Compositions and methods for activation and overexpression of secondary metabolites in microorganisms

Abstract

Methods and compositions herein provide non-naturally occurring γ-butyrolactones (GBLs) in racemic mixtures that increase efficiency and effectiveness of screening for production of antibiotics, and enhance yields and express silent pathways. Non-naturally occurring GBLs were synthesized and found to stimulate antibiotic production in several different streptomycete strains. Antibiotic production by Streptomyces coelicolor was induced by a racemic mixture of non-cognate stereoisomers of VB-D, seven of which are non-naturally occurring. Further, novel A-factor-type GBL analogs stimulated antibiotic production in S. coelicolor. Synthesis in response to the treatment with the non-cognates GBL was observed for known compounds including undecylprodigiosin, desferrioxamine and streptorubin B, as was synthesis of a compound of unknown structure. A group of 37 additional microbial strains was screened by principal component analysis to determine optimal concentrations of each of a panel of four non-cognate synthetic GBLs for addition to cultures with optimal stimulation of secondary metabolites, and large scale fermentations were analyzed and product enhancement by the GBLs was observed.

What is claimed is: 
     
         1 . A composition for upregulating biosynthesis and production of a bioactive microbial product by cells of a sample of microorganisms, the composition comprising:
 at least one synthetic non-naturally occurring derivative of a γ-butyrolactone (GBL) core, in a dose effective to increase expression of the genes and biosynthetic production of the product in the cells.   
     
     
         2 . The composition according to  claim 1 , wherein the bioactive product has at least one activity selected from the group consisting of: anti-bacterial, anti-fungal, anti-viral, anti-helminthic, anti-cancer, anti-malarial, anti-trypanosomal, complement inhibitory, toxin neutralizing, immune stimulant, anti-inflammatory, immune suppressant, diuretic, and herbicidal. 
     
     
         3 . The composition according to  claim 1 , wherein the sample of the microorganism is a mixture of a plurality of strains or species, and the composition is effective to upregulate expression of genes in at least one of the strains or species. 
     
     
         4 . The composition according to  claim 1 , wherein the synthetic non-naturally occurring derivative of the GBL has a chemical structure different from naturally occurring GBLs or is a non-naturally occurring stereoisomer of a GBL, and the composition structure is not the same as:  Streptomyces griseus  A-factor;  S. viridochromogenes  Factor I;  S. lavendulae  IM-2;  S. coelicolor  SCB1, SCB2, and SCB3; anthracyclines from  S.bikiensis; S. cyaneofuscatus  greater length hydrocarbon chains in 2R3R and 2S3S; and  S.virginiae  butanolides VB-A, VB-B, VB-C, VB-D, and VB-E. 
     
     
         5 . The composition according to  claim 1 , wherein the GBL comprises a core which is substituted at the 3 position by a group having the structure methyl-R 1  and at the 2 position by a group having the structure selected from ketone-R 2 , alcohol-R 2 , and carbonyl-R 2 , wherein the substituent at the 2 and 3 position of the GBL are attached to the GBL core by recto (R) or levo (L) bonds, wherein R 1  and R 2  are each independently selected from the group consisting of: a lower alkane, an alkyne, an alkoxyl, an alkoxy, a halogen, a sulfide, an amine, a carbonyl, and an alkene selected from the group consisting of: an ethyl, an ethoxy, an ethoxyl, a propyl, a propoxy, a propoxyl, a butyl, a pentyl, a hexyl, a t-butyl, an s-butyl, an i-butyl, an i-pentyl, an i-hexyl, and an i-heptyl. 
     
     
         6 . The composition according to  claim 5 , wherein the GBL core is substituted at the 2 or 3 position with a lower alkane having a length of 1 carbon to about 8 carbons. 
     
     
         7 . The composition according to  claim 5 , wherein the GBL core is substituted at the 2 or 3 position with an alkane having a length greater than 8 carbons. 
     
     
         8 . The composition according to  claim 5 , wherein the R 1  comprises hydroxyl and is either recto-(R) or levo-(L), and R 2  comprises a hexyl which is R or L. 
     
     
         9 . The composition according to  claim 1 , selected from at least one enantiomer or stereoisomer of the group consisting of 3-(1-hydroxyethyl)-4-(hydroxymethyl)dihydrofuran-2(3 H)-one; 3-(1-hydroxyheptyl)-4-(hydroxymethyl)dihydrofuran-2(3H)-one; 3-acetyl-4-(hydroxymethyl)dihydrofuran-2(3H)-one; and, 3-heptanoyl-4-(hydroxymethyl)dihydrofuran-2(3H)-one. 
     
     
         10 . A method of improving a yield of a microbially-produced bioactive secondary metabolite product, the method comprising
 contacting cells of at least one strain of microorganism with a suitable amount of at least one γ-butyrolactone (GBL) composition wherein the GBL is non-cognate to the strain or is synthetic and non-naturally occurring, and culturing the cells of the strain with the GBL under conditions for production of the product; and,   obtaining the product from the cells by separation of cells and medium or purification of the product from the cells and analyzing the amount of the product, wherein the yield of the product per unit of volume of culture or weight of cells is greater than that from control cells of the strain not contacted with the GBL composition and otherwise identically cultured and analyzed, wherein the yield of the product from the cells cultured with the derivative of the GBL is improved compared to that from the control cells.   
     
     
         11 . The method according to  claim 10 , wherein the strain of microorganism is bacterial. 
     
     
         12 . The method according to  claim 11 , wherein the strain of bacteria is an actinomycete. 
     
     
         13 . The method according to  claim 10 , wherein the strain of microorganism is fungal or algal. 
     
     
         14 . The method according to  claim 10  wherein the derivative of the GBL is synthetic and non-naturally occurring and is at least one of the compositions selected from formulas I-VI in  FIG.  10   . 
     
     
         15 . The method according to  claim 12 , wherein a genus of the actinomycete selected from the group of genera consisting of:  Actinopolyspora, Amycolatopsis, Micromonospora, Nocardia, Pseudonocardia, Saccharothrix, Saccharopolyspora, Salinospora, Streptomyces, Tetinomedara , and  Verrucosispora.    
     
     
         16 . The method according to  claim 10 , wherein the yield of the product from the cells contacted with the GBL is at least about two-fold great, four-fold great, ten-fold greater, or at least about twenty-fold greater than from the control cells. 
     
     
         17 . The method according to  claim 15 , wherein the genus is  Streptomyces  and the species is selected from at least one of the group consisting of:  avermitilis, S. aureofaciens, S. capreolus, S. cattleya, S. clavuligerus, S. coelicolor, S. ,fradiae, S. garyphallus, S. griseus, S. kanamyceticus, S. levoris, S. lincolnensis, S. niveus, S. noursei, S. platensis, S. plicatus, S. pristinaespiralis, S. orientalis, S. ribosidifus, S. rimosus, S. roseosporus, S. scabiei, S. venezuelae, S. vinaceus , and  S. virginiae ; or is at least one  Pseudonocardia  selected from the group of:  P. acacia; P. ailaonensis; P. adelaidensis; P. alaniniphila; P. ammonioxydans; P. carboxydivorans; P. halophobia; P. kujensis; P. nitrificans; P. petroleophila; P. salamisensis; P. sulfoxidans; P. thermophila ; and  P. zigingensis ; or is at least one  Amycolatopsis  selected from the group of:  A. alba, A. azurea, S. balhimycena, A. coloradensis, A. fastidiosa, A. keratiniphila, A. lurida, A. mediterranei, A. orientalis, A. sulphurea, A. tolypomycina , and  A. vancoresmycina.    
     
     
         18 . A method of discovery of a cell-produced secondary metabolic compound in a microbial strain containing putative unexpressed or under expressed genes encoding enzymes for biosynthesis of a chemical entity having a medicinal or industrial biological activity, the method comprising:
 contacting cell samples containing cells from at least one microbial strain with at least one synthetic γ-butyrolactone (GBL) in an amount suitable for inducing secondary metabolite expression, wherein the microbial strains are selected from the group of: fresh isolates from nature, a naturally occurring mixture of unpurified microorganisms, and an established species strain wherein the GBL and the established species are non-cognate;   culturing the cell samples with the GBL derivative under conditions for production of the secondary metabolite chemical compounds;   screening the cultures by at least one detection system for presence of the biological activity, or by at least one detection system for presence of the metabolite not so expressed in control samples not contacted with the GBL and otherwise identical and further screening the metabolite for the biological activity, wherein the presence of the biological activity identifies the producing sample containing at least one strain of microorganism producing the chemical having the activity; and,   characterizing at least one chemical structure having the biological activity, and comparing the structure to a library database of known chemical entities to obtain chemicals not previously known, thereby screening to discover the chemical compounds with the biological activity.   
     
     
         19 . The method according to  claim 18 , wherein the at least one microbial strain contains at least two, at least five, or at least 10 strains. 
     
     
         20 . The method according to  claim 18 , wherein the GBL is a plurality of GBLs having non-identical chemical structures, and the plurality is at least five GBLs. 
     
     
         21 . The method according to  claim 20 , wherein the non-identical chemical structure comprises GBLs which are racemates, enantiomers, stereoisomers, or a racemic mixture. 
     
     
         22 . The method according to  claim 18 , wherein screening further comprises contacting each of the samples to the detection system for the biological activity, the detection system comprising at least one component selected from the group of: an enzyme, an organism, a tissue culture, a cell culture; the method further comprising measuring an activity selected from: anti-bacterial, anti-fungal, anti-viral, anti-helminthic, anti-cancer, anti-malarial, anti-trypanosomal, complement inhibitory, immune stimulant, anti-inflammatory, toxin neutralizing, immune suppressant, diuretic, and herbicidal. 
     
     
         23 . The method according to  claim 18 , wherein culturing cell samples is in a liquid medium, and the method further comprises prior to screening, separating the cells from the medium to obtain a resulting supernatant depleted of the cells and a resulting cell pellet. 
     
     
         24 . The method according to  claim 18 , wherein culturing cell samples is in contact with soil, and the method further comprises prior to screening, separating the cells from the soil and washing the soil and cells, to obtain resulting components of supernatant, cell pellet, and soil, and assaying each for amount of the biological activity. 
     
     
         25 . The method according to  claim 18 , further comprising after identifying, isolating the chemical compound from the producing culture contacted with the GBL. 
     
     
         26 . The method according to  claim 23 , further comprising isolating, from the plurality of GBLs contacted to the sample, the at least one GBL that induces expression of the product. 
     
     
         27 . The method according to  claim 16 , wherein characterizing the chemical structure further comprises analyzing by at least one method selected from the group consisting of: mass spectrometry (MS); gas chromatography; thin layer chromatography; matrix-assisted laser desorption/ionization (MALDI); MALDI-time of flight (MALDI-TOF); moving bed chromatography; and high performance liquid chromatography (HPLC). 
     
     
         28 . The method according to  claim 20 , wherein culturing is growth of the strain or strains on solid medium, and screening further comprises adding cells of a target indicator strain comprising at least one of a bacterium, a fungus, a eukaryotic cell, a white blood cell, and a eukaryotic tissue explant. 
     
     
         29 . The method according to  claim 26 , wherein the method further comprises testing a sample of the supernatant in a test subject which is an experimental animal model of a disease. 
     
     
         30 . The method according to  claim 26 , wherein the method further comprises testing a sample of the supernatant in vivo in cultured cells or tissues of an organism selected from the group consisting of: a mammal; a fungus; a helminth; a plant; and, an insect. 
     
     
         31 . The method according to  claim 16 , further comprising obtaining coordinates of peaks observed by MS, MALDI, MALDI-TOF, or HPLC corresponding to presence of secondary metabolism products, and comparing the location of the peaks to that of known previously characterized products to identify chemical entities. 
     
     
         32 . The method according to  claim 31  further comprising isolating and screening the cell sample strains for production of the novel chemical entities in presence of the GBL. 
     
     
         33 . A method of increasing or accelerating production of a microbial secondary metabolism compound by a producing microorganism, the method comprising:
 contacting a culture of the producing microorganism strain with a GBL at an effective dose to upregulate expression of genes encoding enzymes that synthesize the compound, wherein the GBL is non-cognate to the strain.   
     
     
         34 . The method according to  claim 33 , wherein the GBL is added at or before inoculation of production culture medium cells of the strain. 
     
     
         35 . The method according to  claim 33 , wherein the GBL is added after inoculation or during growth of cells of the strain. 
     
     
         36 . The method according to  claim 33 , wherein the GBL is added at stationary phase or after cessation of growth of the cells of the strain. 
     
     
         37 . The method according to  claim 33 , wherein the GBL is added at a plurality of time points during culture of the micro-organism. 
     
     
         38 . The method according to  claim 33 , further comprising comparing amount of the secondary metabolism compound with that of a control culture not contacted with the GBL and otherwise identical. 
     
     
         39 . The method according to  claim 33 , wherein the effective dose of the GBL is about 0.2 μM-0.8 μM, 0.8 μ-20 μM-100 μM, or is greater than 100 μM. 
     
     
         40 . A chemical entity produced by a culture of  S. coelicolor  treated with at least one enantiomer or stereoisomer of 3-(1-hydroxyheptyl)-4-(hydroxymethyl)dihydrofuran-2(3H)-one, and eluting from an analysis of an extract of the culture from a mass spec time of flight chromatogram at a peak at 1.84.