SEQUENCE LISTING

This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled 222110-2550_ST25.txt, created on Nov. 29, 2018 and having a size of 65 KB. The content of the sequence listing is incorporated herein in its entirety.

BACKGROUND

The thaxtomins are a group of phytotoxins generated by some species ofStreptomycesbacteria, such asStreptomyces scabiei(the main causal organism of potato common scab). The thaxtomins can cause plant cell necrosis of various plant species and thus have broad spectrum herbicidal activity. These potent herbicidal activities, along with their environmentally benign and bio-degradable properties, make thaxtomins good green herbicides

Five toxins, including thaxtomin A and thaxtomin B, that induce the formation of scabs on potato tubers have been isolated fromS. scabiei, and as at least 11 thaxtomin analogs have also been isolated. They are cyclic dipeptides classed as 2,5-Diketopiperazines, with thaxtomin A, the most abundant, having the chemical formula C22H22N4O6. Individual thaxtomins appear to differ only in the presence or absence of N-methyl and hydroxyl groups and their respective substitution sites.

The genes involved in thaxtomin biosynthesis are located on a part of the genome called the pathogenicity island, present in the pathogenicStreptomycesstrains, such as but not limited to,S. scabiei, S. acidiscabiesandS. turgidiscabies. Although more than 800Streptomycesspecies are known to date, only a small number of species have so far been known to be phytopathogenic (Loria et al. 2006). The best characterized pathogenic species areS. scabiei, S. acidiscabies, S. turgidiscabies, andS. ipomoeae(Loria et al. 2008).

Thaxtomins inhibit the synthesis of cellulose, the major component of the plant cell wall, and cause dramatic plant cell hypertrophy and seedling stunting at the nM level (Bischoff et al. 2009; Fry and Loria 2002; King et al. 2001). The attractive bioactivities of thaxtomins makes them desirable candidates for development and use as natural, commercial herbicides for weed control (Koivunen et al. 2013; Leep et al. 2010). However, the limited productivity of thaxtomins in existingStreptomycesspecies as well as limited ability for production on an industrial scale presents problems, such as slow production, low yields, and the need for certain inducers, which are all constraining factors for commercialization. For instance, several pathogenicStreptomycesstrains are known to produce thaxtomins in the presence of cellobiose as the inducer. However, the productivity is generally in the several mg/L range, and the high cost of cellobiose further limits the applications of these strains in large-scale production of these herbicides.

SUMMARY

Briefly described, the present disclosure provides refactored thaxtomin biosynthetic gene clusters for enabling a transformed non-pathogenic bacterial cell to produce of thaxtomin A and/or other thaxtomin compound in the absence of thaxtomin-inducing conditions, such as cellobiose. The present disclosure also includes genetically engineered, non-pathogenicStreptomycesbacterium with exogenous, non-native thaxtomin biosynthetic gene clusters conferring the genetically engineered, non-pathogenicStreptomycesbacterium with the ability to produce thaxtomin compounds, derivatives, and intermediates in the absence of cellobiose. The present disclosure also includes methods of producing thaxtomin and thaxtomin intermediates and the thaxtomin and thaxtomin intermediate compounds produced by the methods of the present disclosure.

Embodiments of genetically engineeredStreptomycesbacteria of the present disclosure include: aStreptomycesbacterium from a non-pathogenicStreptomycesstrain, theStreptomycesbacterium comprising an exogenous, refactored thaxtomin biosynthetic gene cluster comprising at least two thaxtomin modules. In embodiments, each module includes one or more thaxtomin genes from a thaxtomin biosynthetic gene cluster from a pathogenicStreptomycesstrain, and one or more promoters operably linked to the one or more thaxtomin genes. The at least two thaxtomin modules represent a refactored thaxtomin biosynthetic gene cluster such that expression of the refactored thaxtomin biosynthetic gene cluster provides the genetically engineeredStreptomycesbacterium with the ability to produce at least one thaxtomin compound in the absence of thaxtomin-inducing conditions, where the non-pathogenicStreptomycesstrain without the refactored thaxtomin cluster does not have the ability to produce the thaxtomin compound. In some embodiments described in the present disclosure, the at least two thaxtomin modules include a first module, module 1, including a txtE and a txtD biosynthetic gene and a second module, module 2, including a txtA, a txtB, and optionaly txtH biosynthetic gene.

Embodiments according to the present disclosure of a refactored thaxtomin biosynthetic gene cluster include: one or more thaxtomin modules, each module having at least the following: one or more thaxtomin genes from a thaxtomin biosynthetic gene cluster from a pathogenicStreptomycesbacteria, and one or more promoters operably linked to the one or more thaxtomin genes. The one or more thaxtomin modules form a refactored thaxtomin biosynthetic gene cluster such that expression of the refactored thaxtomin biosynthetic gene cluster produces at least one thaxtomin compound in the absence of thaxtomin-inducing conditions. In embodiments, if the thaxtomin biosynthetic gene cluster comprises only one thaxtomin module, that thaxtomin module comprises two or more thaxtomin genes.

The present disclosure also provides methods of providing a genetically engineeredStreptomycesbacterium capable of producing thaxtomin compounds in the absence of thaxtomin-inducing conditions. In embodiments, such methods include: providing aStreptomycesbacterium from a naturally non-pathogenic species that does not produce thaxtomin; and genetically engineering theStreptomycesbacterium to include an exogenous, refactored thaxtomin biosynthetic gene cluster comprising one or more thaxtomin modules that form the refactored thaxtomin biosynthetic gene cluster. In such embodiments, each thaxtomin module includes: one or more thaxtomin genes from a thaxtomin biosynthetic gene cluster from a pathogenicStreptomycesbacteria, and one or more promoters operably linked to the one or more thaxtomin genes in the one or more modules, where the exogenous, refactored thaxtomin biosynthetic gene cluster provides the genetically engineeredStreptomycesbacterium with the ability to produce at least one thaxtomin compound in the absence of thaxtomin-inducing conditions.

Embodiments of methods of producing a thaxtomin compound of the present disclosure include: culturing genetically engineeredStreptomycesbacteria from a non-pathogenicStreptomycesstrain in the absence of thaxtomin-inducing conditions, where the genetically engineeredStreptomycesbacterium have an exogenous, refactored thaxtomin biosynthetic gene cluster including one or more thaxtomin modules, each module having one or more thaxtomin genes from a thaxtomin biosynthetic gene cluster from a pathogenicStreptomycesbacteria, and one or more promoters operably linked to the one or more thaxtomin genes. In such embodiments, the one or more thaxtomin modules form a refactored thaxtomin biosynthetic gene cluster such that expression of the refactored thaxtomin biosynthetic gene cluster provides the genetically engineeredStreptomycesbacterium with the ability to produce at least one thaxtomin compound in the absence of thaxtomin-inducing conditions.

Methods of the present disclosure also include producing a thaxtomin compound analog or intermediate. In embodiments, such methods include: culturing genetically engineeredStreptomycesbacteria from a non-pathogenicStreptomycesstrain in the absence of thaxtomin-inducing conditions, the genetically engineeredStreptomycesbacterium including an exogenous, refactored thaxtomin biosynthetic gene cluster comprising one or more thaxtomin modules, where the one or more thaxtomin modules include a txtE, a txtD a txtA, a txtB, and a txtH biosynthetic gene and one or more promoters operably linked to the biosynthetic genes. In embodiments, the biosynthetic gene cluster does not comprise a txtC biosynthetic gene, and the thaxtomin modules form a refactored thaxtomin biosynthetic gene cluster such that expression of the refactored thaxtomin biosynthetic gene cluster provides the genetically engineeredStreptomycesbacterium with the ability to produce at least one thaxtomin compound analog or intermediate in the absence of thaxtomin-inducing conditions.

The present disclosure also includes embodiments of genetically engineeredStreptomycesbacterium including: aStreptomycesbacterium from a non-pathogenicStreptomycesstrain, theStreptomycesbacterium having an exogenous, refactored thaxtomin biosynthetic gene cluster comprising one or more thaxtomin modules. In such embodiments, each module includes one or more thaxtomin genes from a thaxtomin biosynthetic gene cluster from a plant pathogenicStreptomycesbacterium, where if the thaxtomin biosynthetic gene cluster comprises only one thaxtomin module, that thaxtomin module comprises two or more thaxtomin genes. Each module also includes one or more promoters operably linked to the one or more thaxtomin genes in the one or more thaxtomin modules, such that expression of the thaxtomin genes in the refactored thaxtomin biosynthetic gene cluster provides the genetically engineeredStreptomycesbacterium with the ability to produce at least one thaxtomin compound in the absence of thaxtomin-inducing conditions, where the non-pathogenicStreptomycesstrain without the refactored thaxtomin cluster does not have the ability to produce the thaxtomin compound.

Other systems, methods, features, and advantages of the present disclosure will be or will become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.

