Methods for thaxtomin production and modified streptomyces with increased thaxtomin production

Abstract

In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, embodiments of the present disclosure, in some aspects, relate to genetically modified Streptomyces bacteria capable of increased thaxtomin production, genetically modified Streptomyces bacteria with reduced activity of a CebR protein encoded by a cebR gene and/or reduced activity of a β-glucosidase enzyme encoded by the bglC gene, genetically modified Streptomyces bacteria including a mutation of a native cebR gene and/or a native bglC gene, methods of increasing thaxtomin production in Streptomyces bacteria, methods of suppressing CebR and/or BglC activity, methods of producing thaxtomin, and thaxtomin produced by the methods of the present disclosure.

We claim: 
     
         1 . A genetically modified  Streptomyces  bacterium comprising:
 a mutation that reduces activity of a CebR protein encoded by a cebR gene, such that the genetically modified  Streptomyces  has increased production of a thaxtomin compound as compared to a corresponding wild type  Streptomyces  bacterium, wherein the genetically modified  Streptomyces  bacterium is selected from the group of  Streptomyces  species in which the corresponding wild type  Streptomyces  bacterium is capable of producing one or more thaxtomin compounds under standard thaxtomin-inducing conditions.   
     
     
         2 . The genetically modified  Streptomyces  bacterium of  claim 1 , wherein the genetically modified  Streptomyces  bacterium is selected from the group of  Streptomyces  species consisting of:  Streptomyces  scabies,  Streptomyces acidiscabies , and  Streptomyces turgidiscabies.    
     
     
         3 . The genetically modified  Streptomyces  bacterium of  claim 1 , wherein the  Streptomyces  bacterium is a genetically modified  Streptomyces  scabies bacterium. 
     
     
         4 . The genetically modified  Streptomyces  bacterium of  claim 1 , wherein the mutation comprises a mutation of a native cebR gene, wherein the mutation reduces production or functionality of a CebR protein encoded by the cebR gene. 
     
     
         5 . The genetically modified  Streptomyces  bacterium of  claim 4 , wherein the mutation of cebR is a null mutation. 
     
     
         6 . The genetically modified  Streptomyces  bacterium of  claim 1 , wherein the genetically modified bacterium does not produce functional CebR. 
     
     
         7 . The genetically modified  Streptomyces  bacterium of  claim 1 , wherein the mutation comprises an exogenous nucleic acid sequence introduced into the bacterium, wherein the exogenous nucleic acid sequence reduces activity of a CebR protein encoded by the cebR gene. 
     
     
         8 . The genetically modified  Streptomyces  bacterium of  claim 1 , wherein the genetically modified  Streptomyces  bacterium produces a thaxtomin compound in the absence of at least one carbohydrate that reduces CebR DNA-binding activity. 
     
     
         9 . The genetically modified  Streptomyces  bacterium of  claim 8 , wherein the carbohydrate is cellobiose 
     
     
         10 . The genetically modified  Streptomyces  bacterium of  claim 1 , wherein the thaxtomin compound is thaxtomin A. 
     
     
         11 . The genetically modified  Streptomyces  bacterium of  claim 1 , wherein the cebR gene has a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having about 60% or more sequence identity with SEQ ID NO: 1. 
     
     
         12 . A method of increasing production of a thaxtomin compound in a  Streptomyces  bacterium, the method comprising:
 providing a  Streptomyces  bacterium from a species capable of producing one or more thaxtomin compounds under standard thaxtomin-inducing conditions;   genetically modifying the  Streptomyces  bacterium by creating a mutation in the bacterium, wherein the mutation reduces activity of a CebR protein encoded by a cebR gene, such that the genetically modified  Streptomyces  has increased production of a thaxtomin compound as compared to a corresponding wild type  Streptomyces  bacterium.   
     
     
         13 . The method of  claim 12 , wherein the mutation is a mutation of a native cebR gene, wherein the mutation reduces production or functionality of a CebR protein encoded by the cebR gene. 
     
     
         14 . The method of  claim 13 , wherein the mutation of cebR is a null mutation created by inserting a deletion cassette into the genome to remove the cebR gene, thereby inhibiting production of CebR. 
     
     
         15 . The method of  claim 12 , wherein the mutation comprises an exogenous nucleic acid sequence introduced into the bacterium, wherein the exogenous nucleic acid sequence reduces activity of a CebR protein encoded by the cebR gene. 
     
     
         16 . A method of increasing production of a thaxtomin compound in a  Streptomyces  bacterium capable of producing one or more thaxtomin compounds under standard thaxtomin-inducing conditions, the method comprising suppressing the activity of a CebR protein encoded by a cebR gene. 
     
     
         17 . The method of  claim 16 , wherein suppressing the activity of the CebR protein comprises genetically modifying the  Streptomyces  bacterium to inhibit expression of the cebR gene encoding the CebR protein. 
     
     
         18 . The method of  claim 16 , wherein suppressing the activity of the CebR protein comprises genetically modifying the  Streptomyces  bacterium to introduce an exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence reduces activity of the CebR protein encoded by the cebR gene. 
     
     
         19 . The method of  claim 16 , wherein the gene encoding the CebR protein has a nucleotide sequence of SEQ ID NO: 1 or a sequence having about 60% or more sequence identity with SEQ ID NO: 1. 
     
     
         20 . A method of producing a thaxtomin compound, the method comprising:
 culturing genetically modified  Streptomyces  bacteria, wherein the genetically modified  Streptomyces  bacteria is selected from the group of  Streptomyces  species capable of producing one or more thaxtomin compounds under standard thaxtomin-inducing conditions and wherein the genetically modified  Streptomyces  bacteria comprise a mutation of a native cebR gene, wherein the mutation reduces production or functionality of a CebR repressor encoded by the cebR gene, such that the modified  Streptomyces  bacteria produce a greater amount of the thaxtomin compound as compared to a corresponding wild type  Streptomyces  bacteria.   
     
     
         21 . The method of  claim 20 , further comprising extracting the thaxtomin compound from the culture media. 
     
     
         22 . The method of  claim 20 , further comprising culturing the genetically modified  Streptomyces  bacteria in a culture medium that does not contain cellobiose, wherein the genetically modified  Streptomyces  bacteria produce the thaxtomin compound in the absence of cellobiose.