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

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.

What is claimed is: 
     
         1 . A method for preparing a piperazic acid (Piz)-containing product comprising:
 (i) providing N 5 —OH-Ornithine or derivative thereof;   (ii) providing a suitable enzyme comprising a N 5 —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 N 5  hydroxylase. 
 
     
     
         3 . The method of  claim 1 , wherein
 (i) the N 5 —OH-Ornithine or derivative thereof is an enantiopure L-Ornithine or derivative thereof;   (ii) the enzyme comprising N 5 —OH Ornithine cyclase/dehydratase is a L-N 5 —OH Ornithine cyclase/dehydratase or a PzbB enzyme; or   (iii) the enzyme comprising ornithine N 5  hydroxylase is an L-ornithine N 5 —OHase or a PzbA enzyme.   
     
     
         4 . The method of  claim 1 , wherein the method is carried out in the absence of O 2 , substantially no O 2 , or in the presence of low O 2 . 
     
     
         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: 
       
         
           
           
               
               
           
         
         wherein: 
         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; and 
         each R 3  and R 4  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 R 1  and R 2  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 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  cyclase activity or L-Ornithine N 5  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 N 5  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 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 N 5  cyclase activity, and L-Ornithine N 5  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 O 2 , substantially no O 2 , or in the presence of low O 2 . 
     
     
         19 . The method of  claim 14 , wherein the piperazic acid (Piz)-containing product comprises a compound of formula: 
       
         
           
           
               
               
           
         
         wherein: 
         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; and 
         each R 3  and R 4  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 R 1  and R 2  are not simultaneously hydrogen.