Actinomadura oligospora+; Actinomadura pelletieri+; Actinomadura rifamycini+; Actinomadura rubrobrunea+; Actinomadura rudentiformis; Actinomadura rugatobispora; Actinomadura rupiterrae; Actinomadura scrupuli; Actinomadura sediminis; Actinomadura sp.; Actinomadura sp. 10-124; Actinomadura sp. 10-44; Actinomadura sp. 13670A; Actinomadura sp. 13679C; Actinomadura sp. 171712; Actinomadura sp. 171810; Actinomadura sp. 171812; Actinomadura sp. 171817; Actinomadura sp. 171824; Actinomadura sp. 171828; Actinomadura sp. 171839; Actinomadura sp. 171848; Actinomadura sp. 171849; Actinomadura sp. 172301; Actinomadura sp. 172301y; Actinomadura sp. 172302a; Actinomadura sp. 172315; Actinomadura sp. 172320; Actinomadura sp. 172512; Actinomadura sp. 1A01698; Actinomadura sp. 1g12710; Actinomadura sp. 21G792; Actinomadura sp. 2602GPT1-42; Actinomadura sp. 28a-59-3; Actinomadura sp. 28a-77-2; Actinomadura sp. 2EPS; Actinomadura sp. 3-196; Actinomadura sp. 306D04; Actinomadura sp. 3196; Actinomadura sp. 322C06; Actinomadura sp. 322G01; Actinomadura sp. 334D05; Actinomadura sp. 334E07; Actinomadura sp. 337H02; Actinomadura sp. 387B11; Actinomadura sp. 387H07; Actinomadura sp. 392-1; Actinomadura sp. 40007; Actinomadura sp. 40008; Actinomadura sp. 413D10; Actinomadura sp. 413F04; Actinomadura sp. 413G02; Actinomadura sp. 415A12; Actinomadura sp. 418H03; Actinomadura sp. 419B09; Actinomadura sp. 428G07; Actinomadura sp. 43-45-3; Actinomadura sp. 431D03; Actinomadura sp. 431D09; Actinomadura sp. 6192; Actinomadura sp. 8-104; Actinomadura sp. A16; Actinomadura sp. A17; Actinomadura sp. AC104; Actinomadura sp. AF-555; Actinomadura sp. AML286; Actinomadura sp. AML34; Actinomadura sp. AML691; Actinomadura sp. AMS667; Actinomadura sp. ANSum10; Actinomadura sp. ART34; Actinomadura sp. ART64; Actinomadura sp. AV1; Actinomadura sp. AW310; Actinomadura sp. BK148; Actinomadura sp. CAP 48; Actinomadura sp. CC 0580; Actinomadura sp. CNQ-052_SD01; Actinomadura sp. CNT-075_SF06; Actinomadura sp. CNU-125 PL04; Actinomadura sp. CNU125 PL04; Actinomadura sp. CPCC201357; Actinomadura sp. CPCC202697; Actinomadura sp. DLS-42; Actinomadura sp. DLS-70; Actinomadura sp. DNK540; Actinomadura sp. E6; Actinomadura sp. EGI 80046; Actinomadura sp. EGI 80170; Actinomadura sp. EHA-2; Actinomadura sp. ERI-11; Actinomadura sp. EXM-24-2; Actinomadura sp. EXM-7-1; Actinomadura sp. EYN-10-1; Actinomadura sp. EYN-4-5; Actinomadura sp. FIM95-F26; Actinomadura sp. FXJ1.340; Actinomadura sp. FXJ6.213; Actinomadura sp. FXJ6.337; Actinomadura sp. FXJ7.135; Actinomadura sp. FXJ7.250; Actinomadura sp. FZ04; Actinomadura sp. G08C011; Actinomadura sp. GD15; Actinomadura sp. GKU 128; Actinomadura sp. GKU 147; Actinomadura sp. GKU 154; Actinomadura sp. GKU 157; Actinomadura sp. GKU 505; Actinomadura sp. GKU 822; Actinomadura sp. GMKU359; Actinomadura sp. H590; Actinomadura sp. I43-1; Actinomadura sp. ID05-A0321; Actinomadura sp. IM-1232; Actinomadura sp. IM-1290; Actinomadura sp. IM-2953; Actinomadura sp. IM-3046; Actinomadura sp. IM-3889; Actinomadura sp. IM-5243; Actinomadura sp. IM-5508; Actinomadura sp. IM-5556; Actinomadura sp. IM-5929; Actinomadura sp. IM-6226; Actinomadura sp. IM-6793; Actinomadura sp. IM-6830; Actinomadura sp. IM-6847; Actinomadura sp. IM-6849; Actinomadura sp. IM-6891; Actinomadura sp. IM-6895; Actinomadura sp. IM-6933; Actinomadura sp. IM-6993; Actinomadura sp. IM-7012; Actinomadura sp. IM-7044; Actinomadura sp. IM-7045; Actinomadura sp. IM-7056; Actinomadura sp. IM-7057; Actinomadura sp. IM-7092; Actinomadura sp. IM-7177; Actinomadura sp. IM-7187; Actinomadura sp. IM-7212; Actinomadura sp. IM-7213; Actinomadura sp. IM-7214; Actinomadura sp. IM-7222; Actinomadura sp. IM-7258; Actinomadura sp. IM-7397; Actinomadura sp. IM-7435; Actinomadura sp. IM-8473; Actinomadura sp. J4S16; Actinomadura sp. J4S4; Actinomadura sp. J5S1; Actinomadura sp. J5S10; Actinomadura sp. J5S17; Actinomadura sp. JCM 4674; Actinomadura sp. JSM 082016; Actinomadura sp. K22T; Actinomadura sp. KC-IT-F8; Actinomadura sp. KC-IT-H5; Actinomadura sp. L1958; Actinomadura sp. L2003; Actinomadura sp. L2097; Actinomadura sp. L2187; Actinomadura sp. LZ95; Actinomadura sp. M23; Actinomadura sp. M9; Actinomadura sp. MD49; Actinomadura sp. MNPostmon14; Actinomadura sp. MSSRFDF8; Actinomadura sp. NEAU-Jh1-3; Actinomadura sp. NEAU-Jh2-5; Actinomadura sp. new-30-5s-4-2; Actinomadura sp. new-30-5s-4-5; Actinomadura sp. NN236; Actinomadura sp. NN242; Actinomadura sp. NTRHn4; Actinomadura sp. OS1-43; Actinomadura sp. OS3-82; Actinomadura sp. OS3-83; Actinomadura sp. OS3-87; Actinomadura sp. OS3-89; Actinomadura sp. P3829; Actinomadura sp. P3842; Actinomadura sp. P3874; Actinomadura sp. PM2091; Actinomadura sp. PMPostmon12; Actinomadura sp. PN409; Actinomadura sp. PN414; Actinomadura sp. PN4221; Actinomadura sp. PN4222; Actinomadura sp. PN4223; Actinomadura sp. PN4226; Actinomadura sp. PN425; Actinomadura sp. Postmon13; Actinomadura sp. QAP 98-328-1842; Actinomadura sp. R-Ac152; Actinomadura sp. R10-32; Actinomadura sp. R16-14; Actinomadura sp. R17-27; Actinomadura sp. R39; Actinomadura sp. RD001933; Actinomadura sp. RK2_75; Actinomadura sp. RK59; Actinomadura sp. RK75; Actinomadura sp. RK79; Actinomadura sp. RS-52; Actinomadura sp. RtII23; Actinomadura sp. RtIII29; Actinomadura sp. RtIV13; Actinomadura sp. RtIV2; Actinomadura sp. RY35-68; Actinomadura sp. S14; Actinomadura sp. S19-10; Actinomadura sp. S19-13; Actinomadura sp. S2; Actinomadura sp. S20-30; Actinomadura sp. SBMs009; Actinomadura sp. SBSK-502; Actinomadura sp. Shinshu-MS-02; Actinomadura sp. Shinshu-MS-03; Actinomadura sp. SK74; Actinomadura sp. SpB081030SC-15; Actinomadura sp. SpC090624GE_01; Actinomadura sp. SR-43; Actinomadura sp. T16-1; Actinomadura sp. T3S5; Actinomadura sp. T5S13; Actinomadura sp. T5S5; Actinomadura sp. TCA62003; Actinomadura sp. TF1; Actinomadura sp. TFS 1144; Actinomadura sp. TFS 1200; Actinomadura sp. TFS 455; Actinomadura sp. TP-A0878; Actinomadura sp. UKMCC_L29; Actinomadura sp. VAN305; Actinomadura sp. WMMB 441; Actinomadura sp. WMMB 499; Actinomadura sp. WMMB 616; Actinomadura sp. XM-11-5; Actinomadura sp. XM-17-1; Actinomadura sp. XM-17-10; Actinomadura sp. XM-17-11; Actinomadura sp. XM-17-12; Actinomadura sp. XM-17-13; Actinomadura sp. XM-17-2; Actinomadura sp. XM-17-3; Actinomadura sp. XM-17-4; Actinomadura sp. XM-17-5; Actinomadura sp. XM-17-6; Actinomadura sp. XM-17-7; Actinomadura sp. XM-17-8; Actinomadura sp. XM-18-9; Actinomadura sp. XM-24-1; Actinomadura sp. XM-24-10; Actinomadura sp. XM-24-11; Actinomadura sp. XM-24-12; Actinomadura sp. XM-24-13; Actinomadura sp. XM-24-14; Actinomadura sp. XM-24-15; Actinomadura sp. XM-24-2; Actinomadura sp. XM-24-3; Actinomadura sp. XM-24-4; Actinomadura sp. XM-24-5; Actinomadura sp. XM-24-7; Actinomadura sp. XM-24-8; Actinomadura sp. XM-24-9; Actinomadura sp. XM-4-3; Actinomadura sp. XM-4-4; Actinomadura sp. XM-7-1; Actinomadura sp. XM-7-2; Actinomadura sp. XMU188; Actinomadura sp. Y218; Actinomadura sp. YIM 48842; Actinomadura sp. YIM 61608; Actinomadura sp. YIM 65605; Actinomadura sp. YIM 65650; Actinomadura sp. YIM 65655; Actinomadura sp. YIM 65659; Actinomadura sp. YIM 65663; Actinomadura sp. YIM 65810; Actinomadura sp. YIM 75700; Actinomadura sp. YIM 77502; Actinomadura sp. YIM 77510; Actinomadura sp. YIM M 10855; Actinomadura sp. YIM M 11143; Actinomadura sp. YIM M 11219; Actinomadura sp. YIM M11072; Actinomadura sp. YIM M11327; Actinomadura sp. YN-10-4; Actinomadura sp. YN-5-3; Actinomadura sp. YN-5-4; Actinomadura sp. YN-6-4; Actinomadura sp. YN-7-1; Actinomadura sp. YN-7-10; Actinomadura sp. YN-7-11; Actinomadura sp. YN-7-12; Actinomadura sp. YN-7-13; Actinomadura sp. YN-7-2; Actinomadura sp. YN-7-3; Actinomadura sp. YN-7-6; Actinomadura sp. YN-7-7; Actinomadura sp. YN-7-8; Actinomadura sp. YN-7-9; Actinomadura sp. YN-8-11; Actinomadura sp. ZZY-2013; Actinomadura sputi+; Actinomadura umbrina; Actinomadura verrucosospora; Actinomadura vinacea; Actinomadura viridilutea; Actinomadura viridis; Actinomadura vulgaris+; Actinomadura xylanilytica; Actinomadura yumaensis+; or Excellospora japonica.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Actinosynnema. As an example, the Actinosynnema can be of the species Actinosynnema mirum or Actinosynnema pretiosum.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Amycolatopsis. As an example, the Amycolatopsis can be of the species Amycolatopsis alba; Amycolatopsis albidoflavus; Amycolatopsis azurea; Amycolatopsis balhimycina; Amycolatopsis coloradensis; Amycolatopsis decaplanina; Amycolatopsis eurytherma; Amycolatopsis fastidiosa; Amycolatopsis japonica; Amycolatopsis kentuckyensis; Amycolatopsis keratiniphila; Amycolatopsis lexingtonensis; Amycolatopsis lurida; Amycolatopsis mediterranei; Amycolatopsis methanolica; Amycolatopsis orientalis; Amycolatopsis palatopharyngis; Amycolatopsis pretoriensis; Amycolatopsis rubida; Amycolatopsis rugosa; Amycolatopsis sacchari; Amycolatopsis sulphurea; Amycolatopsis thermoflava; Amycolatopsis tolypomycina; or Amycolatopsis vancoresmycina.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Frankia. As an example, the Frankia can be of the species Frankia brunchorstii or Frankia subtilis.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Kibdelosporangium. As an example, the Kibdelosporangium can be of the species Kibdelosporangium albatum; Kibdelosporangium aridum; or Kibdelosporangium philippinense.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Lentzea. As an example, the Lentzea can be of the species Lentzea albida; Lentzea albidocapillata; Lentzea californiensis; Lentzea flaviverrucosa; Lentzea jiangxiensis; Lentzea kentuckyensis; Lentzea sp. 132; Lentzea sp. 173316; Lentzea sp. 173591; Lentzea sp. 173892; Lentzea sp. 18-3; Lentzea sp. 4_C7_44; Lentzea sp. 4_C7_58; Lentzea sp. 7887; Lentzea sp. 84741; Lentzea sp. ACT-0091; Lentzea sp. BJ36; Lentzea sp. DHS C013; Lentzea sp. G-MN-1; Lentzea sp. GP0204; Lentzea sp. 108A-00410; Lentzea sp. IMER-B1-1; Lentzea sp. IR11-RCA120; Lentzea sp. KLBMP 1096; Lentzea sp. LM 058; Lentzea sp. LM 121; Lentzea sp. mCFU23; Lentzea sp. ML457-mF8; Lentzea sp. MS-15; Lentzea sp. MS-20; Lentzea sp. MS-5; Lentzea sp. MS6; Lentzea sp. SAUK6214; Lentzea sp. YIM 48827; Lentzea sp. YIM 48828; Lentzea sp. YIM 65117; Lentzea sp. YIM 75756; Lentzea sp. YIM 75760; Lentzea sp. YIM 75761; Lentzea sp. YIM 75778; Lentzea sp. YIM 75796; Lentzea sp. YM-11; Lentzea sp. YN-8-6; Lentzea violacea; or Lentzea waywayandensis.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Mycobacterium. As an example, the Mycobacterium can be of the species Mycobacterium abscessus; Mycobacterium africanum; Mycobacterium agri; Mycobacterium aichiense; Mycobacterium alvei; Mycobacterium arupense; Mycobacterium asiaticum; Mycobacterium aubagnense; Mycobacterium aurum; Mycobacterium austroafricanum; Mycobacterium avium+; Mycobacterium boenickei; Mycobacterium bohemicum; Mycobacterium bolletii; Mycobacterium botniense; Mycobacterium bovis +; Mycobacterium branderi; Mycobacterium brisbanense; Mycobacterium brumae; Mycobacterium canariasense; Mycobacterium caprae; Mycobacterium celatum; Mycobacterium chelonae+; Mycobacterium chimaera; Mycobacterium chitae; Mycobacterium chlorophenolicum; Mycobacterium chubuense; Mycobacterium colombiense; Mycobacterium conceptionense; Mycobacterium confluentis; Mycobacterium conspicuum; Mycobacterium cookie; Mycobacterium cosmeticum; Mycobacterium diernhoferi; Mycobacterium doricum; Mycobacterium duvalii; Mycobacterium elephantis; Mycobacterium; Mycobacterium farcinogenes; Mycobacterium flavescens; Mycobacterium florentinum; Mycobacterium fluoranthenivorans; Mycobacterium fortuitum+; Mycobacterium frederiksbergense; Mycobacterium gadium; Mycobacterium gastri; Mycobacterium genavense; Mycobacterium gilvum; Mycobacterium goodie; Mycobacterium gordonae; Mycobacterium haemophilum; Mycobacterium hassiacum; Mycobacterium heckeshornense; Mycobacterium heidelbergense; Mycobacterium hiberniae; Mycobacterium hodleri; Mycobacterium holsaticum; Mycobacterium houstonense; Mycobacterium immunogenum; Mycobacterium interjectum; Mycobacterium intermedium; Mycobacterium intracellulare; Mycobacterium kansasii; Mycobacterium komossense; Mycobacterium kubicae; Mycobacterium lacus; Mycobacterium lentiflavum; Mycobacterium leprae; Mycobacterium lepraemurium; Mycobacterium madagascariense; Mycobacterium mageritense; Mycobacterium malmoense; Mycobacterium marinum; Mycobacterium massiliense; Mycobacterium microti; Mycobacterium montefiorense; Mycobacterium moriokaense; Mycobacterium mucogenicum; Mycobacterium murale; Mycobacterium nebraskense; Mycobacterium neoaurum; Mycobacterium neworleansense; Mycobacterium nonchromogenicum; Mycobacterium novocastrense; Mycobacterium obuense; Mycobacterium palustre; Mycobacterium parafortuitum; Mycobacterium parascrofulaceum; Mycobacterium parmense; Mycobacterium peregrinum; Mycobacterium phlei; Mycobacterium phocaicum; Mycobacterium pinnipedii; Mycobacterium porcinum; Mycobacterium poriferae; Mycobacterium pseudoshottsii; Mycobacterium psychrotolerans; Mycobacterium pulveris; Mycobacterium pyrenivorans; Mycobacterium rhodesiae; Mycobacterium saskatchewanense; Mycobacterium scrofulaceum; Mycobacterium senegalense; Mycobacterium septicum; Mycobacterium shimoidei; Mycobacterium shottsii; Mycobacterium simiae; Mycobacterium smegmatis; Mycobacterium sphagni; Mycobacterium szulgai; Mycobacterium terrae; Mycobacterium thermoresistibile; Mycobacterium tokaiense; Mycobacterium triplex; Mycobacterium triviale; Mycobacterium tuberculosis+; Mycobacterium tusciae; Mycobacterium ulcerans; Mycobacterium vaccae; Mycobacterium vanbaalenii; Mycobacterium wolinskyi; or Mycobacterium xenopi.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Pseudonocardia. As an example, the Pseudonocardia can be of the species Pseudonocardia alaniniphila; Pseudonocardia alni; Pseudonocardia asaccharolytica; Pseudonocardia aurantiaca; Pseudonocardia autotrophica; Pseudonocardia azurea; Pseudonocardia benzenivorans; Pseudonocardia chloroethenivorans; Pseudonocardia compacta; Pseudonocardia halophobica; Pseudonocardia hydrocarbonoxydans; Pseudonocardia kongjuensis; Pseudonocardia nitrificans; Pseudonocardia petroleophila; Pseudonocardia saturnea; Pseudonocardia spinosa; Pseudonocardia spinosispora; Pseudonocardia sulfidoxydans; Pseudonocardia thermophile; Pseudonocardia xinjiangensis; Pseudonocardia yunnanensis; or Pseudonocardia zijingensis.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Rhodococcus. As an example, the Rhodococcus can be of the species Rhodococcus luberonensis; Rhodococcus marchali; Rhodococcus perornatus; Rhodococcus rosaeluteae; Rhodococcus sariuoni; Rhodococcus spiraeae; or Rhodococcus turanicus.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Salinispora. As an example, the Salinispora can be of the species Actinocatenispora; Actinoplanes; Amorphosporangium; Ampullariella; Asanoa; Catellatospora; Catenuloplanes; Couchioplanes; Dactylosporangium; Krasilnikovia; Longispora; Luedemannella; Micromonospora; Myceliochytrium; Pilimelia; Planopolyspora; Planosporangium; Polymorphospora; Salinispora; Spirilliplanes; Verrucosispora; Virgisporangium corrig.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Streptacidiphilus. As an example, the Streptacidiphilus can be of the species Streptacidiphilus albus, Streptacidiphilus carbonis, Streptacidiphilus neutrinimicus, Streptacidiphilus anmyonensis, Streptacidiphilus durhamensis, Streptacidiphilus hamsterleyensis, Streptacidiphilus jiangxiensis, Streptacidiphilus melanogenes, Streptacidiphilus oryzae, or Streptacidiphilus rugosus.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Streptomyces. As an example, the Streptomyces can be of the species Streptomyces coelicolor, S. lividans, S. albicans, S. griseus, or S. plicatosporus. As another example, the Streptomyces can be of the species Streptomyces abietis; Streptomyces abikoensis; Streptomyces aburaviensis; Streptomyces achromogenes; Streptomyces acidiscabies; Streptomyces actinomycinicus; Streptomyces acrimycini; Streptomyces actuosus; Streptomyces aculeolatus; Streptomyces abyssalis; Streptomyces afghaniensis; Streptomyces aidingensis; Streptomyces africanus; Streptomyces alanosinicus; Streptomyces albaduncus; Streptomyces albiaxialis; Streptomyces albidochromogenes; Streptomyces albiflavescens; Streptomyces albiflaviniger Streptomyces albidoflavus; Streptomyces albofaciens; Streptomyces alboflavus; Streptomyces albogriseolus; Streptomyces albolongus; Streptomyces alboniger Streptomyces albospinus; Streptomyces albulus; Streptomyces albus; Streptomyces aldersoniae; Streptomyces alfalfae; Streptomyces alkaliphilus; Streptomyces alkalithermotolerans; Streptomyces almquistii; Streptomyces alni; Streptomyces althioticus; Streptomyces amakusaensis; Streptomyces ambofaciens; Streptomyces amritsarensis; Streptomyces anandii; Streptomyces angustmyceticus; Streptomyces anthocyanicus; Streptomyces antibioticus; Streptomyces antimycoticus; Streptomyces anulatus; Streptomyces aomiensis; Streptomyces araujoniae; Streptomyces ardus; Streptomyces arenae; Streptomyces armeniacus; Streptomyces artemisiae; Streptomyces arcticus; Streptomyces ascomycinicus; Streptomyces asiaticus; Streptomyces asterosporus; Streptomyces atacamensis; Streptomyces atratus; Streptomyces atriruber Streptomyces atroolivaceus; Streptomyces atrovirens; Streptomyces aurantiacus; Streptomyces aurantiogriseus; Streptomyces auratus; Streptomyces aureocirculatus; Streptomyces aureofaciens; Streptomyces aureorectus; Streptomyces aureoverticillatus; Streptomyces aureus; Streptomyces avellaneus; Streptomyces avermitilis; Streptomyces avicenniae; Streptomyces avidinii; Streptomyces axinellae; Streptomyces azureus; Streptomyces bacillaris; Streptomyces badius; Streptomyces bambergiensis; Streptomyces bangladeshensis; Streptomyces baliensis; Streptomyces barkulensis; Streptomyces beijiangensis; Streptomyces bellus; Streptomyces bikiniensis; Streptomyces blastmyceticus; Streptomyces bluensis; Streptomyces bobili; Streptomyces bohaiensis; Streptomyces bottropensis; Streptomyces brasiliensis; Streptomyces brevispora; Streptomyces bullii; Streptomyces bungoensis; Streptomyces burgazadensis; Streptomyces cacaoi; Streptomyces caelestis; Streptomyces caeruleatus; Streptomyces calidiresistens; Streptomyces calvus; Streptomyces canarius; Streptomyces canchipurensis; Streptomyces candidus; Streptomyces cangkringensis; Streptomyces caniferus; Streptomyces canus; Streptomyces capillispiralis; Streptomyces capoamus; Streptomyces carpaticus; Streptomyces carpinensis; Streptomyces castelarensis; Streptomyces catbensis; Streptomyces catenulae; Streptomyces cavourensis; Streptomyces cellostaticus; Streptomyces celluloflavus; Streptomyces cellulolyticus; Streptomyces cellulosae; Streptomyces chartreusis; Streptomyces chattanoogensis; Streptomyces cheonanensis; Streptomyces chiangmaiensis; Streptomyces chrestomyceticus; Streptomyces chromofuscus; Streptomyces chryseus; Streptomyces chilikensis; Streptomyces chlorus; Streptomyces chumphonensis; Streptomyces cinereorectus; Streptomyces cinereoruber; Streptomyces cinereospinus; Streptomyces cinereus; Streptomyces cinerochromogenes; Streptomyces cinnabarinus; Streptomyces cinnamonensis; Streptomyces cinnamoneus; Streptomyces cirratus; Streptomyces ciscaucasicus; Streptomyces clavifer Streptomyces clavuligerus; Streptomyces coacervatus; Streptomyces cocklensis; Streptomyces coelescens; Streptomyces coelicoflavus; Streptomyces coelicolor Streptomyces coeruleoflavus; Streptomyces coeruleofuscus; Streptomyces coeruleoprunus; Streptomyces coeruleorubidus; Streptomyces coerulescens; Streptomyces collinus; Streptomyces colombiensis; Streptomyces corchorusii; Streptomyces costaricanus; Streptomyces cremeus; Streptomyces crystallinus; Streptomyces cuspidosporus; Streptomyces cyaneofuscatus; Streptomyces cyaneus; Streptomyces cyanoalbus; Streptomyces cyslabdanicus; Streptomyces daghestanicus; Streptomyces daliensi; Streptomyces deccanensis; Streptomyces decoyicus; Streptomyces demainii; Streptomyces deserti; Streptomyces diastaticus; Streptomyces diastatochromogenes; Streptomyces djakartensis; Streptomyces drozdowiczii; Streptomyces durhamensis; Streptomyces durmitorensis; Streptomyces echinatus; Streptomyces echinoruber Streptomyces ederensis; Streptomyces emeiensis; Streptomyces endophyticus; Streptomyces endus; Streptomyces enissocaesilis; Streptomyces erythrogriseus; Streptomyces erringtonii; Streptomyces eurocidicus; Streptomyces europaeiscabiei; Streptomyces eurythermus; Streptomyces exfoliatus; Streptomyces faba; Streptomyces fenghuangensis; Streptomyces ferralitis; Streptomyces filamentosus; Streptomyces fildesensis; Streptomyces filipinensis; Streptomyces fimbriatus; Streptomyces finlayi; Streptomyces flaveolus; Streptomyces flaveus; Streptomyces flavofungini; Streptomyces flavotricini; Streptomyces flavovariabilis; Streptomyces flavovirens; Streptomyces flavoviridis; Streptomyces fradiae; Streptomyces fragilis; Streptomyces fukangensis; Streptomyces fulvissimus; Streptomyces fulvorobeus; Streptomyces fumanus; Streptomyces fumigatiscleroticus; Streptomyces galbus; Streptomyces galilaeus; Streptomyces gancidicus; Streptomyces gardneri; Streptomyces gelaticus; Streptomyces geldanamycininus; Streptomyces geysiriensis; Streptomyces ghanaensis; Streptomyces gilvifuscus; Streptomyces glaucescens; Streptomyces glauciniger Streptomyces glaucosporus; Streptomyces glaucus; Streptomyces globisporus; Streptomyces globosus; Streptomyces glomeratus; Streptomyces glomeroaurantiacus; Streptomyces glycovorans; Streptomyces gobitricini; Streptomyces goshikiensis; Streptomyces gougerotii; Streptomyces graminearus; Streptomyces gramineus; Streptomyces graminifolii; Streptomyces graminilatus; Streptomyces graminisoli; Streptomyces griseiniger Streptomyces griseoaurantiacus; Streptomyces griseocarneus; Streptomyces griseochromogenes; Streptomyces griseoflavus; Streptomyces griseofuscus; Streptomyces griseoincarnatus; Streptomyces griseoloalbus; Streptomyces griseolus; Streptomyces griseoluteus; Streptomyces griseomycini; Streptomyces griseoplanus; Streptomyces griseorubens; Streptomyces griseoruber Streptomyces griseorubiginosus; Streptomyces griseosporeus; Streptomyces griseostramineus; Streptomyces griseoviridis; Streptomyces griseus; Streptomyces guanduensis; Streptomyces gulbargensis; Streptomyces hainanensis; Streptomyces haliclonae; Streptomyces halophytocola; Streptomyces halstedii; Streptomyces harbinensis; Streptomyces hawaiiensis; Streptomyces hebeiensis; Streptomyces heilongjiangensis; Streptomyces heliomycini; Streptomyces helvaticus; Streptomyces herbaceus; Streptomyces herbaricolor; Streptomyces himastatinicus; Streptomyces hiroshimensis; Streptomyces hirsutus; Streptomyces hokutonensis; Streptomyces hoynatensis; Streptomyces humidus; Streptomyces humiferus; Streptomyces hundungensis; Streptomyces hyderabadensis; Streptomyces hygroscopicus; Streptomyces hypolithicus; Streptomyces iakyrus; Streptomyces iconiensis; Streptomyces incanus; Streptomyces indiaensis; Streptomyces indigoferus; Streptomyces indicus; Streptomyces indonesiensis; Streptomyces intermedius; Streptomyces inusitatus; Streptomyces ipomoeae; Streptomyces iranensis; Streptomyces janthinus; Streptomyces jamaicensis; Streptomyces javensis; Streptomyces jietaisiensis; Streptomyces jiujiangensis; Streptomyces kaempferi; Streptomyces kanamyceticus; Streptomyces karpasiensis; Streptomyces kasugaensis; Streptomyces katrae; Streptomyces kebangsaanensis; Streptomyces klenkii; Streptomyces koyangensis; Streptomyces kunmingensis; Streptomyces kurssanovii; Streptomyces labedae; Streptomyces lacrimifluminis; Streptomyces lacticiproducens; Streptomyces laculatispora; Streptomyces lanatus; Streptomyces lannensis; Streptomyces lateritius; Streptomyces laurentii; Streptomyces lavendofoliae; Streptomyces lavendulae; Streptomyces lavenduligriseus; Streptomyces leeuwenhoekii; Streptomyces lavendulocolor Streptomyces levis; Streptomyces libani; Streptomyces lienomycini; Streptomyces lilacinus; Streptomyces lincolnensis; Streptomyces litmocidini; Streptomyces litoralis; Streptomyces lomondensis; Streptomyces longisporoflavus; Streptomyces longispororuber Streptomyces lopnurensis; Streptomyces longisporus; Streptomyces longwoodensis; Streptomyces lucensis; Streptomyces lunaelactis; Streptomyces lunalinharesii; Streptomyces luridiscabiei; Streptomyces luridus; Streptomyces lusitanus; Streptomyces lushanensis; Streptomyces luteireticuli; Streptomyces luteogriseus; Streptomyces luteosporeus; Streptomyces lydicus; Streptomyces macrosporus; Streptomyces malachitofuscus; Streptomyces malachitospinus; Streptomyces malaysiensis; Streptomyces mangrovi; Streptomyces marinus; Streptomyces marokkonensis; Streptomyces mashuensis; Streptomyces massasporeus; Streptomyces matensis; Streptomyces mayteni; Streptomyces mauvecolor Streptomyces megasporus; Streptomyces melanogenes; Streptomyces melanosporofaciens; Streptomyces mexicanus; Streptomyces michiganensis; Streptomyces microflavus; Streptomyces milbemycinicus; Streptomyces minutiscleroticus; Streptomyces mirabilis; Streptomyces misakiensis; Streptomyces misionensis; Streptomyces mobaraensis; Streptomyces monomycini; Streptomyces mordarskii; Streptomyces morookaense; Streptomyces muensis; Streptomyces murinus; Streptomyces mutabilis; Streptomyces mutomycini; Streptomyces naganishii; Streptomyces nanhaiensis; Streptomyces nanshensis; Streptomyces narbonensis; Streptomyces nashvillensis; Streptomyces netropsis; Streptomyces neyagawaensis; Streptomyces niger Streptomyces nigrescens; Streptomyces nitrosporeus; Streptomyces niveiciscabiei; Streptomyces niveiscabiei; Streptomyces niveoruber Streptomyces niveus; Streptomyces noboritoensis; Streptomyces nodosus; Streptomyces nogalater Streptomyces nojiriensis; Streptomyces noursei; Streptomyces novaecaesareae; Streptomyces ochraceiscleroticus; Streptomyces olivaceiscleroticus; Streptomyces olivaceoviridis; Streptomyces olivaceus; Streptomyces olivicoloratus; Streptomyces olivochromogenes; Streptomyces olivomycini; Streptomyces olivoverticillatus; Streptomyces omiyaensis; Streptomyces osmaniensis; Streptomyces orinoci; Streptomyces pactum; Streptomyces panacagri; Streptomyces panaciradicis; Streptomyces paradoxus; Streptomyces parvulus; Streptomyces parvus; Streptomyces pathocidini; Streptomyces paucisporeus; Streptomyces peucetius; Streptomyces phaeochromogenes; Streptomyces phaeofaciens; Streptomyces phaeogriseichromatogenes; Streptomyces phaeoluteichromatogenes; Streptomyces phaeoluteigriseus; Streptomyces phaeopurpureus; Streptomyces pharetrae; Streptomyces pharmamarensis; Streptomyces phytohabitans; Streptomyces pilosus; Streptomyces platensis; Streptomyces plicatus; Streptomyces plumbiresistens; Streptomyces pluricolorescens; Streptomyces pluripotens; Streptomyces polyantibioticus; Streptomyces polychromogenes; Streptomyces polygonati; Streptomyces polymachus; Streptomyces poonensis; Streptomyces prasinopilosus; Streptomyces prasinosporus; Streptomyces prasinus; Streptomyces pratens; Streptomyces pratensis; Streptomyces prunicolor Streptomyces psammoticus; Streptomyces pseudoechinosporeus; Streptomyces pseudogriseolus; Streptomyces pseudovenezuelae; Streptomyces pulveraceus; Streptomyces puniceus; Streptomyces puniciscabiei; Streptomyces purpeofuscus; Streptomyces purpurascens; Streptomyces purpureus; Streptomyces purpurogeneiscleroticus; Streptomyces qinglanensis; Streptomyces racemochromogenes; Streptomyces radiopugnans; Streptomyces rameus; Streptomyces ramulosus; Streptomyces rapamycinicus; Streptomyces recifensis; Streptomyces rectiviolaceus; Streptomyces regensis; Streptomyces resistomycificus; Streptomyces reticuliscabiei; Streptomyces rhizophilus; Streptomyces rhizosphaericus; Streptomyces rimosus; Streptomyces rishiriensis; Streptomyces rochei; Streptomyces rosealbus; Streptomyces roseiscleroticus; Streptomyces roseofulvus; Streptomyces roseolilacinus; Streptomyces roseolus; Streptomyces roseosporus; Streptomyces roseoviolaceus; Streptomyces roseoviridis; Streptomyces ruber Streptomyces rubidus; Streptomyces rubiginosohelvolus; Streptomyces rubiginosus; Streptomyces rubrisoli; Streptomyces rubrogriseus; Streptomyces rubrus; Streptomyces rutgersensis; Streptomyces samsunensis; Streptomyces sanglieri; Streptomyces sannanensis; Streptomyces sanyensis; Streptomyces sasae; Streptomyces scabiei; Streptomyces scabrisporus; Streptomyces sclerotialus; Streptomyces scopiformis; Streptomyces scopuliridis; Streptomyces sedi; Streptomyces seoulensis; Streptomyces seranimatus; Streptomyces seymenliensis; Streptomyces shaanxiensis; Streptomyces shenzhenensis; Streptomyces showdoensis; Streptomyces silaceus; Streptomyces sindenensis; Streptomyces sioyaensis; Streptomyces smyrnaeus; Streptomyces sodiiphilus; Streptomyces somaliensis; Streptomyces sudanensis; Streptomyces sparsogenes; Streptomyces sparsus; Streptomyces specialis; Streptomyces spectabilis; Streptomyces speibonae; Streptomyces speleomycini; Streptomyces spinoverrucosus; Streptomyces spiralis; Streptomyces spiroverticillatus; Streptomyces spongiae; Streptomyces spongiicola; Streptomyces sporocinereus; Streptomyces sporoclivatus; Streptomyces spororaveus; Streptomyces sporoverrucosus; Streptomyces staurosporininus; Streptomyces stelliscabiei; Streptomyces stramineus; Streptomyces subrutilus; Streptomyces sulfonofaciens; Streptomyces sulphureus; Streptomyces sundarbansensis; Streptomyces synnematoformans; Streptomyces tacrolimicus; Streptomyces tanashiensis; Streptomyces tateyamensis; Streptomyces tauricus; Streptomyces tendae; Streptomyces termitum; Streptomyces thermoalcalitolerans; Streptomyces thermoautotrophicus; Streptomyces thermocarboxydovorans; Streptomyces thermocarboxydus; Streptomyces thermocoprophilus; Streptomyces thermodiastaticus; Streptomyces thermogriseus; Streptomyces thermolineatus; Streptomyces thermospinosisporus; Streptomyces thermoviolaceus; Streptomyces thermovulgaris; Streptomyces thinghirensis; Streptomyces thioluteus; Streptomyces torulosus; Streptomyces toxytricini; Streptomyces tremellae; Streptomyces tritolerans; Streptomyces tricolor, Streptomyces tsukubensis; Streptomyces tubercidicus; Streptomyces tuirus; Streptomyces tunisiensis; Streptomyces turgidiscabies; Streptomyces tyrosinilyticus; Streptomyces umbrinus; Streptomyces variabilis; Streptomyces variegatus; Streptomyces varsoviensis; Streptomyces verticillus; Streptomyces vastus; Streptomyces venezuelae; Streptomyces vietnamensis; Streptomyces vinaceus; Streptomyces vinaceusdrappus; Streptomyces violaceochromogenes; Streptomyces violaceolatus; Streptomyces violaceorectus; Streptomyces violaceoruber Streptomyces violaceorubidus; Streptomyces violaceus; Streptomyces violaceusniger Streptomyces violarus; Streptomyces violascens; Streptomyces violens; Streptomyces virens; Streptomyces virginiae; Streptomyces viridis; Streptomyces viridiviolaceus; Streptomyces viridobrunneus; Streptomyces viridochromogenes; Streptomyces viridodiastaticus; Streptomyces viridosporus; Streptomyces vitaminophilus; Streptomyces wedmorensis; Streptomyces wellingtoniae; Streptomyces werraensis; Streptomyces wuyuanensis; Streptomyces xanthochromogenes; Streptomyces xanthocidicus; Streptomyces xantholiticus; Streptomyces xanthophaeus; Streptomyces xiamenensis; Streptomyces xinghaiensis; Streptomyces xishensis; Streptomyces yaanensis; Streptomyces yanglinensis; Streptomyces yangpuensis; Streptomyces yanii; Streptomyces yatensis; Streptomyces yeochonensis; Streptomyces yerevanensis; Streptomyces yogyakartensis; Streptomyces yokosukanensis; Streptomyces youssoufiensis; Streptomyces yunnanensis; Streptomyces zagrosensis; Streptomyces zaomyceticus; Streptomyces zhaozhouensis; Streptomyces zinciresistens; or Streptomyces ziwulingensis. As another example, the microorganism can be a streptomyces species with azinothricin as the founding member, Steptomyces flaveolus DSM 9954, Streptomyces MK498-98F14 strain, Steptomyces sp. RJA2928, Streptomyces hygroscopicus strain ATCC 53653, Streptomyces lycidus (strain HKI0343), Streptomyces strain CNQ-593, Streptomyces sp. (A92-308110), or Streptomyces himastatinicus ATCC 53653. As another example, the microorganism can be a Streptomyces strain BB10EC, ES09EC, LM04EC, CS08EC, CM04EC, PF8EC, MRY08EC, LM08EC, JMO5EC, BB04EC, PF1EC, PF5EC, JV594, or JV596.
  • As another example, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus, Corynebacterium. As another example, the Corynebacterium can be of the species Corynebacterium glutamicum. As another example, the Corynebacterium can be of the species Corynebacterium efficiens, Corynebacterium diphtheriae group, Corynebacterium xerosis, Corynebacterium striatum, Corynebacterium minutissimum, Corynebacterium amycolatum, Corynebacterium glucuronolyticum, Corynebacterium argentoratense, Corynebacterium matruchotii, Corynebacterium glutamicum, Corynebacterium sp., Nonfermentative corynebacteria, Corynebacterium afermentans subsp. Afermentans, Corynebacterium auris, Corynebacterium pseudodiphtheriticum, Corynebacterium propinquum, Corynebacterium uropygiale, Corynebacterium jeikeium, Corynebacterium urealyticum, Corynebacterium afermentans subsp. lipophilum, Corynebacterium accolens, Corynebacterium macginleyi, CDC coryneform groups F-1 and G, Corynebacterium bovis, or Corynebacterium kroppenstedtii.
  • As another example, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus, Kutzneria. As another example, the Kutzneria can be of the species Kutzneria spp. 744, Kutzneria albida, Kutzneria kofuensis, Kutzneria viridogrisea), (see e.g., Neuman et al. 2012 13(7) 972-976). Kutzneria were previously known to be in the family of Streptosporangiaceae (suborder Streptosporangineae) and were known as Streptosporangium albidum, Streptosporangium viridogriseum (subspecies kofuense), or Streptosporangium viridogriseum.
  • As described herein, an Actinobacteria that can be used in the biosynthesis of piperazic acid or piperazic acid derivatives can be of the genus Actinomadura. As an example, the Actinomadura can be of the species Actinomadura luzonensis, Actinomadura dassonvillei, Actinomadura madurae, Actinomadura pelletieri, Actinomadura sputi, Actinomadura meyerae, Actinomadura hibisca, Actinomadura pusilla, A. fastidiosa, A. ferruoinea, A. helvata, A. kijaniata, A. libanotica, A. roseola, A. roseoviolacea, A. rubra., A. salmonea, or A. spiralis.
  • As described herein, the microorganism can be a fungi. For example, the gene can be refactored and insterted into eukaryal vectors for yeast or fungal expression. In fact, some fungi also encode functionally orthologous PzbA enzymes (SidA). In some embodiments, the microorganism can be in the Phylum, Ascomycota or the genus, Aspergillus. As an example, the species can be Aspergillus caesiellus, Aspergillus candidus, Aspergillus carneus, Aspergillus clavatus, Aspergillus deflectus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus israelii, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae, Aspergillus sydowii, Aspergillus tamari, Aspergillus terreus, Aspergillus ustus, or Aspergillus versicolor.
  • In some embodiments, transformed microorganisms, as described herein, can accumulate at least about 1 μM to at least about 1 M L-Piz. For example, in some embodiments, transformed microorganisms can accumulate about 1 μM; about 10 μM; about 20 μM; about 30 μM; about 40 μM; about 50 μM; about 60 μM; about 70 μM; about 80 μM; about 90 μM; about 100 μM; about 110 μM; about 120 μM; about 130 μM; about 140 μM; about 150 μM; about 160 μM; about 170 μM; about 180 μM; about 190 μM; about 200 μM; about 210 μM; about 220 μM; about 230 μM; about 240 μM; about 250 μM; about 260 μM; about 270 μM; about 280 μM; about 290 μM; about 300 μM; about 310 μM; about 320 μM; about 330 μM; about 340 μM; about 350 μM; about 360 μM; about 370 μM; about 380 μM; about 390 μM; about 400 μM; about 410 μM; about 420 μM; about 430 μM; about 440 μM; about 450 μM; about 460 μM; about 470 μM; about 480 μM; about 490 μM; about 500 μM; about 510 μM; about 520 μM; about 530 μM; about 540 μM; about 550 μM; about 560 μM; about 570 μM; about 580 μM; about 590 μM; about 600 μM; about 610 μM; about 620 μM; about 630 μM; about 640 μM; about 650 μM; about 660 μM; about 670 μM; about 680 μM; about 690 μM; about 700 μM; about 710 μM; about 720 μM; about 730 μM; about 740 μM; about 750 μM; about 760 μM; about 770 μM; about 780 μM; about 790 μM; about 800 μM; about 810 μM; about 820 μM; about 830 μM; about 840 μM; about 850 μM; about 860 μM; about 870 μM; about 880 μM; about 890 μM; about 900 μM; about 910 μM; about 920 μM; about 930 μM; about 940 μM; about 950 μM; about 960 μM; about 970 μM; about 980 μM; about 990 μM; or about 1000 μM. Recitation of each of these discrete values is understood to include ranges between each value. Recitation of each of a range is understood to include discrete values within the range.
  • In some embodiments, transformed microorganisms, as described herein, can accumulate between at least about 1 mg and at least about 3 mg of Piz or Piz derivatives (e.g., L-Piz, see e.g., Examples 4 or 14) per liter in about 3 days (or at least about 14 μg/L per hour or at least about 0.2 μg/L per minute). In some embodiments, transformed microorganisms can accumulate at least about 0.1 μg up to about 10 μg of a Piz or Piz derivatives (e.g., L-Piz) per minute per L. For example, transformed microorganisms can accumulate at least about 0.1 μg, at least about 0.2 μg, at least about 0.3 μg, at least about 0.4 μg, at least about 0.5 μg, at least about 0.6 μg, at least about 0.7 μg, at least about 0.8 μg, at least about 0.9 μg, or at least about 1 μg of Piz or Piz derivatives (e.g., L-Piz) per minute per L. In other embodiments, various transformed microorganisms accumulate similar amounts of Piz or Piz derivatives (e.g., L-Piz). Recitation of each of these discrete values is understood to include ranges between each value. Recitation of each of a range is understood to include discrete values within the range.
  • Hydroxylase, Cyclase, and Dehydratase
  • A microorganism (e.g., the bacteria, Streptomyces lividans) can be transformed so as to have hydroxylase, cyclase, or dehydratase activity (e.g., L-Ornithine N5-hydroxylase, L-Ornithine cyclase, L-Ornithine dehydratase activity).
  • Hydroxylase (e.g., L-Ornithine N5-hydroxylase) activity can be engineered into a microorganism by way of one or more individual genes encoding a polypeptide having hydroxylase (e.g., L-Ornithine N5-hydroxylase) activity. It is contemplated these activities can likewise be engineered in other microorganisms.
  • Cyclase (e.g., L-Ornithine N5-cyclase) activity or dehydratase (e.g., L-Ornithine N5-dehydratase) activity can be engineered into a microorganism by way of one or more of the individual genes. For example, cyclase (e.g., L-Ornithine N5-cyclase) activity or dehydratase (e.g., L-Ornithine N5-dehydratase) activity can be engineered into a microorganism by way of one or more genes encoding a polypeptide having cyclase (e.g., L-Ornithine N5-cyclase) activity or encoding a polypeptide having dehydratase (e.g., L-Ornithine N5-dehydratase) activity; or by one gene encoding both cyclase (e.g., L-Ornithine N5-cyclase) and dehydratase (e.g., L-Ornithine N5-dehydratase). For example, L-Ornithine N5-cyclase activity and L-Ornithine N5-dehydratase activity can be present in a polypeptide or a fusion polypeptide. It is contemplated these activities can likewise be engineered in other microorganisms.
  • The Piz (e.g., L-Piz) can be endogenous or exogenous to the microorganism. Where the Piz is endogenous, the microorganism can be engineered to produce increased levels of Piz. Where Piz is exogenous, the microorganism can be engineered to produce such exogenous Piz.
  • The microorganism can be engineered to synthesize and accumulate the desired Piz continuously, after some developmental state, or upon being induced to do so. Induction of Piz synthesis can be according to the actions of an inducible promoter associated with the encoded hydroxylase, cyclase, or dehydratase and an inducing agent, as discussed in further detail herein. Also, the promoters as recited herein are only as examples of useful promoters. It is contemplated to adjust copy number (e.g., plasmid as self replicating high copy, low copy, or chromosomally insertional), in conjunction with promoters driving high, medium, or low expression of pzbA and pzbB combinations.
  • Radiolabeled
  • One embodiment of the present disclosure provides for a radiolabeled compound. The composition can be Piz, a Piz derivative, or a Piz-containing compound. According to another embodiment, the radiolabeled compound can be for use as a drug discovery agent or an imaging agent.
  • References herein to “radiolabeled” include a compound where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). One non-limiting exception is 19F, which allows detection of a molecule which contains this element without enrichment to a higher degree than what is naturally occurring. Compounds carrying the substituent 19F may thus also be referred to as “labelled” or the like. The term radiolabeled may be interchangeably used with “isotopically-labelled”, “labelled”, “isotopic tracer group”, “isotopic marker”, “isotopic label”, “detectable isotope”, or “radioligand”.
  • In one embodiment, the compound comprises a single radiolabeled group.
  • Examples of suitable, non-limiting radiolabel groups can include: 2H (D or deuterium), 3H (T or tritium), 11C, 13C, 14C 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl, 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I, or 131I. It is to be understood that an isotopically labeled compound needs only to be enriched with a detectable isotope to, or above, the degree which allows detection with a technique suitable for the particular application, e.g., in a detectable compound labeled with 11C, the carbon-atom of the labeled group of the labeled compound may be constituted by 12C or other carbon-isotopes in a fraction of the molecules. The radionuclide that is incorporated in the radiolabeled compounds will depend on the specific application of that radiolabeled compound. For example, “heavy” isotope-labeled compounds (e.g., compounds containing deuterons/heavy hydrogen, heavy nitrogen, heavy oxygen, heavy carbon) can be useful for mass spectrometric and NMR based studies. As another example, for in vitro labelling or in competition assays, compounds that incorporate 3H, 14C, or 125I can be useful. For in vivo imaging applications 11C, 13C, 18F, 19F, 120I, 123I, 131I, 75Br, or 76Br can generally be useful. In one embodiment, the radiolabel is 11C. In an alternative embodiment, the radiolabel is 14C. In a yet further alternative embodiment, the radiolabel is 13C.
  • Molecular Engineering
  • A gene of particular interest for engineering a microorganism to accumulate Piz or Piz derivative is the active pzbB gene from Streptomyces flaveolus (see e.g., Example 3). Another gene of interest for engineering a microorganism to accumulate Piz is the active pzbA gene. As shown herein, pzbA is natively encoded on the S. lividans chromosome. But pzbA or pzbB can be expressed in another host that does not natively express the pzbA or pzbB gene or the host can be engineered to carry more than one copy of the a non-natively expressed pzbA or pzbB gene.
  • In some embodiments, an pzbA- or pzbB-encoding nucleotide sequence is cloned from its native source (e.g., Streptomyces flaveolus, S. lividans) and inserted into a host microorganism (see e.g., Example 3). In some embodiments, a transformed host microorganism comprises a pzbA or pzbB polynucleotide of SEQ ID NO: 177-SEQ ID NO: 178 (pzbA) or SEQ ID NO: 179-SEQ ID NO: 181 (pzbB). In some embodiments, a microorganism is transformed with a nucleotide sequence encoding pzbA or pzbB polypeptide of SEQ ID NO: 1-SEQ ID NO: 81 or SEQ ID NO: 82-SEQ ID NO: 166. In some embodiments, a transformed host microorganism comprises a pzbA and pzbB polynucleotides of SEQ ID NO: 167-SEQ ID NO: 176.
  • In some embodiments, a transformed host microorganism comprises a nucleotide sequence having at least about 25% sequence identity to SEQ ID NO: 177-SEQ ID NO: 178 or a nucleotide sequence encoding a polypeptide having L-Ornithine N5 hydroxylase activity and at least about 80% sequence identity to SEQ ID NO: 1-SEQ ID NO: 81. As an example, a transformed host microorganism, such as a bacterium, can comprise a nucleotide sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 177-SEQ ID NO: 178, wherein the transformed host exhibits L-Ornithine N5 hydroxylase activity, pzbA activity, and/or accumulation of Piz. As an example, a transformed host microorganism can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 1-SEQ ID NO: 81, wherein the transformed host exhibits L-Ornithine N5 hydroxylase activity, pzbA activity and/or accumulation of Piz. As another example, a transformed host microorganism can comprise a nucleotide sequence that hybridizes under stringent conditions to SEQ ID NO: 177-SEQ ID NO: 178 over the entire length of SEQ ID NO: 177-SEQ ID NO: 178, and which encodes an active pzbA polypeptide. As a further example, a transformed host microorganism can comprise the complement to any of the above sequences.
  • In some embodiments, a transformed host microorganism comprises a nucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 179-SEQ ID NO: 181 or a nucleotide sequence encoding a polypeptide having L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity and at least about 80% sequence identity to SEQ ID NO: 82-SEQ ID NO: 166. As an example, a transformed host microorganism, such as a bacterium, can comprise a nucleotide sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 179-SEQ ID NO: 181, wherein the transformed host exhibits L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity, or pzbB activity and/or accumulation of Piz. As an example, a transformed host microorganism can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 82-SEQ ID NO: 166, wherein the transformed host exhibits L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity, or pzbB activity and/or accumulation of Piz. As another example, a transformed host microorganism can comprise a nucleotide sequence that hybridizes under stringent conditions to SEQ ID NO: 179-SEQ ID NO: 181 over the entire length of SEQ ID NO: 179-SEQ ID NO: 181, and which encodes an active pzbB polypeptide. As a further example, a transformed host microorganism can comprise the complement to any of the above sequences.
  • In some embodiments, L-Ornithine N5 hydroxylase (see e.g., SEQ ID NO: 177-SEQ ID NO: 178 encoding pzbA gene and SEQ ID NO: 1-SEQ ID NO: 81 encoding pzbA polypeptide), or homologue thereof, is engineered to be expressed or overexpressed in a transformed microorganism. For example, a microorganism can be transformed with a nucleotide having a sequence of 1SEQ ID NO: 177-SEQ ID NO: 178 so as to express L-Ornithine N5 hydroxylase. As another example, a microorganism can be transformed with a nucleotide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to SEQ ID NO: 177-SEQ ID NO: 178 encoding a polypeptide having L-Ornithine N5 hydroxylase activity. As another example, a transformed host microorganism can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 1-SEQ ID NO: 81, wherein the transformed host exhibits L-Ornithine N5 hydroxylase activity, pzbA activity, and/or accumulation of Piz.
  • In some embodiments, L-Ornithine N5 cyclase or L-Ornithine N5 dehydratase (see e.g., SEQ ID NO: 179-SEQ ID NO: 181 encoding pzbB gene and SEQ ID NO: 82-SEQ ID NO: 166 encoding pzbB polypeptide), or homologue thereof, is engineered to be expressed or overexpressed in a transformed microorganism. For example, a microorganism can be transformed with a nucleotide having a sequence of SEQ ID NO: 179-SEQ ID NO: 181 so as to express L-Ornithine N5 cyclase or L-Ornithine N5 dehydratase. As another example, a microorganism can be transformed with a nucleotide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to SEQ ID NO: 179-SEQ ID NO: 181 encoding a polypeptide having L-Ornithine N5 hydroxylase activity. As another example, a transformed host microorganism can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 82-SEQ ID NO: 166, wherein the transformed host exhibits L-Ornithine N5 cyclase activity, L-Ornithine N5 dehydratase activity, pzbB activity, and/or accumulation of Piz.
  • In some embodiments, a microorganism (e.g., a bacterium) is engineered to express one or more of pzbA, pzbB, L-Ornithine N5 hydroxylase, L-Ornithine N5 cyclase, or L-Ornithine N5 dehydratase.
  • Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities to an pzbA or pzbB sequence and retaining a required activity of the expressed protein and/or Piz accumulation phenotype is within the skill of the art.
  • The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
  • The terms “heterologous DNA sequence”, “exogenous DNA segment” or “heterologous nucleic acid,” as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
  • Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
  • A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. In some embodiments, the promoter is iducible by an agent selected from the group consisting of temperature, pH, a metabolite, light, an osmotic agent, a heavy metal, and an antibiotic. In some embodiments, the promoter is selected from the group consisting of a constitutive promoter to produce L-Piz.
  • A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • A “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
  • The “transcription start site” or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative.
  • “Operably-linked” or “functionally linked” refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
  • A “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
  • A constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3′ transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3′-untranslated region (3′ UTR). Constructs can include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
  • The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
  • “Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term “untransformed” refers to normal cells that have not been through the transformation process.
  • “Wild-type” refers to a virus or organism found in nature without any known mutation.
  • Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide (e.g. pzbA, pzbB) and/or polypeptide (e.g., pzbA, pzbB) variants having, for example, at least 95%-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
  • Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
  • Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gin by Asn, Val by lie, Leu by lie, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
  • “Highly stringent hybridization conditions” are defined as hybridization at 65° C. in a 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65° C. in the salt conditions of a 6×SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65° C. in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm=81.5° C.+16.6(log10[Na+])+0.41 (fraction G/C content)−0.63(% formamide)−(600/l). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1-1.5° C. for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).
  • Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
  • Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
  • Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, C., et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, Tex.; Sigma Aldrich, Mo.; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3′ overhangs.
  • Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
  • In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
  • In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
  • The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
  • All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
  • Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.
  • Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
    • EXAMPLES
  • The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
    • Example 1: Discovery of the Complete Biosynthetic Pathway to L-Piz from the Central Metabolite L-Orn
  • The following example describes the discovery of the complete biosynthetic pathway to L-Piz from the central metabolite, L-Orn.
  • Select examples of piperazic acid (Piz) family of natural products are shown in FIG. 2. Piz and modified Piz (e.g., dehydropiperazic, chloropiperazic, hydroxypiperazic acid) molecular components are shown in red in FIG. 2. All of these molecules are bioactive, with sanglifehrin (top left of FIG. 2) under consideration as an immunosuppressant and Hepatitis-C antiviral. The small molecule in the center of FIG. 2 (Sch 382583) is a member of an emerging group of Piz containing metalloprotease inhibitors with clinical relevance as metastatic cancer and antibacterial antibiotic leads. All of these molecules are exclusively produced by actinobacteria.
  • Figure US20190002936A1-20190103-C00021
  • Figure US20190002936A1-20190103-C00022
  • Orthologs of both PzbA (yellow) and PzbB (red) were found within biosynthetic gene clusters for known Piz-containing antibiotics (see e.g., FIG. 2). As these clusters encode molecules that are structurally dissimilar except for the incorporation of Piz, parsimony suggests both pzbA (previously known) and pzbB (previously unrecognized) are involved in Piz biosynthesis.
  • In vitro reconstitution of L-Piz production from L-Orn in a coupled enzymatic reaction containing purified PzbA, PzbB, buffer salts, NADPH cofactor, Fe+2 salts, and catalytic FAD (Flavin Adenine Dinucleotide) cofactor according to Scheme 2.
  • FIG. 2 shows the HPLC-ESI-MS detection of products and substrates with assay time points at time 0 min, 15 min, and 30 min showing the consumption of L-Orn, accumulation of the known intermediate N5—OH-Orn, and the concomitant formation of Piz. Data (not shown) in the same assay lacking PzbB, the enzyme product is N5—OH-Orn and no Piz is formed.
    • Example 2: Green Biocatalysis of L-Piz In Vitro
  • L-Piz can be synthesized chemically, but to date a fermentative pathway to the amino acid has eluded researchers. Enantiopure synthetic L-Piz is expensive: ($2800/gram, 95% pure). DL-Piz synthesized as a mix of isomers, which is significantly less chemically desirable, is less expensive ($800/gram, 95% pure), but still of significant cost. Using a coupled enzyme assay containing a suitable L-Ornithine N5—OHase (PzbA), and a suitable PzbB (L-N5—OH Orn cyclase/dehydratase), enantiopure (as currently understood) L-Piz can be made from the inexpensive feedstock enantiopure L-Ornithine ($1.40/gram, >99% pure, Sigma-Aldrich), buffer salts, NADPH cofactor, Fe+2 salts, and catalytic FAD (Flavin Adenine Dinucleotide) cofactor (see e.g., FIG. 3).
    • Example 3: An Enzymatic Route to Heavy Isotope-Labelled Piz
  • Heavy isotope-labeled compounds (e.g., compounds containing deuterons/heavy hydrogen, heavy nitrogen, heavy oxygen, heavy carbon) are valuable tools for mass spectrometric and NMR based studies. Currently, no vendors, custom or otherwise, that offer L-Piz having any combination of these isotopes. Using d7-L-Orn, the feasible production of d7 L-Piz using the reaction described in Example 2 above has been demonstrated. In principle, any heavy isotope labeled L-Orn could yield similarly labeled L-Piz. Coupled PzbA/PzbB enzymatic reactions could be scaled to produce and market variously heavy isotopically labeled or radioisotopically labeled versions of L-Piz, for which there are current no known synthetic paths.
    • Example 4: Green Biocatalysis of L-Piz In Vivo
  • This example shows a greener production of L-Piz (no organic solvents and fewer reagents than conventional methods).
  • Micro-organisms such as bacteria and fungi are preferred producers of amino acids in the biotechnology industry. This is because the cellular enzyme catalysts of life are typically stereospecific, giving enantiopure products. Enantiopurity can be more difficult to achieve in synthetic chemistry. Also, inexpensive feedstocks are provided for growth, significantly reducing the cost of amino acid production in contrast to fine chemical starting points often required for synthetic chemistry. Here, L-Piz fermentation in a heterologous, genetically engineered host (Streptomyces lividans) grown on standard lab media, and with no investment in yield optimization (see e.g., FIG. 4) has been demonstrated.
  • S. lividans (WT parent, no Piz production) is compared against S. lividans harboring a single copy of pzbA (sfaB) alone, pzbB (sfaC) alone, or co-expressing pzbA and pzbB (sfaBC) cloned from the sanglifehrin biosynthetic locus of Streptomyces flaveolus in FIG. 4. LC/MS detection of biosynthetic Piz was compared against an authentic L-Piz standard (top row, FIG. 4). In contrast with the in vitro data in FIG. 4, pzbA is dispensable in the heterologous system because S. lividans encodes a native copy of the gene as part of a siderophore biosynthetic pathway unrelated to Piz production. Thus, pzbA remains required for Piz production, but its role in bacteria is not limited to Piz anabolism. In contrast, to our knowledge, pzbB is only found associated with Piz production.
  • Using a mass-spectrometric (MS/MS) method for sensitive quantification, it was estimated that S. lividans is carrying at minimum a single copy of a suitable pzbB gene (one or more native pzbA's are natively encoded on the S. lividans chromosome, and therefore is not absolutely required for heterologous expression) under a constitutive promoter to produce micromolar L-Piz. Measurably higher (˜1 mM) L-Piz titers can be achieved using a heterologous S. lividans producer carrying one or more copies of a non-native pzbA in conjunction with heterologous pzbB. S. lividans serves as a proof of concept host, not necessarily an industrial endpoint. Much higher L-Piz production can likely be achieved by expressing suitable pzbA and pzbB genes in a heterologous host that overproduces the critical feedstock L-Ornithine. One such candidate host is the actinobacterial industrial producer of L-Orn, Corynebacterium glutamicum (20.8-51.5 grams/liter). Importantly, at least one such industrial L-Orn producing strain is publicly available through the American Type Culture Collection (ATCC), making strain engineering from a high producer feasible.
  • L-Piz Fermentation Production Rate.
  • The following describes the rate of fermented L-Piz in heterologous hosts (Streptomyces lividans), plated in 1 L. S. lividans makes at least 1 mg/L plates in 3 days. This translates to ˜14 μg/L per hour or 0.2 μg/L per minute.
    • Example 5: Directed Discovery of Drugs and Drug-Like Compounds Using Heavy Isotope L-Piz
  • This example shows how newfound ability to recognize biosynthetic genes encoding Piz-derived small molecules (e.g., isotopically labeled Piz compound) can facilitate genomic discovery of new natural products that can be used as drug leads.
  • Current technologies can only enable a rough estimate what the final chemical structures encoded by these biosynthetic genes are. To link biosynthetic genes to the compounds they produce, especially in the case of L-Piz containing compounds, supplying d7-L-Orn to microorganisms of interest can link the biosynthetic compounds to the produced compounds. Some percentage of this labeled compound is expected to become d7 L-Piz in cellulo, and consequently become incorporated into the natural products that will be discovered.
  • Differential mass spectrometry allows for the detection of the labeled compounds in a much more specific way than absence of such a technology. However, L-Orn can be incorporated into many natural compounds, confusing the analyses. Isotopically labeled L-Piz would be a much more useful molecular probe for the specific and directed discovery of L-Piz-containing drug leads compared to labeled L-Orn for the reasons above.
  • Data indicating L-Piz successfully penetrates at least one Piz-compound producing actinomycete was obtained, followed by subsequent incorporation into a Piz drug-like compound sanglifehrin (see e.g., FIG. 5). FIG. 5 shows LC/MS detection of sanglifehrin, a Piz-containing compound produced by Streptomyces flaveolus. Four major isobaric isomers of sanglifehrin A detected in WT S. flaveolus fermentation extracts. As expected from the results in FIG. 5, an unmarked gene deletion of pzbB (sfaC) from S. flaveolus abrogates sanglifehrin production. Genetic complementation of this mutant with an additional copy of pzbB, or exogenously supplied 50 μM authentic L-Piz (top, FIG. 5), restore the production of the four sanglifehrin A isobars. L-Piz is therefore cell penetrant and qualitatively nontoxic. These data (see e.g., FIG. 5) additionally link pzbB function with Piz production in vivo, which agrees with the in vitro assay data.
  • Thus, it is expected that isotopically labeled L-Piz will penetrate cells and label Piz compounds without significant complications from poor cell penetrance, transport, or toxicity.
    • Example 6: Characterization of L-Piperazic Acid Sterochemistry
  • The following example describes the characterization of the synthesized piperazic acid compound. It was shown that the product is an L-Piz and is enantiomerically pure.
  • FIG. 6 shows the Marfey's derivatization analysis of the product of PzbB in an assay with L-N5 hydroxy Ornithine substrate (the product of PzbA). This conclusively shows the product of PzbB has the same stereochemistry (L) and mass as the same derivative produced using L-Piz authentic standard (see e.g., FIG. 6).
    • Example 7: PzbB Ortholog Identity with PzbB Activity
  • The following example shows that a PzbB ortholog can have as little as around 25% sequence identity to another PzbB ortholog and still produce L-Piz or retain PzbB activity.
  • Bioinformatic data showed PzbB orthologs that can be used to produce L-Piz have an estimated protein identity (functional cutoff) to be around 25% (some predicted PzbB orthologs have identity scores in the 30% range and most have 45% or above.
    • Example 8: SfaBC (Co-Expressing pzbA and pzbB) Combined Ornithine In Vitro Assay Method
  • 100 μL of reaction in 50 mM Tris.HCl at pH 8.0 was set up with L-orn (500 μM), FAD (50 μM), His6-SfaB (10 μM), SfaC-His6 (135 μM), NADPH (2 mM), and FeSO4 (10 mM). 30 μL aliquots were removed at 0 min, 15 min, and 30 min, and combined with 30 μL acetonitrile. The cloudy mixture was centrifuged, and 30 μL of the supernatant was acidified with 3 μL 2 M HCl. Sample was analyzed for piperazic acid by HPLC/MS. Analysis was performed using an Imtakt Intrada Amino Acid column (50×3 mm, 3 μm pore size) installed on an Agilent 1260 Infinity HPLC connected to an Agilent 6420 Triple-Quad mass spectrometer using the following method: T=0, 0% B; T=2, 0% B; T=8, 100% B; T=14, 100% B; A: water (30%)/methanol (70%)+0.3% formic acid, B: water+100 mM ammonium formate; 0.4 mL/min. A novel peak at T=5.4 min eluted with a [M+H]+ of 131, corresponding to piperazic acid.
    • Example 9: SfaBC (Co-Expressing pzbA and pzbB) Combined D7-Ornithine In Vitro Assay Method
  • 100 μL of reaction in 50 mM Tris.HCl at pH 8.0 was set up with d7-L-orn (500 μM), FAD (50 μM), His6-SfaB (10 μM), SfaC-His6 (135 μM), NADPH (2 mM), and FeSO4 (10 mM). 30 μL aliquots were removed at 0 min, 15 min, and 30 min, and combined with 30 μL acetonitrile. The cloudy mixture was centrifuged, and 30 μL of the supernatant was acidified with 3 μL 2 M HCl. Sample was analyzed for piperazic acid by HPLC/MS. Analysis was performed using an Imtakt Intrada Amino Acid column (50×3 mm, 3 μm pore size) installed on an Agilent 1260 Infinity HPLC connected to an Agilent 6420 Triple-Quad mass spectrometer using the following method: T=0, 0% B; T=2, 0% B; T=8, 100% B; T=14, 100% B; A: water (30%)/methanol (70%)+0.3% formic acid, B: water+100 mM ammonium formate; 0.4 mL/min. A novel peak at T=5.4 min eluted with a [M+H]+ of 138, corresponding to piperazic acid.
    • Example 10: PzbAB (Amycolatopsis alba) Ornithine In Vitro Assay
  • 100 μL of reaction in 50 mM Tris.HCl at pH 7.0 was set up with L-orn (500 μM), FAD (50 μM), PzbAB (Amycolatopsis alba) (14 μM), NADPH (2 mM), and FeSO4 (10 mM). 30 μL aliquots were removed at 0 min, 15 min, and 30 min, and combined with 30 μL acetonitrile. The cloudy mixture was centrifuged, and 30 μL of the supernatant was acidified with 3 μL 2 M HCl. Sample was analyzed for piperazic acid by HPLC/MS. Analysis was performed using an Imtakt Intrada Amino Acid column (50×3 mm, 3 μm pore size) installed on an Agilent 1260 Infinity HPLC connected to an Agilent 6420 Triple-Quad mass spectrometer using the following method: T=0, 0% B; T=2, 0% B; T=8, 100% B; T=14, 100% B; A: water (30%)/methanol (70%)+0.3% formic acid, B: water+100 mM ammonium formate; 0.4 mL/min. A novel peak at T=5.4 min eluted with a [M+H]+ of 131, corresponding to piperazic acid.
    • Example 11: SfaC (Expressing pzbB) N5—OH-L-Ornithine In Vitro Assay
  • 100 μL of reaction in 50 mM Tris.HCl at pH 8.0 was set up with N5—OH-L-orn (1 mM), His6-SfaC (18 μM). 30 μL aliquots were removed at 0 min, 10 min, and 20 min, and combined with 30 μL 6% 5-sulfosalicylic acid. The cloudy mixture was centrifuged, and the supernatant was used for analysis. Sample was analyzed for piperazic acid by HPLC/MS. Analysis was performed using an Imtakt Intrada Amino Acid column (50×3 mm, 3 μm pore size) installed on an Agilent 1260 Infinity HPLC connected to an Agilent 6420 Triple-Quad mass spectrometer using the following method: T=0, 86% B; T=3, 86% B; T=10, 0% B; T=11, 0% B; T=12, 86% B; T=14, 86% B; A: water+100 mM ammonium formate, B: acetonitrile+0.1% formic acid; 0.6 mL/min. A peak at 5.6 min with a [M+H]+ of 131, corresponded to piperazic acid.
    • Example 12: Marfey's Analysis of SfaC (Expressing pzbB) Product
  • The following example confirms that the product of PzbAB from L-Orn is actually L-Piz. Marfey's analysis was performed on the PzbAB reaction product and compared the results with synthetic L-Piz standard. The data so far are consistent with the PzbAB reaction yielding an enantiopure L-Piz.
  • 100 μL of reaction in 50 mM Tris.HCl at pH 8.0 was set up with N5—OH-L-orn (1 mM), His6-SfaC (20 μM), hemin (20 μM). Reaction was allowed to proceed for a few minutes. A control was also set up in 50 mM Tris.HCl at pH 8.0 with L-Piz (0.25 mg/mL) and hemin (20 μM). To 100 μL of aqueous reaction or control was added 50 μL of 1% FDAA in acetone. The reaction was incubated at 50° C. for 1 hour. 100 μL of 1 M HCl was then added. Finally, 300 μL of water/MeCN (50:50) was added to dissolve the precipitate. The supernatant was filtered (Agilent Captiva Econo Filter, 0.2 μL) into HPLC vials for HPLC/MS analysis.
  • Analysis was performed using a Phenomenex Luna C18 column (75×3 mm, 3 μm pore size) installed on an Agilent 1260 Infinity HPLC connected to an Agilent 6420 Triple-Quad mass spectrometer using the following method: T=0, 10% B; T=5, 10% B; T=25, 100% B; T=27, 100% B, T=29, 10% B, T=30, 10% B; A: water+0.1% formic acid, B: acetonitrile+0.1% formic acid; 0.6 mL/min. 10 μL of the sample was injected per run, and a total ion count chromatogram was obtained for each sample. An extracted ion count chromatogram at m/z 383.1 (monoisotopic mass of protonated FDAA-derivatized piz) was used to detect derivatization. The UV response at 340 nm was also monitored.
    • Example 13: Hemin Influence on SfaC (Expressing pzbB)
  • This example shows that the PzbB's cofactor is now confirmed to be Fe+3-protoporphryin IX (aka hemin). As expected for a bona fide cofactor, adding hemin increases the rate of turnover.
  • 100 μL of reaction in 50 mM Tris.HCl at pH 8.0 was set up with N5—OH-L-orn (1 mM), SfaC-His6 (2 μM), and either hemin in DMSO (10 μM) or just DMSO. The two reactions were incubated at 4° C. for 7 hours. Then, 30 μL aliquots were removed at 30, 60, and 90 sec, and combined with 30 μL 6% 5-sulfosalicylic acid. The cloudy mixture was centrifuged, and the supernatant was used for analysis. Sample was analyzed for piperazic acid by HPLC/MS. Analysis was performed using an Imtakt Intrada Amino Acid column (50×3 mm, 3 μm pore size) installed on an Agilent 1260 Infinity HPLC connected to an Agilent 6420 Triple-Quad mass spectrometer using the following method: T=0, 86% B; T=3, 86% B; T=10, 0% B; T=11, 0% B; T=12, 86% B; T=14, 86% B; A: water+100 mM ammonium formate, B: acetonitrile+0.1% formic acid; 0.6 mL/min. An extracted ion count chromatogram at m/z 131.1 (monoisotopic mass of protonated piperazic acid) was used to detect piperazic acid. For quantification, an SRM transition (m/z 131.1=>56.3; source voltage, 86 V; collision energy, 37 V) was monitored, and a standard curve (second order polynomial, R2=0.9996) was generated between 0.1 μM and 100 μM using a chemically synthesized L-piperazic acid dihydrochloride standard. The concentrations in time were plotted, and fitted to a line. The slope of the line was used as the rate of the reaction. Hemin increased the slope by 14.4 times.
    • Example 14: Fermentative L-Piz Production from Various Streptomyces Strains
  • This example describes L-Piz production from various Streptomyces strains (see e.g., FIG. 7) (methods are as described above unless stated otherwise). Randomly selected environmental Streptomyces isolates were transformed with pYH015 via intergeneric conjugation as described for S. lividans. L-Piz production was quantified via SRM LC/MS from triplicate growths essentially as noted for the S. lividans transformants. Resulting strain to strain Piz production is variable, ranging from very low to nearly the same as S. lividans carrying pYH015 (JV594). Note S. lividans JV 596 expressing both sfaB and sfaC produces more L-Piz, with less titer variability, than all sfaC-alone strains in the panel. L-Piz was not detected in any non-transformed parent strains (not shown).
  • It is noted that the value reported here for for JV596 (˜2.5 mg/L) is higher that what we previously reported (˜1 mg/L).
  • SEQUENCE LISTING
    PzbA
    1. >Mycobacterium_marinum_M
    MQQRLTMWSATGLIFGHALCMNTCRTMVVPRGKPLCIERVPPLPCQPKMGESTMPSGGIA
    DPELALVDRTLSVVGVGFGVTGLALAAALHEAEMTEDALFLESRPKFGWHDDMLIEGSSM
    QVSFLKDIVTMRNPTSRFSFISYLHAMGRLTNFINHGVLTPSRREFADYLRWVARQLDHL
    VRYDVHVTDVRPVYEGATVSALDIVAGENAVVRTRNLVLGTGLRPRMPQGVIPNRRVWHS
    SELLSRLAECGDYLARQIVVVGAGQSAAEIALYLLDRYPDSQVCPVFARYGYSAVDASPF
    ANRIFDPSGVDDFYAASPSVKASLLRYHGNTNYSVVSSDVLGALYRRQYEQSVIGDPRLR
    IFHASRLHLVSFNDDSVVADIEFLPTGEVTRLDTDLVVIYATGYESRDPKHLLTSLAGYL
    RTDELGALRLDRRYRVKTVEGFRCGIFVQGATESTHGIASTLLSVAAVRAGEISQSLMET
    SQARPPAGSVTHRH
    2. >Lentzea_flaviverrucosa_DSM_44664
    VTSEPYDVVGIGFGPSNLSLAIALEETGGLSAAFFEKQDSLRWHSGMLVPGAKMQVSFLK
    DLATPRNPVSSYSFVSYLHDRGRFARFVNNSDFFPTRREFQDYLRWAEARLSPPVHYRAE
    VVSVRRAEGVLRVHVRDTESGATRTVDTRNIVISTGLVPRMPVGLEAGESVWHSSQFLHR
    FHALGDRDVRRVAVVGAGQSAAELVRYLHENLPSAQVFAVLPSYGYAIADSTPFANEVFD
    ADAVDVFYDASDKAKAAIWRYHRNTNYSVVDDEVIRDLYQRAYDDEVRGEPRLRFLPLTR
    VVGAKQDRDGITLLTHSTVDDQARDLPLDLVVCATGYDPMDPGELLAGLGCSVAYDELGR
    HLVGRDHRLVTEPDQDCGIYLQGGTEHTHGLTSSLLSNIAVRGGEITQSILRRRAEQRNG
    APA
    3. >Streptomyces_aureofaciens_ATCC_10762
    VGERQRSGVVAGTGIVDVAGIGFGPSNLALAAAIAEIAGEAPVSARFFEA
    QPRFGWHRGMLIEGATMQVSYLKDLVTMRNPTSPYSFLCYLQARGRLADF
    INTKSPYPLRVEFHDYLEWVAESFADLVSYGARVVSVEPVSAEQGVEFLD
    VHFVAPDGTRQVQRARNLVIAAGIEPRLPAGLPASPRIWHTAKFLPEVDR
    IARQDPRSFVVLGSGQSAAEAIEHLHARFPRAQVHSVHARYGFSVADDSP
    FANQVFNPEAVDRFHTAPDDVRQRLIDYHASTNYSVVDADLLHSLFQQAY
    LEKVAGNPRLNFHNVSRVSEVTETPDGLRIDVESLSSGTSTVIEAQALVC
    ATGYTRTDPAVFLDGLLPHCPLDDQGRLRLDREHRVVTDESVRCGIYVQG
    FGEHSHGLSETLLSLSAVRAGEIGDMLVKALSG
    4. >Streptomyces_diastatochromogenes_NRRL_B-1698
    VNVSEPGSDQVVDVVGIGFGPSNLALAVALGEGGRKASEKPVTSVFFERK
    ERFTWHGGMLIDGATMQISFLKDLVTLRDPRSPYTFLHYLHQVGRLPDFI
    NHKLLFPSRIEFHDYLCWVAESFDHQVRYGADVVDVRPVHSDGAVNHLDV
    VVRHEGPEGERISVQRTRNVVVGTGLEAHMPAGAAPGDRVWHTSELLHKV
    AALKEEPRRIVVVGAGQSAAEATEYLHRRFEAAEICPVFTRYGYSPADDS
    PFANRIFDPLAVDDYYAATPEVKRMLLGYHRNTNYSVVDAELIDELYRRV
    YQEKVQGRHRLKVFNASRLAEVKAGAEGVQVTVESVISRCRTVLDADCVV
    YATGYRPTDVRRLIGGMAGLCKADEMGRLHADRDYRVVTEGDVHCGIYLQ
    GATEHSHGISSSLLSNTAVRAGEIADSIVAGVVGATASE
    5. >Streptomyces_sp._DvalAA-43
    MDASARETYDVVGIGFGPSNLSLAIALEEHEANVPARPISAAFFERQPSF
    GWHRNMLLPAATMQISFLKDLATFRNPVSRYSFIAYLHAADRLVQFVNNQ
    TFFPTRQEFHQYLEWAESSFSDRVSYNSEVTAIRRATGTGPGEPDCLQIE
    VRDGIGGGCRLVHARNVAISTGLVPRMPAGVERDDRVWHSSEFLEKYGQV
    DPNALKSVAVVGAGQSAAEITRFLHDALPHARVFAVVPSYGYSVADDTPF
    ANRVFDPSAVDDYYFGTEQTREAFWRYHRNTNYSVVDDEIIRDLHQRSYD
    EDVRNDRRLHFLNLTRVDDVQRIGTEIRVGLRSLIDVEAQTLDVDALVFA
    TGYGAMQPTGLLGDLDRHCLRDAAGRHRAERDYRLVTTPELSCGIYLQGG
    TEHTHGLTSSLLSNVAVRSGEIADSIVRRRAEEHEPVASLGTSGRTS
    6. >Collimonas_fungivorans_Ter6
    MQVCFLKDLAMLRNPTSPFTFLSYLHDKNRLVDFVNHKILFSSRVEFHDY
    LEWAAAKLKRLVQYDAEVVEVSPVICDGVVKWLDVVVQRDGNPSHHEIYR
    THNLVIAPGLEPTMPPGISRSERVWHSSEVLDRIAHLTEEPQQFTVVGAG
    QSAAEITAYLHDHFKYAKVRSIFSRYGYSAADDSPFTNRIFDPLAVDEYY
    QARDDVKKMLLNFHRNTNYSVVDADLLEDLYRRHYQEMVRGESRLEFMNV
    SKVFGAVADRDSVDLSVEFLPTGDMRKLRSDIVVFGSGYKIADPIRYFSD
    FAGKCIRDSFGQLRVARNYRICTSEDVECGIYLQGTTEHTHGLSSTLLSN
    TAVRAGEILEAMTWERDNKKISSHA
    7. >Streptomyces_reticuli_TUE45
    MTRLAGQAPTAQHSPESEVRDVTGIGFGAANLALAVALHESGAGDRALFL
    EKQKEFGWHRGMLIEGSSLQVSFLKDIATMRNPTSDFGFLSYLQEKGRLV
    DFINQHTLLPSRIEYHDYLQWAADRLGHMVEYGVEATGVRPVTDAGEVVA
    LDVLAGDRVVTRTRNLVIASGLRPRLPEGAETGERVWHSSQLLHRLPAFD
    ERPPRRAVVVGAGQSAAEVAAHLMERYPQAEVCAVFSRYGYSVADSSPFA
    NRVFDPAAVDDFYFAPPEVKQAIMRYHGGTNYAVVDEDVLQGLYRRQYEQ
    KVTGTPRLRVMNASRLVSVEPRGETAAVRVEFLPTGEHADLDADLVVYAT
    GYRSADPAELLGGVAGSLRRDAAGQVLIGRDYRLSTTGDFRCGIYVQGAT
    EATHGIASTLLSMVAVRAGEIAQSIIGGRRDPDRTAGTKAVAGNRG
    8. >Streptomyces_scabiei_NCPPB_4086
    MEAHTDAYEVVGIGFGPSNLSLAIALEEQRGKDEKPLTAAFFEKQASLGW
    HRNMLLPDTKMQISFLKDLATFRNPASQWSFIAYLHAAGRLAQFVNNQNF
    FPTRNEFHDYLDWAESSFSDRVTYNCEVNAVHLPDGYTGGPVDTVRVEVK
    DNTPRGGTRLVEARNLVISTGLVPTMPTGIERGERVWHSSEFLGRFGTLD
    RDRVRRFAVVGAGQSAAEITRYVYDIVPNAEVYAIMPSYGYSIADDTPYA
    NRIFDADAVDDYYGGTDHTRESFWRYHRNTNYGVADDEVIRDLYQRAYDD
    EVARIKRLHLLNLSRVRTVEQTVDGARLTMHSVRDDSTYGLDVDAIVFAT
    GYDSMDPTALLGDLAPHCLRDEEGRLRVERDYRLVTSPDLNVGIYLQGGT
    EHTHGLASALLSNIAIRSGEIADAIAIDLAARQHTTARSTIG
    9. > Kutzneria _albida_DSM_43870
    MQRDYRVVTVPEMRCGIYLQGGTEHTHGLTSSLLSNIVIRTGEITDSIIT
    RRAELNVGERRTVNG
    10. >Streptomyces_albus_ZpM
    MTGPEVYDIVGVGFGPANLALAVALTERGSSTPLRALFLDRNESFSWHPG
    MLIHDATMQVNFLKDLITLRNPASDFSFLSYLKARGRLVDFINHKTFFPT
    RVEFHDYLEWAAGRVGDVVEYGTEVVDVRPVERDGEVVYFDVVGHQQVGG
    VSQAVVCRARNVVVAPGLVPRLPGEASQSERVWHSSELLHRVGDLPTDKR
    MQFVVVGAGQSAAEVVGYLHARYECADVHAVHSRYGYSPADDTPFANRVF
    DPAAVEHFFHAPPSVKDKFFEYHANTNYSVVDVELIEDLYARVYRESVTE
    RRRLHIHGMSELTEVADGPEGLRVSVRFLPDGTTTVLEPDHVVYATGYKP
    ADVNRVIGVVAELCKRDSSGNLRLLHDYRVDMASHVRCGIYLQGGTEHSH
    GITSSLLSNLADRAAEILDSVLAHGGQLSADAAAWEVAS
    11. >Rhodococcus_fascians_02-815
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    12. >Streptomyces_neyagawaensis_NRRL_B-3092
    MEANTEAYEVVGIGFGPANLSLAIALEEQRGKDEKQLTAAFFEKQPSLGW
    HRNMLLPDTKMQISFLKDLATFRNPASQWSFIAYLHAAGRLAQFVNNQNF
    FPTRNEFHDYLEWAESSFSDRVTYNSEVNAVHLPDGHDGGPVDTVRVEVK
    DNGPRGGTRLVEARNLVISTGLVPKMPDGVDRGERVWHSSEFLGRFHTLD
    PSRVRRFAVVGAGQSAAEITRYVYDTIPDAEVYAIMPSYGYSIADDTPYA
    NRIFDADAVDDYYGGTDRTRESFWRYHRNTNYGVADDEVIRDLYQRAYDD
    EVARIKRLHLLNLSRVQRVDQRADGARLTMHSVRDDSVYDLDVDAIVFAT
    GYDSMDPTALLGDLAPYCLRDDEGRLRVERDYRLVTKPELNVGIYLQGGT
    EHTHGLASSLLSNIAIRSGEIADAIAIAIDLASRRHTTV
    13. >Kutzneria_buriramensis_DSM_45791
    MDTRGSETYDVVGIGFGPANLSLAIALEESPQRLTSAFFERQPSLGWHRG
    MLVPAAKMQVAFLKDLVTFRNPTSTFSFVSYLHDRGRLARFVNNQDFFPT
    RREFHDYLEWAESRVSHRVSYQSEVTAMRLPCAQRPGEDDHVEVEVRDRT
    APSGSRTVAARNVVISTGLVPRMPAGLQTDEFVWHSSEFLHKFSRADHSG
    LKRVAVVGAGQSAAEIVRFLYDMLPDANVFAIIPSYGYSIADNTPFANQI
    FDPAAVDDFYAGSDQAKDAIWRYHRNTNYSVVDDEVIKDLYRRQYDDDLG
    RPGRLAFLNLSRVLDVKRVGEDTRVTVHSTATEQAADLDVDVLVCATGYS
    PMEPADLLGDLARYCVYDGDGRYQVDRDYRLVTPDLDCGIYLQGGTEHTH
    GLSSSLLSNIAVRSGEIAASIARRRLSTNGNGVHA
    14. >Streptomyces_yanglinensis_CGMCC_4.2023
    MSNREQTYDVVGIGFGPSNLSLAIALEEFGAHGMENEISSLFLERQPSFG
    WHRNMLLPSATMQISFLKDLVTFRNPTSGFSFIAYLHASGRLPQFVNNQD
    FFPTRQEFHQYLEWAQAQVAGRIEYGAEVTSIRLPSGTAPQEGADRLVLE
    VAEGAGRTGRAVEARNVVISTGLVPSMPAGAERDERVWHSSEFLDKYRRT
    DHRELRRVAVVGAGQSAAEIARFLYDELPHAQVSAIIPSYGYAVADDTPF
    ANRIFDPSAVDDYYFGTEQTRESFWRYHRNTNYSVVDDEVIRDLYRRSYD
    DEVRGVTRLQLLNLTRVTGVKRAGAETRVSLQVGPDAELRELDFDLLVCA
    TGYDGMEPTGLLGELDRYCLRDEAGRYRVERDYRIVTTPELRCGIYLQGG
    TEHTHGLTSSLLSNLAVRSGEIADSIIARRAGYGAEREVLAKIGGDIA
    15. >Streptomyces_griseochromogenes_ATCC_14511
    MSDREHETYDVVGIGFGPSNLSLAIALEEYRANGPENEISALFLERQSAF
    GWHRNMLLPSTTMQISFLKDLVTFRNPTSSFSFIAYLHASGRLPQFVNNQ
    DFFPTRQEFHQYLEWAQARVADRVAYGSEVTSIRLPPGADPERSDRLRLE
    VADATGRNGRVVEARNVVISTGLVPSMPVGTERDERVWHSSEFLEKYRRM
    NPAELRRVAVVGAGQSAAEITRFLYDELPHAEVCAVIPSYGYSVADDTPF
    ANQIFDPGAVDDYYFGTEQTREAFWRYHRNTNYSVVDDEVIRDLYRRSYD
    DEVRGVRRLQFLNLTRVTSVKRVGAETRVSLQVGPDDEVRELDFDALVCA
    TGYSTMEPTDLLGDLDRHCLRDEAGRYRVERDYRIVTAPEMRCGIYLQGG
    TEHTHGLTSSLLSNIAVRSGEIADSIVAGRAGRNAERALLAEVGGDTR
    16. >Streptomyces_incarnatus_NRRL_8089
    MDIAGRPSQEIYDVVGIGFGPSNMSLAIALEEHEASSPQHPLKCHFFERQ
    PTFGWHRNMLLPSTTMQISFLKDLATFRNPTSRFSFISYLHAADRLVQFV
    NNQDFFPTRQEFHQYLEWAAAGLRDRVTYGAEVTSIRPAGEAGSGTSDIL
    EIEVRGGDGTTSVVSARNVVISTGLVPRLPEGVTSDERVWHSSEFLSRFH
    AQAPGDLKSVAVVGAGQSAAEITRFLYDSLPHAQVTAVIPSYGYSVADDT
    PFANQVFDPSAVDEYYFGTERARDSFWRYHRNTNYSVVDADVIRALYQRS
    YDEQVRGSQRLHFRNLTRVDEVERVGSGARVVVRSVLDDRTEELALDALV
    FATGYDGLDPARLLGDFDRHFLRDAAGRHRVERDYRLVPASGLTAGVYLQ
    GGTEHTHGLSSALLSNIAVRSGEIADSIVLRRTERELGSGRPVQAARSAA
    17. >Streptomyces_albulus_PD-1
    MESHRMTGPEVYDIVGVGFGPANLALAVALTERGSSTPLRALFLDRNESFSWHPGMLIHD
    ATMQVNFLKDLITLRNPASDFSFLSYLKARGRLVDFINHKTFFPTRVEFHDYLEWAAGRV
    GDVVEYGTEVVDVRPVERDGEVVYFDVVGHQQVGGVSQAVVCRARNVVVAPGLVPRLPGE
    ASQSERVWHSSELLHRVGDLPTDKRMQFVVVGAGQSAAEVVGYLHARYECADVHAVHSRY
    GYSPADDTPFANRVFDPAAVEHFFHAPPSVKDKFFEYHANTNYSVVDVELIEDLYARVYR
    ESVTERRRLHIHGMSELTEVADGPEGLRVSVRFLPDGTTTVLEPDHVVYATGYKPADVNR
    VIGVVAELCKRDSSGNLRLLHDYRVDMASHVRCGIYLQGGTEHSHGITSSLLSNLADRAA
    EILDSVLAHGGQLSADAAAWEVAS
    18. >Streptomyces_tsukubaensis_NRRL_18488
    MGITGRGKHEVLDLVGIGFGPSNLALAIALDEHGASAPQHPVTSHFFERQPAFGWHRNML
    LPSTTMQISFLKDLATFRNPMSRFSFVSYLHASNRLVQFVNNQDFYPTRQEFHQYLEWAA
    AALGDRVTYGAEVASIRPRTGPGSRTADLLEIEVRRGDGTTGTVTARNVAISTGLVPRLP
    KGVTSGPRVWHSSEFLGRFGAQTPADLRHVAVVGAGQSAAEITRFLHDSLPHAQVSAVIP
    SYGYSIADDTPFANQVFDPGAVDEYYYGTQRARDAFWRYHGNTNYSVVDADVIRDLYRRS
    YDEEVRGGRRLHFRNLTRVVEVEGSASGAWVMLRSLLDDRREELAVDALVFATGYDGMDP
    ARLLGDFDRHFQRDAAGRHRLERDYRLVSASGLTCGVYLQGGTEHSHGLSSSLLSNTAVR
    SGEIADSIVMRRTRQELGRSRSVAESPSAA
    19. >Streptomyces_himastatinicus_ATCC_53653_hmtM
    MAHETEIYDVVGIGFGPSNLSLAIALEESPDPVTSLFFERQPTLGWHRGMLLPSAKMQVS
    FLKDLATFRNPASGFGFISYLHDMGRLTRFVNNQDFFPTRREFHDYLEWAASKLTGRVSY
    DSEVTAVSAVAAAGEGPADRVRVTVRGADGAPRQVEARNVVISTGLVPRMPVNLEAGERV
    WHSSEFLHRFRQREGELTRVAVVGAGQSAAEIVRFLYDTLPEVRVSAVIPSFGYAIADDT
    PFANQVFDPDAVDSYYHGTQASKDAVWQYHKNTNYSVVDDEVIRGLYERAYEDELSGHGR
    LDFRNLARVLDAEPTGDGTRITVYSLVDDASYDLDVDVLICATGYDPMNPARVLGELDKY
    CVHDTEGRHRVDRDYRLVTTSDLTCGIYLQGGTEHTHGLGSSLLSNIAVRSGDIAQSITA
    RCAGAPKKGLTA
    20. >Streptomyces_flaveolus_DSM_9954_sfaB
    MTRLAEQSSTAQQSPESEVLDVTGIGFGAANLALAVALHESEAAGKALFLEKQKEFGWHR
    GMLLGGSSLQVSFLKDIATMRNPTSDFGFLSYLQEKDRLVDFINQHTLLPSRIEYHDYLQ
    WAADRLNHLVEYGVEATGVRPVTEAGEVVALDVLAGDRVVARTRNLVLASGLRPRLPEGA
    ETGERVWHSSQLLHRLPAFDERPPRRAVVVGAGQSAAEVAAHLMDRYPQAEVCAVFARYG
    YSVADSSPFANRVFDPAAVDDFYFAPPEVKQAIMRYHGGTNYAVVDEDVLQGLYRRQYEQ
    KVSGAPRLRVMNASRLVSVEPRQESAAVRVEFLPTGEHTDLDADLVVYATGYDSTDPAEL
    LGGVSGALRRDEAGELLIGRDYRLGTTGDFRCGIYVQGATEATHGIASTLLSMVAVRAGE
    IARSITGGRCDPDRSTGSKAAAGNRG
    21. >Streptomyces_aurantiacus_JA_4570
    MGTREHEIYDIVGIGFGPSNLSLAIALEEHQANSSQQPVRAAFFERQPSFGWHRNMLLPQ
    ATMQISFLKDLATFRNPLSRYSFVSYLHASDRLVQFVNNQDFFPTRQEFHQYLEWAESGF
    RDRVTYNSEVTEIRVSDEGSGGEQLLEIVVRDTVGGGTRVVQARNVTVSTGLVPRMPDGM
    LRDERVWHSSEFLAKYGRMRPEDLKNVAVVGAGQSAAEITKYLHDKLPHAQVSAILPSYG
    YSVADDTPFANQVFDPTAVDHYYFGTENTRDAFWRYHKNTNYSVVDDDVIRELFRRSYEE
    EVAGEKRLHFLNLTRVKEVKRSGNDTRVVLHSLLDGESEQEMDVDALVFATGYSTMDATR
    LLGDLDRFCERDEEGRHRVERDYRVVTSGELSCGIYLQGGTEHTHGLTSSLLSNIAVRSG
    EIADSIVERRGAGQRV
    22. >Streptomyces_sp._RJA2928_padN
    MTDSAPEDRTVDVTGIGFGPSNLALATALAEPSATGPGRPLEAVYFERKNRFSWHGGMLL
    DGATMQISFLKDLVTLRDPRSPYSFLSYLHHAGRLSDFINHKLLFPSRIEFHDYLEWVAG
    FFEEQVVYGSEVVDVRPVAREDAVEHMDVVVRQRTAAGERTVVQRTRDLVVATGLEPSLP
    PGTVCSDRVWHSSELLYRVERLPPTPRRIVVVGAGQSAAEAAEFLHSRFPSTDICAVFSR
    YGYSPSDDSPFANRIFDPAAVDDYCAAAPETRRMLLDYHRNTNYSVVDPELIDELYRRVY
    QEKVRGRPRLNILGASRLMAAEPAGDGVDVVVESLVTGERTPMRADCVVYATGYRPTDAR
    GLLGSMAGLCKADELGRLEADRRYRVITEGDVRCAIYLQGATEHSHGISSSLLSNTAVRA
    GEIADAIRADAVRAGARATTRSQPQPQT
    23. >Frankia_alni_str._ACN14A
    MSAREFDIYDVVGIGFGPSNLSLAVALDEFRVNGMGNVFSNIFFERRSSFAWHPSMLLPS
    ATMQISFLKDLVTFRNPTSSFSFVAYLHESGRLPRFVNNQDFFPTREEFHQYLEWAQARV
    AHRVAYGSEARSLRLPAGVGPERADRLCLQVADAASGTSRMVEARNVVISTGLVPTMPTG
    VERGERVWHSSEFLERFRRTSPARIRRVAVVGAGQSAAEITRFLYDELPHAEVSAIIPSY
    GYCVADDTPFANEVFDPEAIDDYYYATERTREALWRYHSNTNYSVVDDSVIRDLYRRSYE
    DDLRDVGRLRFLRLTRVAGVRSVGAQTRVSLRAGIDGDLRDLDVDVLVCATGYAAMEPTG
    LLGDLDQYCLRDEAGRYRIERDYRIVTAPEMQCGIYLQGGTEHTHGLSSSLLSNIAVRSG
    EIIDSIVARSAERTAPCAVLAEA
    24. >Actinosynnema_mirum_DSM_43827
    MTAVVQGADAPRDVVGVGFGPSNLALAVALAERDGPSSAFFERQPRFGWH
    RGMLLDGATMQVSFLKDLVSMRNPTSPYSFVSYLHARGRMPEFVNAKTLY
    PLRVEFHDYLEWVAGHFAGSVSYGSEITALEPVAEDGVVGHLDVVARRDG
    RTTTTRARNVVVATGLEPRLPDGVTGGERVWHSGELLHRVPWLRERRVRK
    VAVVGAGQSAAEVTEYLHRTLPGAEVIAVFSRFGYSVADDTPFVNEVFDP
    DSVDLFYGSPPSVRQALLAHHGNTNYSVVDADLSLELYRRRYQERVTGSS
    RLRVVNVSRVRSVRERPDGVALQVEYLPTGVVGTLAADAVVCATGYRPAD
    PTPLLRGLAKLDGAGRPVLDRDHRVVTSGSVRAGIYLQGAVTEPTHGLSA
    GLLSTTAVRAGEIVRAILDEGR
    25. >Kutzneria_sp._744_ktzl
    MTVAHAGESPTHDVVGVGFGPANLSLAVALEESPAALTSAFFERRASISWHQGMLLPAAK
    MQVSFLKDLATFRNPASRFSFVSFLHERGRLVRFANNHDFFPTRREFHDYLEWAESKLAH
    EVSYDSEVTAIRPGPGRPVDSVLVDVSTPEATRTVEARNIVISTGLVPRMPAGVQSDEFV
    WHSSRFLDHFRDRDPRSLRRVAVAGGGQSAAEIVRFLHDNRPDTVVHAIMPSYGYVVADN
    TPFANQIFDPAAVDDYFDGSKQAKDAFWRYHRNTNYSVVDDEVIRDLYRRGYDDEVAGAP
    RLNFVNLAHVVGAKRIADDTRVTVYSMAREESYDLDVDVLVCATGYDPMDPGDLLGELAE
    HCVQDAEGRWQVDRDYRMVTTPDLRCGIYLQGGTEHTHGLSSSLLSNLATRSGEIVSSIE
    RRKS
    26. >Kibdelosporangium_sp._MJ126-NF4
    VTDIHDLVGVGFGPSNLALSIAAAEADVPLRAVFLERSERFGWHRDMLIDDATMQVAFLK
    DLATPRNPVSRFGFVPYLWARDRLSAFINQKTLFPTRVEFHDYLEWAAAQVDDVVEYAAE
    VVDIRPVHDNGEVAFLDVVSVRPDGQARVRRTRNVVLALGLQPVVPPGVHPSPRVWHSAD
    LLGRAATLDRAKPLRFAVVGAGQSAAECVSYLHRAFEQAEVHAVFGRYGYSPADDSPFAN
    RIFDPAAVDDYFVSPDQVKQRFFDYHANTNYSAVDTELLEELSHRVYRESLSGRQRLFTH
    HLSAITDLADTDDGVSVSVEFLPTGERTMLRVDHVIHATGYRPTDPIPLLGTTAELCHKD
    TLGRLRVERDYRVVTKPDVRTGIYLQGGTEHSHGISSSLLSNVAVRAGEILASIQERPQR
    RDGDQDERTARAGDDPARRAAALPRR
    27. >Mycobacterium_xenopi_RIVM700367
    MLPGEDDSDLDFIGIGFGPSNLALAVAAEELIPNWRGLFLERSQSFQWHPGMMLEGARMQ
    ISFLKDLATLRNPASRYTFLQYAKARGRLEQFVNINEFRPTRLEYNDYLKWVAESFADRV
    RYGAVVTAVVPLRDSPSPAGRFGRLRVYVRDESTGVETCFSSPNVVYGGGGVPRLLGARN
    TSAVVHSSAFLPNFPNRFNEPDKAYRFAVVGNGQSAAEIAEYLLSHYRRATTHLFISDHT
    LRATDHSPFINEHFFSVNAAEFYDYPPAKRAALRNELRLTNYGVVDADVLQKLYQIAYLD
    EVRGCRRLFLHGESRLSRVEEIDGRVVARFEDRFSGESHEFDFDGAVLATGYDRVLDAEI
    FREVLPHVLRDESGEISLSRSCRVNTGPALTAGLFLQG
    28. >Streptomyces_mirabilis_YR139
    MGITGRRSQEIYDVVGIGFGPSNLSLAIALEEHGASAPQHPVKSLFFERQSRFGWHRNML
    LPSTTMQISFLKDLATYRNPTSRFSFISYLHASNRLVQFVNNQDFYPTRQEFHQYLEWAA
    AGLRDRVTYGAEVTSIRPGTEAGSRTPDLLEVEVRTGDGTTSVVTARNVVISTGLVPRLP
    QGVTSDERVWHSSEFLSRFNAQAPGDLKSVAVVGAGQSAAEITRFLHDSLPHAQVCAVIP
    SYGYSVADDTPFANQVFDPGAVDEYYFGTEQAQDAFWRYHRNTNYAVVDADVIRALYQRS
    YDEQVHGSRRLHFRNLTRVAEVKRTGSGTRVVLRSLLEDRTEELAVDALVFATGYDGLDP
    AHLLGDFDQHFLRDAAGRHRVERDYSLVTASGLTCGVYLQGGTEHSHGLSSSLLSNIAVR
    SGEIADSIVLRRTERELGSTCPVKVASSAA
    29. >Streptomyces_scabrisporus_DSM_41855
    MGMFGHEIHDVVGIGFGPSNLSLAIALEEHQANESARPVTAAFFERQPAFGWHRNMLLPS
    TTMQISFLKDLATFRNPVSRFGFISYLHASGRLPQFVNAQDFFPTRQEFHQYLEWAESSV
    TDRVSYGSDVTSIRPPQGIAARDAKHLEIEVEDLVSGATRLVKARNVIVSTGLVPRLPQG
    IERDERVWHSSEFLEKFGRMDAAGLGSVAVVGAGQSAAEITRFLYDTLPHARVSAILPAY
    GYSVADDTPFANQVFDPGAVDEYYFGSDRTREAFWRYHKNTNYSVVDDEVIRDLYRRSYE
    EEVRGVRRLNFLNLTRVDQVKRSGDETRVSLRSLLDDRVRELDVDALVFATGYDSPEPSG
    LLGDLDRYCLRDEAGRHRVGRDYRLVTSPELSCGIYLQGGTEHTHGLTSSLLSNIAIRSG
    EIADSVIRRRVEHELELERNAALEVARETR
    30. >Streptomyces_sp._TAA040
    MHDLVVVGAGPYGLSIAAHAAAAGLQPRVLGTPMASWRDHMPQGMYLKSEPWSSDLSDPA
    GAHTLAAYCATRGLVAEHGNPLPIEVFTDYGCWFAGRAAPPVEERIVVAVRPHGDGYRVE
    TAEGERITTRTVALAVGVMPFVHHPSALAALPAELATHSSDHRDLARFRGRDVTVVGAGQ
    AALETATLLTEHGARARVLARADRINWNTPPQPLERGLWKSLRDPHCGLGTGWSSWLWSE
    RPSAVRRLPAGLRAAIAGSALGPAGAWWLRERFEQAVPVLLGHRLLAAEQVGGRVRLDVR
    LADGTARNLHTDHVVAATGFTPELDRLGLLALSLTGTLRRVPGTGAPELGRCFESSRPGL
    FFGGLLTAPSFGPAMRFVHGAGFTAGRLVEGVRRRLGSGAASRTRAVPQAAGSVGRAAAE
    RPPG
    31. >Actinoalloteichus_cyanogriseus_DSM_43889
    MYGSVPVDGNQVSDVVGVGFGPSNLALAVAIAEHNETAPPKTRLRAQFLERQPVFGWHRG
    MLLPDTTLQVSFLKDLVTLRNPRSSFGFVSYLHDRNRLVDFVNHQSFFPSRREYHDYLEW
    VAGRFTGSVHYGHEVVDVLPVNEGPDVVAFDVVAAHGGVGATRRVRTRNVVLAPGLEPVL
    PQGITPSDRVWHSSELLHRLDGVRELLPSRPRFVVVGAGQSAAEVMAHLHDAFPTATVRS
    VCSRYGFAPADDSPFVNQLFDPAGVDEFFEAALPARENLLRTHAGTNYSAVDGGLINELY
    RRSYQERVAGEPRLLFERLSRVVATEEGDDEVSVAVRSLADGRVTNRRCDVVVLATGYRP
    RDALRPLGELAALCKLDANGWPRVERDYRITTTETVRAGIYLQGGTEHSHGLSSTLLSNL
    AVRSGEITRALVSR
    32. >Streptomyces_sp._HNS054
    MGITGRRHQEIYDVIGIGFGPSNMSLAIALEEHEASAPQQPLRYHFFERQPTFGWHRNML
    LPSTTMQISFLKDLATFRNPLSRFSFISFLHSSNRLVQFVNNQDFFPTRQEFHQYLEWAA
    AGLSDRVTYGTEVVSIRPGTEGGTLTPDLLEIEVRDGDGTTSVVVTRNVVISTGLVPRLP
    EGVTADERVWHSSQFLSKFHARDPRELKRVAVVGAGQSAAEITRFFYDSLPHAEVLAVIP
    SYGYSVADDTPFANQVFDPGAVDEYYYGTDRARDAFWRYHRNTNYSVVDTDVIRALYQRS
    YDEQVRGTQRLHFRNLTRVVEVGSTGEGTRVVLRSLLDDRREDLAVDALVFATGYDGVDP
    ARLLGDGFDAHFERDAAGRHRVERDYRLVSSSGLTCGVYLQGGTEHSHGLTSSLLSNMAV
    RSGEIADSIVLGRTGRELDRTHSVEEASSAA
    33. >Streptomyces_sp._AW19M42
    VCRGAATFLETTLTTPLETARSAAPHDPADGAPLDVLGVGFGPSNLALAIALSEVERPRP
    RVHFYDRSSRFSWHGGMLLKGATMQVHFLKDLVTLRNPGSPYSFLSYLHDRERLVDFINH
    KALFPSRVEFHDYLEWAAQACSDRVTYGSEVSRIEPEWVDGEVHRFRVHLTHSEPGERGV
    RHEVRSARNVVLAPGLRPHLPEGTAESEHVWHSSRLLSRLEDIPKDAPVRFTVVGAGQSG
    AEVTAYLHGRFPQAQVRAVFSPYGYNPADDSPFANRIFDPAAVDEFFGAPQAVREMLVDR
    HGNTNYSVVDQDLIAELYRIWYQEKVTDERRLIIDNVSRLVGVREASGLRLTIESLATRE
    RHEVDSDYLVYATGYRPVAPDDLVDPEIMKLCRRDAAGGLRVNRDYRVQTEDMVRCGLYV
    QGATEHTHGLSSTLLSNTAVRAGEIASSLLGRM
    34. >Salinispora_pacifica_DSM_45549
    VFDEPSVYDVLGIGFGPSNLSLAIALHEMGDVEGRPLAARFFEQQPSFGWHRNMLLPSAK
    MQVSFLKDLVTFRNPHSRFTFVSYLHEMNRLARFINNCDFFPTREEFHGYLEWAAANFAD
    QVTYGATITSISVPPDSGPGDPIDRVRVNLASGPTGAESSSVEARNVVLGTGLVPRFPAG
    LTSDDRVWHSSEFLGKFQRCDTTKLKRVLVVGGGQSAAEIAHFVYDNVPGVTVTAVIPSY
    GYSIADATPFANRVFDPSAIDDYYYGDENSKDAFWRYHRNTNYAVVDSNLISDLNRKAYD
    EAVTGETRLRFAELSRLSGVRRRDDGVVVSIHSMLSNRTSEVDADIVICATGYEPMEIGD
    MLGPLDRFCIRDEQGRYRVERDYRLATTEHLRCGIYLQGGMEHTHGLSSSLLSNLAVRNG
    DISTSVARRAQSQSHDDGRVLQGLVPTGS
    35. >Salinispora_pacifica_CNT150
    VFDEPSVYDVLGIGFGPSNLSLAIALHEMGDVEGRPLAARFFEQQPSFGW
    HRNMLLPSAKMQVSFLKDLVTFRNPHSRFTFVSYLHEMNRLARFINNCDF
    FPTREEFHGYLEWAAANFADQVTYGATITSISVPPDSGPGDPIDRVRVNL
    ASGPTGAESSSVEARNVVLGTGLVPRFPAGLTSDDRVWHSSEFLGKFQRC
    DTTKLKRVLVVGGGQSAAEIAHFVYDNVPGVTVTAVIPSYGYSIADATPF
    ANRVFDPSAIDDYYYGDENSKDAFWRYHRNTNYAVVDSNLISDLNRKAYD
    EAVTGETRLRFAELSRLSGVRRRDDGVVVSIHSMLSNRTSEVDADIVICA
    TGYEPMEIGDMLGPLDRFCIRDEQGRYRVERDYRLATTEHLRCGIYLQGG
    MEHTHGLSSSLLSNLAVRNGDISTSVARRAQSQSHDDGRVLQGLVPTGS
    36. >Salinispora_tropica_CNB536
    VTGKVHIVFDEPSVYDVLGIGFGPSNLSLAIALHEMGDVEGRPLAARFFEQQPSFGWHRN
    MLLPSAKMQVSFLKDLVTFRNPHSRFTFVSYLHEMNRLARFVNNCDFFPTREEFHGYLEW
    AATNFADQVTYGATITSISVPPDSGPGDPIDRVRVHLASGPTGTESSSVEARNVVLGTGL
    VPRFPAGLTSDDRVWHSSEFLGKFQRCDTTKLKRVLVVGGGQSAAEIAHFVYENVPGATV
    TAVIPSYGYSIADATPFANRVFDPSAIDDYYYGDENSRDAFWRYHRNTNYAVVDSDLISD
    LNRKAYDEAVTGEIRLRFAELSRLSGVRRRDDGVVVSIHSMLSNRTSEVDADIVICATGY
    EPMEIGDMLGPLDRFCIRDEHGRYRVERDYRLATTEHLRCGIYLQGGMEHTHGLSSSLLS
    NLAVRNGDISTSVARRAQSQPHGDGRVLQGLVPTGS
    37. >Salinispora_arenicola_CNH996
    VFDEPSVYDVLGIGFGPSNLSLAIALHEMGDVEGRPLAARFFEQQPSFGWHRNMLLPSAK
    MQVSFLKDLVTFRNPHSRFTFVSYLHEMNRLARFINNCDFFPTREEFHGYLEWAAATFAD
    QVTYGATITSISVPPDSGPGDPIDRVRVHLASGPTGTESSSVEARNVVLGTGLVPRFPAG
    LTSDDRVWHSSEFLGKFQRCDTTKLKRVLVVGGGQSAAEIAHFVYENVPGATVTAVIPSY
    GYSIADATPFANRVFDPSAIDDYYYGDENSKDAFWRYHRNTNYAVVDSDLISDLNRKAYD
    EAVTGETRLRFAELSRLSGVRRRDDGVVVSIHSMLSNRTSEVDADIVICATGYEPMEIGD
    MLGPLDRFCIRDEQGRYRVERDYRLATTEHLRCGIYLQGGMEHTHGLSSSLLSNLAVRNG
    DISTSVARRAQSQPHDDGRVLQGLVPTGS
    38. >Salinispora_arenicola_CNH996B
    VFDEPSVYDVLGIGFGPSNLSLAIALHEMGDVEGRPLAARFFEQQPSFGW
    HRNMLLPSAKMQVSFLKDLVTFRNPHSRFTFVSYLHEMNRLARFINNCDF
    FPTREEFHGYLEWAAATFADQVTYGATITSISVPPDSGPGDPIDRVRVHL
    ASGPTGTESSSVEARNVVLGTGLVPRFPAGLTSDDRVWHSSEFLGKFQRC
    DTTKLKRVLVVGGGQSAAEIAHFVYENVPGATVTAVIPSYGYSIADATPF
    ANRVFDPSAIDDYYYGDENSKDAFWRYHRNTNYAVVDSDLISDLNRKAYD
    EAVTGETRLRFAELSRLSGVRRRDDGVVVSIHSMLSNRTSEVDADIVICA
    TGYEPMEIGDMLGPLDRFCIRDEQGRYRVERDYRLATTEHLRCGIYLQGG
    MEHTHGLSSSLLSNLAVRNGDISTSVARRAQSQPHDDGRVLQGLVPTGS
    39. >Salinispora_tropica_CNY012
    VTGKVHIVFDEPSVYDVLGIGFGPSNLSLAIALHEMGDVEGRPLAARFFEQQPSFGWHRN
    MLLPSAKMQVSFLKDLVTFRNPHSRFTFVSYLHEMNRLARFVNNCDFFPTREEFHGYLEW
    AATNFADQVTYGATITSISVPPDSGPGDPIDRVRVHLASGPTGTESSSVEARNVVLGTGL
    VPRFPAGLTSDDRVWHSSEFLGKFQRCDTTKLKRVLVVGGGQSAAEIAHFVYENVPGATV
    TAVIPSYGYSIADATPFANRVFDPSAIDDYYYGDENSRDAFWRYHRNTNYAVVDSDLISD
    LNRKAYDEAVTGEIRLRFAELSRLSGVRRRDDGVVVSIHSMLSNRTSEVDADIVICATGY
    EPMEIGDMLGPLDRFCIRDEHGRYRVERDYRLATTEHLRCGIYLQGGMEHTHGLSSSLLS
    NLAVRNGDISTSVARRAQSQPHGDGRVLQGLVPTGS
    40. >Salinispora_tropica_CNT261
    VTGKVHIVFDEPSVYDVLGIGFGPSNLSLAIALHEMGDVEGRPLAARFFEQQPSFGWHRN
    MLLPSAKMQVSFLKDLVTFRNPHSRFTFVSYLHEMNRLARFVNNCDFFPTREEFHGYLEW
    AATNFADQVTYGATITSISVPPDSGPGDPIDRVRVHLASGPTGTESSSVEARNVVLGTGL
    VPRFPAGLTSDDRVWHSSEFLGKFQRCDTTKLKRVLVVGGGQSAAEIAHFVYENVPGATV
    TAVIPSYGYSIADATPFANRVFDPSAIDDYYYGDENSRDAFWRYHRNTNYAVVDSDLISD
    LNRKAYDEAVTGEIRLRFAELSRLSGVRRRDDGVVVSIHSMLSNRTSEVDADIVICATGY
    EPMEIGDMLGPLDRFCIRDEHGRYRVERDYRLATTEHLRCGIYLQGGMEHTHGLSSSLLS
    NLAVRNGDISTSVARRAQSQPHGDGRVLQGLVPTGS
    41. >Salinispora_tropica_CNH898
    VTGKVHIVFDEPSVYDVLGIGFGPSNLSLAIALHEMGDVEGRPLAARFFEQQPSFGWHRN
    MLLPSAKMQVSFLKDLVTFRNPHSRFTFVSYLHEMNRLARFVNNCDFFPTREEFHGYLEW
    AATNFADQVTYGATITSISVPPDSGPGDPIDRVRVHLASGPTGTESSSVEARNVVLGTGL
    VPRFPAGLTSDDRVWHSSEFLGKFQRCDTTKLKRVLVVGGGQSAAEIAHFVYENVPGATV
    TAVIPSYGYSIADATPFANRVFDPSAIDDYYYGDENSRDAFWRYHRNTNYAVVDSDLISD
    LNRKAYDEAVTGETRLRFAELSRLSGVRRRDDGVVVSIHSMLSNRTSEVDADIVICATGY
    EPMEIGDMLGPLDRFCIRDEHGRYRVERDYRLATTEHLRCGIYLQGGMEHTHGLSSSLLS
    NLAVRNGDISTSVARRAQSQPHGDGRVLQGLVPTGS
    42. >Streptomyces_sp._PsTaAH-137
    MDTPGSLSQEIYDVVGIGFGPSNLSLAVALEEQGASSAQHPV
    43. >Salinispora_arenicola_CNS296
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFGWHRNMLLPST
    TMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQDFFPTRQEFHQYLEWAEERMA
    GRVAYGSEVTSIRLPSGTVPELSDRLRLEVTDAAGRVGRVVEARNVVISTGLVPRMPEGI
    ERDERVWHSSEFLQKYRRMNPGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYG
    YSVADDTPFANQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCATGYDGMEPTHL
    LGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGTEHTHGLSSSLLSNIAVRSGE
    IADSIVSRRARHNSEYALAAGAEGDTC
    44. >Salinispora_arenicola_CNS299
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFGWHRNMLLPST
    TMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQDFFPTRQEFHQYLEWAEERMA
    GRVAYGSEVTSIRLPSGTVPELSDRLRLEVTDAAGRVGRVVEARNVVISTGLVPRMPEGI
    ERDERVWHSSEFLQKYRRMNPGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYG
    YSVADDTPFANQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCATGYDGMEPTHL
    LGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGTEHTHGLSSSLLSNIAVRSGE
    IADSIVSRRARHNSEYALAAGAEGDTC
    45. >Salinispora_pacifica_CNY363
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFGWHRNMLLPST
    TMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQDFFPTRQEFHQYLEWAEERMA
    GRVAYCSEVTSIRLPSGIVPELSDRLRLEVTDAAGRVGRVVEARNVVISTGLVPRMPEGI
    ERDERVWHSSEFLQKYRRMNPGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYG
    YSVADDTPFANQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCATGYDGMEPTHL
    LGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGTEHTHGLSSSLLSNIAVRSGE
    IADSIVSRRARHNSEYALAAGAEGDTC
    46. >Actinomadura_atramentaria_DSM_43919
    VTGPATDADDILDIVGVGFGPSNLALAVAVREHNADRPAAEHLTQVYFEKQPAFGWHRGM
    LIDGATMQVSFIKDLVTMRNPASEYGFLSYLHDNDRLADFINHKSLFPSRVEFHDYLEWV
    ARRFQDVARYGSEVVAMRPGPGGDHIEVIVRRGGEHRVQRARNVVVAVGQEPALPDDIEL
    GDRIWHCAQLLERVERLTEEPRRAVVVGAGQSAAETTEFLHRRFENAEVSAIFLRYGYSV
    ADDTPFANRIFDPESVDVFYGAPENVKRMLFDYHRNTNYSVVDQELADELYRRVYQERVR
    GVERLRILNASRLHAVRRDVTGDGLRVDVEHLPTGEKRSFGVDLVVYATGYRPIDPANVL
    GEVAEYCRRDAGKRPAITRDYRLETDDRLRAGIYLQGGTEQTHGISAQLLSNTAVRAGEI
    VRSIAGARVGAV
    47. >Streptomyces_drozdowiczii_SCSI0_10141
    MTVNLGSTSVLEVAGIGFGPSNMALAIALEEMHGARANSPGPAMEFFEKQPAFGWHRGML
    MEDATMQVSFLKDLATMRDPQSRYTFMAYLKAKGRIARFINSKTLFPLRVEFHDYLEWVA
    DLLAPVVSYGSDVLAIRPVVEDGVMECLDVVVRTSAGDGEPIVRRARNVVIGTGLTPRLP
    DGTEESARVWHSSRLMDRAASIAAAPRGFVVVGAGQSAAEATEYLHRSFPGTPVSAVFAR
    YGYSVADDSPFTNGIFDPEAVDEFYAASRDVKQDLLDYHGNTNYAVVDLSLTEELYRRAY
    QEEVLGRERLRFHNASRVLKVEEHPDRVRVIVEHLPDRTVETLDADAVVYATGYRPSDPT
    PLLQNLLPECKLDDAGRITLDRDYRIVTSGDVRCGIYLHGASAECTHGLSAGLLSNTAVR
    SGEIADSIIKR
    48. >Streptomyces_sp._RSD-27
    MGITGRRDEEIYDVIGIGFGPSNMSLAIALQEHGAGVPLHPVRSHFFERQ
    PTFGWHRNMLLPSTTMQISFLKDLATFRNPMSRFSFVSYLHASNRLVQFV
    NNQSFIPTRQEFHQYLEWAAAGLRDQVTYGAEVTSVRPVTAAGSRTPDLL
    EVEVRTGDEVSVVTARNVVVSTGLVPRMPEGVPAGERVWHSSEFLARFNA
    QDPAELKSVAVVGAGQSAAEVTRFLYDSLPHAEVSAVIPSYGYSVADDTP
    FANQVFDPDTVDEYYFGTEGARDAFWRYHRNTNYSVVDADVIRSLYQRWY
    DEQVRGVQRLRFRNLTRVDGVEGSGSGARMVLRSLLDDSREELAVDAVVF
    ASGYDGLDPARLLGEDFDRHFQRDAAGRHRVERDYRLVSTSGLTCGVYLQ
    GGTEHSHGLTSALLSNIAIRSGEIADSIVLRRTERELGRHAEEAPSAA
    49. >Actinoalloteichus_spitiensis_RMV-1378
    MDGSFPVDGNQVSDVVGVGFGPSNLALAVAVAEHNEAVGPEERLRARFLERQPDFGWHRG
    MLLPDTTLQVSFLKDLVSLRNPRSSFSFISYLHDRNRLVDFVNHQCFFPSRREYHDYLEW
    VAGRFVDSVHYDHDVVDVLPVHEGPDVVAFDVVAVQGGAGATRRLRTRNVVLAPGLEPVL
    PQGITPSDRVWHSSELLHRLDGFRDRLPDRPRFVVVGAGQSAAEVMAHLHGVFPKATVRS
    VCSRYGFAPADDSPFVNQLFDPAAVDEFFEAALPARENILRVHAGTNYSAVDGDLISELY
    RRSYQERVSGEPRLHFERLARVVATEERDEEVSVSVLSLTDGRVTDRGCDVVVLATGYRP
    RDALRPLGQLAALCKLDANGWPRVERNYRITTTETVRAGIYLQGGTEHSHGLSSTLLSNL
    AVRSGEITRALAAP
    50. >Streptomyces_sp._PBH53
    MTRLAGQAPTAQHSPESEVRDVTGIGFGAANLALAVALHESGAGGRALFLEKQKEFGWHR
    GMLIEGSSLQVSFLKDIATMRNPTSDFGFLSYLQEKGRLVDFINQHTLLPSRIEYHDYLQ
    WAADRLGHMVEYGVEATGVRPVTDAGEVVALDVLAGDRVVTRTRNLVIASGLRPRLPEGA
    ETGERVWHSSQLLHRLPAFDERPPRRAVVVGAGQSAAEVAAHLMERYPQAEVCAVFSRYG
    YSVADSSPFANRVFDPAAVDDFYFAPPEVKQAIMRYHGGTNYAVVDEDVLQGLYRRQYEQ
    KVTGTPRLRVMNASRLVSVEPRGETAAVRVEFLPTGEHADLDADLVVYATGYRSADPAEL
    LGGVAGSLRRDAAGQVLIGRDYRLSTTGDFRCGIYVQGATEATHGIASTLLSMVAVRAGE
    IAQSIIGGRRDPDRTAGTKAVAGNRG
    51. >Salinispora_arenicola_CNS-991_DSM_45545
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFGWHRNMLLPST
    TMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQDFFPTRQEFHQYLEWAEERMA
    GRVAYGSEVTSIRLPSGTVPELSDRLRLEVTDAAGRVGRVVEARNVVISTGLVPRMPEGI
    ERDERVWHSSEFLQKYRRMNPGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYG
    YSVADDTPFANQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCATGYDGMEPTHL
    LGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGTEHTHGLSSSLLSNIAVRSGE
    IADSIVSRRARHNSEYALAAGAEGDTC
    52. >Streptomyces_sp._MNU77
    VEASASVTDVVGVGFGPANLALAIALRELGAGPPGGDGLTAAFLEAQPQFGWHSGMLIED
    STMQVSFLKDLVTPRNPVSPFSFVAYLHAVGRLGRFMDSKMMYPLRIEFHNYLEWVAGHF
    ANQVAYSRRVTALRPVHGQDGVEALDVVARDADGTERVLRARSVVLACGLRPRLPEGLTG
    SDRVWHTADLLPRARRLLESGAAPTSFVVLGAGQSSAEAAHYLHRTFTRSSVSVVHSRYG
    FSVSDDSPFANAVFGAKAVDEFYGAPDEVKRMVLDYHANTNYAVVDEDLIHRLYGDVYRE
    SLTGDDRLRFHHLSRLSTVTPGEDAVRVEVEALHDGRRTVIDADALVCATGYRPSDPADL
    MGDLLPLCARDEQDRLVLDRDRRLVTREPLAGGVYVTGYGEHTHGIAESLLSLTAQRAGE
    LTEALAKTFVT
    53. >Micromonospora_pattaloongensis_DSM_45245
    MSETDSATVRQVVGVGFGPANLALAIAAGEVAGPDGRTLLDECVFLERQP
    SFGWHRGMLLDGATMQVSFLKDLATLRSPSSRYTFTSYLHDVGRLTDFIN
    SKTLYPYRTDFHTYLEWAADRLPADVRYGTEVVSVTPERTDDVVRELLVR
    TGDGRTFRTRNLVIGTGMTPCFPDGVQRGPRVWHSAELLTRLAAPAPTRP
    RTFAVVGAGQSAAEVVEHLHATHPEADVHAIFGRFGYSMSDDSPFANQIF
    DPDSVDEFYHAPGEVRDALMGYHANTNYSVVDLDLIRSLHGTAYREHIAG
    RRRLHFHHASRITRQTVTGEGVHLDVEFLPTGTIRQIDADAIVYATGYRP
    SDPRQLLGDLADECKTDDRGRLALARDYRVITSDGVRCGIYVHGAAAERT
    HGLSAGLLSNVAVRAGEILAAIRSL
    54. >Streptacidiphilus_carbonis_NBRC_100919
    MGARENATYDVVGIGFGPSNLSLAIALEERCANVLTNSITSAFFERQSSF
    GWHRNMLLPSATMQISFLKDLVTFRNPVSRFSFVAFLHAKGRLGQFVNRK
    DFFPTRQEFHQYLEWAAAKMADAVTYDSTVTSVQLPPDHGSGGDGYVQLE
    VRDTAAGSTRRVNTRNVVVSTGLVPRMPDGIARDDRVWHSSEFLTRYGRT
    DPEVLRSVAVVGAGQSAAEITQFFHGRLPHAQVHAIMPSYGYSVADDTPF
    ANQVFDADAVEDYYDGDEPARDAFWRYHRNTNYGVVDSADIQALYQTQYD
    EGVAGAKRLHFHNLTKVRAVERNGSARRVTLQSLRHHEVRQLDVDAIVFA
    TGYASMDPTQLLGDLDRYCLRDESGHHRVTRDYRLVTTPELSCGIYLQGG
    TEHTHGLTSSLLSNIAVRSGEIADSIICRRAESELATIAAEVREAVAERL
    55. >Streptomyces_sp._MnatMP-M27
    MTDSAPGDRTVDVTGIGFGPSNLALATALAEPSATGPGRPLEAVYFERKN
    RFSWHGGMLLDGATMQISFLKDLVTLRDPRSPYSFLSYLHHAGRLSDFIN
    HKLLFPSRIEFHDYLEWVAGFFEEQVVYGSEVVDVRPVAREDAVEHMDVV
    VRQRTAAGERTVVQRTRDLVVATGLEPSLPPGTVCSDRVWHSSELLYRVE
    RLPPTPRRIVVVGAGQSAAEAAEFLHSRFPSTDICAVFSRYGYSPSDDSP
    FANRIFDPAAVDDYCAAAPETRRMLLDYHRNTNYSVVDPELIDELYRRVY
    QEKVRGRPRLNILGASRLTAAEPAGDGVDVVVESLVTGERTPMRADCVVY
    ATGYRPTDARGLLGSMAGLCKADELGRLEADRRYRVITEGDVRCAIYLQG
    ATEHSHGISSSLLSNTAVRAGEIADAIRADAVRAGARATTRSQPQPQT
    56. >Pseudonocardia_sp._EC080625-04
    MCTCKSDVYDVVGIGFGPSNLSLAIALGEHQGNRAGHPVKAAFFERQQSF
    GWHRNMLLPETTMQISFMKDLVTFRNPRSRFSFVNYLHESGRLTQFCNNQ
    DFFPTRQEFHRYLEWVGSSFDDQVSYDSEVLGVTLAPEPCECAQRYLKLE
    ISNGAIGATEIVNARNISISTGLVPKVPDNVATGDRIWHSSQFLEKLRDV
    DPADLRNVAVVGGGQSAAEIARYLHATLPEAQIYAIVPSYGYSVADDTPF
    ANQVFDPEAVDDYYFGSDETRDAFWRYHRNTNYSVVDDDIIRDLHRASYA
    EQVTGERRLHFLNLTRVRAVTRNGATNRVSLHSLIDRETRELDIDALVLA
    TGYTEMTPTGLIGDVDHFCHRDPEGRYRIERDYRLMTDPEFPCGIYLQGG
    TEHTHGLTSSLLSNVAVRGGEIADSVITRTRADAPTMQRSTRRIEQAWER
    AG
    57. >Pseudonocardia_sp._HH130629-09
    MCTCKSDVYDVVGIGFGPSNLSLAIALGEHQGNRAGHPVKAAFFERQQSF
    GWHRNMLLPETTMQISFMKDLVTFRNPRSRFSFVNYLHESGRLTQFCNNQ
    DFFPTRQEFHRYLEWVGSSFDDQVSYDSEVLGVTLAPEPCECAQLYLKLE
    ISNGAIGATEIVNARNISISTGLVPKVPDNVPTGDRIWHSSQFLEKLRDV
    DPADLRNVAVVGGGQSAAEIARYLHATLPEAQIYAIVPSYGYSVADDTPF
    ANQVFDPEAVDDYYFGSDETRDAFWRYHRNTNYSVVDDDIIRDLHRASYA
    EQVTGERRLHFLNLTRVRAVTRNGATNRVSLHSLIDRETRELDIDALVLA
    TGYTEMTPTGLIGDVDHFCHRDPEGRYRIERDYRLMTDPEFPCGIYLQGG
    TEHTHGLTSSLLSNVAVRGGEIADSVITRTRADAPTMQRSTRRIEQAWER
    AG
    58. >Streptomyces_parvulus_2297
    MGITGRRNEEILDVVGIGFGPSNLSLAIALEEHGASAPRHPVTSHFFERQ
    PTFGWHRNMLLPSTTMQISFLKDLATFRNPMSRFSFISYLHASDRLVQFV
    NNQDFFPTRQEFHQYLEWAASGLSDRVTYGAEVTAIRPGSDGNGLSPDLL
    EVEARTADGTTRVVTARNVAISTGLVPRLPEGVTADERVWHSSQFLSRFN
    AQSPDDLKSVAVVGAGQSAAEITRFLHDALPHAQVCAVVPSYGYSVADDT
    PFANQVFDPAAVDDYYFGTDRGRDAFWRYHRNTNYSVVDADVIRDLHQRT
    YDEEVRGTRRLHFRNLTRVAEVERSGSTTRVVLRSLLDDRTEDLSVDALV
    FATGYDGLDPVRLLGDFDRHFRRDAAGRHRLERDYRLVPATDLTCGVYLQ
    GGTEHSHGLSSSLLSNIAVRSGEIADSIVLRRTERELERDRPVEVAPPVA
    59. >Streptomyces_sp._CFMR_7
    MAIRAGSHILDVVGIGFGPSNLALAIALQEMIKADTGRTEYAMAFHERQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNATSRYTFVAYLQEQGRVAEFIN
    SKTLYPLRVEFHDYLEWAAQQFDASVSYGSEIVAVRPVIESGSVEYVDVV
    ARSASGGSSTVVQRARNVVIGMGLTPRLPDGIEESERIWHSSQLLHRADS
    LPYRPRNFVVVGSGQSAAEVADYLHRTFSDANVHTVLSRYGYSVADDSPF
    ANGVFDPEAVDRFYTSSADAKQRLLDYHGNTNYSVVDLEVSQDLYRRSYQ
    EKVLGKQRLRMLNSSRVTSAEEHADGVRVIVEAMDSGSVRTMDADVIVYA
    TGYRPSDAAPLLSELAGECKRDEEGRLAVERDYRVITSEAVRCGIYVHGA
    VTEHSHGLSAGLLSNTAVRSGEIARSILRR
    60. >Streptomyces_sp._DvalAA-19
    MAIRAGSHISDVVGIGFGPSNLALAIALQEMIKADTGRTEYAMAFHERQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNATSRYTFVAYLQEKGRVAEFIN
    SKTLYPLRVEFHDYLEWAAQQFDASVSYGSEIVAVRPVIESGSVEYVDVV
    ARSASGGSSTVVQRARNVVIGMGLTPRLPDGIEESERIWHSSQLLHRADS
    LPYRPRNFVVVGSGQSAAEVADYLHRTFSDANVHTVLSRYGYSVADDSPF
    ANGVFDPEAVDRFYTSSADAKQRLLDYHGNTNYSVVDLEVSQDLYRRSYQ
    EKVLGKQRLRMLNSSRVTSAEEHADGVRVIVEAMDSGSVRTMDADVIVYA
    TGYRPSDAAPLLSELAGECKRDEEGRLAVERDYRVITSEAVRCGIYVHGA
    VTEHSHGLSAGLLSNTAVRSGEIARSILRR
    61. >Rhodococcus_fascians_A3b
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    62. >Rhodococcus_fascians_A73a
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    63. >Rhodococcus_fascians_A76
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    64. >Rhodococcus_fascians_A78
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    65. >Rhodococcus_fascians_D188
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    66. >Rhodococcus_fascians_02-816c
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    67. >Rhodococcus_fascians_05-339-1
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    68. >Rhodococcus_fascians_LMG_3605
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    69. >Rhodococcus_fascians_LMG_3616
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    70. >Rhodococcus_fascians_LMG_3623
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    71. >Rhodococcus_fascians_A22b
    MGAQSGSSVADVVGVGFGPSNLALAIALQESIQPGPVPAKFSMKFYELQP
    RFGWHRGMLMEDATMQVSFLKDLATMRNPMSRYTFVSYLREKERIAEFIN
    SKTLYPLRVEFHDYLEWAASQFQSNVSYGSEIKDIRPVVENGVVEYVDVV
    GPDDVVQRARNIVIGMGLTPRLPDGVNRSERIWHSSQLLGRAAAVTYVPQ
    NFVVVGSGQSAAEVADYLHRTFPRANVHTVLSRYGYSVADDSPYANGIFD
    PEGVDRFFSAPTDEKQRLLEYHANTNYSVVDLDISQSLYLKSYQEKVLGK
    QRLRMINTSRVTSVDEDTDGVRVEVTSSATGLTHTIEADVIVYATGYRPS
    DPAPLLQGLMRECKHDEQGRLSVGRDYRVTTSDAVRAGIYVHGASTEHSH
    GLSAGLLSNTAVRSGEIAQSILRR
    72. >Salinispora_arenicola_CNS848
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFG
    WHRNMLLPSTTMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQD
    FFPTRQEFHQYLEWAEERMAGRVAYGSEVTSIRLPSGTVPELSDRLRLEV
    TDAAGRVGRVVEARNVVISTGLVPRMPEGIERDERVWHSSEFLQKYRRMN
    PGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYGYSVADDTPFA
    NQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCAT
    GYDGMEPTHLLGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGT
    EHTHGLSSSLLSNIAVRSGEIADSIVSRRARHNSEYALAAGAEGDTC
    73. >Salinispora_arenicola_CNY231
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFG
    WHRNMLLPSTTMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQD
    FFPTRQEFHQYLEWAEERMAGRVAYGSEVTSIRLPSGTVPELSDRLRLEV
    TDAAGRVGRVVEARNVVISTGLVPRMPEGIERDERVWHSSEFLQKYRRMN
    PGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYGYSVADDTPFA
    NQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCAT
    GYDGMEPTHLLGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGT
    EHTHGLSSSLLSNIAVRSGEIADSIVSRRARHNSEYALAAGAEGDTC
    74. >Salinispora_arenicola_CNY280
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFG
    WHRNMLLPSTTMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQD
    FFPTRQEFHQYLEWAEERMAGRVAYGSEVTSIRLPSGTVPELSDRLRLEV
    TDAAGRVGRVVEARNVVISTGLVPRMPEGIERDERVWHSSEFLQKYRRMN
    PGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYGYSVADDTPFA
    NQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCAT
    GYDGMEPTHLLGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGT
    EHTHGLSSSLLSNIAVRSGEIADSIVSRRARHNSEYALAAGAEGDTC
    75. >Salinispora_arenicola_CNT005
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFG
    WHRNMLLPSTTMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQD
    FFPTRQEFHQYLEWAEERMAGRVAYGSEVTSIRLPSGTVPELSDRLRLEV
    TDAAGRVGRVVEARNVVISTGLVPRMPEGIERDERVWHSSEFLQKYRRMN
    PGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYGYSVADDTPFA
    NQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCAT
    GYDGMEPTHLLGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGT
    EHTHGLSSSLLSNIAVRSGEIADSIVSRRARHNSEYALAAGAEGDTC
    76. >Salinispora_arenicola_CNY230
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFG
    WHRNMLLPSTTMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQD
    FFPTRQEFHQYLEWAEERMAGRVAYCSEVTSIRLPSGTVPELSDRLRLEV
    TDAAGRVGRVVEARNVVISTGLVPRMPEGIERDERVWHSSEFLQKYRRMN
    PGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYGYSVADDTPFA
    NQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCAT
    GYDGMEPTHLLGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGT
    EHTHGLSSSLLSNIAVRSGEIADSIVSRRARHNSEYALAAGAEGDTC
    77. >Salinispora_arenicola_CNY486
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFG
    WHRNMLLPSTTMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQD
    FFPTRQEFHQYLEWAEERMAGRVAYCSEVTSIRLPSGTVPELSDRLRLEV
    TDAAGRVGRVVEARNVVISTGLVPRMPEGIERDERVWHSSEFLQKYRRMN
    PGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYGYSVADDTPFA
    NQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCAT
    GYDGMEPTHLLGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGT
    EHTHGLSSSLLSNIAVRSGEIADSIVSRRARHNSEYALAAGAEGDTC
    78. >Salinispora_pacifica_CNY331
    MSNQHETYDLVGIGFGPSNLSLAIALKEYEANGQENGISTLFFERQSSFG
    WHRNMLLPSTTMQISFLKDLVTFRNPTSGFSFISYLHASGRLPQFVNNQD
    FFPTRQEFHQYLEWAEERMAGRVAYCSEVTSIRLPSGTVPELSDRLRLEV
    TDAAGRVGRVVEARNVVISTGLVPRMPEGIERDERVWHSSEFLQKYRRMN
    PGDLRRVAVVGAGQSAAEITRFLHDELPHAEVWVVIPSYGYSVADDTPFA
    NQIFDPEAVDDYYFGTEQTRDAFWRYHRNTNYSVVDDEVIRDLYRRVYDA
    EVRGIKRLQILNLTRITGVKRAAAETRVELQVGPDSEVRELDVDALVCAT
    GYDGMEPTHLLGDLDRLCLRDKAGRHQIERDYRIATAPEMRCGIYLQGGT
    EHTHGLSSSLLSNIAVRSGEIADSIVSRRARHNSEYALAAGAEGDTC
    79. >Streptomyces_aureofaciens_NRRL_2209
    VGERQRSGVVAGTGIVDVAGIGFGPSNLALAAAIAEIAGEAPVSARFFEA
    QPRFGWHRGMLIEGATMQVSYLKDLVTMRNPTSPYSFLCYLQARGRLADF
    INTKSPYPLRVEFHDYLEWVAESFADLVSYGARVVSVEPVSAEQGVEFLD
    VHFVAPDGTRQVQRARNLVIAAGIEPRLPAGLPASPRIWHTAKFLPEVDR
    IARQDPRSFVVLGSGQSAAEAIEHLHARFPRAQVHSVHARYGFSVADDSP
    FANQVFNPEAVDRFHTAPDDVRQRLIDYHASTNYSVVDADLLHSLFQQAY
    LEKVAGNPRLNFHNVSRVSEVTETPDGLRIDVESLSSGTSTVIEAQALVC
    ATGYTRTDPAVFLDGLLPHCPLDDQGRLRLDREHRVVTDESVRCGIYVQG
    FGEHSHGLSETLLSLSAVRAGEIGDMLVKALSG
    80. >Streptomyces_sp._OK885
    MGARETEVYDVVGVGFGPSNLSLAVAIQEHNSSTSDRPLTAAFFERQEAF
    GWHRNMLLPAATMQIPFLKDIATFRNPASRYSFVAYLHASGRLAGFVNNQ
    TFFPTRREFHRYLEWVAANFTDQVSYGCEVVGLRLSGQGTGAGAPAHLEI
    EVAGGAGRQRSSVRARNVVVSTGLVPRMPEGVLGDDRVWHSSEFLTRFRG
    LKPVDLRAVAVVGAGQSAAEITRFVHDAAPHAQVYSVIPSYGYALADDTP
    FANQVFDPAAVDDYFFGTDRARQAFWDYHKNTNYSVVDDDVIRDLYRRSY
    DEEVNGARRLHFLNLTRVGEVKRAGDETRVLLMNGERRELEVDLCVFATG
    YHGMEPAGVLGDLAPYCLRDEAGRLRVERDYRLVTGPELPGGIYLQGGTE
    HTHGLSSSLLSNIAVRSGEIAESIVSRHRIERELGQVHPAEPAGKIR
    81. >Pseudonocardia_sp._AL041005-10
    MDTDDMGTYDFVGIGFGPSNLSLAAALRDASSSDASPVRGHFFEAQPSFG
    WHRNMLLPSAKMQVSFLKDLVTFRNPHSRFSFVSYLHEMNRLPQFANNND
    FFPTRREFHQYLEWVAGHFADSVTYGARVTGIEPICGGATAGPHDRFRIT
    IASGKDALATTRVEAYNVVLATGLTPRMPEGSVRDDRVWHSSEFLERFGS
    CSSASLRRVAVVGAGQSAAEIARFCYDHAPNATISAILPSYGYSIADNTP
    FANRVFDPGAVDDYYFSDPLGKDRLWESHRNTNYSVVDDEVIRSLFQRQY
    DDEVRGVERLQIINLARVANIKRSGDETRVTIHSLARDEHFDLDVDVVVC
    ATGYEAMGADGVLAGLDAFCPRDDRGRHRVERDYRLITTDDLTAGIYLQG
    GTEHTHGLTSSLLSNLATRSGEIASSLRSSRRVGSAGGDRW
    PzbB
    82. >Mycobacterium_marinum_M
    MYERPGYSAIEPAAVLDLLTANPLGLVVTIDGARPLATHAPVLFSQGPNGVAQAEVASGD
    APLVGSLLVGHMNADNPQWRGMQKGGRVLVAFQGPHGYVSPSVYGVTPASPTWNFTAVHI
    AGTLEPIADPESTFELVCDTARRLEARFGHGWRQEPSLDYFRRIVSGVGAFEIQVESVQT
    MFKLSQEQPPVLRRRVAEHFESSDSVLHQELADLMRKHVFPKPI
    83. >Lentzea_flaviverrucosa_DSM_44664
    MFVPAQYREPHGHWITDLVRGHPLAQLVSNGPAGSSPYVTHAPIILDPGHPDPHPDDLHG
    AVLWGHLNRANPHWAALGDGTEVTAVFTGPGSYVSPTVYERTPAAPTWDFTAVHVRGTLR
    RVLDAEQTLATVTATVRAFEADHGTGWSMESSLDYFDQLLPGVGAFRLAVTGVDAMFKLS
    QEQPPEVRLRVRDHFAGSERTHHCLIAEMMDRLPVAEH
    84. >Streptomyces_aureofaciens_ATCC_10762
    VFTPKLYQVDGDDWPLRIIERHPLAVLVSNGDPVPNATHVPVIAPPDAAPEDALSGMRLW
    AHLTRANPHWQQLAAAGGGPAKLVFHGPNGYVTPSLYSADMVAPTWNYVAVHLEGTVELA
    GDDETLAIVHTTAQTLEDRFGDGMALAPSLEYHRQIVGAVGGLFFTVTKVDVMFKLSQEK
    DPEVQQRVLDRFAASGSGLHREVADTMRALRLGGSAG
    85. >Streptomyces_diastatochromogenes_NRRL_B-1698
    VYIPDLYRTDDKEWPVRILEENPLGLLTTHASSSAPPFATHLPVIIPSGSRDALLQDEKW
    RGATLLGHMNRANPHWQSLADGTPARIVFQGPGAYVSPSVYHTDPAAPTWDFTAVHVQGT
    LWPVRDEAETLAIVTATATELERKFGTGWCPHSSTEYFRQLLAGVGAFELRVDTMDAMFK
    LSQEKSHEIRNGVVDWFVQGQHGRSRELASLMAEFYKDDRGTGA
    86. >Streptomyces_sp._DvalAA-43
    MFVPSHYREPDGSWMIDLIRANPMAIMAINGSSADGPFATHLPVIPDPAATGRRSADLSG
    ATLLGHMNRANPQWAALESGGVALLIFTGPHGYVSPTVYEMAPAAPTWNFTSVHVHGMVE
    KIDSTEETLGVVKSTVTALETDFGTDWDMSGSVDYFRKIVPAVGAFRFTVSGAEGMFKLS
    QEQPAEVRDRVQTSFSCREQGRYRETAELMGRLPG
    87. >Collimonas_fungivorans_Ter6
    MYVPEYYRVDENTARELVYRHPLALLVCNGNNGLPWATHLPAIFPPETRKLLDQGESIIG
    KTMYGHMNRINPHWNALQAGSALLIFQGPNSYVSPTVYEVTPAAPTWNFTSTHLRGTLRP
    IDERDQILEIVRWTVATFEKEFCTNWDLTESIPYFERIVHGVGAFAFEVESFDSMFKLSQ
    EQPAAIQERVVNSFASSSHCPHKEIADLMQRTNSKNKK
    88. >Streptomyces_reticuli_TUE45
    VYERPLYREDRDGVVLAFLHHHPLALVVTAHEGVPVATHAPVLFRHGPDGADAEAVAAGT
    VPLAGSTLIGHMNVENPQWRRMRSGDQALIVFQGPHGYVSPTVYDVTPAAPTWNFTAVHV
    TGTVEPTAEPADVLDIVSDTARRLEGRFGRGWDQESSLDYFRQIAPGVGAFTLRVESVQT
    MFKLSQEKPTPMRRRVAEQFEASESGTHRALAGMMRAHGLTDADEERETAG
    89. >Streptomyces_scabiei_NCPPB_4086
    MFVPDPYREPDGSWMTELIRLNPFALLVSNGPADADPYATHLPVLRDPEWTGEWTEDLAG
    GRLVGHMNRENPHWTALETGTPVLITFTGPHAYVSPIVYDITPAAPTWDFTSVHVHGVFH
    KIEAAAPGEDTLEVCKDTVKAYERDFGAAKAWDMSRSIDYFATILPAVGAFRVEITGAEG
    MFKLSQEQDQEIRERVQKDFALRDSTQYRETADLMDRMEKTGTVQGCPVHH
    90. >Kutzneria_albida_DSM_43870
    MFVPSHYREPDVSWMVDLMRQNPLALLASNGNPADGPFATHLPVITDPAWDGPPAEKLAG
    WPLLGHMNRANPQWTALENGATVLLTFTGPHAYVSPTVYEISPAAPTWNFTSVHAHGVVE
    KIESIEETLEVVQATVKVFEKFFGDSWDMTESLGYFRKIVPAVGAFRIRVTRADGMFKLS
    QEQKPEVRKRVVTSFSERGCGRHAQTAALMTQLP
    91. >Streptomyces_albus_ZpM
    MFVPPEYRPDDPEWLIEVIRSHPLACLVTNGPDGPRASHVPVIPDPEQFPSGMPAREGEV
    AGRRLFGHMNRLNPHWAALQGGAQALLVFQGPNGYVSPTVYEYTPAAPTWDFTAVHVRGW
    LEPVGDRESSLQIITETVAAYERDLGTGWDMTESLGYFRQLLPGVGAFRLAIDTVDGMFK
    LSQEQSPEVRERVACEFAARAEARGTALAEHIQRTK
    92. >Rhodococcus_fascians_02-815
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPSESEDLEGSTL
    FGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDSVAPTWNYVSVHLRGKLQPVA
    DFEETLKVVQLTVSTYEQKFGSGWEMDSSLDHYRRIGPAVGAFSFEVESADGMFKLSQEQ
    NLETRRRVADHFSANHAGRGKELASFMREYSHGDYNNF
    93. >Streptomyces_neyagawaensis_NRRL_B-3092
    MFVPDPYREPDGSWMTELIRLNPFALLVSNGPADADPYATHLPVIRDPEWTGAWTENLAG
    GRLIGHMNRENPHWTALENGTPVLITFTGPHAYVSPTVYDITPAAPTWDFTSVHVHGVFE
    KIEAAAPGEDSLEVCKDTVKAYERDFGAAKAWDMSRSIDYFATILPAVGAFRVEITGAEG
    MFKLSQEQDEEIRERVREDFALRDSSQYRETAELMDRMEKTGTIKGCPVHH
    94. >Kutzneria_buriramensis_DSM_45791
    MFVPHHYHEPNESWMTDLIRENPLAELVSNGNGPAGPFATHVPVIPDPHDPDRPPGEIVG
    ATLWGHMNRSNPHWAALESETPVVIVFTGPHAYVSPTLYQRTPAAPTWNFTAVHARGLLR
    RVDAEAAGDETLETVMATVRAFEARFGAGWAMSESVEYFRRIVPAVGAFRVTVSHVDGMF
    KLSQEQDADVRARVRESFAERESSNHKAIAAMMGRLADAE
    95. >Streptomyces_yanglinensis_CGMCC_4.2023
    MFVPSQYREPDVSWMVDLMRDNPLALMASNGTAADGPYATHLPVITDPGWEGPPAADLAG
    MLLLGHMNRANPHWSALEDGQTILLTFTGPHAYVSPTVYDITPAAPTWNFTSVHVRGTVE
    KIATTEETLEVVKSTVRAYEKEFGDSWDMNASLDYFRKIVPGVGAFHVRVTRAEAMFKLS
    QEQSPEVRDRVVRSFAGRGCTRHAQAADLMTRLP
    96. >Streptomyces_griseochromogenes_ATCC_14511
    MFVPSHYREPDVSWMVDLMRGNPLALMASNGTPADGPFATHLPVITDPQWEGSPTADLAG
    MPLLGHMNRANPHWAALETGSAILLTFTGPHAYVSPTVYDVTPAAPTWNFTSVHVHGVVE
    KIESTEETLDVVQATVQAFEGEFGDSWDMSESVDYFRKIVTGVGAFRVRVTKAEGMFKLS
    QEQRPEIRERVVQSFAGRECTRHVQTADLMNRLP
    97. >Frankia_sp._Avcl.1
    MFVPCHYRAPNVSMMVDLMRENPLALMVSNGAPGAVPFATHLPVITDPCWDGQAGPDLGG
    MVLLGHLNRANPHWXALETGSMILLTFTGPHAYVSPTVYGLTPAAPTWDFTSVHVHGVVE
    KLTTTEETLEVVRATVLAFEQEFGDGWDMTDSLGYFRRIVPRVGAFRLRVTGAQGMFKLS
    QEQTPEIRERVARSFAAHGSTRHAQTAELISRLPH
    98. >Streptomyces_incarnatus_NRRL_8089
    MFVPSFYREPDSAWMVDLIRGNPLALAVTNGSPEDGPFATHLPVIFDPETSGDWSGELPG
    ATLLGHMNRANPHWAALETGSVLLLTFTGPHSYVSPTVYETTPAAPTWNFTAVHVRGVVE
    KISSTEETLGVVQSTVRAYEGAFGDGWDMSESLDYFRKIVPAVGAFRFTVTGAEGMFKLS
    QEQPGEVRERVRDAFGQSGCAYRREVAGLMSRLP
    99. >Streptomyces_sp._MUSC136T
    MFVPPQYREPDGSWMVDLMRRNPLALCVTNGDAADGPYATHLPVIRDPGMTGEWAEDLSG
    GTLLGHMNLQNPHWAALRDGQSVLLVFTGPHAYVSPTVYEKSPAAPTWDFTAVHVHGTVE
    KLTSAQDTLDVVKSTVRAFESDLGTGWDMTESEAYFDQLLPGVGAFRVEVTGAEGMFKLS
    QEQQPHVRDRVHDAFAERPCGRHRETAELMARLP
    100. >Streptomyces_albulus_PD-1
    MFVPPEYRPDDPEWLIEVIRSHPLACLVTNGPDGPRASHVPVIPDPEQFPSGMPAREGEV
    AGRRLFGHMNRLNPHWAALQGGAQALLVFQGPNGYVSPTVYEYTPAAPTWDFTAVHVRGW
    LEPVGDRESSLQIITETVAAYERDLGTGWDMTESLGYFRQLLPGVGAFRLAIDTVDGMFK
    LSQEQSPEVRERVACEFAARAEARGTALAEHIQRTK
    101. >Streptomyces_tsukubaensis_NRRL_18488
    MFVPSMYRAPDSSWMVNLIRENPLALAVANGSPENGPFATHLPVVFDPETSADPAGELPG
    TTLLGHMNRANPHWAALETGSVLLLTFTGPNSYVSPSVYGVTPAAPTWNFTAVHVRGVVE
    KISSLEESLDVVQSTVRAFEGAFGNGWDMTESLGYFRRIAPAVGAFRLTVTGAEGMFKLS
    QEQPGDVRRRVRESFGQSACRYRRETAGLMSRLP
    102. >Streptomyces_himastatinicus_ATCC_53653_hmtC
    MFVPSHYREPDSSWMVDIIRGNPLALMMSNGAAGEPPFATHLPVIPDPAMTGDWSERLSE
    ATLLGHMNRDNPQWQALEDGAVVRIAFSGPHAYVSPTLYGVTPAAPTWNFTSVHVRGVVE
    RIPSTEETLEVVKSTVRAFEADFGEGWDMAASIDYFRKIVPGVGAFRIMVRNVDGMFKLS
    QEQQPEVRDRVRKSFAGRECGRHQETAAYMSRLP
    103. >Streptomyces_flaveolus_DSM_9954_sfaC
    MYERPLYREDCDGVVLAFLRHNPLAMVVTSHDDVPVATHAPVLFRHGPDGADAEAVAAGT
    VPLAGSTLIGHMNVENPQWRRMRSGDRALIVFQGPHGYVSPTVYGVTPAAPTWDFIAVHV
    NGTVEPTADPAAVLDIVSDTARRLESGFGRGWDQESSLDYFRQIAPGVGAFTLRVDSVQT
    MFKLSQEKPAPMRRRVVEQFEASESGTHRALASVMRDRGLTEADEERETAG
    104. >Streptomyces_auranticaus_JA_4570
    MFVPSQYRQPDSSWMLDLIHGNPLALFVSNGSPEAGPFATHLPVIQDPEWTGEWSDDLSG
    GRLLGHMNRANPHWKALESGTVNLLTFTGPHGYVSPTVYRTTPAAPTWNFTSVHVHGVVE
    KIDGIENTLEVVKATVRAYEGAFGAGWDMTESLDYFRKIVPAVGAFQFRVTGAEGMFKLS
    QEQPDDVQERVRESFGGRECTRHQAAAQLMDKLR
    105. >Streptomyces_sp._RJA2928_padO
    MFVPQHYRTDDRRWPVRIVQDNPLALLMSTRDGRAPFASHVPVIVLPRQREELERTGRWQ
    GAVLHGHMNRANPHWKSLADGQPAGLVFQGPAGYVSPAVYNTSPAVPTWNFTAVHVQGRL
    KLVADEEATLGVVSATARQLEERFGARWTVEPSVDHFRQILPGVGAFELRVEECDSMFKL
    SQEKEHEVRHAVMDWCARSPRGRSNDLAAVMRDYYPPTTTWPS
    106. >Frankia_alni_str._ACN14A
    MFVPCHYRAPNVSMMVDLMRENPLALMVSNGAPGAVPFATHLPVITDPCWDGQAGPDLGG
    MVLLGHLNRANPHWAALETGSMILLTFTGPHAYVSPTVYGLTPAAPTWDFTSVHVHGVVE
    KLTTTEETLEVVRATVLAFEQEFGDGWDMTDSLGYFRRIVPRVGAFRLRVTGAQGMFKLS
    QEQTPEIRERVARSFAAHGSTRHAQTAELISRLPH
    107. >Actinosynnema_mirum_DSM_43827
    MHVPPMYRADDEDRARQVVHDYPLATLVSNGPRVPHATHLPVVAAPGAPQVGGLAGSTLW
    GHLNRANAHWRALAGGVPAVLVFTGPHAYITPAIYRTTPAVPTWDFVSVHLHGRVEPIDG
    EAGTLEVVKRTAELFESAFGAGWAAEPSHGHFARIVSGVGAFRFHVESVDSMFKLSQEKD
    RDVRVRIIASLREASGPAAELGRIMHEHGLGGRGAEGA
    108. >Kutzneria_sp._744_orf4
    MFVPGPYHAPEDRWLVDLVRGHPLAQLASNGAGGAAPHITHVPIIVDPELDGPVDRLVGI
    TLWGHMNRANPHWAALGGAANVVATFAGPNAYVSPAVYRTAPAAPTWNFTSVQVRGELRK
    VESADDTLATVRATVAALESRFGAGWDMTGSLDYFRRILPGVGAFRLRVAEADGMFKLSQ
    EQQPAIRRRVRHSFGGCEATRAVAGLMDRLPTE
    109. >Kibdelosporangium_sp._MJ126-NF4
    MHVPPMYEAPDPAWIPALIRAHPLATLVTAPDGIPAASHVPMIIRRTPDDERLTLVGHMN
    RMNPQFKAIGDGCPALLVFTGPHGYVSPTVYGFTPAAPTWNFAVVHASGTLSPLPAGPDT
    LEVIIDTVTALEGQLGNGWQMRDSLEYFDQLLPGVGAFSVQVDRVEAMYKLSQEQEPTTR
    ETVAAAFEARSSDLAAMMRVCLDVERSTLGNRVG
    110. >Mycobacterium_xenopi_RIVM700367
    MLSLLPFRAQAIAQEIAASRHRDAVTVRQRPVGDYPPKRYLETDPDRLRAVIERYRFATL
    ISARATDEPVVTQLPLTLDTSRGSHGVLFGHMDLANPHAELLDGRPVLALFHGPNGYIPP
    HQSNQLPTWNSITVEVRGRARILRDKDAVVDGLRGIAAAADPSPGGFRLTREAASDERLF
    PFLVGFEIDIDEMVGRFKLSQDRDDRDRWLAARTLAHGLEQDDRDLIASIVELPLDRDDD
    PIPLRRARTSGT
    111. >Streptomyces_mirabilis_YR139
    MFVPSFYREPDSSWMVDLIRGNPLALAAANGSPEEGPFATHLPVIFDPETSGDWSGELPG
    ATLLGHMNRANPHWAALATGSVLLLTFTGPHSYVSPTVYEVTPAAPTWNFTAVHVRGVVE
    KIDSIEETLGVVQSIVRAFEGAFGDGWDMTESLGYFRKIVPDVGAFRFTVTGAEGMFKLS
    QEQPGEVRERVRESFGHSACAYKRETAGLMSRLP
    112. >Streptomyces_scabrisporus_DSM_41855
    MFVPRHYREPDSSWMVDLIRANPLALAVMNGDPSAGPFATHLPVIPDPQMTPSWSDDLSG
    ATLLGHMNRANPHWKALETGTVLLLTFTGPHGYVSPTVYEVTPAAPTWNFTSVHVRGVVE
    RIDSLEETLGVVRATALAFESEFGAGWDQTESVDYFRKIVPGVGAFRVTVTGAEGMFKLS
    QEQPAEVRERVRQSFSTRACSLQRETAELMTRLP
    113. >Streptomyces_sp._TAA040
    VFVPTHYREPDGSWMADLMRENPLALAVTDGGAGDGPFATHLPVVPDPGTTGDWPNGLKG
    ATLLGHMNRANPHWRALETGGVVLLAFTGPHAYVSPTVYEVTPAAPTWNFTSVHVRGVVD
    RIDSPEETLDVVRTTALVYEARFGAGWDQAASLDYFRRIVPAVGAFRIAVTSAEGMFKLS
    QEQPAEVRERVHRSFSGRECGRHRDTAALMERLPRTGAEPPVGR
    114. >Actinoalloteichus_cyanogriseus_DSM_43889
    MFVPHQYRAADTRPLVELIRSFPLATLVSHADGALFATHVPVLLAADADAGRDVPDPADL
    TILGHLNRLNPHRDALAGGGACLLTFTGPHSYVSPAHYGRDTAAPTWNFTSVHVHGHLTP
    LDSTEDTRHVVRSTALLYERRFGAGWDMTGSLDYFEQLLPGVSAFRVDVGTVEGMFKLGQ
    EQPGHARQGVLAAFTSPGAPPHQRAVAELMRRFPPDAAGGVPGCPAQSAARMSPPADAIR
    GEH
    115. >Streptomyces_sp._HNS054
    MFVPNFYREPDASWMVDLVRGNPLALAVSNGCPEDGPFATHLPVIFDPARYGDLPGELAG
    ATLLGHMNRANPHWPALQTGGILLLTFTGPHSYVSPTAYGTTPAAPTWNFTAVHARGVVE
    KIDSTEETLDVVKATVRAYEGEFGDGWDMTESLGYFRKIVPAVGAFRLTVTRAEGMFKLS
    QEQPAEVRERVRESFEQSACRYKRETAGLMSRLP
    116. >Streptomyces_sp._AW19M42
    MYVPDHYQGSPEAALTVVRAGPLATLVTGADPWPLATHLPVVVPADVEAALEHGPVDLRG
    HRLIGHLNRANPHWRQLSAGEQPSLLIFRGPHGYISPVVYESTPAAPTWNFTAVHVHGTI
    RPLPAGKETLDVIHRTVEVLEGGFGHGWDMRGSLEYFEKIVPHVGAFEFQVAEVDGMFKL
    SQELDEETRERTTHHFATSAHGTHRELACEMARLSTAAETKDGASEGASGSSSKRGTA
    117. >Salinispora_pacifica_DSM_45549
    MFVPSPYREPDGSWTVDLMRRNPLALLVTSSDKTDVPYATHLPVIFDPCMPEEDYSDPAR
    FVLLGHMNRANPHWKALATGMPTLVVFSGSHAYVSPTVYDKSPAAPTWNFTAAHARGVLE
    KIESAEETLGVIGSTVRAFEADFGTDWDMTQSVGYFRKILPGVGAFRIAVSSIDSMFKLS
    QEQPPEVRDRVGCAFAESASTRHREVAGLMNRLAVPKQVTV
    118. >Salinispora_pacifica_CNT150
    MFVPSPYREPDGSWTVDLMRRNPLALLVTSSDKTDVPYATHLPVIFDPCM
    PEEDYSDPARFVLLGHMNRANPHWKALATGMPTLVVFSGSHAYVSPTVYD
    KSPAAPTWNFTAAHARGVLEKIESAEETLGVIGSTVRAFEADFGTDWDMT
    QSVGYFRKILPGVGAFRIAVSSIDSMFKLSQEQPPEVRDRVGCAFAESAS
    TRHREVAGLMNRLAVPKQVTV
    119. >Salinispora_tropica_CNB536
    MFVPSPYREPDGSWTVDLMRRNPLALLVTSSDKTDIPYATHLPVIFDPRMPEEDYSDPAR
    FVLLGHMNRANPHWKALATGMPTLVVFSGSHAYVSPTVYDKSPAAPTWNFTAAHARGVLE
    KIESAEETLGVIGSTVRAFEADFGADWDMAQSVGYFRKILPGVGAFRIAVSSIDSMFKLS
    QEQSPEVRDRVGCAFAESASTRHREVADLMNRLAVPKQVTV
    120. >Salinispora_arenicola_CNH996B
    MFVPSPYREPDGSWTVDLMRRNPLALLVTSSDKTDVPYATHLPVIFDPCM
    PEEDYSDPARFVLLGHMNRANPHWKALATGMPTLVVFSGSHAYVSPTVYD
    KSPAAPTWNFTAAHARGVLEKIESAEEALGVIGSTVRAFEADFGTDWDMT
    QSVGYFRKILPGVGAFRIAVSSIDSMFKLSQEQPPEVRDRVGCAFAESAS
    TRHREVAGLMNRLAVPKRVIV
    121. >Salinispora_arenicola_CNH996
    MFVPSPYREPDGSWTVDLMRRNPLALLVTSSDKTDVPYATHLPVIFDPCMPEEDYSDPAR
    FVLLGHMNRANPHWKALATGMPTLVVFSGSHAYVSPTVYDKSPAAPTWNFTAAHARGVLE
    KIESAEEALGVIGSTVRAFEADFGTDWDMTQSVGYFRKILPGVGAFRIAVSSIDSMFKLS
    QEQPPEVRDRVGCAFAESASTRHREVAGLMNRLAVPKRVTV
    122. >Salinispora_tropica_CNY012
    MFVPSPYREPDGSWTVDLMRRNPLALLVTSSDKTDIPYATHLPVIFDPRMPEEDYSDPAR
    FVLLGHMNRANPHWKALATGMPTLVVFSGSHAYVSPTVYDKSPAAPTWNFTAAHARGVLE
    KIESAEETLGVIGSTVRAFEADFGTDWDMAQSVGYFRKILPGVGAFRIAVSSIDSMFKLS
    QEQSPEVRDRVGCAFAESASTRHREVADLMNRLAVPKQVTV
    123. >Salinispora_tropica_CNT261
    MFVPSPYREPDGSWTVDLMRRNPLALLVTSSDKTDIPYATHLPVIFDPRMPEEDYSDPAR
    FVLLGHMNRANPHWKALATGMPTLVVFSGSHAYVSPTVYDKSPAAPTWNFTAAHARGVLE
    KIESAEETLGVIGSTVRAFEADFGTDWDMAQSVGYFRKILPGVGAFRIAVSSIDSMFKLS
    QEQSPEVRDRVGCAFAESASTRHREVADLMNRLAVPKQVTV
    124. >Salinispora_tropica_CNH898
    MFVPSPYREPDGSWTVDLMRRNPLALLVTSSDKTDIPYATHLPVIFDPRMPEEDYSDPAR
    FVLLGHMNRANPHWKALVTGMPTLVVFSGSHAYVSPTVYDKSPAAPTWNFTAAHARGVLE
    KIESAEETLGVIGSTVRAFEADFGTDWDMAQSVGYFRKILPGVGAFRIAVSSIDSMFKLS
    QEQSPEVRDRVGCAFAESASTRHREVADLMNRLAVPKQVTV
    125. >Streptomyces_sp._PsTaAH-137
    MFVPSFYREPDSSWMVDLIRGNPLALAVANGPAEDGPFATHLPVIFDPETSADVSGELPG
    VTLLGHMNRANPHWSALQDGGVLLLTFTGPHSYVSPTVYEKSPAAPTWNFTSVHVRGVVE
    KISSIEETLEVVQATVRAFEGAFGDGWDMTGSLDYFRKIVPAVGAFRFTVTGAEGMFKLS
    QEQPGEVRERVRESFGQSACTYKRETAGLMNRLAQTEDVTVSSGA
    126. >Salinispora_arenicola_CNS296
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQTGPGSVLLGH
    MNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAPTWDFVVVHVSGRVMPLDAGE
    PTLAVVQRTAATLEGAFGAGWDHTGSIDYFRSIVGGVGAFEFVVEQVESMFKLSQEKDHT
    VRQRLIDDFTSAPRNGSAQVGQLMSDLNLGVAP
    127. >Salinispora_arenicola_CNS299
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQTGPGSVLLGH
    MNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAPTWDFVVVHVSGRVMPLDAGE
    PTLAVVQRTAATLEGAFGAGWDHTGSIDYFRSIVGGVGAFEFVVEQVESMFKLSQEKDHT
    VRQRLIDDFTSAPRNGSAQVGQLMSDLNLGVAP
    128. >Salinispora_pacifica_CNY363
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQTGPGSVLLGH
    MNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAPTWDFVVVHVSGRVMPLDAGE
    PTLAVVQRTAATLEGAFGAGWDHTGSIDYFRSIVGGVGAFEFVVEQVESMFKLSQEKDHT
    VRQRLIDDFTSAPRNGSTQVGQLMSDLNLGVAP
    129. >Actinomadura_atramentaria_DSM_43919
    VFVPPQYRPRGRSWTLETVRSNPLAMLVTRGERALPWITHLPVITHPERPPAELPGATLL
    GHMNAANPHWAAVASGGPGTLVFTGPHGYVSPTVYELPVAAPTWDFVAVHVHGTLRPLDT
    PEDARRVVRWTVEAYEGTHGTGWDPEGSLDYFDKILPGVRAFEFHVESVDGMYKLSQEQE
    PETRRRVVRSFAASGRGAHAELSALIDRFGDPGPGAPATGCPAAREAGDGAR
    130. >Streptomyces_drozdowiczii_SCSIO_10141
    MFVPPMYRTENEGRLRQVMERYPLAMLVTNGEPTPYATHLPVIFDQNGAPGTDGPVGATL
    LGHLNRNNPHWRTLTDGLAAKLVFTGPHSYITPTLYETTPAAPTWNFVTVHLEGTLHPVT
    DLEETLGVLQATVETFESAFGNKWEMDSSLDYFRHIGPAVGAFRFVVTSADGMFKLSQEK
    TPEIQHRIADRLIGTETGTRHELGALMAELTLGDRDGV
    131. >Streptomyces_sp._RSD-27
    MVDLVRGHPMALAVANGSPEDGPFATHLPVIFDPVTSGQWTGELPGATLLGHMNRANPHW
    AALETGGVLLLTFTGPHSYVSPTVYAKSPAAPTWNFTSVHVRGVVEKIDSIEETLEVVQS
    TVRAFEGAFGDGWDMTGSLDYFRKIVPDVGAFRLTVTGAEGMFKLSQEQPGEVRERVRES
    FGQSACTYRRETAGLMG RLP
    132. >Streptomyces_sp._YR375
    MVDLLRNNPLALMVSNGDAAAAPFATHLPVIPDPAMTDEWSADLSGATLLGHMNRGNPHW
    KALETGDVVLLTFTGPHAYVSPTVYEVTPAAPTWNFTSVHVRGVVEKIDSAEETLEVVQS
    TVRAFEADFGDDWDMTESLGYFRRIVPAVGAFRLTVSGAEGMFKLSQEQKPEVRERVQKA
    FSGRECGRHRETASFMSRLP
    133. >Actinoalloteichus_spitiensis_RMV-1378
    MFVPDQYRAADNRPLVELIRSFPLATLVSHAEGTLFATHVPVLLAADADAGRDVPEPADL
    TILGHLDRRNPHRAALAAGGPCLLTFTGPHSYVSPAHYGRETAAPTWNFTAVHVHGRLTP
    LDGAEDTRHVVRSTALLYERRFGAGWDTTGSLDYFEQLLPGVSAFRVDVSTVEGMFKLGQ
    EQPGYARQGVVAAFTSPGAPPHQRAVAELMRRFAPDSPDDGGPGCPVRAPAKPEPATRGE
    R
    134. >Streptomyces_sp._Ncost-T6T-1
    MVDLMRSNPLALMVSNGSPEASPFATHLPVIFDPGDAADLAEDLARLPLLGHMNRANPHW
    SALQDDAVVLLSFTGPHAYVSPTVYDVTPAAPTWNFTSVHVHGVVEKFDSTEETLEVVQA
    TVRAFEEKFGNNWDMTDSIDYFRKIVHDVGAFRIRVTKAEGMFKLSQEQEPEIRDRVVQS
    FTGRGCTRHAQTATLMSRLP
    135. >Streptomyces_sp._PBH53
    VYERPLYREDRDGVVLAFLHHHPLALVVTAHEGVPVATHAPVLFRHGPDGADAEAVAAGT
    VPLAGSTLIGHMNVENPQWRRMRSGDRALIVFQGPHGYVSPTVYDVTPAAPTWNFTAVHV
    TGTVEPTAEPADVLDIVSDTARRLEGRFGRGWDQESSLDYFRQIAPGVGAFTLRVESVQT
    MFKLSQEKPTPMRRRVAEQFEASESGTHRALAGMMRAHGLTDADEERETAG
    136. >Salinispora_arenicola_CNS-991_DSM_45545
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQTGPGSVLLGH
    MNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAPTWDFVVVHVSGRVMPLDAGE
    PTLAVVQRTAATLEGAFGAGWDHTGSIDYFRSIVGGVGAFEFVVEQVESMFKLSQEKDHT
    VRQRLIDDFTSAPRNGSAQVGQLMSDLNLGVAP
    137. >Streptomyces_sp._MNU77
    MFVPRIYQVDGEHWPSEIIDRHPLALLTTNGDDVPHATHVPVIRPPHDEQLVGSELLVHM
    NRANPHWAALSDHDAAKLVFQGPDGYVTPSVYHVEPAVPTWDFVTVHLTGTLRISEDVDE
    VLSIVTATARTLERRFGAGFDVDRAADHHARIASGVGAIRFRVTKAEAMFKFSQEKDAEI
    RDRVMQWFEDSDIGEYADLGRLMRQFLDRPDITAPAAAG
    138. >Micromonospora_halophytica_DSM_43171
    MFVPRSFAVEDAGPVVELMRSNPLACFVLGGESPSVSHLPVVFADDDERDDLAGITLLTH
    MNRQNPLWGSLSDGARVLVVFQGPHGYVSPTVYGVSPAAPTWNFTVVHAHGVVRLLGAGE
    PALRVVKRTVQVLEGRFGAGWDMTGSLGYFERIVHAVGALEIHVDAVQSMFKLSQDQPVE
    LQSKVAAAFAGSGRGTHRELAEQMYTHLRLKADVDGF
    139. >Streptacidiphilus_carbonis_NBRC_100919
    MFVPPPYRPPDGSWTAELIRSNPLAILASNGSTADGPFATHLPVIPDPGT
    PDLLSAELTGAVLLGHMNRANPHWAALAEGGTSLLTFTGPHAYVSPTVYG
    VTPAAPTWNFTSVHARGTIERIESSEETLEVVKATVRAFEERFGAEWDMS
    ESISYFRQILPGVGGFRFTVTGTDGMFKLSQEQAPEIRCRVQRSFTGREC
    SRH RETAALMGSLP
    140. >Streptomyces_sp._MnatMP-M27
    MFVPQHYRTDDRRWPVRIVQDNPLALLMSTRDGRAPFASHVPVIVLPRQR
    EELERTGRWQGAVLHGHMNRANPHWKSLADGQPAGLVFQGPAGYVSPAVY
    NTSPAVPTWNFTAVHVQGRLKLVADEEATLGVVSATARQLEERFGARWTV
    EPSVDHFRQILPGVGAFELRVEECDSMFKLSQEKEHEVRHAVMDWCARSP
    RGRSNDLAAVMRDYYPPTTAWPS
    141. >Pseudonocardia_sp._EC080625-04
    MFVPEQYREQDSNWMLDIVRSNPLALMASDGTPEGCGPAATHLPCIPDPS
    APHDWSDGPRGAVLLGHMNRANPQWRHLHDGQIVLLVFTGPHAYVSPAVY
    DTTPAAPTWDFTAVHVHGVVTKLEPHKAERTTLDVVTDTVTALEGRFGAG
    WDMTDSIEYFHRLLPGVGAFRVRVGSAEGMFKLSQEQPSDIRDRVRCHFA
    AAQHGRSSEIAHLMTTLDGH
    142. >Pseudonocardia_sp._HH130629-09
    MFVPEQYREQDSNWMLDIVRSNPLALMASDGTPEGCGPAATHLPCIPDPS
    APHDWSDGPRGAVLLGHMNRANPQWRHLHDGQIVLLVFTGPHAYVSPAVY
    DTTPAAPTWDFTAVHVHGVVTKLEPHKAERTTLDVVTDTVTALEGRFGAG
    WDMTDSIEYFHRLLPGVGAFRVRVGSAEGMFKLSQEQPSDIRDRVRCHFA
    AAQHGRSSEIAHLMTTLDGH
    143. >Streptomyces_paryulus_2297
    MFVPSFYREPSNSWMVDLIRGNPLALAVANGQPDEGPFATHLPVIFDPDH
    PLDRDDDLTGATLLGHMNRANPHWGSLETGGVLLLTFTGPHSYVSPTVYE
    VTPAAPTWNFTAVHVRGVVEKLDSTDETLAVVQSTVRAFEGEFGNGWDMT
    DSLGYFRKIAPGVGAFRFTVTGAEGMFKLSQEQPGEVRDRVRESFGQSGC
    VHKRGTAGLMSRLP
    144. >Streptomyces_sp._OK885
    MFVPDPYREPNTTWMVDLIRRNPLALLTTNGPAECGPFATHLPVIQDPGM
    TAEWSADLSGSLLLGHMNAQNPHWSALRDGDSVLLAFTGPHAYVSPTVYQ
    KIPAAPTWNFTSVHVHGVIEKIESEEETLTVVRSTVRAFEEEFGTDWNME
    GSVDYFRKILPGVGAFRITVSRADGMFKLSQEQEPQIRDRVRQSFAQRKC
    SLHRETADLMGRLP
    145. >Streptomyces_sp._CFMR7
    MYVPSIYQAEDRAWLRHVVERYPLATVITNGPQAPYATHVPVIPAPDTTS
    WNDGPEGATLLGHMNRANSHWGSLTDGTHAQLVFTGPNGYVSPTVYETSP
    AAPTWNFVSVHLRGRLRPISDFEETLEVVRLTVEAYEKNFGDGWEMDSSL
    EYFRNIGPAVGGFRFDVESADGMFKLSQEKHPETRRRIADRFGGRRSGRA
    TELAFFMRQFTSADHHAS
    146. >Streptomyces_sp._DvalAA-19
    MYVPSIYQAEDRAWLRHVVERYPLATVITNGPQAPYATHVPVIPAPDTTS
    WNDGPEGATLLGHMNRANSHWGSLTDGTHAQLVFTGPNGYVSPTIYETSP
    AAPTWNFVSVHLRGRLRPISDFEETLEVVRLTVEAYEKNFGDGWEMDSSL
    EYFRNIGPAVGGFRFDVESADGMFKLSQEKHPETRRRIADRFGGRRSGRA
    TELAFFMRQFTSADRHAS
    147. >Rhodococcus_fascians_A3b
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    148. >Rhodococcus_fascians_A73a
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    149. >Rhodococcus_fascians_A76
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    150. >Rhodococcus_fascians_A78
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    151. >Rhodococcus_fascians_D188
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    152. >Rhodococcus_fascians_02-816c
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    153. >Rhodococcus_fascians_05-339-1
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLIVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    154. >Rhodococcus_fascians_LMG_3605
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    155. >Rhodococcus_fascians_LMG_3616
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    156. >Rhodococcus_fascians_LMG_3623
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    157. >Rhodococcus_fascians_A22b
    MYVPRIYKASDRTWLRRVVAQYPFAALISNGPKAPYATHLPVICAPCAPS
    ESEDLEGSTLFGHMNRANPHWDSLVDGADAQLIFTGPHGYVTPSVYQRDS
    VAPTWNYVSVHLRGKLQPVADFEETLKVVQLTVSTYEQKFGSGWEMDSSL
    DHYRRIGPAVGAFSFEVESADGMFKLSQEQNLETRRRVADHFSANHAGRG
    KELASFMREYSHGDYNNF
    158. >Streptomyces_sp._CNT360
    MYVPQHFAVDETEPVVELIRANPLAVFVTTQGGVPVASHIPVVFASEDEA
    EQADDLVGVTLFGHLNVQNPQYGVLADGDRVLVVFQGSHGYISPTVYDTV
    PAAPTWNFSAVHVTGTVRLLGPGEPALKVVRRIVTALERRFGAGWDMTES
    LPYFERIVPGVGAFEIAVEAVDSIFKLSQDQPAELRDKAECAFRNSDAGV
    HRELAAQMRRHNGAACSHQERTARDGD
    159. >Salinispora_arenicola_CN5848
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQ
    TGPGSVLLGHMNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAP
    TWDFVVVHVSGRVMPLDAGEPTLAVVQRTAATLEGAFGAGWDHTGSIDYF
    RSIVGGVGAFEFVVEQVESMFKLSQEKDHTVRQRLIDDFTSAPRNGSAQV
    GQLMSDLNLGVAP
    160. >Salinispora_arenicola_CNY231
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQ
    TGPGSVLLGHMNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAP
    TWDFVVVHVSGRVMPLDAGEPTLAVVQRTAATLEGAFGAGWDHTGSIDYF
    RSIVGGVGAFEFVVEQVESMFKLSQEKDHTVRQRLIDDFTSAPRNGSAQV
    GQLMSDLNLGVAP
    161. >Salinispora_arenicola_CNY280
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQ
    TGPGSVLLGHMNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAP
    TWDFVVVHVSGRVMPLDAGEPTLAVVQRTAATLEGAFGAGWDHTGSIDYF
    RSIVGGVGAFEFVVEQVESMFKLSQEKDHTVRQRLIDDFTSAPRNGSAQV
    GQLMSDLNLGVAP
    162. >Salinispora_arenicola_CNT005
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQ
    TGPGSVLLGHMNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAP
    TWDFVVVHVSGRVMPLDAGEPTLAVVQRTAATLEGAFGAGWDHTGSIDYF
    RSIVGGVGAFEFVVEQVESMFKLSQEKDHTVRQRLIDDFTSAPRNGSAQV
    GQLMSDLNLGVAP
    163. >Salinispora_arenicola_CNY230
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQ
    TGPGSVLLGHMNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAP
    TWDFVVVHVSGRVMPLDAGEPTLAVVQRTAATLEGAFGAGWDHTGSIDYF
    RSIVGGVGAFEFVVEQVESMFKLSQEKDHTVRQRLIDDFTSAPRNGSTQV
    GQLMSDLNLGVAP
    164. >Salinispora_arenicola_CNY486
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQ
    TGPGSVLLGHMNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAP
    TWDFVVVHVSGRVMPLDAGEPTLAVVQRTAATLEGAFGAGWDHTGSIDYF
    RSIVGGVGAFEFVVEQVESMFKLSQEKDHTVRQRLIDDFTSAPRNGSTQV
    GQLMSDLNLGVAP
    165. >Salinispora_pacifica_CNY331
    MLVPHMYEAPSAAQVDAVITGHPMAVLVTNGPDVPHATHLPVIRTVDTEQ
    TGPGSVLLGHMNRTNPHWSALTSGTPGKLIFTGPNTYVCPVLYQTEPAAP
    TWDFVVVHVSGRVMPLDAGEPTLAVVQRTAATLEGAFGAGWDHTGSIDYF
    RSIVGGVGAFEFVVEQVESMFKLSQEKDHTVRQRLIDDFTSAPRNGSTQV
    GQLMSDLNLGVAP
    166. >Streptomyces_aureofaciens_NRRL_2209
    VFTPKLYQVDGDDWPLRIIERHPLAVLVSNGDPVPNATHVPVIAPPDAAP
    EDALSGMRLWAHLTRANPHWQQLAAAGGGPAKLVFHGPNGYVTPSLYSAD
    MVAPTWNYVAVHLEGTVELAGDDETLAIVHTTAQTLEDRFGDGMALAPSL
    EYHRQIVGAVGGLFFTVTKVDVMFKLSQEKDPEVQQRVLDRFAASGSGLH
    REVADTMRALRLGGSAG
    PzbAB
    167. >Streptomyces_sp._CFMR_7
    VRNAHATHPDDDPVGTTTERPYDLLGIGFGPSNLALAVCAREQKLPLSCL
    FVERQDTVAWHPGMLIDGARMQISFLKDLVSLRNPSSPYSFLQYTKAKGR
    LERFVNLNESRPTRIEYDDYLKWVAQDFADQVRFGSQVDRVTPVQGPDGG
    DLSLFRVETQDVATGRHSVHYARNVVHAGGGRPPARTAGVAEVSSVVHSS
    EFLTRFPDQFKDHDGAYRFVVVGGGQSAGEISEYLLDHYDRAEVHVVVSG
    YTLLPTDNSPFVNEQFYSGNADAFYRMRPEQRAAVSGRLRAANYGVVRED
    LLERLFNTDYLDQVKGRKRLHIHPFSRLSEVRENGDALAVTLRQHLDEGP
    EEPLRCDGVVLATGYDRSLDPAVFGDVLPHLTAGEGEGVGGVALSRHYRA
    RTSPELRAGLYLQGFGEAQFGLGDTLLSLLPFRSQEIVEDIADRVPVAGV
    GGCPVMSPYGSGVVSTSPHGPARSAVYPPKWYLEHDREKLYGLMERFRFA
    TLISARSGDQPFATHLPLILDRSRGANGVLFGHLDRGNEHADLIDGRHML
    AVFHGPNAYMPPGVFESDPLPTWNSMSVHVRGRVRVVRDRDALVHGLIGI
    AERSQPDNRLAADDPRIDRIIGSIVGFEFEVEELVGRFKLSQDRDETDRR
    HAAVALARATERGERDFIEYVVGLSLITEDDPRDLAGRPLSPLAIGGVHE
    168. >Micromonospora_tulbaghiae_DSM_45142
    MRNDPAPDARSSEPGSEQNPYDLIGVGFGPSNLALAIAAEELDGERTCLF
    FERSPSLQWHPGMLLEGSRMQISFLKDLVSLRNPASPYTFLQYAKAKDRL
    ERFVNLSEFRPTRLEYQDYLRWVAEFFAGQVRYHTEVTRVSPVRRPGEDV
    HRLFRVEARDIRTGETTVHHAANVVHAAGGRPRLPPGGVCASPAVIHSSD
    FLPHFPERFADRSRPYEFAVAGDGQSAGEVALYLMRTYPESRVHLFLSGQ
    ALRATDNSPFVNEQFFESSANAFSARPRDERTALRAELRNTNYGVVEAGT
    LDDLYRTVYDDEVRGRHRLIVHPATRVVAVREGDEGPLVAILDRRSGAEG
    EIRCDGVVLATGYVRALDESIFSELTPFLRTESDKLLLSGYRVRTTAEVA
    GGFYVQGYGEQHFGLGDTLLSLLPFRSRQIFTDICRRTPPPRQAVAVSDA
    SAYPPPHYLEHDPEKLYAVMERFNFATVISARAAEDPVVTHVPLTLDRSR
    GAHGVLFGHLDRANPHAQLIDGKQVTVVFHGPNTYLSPYALETDALPTWN
    SMNVHVGGRGRLLADRAALVTGLSGICEKSDPGVDSYRLDPDDPRIDRLV
    DYVVGFEIEIQALVGRFKLSQELDDRNRRLAASALMATARRDESEVIGKV
    FGMSPVNGRQNGSSALWSAHSR
    169. >Amycolatopsis_alba_DSM_44262
    MRNDAPPNPLTAELGAEGNPYDLIGVGFGPSNLALAIAAEELDSERNCLF
    FERSSRLRWHPGMLIDGSRMQISFLKDLVSLRNLASPYTFLQYTKAKGRL
    EQFVNLNDFRPTRLEYQDYLEWVAESFSGQVRYNSEVTRVTPVRRTGEDA
    HRLFRVEARDVVTGQTTVRYAANVVHAAGGRPRLPDGGVCDSPAVVHSSD
    FLPRFPGHFADRSRPYEFGVAGDGQSAGEIAAYLLSRYPASRVHLLLSGS
    ALRAADSNPFVNEQFFEGRANHFHARTKPDRTGLLAELRNTNYAVVEPGF
    LDDLYRLVYDDEVRGTRRLIVHPGTKVTAVGADGASLRVAVTDRRGGDEE
    MRCDGVVLATGYVRALDESMFADLLPFLREESGDLVLSPDYRVGTTAELE
    GGFYVQGYGESSFGLGDTLLSLLPFRAKQIFTDICKQTPPPVRTRRPVEV
    SKASAYPPPHYVETDPKKIYAVMERFSFATLISARGAEDPVVTHLPLTLD
    RARGAHGVLFGHLDRANPHVQLIDGHQLTVLFHGPNAYLSPQVFETSVLP
    TWNSMNVHVRGRGRLLPDRAALLAGLSGICVKSDPGDDSYRLDLDDPRID
    RMIEHIVGFEIEIHELVGRFKLSQELDDQNRMLAASALSATARRGELELI
    EEVVGLNVVQG
    170. >Mycobacterium_sp._IS-1556
    MTSMPPGEGHDSDLDFIGIGFGPSNLALAVAADEIVPDRKGLFFERSGTF
    QWHPGMLLDGTKMQISFLKDLATLRNPASRYTFLQYAKARGRLEQFVNLH
    EFHPSRLEYNDYLRWVAEFFTDRVCYNTIVTAVVPVGHSPSSNGHLTRFR
    VHVRDMATGAESCFFTANVIFGGGGVPRLLGARADASAVLHSSAFLPNFT
    NRFNESQKPYRFAVIGNGQSAAEIVDYLLNHYPGATIHLFISDCTLRATD
    HSPFINEHFFSTSAADFYNHPPAQRVALRSALRSTNYGVVDADLLQKLYQ
    ITYLDEVKGCRRLLLHRESRLSQIEEIDDQVVASFEDRFSGDSSEFHFDG
    AVLATGYERVLDAEVFRHVLPHVLWDESGAISLTRSCRVNIVPAVTARLF
    LQGYGEAWFGIGDTLLSLLPFRAQAIAQEIGNAPSGAPIRRKQRVHGEYP
    PKRYLETDPDRLHDVINRYRFATLVSASGVDEPVVTQLPLTLDTSRGSLG
    VLFGHMDFANPHTELLDGRRVLVLFHGPNGYISPHVYESAQLPTWNSITV
    EVRGRARILRDKDAVVNGLRGIAAAADPTPGGFRLTREAASDQRLFPLLV
    GFEIDIDDMRGRFKLSQERDDRDRWHAAHALANGVEQDDRDLISSIVGLP
    LDVDEEPKPQQQAQIHQYGNAPADTAYRRVDG
    171. >Streptomyces_sp._Root55
    MSSEAGAVFPCANGRPAAEVAPGPSRGSHPADPYDLIGVGFGPSNMALAI
    AVEELDPGRSCLFLERNTGVRWHPGMLIEGARMQISYLKDLVSLRNLASP
    YTFLSYLKAKGRLEKFINVGASRPTRLEYQDYLSWVAEDFGHVVRYESEV
    VAVVPVAGPGSETLDLLRVRVRDAGSAEFHDLYARNVVHAGGGTPRRGAP
    GQICDASSVIHSSTFLDAFPARFPDHDAALDLGVVGDGQSAAEITSHVLK
    GYPNARVHLFVPGYALRATDNNPFANEQFYQRNAGEFYASGARRRTILRT
    ELRNTNYGAVEAGHLDELYDITYADEVRGAPRLVVHRASHVSRVVEDGER
    LSVEVRDRTDGPDRTMVCDGLVLATGYTRELHPAVFGELTPLLSRDDSGE
    LLVTADCRVRTDERVTAGFYVQGYAESAYGIGDTLLSLLPFRSQQIVDDI
    RGRLPAGRPVAVEESAPYPPSHYVETDLDRIRSLMERFNFATVISVARDA
    RVLVTHVPLVVERDRGGEHGMLIGHLDRSNPQVELLRDRPVTVVFHGPDA
    YLSPDVLKTDRLPTWNSMSVHVRGHARLFSGRDELMRVFNGLCEQAEGES
    GSYWLRPDDTRIEQLRGQVVGFEVDIHELTGRFKLSQELDEANRELAAAD
    MARGTSAERQAFIERAFDLQPRPDVLGPPGGPGVGGCPVGGARAAGGTTA
    VADNERETAR
    172. >Streptomyces_sp._2AW
    MLDLLGIGFGPSNVALAAAMAEGGKPPRALFLEAKERFGWHPGMLLDGAR
    MQISFLKDLVTLRNPESPYSFLAYLKAKGRLEEFANLREFYPSRIEFQDY
    LRWVAGHFEHQAVFGARVASVSPDFGIDGMARSFTVRAELADSGEYVTYQ
    ARNVVYAPGGTPNRVAGVAPRDERVIHTAEFLERFPKSFPDHSADLSFAV
    VGGGQSAAEIIEYILAKYPLSRVHAILPGYSFRPADDSPYSNEVFFSAEV
    DDHFTAHDQAARLAEARSTNYGVVDLDLIEDLYRMGYEDQVRGNVPRLTF
    CRSSRLLSADAGPSGIEVTVGGPEGSRSLNLDGLVLATGYHRELDPEMFR
    DVIPHLQRNESGNFLVSRAYRADSVPELTAGIYFQGLTELSHGIGDTLLS
    LLSFRSAEIAEDVRKRSEVPSADEVEYPPARHIEPYRAAILETLQRFPLA
    TLISSDDESEVFATHLPLILDRERGEQGVLFGHLDVGNPQVPNLNGRRVL
    AVFHGPNSYISPRTYTTDQLPTWNYVAVHVRGHVRVLENQDQVVSGLASI
    SEKADRSDGAYRLDENDSRIEKLIGGIVGFELDIESLTGRFKLSQDRSDE
    DRKRAMAVLREGAGDEHHDFVARIHQQ
    173. >Streptomyces_sp._SolWspMP-5a-2
    MPKKGGAVTPRAQGLPSGEAGPAPRRGTDPADPLDLIGIGFGPSNLALAI
    AAEELDPAADRLFLERNAGVHWHPGMLLEGARMQISYLKDLVSLRNLASP
    YTFLSYLKAKGRLEKFINIGVTRPTRLEYQDYLTWVAGHFADVVRYRSEV
    VSVTPVSGPGSTALDLLHVRVRDTATGTPYSLYARNVVHAGGGTPRRGTP
    DRICDTPSVIHSSRFLPAFPRRFPDHDAALDLGVVGDGQSAAEIAAHMLT
    HYPDATVHLFVPGYALRATDNNPFVNEQFYRHNADAFYADEPHRRALLRT
    ELRNTNYGAVEAGYLDTLYDITYADEVRGAPRLLVHRGCDVTRITEDGPR
    LDVLVRDRTGGPDRTVRCDGVVLATGYTRALDPAVFAGLDPLLRRDESGA
    LLVSADCRVDAEAPLTAGFYVQGYAEGAYGIGDTLLSLLPFRSQRIIDDL
    RARRPEDLPSGGPYPPDHYVEKDLERVRAVMERFNFATVISADRDARVLV
    THVPLVVERDRGGEHGTLIGHLDRSNPQVELLRDRPVTVVFHGPNSYLSP
    DVLTTDKLPTWNSMSVHVRGHARLFSGRDELMRVFNGLCEQAEPGPGSYR
    LRPDDERIDQLLGHVVGFEVDIQEVTGRFKLSQDLDEDNRALAAADMQRD
    LGEERRTFVADVFDLAPRPDGPEAGPRACGCPLGGPPAGTGAALAEEAGQ
    TVR
    174. >Streptomyces_sp._ScaeMP-e83
    VRNAHATHPDDDPVGTTTERPYDLLGIGFGPSNLALAVCAREQKLPLSCL
    FVERQDTVAWHPGMLIDGARMQISFLKDLVSLRDPSSPYSFLRYTKAKGR
    LERFVNLNESRPTRIEYDDYLKWVAQDFADQVRFGSQVDRVTPVQGPDGG
    DLSLFRVETEDVATGRRSVHYARNVVHAGGGRPPTRTAGVAEVPSVVHSS
    EFLTRFPGQFKDHDGAYRFVVVGGGQSAGEISEYLLDHYDRAEVHVVVPG
    YTLLPTDNSPFVNEQFYSGNADAFYRMRPEQRAAVSGRLRAANYGVVRED
    LLERLFNTDYLDQVKGRKRLHIHSFSRLSEVREDGEALAVTLQPRLDEGP
    EESLRCDGVVLATGYDRSLDPAVFGDVLPHLTPGEGEGAAGVVLSRHYRA
    RTSPELRAGLYLQGFGEAQFGLGDTLLSLLPFRSQEIVEDIADRVPAAGV
    GGCPVMSPYGSGVVSTSPHGPVPSAVYPPKWYLEHDREKLYGLMERFRFA
    TLISARSGDEPFATHLPLILDRSRGANGVLFGHLDRGNEHAELIDGRHML
    AVFHGPNAYMPPGVFESDPLPTWNSMSVHVRGRVRAVRDQDALVRGLIGI
    AERSQPDNRLAADDPRIDRIIGSIVGFEFEVEELVGRFKLSQDRDETDRR
    HAAVALARATERGERDFIEYVVGLSLITEDDPRDLAGRPLSPSP
    175. >Mycobacterium_sp._GA-0227b
    MTSMPPGEGHDSDLDFIGIGFGPSNLALAVAADEIVPDRKGLFFERSGTF
    QWHPGMLLDGTKMQISFLKDLATLRNPASRYTFLQYAKARGRLEQFVNLH
    EFHPSRLEYNDYLRWVAEFFTDRVCYNTIVTAVVPVGHSPSSNGHLTRFR
    VHVRDMATGAESCFFTANVIFGGGGVPRLLGARADASAVLHSSAFLPNFT
    NRFNESQKPYRFAVIGNGQSAAEIVDYLLNHYPGATIHLFISDCTLRATD
    HSPFINEHFFSTSAADFYNHPPAQRVALRSALRSTNYGVVDADLLQKLYQ
    ITYLDEVKGCRRLLLHRESRLSQIEEIDDQVVASFEDRFSGDSSEFHFDG
    AVLATGYERVLDAEVFRHVLPHVLWDESGAISLTRSCRVNIVPAVTARLF
    LQGYGEAWFGIGDTLLSLLPFRAQAIAQEIGNAPSGAPIRRKQRVHGEYP
    PKRYLETDPDRLHDVINRYRFATLVSASGVDEPVVTQLPLTLDTSRGSLG
    VLFGHMDFANPHTELLDGRRVLVLFHGPNGYISPHVYESAQLPTWNSITV
    EVRGRARILRDKDAVVNGLRGIAAAADPTPGGFRLTREAASDQRLFPLLV
    GFEIDIDDMRGRFKLSQERDDRDRWHAAHALANGVEQDDRDLISSIVGLP
    LDVDEEPKPQQQAQIHQYGNAPADTAYRRVDG
    176. >Mycobacterium_sp._GA-1999
    MTSMPPGEGHDSDLDFIGIGFGPSNLALAVAADEIVPDRKGLFFERSGTF
    QWHPGMLLDGTKMQISFLKDLATLRNPASRYTFLQYAKARGRLEQFVNLH
    EFHPSRLEYNDYLRWVAEFFTDRVCYNTIVTAVVPVGHSPSSNGHLTRFR
    VHVRDMATGAESCFFTANVIFGGGGVPRLLGARADASAVLHSSAFLPNFT
    NRFNESQKPYRFAVIGNGQSAAEIVDYLLNHYPGATIHLFISDCTLRATD
    HSPFINEHFFSTSAADFYNHPPAQRVALRSALRSTNYGVVDADLLQKLYQ
    ITYLDEVKGCRRLLLHRESRLSQIEEIDDQVVASFEDRFSGDSSEFHFDG
    AVLATGYERVLDAEVFRHVLPHVLWDESGAISLTRSCRVNIVPAVTARLF
    LQGYGEAWFGIGDTLLSLLPFRAQAIAQEIGNAPSGAPIRRKQRVHGEYP
    PKRYLETDPDRLHDVINRYRFATLVSASGVDEPVVTQLPLTLDTSRGSLG
    VLFGHMDFANPHTELLDGRRVLVLFHGPNGYISPHVYESAQLPTWNSITV
    EVRGRARILRDKDAVVNGLRGIAAAADPTPGGFRLTREAASDQRLFPLLV
    GFEIDIDDMRGRFKLSQERDDRDRWHAAHALANGVEQDDRDLISSIVGLP
    LDVDEEPKPQQQAQIHQYGNAPADTAYRRVDG
    Plasmids:
    177. SfaB (PzbA) expression
       1 tatggctgcc gcgcggcacc aggccgctgc tgtgatgatg atgatgatgg ctgctgccca
      61 tggtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct
     121 cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg
     181 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
     241 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
     301 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
     361 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
     421 tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc
     481 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa
     541 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     601 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     661 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     721 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     781 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     841 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     901 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     961 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
    1021 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
    1081 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
    1141 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
    1201 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
    1261 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
    1321 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
    1381 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
    1441 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
    1501 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
    1561 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
    1621 caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt
    1681 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
    1741 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt
    1801 cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt
    1861 attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt
    1921 cggcgagaag caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct
    1981 ggcgttcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg
    2041 catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg
    2101 acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcactg gaccgctgat
    2161 cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg
    2221 cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac
    2281 ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga
    2341 gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc
    2401 atcgcgtccg ccatctccag cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg
    2461 ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
    2521 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
    2581 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
    2641 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
    2701 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
    2761 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
    2821 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
    2881 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
    2941 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
    3001 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
    3061 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
    3121 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
    3181 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
    3241 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
    3301 gagtgcacca tatatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
    3361 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
    3421 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
    3481 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
    3541 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
    3601 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
    3661 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
    3721 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
    3781 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
    3841 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
    3901 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
    3961 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
    4021 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
    4081 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
    4141 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
    4201 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
    4261 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
    4321 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
    4381 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
    4441 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
    4501 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
    4561 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
    4621 tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
    4681 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
    4741 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
    4801 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
    4861 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
    4921 gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
    4981 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
    5041 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
    5101 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
    5161 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
    5221 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
    5281 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
    5341 aaataggcgt atcacgaggc cctttcgtct tcaagaattc tcatgtttga cagcttatca
    5401 tcgataagct ttaatgcggt agtttatcac agttaaattg ctaacgcagt caggcaccgt
    5461 gtatgaaatc taacaatgcg ctcatcgtca tcctcggcac cgtcaccctg gatgctgtag
    5521 gcataggctt ggttatgccg gtactgccgg gcctcttgcg ggatatccgg atatagttcc
    5581 tcctttcagc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt atgctagtta
    5641 ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg gctttgttag cagccggatc
    5701 ctcagcccct gttccccgct gctgccttgc ttccggtgga gcggtccggg tcgcaccggc
    5761 cgccggtgat cgaccgggcg atctcgcccg cgcggaccgc caccatggac agcagggtgg
    5821 aggcgatgcc gtgggtcgcc tcggtggcgc cctggacgta gatgccgcac cggaaatccc
    5881 cggtggtgcc gagccggtag tcgcggccga tcagcaactc ccccgcctcg tcccggcgga
    5941 gggcgccgga gacgccgccg agcagttcgg ccgggtcggt ggagtcgtac ccggtggcgt
    6001 acacgaccag gtcggcgtcc aggtcggtgt gttcgcccgt gggcaggaac tccacgcgta
    6061 cggcggcgga ttcctggcgc ggttcgacgg acaccaggcg ggaggcgttc atcacccgca
    6121 gccgcggggc gccggacacc ttctgctcgt actggcggcg gtagaggccc tggaggacgt
    6181 cctcgtcgac gacggcgtag ttggtgccgc cgtggtagcg catgatggcc tgcttgacct
    6241 cgggcggggc gaagtagaag tcgtccacgg cggccgggtc gaagacgcgg ttggcgaacg
    6301 ggctggagtc ggcgacgctg tagccgtagc gggcgaacac cgcgcacacc tcggcctgcg
    6361 ggtagcggtc catgaggtgc gcggcgacct cggccgcgct ctggccggcg ccgaccacga
    6421 cggcccggcg gggcgggcgt tcgtcgaacg cgggcagccg gtgcagcaac tgggagctgt
    6481 gccagacgcg ttcgccggtc tccgcgccct cgggcagccg ggggcgcagg ccggaggcga
    6541 ggacgaggtt tctggtccgg gcgaccaccc ggtccccggc gagcacgtcg agcgcgacga
    6601 cctcaccggc ttcggtcacc ggccgcacac cggtggcctc cacgccgtac tcgaccaggt
    6661 ggttcagccg gtcggcggcc cactggaggt agtcgtggta ctcgatccgg gagggcagca
    6721 gggtgtgctg gttgatgaag tcgaccagcc ggtccttctc ctggagatag gacaggaatc
    6781 cgaaatcact ggtgggattg cgcatcgtgg cgatgtcctt gagaaaggac acctggagcg
    6841 aggagccccc caggagcatc ccccgatgcc agccgaattc cttctgcttc tccaggaaaa
    6901 gggccttccc ggcggcttcg gattcatgga gcgccaccgc cagggcgaga ttcgcggcac
    6961 cgaatccgat tccggtgacg tccagtactt ctgattccgg gctctgctgc gcagtggatg
    7021 attgctctgc gagccgggtc a
    178. SfaB (PzbA) in vivo expression
       1 gtaggagggc gtggatatgt cctgcgggta aactatagtc gttgagagga ggagtctgac
      61 tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg
     121 gttgccgccg ggcgtttttt attggtgaga ataggtcttg acggctggcg agaggtgcgg
     181 ggaggatctg accgacgcgg tccacacgtg gcaccgcgat gctgttgtgg gcacaatcgt
     241 gccggttggt aggatccggt taattaagca gtaccagatc tgactgagtg accaaaggag
     301 gcggacatat gacccggctc gcagagcaat catccactgc gcagcagagc ccggaatcag
     361 aagtactgga cgtcaccgga atcggattcg gtgccgcgaa tctcgccctg gcggtggcgc
     421 tccatgaatc cgaagccgcc gggaaggccc ttttcctgga gaagcagaag gaattcggct
     481 ggcatcgggg gatgctcctg gggggctcct cgctccaggt gtcctttctc aaggacatcg
     541 ccacgatgcg caatcccacc agtgatttcg gattcctgtc ctatctccag gagaaggacc
     601 ggctggtcga cttcatcaac cagcacaccc tgctgccctc ccggatcgag taccacgact
     661 acctccagtg ggccgccgac cggctgaacc acctggtcga gtacggcgtg gaggccaccg
     721 gtgtgcggcc ggtgaccgaa gccggtgagg tcgtcgcgct cgacgtgctc gccggggacc
     781 gggtggtcgc ccggaccaga aacctcgtcc tcgcctccgg cctgcgcccc cggctgcccg
     841 agggcgcgga gaccggcgaa cgcgtctggc acagctccca gttgctgcac cggctgcccg
     901 cgttcgacga acgcccgccc cgccgggccg tcgtggtcgg cgccggccag agcgcggccg
     961 aggtcgccgc gcacctcatg gaccgctacc cgcaggccga ggtgtgcgcg gtgttcgccc
    1021 gctacggcta cagcgtcgcc gactccagcc cgttcgccaa ccgcgtcttc gacccggccg
    1081 ccgtggacga cttctacttc gccccgcccg aggtcaagca ggccatcatg cgctaccacg
    1141 gcggcaccaa ctacgccgtc gtcgacgagg acgtcctcca gggcctctac cgccgccagt
    1201 acgagcagaa ggtgtccggc gccccgcggc tgcgggtgat gaacgcctcc cgcctggtgt
    1261 ccgtcgaacc gcgccaggaa tccgccgccg tacgcgtgga gttcctgccc acgggcgaac
    1321 acaccgacct ggacgccgac ctggtcgtgt acgccaccgg gtacgactcc accgacccgg
    1381 ccgaactgct cggcggcgtc tccggcgccc tccgccggga cgaggcgggg gagttgctga
    1441 tcggccgcga ctaccggctc ggcaccaccg gggatttccg gtgcggcatc tacgtccagg
    1501 gcgccaccga ggcgacccac ggcatcgcct ccaccctgct gtccatggtg gcggtccgcg
    1561 cgggcgagat cgcccggtcg atcaccggcg gccggtgcga cccggaccgc tccaccggaa
    1621 gcaaggcagc agcggggaac aggggctgag gatccccggg taccttcgaa aaaaaaaggc
    1681 tccaaaagga gcctttaatt gttcctccag accttacttg accggcgctc actgcccgct
    1741 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
    1801 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
    1861 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
    1921 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
    1981 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
    2041 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
    2101 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
    2161 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
    2221 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
    2281 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
    2341 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
    2401 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
    2461 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
    2521 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
    2581 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
    2641 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
    2701 ttggttcatg tgcagctcca ctgctttaga ctctacatct gtatgaagtc ttcagatcct
    2761 ctacgccgga cgcatcgtgg ccggatctaa aaaaaagccc gctcattagg cgggctgaca
    2821 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
    2881 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
    2941 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
    3001 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
    3061 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
    3121 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
    3181 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
    3241 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
    3301 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
    3361 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
    3421 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
    3481 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
    3541 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
    3601 gcgtttctgg gtgagcaaaa acaggaaggc aaagtgccgc aaaaaaggga ataagggcga
    3661 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
    3721 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
    3781 ttccgcgcac atttccccga aaagtgccac ctggcgcgcc acaaaacagc agggaagcag
    3841 cgcttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca
    3901 tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt
    3961 atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc
    4021 ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc
    4081 tggccggcta ccgccggcgt aacagatgag ggcaagcgga tggctgatga aaccaagcca
    4141 accaggaagg gcagcccacc tatcaaggtg tactgccttc cagacgaacg aagagcgatt
    4201 gaggaaaagg cggcggcggc cggcatgagc ctgtcggcct acctgctggc cgtcggccag
    4261 ggctacaaaa tcacgggcgt cgtggactat gagcacgtcg gcgcgcctct agtatgcagg
    4321 agtggggagg cacgatggcc gctttggtcg acctcaacga gacgatgaag ccgtggaacg
    4381 acaccacccc ggcggccctg ctggaccaca cccggcacta caccttcgac gtctgatcat
    4441 cactgacgaa tcgaggtcga ggaaccgagc gtccgaggaa cagaggcgct tatcggttgg
    4501 ccgcgagatt cctgtcgatc ctctcgtgca gcgcgattcc gagggaaacg gaaacgttga
    4561 gagactcggt ctggctcatc atggggatgg aaaccgaggc ggaagacgcc tcctcgaaca
    4621 ggtcggaagg cccacccttt tcgctgccga acagcaaggc cagccgatcc ggattgtccc
    4681 cgagttcctt cacggaaatg tcgccatccg ccttgagcgt catcagctgc ataccgctgt
    4741 cccgaatgaa ggcgatggcc tcctcgcgac cggagagaac gacgggaagg gagaagacgt
    4801 aacctcggct ggccctttgg agacgccggt ccgcgatgct ggtgatgtca ctgtcgacca
    4861 ggatgatccc cgacgctccg agcgcgagcg acgtgcgtac tatcgcgccg atgttcccga
    4921 cgatcttcac cccgtcgaga acgacgacgt ccccacgccg gctcgcgata tcgccgaacc
    4981 tggccgggcg agggacgcgg gcgatgccga atgtcttggc cttccgctcc cccttgaaca
    5041 actggttgac gatcgaggag tcgatgaggc ggaccggtat gttctgccgc ccgcacagat
    5101 ccagcaactc agatggaaaa ggactgctgt cgctgccgta gacctcgatg aactccaccc
    5161 cggccgcgat gctgtgcatg aggggctcga cgtcctcgat caacgttgtc tttatgttgg
    5221 atcgcgacgg cttggtgaca tcgatgatcc gctgcaccgc gggatcggac ggatttgcga
    5281 tggtgtccaa ctcagtcatg gtcgtcctac cggctgctgt gttcagtgac gcgattcctg
    5341 gggtgtgaca ccctacgcga cgatggcgga tggctgccct gaccggcaat caccaacgca
    5401 aggggaagac tacgccttcc actagaccgg tcgacctgca ggcctgctgg cgccggacgg
    5461 ggcttcagac gtttcgggtg ctgggttgtt gtctctggac agtgatccat gggaaactac
    5521 tcagcaccac caatgttccc aaaagaaagc gcaggtcagc gcccatgagc caatatctag
    5581 gcatgtcgcc cttcatcgct cccgaggtcc ctgagcacct tctcgacact gttcgcgtct
    5641 tcctgtacgc gcgtcagtct aagggccggt ccgacggctc agacgtgtcg accgaagcac
    5701 agctcgcggc cggtcgtgcg ttggtcgcgt ctcgcaacgc ccaggggggt gcgcgctggg
    5761 tcgtggcagg tgagttcgtg gacgtcgggc gctccggctg ggacccgaac gtgacccgtg
    5821 ccgacttcga gcgcatgatg ggcgaagtcc gcgccggcga aggtgacgtt gtcgttgtga
    5881 atgagctttc ccggctcact cgcaagggcg cccatgacgc gctcgaaatc gacaacgaat
    5941 tgaagaagca cggcgtgcgc ttcatgtcgg ttcttgagcc gttccttgac acgtctaccc
    6001 ctatcggcgt cgccattttc gcgctgatcg ctgcccttgc gaaacaggac agtgacctga
    6061 aggcggagcg cctgaagggt gcgaaagacg agattgccgc gctgggtggc gttcactcgt
    6121 cttccgcccc gttcggaatg cgcgccgtgc gcaagaaggt cgataatctc gtgatctccg
    6181 ttcttgagcc ggacgaagac aacccggatc acgtcgagct agttgagcgc atggcgaaaa
    6241 tgtcgttcga gggcgtgtcc gacaacgcca ttgcaacgac cttcgagaag gaaaagatcc
    6301 cgtcgcccgg aatggctgag agacgcgcca cggaaaagcg tcttgcgtcc atcaaggcac
    6361 gtcgcctgaa cggcgctgaa aagccgatca tgtggcgcgc tcaaacggtc cgatggattc
    6421 tcaaccatcc cgcaatcggc ggtttcgcat tcgagcgtgt gaagcacggt aaggcgcaca
    6481 tcaacgtcat acggcgcgac cccggcggca agccgctaac gccccacacg ggcattctca
    6541 gcggctcgaa gtggcttgag cttcaagaga agcgttccgg gaagaatctc agcgaccgga
    6601 agcctggggc cgaagtcgaa ccgacgcttc tgagcgggtg gcgtttcctg gggtgccgaa
    6661 tctgcggcgg ctcaatgggt cagtcccagg gtggccgtaa gcgcaacggc gaccttgccg
    6721 aaggcaatta catgtgcgcc aacccgaagg ggcacggcgg cttgtcggtc aagcgcagcg
    6781 aactggacga gttcgttgct tcgagggtgt gggcacggct ccgcacagcc gacatggaag
    6841 atgaacacga tcaggcatgg attgccgccg ctgcggagcg cttcgccctt cagcacgacc
    6901 tagcgggggt ggccgatgag cggcgcgaac aacaggcgca cctagacaac gtgcggcgct
    6961 ccatcaagga ccttcaggcg gaccgtaagg ccggtctgta cgtcgggcgt gaagagctgg
    7021 aaacgtggcg ctcaacggtg ctgcaatacc ggtcctacga agcggagtgc acgacccgac
    7081 tcgctgagct tgacgagaag atgaacggca gcacccgcgt tccgtctgag tggttcagcg
    7141 gcgaagaccc gacggccgaa gggggcatct gggcaagctg ggacgtgtac gagcgtcggg
    7201 agttcctgag cttcttcctt gactccgtca tggtcgaccg ggggcgccac cctgagacga
    7261 agaaatacat ccccctgaag gaccgtgtga cgctcaagtg ggcggagctg ctgaaggagg
    7321 aagacgaagc gagcgaagcc actgagcggg agcttgcggc gctgtaggta caatcataat
    7381 gaggctagac tacagacgcg aagaatctcg tgctttcagc ttcgat
    179. SfaC (PzbB) expression
       1 tatgtacgaa cgtccgctgt accgggagga ttgcgacggc gtcgtcctgg cgtttctgcg
      61 acacaaccca ctggcaatgg tcgtcacctc gcacgacgac gtcccggtgg ccacccacgc
     121 gccggtgctg ttccggcacg gacccgacgg cgccgacgcc gaggccgtcg ccgcgggcac
     181 cgtcccgctc gccggctcca ccctgatcgg ccacatgaac gtcgagaacc cgcagtggcg
     241 ccggatgcgc tccggcgacc gggcgctcat cgtcttccag ggcccgcacg gctatgtctc
     301 gccgacggtc tacggggtca cgcccgcggc ccccacctgg gacttcatcg ccgtccacgt
     361 gaacggcaca gtggagccca ccgccgaccc cgccgccgtg ctggacatcg tctccgacac
     421 cgcccggcgg ctggagtccg gcttcgggcg cggctgggac caggagtcct ccctcgacta
     481 cttccgccag atcgcgcccg gcgtgggcgc cttcaccctg cgggtcgatt ccgtgcagac
     541 gatgttcaag ctcagccagg agaagcccgc cccgatgcgg cggcgcgtgg tcgagcagtt
     601 cgaagcaagc gagtccggca cccaccgcgc cctggccagc gtgatgcgcg accgcggact
     661 caccgaagcc gacgaggagc gggagacagc cggatgagga tccggctgct aacaaagccc
     721 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     781 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatcccg
     841 caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt
     901 gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca
     961 atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga
    1021 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
    1081 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
    1141 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
    1201 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
    1261 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
    1321 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
    1381 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
    1441 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
    1501 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
    1561 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
    1621 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
    1681 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
    1741 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
    1801 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
    1861 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
    1921 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
    1981 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
    2041 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
    2101 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
    2161 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
    2221 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
    2281 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
    2341 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
    2401 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
    2461 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
    2521 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
    2581 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
    2641 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
    2701 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
    2761 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
    2821 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
    2881 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
    2941 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
    3001 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
    3061 atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc
    3121 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
    3181 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
    3241 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
    3301 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
    3361 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
    3421 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
    3481 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
    3541 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
    3601 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
    3661 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
    3721 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
    3781 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
    3841 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
    3901 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
    3961 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
    4021 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
    4081 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
    4141 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
    4201 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca gtgatcgaag
    4261 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
    4321 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
    4381 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
    4441 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
    4501 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
    4561 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
    4621 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
    4681 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
    4741 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
    4801 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
    4861 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
    4921 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
    4981 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
    5041 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
    5101 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
    5161 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
    5221 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
    5281 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
    5341 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
    5401 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
    5461 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
    5521 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
    5581 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
    5641 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
    5701 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
    5761 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
    5821 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
    5881 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
    5941 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
    6001 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
    6061 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
    6121 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
    6181 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
    6241 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
    6301 gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca
    6361 tcatcacagc agcggcctgg tgccgcgcgg cagcca
    180. SfaC (PzbB) complementation of flaveolus
       1 gtaggagggc gtggatatgt cctgcgggta aactatagtc gttgagagga ggagtctgac
      61 tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg
     121 gttgccgccg ggcgtttttt attggtgaga ataggtcttg acggctggcg agaggtgcgg
     181 ggaggatctg accgacgcgg tccacacgtg gcaccgcgat gctgttgtgg gcacaatcgt
     241 gccggttggt aggatccggt taattaagca gtaccagatc tgactgagtg accaaaggag
     301 gcggacatat gtacgaacgt ccgctgtacc gggaggattg cgacggcgtc gtcctggcgt
     361 ttctgcgaca caacccactg gcaatggtcg tcacctcgca cgacgacgtc ccggtggcca
     421 cccacgcgcc ggtgctgttc cggcacggac ccgacggcgc cgacgccgag gccgtcgccg
     481 cgggcaccgt cccgctcgcc ggctccaccc tgatcggcca catgaacgtc gagaacccgc
     541 agtggcgccg gatgcgctcc ggcgaccggg cgctcatcgt cttccagggc ccgcacggct
     601 atgtctcgcc gacggtctac ggggtcacgc ccgcggcccc cacctgggac ttcatcgccg
     661 tccacgtgaa cggcacagtg gagcccaccg ccgaccccgc cgccgtgctg gacatcgtct
     721 ccgacaccgc ccggcggctg gagtccggct tcgggcgcgg ctgggaccag gagtcctccc
     781 tcgactactt ccgccagatc gcgcccggcg tgggcgcctt caccctgcgg gtcgattccg
     841 tgcagacgat gttcaagctc agccaggaga agcccgcccc gatgcggcgg cgcgtggtcg
     901 agcagttcga agcaagcgag tccggcaccc accgcgccct ggccagcgtg atgcgcgacc
     961 gcggactcac cgaagccgac gaggagcggg agacagccgg atgaggatcc ccgggtacct
    1021 tcgaaaaaaa aaggctccaa aaggagcctt taattgttcc tccagacctt acttgaccgg
    1081 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
    1141 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
    1201 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
    1261 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
    1321 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
    1381 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
    1441 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
    1501 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
    1561 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
    1621 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
    1681 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
    1741 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
    1801 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
    1861 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
    1921 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
    1981 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
    2041 ttcacctaga tccttttggt tcatgtgcag ctccactgct ttagactcta catctgtatg
    2101 aagtcttcag atcctctacg ccggacgcat cgtggccgga tctaaaaaaa agcccgctca
    2161 ttaggcgggc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    2221 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    2281 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    2341 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    2401 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    2461 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    2521 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    2581 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    2641 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    2701 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    2761 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    2821 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    2881 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    2941 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaagt gccgcaaaaa
    3001 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    3061 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    3121 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggc gcgccacaaa
    3181 acagcaggga agcagcgctt ttccgctgca taaccctgct tcggggtcat tatagcgatt
    3241 ttttcggtat atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta
    3301 gactttcctt ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc
    3361 gcgagcgggt gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat
    3421 cctgctctgc gaggctggcc ggctaccgcc ggcgtaacag atgagggcaa gcggatggct
    3481 gatgaaacca agccaaccag gaagggcagc ccacctatca aggtgtactg ccttccagac
    3541 gaacgaagag cgattgagga aaaggcggcg gcggccggca tgagcctgtc ggcctacctg
    3601 ctggccgtcg gccagggcta caaaatcacg ggcgtcgtgg actatgagca cgtcggcgcg
    3661 cctctagtat gcaggagtgg ggaggcacga tggccgcttt ggtcgacctc aacgagacga
    3721 tgaagccgtg gaacgacacc accccggcgg ccctgctgga ccacacccgg cactacacct
    3781 tcgacgtctg atcatcactg acgaatcgag gtcgaggaac cgagcgtccg aggaacagag
    3841 gcgcttatcg gttggccgcg agattcctgt cgatcctctc gtgcagcgcg attccgaggg
    3901 aaacggaaac gttgagagac tcggtctggc tcatcatggg gatggaaacc gaggcggaag
    3961 acgcctcctc gaacaggtcg gaaggcccac ccttttcgct gccgaacagc aaggccagcc
    4021 gatccggatt gtccccgagt tccttcacgg aaatgtcgcc atccgccttg agcgtcatca
    4081 gctgcatacc gctgtcccga atgaaggcga tggcctcctc gcgaccggag agaacgacgg
    4141 gaagggagaa gacgtaacct cggctggccc tttggagacg ccggtccgcg atgctggtga
    4201 tgtcactgtc gaccaggatg atccccgacg ctccgagcgc gagcgacgtg cgtactatcg
    4261 cgccgatgtt cccgacgatc ttcaccccgt cgagaacgac gacgtcccca cgccggctcg
    4321 cgatatcgcc gaacctggcc gggcgaggga cgcgggcgat gccgaatgtc ttggccttcc
    4381 gctccccctt gaacaactgg ttgacgatcg aggagtcgat gaggcggacc ggtatgttct
    4441 gccgcccgca cagatccagc aactcagatg gaaaaggact gctgtcgctg ccgtagacct
    4501 cgatgaactc caccccggcc gcgatgctgt gcatgagggg ctcgacgtcc tcgatcaacg
    4561 ttgtctttat gttggatcgc gacggcttgg tgacatcgat gatccgctgc accgcgggat
    4621 cggacggatt tgcgatggtg tccaactcag tcatggtcgt cctaccggct gctgtgttca
    4681 gtgacgcgat tcctggggtg tgacacccta cgcgacgatg gcggatggct gccctgaccg
    4741 gcaatcacca acgcaagggg aagactacgc cttccactag accggtcgac ctgcaggcct
    4801 gctggcgccg gacggggctt cagacgtttc gggtgctggg ttgttgtctc tggacagtga
    4861 tccatgggaa actactcagc accaccaatg ttcccaaaag aaagcgcagg tcagcgccca
    4921 tgagccaata tctaggcatg tcgcccttca tcgctcccga ggtccctgag caccttctcg
    4981 acactgttcg cgtcttcctg tacgcgcgtc agtctaaggg ccggtccgac ggctcagacg
    5041 tgtcgaccga agcacagctc gcggccggtc gtgcgttggt cgcgtctcgc aacgcccagg
    5101 ggggtgcgcg ctgggtcgtg gcaggtgagt tcgtggacgt cgggcgctcc ggctgggacc
    5161 cgaacgtgac ccgtgccgac ttcgagcgca tgatgggcga agtccgcgcc ggcgaaggtg
    5221 acgttgtcgt tgtgaatgag ctttcccggc tcactcgcaa gggcgcccat gacgcgctcg
    5281 aaatcgacaa cgaattgaag aagcacggcg tgcgcttcat gtcggttctt gagccgttcc
    5341 ttgacacgtc tacccctatc ggcgtcgcca ttttcgcgct gatcgctgcc cttgcgaaac
    5401 aggacagtga cctgaaggcg gagcgcctga agggtgcgaa agacgagatt gccgcgctgg
    5461 gtggcgttca ctcgtcttcc gccccgttcg gaatgcgcgc cgtgcgcaag aaggtcgata
    5521 atctcgtgat ctccgttctt gagccggacg aagacaaccc ggatcacgtc gagctagttg
    5581 agcgcatggc gaaaatgtcg ttcgagggcg tgtccgacaa cgccattgca acgaccttcg
    5641 agaaggaaaa gatcccgtcg cccggaatgg ctgagagacg cgccacggaa aagcgtcttg
    5701 cgtccatcaa ggcacgtcgc ctgaacggcg ctgaaaagcc gatcatgtgg cgcgctcaaa
    5761 cggtccgatg gattctcaac catcccgcaa tcggcggttt cgcattcgag cgtgtgaagc
    5821 acggtaaggc gcacatcaac gtcatacggc gcgaccccgg cggcaagccg ctaacgcccc
    5881 acacgggcat tctcagcggc tcgaagtggc ttgagcttca agagaagcgt tccgggaaga
    5941 atctcagcga ccggaagcct ggggccgaag tcgaaccgac gcttctgagc gggtggcgtt
    6001 tcctggggtg ccgaatctgc ggcggctcaa tgggtcagtc ccagggtggc cgtaagcgca
    6061 acggcgacct tgccgaaggc aattacatgt gcgccaaccc gaaggggcac ggcggcttgt
    6121 cggtcaagcg cagcgaactg gacgagttcg ttgcttcgag ggtgtgggca cggctccgca
    6181 cagccgacat ggaagatgaa cacgatcagg catggattgc cgccgctgcg gagcgcttcg
    6241 cccttcagca cgacctagcg ggggtggccg atgagcggcg cgaacaacag gcgcacctag
    6301 acaacgtgcg gcgctccatc aaggaccttc aggcggaccg taaggccggt ctgtacgtcg
    6361 ggcgtgaaga gctggaaacg tggcgctcaa cggtgctgca ataccggtcc tacgaagcgg
    6421 agtgcacgac ccgactcgct gagcttgacg agaagatgaa cggcagcacc cgcgttccgt
    6481 ctgagtggtt cagcggcgaa gacccgacgg ccgaaggggg catctgggca agctgggacg
    6541 tgtacgagcg tcgggagttc ctgagcttct tccttgactc cgtcatggtc gaccgggggc
    6601 gccaccctga gacgaagaaa tacatccccc tgaaggaccg tgtgacgctc aagtgggcgg
    6661 agctgctgaa ggaggaagac gaagcgagcg aagccactga gcgggagctt gcggcgctgt
    6721 aggtacaatc ataatgaggc tagactacag acgcgaagaa tctcgtgctt tcagcttcga
    6781 t
    181. SfaC (PzbB) in vivo expression
       1 atctacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
      61 gctctcgcag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     121 cctgcgggta aactatagtc gttgagagga ggagtctgac tcctgttgat agatccagta
     181 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     241 attggtgaga ataggtcttg acggctggcg agaggtgcgg ggaggatctg accgacgcgg
     301 tccacacgtg gcaccgcgat gctgttgtgg gcacaatcgt gccggttggt aggatccggt
     361 taattaagca gtaccagatc tgactgagtg accaaaggag gcggacatat gtacgaacgt
     421 ccgctgtacc gggaggattg cgacggcgtc gtcctggcgt ttctgcgaca caacccactg
     481 gcaatggtcg tcacctcgca cgacgacgtc ccggtggcca cccacgcgcc ggtgctgttc
     541 cggcacggac ccgacggcgc cgacgccgag gccgtcgccg cgggcaccgt cccgctcgcc
     601 ggctccaccc tgatcggcca catgaacgtc gagaacccgc agtggcgccg gatgcgctcc
     661 ggcgaccggg cgctcatcgt cttccagggc ccgcacggct atgtctcgcc gacggtctac
     721 ggggtcacgc ccgcggcccc cacctgggac ttcatcgccg tccacgtgaa cggcacagtg
     781 gagcccaccg ccgaccccgc cgccgtgctg gacatcgtct ccgacaccgc ccggcggctg
     841 gagtccggct tcgggcgcgg ctgggaccag gagtcctccc tcgactactt ccgccagatc
     901 gcgcccggcg tgggcgcctt caccctgcgg gtcgattccg tgcagacgat gttcaagctc
     961 agccaggaga agcccgcccc gatgcggcgg cgcgtggtcg agcagttcga agcaagcgag
    1021 tccggcaccc accgcgccct ggccagcgtg atgcgcgacc gcggactcac cgaagccgac
    1081 gaggagcggg agacagccgg atgaggatcc ccgggtacct tcgaaaaaaa aaggctccaa
    1141 aaggagcctt taattgttcc tccagacctt acttgaccgg cgctcactgc ccgctttcca
    1201 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
    1261 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
    1321 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
    1381 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
    1441 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
    1501 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
    1561 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
    1621 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
    1681 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
    1741 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
    1801 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
    1861 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
    1921 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
    1981 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
    2041 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
    2101 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttggt
    2161 tcatgtgcag ctccatcagc aaaaggggat gataagttta tcaccaccga ctatttgcaa
    2221 cagtgccgtt gatcgtgcta tgatcgactg atgtcatcag cggtggagtg caatgtcgtg
    2281 caatacgaat ggcgaaaagc cgagctcatc ggtcagcttc tcaaccttgg ggttaccccc
    2341 ggcggtgtgc tgctggtcca cagctccttc cgtagcgtcc ggcccctcga agatgggcca
    2401 cttggactga tcgaggccct gcgtgctgcg ctgggtccgg gagggacgct cgtcatgccc
    2461 tcgtggtcag gtctggacga cgagccgttc gatcctgcca cgtcgcccgt tacaccggac
    2521 cttggagttg tctctgacac attctggcgc ctgccaaatg taaagcgcag cgcccatcca
    2581 tttgcctttg cggcagcggg gccacaggca gagcagatca tctctgatcc attgcccctg
    2641 ccacctcact cgcctgcaag cccggtcgcc cgtgtccatg aactcgatgg gcaggtactt
    2701 ctcctcggcg tgggacacga tgccaacacg acgctgcatc ttgccgagtt gatggcaaag
    2761 gttccctatg gggtgccgag acactgcacc attcttcagg atggcaagtt ggtacgcgtc
    2821 gattatctcg agaatgacca ctgctgtgag cgctttgcct tggcggacag gtggctcaag
    2881 gagaagagcc ttcagaagga aggtccagtc ggtcatgcct ttgctcggtt gatccgctcc
    2941 cgcgacattg tggcgacagc cctgggtcaa ctgggccgag atccgttgat cttcctgcat
    3001 ccgccagagg cgggatgcga agaatgcgat gccgctcgcc agtcgattgg ctgagctcat
    3061 gagcggagaa cgagatgacg ttggaggggc aaggtcgcgc tgattgctgg ggcaacacgt
    3121 ggagcggatc ggggattgtc tttcttcagc tcgctgatga tatgctgacg ctcaatgccg
    3181 tttggcctcc gactaacgaa aatcccgcat ttggacggct gatccgattg gcacggcgga
    3241 cggcgaatgg cggagcagac gctcgtccgg gggcaatgag atatgaaaaa gcctgaactc
    3301 accgcgacgt atcgggccct ggccagctag ctagagtcga cctgcaggtc cccggggatc
    3361 ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc gcgaagtcgc tcttcttgat
    3421 ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc cggccgtttt agcggctaaa
    3481 aaagtcatgg ctctgccctc gggcggacca cgcccatcat gaccttgcca agctcgtcct
    3541 gcttctcttc gatcttcgcc agcagggcga ggatcgtggc atcaccgaac cgcgccgtgc
    3601 gcgggtcgtc ggtgagccag agtttcagca ggccgcccag gcggcccagg tcgccattga
    3661 tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc cgtgattttg tagccctggc
    3721 cgacggccag caggtaggcc gacaggctca tgccggccgc cgccgccttt tcctcaatcg
    3781 ctcttcgttc gtctggaagg cagtacacct tgataggtgg gctgcccttc ctggttggct
    3841 tggtttcatc agccatccgc ttgccctcat ctgttacgcc ggcggtagcc ggccagcctc
    3901 gcagagcagg attcccgttg agcaccgcca ggtgcgaata agggacagtg aagaaggaac
    3961 acccgctcgc gggtgggcct acttcaccta tcctgcccgg ctgacgccgt tggatacacc
    4021 aaggaaagtc tacacgaacc ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat
    4081 ataccgaaaa aatcgctata atgaccccga agcagggtta tgcagcggaa aagatccgtc
    4141 gacctgcagg catgcaagct ctagcgattc cagacgtccc gaaggcgtgg cgcggcttcc
    4201 ccgtgccgga gcaatcgccc tgggtgggtt acacgacgcc cctctatggc ccgtactgac
    4261 ggacacaccg aagccccggc ggcaaccctc agcggatgcc ccggggcttc acgttttccc
    4321 aggtcagaag cggttttcgg gagtagtgcc ccaactgggg taacctttga gttctctcag
    4381 ttgggggcgt agggtcgccg acatgacaca aggggttgtg accggggtgg acacgtacgc
    4441 gggtgcttac gaccgtcagt cgcgcgagcg cgaaaattcg agcgcagcaa gcccagcgac
    4501 acagcgtagc gccaacgaag acaaggcggc cgaccttcag cgcgaagtcg agcgcgacgg
    4561 gggccggttc aggttcgtcg ggcatttcag cgaagcgccg ggcacgtcgg cgttcgggac
    4621 ggcggagcgc ccggagttcg aacgcatcct gaacgaatgc cgcgccgggc ggctcaacat
    4681 gatcattgtc tatgacgtgt cgcgcttctc gcgcctgaag gtcatggacg cgattccgat
    4741 tgtctcggaa ttgctcgccc tgggcgtgac gattgtttcc actcaggaag gcgtcttccg
    4801 gcagggaaac gtcatggacc tgattcacct gattatgcgg ctcgacgcgt cgcacaaaga
    4861 atcttcgctg aagtcggcga agattctcga cacgaagaac cttcagcgcg aattgggcgg
    4921 gtacgtcggc gggaaggcgc cttacggctt cgagcttgtt tcggagacga aggagatcac
    4981 gcgcaacggc cgaatggtca atgtcgtcat caacaagctt gcgcactcga ccactcccct
    5041 taccggaccc ttcgagttcg agcccgacgt aatccggtgg tggtggcgtg agatcaagac
    5101 gcacaaacac cttcccttca agccgggcag tcaagccgcc attcacccgg gcagcatcac
    5161 ggggctttgt aagcgcatgg acgctgacgc cgtgccgacc cggggcgaga cgattgggaa
    5221 gaagaccgct tcaagcgcct gggacccggc aaccgttatg cgaatccttc gggacccgcg
    5281 tattgcgggc ttcgccgctg aggtgatcta caagaagaag ccggacggca cgccgaccac
    5341 gaagattgag ggttaccgca ttcagcgcga cccgatcacg ctccggccgg tcgagcttga
    5401 ttgcggaccg atcatcgagc ccgctgagtg gtatgagctt caggcgtggt tggacggcag
    5461 ggggcgcggc aaggggcttt cccgggggca agccattctg tccgccatgg acaagctgta
    5521 ctgcgagtgt ggcgccgtca tgacttcgaa gcgcggggaa gaatcgatca aggactctta
    5581 ccgctgccgt cgccggaagg tggtcgaccc gtccgcacct gggcagcacg aaggcacgtg
    5641 caacgtcagc atggcggcac tcgacaagtt cgttgcggaa cgcatcttca acaagatcag
    5701 gcacgccgaa ggcgacgaag agacgttggc gcttctgtgg gaagccgccc gacgcttcgg
    5761 caagctcact gaggcgcctg agaagagcgg cgaacgggcg aaccttgttg cggagcgcgc
    5821 cgacgccctg aacgcccttg aagagctgta cgaagaccgc gcggcaggcg cgtacgacgg
    5881 acccgttggc aggaagcact tccggaagca acaggcagcg ctgacgctcc ggcagcaagg
    5941 ggcggaagag cggcttgccg aacttgaagc cgccgaagcc ccgaagcttc cccttgacca
    6001 atggttcccc gaagacgccg acgctgaccc gaccggccct aagtcgtggt gggggcgcgc
    6061 gtcagtagac gacaagcgcg tgttcgtcgg gctcttcgta gacaagatcg ttgtcacgaa
    6121 gtcgactacg ggcagggggc agggaacgcc catcgagaag cgcgcttcga tcacgtgggc
    6181 gaagccgccg accgacgacg acgaagacga cgcccaggac ggcacggaag acgtagcggc
    6241 gtagcgagac acccgggaag cctg

Claims (20)

What is claimed is:
1. A method for preparing a piperazic acid (Piz)-containing product comprising:
(i) providing N5—OH-Ornithine or derivative thereof;
(ii) providing a suitable enzyme comprising a N5—OH Ornithine cyclase/dehydratase; and
(iii) optionally, buffer salts, a NADPH cofactor, Fe+2 salts, and a catalytic Flavin Adenine Dinucleotide (FAD) cofactor.
2. The method of claim 1 further comprising:
(i) providing an ornithine or a derivative thereof; and
(ii) providing a suitable enzyme comprising an ornithine N5 hydroxylase.
3. The method of claim 1, wherein
(i) the N5—OH-Ornithine or derivative thereof is an enantiopure L-Ornithine or derivative thereof;
(ii) the enzyme comprising N5—OH Ornithine cyclase/dehydratase is a L-N5—OH Ornithine cyclase/dehydratase or a PzbB enzyme; or
(iii) the enzyme comprising ornithine N5 hydroxylase is an L-ornithine N5—OHase or a PzbA enzyme.
4. The method of claim 1, wherein the method is carried out in the absence of O2, substantially no O2, or in the presence of low O2.
5. The method of claim 2 wherein the method comprises a coupled enzyme assay.
6. The method of claim 1, wherein the piperazic acid (Piz)-containing product comprises a compound of formula:
Figure US20190002936A1-20190103-C00023
wherein:
R5 is a hydrogen, an alkyl, a piperazic acid, an acetyl, or a carboxyl protecting group;
each R1 and R2 are independently selected from hydrogen or an amino protecting group, wherein R1 and R2 may be taken together to form a fused bicyclic or tricyclic amino protecting group; and
each R3 and R4 are independently selected from a hydrogen, a halo (optionally, a chloro, a fluoro, a bromo, or a iodo), or a hydroxyl.
7. The method of claim 1, wherein R1 and R2 are not simultaneously hydrogen.
8. The method of claim 1, wherein the piperazic acid (Piz)-containing product is used as a starting material in a synthetic method of making a bioactive Piz-containing composition selected from the group consisting of:
(i) an antibacterial agent, an antibiotic agent, an antitumor agent, an antiviral agent, an immunomodulatory agent, or an anti-inflammatory agent;
(ii) a molecular probe, anticancer drug, or drug lead;
(iii) a metalloprotease inhibitor, a caspase inhibitor, an angiotensin converting enzyme (ACE) inhibitor, an inflammatory peptide C5a antagonist, an oxytocin receptor antagonist, or a matylastin type-IV collagenase inhibitor;
(iv) a dehydropiperazic acid; a chloropiperazic acid; a hydroxypiperazic acid; a monamycin, an aurantimycin, an antrimycin, an azinothricin, a luzopeptin, a kettapeptin, a quinoxapeptin, a lydiamycin, a piperazimycin, or a sangamide; or
(v) sanglifehrin A, pandanamide A, azinothricin, Sch392583, luzopeptin A, kutzernide 2, piperazic acid, L-piperazic acid, antrimycin, kettapeptin, GE3, A83586C, chloptosin, himastatin, luzopeptin, quinoxapeptin, lydiamycin, piperazimycin, sanglifehrin, sangamide NVP018, sangamide NVP019, sanglifehrin, Sch 382583; chloptosin, himastatin, verucopeptin, luzopeptin A, L-156,602, aurantimycin A, or L-156,373.
9. A transgenic microorganism comprising an artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription:
(I) (a) a promoter functional in the microorganism;
(b) (i) a first polynucleotide comprising a nucleotide sequence encoding a first polypeptide having a L-Ornithine N5 hydroxylase activity;
(ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide having a L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity; or
(iii) a third polynucleotide comprising a nucleotide sequence encoding a third polypeptide having a L-Ornithine N5 hydroxylase activity and a L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity; and
(c) a transcriptional termination sequence; or
(II) (a) a promoter functional in the microorganism;
(b) (i) a first polynucleotide comprising a nucleotide sequence encoding a first polypeptide having PzbA activity;
(ii) a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide having PzbB activity; or
(iii) a third polynucleotide comprising a nucleotide sequence encoding a first polypeptide having PzbA activity and PzbB activity; and
(c) a transcriptional termination sequence;
wherein,
the transgenic microorganism accumulates increased levels of a piperazic acid (Piz)-containing product, optionally L-Piz, compared to a microorganism not comprising the DNA construct.
10. The transgenic microorganism of claim 9, wherein the microorganism comprises:
(a) (i) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 1-SEQ ID NO: 81 or SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N5 hydroxylase activity; and
(ii) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 82-SEQ ID NO: 166 or SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N5 cyclase activity and L-Ornithine N5 dehydratase activity; or
(b) a nucleotide sequence encoding a polypeptide selected from SEQ ID NO: 167-SEQ ID NO: 176 or a sequence at least 25% identical thereto having L-Ornithine N5 hydroxylase activity, L-Ornithine N5 cyclase activity, and L-Ornithine N5 dehydratase activity.
11. The transgenic microorganism of claim 9 comprising:
(i) a PzbA ortholog with at least about 25% identity to SEQ ID NO: 1-SEQ ID NO: 81 or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbA activity to produce a piperazic acid (Piz)-containing product;
(ii) a PzbB ortholog with at least about 25% identity to SEQ ID NO: 82-SEQ ID NO: 166 or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbB activity to produce a piperazic acid (Piz)-containing product; or
(iii) a PzbAB ortholog with at least about 25% identity to or SEQ ID NO: 167-SEQ ID NO: 176 and has PzbA and PzbB activity to produce a piperazic acid (Piz)-containing product.
12. The transgenic microorganism of claim 9, wherein the microorganism is an Actinobacteria selected from the group consisting of Streptomyces, Corynebacterium, Kutzneria, and Actinomadura;
is a heterologous population of microorganisms;
is an Actinobacteria (optionally, an actinomycete); or
is selected from the group consisting of Streptomyces lividans or Corynebacterium glutamicum, optionally carrying one or more copies of a native or non-native pzbA and optionally carrying one or more copies of pzbB.
13. The transgenic microorganism of claim 9, wherein
the transgenic microorganism overproduces L-Ornithine;
the pzbA or the pzbB are cloned from a sanglifehrin biosynthetic locus of Streptomyces flaveolus; or
a piperazic acid (Piz)-containing product accumulates within the microorganism.
14. A method for producing a piperazic acid (Piz)-containing product comprising:
(i) providing a transgenic microorganism capable of accumulating a piperazic acid (Piz)-containing product;
(ii) cultivating the microorganism; and
(iii) isolating accumulated piperazic acid (Piz)-containing product.
15. The method of claim 14, comprising:
providing a transgenic microorganism and providing a feedstock, wherein
the transgenic microorganism comprises at least one copy of pzbA and at least one copy of pzbB under a constitutive promoter; and
the at least one pzbA is optionally a native copy.
16. The method of claim 14, wherein the transgenic microorganism is
(i) a heterologous population of microorganisms;
(ii) an Actinobacteria (optionally, an actinomycete); or
(ii) selected from the group consisting of Streptomyces lividans or Corynebacterium glutamicum, optionally carrying one or more copies of a native or non-native pzbA and optionally carrying one or more copies of pzbB.
17. The method of claim 14, wherein
pzbA or pzbB are cloned from a sanglifehrin biosynthetic locus of Streptomyces flaveolus; or
a piperazic acid (Piz)-containing product accumulates within the microorganism.
18. The method of claim 14, wherein the method is carried out in the absence of O2, substantially no O2, or in the presence of low O2.
19. The method of claim 14, wherein the piperazic acid (Piz)-containing product comprises a compound of formula:
Figure US20190002936A1-20190103-C00024
wherein:
R5 is a hydrogen, an alkyl, a piperazic acid, an acetyl, or a carboxyl protecting group;
each R1 and R2 are independently selected from hydrogen or an amino protecting group, wherein R1 and R2 may be taken together to form a fused bicyclic or tricyclic amino protecting group; and
each R3 and R4 are independently selected from a hydrogen, a halo (optionally, a chloro, a fluoro, a bromo, or a iodo), or hydroxyl.
20. The method of claim 14, wherein R1 and R2 are not simultaneously hydrogen.
US16/024,077 2017-06-30 2018-06-29 Transgenic microorganisms and synthesis of piperazic acid, piperazic acid containing products, and derivatives thereof Abandoned US20190002936A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112359048A (en) * 2020-08-27 2021-02-12 浙江大学 Preparation method of strychnos ignatii C
CN112662573A (en) * 2020-12-25 2021-04-16 中国海洋大学 Microbial strain for efficiently synthesizing L-piperazinic acid and construction method and application thereof
CN116064288A (en) * 2022-08-30 2023-05-05 内蒙古农业大学 Streptomyces roseoformis HC7-22 for plant iron-removing and growth-promoting and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112359048A (en) * 2020-08-27 2021-02-12 浙江大学 Preparation method of strychnos ignatii C
CN112662573A (en) * 2020-12-25 2021-04-16 中国海洋大学 Microbial strain for efficiently synthesizing L-piperazinic acid and construction method and application thereof
CN116064288A (en) * 2022-08-30 2023-05-05 内蒙古农业大学 Streptomyces roseoformis HC7-22 for plant iron-removing and growth-promoting and application thereof

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