Patent Publication Number: US-11655477-B2

Title: Methods for thaxtomin production and engineered non-native Streptomyces with increased thaxtomin production

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is the 35 U.S.C. § 371 national stage application of PCT Application No. PCT/US2018/034071, filed May 23, 2018, where the PCT claims priority to, and the benefit of, U.S. provisional application entitled “METHODS FOR THAXTOMIN PRODUCTION AND ENGINEERED NON-NATIVE  STREPTOMYCES  WITH INCREASED THAXTOMIN PRODUCTION” having Ser. No. 62/646,626, filed Mar. 22, 2018, and U.S. provisional application entitled “METHODS FOR THAXTOMIN PRODUCTION AND ENGINEERED NON-NATIVE  STREPTOMYCES  WITH INCREASED THAXTOMIN PRODUCTION” having Ser. No. 62/509,792, filed May 23, 2017, all of which are herein incorporated by reference in their entireties. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under Grant No. FA9550-16-1-0186 awarded by the US Air Force Office of Scientific Research and Grant No. 2010-65110-20416 awarded by the United States Department of Agriculture. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled 222110-2290_ST25.txt, created on May 23, 2018 and having a size of 77 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 of  Streptomyces  bacteria, such as  Streptomyces 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. 
     Thaxomins, also known as virulence factor in common scab potato disease, induce the formation of scabs on potato tubers and have been isolated from  S. scabiei . These toxins, including thaxtomin A, thaxtomin B and at least 10 other analogs, are cyclic dipeptides classed as 2,5-Diketopiperazines, with thaxtomin A, the most abundant, having the chemical formula C 22 H 22 N 4 O 6 . Individual thaxtomins appear to differ only in the presence or absence of N-methyl and hydroxyl groups and their respective substitution sites. 
     The pathogenicity of  Streptomyces  strains is believed to be related to the production of thaxtomin. The genes involved in thaxtomin biosynthesis are located on a part of the genome called the pathogenicity island, present in the pathogenic  Streptomyces  strains  S. acidiscabies  and  S. turgidiscabies . Although more than 800  Streptomyces  species are known to date, only about ten species are known to be phytopathogenic (Loria et al. 2006). The best characterized pathogenic species are  S. scabiei, S. acidiscabies, S. turgidiscabies , and  S. 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). Thaxtomins have been explored as effective herbicides to control the germination and growth of broadleaved, sedge, and grass weeds (Koivunen et al. 2013). In addition, thaxtomins have also been applied to control algae growth in algae contaminated environments (Kang et al. 2008). However, the limited productivity of thaxtomins in existing  Streptomyces  species, with isolation yields of less than 10 mg/L from native producers, results in a costly production process. Current synthetic routes to thaxtomin compounds and analogs are lengthy and inefficient for production on an industrial scale. 
     SUMMARY 
     Briefly described, the present disclosure provides genetically engineered, non-pathogenic  Streptomyces  bacterium with exogenous, non-native thaxtomin biosynthetic gene clusters conferring the genetically engineered, non-pathogenic  Streptomyces  bacterium with the ability to produce thaxtomin compounds, derivatives, and intermediates such as thaxtomin A, thaxtomin C, thaxtomin D, nitrotryptophan compounds, and fluorinated derivatives of these compounds. Also included are methods of providing thaxtomin-producing capability in non-native  Streptomyces  bacterial strains, methods of producing thaxtomin compounds with the genetically engineered  Streptomyces  bacteria of the present disclosure, and methods of producing thaxtomin compounds and nitrotryptophan analogs, and fluorinated thaxtomin compounds, analogs, and intermediates 
     Embodiments of genetically engineered  Streptomyces  bacteria of the present disclosure include: a  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain, the  Streptomyces  bacterium having an exogenous, non-native Thaxtomin A (ThxA) biosynthetic gene cluster from a pathogenic  Streptomyces  strain, where the presence of the non-native ThxA biosynthetic cluster provides the genetically engineered  Streptomyces  bacterium with the ability to produce thaxtomin A, where the non-pathogenic  Streptomyces  strain without the exogenous non-native ThxA biosynthetic cluster does not have the ability to produce thaxtomin. 
     The present disclosure also provides, in embodiments, genetically engineered  Streptomyces  bacteria of the including: a  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain, the  Streptomyces  bacterium having an exogenous, non-native engineered thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, where the engineered thaxtomin biosynthetic gene cluster does not include the txtC gene, where the presence of the non-native, engineered thaxtomin biosynthetic cluster provides the genetically engineered  Streptomyces  bacterium with the ability to produce thaxtomin biosynthetic intermediates selected from thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan, where the non-pathogenic  Streptomyces  strain without the exogenous non-native, engineered thaxtomin biosynthetic cluster does not have the ability to produce the thaxtomin intermediates. 
     Embodiments of methods of the present disclosure include methods for providing a non-native  Streptomyces  bacterium with the ability to product thaxtomin compounds, derivatives thereof, or intermediates thereof. Embodiments of such methods include: providing a  Streptomyces  bacterium from a naturally non-pathogenic species that does not produce thaxtomin, and genetically engineering the  Streptomyces  bacterium by introducing a non-native nucleic acid molecule encoding for a non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, such that the non-native thaxtomin biosynthetic gene cluster is integrated into the genome of the  Streptomyces  bacterium such that the genetically engineered  Streptomyces  bacterium is capable of producing one or more thaxtomin compounds, intermediates thereof, or derivatives thereof. In some embodiments, introducing the non-native nucleic acid molecule includes: providing a genetically engineered pathogenic  Streptomyces  bacterium having an exogenous nucleic acid encoding a selectable marker operably linked to a TR nucleic acid encoding a native TR1 and TR2; performing conjugal mating of the genetically engineered pathogenic  Streptomyces  bacterium with the naturally non-pathogenic  Streptomyces  bacterium such that the selectable marker, TR1, and TR2 are transferred from the pathogenic  Streptomyces  bacterium to the naturally non-pathogenic  Streptomyces  bacterium; and selecting for transconjugants and detecting for integration of the TR into a genome of the naturally non-pathogenic  Streptomyces  bacterium to produce a genetically engineered  Streptomyces  bacterium capable of producing thaxtomin. In some other embodiments, introducing the non-native nucleic acid molecule includes: cloning a thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, transforming the thaxtomin biosynthetic gene cluster into the  Streptomyces  bacterium from the naturally non-pathogenic species, and selecting for transformants to provide genetically engineered  Streptomyces  bacterium capable of producing thaxtomin. The present disclosure also provides genetically engineered  Streptomyces  bacteria produced by the methods of the present disclosure. 
     Methods of producing thaxtomin compounds, thaxtomin derivatives, and thaxtomin intermediates of the present disclosure, in embodiments, include: culturing genetically engineered  Streptomyces  bacteria from a non-pathogenic  Streptomyces  strain, the genetically engineered  Streptomyces  bacterium having an exogenous, non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, such that the genetically engineered  Streptomyces  bacteria produce thaxtomin compounds, derivatives or intermediates. In embodiments, the genetically engineered  Streptomyces  bacteria of the present disclosure have about the same or increased production of a thaxtomin compound, derivative, or intermediate as compared to a wild type  S. scabiei  bacteria under the same culture conditions. 
     In embodiments of the present disclosure, methods of producing thaxtomin compounds and nitrotryptophan analogs include: providing a  Streptomyces  bacterium from a naturally non-pathogenic species that does not produce thaxtomin and genetically engineering the  Streptomyces  bacterium by introducing a non-native nucleic acid molecule encoding for a genetically modified, non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, where the genetically modified, non-native thaxtomin biosynthetic gene cluster is integrated into the genome of the  Streptomyces  bacterium such that the genetically engineered  Streptomyces  bacterium is capable of producing thaxtomin, thaxtomin intermediates, and/or thaxtomin analogs. 
     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. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views. 
         FIGS.  1 A-C  illustrate  Streptomyces  scabies and thaxtomin A.  FIG.  1 A  is an image of  Streptomyces scabiei ,  FIG.  1 B  shows potato scab lesions caused by  Streptomyces scabiei , and  FIG.  1 C  shows the structure of thaxtomin A. 
         FIG.  2    is a schematic of genetic organization of genomic islands from different plant pathogenic  Streptomyces  species. Arrowed boxes represent the location and orientation of the open reading frames (ORFs) of thaxtomin biosynthetic genes, integrase and recombination directionality factor (RDF), lantibiotic biosynthetic genes, conjugative system genes, and aviX1. The sequences of attachment (att) sites (either TTGAAGCGGAAC (SEQ ID NO: 62) or TTGAACCGGAAC (SEQ ID NO: 63), or TTGAACCTGAAC (SEQ ID NO: 64) at the junctions for TR1/TR2 are also shown. 
         FIGS.  3 A- 3 B  illustrate the excision and site-specific integration of the  S. scabiei  thaxtomin biosynthetic cluster into the  S. diastatochromogenes  chromosome.  FIG.  3 A  is a schematic representation of the mating experiments between  S. scabiei  87-22 deletion mutants and  S. diastatochromogenes .  FIG.  3 B  illustrates the three excision forms and two site-specific integration forms of  S. scabiei  thaxtomin biosynthetic cluster: TR1 alone, TR2 alone, and the whole TR (TR1 and TR2) can excise from the  S. scabiei  chromosome, but only TR2 alone and the whole TR can integrate into the  S. diastatochromogenes  chromosome. 
         FIG.  4    illustrates a graph of the production of thaxtomin by heterologous hosts.  Streptomyces  strains were grown in triplicate TDM medium with 1% cellobiose for 6 days at 30° C. Spores were cultured on TSB at 30° C. for 48 hr. Vegetative culture was diluted to OD600=1 and 0.5 ml portion of the diluted culture was inoculated into 50 ml TDMc liquid medium and incubated at 30° C. with shaking (250 rpm). The average thaxtomin production of  S. scabiei  87-22 is set to 100%. The average % production for each strain relative to  S. scabiei  87-22 is shown, and the errors bars represent the standard deviation from the mean. Letters represent results of a one-way ANOVA with Tukey&#39;s HSD test; bars not sharing letters are significantly different at P&lt;0.05. 
         FIG.  5    illustrates a graph of the thaxtomin production of  S. albus  J1074 and  S. scabiei  87-22, in TDMc liquid medium. The final yield of thaxtomin of  S. scabiei  87-22 is set to 100%. The average % production of thaxtomin for two strains at each time points relative to  S. scabiei  87-22 final yield is shown, and the errors bars represent the standard deviation from the mean. Spores were cultured on TSB at 30° C. for 48 hours. Vegetative culture was diluted to OD600=1 and 1 ml portion of the diluted culture was inoculated into 100 ml TDMc liquid medium and incubated at 30° C. with shaking (250 rpm). Samples (3 ml) of cultures were collected every 24 hours to determine the concentration of thaxtomin. 
         FIGS.  6 A- 6 B  are graphs illustrating the nitrated precursors of  S. albus  J1074 and  S. scabiei  87-22, in TDMc liquid medium.  FIG.  6 A  illustrates HPLC traces of C18 SPE, 25% methanol washes of TDMc medium for analysis of nitrated precursors of thaxtomin A. The top chromatogram is from the  S. albus  J1074 extract seven days after inoculation, and the bottom chromatogram is from the  S. scabiei  87-22 extract seven days after inoculation.  FIG.  6 B  is a set of graphs illustrating accumulation of 4-nitrotryptophan of  S. albus  J1074 (left) and  S. scabiei  87-22 (right), in TDMc liquid medium. The final yield of N-methyl-4-nitrotryptophan of  S. scabiei  87-22 is set to %100. The average % production of nitrated precursors for two strains at each time points relative to  S. scabiei  87-22 final yields of N-methyl-4-nitrotryptophan is shown, and the errors bars represent the standard deviation from the mean. Spores were cultured on TSB at 30° C. for 48 hours. Vegetative culture was diluted to OD600=1 and 1 ml portion of the diluted culture was inoculated into 100 ml TDMc liquid medium and incubated at 30° C. with shaking (250 rpm). Samples (3 ml) of cultures were collected every 24 hours to determine the concentration of nitrated precursors. Absorbance was monitored at 380 nm. 
         FIG.  7    is a schematic of the process of heterologous expression of thaxtomins and nitro-tryptophans in  S. albus  J1074 using TAR based method. I) Construction of the capture vector by inserting the fused upstream and downstream regions (I kb/each) of thaxtomin biosynthetic gene cluster into pTARa vector; II) linearizing capture vector by using the inserted digested site (HpaI); III) linearizing the cosmid containing thaxtomin gene cluster by DraI; IV) TAR capture in yeast CRY 1-2; V) transformation of the constructed plasmid into conjugative  E. coli  strain S17-1; VI) conjugation of  E. coli  S17-1 with  S. albus  J1074; and VII) fermentation and isolation of thaxtomins and nitro-tryptophans from the culturing media of the engineered strain. 
         FIG.  8 A  is a schematic representation of an 18-kb thaxtomin gene cluster, and  FIG.  8 B  illustrates the biosynthetic pathway of thaxtomin leading to the production of multiple analogs (1-5). 
         FIG.  9 A  illustrates representative HPLC traces of culture extracts of  S. albus  J1074,  S. scabiei  87.22,  S. albus -thx1 and  S. albus -thx2. Two engineered  S. albus  strains had the same product profiles with multiple thaxtomin metabolites.  FIG.  9 B  is a bar graph illustrating that  S. albus -thx1 and  S. albus -thx2 produced about 20 times more thaxtomin A (1) per g of dried weight of biomass than  S. scabiei  87.22. Data represented mean±s. d. (n≥3). Significant differences between  S. scabiei  87.22 and two engineered  S. albus  strains were determined by the student&#39;s t-test analysis and was shown (***P&lt;0.001). 
         FIGS.  10 A- 10 N  are HR-MS ( FIGS.  10    A, C, E, G, I, K, and M) and MS/MS ( FIGS.  10    B, D, F, H, J, L, and N) spectra of compounds 1-5 and 7-8, as indicated. Chemical structures of these compounds were included in corresponding spectra. There was a relatively low abundance of compound 8 in the crude extract led for the HR-MS spectra in  FIG.  10 M . 
         FIG.  11    is a schematic illustration of the creation of  S. albus -thx2 strain. I) Construction of the plasmid pLST9828-thx based on pTARa-thx using NEBuilder® HiFi assembling kit; II) transformation of pLST9828-thx into  E. coli  EPI300; III) analysis of pLST9828 and transformation into  E. coli  S17-1; and IV) conjugation of  E. coli  S17-1-pLST9828-thx with  S. albus  J1074. 
         FIG.  12    is a graph illustrating accumulation of thaxtomin D (5), N-acetyl-4-nitrotryptophan (7) and N-methyl-4-nitrotrptophan (8) when culturing  S. albus -thx2 in TDMc at different time points. Vegetative culture (1 mL) of  S. albus -thx2 with OD 600 =1 was inoculated into 75 mL TDMc, which was then incubated at 28° C. with shaking (250 rpm). Culture samples (3 ml) were collected at eight time points (0.5, 1, 1.5, 2.0, 3.0, 4.0, 5.0 and 6.0) to determine the concentrations of three intermediates by HPLC. The highest concentration of each intermediate (areas under peak) was set as 100% and all other data points were then normalized. Data represented mean±s. d. (n=3). 
         FIGS.  13 A- 13 B  are schematic illustrations of the creation of  S. albus -thx2-ΔC ( FIG.  13 A ) and  S. albus -thx2-ΔABCH ( FIG.  13 B ) strains. I) Construction of plasmids based on pTARa-thx using NEBuilder® HiFi assembling kit; II) transformation of plasmids into  E. coli  EPI300; III) Analysis of plasmids and transformation into  E. coli  S17-1; and IV) Conjugation with  S. albus  J1074. 
         FIG.  14    illustrates representative HPLC analysis of culture extracts of  S. albus -thx2-ΔC strain cultured in different media. 
         FIG.  15    illustrates representative HPLC analysis of culture extracts of  S. albus -thx2-ΔBCH cultured in different media. 
         FIGS.  16 A- 16 C  illustrate chemical analysis of culture extract of  S. albus -thx2 and compound 9.  FIG.  16 A  is HPLC analysis of culture extract of  S. albus -thx2 cultured in TDMc supplied with 5-F-I-tryptophan or sterile water (control).  FIG.  16 B  is an HR-MS of compound 9, and  FIG.  16 C  is an HR-MS/MS spectra of compound 9. 
         FIG.  17    illustrates selected key COSY (thick, bold bonds), HMBC (thin, solid, single-headed arrows) and NOESY (thick, dashed, double-headed arrows) correlations of isolated compound by 2D NMR analyses, leading to the elucidation of its structure as 5-F-thaxtomin A (9). 
         FIGS.  18 A- 18 C  illustrated NMR analysis of isolated compound 9:  FIG.  18 A ,  1 H NMR (500 MHz, CD 3 OD);  FIG.  18 B ,  13 C NMR (125 MHz, CD 3 OD); and  FIG.  18 C ,  19 F NMR (500 MHz, CD 3 OD). 
         FIG.  19    is a graph illustrating characterization of herbicidal activity of thaxtomin analogs. Thaxtomin A (1), ortho-thaxtomin A (2) and 5-F-thaxtomin A (9) demonstrated potent growth inhibition activity toward radish seedling. DMSO was used as the negative control for normalizing the herbicidal activities of thaxtomins. Each data point represented mean±s. d. (n=18). 
         FIGS.  20 A- 20 B  are graphs illustrating that thaxtomin A (1), ortho-thaxtomin A (2), thaxtomin B (3) and 5-F-thaxtomin A (9) possessed weak cytotoxicity toward Jurkat ( FIG.  20 A ) and PC-3 ( FIG.  20 B ) prostate cell lines. Each data point represented mean±s. d. (n≥3). In both assays, PBS was used as the negative control to normalize the bioactivities of thaxtomins. 
     
    
    
     DETAILED DESCRIPTION 
     Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. 
     Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. 
     As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. 
     Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, botany, biochemistry, biology, molecular biology, genetics, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere. 
     Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible. 
     All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications and patents that are incorporated by reference, where noted, are incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. Any terms not specifically defined within the instant application, including terms of art, are interpreted as would be understood by one of ordinary skill in the relevant art; thus, is not intended for any such terms to be defined by a lexicographical definition in any cited art, whether or not incorporated by reference herein, including but not limited to, published patents and patent applications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. 
     It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of cells. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent. 
     As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like. In this disclosure, “consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. “Consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. 
     Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated. 
     Definitions 
     In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below. 
     The terms “nucleic acid” and “polynucleotide” are terms that generally refer to a string of at least two base-sugar-phosphate combinations. As used herein, the terms include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and generally refer to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes. Thus, for instance, polynucleotides as used herein refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The terms “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above. 
     In addition, polynucleotide as used herein refers to double-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a double-helical region often is an oligonucleotide. 
     It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. 
     The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotides” as that term is intended herein. 
     A “gene” typically refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. 
     As used herein, the term “transfection” refers to the introduction of an exogenous and/or recombinant nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus, or chloroplast. The nucleic acid may be in the form of naked DNA or RNA, it may be associated with various proteins or regulatory elements (e.g., a promoter and/or signal element), or the nucleic acid may be incorporated into a vector or a chromosome. A “transformed” cell is thus a cell transfected with a nucleic acid sequence. The term “transformation” refers to the introduction of a nucleic acid (e.g., DNA or RNA) into cells in such a way as to allow expression of the coding portions of the introduced nucleic acid. 
     As used herein, “transformation” or “transformed” refers to the introduction of a nucleic acid (e.g., DNA or RNA) into cells in such a way as to allow expression of the coding portions of the introduced nucleic acid. 
     As used herein a “transformed cell” is a cell transfected with a nucleic acid sequence. As used herein, a “transgene” refers to an artificial gene or portion thereof that is used to transform a cell of an organism, such as a bacterium or a plant. 
     As used herein, “transgenic” refers to a cell, tissue, or organism that contains a transgene. 
     As used herein, “exogenous nucleic acid sequence” or “exogenous polynucleotide” refers to a nucleic acid sequence that was introduced into a cell, organism, or organelle via transfection. Exogenous nucleic acids originate from an external source, for instance, the exogenous nucleic acid may be from another cell or organism and/or it may be synthetic and/or recombinant, e.g., a “non-native” nucleic acid. While an exogenous nucleic acid sometimes originates from a different organism or species, it may also originate from the same species (e.g., an extra copy or recombinant form of a nucleic acid that is introduced into a cell or organism in addition to or as a replacement for the naturally occurring nucleic acid). Typically, the introduced exogenous sequence is a recombinant sequence. 
     The term “recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a “fusion protein” (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments)), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally). Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man. 
     As used herein, “isolated” means removed or separated from the native environment. Therefore, isolated DNA can contain both coding (exon) and noncoding regions (introns) of a nucleotide sequence corresponding to a particular gene. An isolated peptide or protein indicates the protein is separated from its natural environment. Isolated nucleotide sequences and/or proteins are not necessarily purified. For instance, an isolated nucleotide or peptide may be included in a crude cellular extract or they may be subjected to additional purification and separation steps. 
     With respect to nucleotides, “isolated nucleic acid” refers to a nucleic acid with a structure (a) not identical to that of any naturally occurring nucleic acid or (b) not identical to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes, and includes DNA, RNA, or derivatives or variants thereof. The term covers, for example but not limited to, (a) a DNA which has the sequence of part of a naturally occurring genomic molecule but is not flanked by at least one of the coding sequences that flank that part of the molecule in the genome of the species in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic nucleic acid of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any vector or naturally occurring genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, e.g., a gene encoding a fusion protein, and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is not naturally occurring. Isolated nucleic acid molecules of the present disclosure can include, for example, natural allelic variants as well as nucleic acid molecules modified by nucleotide deletions, insertions, inversions, or substitutions. 
     It is advantageous for some purposes that a nucleotide sequence is in purified form. The term “purified” in reference to nucleic acid represents that the sequence has increased purity relative to the natural environment. 
     The term “polypeptides” and “protein” include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V). 
     “Variant” refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. 
     Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide&#39;s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties. 
     In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). 
     It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. 
     Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. 
     As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest. 
     As used herein “functional variant” refers to a variant of a protein or polypeptide (e.g., a thaxtomin compound) that can perform the same functions or activities as the original protein or polypeptide, although not necessarily at the same level (e.g., the variant may have enhanced, reduced or changed functionality, so long as it retains the basic function). 
     “Identity,” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). 
     Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure. 
     By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide. 
     The term “expression” as used herein describes the process undergone by a structural gene to produce a polypeptide. It can refer to transcription or the combination of transcription and translation. Expression generally refers to the transcription of a gene to produce messenger RNA, as used herein expression may refer to the entire process of “expression” of a nucleic acid to produce a polypeptide (e.g., transcription plus translation). If “expression” is used in reference to a polypeptide, it indicates that the polypeptide is being produced via expression of the corresponding nucleic acid. 
     As used herein, the term “over-expression” and “up-regulation” or “increasing” production of a polypeptide refers to the expression of a nucleic acid encoding a polypeptide (e.g., a gene) in a modified cell at higher levels (therefore producing an increased amount of the polypeptide encoded by the gene) as compared to a “wild type” cell (e.g., a substantially equivalent cell that is not modified in the manner of the modified cell) under substantially similar conditions. Thus, to over-express or increase expression of thaxtomin refers to increasing or inducing the production of the thaxtomin dipeptide by one or more enzymes encoded by the thaxtomin biosynthetic genes, which may be done by a variety of approaches, such as, but not limited to: increasing the transcription of the genes (such as by placing the genes under the control of a constitutive promoter) responsible for synthesis of thaxtomin, or increasing the translation of such genes, inhibiting or eliminating a repressor of thaxtomin production (e.g., CebR or β-glucosidase enzyme), or a combination of these and/or other approaches. 
     Conversely, “under-expression” and “down-regulation” refers to expression of a polynucleotide (e.g., a gene) at lower levels (producing a decreased amount of the polypeptide encoded by the polynucleotide) than in a “wild type” cell. As with over-expression, under-expression can occur at different points in the expression pathway, such as by decreasing the number of gene copies encoding for the polypeptide; removing, interrupting, or inhibiting (e.g., decreasing or preventing) transcription and/or translation of the gene (e.g., by the use of antisense nucleotides, suppressors, knockouts, antagonists, etc.), or a combination of such approaches. “Suppression” refers to the inhibition of production and/or activity functional gene product. Thus, the suppression of a gene or protein may indicate that the expression of the gene and/or activity of the encoded peptide has been inhibited such as by transcription and/or translation being inhibited, thus resulting in low to no production of the encoded protein, or production of a non-functional product, or production of an interfering nucleic acid that otherwise suppresses activity of the target protein. 
     Similarly, with respect to a gene product, such as a protein, “reduced activity” indicates that the activity of the protein is reduced relative to activity in a “wild type cell”. Such reduction in activity can be the result of inhibition/suppression/down-regulation/under-expression of the gene encoding the protein, the result of inhibition of translation of the messenger RNA into a functional gene product, or the result of production of a non-functional protein with reduced or no activity, or the direct suppression of the protein activity (e.g., preventing binding to a target), or the like. “Reduced production” of a gene product (e.g., a protein), such as by suppression, interruption, or other inhibition of transcription or translation, may result in reduced activity, but “reduced activity” of a protein or other gene product may result from other causes other than “reduced production”, such as set for the above. 
     As used herein, the term “genetically engineered,” with respect to a living organism, refers to an organism that has had its genetic makeup directly manipulated by techniques of biotechnology (as opposed to random changes occurring in nature). Genetically engineered organisms can include mutations involving changes only of the genetically engineered organisms&#39; own genetic material as well as mutations involving insertions of exogenous genetic material, such as insertions resulting in either cisgenic (including exogenous genetic material from the same or a closely related organism) or transgenic (including exogenous genetic material from a non-closely related organism) organisms. 
     As used herein a “mutation” refers to a heritable change in genetic material, which may include alteration of single base pairs of a nucleic acid, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes. An “engineered mutation” refers to a mutation created by human design (e.g., the mutation did not spontaneously occur by natural causes and/or was the result of intentional human manipulation). A “genetically modified” organism is an organism whose genetic material has been altered by one or more engineered mutations (e.g., human induced mutations). 
     Similarly, with respect to genes or other nucleic acids, “silencing” or “deletion” of a gene may include complete deletion of the nucleic acid/gene encoding a target peptide, complete suppression of translation or transcription of the target nucleic acid such that the target peptide is not produced, but the terms may also include some of the methods for “suppression” and “down-regulation” discussed above, where the “suppression” is significant enough to reduce expression of the target gene to the extent that the resulting peptide is inactive or the activity of the resulting peptide is so minimal as to be virtually undetected. 
     The term “null mutation” refers to a mutation in which the gene product (e.g., the protein encoded by the gene) is either not produced (or produced at significantly reduced levels, so as to be negligible) or is non-functional. Typically, a null mutation will involve a mutation of the native gene, such that the gene is not transcribed into RNA, the RNA product cannot be translated, or the protein produced by gene expression is non-functional. 
     The term “plasmid” as used herein refers to a non-chromosomal double-stranded DNA sequence including an intact “replicon” such that the plasmid is replicated in a host cell. A plasmid may include exogenous nucleic acid sequences and/or recombinant sequences. 
     As used herein, the term “vector” or “expression vector” is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may include a DNA molecule, linear or circular, which includes a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. As such, expression vectors typically contain recombinant nucleic acid sequences having different sequences linked together to effect expression of a target sequence. Expression vectors are generally derived from yeast DNA, bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of more than one of these. 
     As used herein, the term “expression system” includes a biologic system (e.g., a cell based system) used to express a polynucleotide to produce a protein. Such systems generally employ a plasmid or vector including the polynucleotide of interest (e.g., an exogenous nucleic acid sequence, a recombinant sequence, etc.), where the plasmid or expression vector is constructed with various elements (e.g., promoters, selectable markers, etc.) to enable expression of the protein product from the polynucleotide. Expression systems use the host system/host cell transcription and translation mechanisms to express the product protein. Common expression systems include, but are not limited to, bacterial expression systems (e.g.,  Streptomyces  strains), yeast expression systems, viral expression systems, animal expression systems, and plant expression systems. 
     As used herein, the term “promoter” or “promoter region” includes all sequences capable of driving transcription of a coding sequence. In particular, the term “promoter” as used herein refers to a DNA sequence generally described as the 5′ regulator region of a gene, located proximal to the start codon. The transcription of an adjacent coding sequence(s) is initiated at the promoter region. The term “promoter” also includes fragments of a promoter that are functional in initiating transcription of the gene. 
     The term “operably linked” indicates that the regulatory sequences necessary for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements), and/or selectable markers in an expression vector. 
     As used herein, the term “selectable marker” refers to a gene whose expression allows one to identify cells that have been transformed or transfected with a vector containing the marker gene (e.g., by antibiotic resistance on antibiotic medium, fluorescence, color generation, or other detectable signal). For instance, a recombinant nucleic acid may include a selectable marker operably linked to a gene of interest and a promoter, such that expression of the selectable marker indicates the successful transformation of the cell with the gene of interest. 
     The terms “native,” “wild type”, or “unmodified” in reference to a polypeptide/protein/enzyme, polynucleotide, cell, or organism, are used herein to provide a reference point for a variant/mutant of a polypeptide/protein/enzyme, polynucleotide, cell, or organism prior to its mutation and/or modification/engineering (whether the mutation and/or modification occurred naturally or by human design). On the other hand, “non-native” refers to a polypeptide/protein/enzyme, polynucleotide, cell, or organism that has a sequence or feature not found naturally in that peptide/cell/organism/etc., but that has been genetically engineered/manipulated to have such non-native feature. 
     As used herein, “thaxtomin” or “thaxtomin compound” refers to one or more compounds from a family of cyclic dipeptide phytotoxins, 4-nitroindol-3-yl-containing 2,5-dioxopiperazines, generated by some species of  Streptomyces  bacteria (and possibly by other actinomycetes) and exhibiting toxicity to various plant species. Thaxtomin compounds of the present disclosure have the general formula of Formula I below, and variants thereof. At least 5 thaxtomin compounds have been characterized, including thaxtomin A, A ortho analog, B, C, and D, and up to at least 12 different variants identified. Thaxtomin A, the most abundant of the thaxtomins and also believed to be the most physiologically active, has the chemical formula C 22 H 22 N 4 O 6  (chemical structure illustrated in  FIG.  1 C ). The thaxtomins, such as those isolated from  S. scabiei  ( FIG.  1 A ), can cause plant cell necrosis of various plant species and can induce the formation of scabs on potato tubers ( FIG.  1 B ). As used herein “thaxtomin” and “thaxtomin compound” refers generally to any of the members of this chemical group. Much of the discussion of thaxtomin in the present disclosure is in reference to thaxtomin A. However, as thaxtomin A may be a precursor to other thaxtomin compounds and/or the production of thaxtomin A is interwoven with production of other thaxtomin compound&#39;s, to the extent the methods and compositions of the present disclosure also modulate the production of other thaxtomin compounds, this is also intended to fall within the scope of the present disclosure. The general structure of a thaxtomin compound is shown below as Formula I, where R1 and R3 are independently selected from methyl or H and where R2, R4, R5, and R6 are each independently selected from hydroxyl or H. 
     
       
         
         
             
             
         
       
     
     The term “thaxtomin A (ThxA) biosynthetic gene cluster” refers to a gene cluster responsible for production of thatxtomin A (ThxA) in a pathogenic organism. In pathogenic  Streptomyces  species, the native ThxA gene cluster includes genes involved in ThxA production, such as, but not limited to, TxtA (SEQ ID NO: 1), TxtB (SEQ ID NO: 3), TxtC (SEQ ID NO: 5), TxtD (SEQ ID NO: 7), TxtE (SEQ ID NO: 9), TxtH (SEQ ID NO: 11), TxtR (SEQ ID NO: 13), which encode the peptides TxtA (SEQ ID NO: 2), TxtB (SEQ ID NO: 4), TxtC (SEQ ID NO: 6), TxtD (SEQ ID NO: 8), TxtE (SEQ ID NO: 10), TxtH (SEQ ID NO: 12), and TxtR (SEQ ID NO: 14), respectively). The sequences listed above are from  Streptomyces scabiei  and are representative of the ThxA genes of pathogenic  Streptomyces  species; however, the genes encoding the thaxtomin peptides may vary slightly in different species. Thus, in the present disclosure, the genes involved in production of thaxtomin A and the encoded peptides in different species are also intended to be included in the scope of the disclosure, such as polynucleotide sequences having sequence similarity with the sequences above from  Streptomyces scabiei  and still having the same function (e.g., sequences having about 70% or greater, 80% or greater, 90% or greater, 95% or greater, 99% or greater sequence identity with SEQ ID Nos: 1, 3, 5, 7, 9, 11, and 13) and peptide sequences having sequence identity with the peptide sequences from  Streptomyces scabiei  and still having the same function (e.g., sequences having about 70% or greater, 80% or greater, 90% or greater, 95% or greater, 99% or greater sequence identity with SEQ ID Nos: 2, 4, 6, 8, 10, 12, and 14). 
     The ThxA gene cluster of the present disclosure may include all of the above genes or any subset/variant of these needed to confer a recipient organism with the ability to produce thaxtomin. Thus the ThxA gene cluster or thaxtomin gene cluster can be engineered to include a subset or variant of the thaxtomin genes. The term “minimal thaxtomin biosynthetic gene cluster” refers to the minimal subset of the Thx A biosynthetic gene cluster needed to confer a recipient organism with the ability to produce thaxtomin. A minimal thaxtomin A biosynthetic gene cluster includes a minimal subset of the ThxA gene cluster needed to confer the ability to produce thaxtomin A, whereas other engineered thaxtomin biosynthetic gene clusters may include subsets of the ThxA gene cluster that confer the ability to produce other thaxtomin compounds or variants (e.g., thaxtomin C, thaxtomin D) or intermediates, such as nitrotryptophan intermediates (e.g., N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan), such engineered thaxtomin biosynthetic gene clusters may or may not also confer the ability to produce thaxtomin A. 
     The term “toxigenic region (TR)”, refers to a mobile genomic island in some  Streptomyces  species that can include “toxigenic region 1 (TR1)” (see  FIG.  2   ), that includes the thaxtomin A biosynthetic cluster, described above, and typically confers pathogenicity, and “toxigenic region 2 (TR2)” (see  FIG.  2   ) that includes integrative and conjugative elements that can mobilize itself (TR2) or an entire TR element (TR1 and TR2) between species (e.g., between pathogenic and non-pathogenic species) as described in greater detail below). 
     The terms “thaxtomin-inducing conditions” indicates certain environmental conditions (e.g., natural or cell culture conditions) known to induce thaxtomin production in wild-type  Streptomyces  bacterial species known to be capable of thaxtomin production. For instance, wild type  Streptomyces  are induced to produce thaxtomin in the presence of certain products of cellulose degradation, such as, but not limited to, cellobiose, as well as xylan-degradation products (Wach et al. 2007), such as, but not limited to suberin (Lauzier et al. 2008). In embodiments, “thaxtomin-inducing conditions” may include specific conditions or cell culture media (such as but not limited to, Oat Bran Broth (OBB), Oat Bran Agar (OBA), etc.) known to induce thaxtomin production in cell culture of wild-type  Streptomyces  species (such as, but not limited to  S. scabiei, S. acidiscabies , and  S. turgidiscabies ). In embodiments, “thaxtomin-inducing conditions” may also include a standard cell culture growth medium supplemented with a known thaxtomin-inducing compound, such as, but not limited to cellobiose. 
     The term “cebR gene” as used in the present disclosure indicates a nucleic acid sequence encoding a CebR protein in a  Streptomyces  or other thaxtomin-producing Actinomycete species that is a modulator of thaxtomin production. In embodiments, “cebR genes” include the cebR gene of  S. scabiei  (SEQ ID NO: 15) and variants and/or homologs (e.g., orthologs and paralogs) thereof retaining the function of modulation of thaxtomin production. In embodiments “cebR genes” include nucleic acids having a sequence of SEQ ID NO: 15 as well as sequences having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 99% or more (including any intervening ranges) sequence identity with SEQ ID NO: 15. In embodiments, cebR genes include nucleic acids having a sequence identity to SEQ ID NO: 15 of about 60% or more and having a sequence coverage to SEQ ID NO: 15 of about 70% or more, where sequence coverage indicates the percent of the total length of nucleic acids that are aligned. 
     The term “bglC gene” as used in the present disclosure indicates a nucleic acid sequence encoding a β-glucosidase enzyme in a  Streptomyces  or thaxtomin-producing Actinomycete species that is a modulator of thaxtomin production. In embodiments, “bglC genes” include the bglC gene of  S. scabiei  (SEQ ID NO: 17) and variants and/or homologs (e.g., orthologs and paralogs) thereof retaining the function of modulating thaxtomin production. In embodiments “bglC genes” include nucleic acids having a sequence of SEQ ID NO: 17 as well as sequences having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 99% or more (including any intervening ranges) sequence identity with SEQ ID NO: 17. In embodiments, bglC genes include nucleic acids having a sequence identity to SEQ ID NO: 17 of about 60% or more and also having a sequence coverage of about 70% with SEQ ID NO: 17. 
     Discussion 
     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 engineered  Streptomyces  bacteria from a naturally non-pathogenic  Streptomyces  species capable of increased thaxtomin production, genetically engineered  Streptomyces  bacteria with non-native thaxtomin pathogenicity clusters capable of producing about the same or a greater amount of thaxtomin than  S. scabiei  under the same culture conditions. In aspects, embodiments of the present disclosure also relate to methods of producing thaxtomins, thaxtomin analogs, nitro-tryptophan analogs, fluorinated thaxtomin compounds, analogs and intermediate, and the thaxtomin compounds, analogs, and intermediates produced by the methods of the present disclosure. 
       Streptomyces  is a very large genus of filamentous Gram-positive, high G+C content bacteria that are mostly saprophytes and best known for the production of pharmaceutically- and agriculturally-important secondary metabolites, including two-thirds of currently-used antibiotics (Hopwood 2007, Bentley et al. 2002). Although several hundred species are known to date, only about ten are known to be phytopathogenic (Loria et al. 2006). The best characterized pathogens are  Streptomyces scabiei, S. acidiscabies, S. turgidiscabies  and  S. ipomoeae , which cause raised or pitted scab lesions on economically-important root and tuber crops like potato, radish, beet, peanut, and sweet potato ( FIG.  1 A,  1 B ). The primary virulence determinant of  S. scabiei, S. acidiscabies  and  S. turgidiscabies  is the phytotoxin thaxtomin A (Loria et al. 2008) ( FIG.  1 C ). It is member of a family of nitrated 2,5-diketopiperazines formed by non-ribosomal peptide synthases out of the main components tryptophan, phenylalanine and nitric oxide derived from arginine (Loria et al. 2008; Barry et al. 2012), with 11 members of the family differing only in the presence or absence of hydroxyl and N-methyl groups at specific sites. 
     The biosynthesis of ThxA involves two nonribosomal peptide synthetases (NRPSs) encoded by the txtA and txtB genes, a P450 monooxygenase (TxtC), a nitric oxide synthase (TxtD), and a novel cytochrome P450 (TxtE) that site specifically nitrates tryptophan prior to cyclization (Bignell et al. 2014b). The thaxtomin biosynthetic cluster also contains a txtH gene encoding a 65-amino-acid MbtH-like protein potentially regulating NRPS activity (Herbst et al. 2013; Stegmann et al. 2006). The production of thaxtomin A is under strict controls, including both the pathway-specific transcriptional activator TxtR (Joshi et al. 2007) and global regulators belonging to the bld gene family (Bignell et al. 2014a) and the cellulose utilization repressor CebR (Francis et al. 2015). This group of genes, called the thaxtomin A biosynthetic gene cluster, resides on a portion of a mobile genomic island in pathogenic  Streptomyces  species, known as toxigenic region 1 (TR1). Many  Streptomyces  species also include another toxigenic region (TR2) that has integrative and conjunctive elements and has the ability to mobilize TR2 alone or the whole TR element (TR1 and TR2) between  Streptomyces  species as described in Zhang &amp; Loria 2016 (“Emergence of Novel Pathogenic  Streptomyces  Species by Site-Specific Accretion and cis-Mobilization of Pathogenicity Islands”, December 2016) and Zhang, et al. 2016 (“Promiscuous Pathogenicity Islands and Phylogeny of Pathogenic  Streptomyces  spp.” July 2016), both of which are hereby incorporated by reference herein in their entirety. 
     Thaxtomin A (and other toxic thaxtomin compounds) primarily targets the cell wall in dividing and expanding plant cells through an alteration of expression of cell wall biosynthesis-related genes and depletion of cellulose synthase complexes from the plasma membrane. This causes extensive cell wall remodeling, characterized by reduced incorporation of crystalline cellulose into the plant cell wall, and is compensated by an increased amount of pectins and hemicelluloses (Scheible et al. 2003; Bischoff et al. 2009). Data have shown that thaxtomin provokes the same effects on plants, qualitatively as well as quantitatively, as the synthetic cellulose biosynthesis inhibitor isoxaben, making thaxtomin an excellent candidate as a natural herbicide (Heim et al. 1990; Bischoff et al. 2009). In 2001, King and Lawrence reported a study in collaboration with James A. Gray from Dow Agrosciences, Inc. to evaluate the potential of thaxtomin for use as a commercial herbicide. The biological properties of this novel phytotoxin raised an interest in using thaxtomin as a biological compound to control weeds (Marrone Bio Innovations 2009, 2010; Novozymes Biologicals 2011, 2012); however, thaxtomin production in wild type  Streptomyces  requires specialized cell culture media (such as media supplemented with cellobiose or other thaxtomin-inducing compounds), which can be expensive. Thus, these methods of production of thaxtomin in wild type  Steptomyces , such as  S. scabiei , are insufficient. The present disclosure provides genetically engineered non-native  Streptomyces  bacteria with the ability to produce thaxtomin compounds at an increase over wild type bacteria and/or in species that do not naturally produce thaxtomin and are not naturally pathogenic to plants. In embodiments, the present disclosure provides genetically engineered bacteria as well as methods to produce thaxtomin in non-native species and at greater amounts than in wild type, native, thaxtomin-producing  Steptomyces , such as  S. scabiei , as well as methods for producing thaxtomin using such genetically modified bacteria. 
     Mobilization of pathogenicity islands (PAIs) can drive the evolution of plant pathogenic  Streptomyces  species (Loria et al. 2006). This disclosure describes the purposeful mobilization of a thaxtomin biosynthetic cluster (the full thaxtomin biosynthetic gene cluster, a subset of the cluster, or minimal thaxtomin biosynthetic gene cluster) of  S. scabiei , and its application for the heterologous production of thaxtomin in non-pathogenic  Streptomyces  species. The present disclosure describes that the thaxtomin biosynthetic cluster is located on a mobile genomic island and that the mating of  S. scabiei  with non-pathogenic  Streptomyces  species resulted in the acquisition of the thaxtomin biosynthetic cluster by non-pathogenic  Streptomyces  species. Some of the recipient  Streptomyces  spp. do not produce thaxtomins, or produce lower amounts than  S. scabiei  upon the acquisition of the thaxtomin cluster, indicating that the genetic backgrounds of recipients affect the thaxtomin production considerably. 
     Acquisition of genes required for virulence is one step on the way to pathogenicity. Indeed, more subtle genetic changes are involved in adapting the expression of newly acquired genes to the environment and the life cycle of the recipient microorganism. For instance, a limited number of mutations in intergenic regulatory regions can transform a harmless strain into a pathogen. The distribution of cis-acting elements in the gene is an element involved in development of a strain-specific transcriptional response. These DNA motifs are targeted by transcription factors, which themselves are informed of the presence of environmental signals through direct interaction with membrane sensors or indirect association with elicitor transporters. The production of thaxtomin A itself is under transcriptional regulatory control including at least five global regulators belonging to the bld gene family involved in secondary metabolism and/or morphological differentiation of  Streptomyces  (Bignell et al. 2014) in addition to the thaxtomin biosynthesis pathway-specific transcriptional activator, TxtR. The multiplicity of global and specific regulators associated with thaxtomin production suggests that  S. scabiei  may respond to multiple triggers that originate from plant material such as xylan-degradation products (Wach et al. 2007), suberin (Lauzier et al. 2008), and cellobiose, a product of cellulose degradation and the best-known elicitor of thaxtomin biosynthesis (Wach et al. 2007; Johnson et al. 2009) by directly targeting TxtR (Joshi et al. 2007). 
     Remarkably,  S. albus  J1074 acquires the cluster via mating with  S. scabiei  and produces a significantly higher concentration of thaxtomins in comparison to  S. scabiei . Furthermore, in embodiments, described in greater detail in the Examples below, a cloned thaxtomin cluster from the mobile genomic island is inserted, via a transformation-associated recombination (TAR) cloning approach (Kouprina et al. 2006; Mathee et al. 2008), into  S. albus  J1074 conferred a similar increase in thaxtomin production over wild type  S. scabiei . The growth rate and thaxtomin production of the strain carrying the cloned thaxtomin cluster were similar to the  S. albus  strain created by the mobilization of the genomic island. The data provided in the Examples below indicated that non-pathogenic  Streptomyces  strains can be genetically engineered to produce thaxtomin and that these engineered strains, such as  S. albus  J1074, can provide a premium host for the heterologous production of thaxtomin for commercial applications. 
     Thus, as described in more detail in the discussion and the examples below, the present disclosure includes genetically engineered  Streptomyces  bacterium from non-pathogenic species (or other non-pathogenic gram-positive bacteria) that have been genetically engineered to express an exogenous, non-native thaxtomin gene from a pathogenic  Streptomyces  strain. The present disclosure also provides methods of providing thaxtomin-producing capability in non-native  Streptomyces  bacterium that are naturally non-pathogenic, and methods of making the genetically engineered  Streptomyces  bacterium. Additionally, the present disclosure provides methods of producing thaxtomin compounds by culturing the genetically engineered  Streptomyces  bacteria of the present disclosure. 
     In additional embodiments, and as described in application PCT/US2015/050582 (WO 2016/044527 A1), which is hereby incorporated by reference in its entirely, the genetically engineered non-native, thaxtomin producing  Streptomyces  of the present disclosure can also include additional engineered mutations affecting the production of proteins involved in regulation of thaxtomin regulation, such as cebR and bglC (e.g., by engineered mutations in the genes encoding the proteins and/or by inhibition of the proteins themselves), to further increase thaxtomin production. In embodiments, genetically engineered  Streptomyces  bacteria have non-native thaxtomin producing gene clusters as well as modifications to cebR and bglC to decrease production of and/or activity of CebR protein and/or β-glucosidase enzyme to further increase production of thaxtomin compounds, such as, but not limited to, thaxtomin A. In an embodiment, the genetically engineered non-native  Streptomyces  bacteria are capable of thaxtomin production in non-inducing conditions and on non-inducing media (e.g., conditions in which wild type thaxtomin producing  Streptomyces  do not produce thaxtomin or produce only trace amounts of thaxtomin). 
     The genetically modified bacteria of the present disclosure, methods of increasing production of thaxtomin compounds in  Streptomyces  bacteria, methods of producing thaxtomin, and thaxtomin produced by methods of the present disclosure are described in greater detail in the discussion below and following examples. 
     Genetically Modified Bacteria 
     Embodiments of the present disclosure include genetically engineered  Streptomyces  bacterium including a  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain (e.g.,  Streptomyces  strain that, in its native, unmodified form, does not produce thaxtomin and is not pathogenic to plants), that has been engineered to have and express an exogenous, non-native Thaxtomin A (ThxA) biosynthetic gene cluster from a pathogenic  Streptomyces  strain. While the present disclosure primarily discusses the engineering of non-pathogenic  Streptomyces  bacteria, the same approach could be extended to other types of bacterial hosts, such as other non-pathogenic gram-positive bacteria. In embodiments, the presence of the non-native ThxA biosynthetic cluster in the modified genome of the genetically engineered  Streptomyces  bacterium provides the naturally non-pathogenic strain with the ability to produce thaxtomin A, unlike corresponding wild type strains of the bacterium. Thus, although a wild-type version of this non-pathogenic  Streptomyces  strain without the exogenous non-native ThxA biosynthetic cluster does not have the ability to produce thaxtomin, the genetically engineered strain that harbors the exogenous ThxA cluster can express the ThxA biosynthetic genes and thus produce thaxtomin. 
     As discussed above, all known pathogenic  Streptomyces  species carry TR1, which carries the thaxtomin biosynthetic cluster, while some pathogenic and non-pathogenic species carry TR2. TR1 and TR2 together are referred to herein as the toxigenic region (TR). Some species, such as, but not limited to  Streptomyces scabiei  (also referred to herein as  S. scabiei ), include both TR1 and TR2. Some non-pathogenic species include TR2, but naturally non-pathogenic species do not contain TR1. Thus, in embodiments, the exogenous, non-native ThxA biosynthetic cluster includes toxigenic region 1 (TR1). In some embodiments the exogenous, non-native thaxtomin biosynthetic gene cluster includes a subset of TR1 capable of conferring thaxtomin-producing ability to the recipient species. In some embodiments, the exogenous thaxtomin biosynthetic gene cluster includes a minimal thaxtomin biosynthetic gene cluster (e.g., the minimal portion of the thaxtomin biosynthetic gene cluster needed to confer the ability to produce thaxtomin). In some embodiments, the genetically engineered  Streptomyces  bacterium also includes exogenous, non-native TR2. 
     In embodiments, the non-native ThxA biosynthetic cluster is from a pathogenic  Streptomyces  strain including, but not limited to,  Streptomyces scabiei, Streptomyces acidiscabies , and  Streptomyces turgidiscabies . In embodiments, the  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain can be, but is not limited to, one of the following strains:  S. albus, S. diastatochromogenes, S. lividans, S. coelicolor , and  S. avermitilis . In embodiments, the non-pathogenic bacteria may be from a different gram positive bacterial species, such as  Norcardia , or other species compatible with receipt of the ThxA biosynthetic cluster from the pathogenic  Streptomyces  species. In embodiments, the pathogenic  Streptomyces  strain is  S. scabiei  and the non-pathogenic strain is  S. albus  J1074. 
     In order to select for successful integration of the exogenous, non-native ThxA biosynthetic gene cluster from the pathogenic strain into the genome of the recipient non-pathogenic  Streptomyces  bacteria, in embodiments, the ThxA biosynthetic gene cluster from a pathogenic  Streptomyces  strain is operably linked to a nucleotide encoding a selectable marker. Thus, in such embodiments, the expression of the selectable marker indicates integration and expression of the genes in the non-native ThxA gene cluster. In embodiments, the selectable marker is antibiotic resistance, such as known to those of skill in the art, and described in greater detail in the examples below. 
     In embodiments the genetically engineered  Streptomyces  bacterium of the present disclosure produces about the same or a greater amount of thaxtomin than  S. scabiei  under the same culture conditions. Thus, in some conditions the genetically engineered  Streptomyces  bacterium of the present disclosure “overproduces” thaxtomin, as compared to a wild-type, thaxtomin producing, pathogenic species, such as  S. scabiei.    
     In embodiments, the genetically engineered  Streptomyces  bacterium also includes at least one additional mutation, in addition to the addition of the exogenous ThxA gene cluster, to increase thaxtomin production, such as those discussed above and described in detail in PCT/US2015/050582 (WO 2016/044527 A1). In embodiments, this mutation is one or more mutations, affecting the production of proteins involved in regulation of thaxtomin regulation, such as cebR and bglC. For instance, in embodiments, the genetically engineered  Streptomyces  bacterium of the present disclosure further includes at least one of a mutation of a native cebR gene and a mutation of a native bglC gene, where the mutation reduces production or functionality of at least one of a CebR repressor encoded by the cebR gene and a β-glucosidase enzyme encoded by the bglC gene. In embodiments the cebR gene has a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having about 60% or more sequence identity with SEQ ID NO: 15. In embodiments, the bglC gene has a nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having about 60% or more sequence identity with SEQ ID NO: 17. 
     Methods of Providing Thaxtomin-Production in Non-Native  Streptomyces    
     The present disclosure also provides methods of providing thaxtomin-producing capability in a non-native  Streptomyces  bacterium (or other Actinomycete capable of receiving and integrating the thaxtomin gene cluster into its genome). In general, methods of the present disclosure for providing thaxtomin-producing capability in a non-native  Streptomyces  bacterium include providing a  Streptomyces  bacterium from a naturally non-pathogenic species that does not produce thaxtomin and genetically engineering the bacterium for thaxtomin production. In embodiments the genetic engineering includes introducing a non-native nucleic acid molecule encoding for non-native Thaxtomin A (ThxA) biosynthetic gene cluster from a pathogenic  Streptomyces  strain, such that the non-native ThxA is integrated into the genome of the  Streptomyces  bacterium such that the genetically engineered  Streptomyces  bacterium is capable of producing thaxtomin. In embodiments, the exogenous, non-native nucleic acid molecule encoding for the non-native ThxA gene cluster includes all of TR1, as described above. In embodiments the exogenous, non-native nucleic acid molecule also includes exogenous, TR2, where TR1 and TR2 are operably linked. In other embodiments, as described above, the exogenous, non-native nucleic acid molecule encoding for the non-native ThxA gene cluster includes a subset of TR1. 
     In embodiments, the process of introducing the non-native nucleic acid molecule is done via conjugal mating of a recombinant pathogenic strain and a naturally non-pathogenic strain, such that the exogenous, non-native nucleic acid molecule encoding for the non-native ThxA gene cluster is transferred to the naturally non-pathogenic strain. In embodiments the method includes providing a pathogenic  Streptomyces  bacterium that includes a TR nucleic acid encoding a native TR1, a subset thereof, and TR2 (TR) and conjugally mating this pathogenic  Streptomyces  bacterium with a naturally non-pathogenic  Streptomyces  bacterium lacking TR1, such that TR1 and TR2 are transferred from the pathogenic  Streptomyces  bacterium to the naturally non-pathogenic  Streptomyces  bacterium. In embodiments, the process includes providing a genetically engineered pathogenic  Streptomyces  bacterium that has been engineered to include an exogenous nucleic acid encoding a selectable marker operably linked to a TR nucleic acid encoding a native TR1, a subset thereof, and TR2 (TR). Then this genetically engineered pathogenic  Streptomyces  bacterium is conjugally mated with a naturally non-pathogenic  Streptomyces  bacterium lacking TR1, such that the selectable marker, TR1, and TR2 are transferred from the pathogenic  Streptomyces  bacterium to the naturally non-pathogenic  Streptomyces  bacterium. Then the selectable marker can be used to select for transconjugants that harbor the TR. Additional methods (such as PCR) can be used to confirm integration of the TR into a genome of the naturally non-pathogenic  Streptomyces  bacterium. The resulting genetically engineered transformants express the ThxA gene cluster (e.g., the whole cluster or subset thereof) and are able to produce thaxtomin. In embodiments the genetically engineered  Streptomyces  bacterium made according to the methods of the present disclosure are able to produce about as much or more thaxtomin than a wild type pathogenic species, such as the species from which the ThxA gene cluster originated. 
     In other methods, introducing the non-native nucleic acid molecule does not include bacterial conjugation, due to possible instability of the conjugates. In embodiments, the method includes cloning a ThxA biosynthetic gene cluster from a pathogenic  Streptomyces  strain, optionally operably linking the ThxA biosynthetic gene cluster to a selectable marker, and then transforming the ThxA biosynthetic gene cluster into the  Streptomyces  bacterium from the naturally non-pathogenic species using methods such as yeast cloning systems and bacterial transforming systems, such as  E. coli  (as described in greater detail in Examples 1 and 2, below). In embodiments, after transforming, the recipient, naturally non-pathogenic species includes the ThxA biosynthetic cluster and optional selectable marker, and successful transformants can be identified using the selectable marker or other method known to those of skill in the art. The successful transformants can be further tested (e.g., genetic analysis or thaxtomin production tests) to confirm integration of the ThxA gene cluster into the recipient bacterial genome. Other methods known to those of skill in the art can be used to introduce the exogenous non-native ThxA gene cluster into the  Streptomyces  bacteria resulting in integration of the ThxA gene cluster and consequent production of thaxtomin. Such methods are intended to be included in the scope of the present application. 
     These methods provide genetically engineered  Streptomyces  bacterium capable of producing thaxtomin. In embodiments, the genetically engineered  Streptomyces  bacterium are capable of producing thaxtomin produces in about the same or a greater amount of thaxtomin than  S. scabiei  under the same culture conditions. 
     In the various methods described herein, in embodiments, the non-native ThxA biosynthetic cluster is from a pathogenic  Streptomyces  strain including, but not limited to,  Streptomyces scabiei, Streptomyces acidiscabies , and  Streptomyces turgidiscabies . In embodiments, the  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain can be, but is not limited to, one of the following strains:  S. albus, S. diastatochromogenes, S. lividans, S. coelicolor , and  S. avermitilis . As mentioned above, in some embodiments the non-pathogenic recipient bacterial species is another non-pathogenic species compatible with transformation with and expression of the ThxA biosynthetic cluster from a pathogenic  Streptomyces  strain, such as, but not limited to  Norcardia . In embodiments, the pathogenic  Streptomyces  strain is  S. scabiei  and the non-pathogenic strain is  S. albus  J1074. 
     In some embodiments, the methods also include introducing a non-naturally occurring mutation into the  Streptomyces  bacterium, such as discussed above that affect the production of CebR protein β-glucosidase enzyme, and thus the production of thaxtomin. In embodiments, the non-naturally occurring mutation can include a mutation of a cebR gene that reduces production or functionality of a CebR protein encoded by the cebR gene, a mutation of a bglC gene that reduces production or functionality of a β-glucosidase enzyme encoded by the bglC gene, or both. 
     The present disclosure also includes genetically engineered  Streptomyces  bacterium produced by the methods described in the present disclosure. 
     Methods of Producing Thaxtomin 
     The present disclosure also includes methods of producing thaxtomin. Embodiments of such methods include culturing the genetically engineered  Streptomyces  bacteria of the present disclosure described above, so that the modified  Streptomyces  bacteria produce thaxtomin. 
     In the methods of producing thaxtomin of the present disclosure, the genetically engineered  Streptomyces  bacteria exhibit about the same or increased production of thaxtomin compounds as compared to a wild type  Streptomyces scabiei  bacteria. As described above, in some embodiments, the genetically engineered  Streptomyces  bacteria of the present disclosure produce thaxtomin in environmental conditions (e.g., standard growth medium) where the native or wild type  Streptomyces  bacteria would not be able to produce thaxtomin or may only produce trace amounts. In embodiments of the methods of the present disclosure for producing thaxtomin, the thaxtomin produced by the genetically engineered  Streptomyces  bacteria is collected and/or extracted from the cell culture. After collection/extraction of the thaxtomin from the cell culture, the thaxtomin may be further extracted/separated from the culture media, and/or the extracted thaxtomin may then be subject to further isolation and/or purification steps as needed or desired. 
     The present disclosure also includes the thaxtomin compounds produced by the methods and genetically engineered bacteria of the present disclosure described above. The isolated and/or purified thaxtomin compound isolated from the genetically engineered  Streptomyces  bacteria of the present disclosure can then be used for various purposes, such as in the production of certain herbicides. Thus, the methods of the present disclosure also include methods of making herbicides including thaxtomin by producing thaxtomin according to the methods of the present disclosure and using the thaxtomin to produce the herbicide. The present disclosure also includes thaxtomin compounds produced by the methods of making thaxtomin of the present disclosure described above. 
     Methods of Producing Thaxtomin Analogs and Intermediates 
     The present disclosure also provides methods of producing thaxtomin derivatives, analogs and/or intermediates and genetically engineered  Streptomyces  bacterium capable of producing the thaxtomin derivatives, analogs and/or intermediates. In embodiments, methods of the present disclosure, include methods such as those described above for producing thaxtomin compounds, by providing a  Streptomyces  bacterium from a naturally non-pathogenic species that does not produce thaxtomin compounds or intermediates and genetically engineering the  Streptomyces  bacterium by introducing a non-native nucleic acid molecule encoding for a genetically modified, non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, where the non-native thaxtomin biosynthetic gene cluster can confer the ability to produce thaxtomin compounds, derivatives, or intermediates. In embodiments, the genetically modified, non-native thaxtomin biosynthetic gene cluster is integrated into the genome of the  Streptomyces  bacterium such that the genetically engineered  Streptomyces  bacterium is capable of producing thaxtomin, thaxtomin intermediates, and/or thaxtomin analogs. 
     In embodiments for making thaxtomin intermediates or other thaxtomin compounds other than thaxtomin A, such as thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan, the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain is engineered such that instead of producing thaxtomin A, the genetically engineered  Streptomyces  bacterium produces one or more of the intermediates. Although other native, pathogenic  Streptomyces  bacterium and other genetically engineered  Streptomyces  bacterium of the present disclosure may produce some of these intermediates, in embodiments, the genetically engineered  Streptomyces  bacterium can produce a greater amount of intermediates/derivatives than a native pathogenic or other engineered strain. 
     In embodiments, the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises an engineered thaxtomin biosynthetic gene cluster that has a deletion or other silencing mutation of the txtC gene and therefore does not include/does not include txtC and/or does not produce TxtC. This deletion of txtC provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin biosynthetic intermediates such as, but not limited to, thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan. In some embodiments the engineered thaxtomin biosynthetic gene cluster also has a deletion or other silencing mutation of other txt genes from the TR1 region, such as deletion of one or more of txtC, txtA, txtB, and txtH. In embodiments these engineered thaxtomin biosynthetic gene clusters that do not include one or more of txtC, txtA, txtB, and txtH, confer the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin biosynthetic intermediates such as, but not limited to, thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan. 
     The present disclosure also includes the genetically engineered  Streptomyces  bacterium that include these exogenous, non-native engineered thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, wherein the engineered thaxtomin biosynthetic gene cluster does not include one or more of txtC, txtA, txtB, and txtH. 
     In yet other embodiments, methods of the present disclosure also include methods of making halogenated derivatives of thaxtomin compounds, intermediates, and/or analogs. In embodiments, such halogenated derivatives include, but are not limited to, fluorinated derivatives. In embodiments, methods include culturing any of the genetically engineered  Streptomyces  bacterium of the present disclosure having the non-native thaxtomin biosynthetic gene clusters in a culture media including a halogenated compound, (e.g., fluorinated compound 5-F-L-tryptophan or other fluorinated enzyme substrate, or other halogenated compound), such that the genetically engineered  Streptomyces  bacterium incorporates the halogenated substrates into the thaxtomin compounds/intermediates to produce fluorinated derivatives of these compounds, such as, but not limited to, 5-F-thaxtomin A. 
     Additional details regarding the methods, compositions, and organisms of the present disclosure are provided in the Examples below. The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety. 
     It should be emphasized that the embodiments of the present disclosure, particularly, any “preferred” embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and protected by the following claims. 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere. 
     It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt % to about 5 wt %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. In an embodiment, the term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”. 
     Aspects 
     The following listing of exemplary aspects supports and is supported by the disclosure provided herein. 
     Aspect 1. A genetically engineered  Streptomyces  bacterium comprising: 
     a  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain, the  Streptomyces  bacterium comprising an exogenous, non-native Thaxtomin A (ThxA) biosynthetic gene cluster from a pathogenic  Streptomyces  strain, wherein the presence of the non-native ThxA biosynthetic cluster provides the genetically engineered  Streptomyces  bacterium with the ability to produce thaxtomin A, wherein the non-pathogenic  Streptomyces  strain without the exogenous non-native ThxA biosynthetic cluster does not have the ability to produce thaxtomin. 
     Aspect 2. The genetically engineered  Streptomyces  bacterium of aspect 1, wherein the exogenous, non-native ThxA biosynthetic cluster comprises toxigenic region 1 (TR1). 
     Aspect 3. The genetically engineered  Streptomyces  bacterium of aspect 1 or 2, wherein the non-native ThxA biosynthetic cluster is from a pathogenic  Streptomyces  strain selected from the group of pathogenic  Streptomyces  species consisting of:  Streptomyces scabiei, Streptomyces acidiscabies , and  Streptomyces turgidiscabies.    
     Aspect 4. The genetically engineered  Streptomyces  bacterium of aspect 3, wherein the pathogenic  Streptomyces  species is  S. scabiei  87-22 or other strain of  S. scabiei.    
     Aspect 5. The genetically engineered  Streptomyces  bacterium of any of aspects 1-4, wherein the  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain is selected from the group of non-pathogenic  Streptomyces  species consisting of:  S. albus, S. diastatochromogenes, S. lividans, S. coelicolor , and  S. avermitilis.    
     Aspect 6. The genetically engineered  Streptomyces  bacterium of aspect 5, wherein the  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain is  S. albus  J1074. 
     Aspect 7. The genetically engineered  Streptomyces  bacterium of any of aspects 1-6, wherein the genetically engineered  Streptomyces  bacterium further comprises exogenous, non-native toxigenic region 2 (TR2). 
     Aspect 8. The genetically engineered  Streptomyces  bacterium of any of aspects 1-7, wherein the exogenous, non-native Thaxtomin A (ThxA) biosynthetic gene cluster from a pathogenic  Streptomyces  strain is operably linked to a nucleotide encoding a selectable marker. 
     Aspect 9. The genetically engineered  Streptomyces  bacterium of aspect 8, wherein the selectable marker is antibiotic resistance. 
     Aspect 10. The genetically engineered  Streptomyces  bacterium of any of aspects 1-9, wherein the genetically engineered  Streptomyces  bacterium produces about the same or a greater amount of thaxtomin than  S. scabiei  under the same culture conditions. 
     Aspect 11. The genetically engineered  Streptomyces  bacterium of any of aspects 1-10, wherein the non-native ThxA biosynthetic cluster is from a pathogenic  Streptomyces  strain of  S. scabiei  and wherein the  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain of  S. albus.    
     Aspect 12. A genetically engineered  Streptomyces  bacterium comprising: a  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain, the  Streptomyces  bacterium comprising an exogenous, non-native engineered thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, wherein the engineered thaxtomin biosynthetic gene cluster does not include the txtC gene, wherein the presence of the non-native, engineered thaxtomin biosynthetic cluster provides the genetically engineered  Streptomyces  bacterium with the ability to produce thaxtomin biosynthetic intermediates selected from thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan, wherein the non-pathogenic  Streptomyces  strain without the exogenous non-native, engineered thaxtomin biosynthetic cluster does not have the ability to produce the thaxtomin intermediates. 
     Aspect 13. The genetically engineered  Streptomyces  bacterium of aspect 12, wherein the engineered thaxtomin biosynthetic gene cluster further does not include any of the txtA, txtB, and txtH genes. 
     Aspect 14. A method of providing a non-native  Streptomyces  bacterium with the ability to product thaxtomin compounds, derivatives thereof, or intermediates thereof, the method comprising: providing a  Streptomyces  bacterium from a naturally non-pathogenic species that does not produce thaxtomin; and genetically engineering the  Streptomyces  bacterium by introducing a non-native nucleic acid molecule encoding for a non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, such that the non-native thaxtomin biosynthetic gene cluster is integrated into the genome of the  Streptomyces  bacterium such that the genetically engineered  Streptomyces  bacterium is capable of producing one or more thaxtomin compounds, intermediates thereof, or derivatives thereof. 
     Aspect 15. The method of aspect 14, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises toxigenic region 1 (TR1) and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin A. 
     Aspect 16. The method of aspect 14 or 15, wherein the non-native nucleic acid molecule further comprises exogenous, toxigenic region 2 (TR2), wherein TR1 and TR2 are operably linked. 
     Aspect 17. The method of aspect 16, wherein introducing the non-native nucleic acid molecule includes: providing a genetically engineered pathogenic  Streptomyces  bacterium comprising an exogenous nucleic acid encoding a selectable marker operably linked to a TR nucleic acid encoding a native TR1 and TR2; performing conjugal mating of the genetically engineered pathogenic  Streptomyces  bacterium with the naturally non-pathogenic  Streptomyces  bacterium such that the selectable marker, TR1, and TR2 are transferred from the pathogenic  Streptomyces  bacterium to the naturally non-pathogenic  Streptomyces  bacterium; and selecting for transconjugants and detecting for integration of the TR into a genome of the naturally non-pathogenic  Streptomyces  bacterium to produce a genetically engineered  Streptomyces  bacterium capable of producing thaxtomin. 
     Aspect 18. The method of any of aspects 14-16, wherein introducing the non-native nucleic acid molecule comprises: cloning a thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain; transforming the thaxtomin biosynthetic gene cluster into the  Streptomyces  bacterium from the naturally non-pathogenic species; and selecting for transformants to provide genetically engineered  Streptomyces  bacterium capable of producing thaxtomin. 
     Aspect 19. The method of aspect 18 further comprising: operably linking the thaxtomin biosynthetic gene cluster to a selectable marker and using the selectable marker to select for transformants having the thaxtomin biosynthetic gene cluster. 
     Aspect 20. The method of any of aspects 14-19, wherein the pathogenic  Streptomyces  strain is selected from the group of pathogenic  Streptomyces  species consisting of:  Streptomyces scabiei, Streptomyces acidiscabies , and  Streptomyces turgidiscabies.    
     Aspect 21. The method of any of aspects 14-20, wherein the  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain is selected from the group of non-pathogenic  Streptomyces  species consisting of:  S. albus, S. diastatochromogenes , and  S. avermitilis.    
     Aspect 22. The method of aspect 21, wherein the  Streptomyces  bacterium from a non-pathogenic  Streptomyces  strain is  S. albus  J1074. 
     Aspect 23. The method of any of aspects 14-22, wherein the genetically engineered  Streptomyces  bacterium capable of producing thaxtomin produces about the same or a greater amount of thaxtomin than  S. scabiei  under the same culture conditions. 
     Aspect 24. The method of any of aspects 18-23, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises an engineered ThxA biosynthetic gene cluster comprising less than the full toxigenic region 1 (TR1) and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin A. 
     Aspect 25. The method of any of aspects 18-23, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises an engineered thaxtomin biosynthetic gene cluster that does not include txtC gene and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin biosynthetic intermediates selected from thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan. 
     Aspect 26. The method of aspect 25, wherein the engineered thaxtomin biosynthetic gene cluster also does not include any of the txtA, txtB, and txtH genes and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin biosynthetic intermediates selected from thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan. 
     Aspect 27. A genetically engineered  Streptomyces  bacterium produced by the method of any of aspects 14-26. 
     Aspect 28. A method of producing thaxtomin compounds, thaxtomin derivatives, and thaxtomin intermediates, the method comprising: culturing genetically engineered  Streptomyces  bacteria from a non-pathogenic  Streptomyces  strain, the genetically engineered  Streptomyces  bacterium comprising an exogenous, non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, such that the genetically engineered  Streptomyces  bacteria produce thaxtomin compounds, derivatives or intermediates, wherein the genetically engineered  Streptomyces  bacteria have about the same or increased production of a thaxtomin compound, derivative, or intermediate as compared to a wild type  S. scabiei  bacteria under the same culture conditions. 
     Aspect 29. The method of aspect 28, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises toxigenic region 1 (TR1) and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin A. 
     Aspect 30. The method of aspect 28, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises an engineered ThxA biosynthetic gene cluster comprising less than the full toxigenic region 1 (TR1) and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin A. 
     Aspect 31. The method of aspect 28, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises an engineered thaxtomin biosynthetic gene cluster that does not include txtC gene and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin biosynthetic intermediates selected from thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan. 
     Aspect 32. The method of aspect 31, wherein the engineered thaxtomin biosynthetic gene cluster also does not include any of the txtA, txtB, and txtH genes and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin biosynthetic intermediates selected from thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan. 
     Aspect 33. The method of any of aspects 28-33, wherein culturing the genetically engineered  Streptomyces  bacterium comprising the non-native thaxtomin biosynthetic gene cluster comprises culturing in a culture media comprising a halogenated compound, such that the genetically engineered  Streptomyces  bacterium produces halogenated thaxtomin derivatives, halogenated thaxtomin intermediates, and/or halogenated thaxtomin analogs. 
     Aspect 34. The method of aspect 33, wherein the halogenated thaxtomin analog comprises 5-F-thaxtomin A. 
     Aspect 35. The method of any of aspects 27-34, further comprising extracting the thaxtomin compound from the culture media. 
     Aspect 36. A method of producing thaxtomin compounds and nitrotryptophan analogs, the method comprising: providing a  Streptomyces  bacterium from a naturally non-pathogenic species that does not produce thaxtomin; and genetically engineering the  Streptomyces  bacterium by introducing a non-native nucleic acid molecule encoding for a genetically modified, non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain, such that the genetically modified, non-native thaxtomin biosynthetic gene cluster is integrated into the genome of the  Streptomyces  bacterium such that the genetically engineered  Streptomyces  bacterium is capable of producing thaxtomin, thaxtomin intermediates, and/or thaxtomin analogs. 
     Aspect 37. The method of aspect 36, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises toxigenic region 1 (TR1) and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin A. 
     Aspect 38. The method of aspect 36, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises an engineered ThxA biosynthetic gene cluster comprising less than the full toxigenic region 1 (TR1) and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin A. 
     Aspect 39. The method of aspect 36, wherein the non-native thaxtomin biosynthetic gene cluster from a pathogenic  Streptomyces  strain comprises an engineered thaxtomin biosynthetic gene cluster that does not include txtC gene and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin biosynthetic intermediates selected from thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan. 
     Aspect 40. The method of aspect 39, wherein the engineered thaxtomin biosynthetic gene cluster also does not include any of the txtA, txtB, and txtH genes and provides the genetically engineered  Streptomyces  bacterium with the capability of producing thaxtomin biosynthetic intermediates selected from thaxtomin D, thaxtomin C, N-acetyl-nitro-L-tryptophan, and N-methyl-nitro-L-tryptophan. 
     Aspect 41. The method of any of aspects 36-40, further comprising culturing the genetically engineered  Streptomyces  bacterium comprising the non-native thaxtomin biosynthetic gene cluster in a culture media comprising a halogenated compound, such that the genetically engineered  Streptomyces  bacterium produces halogenated thaxtomin compounds, halogenated thaxtomin intermediates, and/or halogenated thaxtomin analogs. 
     From the foregoing, it will be seen that aspects herein are well adapted to attain the ends and objectives hereinabove set forth together with other advantages which are obvious and which are inherent to the systems and methods. 
     It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the aspects. 
     While specific elements and steps are discussed in connection to one another, it is understood that any element and/or steps provided herein is contemplated as being combinable with any other elements and/or steps regardless of explicit provision of the same while still being within the scope provided herein. Since many possible aspects may be made of the disclosure without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense. 
     EXAMPLES 
     Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. 
     Example 1—Overproduction of Thaxtomin in Non-Native  Streptomyces  Species 
     Thaxtomins are metabolites of several plant pathogenic  Streptomyces  species and have broad spectrum herbicidal activity. This example describes the overproduction of thaxtomins in nonpathogenic  Streptomyces  species that carry the genomic island of the plant pathogen  S. scabiei  87-22; this island contains the thaxtomin biosynthetic cluster. Also provided herein are strategies for enhancing thaxtomin production in microbial fermentation. 
     Materials and Methods 
     Bacterial Strains and Culture Conditions 
       Escherichia coli  strains were cultured in Luria-Bertani (LB) medium at 37° C.  Streptomyces  strains were cultured at 30° C. on the International  Streptomyces  Project medium 4 (ISP4) agar medium or in tryptic soy broth (TSB; BD Biosciences). All liquid cultures were shaken at 250 rpm. When required, media were supplemented with the following antibiotics at the indicated final concentrations: hygromycin B (100 μg/ml), apramycin (50 μg/ml), kanamycin (50 μg/ml), chloramphenicol (25 μg/ml), and nalidixic acid (50 μg/ml). All  S. scabiei  87-22 deletion mutants used in this study were generated by intergeneric conjugation from the non-methylating  E. coli  strain ET12567/pUZ8002. Strains and plasmids used in this study are described in Table 2. 
     Insertion of Antibiotic Resistance Markers in the PAI 
     Insertion of the antibiotic resistance markers into the TR1 or TR2 region of  S. scabiei  87-22 was accomplished by replacing txtH (SCAB_31771) with the apramycin resistance gene or replacing the lantibiotic biosynthesis genes lanA (SCAB_32021) and lanB (SCAB_32031) with the hygromycin B resistance gene using the Redirect PCR targeting system (Gust et al. 2003). 
     The cosmid 1989 (harboring the txtH gene) or the cosmid 2757 (harboring the lantibiotic biosynthesis genes) was introduced to the  Escherichia coli  BW25113 strain harboring the arabinose-inducible λ red expression plasmid pIJ790 (aac(3)IV+oriT) or pIJ10700 (hyg+oriT) (Table 2). The deletion cassette for txtH was PCR amplified using pIJ773 as the template and using primers DRB201 and DRB202 (Table 7); the deletion cassette for lanA and lanB was PCR amplified using pIJ10700 as the template and using primers DRB431 and DRB432 (Table 7). The gel-purified deletion cassettes were electroporated into the  E. coli  BW25113 cells that contained either the cosmid 1989 or cosmid 2757 and which had been induced with arabinose (20 mM final concentration). The ΔtxtH and ΔlanAB mutant cosmids were then isolated and confirmed by PCR and sequencing. Following transformation of each cosmid into the non-methylating  E. coli  ET12567 strain (Table 2) (MacNeil et al. 1992), which contains the plasmid pUZ8002 as a driver for transfer (Bierman et al. 1992), the cosmids were conjugated into  S. scabiei  87-22 on soy flour mannitol (SFM) agar (Kieser et al. 2000). Resulting exconjugants for ΔtxtH were selected for resistance to apramycin (apr R ) and sensitive to kanamycin, and exconjugants for ΔlanAB were selected for resistance to hygromycin B (hyg R ) and sensitive to kanamycin. The mutant strains were confirmed using PCR and were stored as spore stocks in 20% vol/vol glycerol at −80° C. 
     Selection of Transconjugant Strains 
     Conjugal mating was carried out by co-culturing donor and recipient strains on SFM plates (Kieser et al. 2000) for 2 days at 30° C. A soft nutrient agar overlay containing antibiotics was used to select for transconjugants. Qualitative PCR assays were conducted to detect the integration of  S. scabiei  TR into the chromosome of non-pathogenic strains (Table 7). Primers a+b flanking the att site located at the 3′ end of the aviX1 gene were used to detect the site-specific integration of TR in aviX1. Primers e+f were used to amplify the chromosomal junction formed by integration of the whole TR or TR2 into aviX1. Primers c+d that are located within the integrase gene (SCAB_31871) of TR2 were used to confirm TR2 integration. Primers g+h flanking txtH (SCAB_31771) were used to confirm TR1 integration. All PCR products were sequenced. 
     The Procedure of Fermentation and Isolation Thaxtomins and Nitro-Tryptophans 
     Mycelial suspensions of  S. scabiei  and  S. albus  J1074 strains were prepared from 48-hour-old 20 ml TSB-grown cultures by pelleting the mycelia, washing twice with sterile water, and resuspending in 20 ml sterile water. For analysis of thaxtomin production, TDM medium with 1% cellobiose (Johnson et al. 2007) was used. Three times 500 ml medium in 2 Liter flasks were inoculated with 15 ml of mycelial suspension of OD 600 nm  1.0. After incubation for 7 days at 30° C. with shaking at 250 rpm, the culturing media were centrifuged with 5000 rpm to pellet the mycelium. The supernatants of centrifugation were added to C18 columns (waters-2 g). Nitrotrptophans were eluted with 25% methanol while thaxtomins were eluted with 100% methanol. The eluted solvents containing targeted compounds were further evaporated to dryness and then were further purified by semi-prep HPLC column to give isolation yields in Table 1. Experiments were repeated with at least three technical replicates per strain and per medium. Thaxtomins could also be extracted by ethyl acetate directly from supernatant which could provide the same amount of thaxtomins from the same volume of supernatant. Experiments were repeated using different biological replicates of the  Streptomyces  strains with three technical replicates per strain. 
     Analytical HPLC Analysis 
     Thaxtomins: the HPLC column (ZORBAX RX-C18, 5.0 μm, 4.6×250 mm) kept at 30° C. was eluted with 40% solvent B acetonitrile for 15 min. The flow rate was set as 1.0 ml/min, and the products were detected at 380 nm with a PDA detector. 
     Tryptophans: the HPLC column (Agilent Poroshell 120 EC-C18, 2.7 μm, 4.6×50 mm) kept at 30° C. was eluted first with 10% solvent B (acetonitrile with 0.1% formic acid) for 2 min and then with a linear gradient of 10-50% solvent B in 8 min, followed by another linear gradient of 50-99% solvent B in 5 min. After eluting in 99% solvent B for 3 min, the liner gradient of 99-10% solvent B in 1 min was used. The column was further re-equilibrated with 10% solvent B for 1 min. The flow rate was set as 0.5 ml/min, and the products were detected at 380 nm with a PDA detector. 
     Semi-Preparative HPLC Isolation 
     For semi-preparative analysis, the column (ZORBAX SB-C18, 5.0 μm, 9.4×250 mm) kept at 40° C. was eluted first with 10% solvent B (acetonitrile with 0.1% formic acid) for 2 min and then with a linear gradient of 10-50% solvent B for 8 min, followed by a linear gradient of 50-99% solvent B for 5 min. The column was then cleaned by 99% solvent B for 3 min and re-equilibrated with 10% solvent B for 1 min. The flow rate was set at 3 ml/min, and the products were detected at 380 nm with a PDA detector. All isolates were combined, concentrated, freeze-dried, and then weighed. 
     LC-MS Analysis of Isolated Products 
     A SHIMADZU Prominence UPLC system fitted with an Agilent Poroshell 120 EC-C18 column (2.7 μm, 4.6×50 mm) coupled with a Linear Ion Trap Quadrupole LC/MS/MS Mass Spectrometer system was used in the studies. The column was eluted with 10% solvent B (acetonitrile with 0.1% formic acid) for 2 min and then with a linear gradient of 10-50% solvent B in 8 min, followed by another linear gradient of 50-99% solvent B in 5 min. After eluting in 99% solvent B for 3 min, the liner gradient of 99-10% solvent B in 1 min was used. The column was further re-equilibrated with 10% solvent B for 1 min. The flow rate was set as 0.5 ml/min. For MS detection, the turbo spray conditions were identical for all chemicals (curtain gas: 30 psi; ion spray voltage: 5500 V; temperature: 600° C.; ion source gas 1: 50 psi; ion source gas 2: 60 psi). 
     Results 
     Comparative Genomic Analysis Suggests the Thaxtomin Cluster is Located on a Genomic Island 
     To understand the emergence and evolution of thaxtomin biosynthetic cluster, we sequenced the genomes of 10 isolates of plant pathogenic streptomycetes from different locations. Comparative genomic analysis of these newly sequenced pathogenic  Streptomyces  genomes together with those available in the public database indicates that pathogenic isolates share a 20 Kb genomic region, designated TR1. TR1 contains the thaxtomin biosynthetic cluster and is flanked by two identical attachment (att) sites ( FIG.  2   ). Moreover, a nearby 157 Kb genomic region is conserved only in  S. scabiei  and S. sp. 96-12, which belongs to a novel pathogenic species (Zhang et al. 2016). This region has been named TR2 by a previous study (Chapleau et al. 2016). TR2 is also flanked by two identical att sites, and includes putative integrative and conjugative elements (ICEs). ICEs are genomic islands that can be excised from chromosome to form a circular molecule, which is then transferred by conjugation and integrated into another genome by site-specific recombination (Burrus et al. 2002). Since S. sp. 96-12 is phylogenetically distant from  S. scabiei  (Zhang et al. 2016), the same organization and contents of TR1 and TR2 between S. sp. 96-12 and  S. scabiei  suggested an in vivo mobilization of the TR1 and TR2. 
     The  S. scabiei  Thaxtomin Biosynthetic Cluster is Site-Specifically Mobilized into the Chromosome of Non-Pathogenic Species 
     To investigate the mobilization of thaxtomin biosynthetic cluster from  S. scabiei  to non-pathogenic species, two  S. scabiei  87-22 deletion mutants were created, one with the apramycin resistance (apr R ) marker inserted in its TR1 [ S. scabiei  ΔtxtH (apr R )] and the other with the hygromycin B (hyg R ) resistance marker inserted in its TR2 [ S. scabiei  ΔlanAB (hyg R )]. In mating experiments, these mutants served as the genomic island donors. A naturally streptomycin resistant (str R ) nonpathogenic strain,  S. diastatochromogenes  ATCC 12309, was used as the recipient ( FIG.  3 A ). Following selection with apramycin or hygromycin B, together with streptomycin, a number of  S. diastatochromogenes  mutants were isolated. Using PCR-based genotypic diagnosis with primers shown in Table 7, integration of  S. scabiei  TR1 and TR2 regions into the att site (TTGAAGCGGAAC, SEQ ID NO: 62) of the  S. diastatochromogenes  aviX1 gene ( FIG.  3 B ) was detected. Mating of  S. scabiei  ΔlanAB with  S. diastatochromogenes  resulted in the acquisition of TR2 alone or of the complete TR (TR1 and TR2) element by  S. diastatochromogenes ; however, all transconjugants from the mating of  S. scabiei  ΔtxtH with  S. diastatochromogenes  acquired the entire TR element, but not TR1 alone. These observations suggested that the integrative and conjugative element of the TR2 region is important for the mobilization and site-specific integration of TR2 or the entire TR region into a new host chromosome via conjugation. In contrast, the TR1 region carrying the thaxtomin biosynthetic cluster does not appear to be mobile alone; its transfer appears to depend on the integrative and conjugative elements in the TR2 region. Acquisition of both TR1 and TR2, but not TR2 alone, conferred a pathogenic phenotype and thaxtomin production ability on  S. diastatochromogene  (data not shown). 
     Engineering of Non-Pathogenic  Streptomyces  Species for the Heterologous Production of Thaxtomin 
     After characterizing the mobilization of the  S. scabiei  genomic island containing the thaxtomin biosynthetic cluster, the extent to which non-pathogenic  Streptomyces  sp. can act as the heterologous hosts for thaxtomin production was investigated. First, the apramycin-resistant (apr R ) integrative plasmid pSET152 from  Escherichia coli  was transferred into  S. albus  J1074,  S. coelicolor  M145,  S. lividans  1326, and  S. avermitilis  NRRL 8165 to provide them with the apramycin-resistance for selection during the mating experiment. These non-pathogenic  Streptomyces  isolates comprising pSET52 (apr R ) were mated with  S. scabiei  ΔlanAB (hyg R ), which contains the hygromycin B (hyg R ) resistance marker in its TR2 region. After selection with apramycin together with hygromycin B,  S. albus  J1074,  S. coelicolor  M145,  S. lividans  1326, and  S. avermitilis  NRRL 8165 transconjugants with the  S. scabiei  thaxtomin biosynthetic cluster were identified by the PCR screening with primers within TR1 and TR2 (Table 7). 
     Thaxtomin production of these four newly created heterologous hosts was then assessed along with the engineered  S. diastatochromogenes  ATCC 12309 created in the above studies in TDM medium supplemented with cellobiose (TDMc). The five heterologous hosts ( S. albus  J1074,  S. coelicolor  M145,  S. lividans  1326,  S. avermitilis  NRRL 8165, and  S. diastatochromogenes  ATCC 12309) carrying the thaxtomin cluster produce thaxtomin at different levels ( FIG.  4   ).  S. diastatochromogenes  and  S. avermitilis  produced a similar level of thaxtomin to  S. scabiei ; however,  S. coelicolor  only produced a trace amount of thaxtomin and  S. lividans  did not produce a detectable level of thaxtomin ( FIG.  4   ). These data indicate that subtle differences in genetic backgrounds of these recipients can influence thaxtomin production. Remarkably,  S. albus  produced a significantly higher level of thaxtomin (about 9-fold) than  S. scabiei.    
     Early Metabolic Switch of  S. albus  J1074 Leads to Early Thaxtomin Production 
     To further characterize the overproduction of thaxtomins in  S. albus , the production of thaxtomin and its precursor 4-nitrotryptophan in TDMc medium was quantified at different time points ( FIG.  5   ).  S. scabiei  was used as the control. Thaxtomin A was detected from the  S. albus  fermentation medium within 24 hours by HPLC analysis. By contrast,  S. scabiei  produced thaxtomin A two days after inoculation. The thaxtomin A level in the  S. albus  fermentation medium linearly increased until day five and remained stable from day five to seven ( FIG.  5   ). At day two,  S. albus  already produces the same level of thaxtomin A as the final yield of  S. scabiei.    
     The accumulation of the biosynthetic intermediate in the thaxtomin biosynthetic pathway, 4-nitrotryptophan, was also monitored during the fermentation of  S. albus  J1074 and  S. scabiei  87-22. A significant peak with the retention time of 7.93 min in the HPLC analysis was observed in the  S. albus  J1074 culture extract; this peak occurred in the  S. scabiei  87-22 extract but was much smaller ( FIG.  6 A ; t R =7.93 min). LC-MS analysis revealed the peak content as N-acetyl-4-nitrotryptophan. HPLC analysis further detected a minor peak ( FIG.  6 A ; t R =5.77 min) in both extracts, which was determined as N-methyl-4-nitrotryptophan in the LC-MS analysis. The concentration of N-Methyl-4-nitrotryptophan was low in both transformed  S. albus  J1074 and  S. scabiei  87-22 extracts ( FIG.  6 B ); however, N-acetyl-4-nitrotryptophan accumulated in the culture of transformed  S. albus  J1074 over time ( FIG.  6 B ). The level of N-acetyl-4-nitrotryptophan was about 100-fold of that of N-methyl-4-nitrotryptophan in the transformed  S. albus  J1074 and about 300-fold of that of N-methyl-4-nitrotryptophan in  S. scabiei  87-22 seven days after inoculation. 
     Creating a Genetically Stable  S. albus  Strain for Thaxtomin Production 
     The mobility of the genomic island was leveraged as described above to engineer  S. albus  J1074 for thaxtomin overproduction. However, given the inherent instabilities of genomic islands, use of this strain for commercial production could raise concerns about this strain&#39;s genetic stability. In this regard, it was desired to engineer a strain to express only the thaxtomin gene cluster in  S. albus  J1074. Specifically, as illustrated in  FIG.  7   , a transformation-associated recombination (TAR) approach was employed to clone the thaxtomin gene cluster into pTARa vector using yeast as the assembly host. The pTARa plasmid carrying the thaxtomin cluster was transformed into  E. coli  S17 cells. The conjugation of the transformed  E. coli  S17 with  S. albus  J1074 enabled the transfer of the thaxtomin biosynthetic cluster into  S. albus  J1074. Detailed characterization of the strain carrying only the thaxtomin gene cluster revealed yields of thaxtomins and nitro-tryptophan derivatives to the strain with the genomic island, in the tested media, as described in greater detail in Example 2, below. 
     Quantification of Thaxtomin and 4-Nitrotryptophan Production in Different Media 
     To evaluate the effects of culture media on thaxtomin production, the transformed  S. albus  J1074 was cultured in ISP4, TDM, R5 and PDB with sea salt (PDBS) media supplemented with 1% cellobiose. ISP4c (ISP4 with 1% cellobiose), R5c (R5 with 1% cellobiose) and PDBSc (Potato Dextrose Broth with 3.7% sea salt and 1% cellobiose) supported higher production of thaxtomins by the transformed  S. albus  (ThxA, ˜150 to ˜170 mg/L) than did TDMc (ThxA, ˜90 mg/L) (Table 1). Among these media, PDBSc can be prepared directly from raw potato and thus is a cost-effective option for the commercial production of thaxtomins. 
     Example 2—High-Yield Production of Herbicidal Thaxtomins and Analogs in a Nonpathogenic  Streptomyces  Strain 
     The present example describes heterologous expression of a thaxtomin gene cluster in  S. albus  J1074, which resulted in the highest yield of thaxtomins ever reported. Furthermore, current synthetic routes to thaxtomins and analogs are lengthy, and two thaxtomin biosynthetic intermediates produced at high yields in this example can provide precursors and building blocks to advanced synthetic routes. The production of 5-F-thaxtomin A in engineered  S. albus  J1074 in this example demonstrated a viable alternative to chemical methods in the synthesis of new thaxtomin analogs. This work represents an attractive synthetic biology strategy to improve the supply of herbicidal thaxtomins. 
     Herbicides play a crucial role in agricultural production all over the world but their extensive and broad uses have resulted in the herbicide resistance in weeds. The situation is further exaggerated by the fact that no new class of herbicides has been commercialized in the past decades. Known as a virulence factor in the common scab potato disease, thaxtomins, including the major metabolite thaxtomin A and 10 other analogs, are produced by tens of pathogenic  Streptomyces  strains ( FIGS.  8 A- 8 B ). Thaxtomin A (1) inhibits cellulose biosynthesis in the nM range, a unique mechanism of plant pathogenicity. As weeds have a significantly higher demand of cellulose for the rapid growth, a bioherbicide with 1 as the main active ingredient has been approved by the United States Environmental Protection Agency for pre- and post-emergence weed control on various crops at a ppm concentration. Economically and environmentally acceptable production of thaxtomins will foster and sustain their agricultural applications. Accordingly, several synthetic routes have been developed but generally generate environmentally damaging wastes, produce racemic products, and experience a low overall yield, therefore being impractical to industrial production. On the other hand, the isolation yield of 1 from its native producers (e.g.,  S. scabiei  and  S. acidiscabie ) is lower than 10 mg/L, rendering a costly production process. 
     The gene cluster of 1 has been elucidated and contains seven genes encoding two P450s (TxtC and TxtE), two nonribosomal peptide synthetases (NRPSs, TxtA and TxtB), one MbtH-like protein (TxtH), one positive regulator (TxtR) and one nitric oxide synthase (TxtD) ( FIG.  8 A ). TxtD generates nitric oxide (NO) from I-arginine, which is then used by TxtE to nitrate C4 of I-tryptophan, resulting in 4-NO 2 -I-tryptophan (6). Compound 6 is then cyclized with I-phenylalanine by TxtA and TxtB to form a diketopiperazine thaxtomin D (5), which is further hydroxylated twice to produce thaxtomin A, likely by TxtC ( FIG.  8 B ). The expression of the thaxtomin gene cluster is regulated by TxtR, which is known to be activated by cellobiose. 
     This disclosure seeks to address the thaxtomin supply issue, by expressing the thaxtomin cluster in capable hosts to achieve overproduction.  Streptomyces  strains including  S. coelicolor, S. lividans, S. avermitilis , and  S. albus  J1074 have been used to express active and silent natural product gene clusters.  S. albus  J1074 has the smallest genome within the genus streptomycetes, indicating fewer competing pathways for efficient heterologous expression. Indeed,  S. albus  J1074 has been used to produce structurally diverse natural products of actinomycete and nonstreptomycete origins, and can demonstrate high productivity. 
     Example 1, above, and Zhang, Y., et al., (2018, incorporated by reference herein) described expression of mobile pathogenicity islands (PAIs, 177 kb and 674 kb) from two pathogenic  Streptomyces  strains, which contain the thaxtomin gene cluster, mobile elements and others, in five nonpathogenic  Streptomyces  hosts, including  S. albus  J1074,  S. avermitilis  NRRL8165,  S. coelicolor  M145,  S. diastatochromogenes  ATCC12309, and  S. lividans  1326. Strikingly,  S. albus  J1074 expressing the 177-kb PAI from  S. scabiei  produced the highest level of thaxtomins among all engineered strains and two native producers when cultured in TDMc. This result indicates the influences of genetic backgrounds of expression hosts on the gene cluster expression and illustrates the attractive capability of  S. albus  J1074 in heterologous production of natural products. However, the PAI-based production of thaxtomins in  S. albus  J1074 has potential limitations to developing industrial processes, e.g., the mobility nature of PAI, leading to the possible genetic instability of production strain, technique difficulties in engineering the biosynthetic gene cluster for further improving the production yield, and the cost and safety concerns on the use of two antibiotics in the fermentation media. 
     This example describes the high-yield production of thaxtomins in  S. albus  J1074 after chromosomal integration of only its biosynthetic gene cluster. The yield of thaxtomins was improved by about 12 times in comparison with the native producer in TDMc. Further engineering of the cluster produced key biosynthetic intermediates, which can be used as precursors for chemical synthesis of thaxtomin analogs. Moreover, feeding 5-F-I-tryptophan into the culture medium led to the generation of one unnatural analog 5-F-thaxtomin A. Natural and unnatural thaxtomins demonstrated potent herbicidal activity and possessed weak cytotoxicity. 
     Materials and Methods 
     Microorganisms, Fermentation and Analysis. 
     Molecular biology reagents and enzymes were purchased from Fisher Scientific. Primers were ordered from Sigma-Aldrich. Other chemicals and solvents were purchased from Sigma-Aldrich and Fisher Scientific.  E. coli  EP1300 competent cells were purchased from Epicenter. DNA sequencing was performed at Eurofins. The plasmids and strains used in this study were listed in Table 6.  Streptomyces  strains were cultivated on soybean flour-mannitol agar plates and ISP4 agar plates (BD Biosciences, San Jose, Calif., USA) for sporulation and conjugation, respectively. Spores were collected, suspended in 20% (v/v) glycerol and stored at −80° C. Trypticase soy broth (TSB) was used to prepare seed culture for fermentation. A Shimadzu Prominence UHPLC system (Kyoto, Japan) fitted with an Agilent Poroshell 120 EC-C18 column (2.7 μm, 4.6×50 mm), coupled with a PDA detector was used for HPLC analysis. Agilent ZORBAX SB-C18, (5 μm, 9.4×250 mm) or YMC-Pack Ph column (5 μm, 4.6×250 mm) was used for semi-preparative HPLC analysis to isolate the metabolite. 1D and 2D NMR spectra were recorded in CDOD 3  on Bruker 400 MHz or Bruker 500 MHz instruments at the University of Florida, Gainesville, Fla., USA. Spectroscopy data were collected using Topspin 3.5 software. HRMS data were obtained using a Thermo Fisher Q Exactive Focus mass spectrometer equipped with electrospray probe on Universal Ion Max API source. Unless special instruction, all the samples were analyzed at the end of 6-day fermentation. 
     Preparation of Thaxtomin-Producing  S. albus  Strains 
     The transformation-associated recombination (TAR) approach (Kim, J H, et al., 2010, and Kallifidas, et al., 2012, both of which are hereby incorporated by reference herein) was followed to clone the thaxtomin gene cluster into pTARa vector as shown in  FIG.  7   . Primers used were included in Table 8. The resultant pTARa-thx was transformed into  E. coli  S17-1. The conjugation of the transformed  E. coli  S17-1 with  S. albus  J1074 led to the creation of S. albus-thx1. To create  S. albus -thx2 and strains with the engineered thaxtomin clusters, end-overlapped DNA fragments (1 kb/each) of whole or partial thaxtomin synthetic gene clusters were PCR amplified using the cosmid 1989 as template and assembled with the conjugative vector pLST9828 following the protocols of NEBuilder HiFi DNA Assembly Cloning Kit. The assembled mixtures were transformed into  E. coli  EP1300. The constructed plasmids were isolated and confirmed by restriction digestion and DNA sequencing. The validated construct was then transformed into  E. coli  S17-1 for conjugating with  S. albus  J1074 to create  S. albus -thx2 , S. albus -thx2-ΔC and  S. albus -thx2-ΔABCH. 
     Isolation of Thaxtomins and Nitro-Tryptophan Analogs 
       Streptomyces  strains were cultured in TSB for 2 days. Mycelial pellets were collected after centrifugation, washed twice with sterile water, and then resuspended in an equal volume of sterile water to prepare mycelial suspension solutions. Fermentation medium (500 mL) in one 2-L flask was inoculated with 15 mL of mycelial suspension solutions and then incubated at 30° C., 250 rpm for 6 days. Clean supernatants were collected after centrifugation at 5000 rpm for 10 min and then passed through Sep-Pak C18 columns (Waters, 2 g). The columns were washed with one volume of water. Nitrotryptophan analogs were eluted with 25% MeOH while thaxtomins were eluted with 100% MeOH. The eluted solvents containing targeted compounds were further dried in vacuo evaporation. Microscale balance (Mettler Toledo) was used to measure the weights of isolated compounds. To determine the yields of thaxtomin A and other analogs, thaxtomins were further purified by semi-prep HPLC. After drying corresponding fractions in vacuo evaporation, the weights of thaxtomins were measured by microscale balance. Alternatively, the concentrations of thaxtomins were calculated on the basis of their areas under peak in HPLC traces using an established standard curve of authentic thaxtomin A. Experiments were repeated with at least three technical replicates per strain and per medium. Isolated thaxtomins and nitro-tryptophans were analyzed in HR-MS and MS/MS studies. 
     HPLC Analysis 
     The HPLC program included the column elution first with 10% solvent B (acetonitrile with 0.1% formic acid, FA) for 2 min and then with a linear gradient of 10-50% solvent B in 8 min, followed by another linear gradient of 50-99% solvent B in 5 min. The column was further cleaned with 99% solvent B for 3 min and then re-equilibrated with 10% solvent B for 1 min. Solvent A was water with 0.1% FA. The flow rate was set as 0.5 mL/min, and the products were detected at 254 nm with a PDA detector. For semi-preparative HPLC analysis, the column at 40° C. was first eluted with 10% solvent B (acetonitrile with 0.1% FA) for 2 min and then with a linear gradient of 10-50% solvent B for 8 min, followed by a linear gradient of 50-99% solvent B for 5 min. The column was then cleaned by 99% solvent B for 1 min and re-equilibrated with 10% solvent B for 1 min. The flow rate was set at 3 mL/min, and the products were detected at 380 nm with a PDA detector. All metabolites were well separated, and corresponding fractions were combined, concentrated, dried, and then weighed. 
     LC-MS Analysis 
     A SHIMADZU Prominence UPLC system fitted with an Agilent Poroshell 120 EC-C18 column (2.7 μm, 4.6×50 mm) coupled with a Linear Ion Trap Quadrupole LC/MS/MS Mass Spectrometer system was used in the studies. The HPLC conditions were the same as described above. For MS detection, the turbo spray conditions included curtain gas: 30 psi; ion spray voltage: 5500 V; temperature: 600° C.; ion source gas 1: 50 psi; ion source gas 2: 60 psi). For MS/MS analysis, the collision energy was 12 eV. LC-HR-MS analysis was performed on a Thermo Fisher Q Exactive Focus mass spectrometer. Acetonitrile (B)/water (A) containing 0.1% FA were used as mobile phases with a linear gradient program (10-90% solvent B over 15 min) to separate chemicals at a flow rate of 0.3 mL/min. A pre-wash phase of 15 min with 10% solvent B was added at the beginning of each run, in which the elute was diverted to the waste by a diverting valve. MS1 were acquired under Full Scan mode of Orbitrap, in which a mass range of m/z 150-2000 was covered and data were collected in the positive ion mode. Fragmentation was introduced by HCD technique with optimized collision energy ranging from 6 to 15 eV. Other settings for the Orbitrap scan were as follows: resolution 15000, AGC target 5×10 5 . Full scan mass spectra and targeted MS/MS spectra for each of the pre-selected parental ion were extracted from the raw files of the HPLC-MS/MS Experiment II using Xcalibur™ 2.1 (Thermo Scientific). 
     Feeding Experiment with 5-F-I-tryptophan 
       S. albus -thx2 were cultured in TDMc under the same conditions described above for 2 days, and filter sterilized 5-F-I-tryptophan solutions (0 to 50 μM final concentrations) were added to the culture medium. After fermentation for 5 additional days, the cultures were centrifuged to prepare clean supernatants that were extracted with the equal volume of ethyl acetate three times. The combined organic layers were washed, dried over sodium sulfate, and then evaporated to the dryness in vacuo. The residues were suspended in methanol for HPLC analysis as described above. To purify 5F-thaxtomin A (compound 9) for structural determination, the mixture was separated on one C18 column (Agilent ZORBAX SB-C18, 5 μm, 9.4×250 mm). The column was first eluted with 30% solvent B (acetonitrile with 0.1% FA) for 18 min, followed by another linear gradient of 50-99% solvent B in 0.5 min. After eluting in 99% solvent B for 0.5 min, the liner gradient of 99-10% solvent B in 0.5 min was used. The column was further re-equilibrated with 30% solvent B for 0.5 min. The flow rate was set at 3 mL/min, and the products were detected at 380 nm with a PDA detector. The fraction 8 (F8) with a retention time of 18.1 min was collected and dried for further purification with one analytical column (YMC-Pack Ph column, 5 μm, 4.6×250 mm). The column at 30° C. was eluted with 50% solvent B (methanol with 0.1% FA) for 13.5 min and then with a linear gradient of 50-99% solvent B in 0.5 min, followed by another linear gradient of 50-99% solvent B in 0.5 min. After eluting in 99% solvent B for 0.5 min, the liner gradient of 99-50% solvent B in 1.0 min was used. The flow rate was set at 1 mL/min, and the product was detected at 380 nm with a PDA detector. The compound 9 was eluted and collected for NMR analysis. 
     5F-thaxtomin A (9): yellow solid; [α] 20   D +148.3 (c 0.0022, MeOH); HRMS (ESI-TOF) m/z 457.1497 [M+H] +  (calcd. for C 22 H 21 FN 4 O 6 , 457.1523);  19 F NMR (500 MHz, CD 3 OD) δ−137.41 (dd, J=10.9 Hz, 4.5 Hz, 1F);  1 H and  13 C NMR data were described in Table 4. 
     Herbicidal Activity Assay of Thaxtomins 
     Serial concentrations (0 to 4 μM) of thaxtomins in DMSO were added into 20 mL of 1.5% warm agar solution with gentle agitation. DMSO was included as the negative control. The solution was then poured into the plate for solidification at room temperature for 30 min. Radish seeds (Burpee) were surface disinfested, pregerminated and selected when the radicle was 1±2 mm and just emerged from the seed coat. Six radish seedlings were equally located on the surface of each plate with the root ends all pointed in the same direction. Agar plates were covered, sealed with Parafilm. The seedlings in the agar plates grew at room temperature under fluorescent lighting (12 h per day for 6 days), and total seedling lengths were then recorded. Three plates were set up for each dosage of each compound. Percent inhibition relative to the mean growth response in DMSO treated control plates was then calculated. Dose-response curves were fit to a four-parameter logistic model as described in Streibig, J C, et al., 1993 (which is hereby incorporated by reference herein), and Iso values were estimated from these curves. 
     MTT Assay to Characterize Cytotoxicity of Thaxtomins 
     PC-3 and Jurkat cells were cultured in DMEM or RPM11640 medium containing 10% fetal bovine serum and 100 U/mL penicillin and streptomycin, and maintained at 37° C. in a humidified incubator under 5% CO 2 . The cells (1×10 4  for Jurkat, and 5,000 for PC-3 in 100 μL) were seeded into 96-well plates and incubated overnight. Varying concentrations of purified compounds (0 μM, 12.5 μM, 25 μM, 50 μM and 100 μM) were added to the wells. After incubation at 37° C. for 48 to 72 h, 10 μL of MTT (5 mg/mL) in PBS was added and incubated for 4 h, followed by aspiration of the medium. DMSO (100 μL) was added to each well to dissolve the MTT in the wells, and the plates were agitated for 1 h for recording the absorbance at 570 nm using UV/vis microplate spectrophotometer (BioTek). Three to six replications were performed per treatment of each sample. 
     Statistic Analysis 
     Statistical significance among multiple groups was analyzed by one-way ANOVA followed by Student&#39;s t-test for comparison of the results between two groups using Prism 5 (Graphpad Software, Inc). P value of &lt;0.05 is considered to be statistically significant. 
     Results and Discussion 
     Thaxtomin Production in  S. albus    
     Transformation-associated recombination approach described in Kim, J H, et al., 2010 and Kallifidas, D. et al., 2012, hereby incorporated by reference herein) was used to capture the 18-kb thaxtomin gene cluster of  S. scabiei  87.22. The resultant integrative construct pTARa-thx was then conjugated into  S. albus  J1074 to create  S. albus -thx1 ( FIG.  7   ). This strain was cultured along with the untransformed  S. albus  J1074 as a control in the minimal medium TDMc. TDMc contains 1% cellobiose that induces the production of thaxtomin in native producers as described in Joshi, M V, et all, 2007 and Francis, I M, 2015 (hereby incorporated by reference herein) presumably by its binding to the pathway-specific regulator TxtR and likely one global regulator CebR. 
     After 6-day fermentation,  S. albus -thx1 produced thaxtomin A (1), four thaxtomin analogs as minor metabolites (2-5), N-acetyl-4-nitrotryptophan (7) and a trace amount of N-methyl-4-nitrotryptophan (8) as shown in HPLC trace ( FIG.  9 A ). The identities of these compounds were revealed by high resolution-mass spectrometry (HR-MS) analysis including the fragmentation of target ions ( FIGS.  10 A- 10 N ). None of these metabolites were detected from the culture medium extract of wild type  S. albus  J1074. The production of these thaxtomin-related metabolites suggested that  S. albus  J1074 properly recognized the native promoters of the thaxtomin gene cluster of  S. scabiei  87.22. 
     Remarkably, the yield of the most abundant metabolite thaxtomin A (1) reached about 23 mg/L after only 24 hours, highlighting a rapid and highly efficient production. Compounds 7 and 8 are the derivatives of 4-NO 2 -I-tryptophan, and have been isolated from the culture medium of  S. scabiei  87.22 at low titers. Nitrotryptophan analogs can be valuable building blocks for the synthesis of bioactive chemicals such as the protein kinase C activator indolactam V and new analogs of numerous tryptophan-containing compounds. However, chemical synthesis of optically pure 4-NO 2 -I-tryptophan (6) is complicated.  S. albus -thx1 produced a high amount of N-acetyl-nitrotryptophan (7, &gt;50.0 mg/L) and can provide a green route to nitrotryptophan analogs. 
     The integrative bacterial artificial chromosome (BAC) vector pTARa is specific for assembling natural product gene clusters and is over 15 kb in size. To assess the potential size effect of integrative vectors on the production of thaxtomins and to ease the cluster engineering as detailed below, the assembled thaxtomin gene cluster was cloned from pTARa-thx into a smaller integrative vector pLST9828 (˜5.7 kb) to generate pLST9828-thx via Gibson assembly approach ( FIG.  11   ) (see gibson, DG., et al., 2009 and Butler, A R, et al., 1999, which are hereby incorporated by reference herein). This new construct was used to create  S. albus -thx2. The metabolite profile of  S. albus -thx2 cultured in TDMc was identical to  S. albus -thx1 ( FIG.  9 A ), including the increasing accumulation of compounds 7 and 8 during the fermentation ( FIG.  12   ). This result suggested that the vector size minimally affects the thaxtomin production. 
     Compared with  S. scabiei  87.22 cultured under the same conditions,  S. albus -thx1 and -thx2 produced significantly higher concentrations of 1, 2 and 7 ( FIG.  9 A ). Of note, the engineered  S. albus  strains produced about 10 times more thaxtomin A (1) than  S. scabiei  87.22 (91.2±6.8 mg/L vs 9.1±0.4 mg/L, Table 3) after 6-day fermentation in TDMc. The productivity of thaxtomin A (1) was determined per biomass, and two  S. albus  strains demonstrated even greater improvement ( FIG.  9 B ).  S. albus -thx2 showed a slightly, statistically insignificantly higher yield of thaxtomin A (1) than  S. albus -thx1 (142±4 mg/g vs 137±3 mg/g of dry weight, DW,  FIG.  9 B ), both of which were about 20 times higher than the native producer (7.2±0.7 mg/g DW). Collectively, these results indicate the superior production of thaxtomins in  S. albus.    
     Screening of Culture Media for Improving Thaxtomin Yield 
     To further improve the productivity of thaxtomins in  S. albus , we screened a total of seven fermentation media. Specifically, both oat bran broth (OBB) and TDMc support the production of thaxtomins by pathogenic  Streptomyces  strains, while TSB, ISP4 and R5 are common media for growing a number of  Streptomyces  strains. Additionally, although potato dextrose broth (PDB) is a fungal culture medium, it was prepared in water or artificial seawater (PDBS) for screening because it contains ingredients of plant origin, as does OBB.  S. albus -thx2 was selected for the culturing in these media under the same conditions. HPLC analysis revealed that in addition to TDMc, only PDBS was able to induce the production of thaxtomin A (1) at a low level (about 0.5 mg/L) (Table 3). This result presumably suggested that cellobiose remains indispensable for the induction of thaxtomin biosynthesis in  S. albus . Consequently, these media (except TDMc) were supplemented with 1% cellobiose, which is indicated by a lowercase c (e.g., ISP4c). It was found that ISP4c, R5c and PDBSc supported the production of ˜150 to ˜170 mg/L of thaxtomin A (1) by  S. albus -thx2 after 6-day fermentation, 1.7 to 1.9 times higher than TDMc (Table 3). Furthermore, when PDBSc was prepared directly from raw potato materials,  S. albus -thx2 was able to produce˜142 mg/L of thaxtomin A (1), providing a highly economical option for the production of thaxtomins. In these cellobiose-containing media,  S. albus -thx2 also produced compounds 2-5, whose total productivity was about one third of thaxtomin A (1), 50 to 80 mg/L of N-acetyl-4-nitrotryptophan (7) and a trace amount of 8 (Table 3). 
     Engineering the Thaxtomin Gene Cluster by Gene Deletion to Produce Biosynthetic Intermediates 
     Thaxtomin biosynthetic intermediates can be valuable for developing advanced synthetic approaches (e.g., semi-synthesis) to produce thaxtomins and analogs. Thaxtomin D (5) is an advanced intermediate carrying a featured diketopiperazine core ( FIG.  8 B ), but only a trace amount was produced by both native producers and two engineered  S. albus  strains ( FIG.  9 A ). To overproduce this compound, the txtC gene was deleted from the thaxtomin cluster cloned in pLST9828 and then created  S. albus -thx2-ΔC ( FIG.  13 A ). This new strain was cultured in thaxtomin production media TDMc, ISP4c, R5c and PDBSc for 6 days. All selected media supported the production of thaxtomin D (5) ( FIG.  14   ), and the highest yield at 76 mg/L was achieved in ISP4c (Table 5). This strain also produced a low concentration of thaxtomin C (4) in all media ( FIG.  14   ) and 55 to 65 mg/L of 7-8 in TDMc, R5c and ISP4c (Table 5). However, only about 11 mg/L of nitrotryptophans were produced when the stain was cultured in PDBSc (Table 5), strikingly different compared to that of  S. albus -thx2, which produced˜80 mg/L of 7-8 in PDBSc (Table 3). It remains unclear how the removal of txtC influences the production of nitrotryptophan analogs in some media. 
     Next, txtABCH genes were deleted from the thaxtomin gene cluster ( FIG.  13 B ), and  S. albus -thx2-ΔABCH was created, which can potentially overproduce nitrotryptophans. Indeed, about 50 mg/L of 7 was produced by  S. albus -thx2-ΔABCH when cultured in TMDc and ISP4c for 6 days (Table 5 and  FIG.  15   ). R5c and PDBSc were less effective production media (about less than 18 mg/L). Interestingly, no 8 was detected from any extract of  S. albus -thx2-ΔABCH ( FIG.  15   ). Future studies can investigate the potential role of the methyltransferase domain of TxtB/TxtA in the formation of 8.  S. albus -thx2-ΔABCH is not able to produce thaxtomin analogs, therefore having no competing pathway using 4-NO 2 -I -tryptophan (6) as substrate ( FIG.  8 B ). The comparable amounts of 7 with  S. albus -thx2 and  S. albus -thx2-ΔC indicated that the cellular availability of 4-NO 2 -I-tryptophan (6) may not limit the productivity of thaxtomins in  S. albus , suggesting potential synthetic biology approaches to further overproduce thaxtomins, e.g., the overexpression of txtABCH genes under stronger promoters. 
     Production of 5-F-thaxtomin A 
     The impressive substrate promiscuity of TxtE was previously demonstrated in in vitro studies, and 5-F-I-tryptophan was identified as the best enzyme substrate as described in Zuo, R. et al., 2016 and Zuo, R. et al., 2017, incorporated herein by reference. To further probe the feasibility of the thaxtomin biosynthetic system as an alternative to chemical methods in synthesizing thaxtomin analogs, it was desired to produce unnatural thaxtomin analogs by feeding tryptophan analogs. Specifically, serial concentrations (0 to 50 μM) of 5-F-I-tryptophan were included in TDMc to culture  S. albus -thx2. HPLC analysis of culture extract revealed the appearance of a new peak with a retention time of 18.1 min ( FIG.  16 A ). The peak content showed a molecular ion of 457.1497 in HRMS analysis ( FIG.  16 B ), matching the value of 5-F-thaxtomin A (9, calculated: 457.1523) (HR-MS/MS analysis shown in  FIG.  16 C ). This compound (1.8 mg/L) was further isolated by semi-prep HPLC analysis and its structure as 5-F-thaxtomin A (9) ( FIG.  17   ) was determined by multiple 1D ( 1 H,  19 F, and  13 C) ( FIGS.  18 A- 18 C ) and 2D (COSY, HMBC, HSQC, and NOESY) NMR analyses (data not shown). Of note, the purified compound showed a chemical shift (δ) of −137.41 ppm (dd, J=10.9 Hz and 4.5 Hz, 1F) in  19 F NMR analysis, confirming the aromatic C5-F substitution. Compared with reported NMR spectra of thaxtomin A (1), this new analog lost a proton signal at its C5 and possessed an increased chemical shift of its C5 (δ=151.7 ppm vs 119.2 ppm in thaxtomin A) caused by the deshielding of C5-F substitution (Table 4). These results illustrated the remarkable plasticity of thaxtomin biosynthetic system and the use of  S. albus  as host to produce new thaxtomin analogs. In addition, fluorine substitution is an effective strategy to improve other properties (e.g., metabolism) of bioactive compounds. 
     Bioactivities of Natural and Unnatural Thaxtomin Analogs 
     Thaxtomins have potent herbicidal activity, and their analogs can possess additional bioactivities including antifungal and antiviral activities. So, the herbicidal activity of 5-F-thaxtomin A (9) was measured in a radish seedling assay ( FIG.  19   ). Its I 50  at 0.56±0.03 μM was statistically insignificantly weaker than thaxtomin A (1) (I 50 =0.45±0.05 μM, P value=0.04) but significantly stronger than ortho-thaxtomin A (2) (I 50 =0.81±0.04 μM, P value=0.0005). The cytotoxicity of compound 9 was further examined along with compounds 1-3, and weak growth inhibition toward human T-cell leukemia Jurkat and prostate cancer PC-3 cell lines was observed in a dose-dependent manner ( FIGS.  20 A- 20 B ). IC 30  values (i.e., 30% inhibitory concentrations) of compounds 1, 2, 3, and 9 toward Jurkat cells were 70.84±16.68 μM, 56.46±12.04 μM, 50.92±13.17 μM and 63.26±9.29 μM, respectively ( FIG.  20 A ). For PC-3 cells, the corresponding values were 80.28±10.50 μM, 42.38±13.62 μM, 37.87±13.46 μM and 56.76±17.12 μM ( FIG.  20 B ). The IC 30  values of 2 and 3 were significantly different to IC 30  value of 1, with P values=0.019 and 0.013, respectively, while IC 30  value difference of 9 with 1 was statistically insignificant (P value=0.112). 
     In conclusion, despite the promising agricultural applications of thaxtomins, there has, thus far, been a lack of cost-effective production approaches. The present examples describes the heterologous production of thaxtomins in  S. albus . Through medium optimization, the expression of the chromosomally integrated biosynthetic gene cluster resulted in the highest yield of thaxtomin A ever reported, at about 170 mg/L. This significant achievement can lay a solid basis toward the wide agricultural use of this novel class of bioherbicide. Furthermore, the thaxtomin biosynthetic gene cluster was engineered to produce two valuable biosynthetic intermediates at high yields, both of which can act as precursors and building blocks for the development of advanced synthetic strategies of chemicals. This work also demonstrated the versatility and feasibility of the thaxtomin biosynthetic system by the production of 5-F-thaxtomin A (9), which showed potent herbicidal activity similar to thaxtomin A (1) and ortho-thaxtomin A (2). Overall, this disclosure, as evidenced by this Example, presents an attractive synthetic biology strategy to address the insufficient supply of herbicidal thaxtomins and analogs, which can find general applications in translating genomes into chemicals. 
     It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, and are merely set forth for a clear understanding of the principles of this disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Tables and Sequences from Examples 
               
               
                 Production of thaxtomins and nitrotryptophan derivatives by  S .  albus   
               
               
                 J1074 transformed with a minimal gene cluster in different media. 
               
            
           
           
               
               
               
               
            
               
                   
                 Thaxtomin A 
                 Other 
                 4-Nitrotryptophans 
               
               
                 Media 
                 (mg/L) 
                 thaxtomins (mg/L) 
                 (mg/L) 
               
               
                   
               
               
                 TDMc 
                 91.2 ± 6.8 
                 33.6 ± 2.4 
                 58.2 ± 1.4 
               
               
                 ISP4c 
                 151.2 ± 12.2 
                 48.4 ± 3.6 
                 50.4 ± 2.1 
               
               
                 R5c 
                 168.2 ± 13.2 
                 54.2 ± 3.8 
                 56.6 ± 2.3 
               
               
                 PDBSc 
                 157.6 ± 8.8  
                 48.0 ± 2.2 
                 80.2 ± 3.2 
               
               
                   
               
               
                 mean ± standard deviation, n = 3 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Bacterial strains, plasmids, and cosmids used in this study 
               
            
           
           
               
               
               
            
               
                   
                   
                 Source or 
               
               
                 Strain or plasmid 
                 Description †   
                 reference 
               
               
                   
               
               
                   E .  coli  strains 
                   
                   
               
               
                 DH5α 
                 General cloning host 
                 Gibco-BRL 
               
               
                 BW25113 
                 Host for the REDIRECT © PCR targeting system 
                 (Gust et al. 
               
               
                   
                   
                 2003) 
               
               
                 ET12567 
                 dam − , dcm − , hsdS − ; non-methylating host for 
                 (MacNeil et al. 
               
               
                   
                 transfer of DNA into  Streptomyces  spp. (cml R , tet R ) 
                 1992) 
               
               
                 S 17-1 
                 General cloning and conjugation donor strain (tmp R ) 
                 (Simon et al. 
               
               
                   
                   
                 1983) 
               
               
                 Plasmids or 
                   
                   
               
               
                 cosmids 
                   
                   
               
               
                 pIJ790 
                 λ Red plasmid (t S , cml R ) 
                 (Gust et al. 
               
               
                   
                   
                 2003) 
               
               
                 pUZ8002 
                 Supplies transfer functions for mobilization of 
                 (Kieser et al. 
               
               
                   
                 oriT-containing vectors from  E .  coli  to 
                 2000) 
               
               
                   
                   Streptomyces  (kan R ) 
                   
               
               
                 pIJ773 
                 Template for the REDIRECT © PCR targeting 
                 (Gust et al. 
               
               
                   
                 system, contains the [aac(3)IV + oriT] disruption 
                 2003) 
               
               
                   
                 cassette (amp R , apr R ) 
                   
               
               
                 Cosmid 1989 
                 SuperCos1 derivative containing the  S .  scabiei   
                 This study 
               
               
                   
                 87-22 txtH locus (kan R , amp R ) 
                   
               
               
                 Cosmid 2757 
                 SuperCos1 derivative containing the  S .  scabiei   
                 This study 
               
               
                   
                 87-22 lanAB locus (kan R , amp R ) 
                   
               
               
                 pTARa 
                 Bacterial artificial chromosome (BAC) plasmid 
                 (Dimitris 
               
               
                   
                 (chl R , amp R ) 
                 Kallifidas 2012) 
               
               
                   
               
               
                   † apr R , apramycin resistance; cml R , chloramphenicol resistance; tet R , tetracylcin resistance; t S , temperature sensitive; kan R , kanamycin resistance; amp R , ampicillin resistance; thio R , thiostrepton resistance; chl R , chloramphenicol resistance; tmp R , Trimethoprim resistance. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 The yields of thaxtomins (1-5) and nitrotryptophan derivatives (7, 8)  
               
               
                 by  S .  albus -thx2 in different media and by  S .  scabiei  87.22 in TMDc a   
               
            
           
           
               
               
               
               
            
               
                 Media 
                 1 
                 2-5 
                 7, 8 
               
               
                   
               
               
                 TDMc b   
                 9.1 ± 0.4 
                 1.5 ± 0.1 
                 2.1 ± 0.1 
               
               
                 TDMc 
                 91.2 ± 6.8  
                 33.6 ± 2.4  
                 58.2 ± 1.4  
               
               
                 PDBS 
                 0.50 ± 0.03 
                 0.40 ± 0.02 
                 Trace 
               
               
                 ISP4c 
                 151.2 ± 12.2  
                 48.4 ± 3.6  
                 50.4 ± 2.1 
               
               
                 R5c 
                 168.2 ± 13.2  
                 54.2 ± 3.8  
                 56.6 ± 2.3 
               
               
                 PDBSc 
                 157.6 ± 8.8  
                 48.0 ± 2.2  
                 80.2 ± 3.2 
               
               
                 PDBSc c   
                 142.1 ± 5.4  
                 44.1 ± 1.3  
                 76.2 ± 2.1 
               
               
                   
               
               
                   a data represented mean ± s. d. (n = 3). The concentration unit was mg/L; 
               
               
                   b   S .  scabiei  87.22 was cultured in TDMc; 
               
               
                   c home-made PDBS with 1% cellobiose. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                   1 H and  13 C NMR data comparison of compounds 1 and 9 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Posi- 
                 Thaxtomin A a   
                 9 b   
               
            
           
           
               
               
               
               
               
            
               
                 tion 
                 δ C , type 
                 δ H  (J in Hz) 
                 δ C , type 
                 δ H  (J in Hz) 
               
               
                   
               
               
                  2 
                 132.5, CH 
                 6.95, s 
                 132.6, CH 
                 , 6.97, s 
               
               
                  3 
                 110.5, C 
                   
                 110.3, C 
                   
               
               
                  4 
                 143.6, C 
                   
                 149.7, C 
                   
               
               
                  5 
                 119.2, CH 
                 7.84 (7.9, 1.0, dd) 
                 151.7, C 
                   
               
               
                  6 
                 121.0, CH 
                 7.19 (8.0, t) 
                 110.4, CH 
                 7.05, (12.0, t) 
               
               
                  7 
                 118.6, CH 
                 7.68 (8.1, 1.0, dd) 
                 117.3, CH 
                 7.56 (12.0, 4.0, dd) 
               
               
                  8 
                 119.8, C 
                   
                 119.3, C 
                   
               
               
                  9 
                 141.1, C 
                   
                 136.3, C 
                   
               
               
                 10 
                  33.5, CH 2   
                 1.62 (14.2, 8.9, dd); 
                  31.8, CH 2   
                 1.40 (16.0, 8.0, dd); 
               
               
                   
                   
                 2.60 (14.1, 6.2, 0.5,  
                   
                 2.21 (12.0, 4.0, dd) 
               
               
                   
                   
                 ddd) 
                   
                   
               
               
                 11 
                  64.6, CH 
                 3.86 (8.9, 6.3, dd) 
                  64.0, CH 
                 3.69 (8.0, 4.0, dd) 
               
               
                 13 
                 168.3, C 
                   
                 168.4, C 
                   
               
               
                 14 
                  88.0, C 
                   
                  88.0, C 
                   
               
               
                 16 
                 166.8, C 
                   
                 166.4, C 
                   
               
               
                 17 
                  45.4, CH 2   
                 3.11 (13.4, d);  
                  43.6, CH 2   
                 3.11 (12.0, d);  
               
               
                   
                   
                 3.32 (13.5, d) 
                   
                 3.29 (12.0, d) 
               
               
                 18 
                 137.4, C 
                   
                 137.4, C 
                   
               
               
                 19 
                 118.4, CH 
                 6.71, m 
                 118.6, CH 
                 6.67, m 
               
               
                 20 
                 159.1, C 
                   
                 159.2, C 
                   
               
               
                 21 
                 115.8, CH 
                 6.71, m 
                 115.9, CH 
                 6.74, m 
               
               
                 22 
                 131.2, CH 
                 7.23 (8.1, t) 
                 131.3, CH 
                 7.21 (8.0, t) 
               
               
                 23 
                 122.7, CH 
                 6.71, m 
                 122.7, CH 
                 6.67, m 
               
               
                 N-12 
                  28.5, CH 3   
                 3.03, s 
                  28.5, CH 3   
                 3.03, s 
               
               
                 N-15 
                  34.2, CH 3   
                 2.81, s 
                  34.2, CH 3   
                 2.77, s 
               
               
                   
               
               
                   a NMR data were reported in King, RR., et al., 1992; 
               
               
                   b NMR spectra were recorded in CDOD 3 . 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 The yields of thaxtomin biosynthetic intermediates 5, 7,  
               
               
                 and 8 by two engineered strains in different media a   
               
            
           
           
               
               
               
               
            
               
                   
                   
                   S .  albus -thx2-ΔC 
                   S .  albus -thx2-ΔABCH 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Media 
                 5 
                 7, 8 
                 7 
               
               
                   
                   
               
               
                   
                 TDMc 
                 36.1 ± 0.3 
                 54.9 ± 1.5 
                 48.2 ± 2.2 
               
               
                   
                 ISP4c 
                 75.9 ± 1.1  
                 65.8 ± 2.1 
                 56.2 ± 3.5 
               
               
                   
                 R5c 
                 63.9 ± 1.2 
                 64.8 ± 1.3 
                 17.5 ± 0.8 
               
               
                   
                 PDBSc 
                 27.2 ± 0.4 
                 11.3 ± 0.3 
                 11.5 ± 0.4 
               
               
                   
                   
               
               
                   
                   a data represented mean ± s. d. (n = 3). The concentration unit was mg/L. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Microbial strains, plasmids, and cosmid used in this study 
               
            
           
           
               
               
               
            
               
                 Strains or plasmids  
                 Features †   
                 Source 
               
               
                   
               
               
                   E .  coli  strains 
                   
                   
               
               
                 EPI300 
                 General cloning host 
                 Epicentre 
               
               
                 S17-1 
                 General cloning and conjugation donor strain (tmp R )  
                 59 
               
               
                   Streptomyces  strains 
                   
                   
               
               
                   S .  albus  J1074 
                 Isoleucine and valine auxotrophic derivative of  S . 
                 60 
               
               
                   
                   albus  G lacking Sa/l-restriction activity 
                   
               
               
                   S .  albus -thx1 
                   S .  albus  J1074 transformed with pTARa-thx 
                 This study 
               
               
                   S .  albus -thx2 
                   S .  albus  J1074 transformed with pLST9828-thx 
                 This study 
               
               
                   S .  albus -thx2-ΔC 
                 TxtC gene was deleted from the thaxtomin gene 
                 This study 
               
               
                   
                 cluster 
                   
               
               
                   S .  albus -thx2- 
                 TxtA-C and H genes were deleted from the 
                 This study 
               
               
                 ΔABCH 
                 thaxtomin gene cluster 
                   
               
               
                   S .  scabiei  87.22 
                 Native thaxtomin producer 
                 18 
               
               
                 Yeast strain 
                   
                   
               
               
                 
                   Saccharomyces 
                 
                 Uracil-deficient (MATa, ura3Δ, cyh2 R ) 
                 43 
               
               
                   cerevisiae  CRY1-2 
                   
                   
               
               
                 Plasmids 
                   
                   
               
               
                 Cosmid 1989 
                 SuperCos1 derivative containing the thaxtomin 
                 This study 
               
               
                   
                 gene cluster of  S .  scabiei  87-22 (kan R , amp R ) 
                   
               
               
                 pTARa 
                 Bacterial artificial chromosome vector (chl R , amp R )  
                 42 
               
               
                 pTARa-thx 
                 pTARa was cloned with the thaxtomin gene cluster  
                 This study 
               
               
                 pLST9828 
                 E.coli-Streptomyces integrative shuttle vector (apr R ) 
                 52 
               
               
                 pLST9828-thx 
                 pLST9828 was cloned with the thaxtomin gene 
                 This study 
               
               
                   
                 cluster 
                   
               
               
                 pLST9828-thx-ΔC 
                 TxtC gene was deleted from the thaxtomin cluster 
                 This study 
               
               
                   
                 in pLST9828 
                   
               
               
                 pLST9828-thx- 
                 TxtA-C and H genes were deleted from the 
                 This study 
               
               
                 ΔABCH 
                 thaxtomin cluster in pLST9828 
               
               
                   
               
               
                   † apr R , apramycin resistance; cml R , chloramphenicol resistance; kan R , kanamycin resistance; amp R , ampicillin resistance; chl R , chloramphenicol resistance; tmp R , Trimethoprim resistance; cyh2 R , cycloheximide resistance. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                   
               
               
                 LISTING OF SEQUENCES: 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 txtA (scab31791) (SEQ ID NO: 1) 
               
               
                 GTGTCGCACCTGACCGGTGAAGATCTCCCGGAGGGAGCGCTCGCCACGACGTGGCCGAGTCTCCTCGAAGCGCGGGTGGCC 
               
               
                 GACACACCTGACGCCATCGCGCTCGTCGCCGGGGACACGGCGCTCACGTACGCGCAGTTCAATGCCCGTGCGAACCGGCTC 
               
               
                 GCCCGGTGGCTGAAGTACCTCGGCGCCGGGCCGGAGCGGTCGGTCGGGCTGGTGCTGGGCAGGTCCGCGGACTTCTTCCTG 
               
               
                 TGCGCGACGGCCGTGCTCAAGTGCGGGGCCGCGTACCTGCCGCTGGATCCGAACTACCCCGTGGAGCGACTGTCCTTCATG 
               
               
                 GCCCGGGACGCAGCACCCGTGGTGCTGGTGACGACGTCGGACGTCCGGGGCGACCTTCTGGGCCAGCTGCCCACCGGCAGC 
               
               
                 CTCGTGGTACTGGACGACGAGGCCACCGAGGACGTACTGCGCCGTCTGCCGGACCACGACATGGAGGACGGGGAACGTTTG 
               
               
                 GAGCCACTGCGCCCCGCGAGTCCCGCCTACATCATCTACACCTCCGGCTCCACGGGGATCCCCAAGGGAGTCGTCGTCACC 
               
               
                 CACCAAGGCGTCGCGAGCCTGATCGCGACCCAGCGTCGTCGCCTCGCCGTCACCGGCGCCTCACGCGTGCTCGCCTTCTCG 
               
               
                 TCCCCGAGTTTCGACGCCAGTTTCTGGGAGATGTCGATGGCGCTGCTGGCCGGGGCCGCGCTCGTGGTCGGCAGGCCGGGG 
               
               
                 CGGCTGCTGCCCGACGCCGAACTGGCCGCGTTGATCGCGGACCACGGAGTCACTCATGTCACTCTCCCGCCCTCGGTCGCG 
               
               
                 GGTGCGCTGGGCCCCGACATGCTGCCTCCGAGCGTGACGCTGGTCGTCGCGGGCGAAGCGTGCCCGGCGGCTCTCGTGCAG 
               
               
                 CGCTGGCGCCCGCACCGGACGATGGTGAACGCCTACGGCCCGACGGAGTCCACCGTCTGCGCCACCATGAGCGATCCGCTG 
               
               
                 GCCGACGACGTGGCGCCGCCGGTCGGCCGGGCGGTGGACGGCACCCGGATCCATGTCCTCGACGACCGCCTCGCACCGGTT 
               
               
                 GTGCCGGGAGCGGTCGGCGAGATCTACATCGCGGGGCACAGCCTGGCACGCGGGTACCTCGAGCGGCCGGGTCTGACCGCG 
               
               
                 CAGCGGTTCGTGGCGGACCCCTTCGGCCCGGCCGGCAGCCGTATGTACCGCAGCGGTGACCTCGGCCGCTGGACCCGTTCA 
               
               
                 GGAGACCTGGAGTTCGTCGGCAGGGCGGACGACCAGGTCAAGGTACGCGGCTTCCGTATCGAGCCGGGCGAGATCGAATCC 
               
               
                 GTCATCGCCGGGTGCCGCGGGGTCCGGCAGGCCGCCGTCGTCCTGCGTGAGGACCGGCCCGGAGAGCCATACCTCGCCGCC 
               
               
                 TACGTCATACCCGAGAACGCGGCCGCCGACGAGGCGGCCGGCGAGGAACCGGACGGTCAACTCGATGCCTGGCGACGGCTC 
               
               
                 TACGACGATCTGTACGGCCGAGCCGACACCGCCGACTTCGGCGAGGACTTCTCCGGCTGGGTGAGCAGTTATGGCGGGCGG 
               
               
                 CCGATCGAGGGGATGCGCGAATGGCGTGAGCAGACCGTGCGGCAGATCCGCGAACTGGCTCCGCGCCGCGTACTGGAGATC 
               
               
                 GGCTGCGGTTCCGGTCTGCTGCTCTCGCAGCTGGCGGGTGACTGCGAAAGCTACTGGGGCACCGACATCTCCGGGGCCCTG 
               
               
                 ATCGAGCGGCTGCGCGGGCAGGTCGCCGAGCGCCCCGGCCTCGCGGACCGGGTCGTCCTGCATCAGCTCTCCGCCCATGAG 
               
               
                 CTGGGGAGTCTGCCCAGCGGCGGCTTCGACACCGTCGTGCTCAACTCCGTGATCCAGTACTTTCCCTCAGGCGATTACCTG 
               
               
                 TTCGACCTACTGCGCGAGGTGTCCCGGCTCCTGGTACCCGGGGGCGCGGTGTTCCTCGGCGACGTCCGTAACCTTCGTCTG 
               
               
                 CTGCGCACCTTCCACGCCGGGGGGCTGCTGGCGGCGGCCACGCACACCGACACTCCGCAGACGGTCTGCGCGGCGATCGAC 
               
               
                 CGGGCCATGGCGCAGGAGAAGGAACTGCTCGTGGACCCGGAGTTCTTCACGACGGCCGTCGGCGCGCTGCCCGGCATGACG 
               
               
                 CTGGAGTCGTGCACGCTCAAACGGGGCGGGTACGACAACGAACTCAGCCGCTATCGCTACGAGGTGGTGCTGCGCAAGCAT 
               
               
                 GCCGGGCCTGCCGATGACACCGGGCCCACGGACGACGCGGGGCCGGTCGTGCGACTGCGGTGGGACGGCGAGATGGCGAGC 
               
               
                 CTGGCCGACGTCGCCGATCGGCTGCGTCGTGGGAAACCGGAGCGGTTGTGCGTCACCGGGATCCCCAACGGCCGGGTGGCC 
               
               
                 GGCGAGCATGCCGCGACACTCGCGCTGTTCGACCGGCGCCCCCTGCACGAGGTGCTGTCCCTGGGGCAGGCTCCGGCGGGC 
               
               
                 GTGGCACCGGAGGACCTGCGCCGGCTGGGCGCGGAACTGGGCTACCGGGTCGACTGCACCTGGTCGTCCGAGGACGACGCC 
               
               
                 CTGATCGACGCTTCCTTCACACGCGCCGGAGCGCTCGTGCCGCGTCCCGCCCCCCGGACCGACGCGGAGCCGGACGGTTTC 
               
               
                 TCCCCGGCCCGGTTCACCAACAGGCCGGCGTTCGCCCGCCCCGACTCCCAGACGATGGCCTCTCTTCCCGGGCAGGTCGCG 
               
               
                 GCGAAGCTGCCGGCCTTCATGGTCCCGGAGGTCTTCGTCCCGCTCGACAGGCTGCCGGTCACGGTGAACGGAAAGCTCGAC 
               
               
                 CGCGGCGCCCTGCCCCGGCCGCGGCGCGCCGCCCATGCCTCGGGACGTCCGCCCAGGACCGCCCGCGAGGAGGTACTGGCG 
               
               
                 GCGATCTTCGCCGACGTACTCGCGACAGCCGACGTCACAGCCGACAGCGACTTCTTCGCCGTCGGCGGCAACTCCCTGCTG 
               
               
                 GCCACCCGACTCGCCGCCGAGGTCCGGCGGCGCCTGAACACCGAGATGCCGCTGTCGTGGCTGTTCGAGTCGCCCACCGTC 
               
               
                 GGCGCGCTCGCCGCCCGCTTCGACGCGGGGGACGAGGCCAGGCCGCTGCCCGTGCCGAGCGAGTACGCCTCCGGCAGCACG 
               
               
                 GCGCCGTTGTCGGCCCAGCAGATGCAGATGTGGCACGAGTACCGCCGAAGCCTGTGTCGCGACATGTTCAACGTGCCGCTG 
               
               
                 TCGCAGCGGCTGACCGGTGCCGTCGACGCCGAGGCACTGCGCGCCGCCCTCGCCGATGTCGTCACCCGGCACGTTCCGCTG 
               
               
                 CGCACGCTCGTCCAGGACGACGGCAGCGGTCCGTGTGCGGTGATCACGGAAGCCACCGCGGACGACATCCCATGGACGGAG 
               
               
                 ACCAGGACCACGCCCGAGCGGCTGTCCGAGGATCTCGCGCACGCCGCCCGCCGCCACTTCGACCTCGAGACCGAGATCCCG 
               
               
                 CTGCGGGCCGTACTGTTCACGCTCGGCCCGGACGAGTCCGTACTGCTGCTGGTCATGCATCACATCGCCGCCGACGGCTGG 
               
               
                 TCCTTCGGCCCCCTGCTGGAGGACCTGGTCCGCGCCTACCGCGCCCGGACCGAGGGGCGCGCACCACAGTGGGAGCCGCTG 
               
               
                 TCCTTCGGCTACCTCGACTACGTCGCCTGGCAGCGCCGGCTGCTCGGCGCCACGGACGACCCGAGCGACGTCGCGCTGCGC 
               
               
                 CAGGCGGAGTACTGGAGGAAGACGCTGCACGGTGCCGACGACAGGCCGGTCCTGGAGACCGACAGCCCGGCGCCGGCCCAG 
               
               
                 CAGGACTTTGCCGGCAGGTCCCTCGATCTTCCGCTCGAAGTCGGCGGCCACCGGGTGCTGACAGCCGCGGCCCGTGAGCAC 
               
               
                 GGTGTCACCGTCTTCATGATCCTGCACGCCGCGCTCGTCGCACTGCTCGCCCGCAGGGGAGCAGGAGGGGACGTCACCGTC 
               
               
                 GTGACCGCGGTGGCCGGCCGGACCGACACCCAGTTCGAACCGCTGGTGGGCCTCTTCGCCAACACCTTGGCGCTGCGCACC 
               
               
                 GACACGTCGGGCAACCCCACCTTCCGCGAACTGCTGGACCGGGTCCGCGTGACCGATCTCGGTGCCTATGCCCACCAGGAC 
               
               
                 CTGCTCTTCGAGCGCCTGGCCGACGTGCCACCGCCCCAGGTGTCACTCGTCCTGCGCACGGTCGCAGCTCCGCCGGCCGAC 
               
               
                 CTGCCGGGCCTCACCATCAGTCCCGGCCCACGGCCGGCGAGCGAATCCGCCCGCTATCCGGTGCTGTGGACCGTGGAGCAT 
               
               
                 CTGGCCTCCGCCGCGGACGGCGGGACGCTGCGCAGCCACATCCAGTACCAGAGCGGGCTGCTGCGCGACGACACGGTCGTC 
               
               
                 CGGCTCGCCCAGCAGTACGAAGTCGTGCTGTCCCTGTTGTTGAAGGATCCCGATCTCCGCGTCCAGGACCTCCCACTGCAG 
               
               
                 TGA 
               
               
                   
               
               
                 TxtA (scab31791) (SEQ ID NO: 2) 
               
               
                 VSHLTGEDLPEGALATTWPSLLEARVADTPDAIALVAGDTALTYAQFNARANRLARWLKYLGAGPERSVGLVLGRSADFFL 
               
               
                 CATAVLKCGAAYLPLDPNYPVERLSFMARDAAPVVLVTTSDVRGDLLGQLPTGSLVVLDDEATEDVLRRLPDHDMEDGERL 
               
               
                 EPLRPASPAYIIYTSGSTGIPKGVVVTHQGVASLIATQRRRLAVTGASRVLAFSSPSFDASFWEMSMALLAGAALVVGRPG 
               
               
                 RLLPDAELAALIADHGVTHVTLPPSVAGALGPDMLPPSVTLVVAGEACPAALVQRWRPHRTMVNAYGPTESTVCATMSDPL 
               
               
                 ADDVAPPVGRAVDGTRIHVLDDRLAPVVPGAVGEIYIAGHSLARGYLERPGLTAQRFVADPFGPAGSRMYRSGDLGRWTRS 
               
               
                 GDLEFVGRADDQVKVRGFRIEPGEIESVIAGCRGVRQAAVVLREDRPGEPYLAAYVIPENAAADEAAGEEPDGQLDAWRRL 
               
               
                 YDDLYGRADTADFGEDFSGWVSSYGGRPIEGMREWREQTVRQIRELAPRRVLEIGCGSGLLLSQLAGDCESYWGTDISGAL 
               
               
                 IERLRGQVAERPGLADRVVLHQLSAHELGSLPSGGFDTVVLNSVIQYFPSGDYLFDLLREVSRLLVPGGAVFLGDVRNLRL 
               
               
                 LRTFHAGGLLAAATHTDTPQTVCAAIDRAMAQEKELLVDPEFFTTAVGALPGMTLESCTLKRGGYDNELSRYRYEVVLRKH 
               
               
                 AGPADDTGPTDDAGPVVRLRWDGEMASLADVADRLRRGKPERLCVTGIPNGRVAGEHAATLALFDRRPLHEVLSLGQAPAG 
               
               
                 VAPEDLRRLGAELGYRVDCTWSSEDDALIDASFTRAGALVPRPAPRTDAEPDGFSPARFTNRPAFARPDSQTMASLPGQVA 
               
               
                 AKLPAFMVPEVFVPLDRLPVTVNGKLDRGALPRPRRAAHASGRPPRTAREEVLAAIFADVLATADVTADSDFFAVGGNSLL 
               
               
                 ATRLAAEVRRRLNTEMPLSWLFESPTVGALAARFDAGDEARPLPVPSEYASGSTAPLSAQQMQMWHEYRRSLCRDMFNVPL 
               
               
                 SQRLTGAVDAEALRAALADVVTRHVPLRTLVQDDGSGPCAVITEATADDIPWTETRTTPERLSEDLAHAARRHFDLETEIP 
               
               
                 LRAVLFTLGPDESVLLLVMHHIAADGWSFGPLLEDLVRAYRARTEGRAPQWEPLSFGYLDYVAWQRRLLGATDDPSDVALR 
               
               
                 QAEYWRKTLHGADDRPVLETDSPAPAQQDFAGRSLDLPLEVGGHRVLTAAAREHGVTVFMILHAALVALLARRGAGGDVTV 
               
               
                 VTAVAGRTDTQFEPLVGLFANTLALRTDTSGNPTFRELLDRVRVTDLGAYAHQDLLFERLADVPPPQVSLVLRTVAAPPAD 
               
               
                 LPGLTISPGPRPASESARYPVLWTVEHLASAADGGTLRSHIQYQSGLLRDDTVVRLAQQYEVVLSLLLKDPDLRVQDLPLQ 
               
               
                   
               
               
                 txtB (scab31781) (SEQ ID NO: 3) 
               
               
                 ATGTCCATGCTGCCGCCGGGGCGAAGCCGCACCACGGCCTCGCCCGCCGGGGCCCAGGCCGGCCCCGAGTTCACCCCCGGC 
               
               
                 CTATGGGGACGGCTCTTCGAAGCCCGTGTCGACGCCGCCCCCGAATCCACCGCGATCAACTCCGCGAGCGAGCGGCTGAGT 
               
               
                 TACGCCGAACTGAACCGGCGGGCCAACCGCCTCGCCAGGTTGCTCATCGCACGTGGCGCCGGCCCGGAGAGCCTGGTCGGT 
               
               
                 CTCGCCCTGCCGCGCTCGACCGACTTCGTGGTGGCCGTGGCGGCCGTACTGAAATCGGGCGCCGGCTACTTCCCGATGGAT 
               
               
                 CCGGACTATCCTCCGCAGCGGCTGGCGTTCATGCTCGCCGACGCCGCTCCCATGCTGGTGCTGACCAGGAGTGACATCGAG 
               
               
                 CCCGAGCTGCCGGCCGAGGCGGCCTCCCGCACGGTGGTGCTGGACGACCCGGCCGTCGTACGGACCCTGGCCGACTGCTCC 
               
               
                 GCGGCGGATGTGGCGGACGACGAACGCGGCGCCCCGCTCCGGACCCGGCATCCGGCCTATGTCATCTACACCTCGGGTTCC 
               
               
                 ACCGGTACTCCCAAAGGAGTGGTCCTCACCCACCACGGCATCGCCAGCCTGGTGGGCAGCCATGCGCGGGACCTGGGGATC 
               
               
                 GGGCCGTCCAGCCGGTTGCTGCTCTTTTCCTCGCCGAGTTTCGACGGCGCCTTCTGGGACGTGTCGATGGCCTTGCTCACT 
               
               
                 GGCGCCACGCTGGTGGTCGCACCGCGTGAACGGCTCCTGCCCGGACCGGAGTTCAGTGCGCTCGCCGCCGAGGAGGGCATC 
               
               
                 ACCCACTTCACCCTGCCGGCCTCCACGCTCGCCGCCCTGCCGGACGGCGCTCTGCCCGCCGGGGCCACCGTCGTCAATGTG 
               
               
                 GGCGAGGCCTGCAACAGCGAGCTGGTGCGCCGCTGGTCGCCGGGCCGGCTGCTGGTGAACGCGTACGGACCGACCGAATCG 
               
               
                 ACCGTCTCCGCGACCATGAGCGGACCGTTGGCCGGGGCAGGCATCCCGCCCATCGGCCGTCCGCTCTCGGACACCCGCATC 
               
               
                 CACGTCCTCGACGAGCGGCTCCGGCCGGTACCGCCGGGAGCGGTCGGAGAGATCCACATCGCCGGAGCGGGGCTGGCCCGC 
               
               
                 GGGTACCTGGGGCGGCCCGCGCTGACCGCCGAGCGGTTCGTGGCCGACCCCTTCGGGACGCCGGGCGAGCGGATGTACCGG 
               
               
                 ACCGGCGACCGGGTGAGGGTGCGTGACGACGGGCAACTGGAGTTCGTGGGCCGGGTCGACGACCAGGCGAAGATACGGGGT 
               
               
                 TTCCGAGTGGAGCCCGGCGAGGTCGAAGCCGTGCTGCGCGACCATCCCGAGGTCGCGCAGGCCGCGGTGGTGGTCCGGGAG 
               
               
                 GACACTCCGGGAGACCAGAGGCTCGTCGCCTATGTCGTGCCGGACCACCCGGCCGTGCGGCAGGCCGACGACACCACCTCG 
               
               
                 GAGCACGTCGAGGAATGGCAACGGCTCTACGACGAGGTCTACAGCGCAGTGGGAGCGCTCCCCCTGGGGGAGGACTTCAGC 
               
               
                 GGCTGGAACAGCACCTACGACGGCGAGCCCATTCCCGTGCCTCAGATGCAAGCCTGGCGGGACGCCACGGTCGACAGCATC 
               
               
                 CGTGCCCTGCGACCGCGCCGGGTACTGGAGATCGGCGTGGGCACCGGGCTGCTGCTGTCCCGCCTCGCCGGTGACTGCGAG 
               
               
                 GCGTACTGGGCCACCGACTTCTCCGCCGAGGTGATCGAGACACTCGGCAAGAAGGTGGACGTCGACCCGGTGCTGCGGGAG 
               
               
                 AAGGTCCACCTGCTGCACGGTCCCGCACACGACCTCCCCGGCCTCCCCGAGGGGTACTTCGACACCGTTGTCCTCAACTCG 
               
               
                 GTGATCCAGTACTTTCCGTCGGCCGACTACCTGGTGAGCGTCCTGCGCGAGGCGGCACGCCTGCTGGCGCCGGGCGGCCGG 
               
               
                 GTGTTCGTCGGCGACATCCGGCACCTGCGTCTGCTGCGCCCGCTGCGCAGCGCCGTCCGGCTGCGCTCCGCCACCCGGCGG 
               
               
                 GAAGCCTCTGCCTCCGCCGTCCGCGCGGCGGTCGAGCAGGACCTGGTGGATGAGAAGGAGTTGCTCCTCGACCCCGCGTTC 
               
               
                 TTCGCCGCGGTACCCCGGTGGATCCCGCAGCTCCGCGGGGTACGCACAGCGGTGCAGCGCGGCACGCACCACAACGAACTG 
               
               
                 ACCCGCTACCGGTACGACGCGGTGCTCATCAAGGAGCCGGTGGAAACCGGTACCGCTGCGCCGGACGCGCAGACACTGACC 
               
               
                 TGGGGCACGGATGTCAGCGGACTGCAGGAGTTGTCCGGCCTGCTGGCCCGCACCCGCACGTCGCTGCTGCTGCGCGGCGTG 
               
               
                 CCGAACAGCCGGATCCTCGGTGAGGCATCGGCCGCGACGGCGCTGACCACGGCCCGGTCGCTCGACGAGCCGTTGCGGTTG 
               
               
                 CTGCAAGAACCGGCGGCAGGGATCGACCCCGAGGAACTGCACGCCCTGGGCGGGGGCGCCGGCTGCGAGGTCCACCTCACG 
               
               
                 TGGTCGGCGCAGGACCCCACGCGACTGGACGCCTGTTTCACACCCGTGGGCGGTGAACCGGGCGCCGTCCCGCTGGCGGAG 
               
               
                 TCCGCCGACAGCGGCAGGACGTCGCCCGGTGACCACGCCAACCAGCCGACCACGCACCGGACCGGCAACGCCCTGATGGGC 
               
               
                 AAGCTCCCCGGCTATCTGGCCGCCAGGCTCCCCGCGTACCTGCGGCCCAGCGCCGTGGTACGCATCGCGTCGCTCCCCCTC 
               
               
                 ACCGTCAACGGCAAGCTCGACCGCACGGCGCTGCCCCGTCCCGCCCTGTTCCCGCGGGCTGACGGGCAGGCGCCCCGCACT 
               
               
                 CCGCGCGAGGAGATCCTCGCCAATCTCTTCGCCGATGTGCTCGGCCTGCCCGGGGTGCCGAGGGACGCCGACTTCTTCGCC 
               
               
                 CTGGGCGGCAACTCGCTACTGGCCACGCGCCTCGTCGGCCGTATCGCGAAACACCTCGAAGTCGATGTTCCGATCGCCTGG 
               
               
                 ATCTTCGAGACACCGACCGTCGAGGGCCTGGCCGGGCGTACCGCTCCGGCGAGCAGGCTCCGCCCGCTGTTGCTCTGCCGC 
               
               
                 GACGAGAACCACGCGGCGGTGCCGCTCTCGCACAGCCAGTACGGCATGTGGTTCATCAACCAACTCGGCGGACCCGCGAGC 
               
               
                 CGGATCTACAACGTGCCGTACTGCCTGCGGATCACGGGCCGGGTGGACACCGGGGCGCTGCGGACCGCACTCGATGACGTC 
               
               
                 GTGGCTCGTCACGAACCCCTGCGTACCGTCTTCCCCGATGACGGTGACGGCCCCCGCCAACGGGTCCTCGCCCCCGAGGAC 
               
               
                 GCCGCGGTGGTCCTTCATGAGACCGACGCCGCCGAAGACCGTCTGGCCGGCCACCTGGCGCGGGCCGCGGCGGAACCCTTC 
               
               
                 GAGCTCAGGACGGACCTTCCCCTGCGCGCACGCCTGTTCCGGCACGGACAGGACCGGTACACGCTCCTGCTCCTGATGCAC 
               
               
                 CACATCACCGTGGACGCCTGGTCGCTGGCCCCCCTGACGGCGGACCTGGCGCACGCCTACCGGGCGCGGCTGGGGCAGCGG 
               
               
                 GCCCCGCAGTGGCAGCCGCTGCCGGTTCACTATCGCGACTACGCCGTATGGCACAACGAGCAAGCAGCCGAGGCGCAGGAC 
               
               
                 CGCGGCAGCGGCTTCGGGCGCCAGCTCGCCTTCTGGGAGCGGACGCTGCGCGGTCTTCCGGTCGAGACGCGGCTGCCGGCC 
               
               
                 GACCGGAGCCGTCCGGCCAGACCTACCTATCGTGGCGGCACCGTCCACACCCACGTCGAAGCCTCCCTCCATCAGGAACTG 
               
               
                 CTCAACTGCGCGCGGGAGACGGGCGCGACGCTCTTCATGGTGCTGCACGCCGCGCTCGCGGCACTGCTGACCCGGCTGGGC 
               
               
                 GGCGGCACCGACATCGTCGTCGGCACCGCTGCCGCGGCGCGCACGGACCCCGCGCTGGACGACCTCGTCGGGCTGTTCGCC 
               
               
                 AACAGTGTCGTCCTTCGCGTCGACACCTCGGGCGACCCGACGTTCCGCACCCTGCTCGCTCGGACCCGGGCCGTGGACCTC 
               
               
                 GACGCCTTCACCCACCAGGAGGTCCCGTTCGACCAGGTGGTGGATCGCGTCAACCCGGCACGCCACCCGGCACGTCACCCG 
               
               
                 CTCTACCAGACGGCCCTCGTCCTGCACGCACCGCCCGGCGACGGCCATCGGGCCGACTCCGTCACCCTCACCCCCGAACCG 
               
               
                 CCCCCGAACACCGGAACGGCCCGCTTCGATCTGATGTTCAACTGGGACGAGAGCCGGGACAGCGCCGGCCTCGCCCAGGGC 
               
               
                 CTCACCGGCCGTACCGAGTACAGCTCGGACCTCTTCTCCCAGGAGACAGTCGAACTGCTCCTGGAGCGGTATCTCCTGCTG 
               
               
                 CTGTCCGCCGCGGTCCGCGACCCGGACGCACGCCTTCACACCCTGGACATCCTCACCGAGCCGGAACGGCGGGCCTTCTCA 
               
               
                 CCACGGCCGTAG 
               
               
                   
               
               
                 TxtB (scab31781) (SEQ ID NO: 4) 
               
               
                 MSMLPPGRSRTTASPAGAQAGPEFTPGLWGRLFEARVDAAPESTAINSASERLSYAELNRRANRLARLLIARGAGPESLVG 
               
               
                 LALPRSTDFVVAVAAVLKSGAGYFPMDPDYPPQRLAFMLADAAPMLVLTRSDIEPELPAEAASRTVVLDDPAVVRTLADCS 
               
               
                 AADVADDERGAPLRTRHPAYVIYTSGSTGTPKGVVLTHHGIASLVGSHARDLGIGPSSRLLLFSSPSFDGAFWDVSMALLT 
               
               
                 GATLVVAPRERLLPGPEFSALAAEEGITHFTLPASTLAALPDGALPAGATVVNVGEACNSELVRRWSPGRLLVNAYGPTES 
               
               
                 TVSATMSGPLAGAGIPPIGRPLSDTRIHVLDERLRPVPPGAVGEIHIAGAGLARGYLGRPALTAERFVADPFGTPGERMYR 
               
               
                 TGDRVRVRDDGQLEFVGRVDDQAKIRGFRVEPGEVEAVLRDHPEVAQAAVVVREDTPGDQRLVAYVVPDHPAVRQADDTTS 
               
               
                 EHVEEWQRLYDEVYSAVGALPLGEDFSGWNSTYDGEPIPVPQMQAWRDATVDSIRALRPRRVLEIGVGTGLLLSRLAGDCE 
               
               
                 AYWATDFSAEVIETLGKKVDVDPVLREKVHLLHGPAHDLPGLPEGYFDTVVLNSVIQYFPSADYLVSVLREAARLLAPGGR 
               
               
                 VFVGDIRHLRLLRPLRSAVRLRSATRREASASAVRAAVEQDLVDEKELLLDPAFFAAVPRWIPQLRGVRTAVQRGTHHNEL 
               
               
                 TRYRYDAVLIKEPVETGTAAPDAQTLTWGTDVSGLQELSGLLARTRTSLLLRGVPNSRILGEASAATALTTARSLDEPLRL 
               
               
                 LQEPAAGIDPEELHALGGGAGCEVHLTWSAQDPTRLDACFTPVGGEPGAVPLAESADSGRTSPGDHANQPTTHRTGNALMG 
               
               
                 KLPGYLAARLPAYLRPSAVVRIASLPLTVNGKLDRTALPRPALFPRADGQAPRTPREEILANLFADVLGLPGVPRDADFFA 
               
               
                 LGGNSLLATRLVGRIAKHLEVDVPIAWIFETPTVEGLAGRTAPASRLRPLLLCRDENHAAVPLSHSQYGMWFINQLGGPAS 
               
               
                 RIYNVPYCLRITGRVDTGALRTALDDVVARHEPLRTVFPDDGDGPRQRVLAPEDAAVVLHETDAAEDRLAGHLARAAAEPF 
               
               
                 ELRTDLPLRARLFRHGQDRYTLLLLMHHITVDAWSLAPLTADLAHAYRARLGQRAPQWQPLPVHYRDYAVWHNEQAAEAQD 
               
               
                 RGSGFGRQLAFWERTLRGLPVETRLPADRSRPARPTYRGGTVHTHVEASLHQELLNCARETGATLFMVLHAALAALLTRLG 
               
               
                 GGTDIVVGTAAAARTDPALDDLVGLFANSVVLRVDTSGDPTFRTLLARTRAVDLDAFTHQEVPFDQVVDRVNPARHPARHP 
               
               
                 LYQTALVLHAPPGDGHRADSVTLTPEPPPNTGTARFDLMFNWDESRDSAGLAQGLTGRTEYSSDLFSQETVELLLERYLLL 
               
               
                 LSAAVRDPDARLHTLDILTEPERRAFSPRP 
               
               
                   
               
               
                 txtC (scab31761) (SEQ ID NO: 5) 
               
               
                 ATGGAATCTCCGGCCACCCAGGTCGACCCGGCGAACTCGCCGTTGGAGCCCTATCACATCTACCCGGAGGCCAAGTCCTGC 
               
               
                 CCGGTGGCGAAGGTCGGTCTGTGGAACGGCACGCCGGCGCACGTGTTCTCCGGGTATGAGGATGTGCGGACCGTGCTGCAG 
               
               
                 GACAGGCGGTTCAGCTCGGACTCGCGCCGACCCAACTTCACCGAACTCACTCCGACGCTCCAGTCGCAGGCCGCGGCACCG 
               
               
                 CCGTTCGTACGCACCGACAATCCTGATCACCGGCGCCTGCGAGGCACCATCGCACGCGAGTTCCTGCCCAAGCACATCGAG 
               
               
                 CTGCTGCGCCCCGCGATCCGCGAGATCGTCCAGGGTGTGCTCGACGGGCTCGCCGAGACCGCGCCTCCCCAGGACATGCTC 
               
               
                 GAGGCCTTCGCCGTACCGGTCGCGTCCGCGACCGTCTTCCGGCTGCTGGGGATTCCGGCCGAGGACCGCGCGTTGCTCACC 
               
               
                 CGATGCGTCAAGGGCGTGGTCTCGGCGGTGGGGAGCGAGGACGAAGGTGCCGAGGTGTTCCGGACACTCGGCGAGTACATC 
               
               
                 GGCGGGCTCGTCCAGGACCCCTCCGAACTGCCCGAGGACAGCCTGATCCGGCGCCTGGTGACGGGCCCGTACCAGGAGAAG 
               
               
                 CAGCTCACCTTCCACGAGACCATCGGCGTGATCCTCATGCTCATCGTCGGGGGCTACGACACGACGGCCAGCACCATCTCG 
               
               
                 CTGTCCTTGGTGAGTTATGCACTGCAGCCGGAGAAGTTCTCCGTCGTCCACGAACACCCGGAGCGGATACCCCTGCTCGTC 
               
               
                 GAGGAGTTGCTGCGCTATCACACCGTCTCGCAGCTCGGACTGGGCAGGATCGCCACCGAGGACGTCGAGGTGGGCGGCGTC 
               
               
                 ACGGTGCGGGCCGGCCAGATGGTGGTGGCGGCGCTCCCCCTGGCCAACCGGGACGAGAGTGTCTTCCCGAACCCGGACGAA 
               
               
                 CTCGACTTCGACCGCCCGTCCGTGCCCCATGTCGGCTTCGGTTACGGACCCCACCAGTGCGTCGGCCAGGCACTGGCCCGA 
               
               
                 GTCGAACTCCAGGAGGCCATTCCCGCGGTGATCCGACGGCTGCCCGGCATGCGGCTCGCCTGCGCTCTGGAAGACCTGCCG 
               
               
                 TTCCGGCACGACATGGCCACCTACGGCATCCATGAGCTGCCCATGACCTGGTGA 
               
               
                   
               
               
                 TxtC (scab31761) (SEQ ID NO: 6) 
               
               
                 MESPATQVDPANSPLEPYHIYPEAKSCPVAKVGLWNGTPAHVFSGYEDVRTVLQDRRFSSDSRRPNFTELTPTLQSQAAAP 
               
               
                 PFVRTDNPDHRRLRGTIAREFLPKHIELLRPAIREIVQGVLDGLAETAPPQDMLEAFAVPVASATVFRLLGIPAEDRALLT 
               
               
                 RCVKGVVSAVGSEDEGAEVFRTLGEYIGGLVQDPSELPEDSLIRRLVTGPYQEKQLTFHETIGVILMLIVGGYDTTASTIS 
               
               
                 LSLVSYALQPEKFSVVHEHPERIPLLVEELLRYHTVSQLGLGRIATEDVEVGGVTVRAGQMVVAALPLANRDESVFPNPDE 
               
               
                 LDFDRPSVPHVGFGYGPHQCVGQALARVELQEAIPAVIRRLPGMRLACALEDLPFRHDMATYGIHELPMTW 
               
               
                   
               
               
                 txtD (scab31841) (SEQ ID NO: 7) 
               
               
                 TTGCCCGCCCCGTCCCCGACAGCGTGCCCGGCACTGGGGCCCGATTCGTCCCTTGGCCCGGTCCCGTCGGCGGAACCGGCG 
               
               
                 ACGCCGCAGTCCTGCGGCGTCGCCGATCCAAATGAGGCTGAGGAGTTCCTGCGCCAGTTCCACGCGGAGCAGTCCGATCAG 
               
               
                 CCCGTCCCGCTCGCCCGGCGCCTGGAGCAGGTCCGCGCCGCCATCGACGCCACGGGCACCTACCGGCACACCACCGCCGAG 
               
               
                 CTCGTGTACGGTGCCCGCGTCGCGTGGCGCAACTCCAGTCGCTGCATCGGCCGCCTGTACTGGAACAGCCTGCGCGTCCTG 
               
               
                 GACCGCCGGGACGCCACAGCCCCCGATGAGATCCACCGGCACTTGTGCACGCACCTGCGCCAGGCGACCAACGGCGGGCGC 
               
               
                 ATCAGGCCGGTGATTTCGGTCTTCGCCCCGGACTCCCCCGGCCGGCCCGGCCCGCAGGTGTGGAACGAGCAGCTCATCCGG 
               
               
                 TACGCCGGCTACCGCCGCGACGACGGCACCGTGCTCGGTGACCCGCGCACCGCCGACCTCACCGAGGCCATCCTCCGCCTC 
               
               
                 GGCTGGCAGGGCTGCCCCCAAGGGCCGTTCGACGTCCTGCCCCTGGTCATCGACACCCCCGACGACAAACCCCGGTTCTTC 
               
               
                 GAGCTGCCGCGGGAGCTGGTCTTGGAGGTCCCTATCACCCACCCCGACGTCCCACGCCTGGCCGAACTGGGCCTGCGCTGG 
               
               
                 CACGCCGTACCCGTCATCTCCAACATGCGCCTACGCATCGGCGGGATGGACTACCCGCTCGCCCCGTTCAACGGCTGGTAC 
               
               
                 ATGGGCACGGAGATCGGCGCCCGCAACCTCGTCGACGAGGACCGCTACAACATGCTCCCCGCCGTCGCCGCCTGCCTCCAG 
               
               
                 CTGGACACCACCAGCGAGTCAACCCTGTGGCGCGACCGCGCCCTGGTCGAGCTCAACGTCGCCGTCCTGCACTCCTTCGAG 
               
               
                 GCCGCAGGTGTCCGGATCAGCGACCACCACGAGGAGTCCCGGCGCTTCCTCGCCCACCTGGCCAAGGAGGAACGCCAGGGC 
               
               
                 CGCACCGTATCCGCAGACTGGAGCTGGATCGTCCCCCCGCTCTCCGGCGGCATCACCCCCGTGTTCCACCGTTACTACGAC 
               
               
                 AACGTCGACCAGCGCCCCAACTTCTACCCCCACCAGTGA 
               
               
                   
               
               
                 TxtD (scab31841) (SEQ ID NO: 8) 
               
               
                 LPAPSPTACPALGPDSSLGPVPSAEPATPQSCGVADPNEAEEFLRQFHAEQSDQPVPLARRLEQVRAAIDATGTYRHTTAE 
               
               
                 LVYGARVAWRNSSRCIGRLYWNSLRVLDRRDATAPDEIHRHLCTHLRQATNGGRIRPVISVFAPDSPGRPGPQVWNEQLIR 
               
               
                 YAGYRRDDGTVLGDPRTADLTEAILRLGWQGCPQGPFDVLPLVIDTPDDKPRFFELPRELVLEVPITHPDVPRLAELGLRW 
               
               
                 HAVPVISNMRLRIGGMDYPLAPFNGWYMGTEIGARNLVDEDRYNMLPAVAACLQLDTTSESTLWRDRALVELNVAVLHSFE 
               
               
                 AAGVRISDHHEESRRFLAHLAKEERQGRTVSADWSWIVPPLSGGITPVFHRYYDNVDQRPNFYPHQ 
               
               
                   
               
               
                 txtE (scab31831) (SEQ ID NO: 9) 
               
               
                 GTGACCGTCCCCTCGCCGCTCGCCGACCCGTCCATCGTGCCCGACCCCTACCCTGTCTACGCCGACCTGGCCCAGCGCCGC 
               
               
                 CCCGTCCACTGGGTCGAGCGCCTGAACGCCTGGGCGGTCTTGACGTACGCCGACTGCGCCGCCGGGCTGAAGGATCCCCGG 
               
               
                 CTCACCGCCGACCGGGGGACGGAAGTGCTGGCCGCGAAGTTCCCCGGACAGCCGCTGCCGCCGGACAACATCTTCCACCGC 
               
               
                 TGGACCAAGAACGTGGTGATGTACACGGACCCGCCGCTCCACGACGCGCTACGCCGGTCCGTCCGCGCAGGCTTCACCCGT 
               
               
                 GCCGCGCACCAGCACTACGACCAAGTCCTCCAGAAGGTCGCGCACGACCTGGTCGCTTCCATCCCGGCCGGTGCCACCGAG 
               
               
                 ATCGACGCCGTCCCCGCCCTGGCTGCCGAACTCCCCGTACGCTCCGCCGTGCACGCCTTCGGGGTCCCCGAGGAGGACCTC 
               
               
                 GGATTCCTCATCCCGCGCGTGAATACGATCATGACGTACCACTCCGGTCCGAAGGATCAGCCGGTGACGCAGGAGATAATC 
               
               
                 CTGGAAAAGCTCACCGACCTGCACACGTACGCCTCCGAACTCCTCCAGGGCATGCGGGGCAAGGTCCTGCCGGACACCGTC 
               
               
                 ATCGCCCGCCTGGCAGCCGCCCAGGACGGCCTGACCGAGACCACGCCGGAACAGACCGTGCACCAGCTGGCGCTGGTGTTC 
               
               
                 ATCGCGTTGTTCGCGCCCACGACGCCGGGCTCTCTCAGCAGCGGCACGCTCGCGTTCGCCCGCAACCCGCGGCAGGTCGAA 
               
               
                 CGCTTCCTGGCGGACCAGGCGTGCGTGGACAACACGGCGAACGAGGTCCTCCGCTACAACGCCTCGAACCAGTTCACCTGG 
               
               
                 CGCGTCGCGGCCAAGGACGTCGAGATGGGCGGCGTACGGATCGAGGCCGGGCAGACTCTCGCCCTGTTCCTGGGCTCGGCC 
               
               
                 AACCGGGACGCCAACATGTTCGAGCGACCGAACGACTTCGACCTCGACCGTCCCAACAGCGCTCGGCACCTGTCGTTCGGC 
               
               
                 CAAGGGGTGCACGCCTGTCTCGCCGCGCAGCTCATCTCCCTGCAGCTGAAGTGGTTCTACGTCGCCCTGCTGAACCGCTTC 
               
               
                 CCGGGCATCCGGACGGCGGGCGAGCCGATCTGGAACGAGAACCTCGAATTCCGCTCCCTTCGCTCCCTGCCGCTCAGCCTC 
               
               
                 CGCTGA 
               
               
                   
               
               
                 TxtE (scab31831) (SEQ ID NO: 10) 
               
               
                 VTVPSPLADPSIVPDPYPVYADLAQRRPVHWVERLNAWAVLTYADCAAGLKDPRLTADRGTEVLAAKFPGQPLPPDNIFHR 
               
               
                 WTKNVVMYTDPPLHDALRRSVRAGFTRAAHQHYDQVLQKVAHDLVASIPAGATEIDAVPALAAELPVRSAVHAFGVPEEDL 
               
               
                 GFLIPRVNTIMTYHSGPKDQPVTQEIILEKLTDLHTYASELLQGMRGKVLPDTVIARLAAAQDGLTETTPEQTVHQLALVF 
               
               
                 IALFAPTTPGSLSSGTLAFARNPRQVERFLADQACVDNTANEVLRYNASNQFTWRVAAKDVEMGGVRIEAGQTLALFLGSA 
               
               
                 NRDANMFERPNDFDLDRPNSARHLSFGQGVHACLAAQLISLQLKWFYVALLNRFPGIRTAGEPIWNENLEFRSLRSLPLSL 
               
               
                 R 
               
               
                   
               
               
                 txtH (scab31771) (SEQ ID NO: 11) 
               
               
                 GTGCCCTCACCCTTCGACGACCATGACGGGCAGTTCCATGTGCTCCGCAACGAGGAAGGCCAGTTCTCACTCTGGCCGAAT 
               
               
                 TTCGCCGACATCCCCTCCGGGTGGCGTTCCGTGAGCGGGCCGAGCCCCCGCGGAAGCGCCCTTGAGTACATCGAGAAGGAA 
               
               
                 TGGACGGACATGCGCCCGGCGTCCGTCCGTGAATGA 
               
               
                   
               
               
                 TxtH (scab31771) (SEQ ID NO: 12) 
               
               
                 VPSPFDDHDGQFHVLRNEEGQFSLWPNFADIPSGWRSVSGPSPRGSALEYIEKEWTDMRPASVRE 
               
               
                   
               
               
                 txtR (scab31801) (SEQ ID NO: 13) 
               
               
                 ATGCAGATAAAATCTTTCAAGGCCGGCGGGGTCAAGGTGACGATCATAGATTCCGGTCCAGCCGTCATCGAGTTCGAGGCA 
               
               
                 ATCAACTCGGAGGCGGCCTTGACGCCGCAGAGAACAGTCATATGCGTACTGTCAGGAATGGCGTTCATCGCTGGTACCGGA 
               
               
                 AACGGTACGGAGATCGACGCGGGGACGCTGGTTATGACGGACGGCGACGTTCCCTTTTCGATGAATGTGCCCGTTGCTTCG 
               
               
                 CGACTCCTCGTACTGCGTTTCGCCGACGAAGCGAAGGATGGACTCCCGGTGTCGCCTCGGGGGACTTTTATCGTGACGGAT 
               
               
                 GCTGCCAAGGGTCCCGGATCCGGATTTCTTTTTTCGTTCTTGAATACCCTGGCTGTGGAGATGATGAAAACCGATGGGATT 
               
               
                 CTGTCCTCGTATATGGAGGAGGTCGTGCGCATCCTGGCGATCTCCGCGACGCGAATCGCATATGCCGAGCTCGGAAAGCAT 
               
               
                 TACTCTGGGGGATGCGATCCACTTCTGATCGCGGTTCAGGAGTCGATCGACCGGCAGTTGGCCGACCCCGAGATCAGCCCG 
               
               
                 GCGACCCTCGCGGCCGAACACAACATATCGGTGCGTCAGTTACATCGAGTTTTCGGACCGATCGGGGAAAGCGTCATGAGC 
               
               
                 TATGTCAAACGCCGTCGCCTGGAGCGTTTCGCATGCGATCTGAGGGATCCGAGCCTGGGGCACCGGAAGATCAATGAGCTG 
               
               
                 GCGGCGGACTGGGGGATGCTGGATGCCGCGATGCTGAGCAGACACTTCCGCTGCGCCTACGGAATGTCGCCCCGCGATTAC 
               
               
                 CGGAAGCAGCACTGTTTCACCTGA 
               
               
                   
               
               
                 TxtR (scab31801) (SEQ ID NO: 14) 
               
               
                 MQIKSFKAGGVKVTIIDSGPAVIEFEAINSEAALTPQRTVICLVLSGMAFIAGTGNGTEIDAGTLVMTDGDVPFSMNVPVA 
               
               
                 SRLLVLRFADEAKDGLPVSPRGTFIVTDAAKGPGSGFLFSFLNTLAVEMMKTDGILSSYMEEVVRILAISATRIAYAELGK 
               
               
                 HYSGGCDPLLIAVQESIDRQLADPEISPATLAAEHNISVRQLHRVFGPIGESVMSYVKRRRLERFACDLRDPSLGHRKINE 
               
               
                 LAADWGMLDAAMLSRHFRCAYGMSPRDYRKQHCFT 
               
               
                   
               
               
                 cebR (scab57761) (SEQ ID NO: 15) 
               
               
                 ATGGTGACAGGCCACGGGGCACGGGGCCGGAGCGGTGGGCGGCCGACGTTGGAGGAGGTCGCCGCACGGGCCGGAGTGGGC 
               
               
                 CGGGGGACGGTGTCCCGGGTGATCAACGGCTCGCCCCCGGGTGAGCGACGCGACCCGCGCGGCGGTCGAGGCGGCCGTCGC 
               
               
                 GGAGCTGGGTTACGTCCCGAACACGGCGGCCCGCGCGCTCGCGGCGAACCGTACCGACGCGATCGCGATGGTCGTGCCCGA 
               
               
                 ACCGGAGACCCGCTTCTTCTCGGAGCCGTACTTCTCCGACATCCTCAAGGGTGTCGGAGCGCAACTGTCCGACACCGAGAT 
               
               
                 GCAGCTCCTGCTGATCTTCGCGGGCAACGACCGGGAGCGCCGGCGCCTCGCCCAGTACCTGGCCGCGCACCGCGTCGACGG 
               
               
                 TGTCCTCCTGGTCTCCGTCCACGCGGACGACCCGCTCCCCGATCTGCTGTCGCAACTGGAAATCCCGGCCGTCATCAGCGG 
               
               
                 CCCCCGCTCCGAGCACGAGACGCTCCCCTCGGTCGACTCCGACAACTACGGCGGCGGCCGCTCGGCGGTCGAGCACCTCAT 
               
               
                 CGCACGGGGGCGCGCCCGGATCGCCACGATCACCGGCCGGCTGGACGTCTACGGCGCCCAGCGGCGCATCGAGGGCTACCG 
               
               
                 CGACGCCCTGGAGGACGCGGGCCGCGAGGTGGACGAGCGCCTGATCGCCCCCGGTGACTTCACGGAGGAGGGCGGCCGCCG 
               
               
                 AGCGATGCGCGAACTCCTGGCCCGCTGCCCCGACCTCGACGCGGTCTTCGCCGAGTCGGACGTCATGGCCGCCGGCGCCCG 
               
               
                 CCAGGTGCTCCGCGAGGAGGGCCGCCGCATACCCGACGACGTGGCGCTGGTCGGCTACGACGACTCGGCGATCGCCCGCCA 
               
               
                 CATGGACCCGCCGCTCACCAGCGTCCGCCAGCCGATAGAGGAGATGGGCCGCGCGATGATCGACCTCCTCCTGGACGAGAT 
               
               
                 CGCGGACCGCCGCCCGGCGGTGTCGAGGGGCTTGGAACGACGCCAGGTGGTGCTGCCGACGGAGCTGGTGGGGCGGGATTC 
               
               
                 TTCCTGA 
               
               
                   
               
               
                 CebR (SCAB57761) (SEQ ID NO: 16) 
               
               
                 MVTGHGARGRSGGRPTLEEVAARAGVGRGTVSRVINGSPRVSDATRAAVEAAVAELGYVPNTAARALAANRTDAIAMVVPE 
               
               
                 PETRFFSEPYFSDILKGVGAQLSDTEMQLLLIFAGNDRERRRLAQYLAAHRVDGVLLVSVHADDPLPDLLSQLEIPAVISG 
               
               
                 PRSEHETLPSVDSDNYGGGRSAVEHLIARGRARIATITGRLDVYGAQRRIEGYRDALEDAGREVDERLIAPGDFTEEGGRR 
               
               
                 AMRELLARCPDLDAVFAESDVMAAGARQVLREEGRRIPDDVALVGYDDSAIARHMDPPLTSVRQPIEEMGRAMIDLLLDEI 
               
               
                 ADRRPAVSRGLERRQVVLPTELVGRDSS 
               
               
                   
               
               
                 bgIC (scab57721) (SEQ ID NO: 17) 
               
               
                 ATGCCTGAACCCGTGAATCCGGCCACCCCGGTGACCTTTCCTCCCGCCTTCCTCTGGGGCGCGGCCACCTCCGCGTACCAG 
               
               
                 ATCGAGGGGGCGGTGCGGGAGGACGGCCGTACGCCCTCCATCTGGGACACCTTCAGTCACACGCCGGGCAAGACCGCCGGC 
               
               
                 GGCGAGAACGGTGACATCGCTGTCGACCACTACCACCGCTACCGCGACGACGTGGCGATGATGGCGGACCTGGGCCTCAAC 
               
               
                 GCGTACCGCTTCTCCGTCTCCTGGTCGCGGGTGCAGCCGACGGGGCGGGGCCCGGCCGTCCAGAAGGGGCTCGACTTCTAC 
               
               
                 CGACGGCTGGTCGACGAGCTGCTGGCCAAGGGCATCAAGCCCGCCGTCACCCTCTACCACTGGGACCTCCCGCAGGAGCTG 
               
               
                 GAGGACGCCGGCGGCTGGCCCGAGCGGGACATCGTGCACCGGTTCGCCGAGTACGCGCGGATCATGGGCGAGGCGCTCGGC 
               
               
                 GACCGCGTCGAGCAGTGGATCACCCTCAACGAGCCGTGGTGCACCGCGTTCCTGGGCTACGGCTCCGGGGTGCACGCGCCG 
               
               
                 GGCCGTACGGACCCGGTGGCGTCCCTGCGCGCGGCCCACCATCTGAACGTGGCGCACGGCCTCGGCGTCTCGGCGCTGCGG 
               
               
                 TCGGCGATGCCCGCCCGCAACTCGATCGCGGTGAGCCTCAACTCCTCGGTGGTGCGGCCGATCACCAGCTCCCCGGAGGAC 
               
               
                 CGGGCCGCGGCCCGGAAGATCGACGACCTCGCGAACGGCGTCTTCCACGGACCGATGCTGCACGGGGCCTACCCGGAGACC 
               
               
                 CTGTTCGCCGCGACCTCGTCGCTGACGGACTGGTCGTTCGTGCGGGACGGTGACGTGGCGACGGCCCATCAGCCGCTGGAC 
               
               
                 GCTCTGGGGCTGAACTACTACACGCCGGCGCTGGTCGGCGCGGCGGACGCCGGCCTGGAGGGCCCCCGCGCGGACGGCCAC 
               
               
                 GGGGCGAGCGAGCACTCGCCGTGGCCGGCCGCGGACGACGTCCTGTTCCACCAGACCCCGGGCGAGCGTACGGAGATGGGC 
               
               
                 TGGACCATCGACCCGACGGGCCTGCACGAGCTGATCATGCGGTACGCGCGGGAGGCTCCGGGCCTGCCGATGTACGTGACG 
               
               
                 GAGAACGGCGCCGCGTACGACGACAAGATGGACGCGGACGGCCGTGTCCACGACCCCGAGCGCATCGCCTACCTGCACGGC 
               
               
                 CACCTGCGGGCGGTCCGGCGCGCGATCGCCGAGGGGGCGGACGTGCGCGGGTACTACCTGTGGTCCCTGATGGACAACTTC 
               
               
                 GAGTGGGCGTACGGCTACGGCAAGCGCTTCGGCGCGGTGTACGTCGACTACGCGACCCTGACCCGCACACCGAAGTCGAGC 
               
               
                 GCGCACTGGTACGGGCAGGCGGCGAAGACGGGCGCCCTCCCGCCGCTGGCGCCGGCGCCGGCGTAG 
               
               
                   
               
               
                 BgIC (SCAB57721) (SEQ ID NO: 18) 
               
               
                 MPEPVNPATPVTFPPAFLWGAATSAYQIEGAVREDGRTPSIWDTFSHTPGKTAGGENGDIAVDHYHRYRDDVAMMADLGLN 
               
               
                 AYRFSVSWSRVQPTGRGPAVQKGLDFYRRLVDELLAKGIKPAVTLYHWDLPQELEDAGGWPERDIVHRFAEYARIMGEALG 
               
               
                 DRVEQWITLNEPWCTAFLGYGSGVHAPGRTDPVASLRAAHHLNVAHGLGVSALRSAMPARNSIAVSLNSSVVRPITSSPED 
               
               
                 RAAARKIDDLANGVFHGPMLHGAYPETLFAATSSLTDWSFVRDGDVATAHQPLDALGLNYYTPALVGAADAGLEGPRADGH 
               
               
                 GASEHSPWPAADDVLFHQTPGERTEMGWTIDPTGLHELIMRYAREAPGLPMYVTENGAAYDDKMDADGRVHDPERIAYLHG 
               
               
                 HLRAVRRAIAEGADVRGYYLWSLMDNFEWAYGYGKRFGAVYVDYATLTRTPKSSAHWYGQAAKTGALPPLAPAPA 
               
               
                   
               
               
                 Apramycin resistance gene deletion cassette (SEQ ID NO: 19) 
               
               
                 ATTCCGGGGATCCGTCGACCTGCAGTTCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAAGTTCCCGCCAGCCTCG 
               
               
                 CAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTAC 
               
               
                 TTCACCTATCCTGCCCGGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCG 
               
               
                 TGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAATGCAGCTCACGG 
               
               
                 TAACTGATGCCGTATTTGCAGTACCAGCGTACGGCCCACAGAATGATGTCACGCTGAAAATGCCGGCCTTTGAATGGGTTC 
               
               
                 ATGTGCAGCTCCATCAGCAAAAGGGGATGATAAGTTTATCACCACCGACTATTTGCAACAGTGCCGTTGATCGTGCTATGA 
               
               
                 TCGACTGATGTCATCAGCGGTGGAGTGCAATGTCGTGCAATACGAATGGCGAAAAGCCGAGCTCATCGGTCAGCTTCTCAA 
               
               
                 CCTTGGGGTTACCCCCGGCGGTGTGCTGCTGGTCCACAGCTCCTTCCGTAGCGTCCGGCCCCTCGAAGATGGGCCACTTGG 
               
               
                 ACTGATCGAGGCCCTGCGTGCTGCGCTGGGTCCGGGAGGGACGCTCGTCATGCCCTCGTGGTCAGGTCTGGACGACGAGCC 
               
               
                 GTTCGATCCTGCCACGTCGCCCGTTACACCGGACCTTGGAGTTGTCTCTGACACATTCTGGCGCCTGCCAAATGTAAAGCG 
               
               
                 CAGCGCCCATCCATTTGCCTTTGCGGCAGCGGGGCCACAGGCAGAGCAGATCATCTCTGATCCATTGCCCCTGCCACCTCA 
               
               
                 CTCGCCTGCAAGCCCGGTCGCCCGTGTCCATGAACTCGATGGGCAGGTACTTCTCCTCGGCGTGGGACACGATGCCAACAC 
               
               
                 GACGCTGCATCTTGCCGAGTTGATGGCAAAGGTTCCCTATGGGGTGCCGAGACACTGCACCATTCTTCAGGATGGCAAGTT 
               
               
                 GGTACGCGTCGATTATCTCGAGAATGACCACTGCTGTGAGCGCTTTGCCTTGGCGGACAGGTGGCTCAAGGAGAAGAGCCT 
               
               
                 TCAGAAGGAAGGTCCAGTCGGTCATGCCTTTGCTCGGTTGATCCGCTCCCGCGACATTGTGGCGACAGCCCTGGGTCAACT 
               
               
                 GGGCCGAGATCCGTTGATCTTCCTGCATCCGCCAGAGGCGGGATGCGAAGAATGCGATGCCGCTCGCCAGTCGATTGGCTG 
               
               
                 AGCTCATAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAAGCAGCTCCAGCCTACA 
               
               
                   
               
               
                 Attachment site (att) sequence for  S. scabiei ,  S. europaeiscabiei , and  S . 
               
               
                   stelliscabiei , and  S . sp. 96-12 (SEQ ID NO: 62) 
               
               
                 TTGAAGCGGAAC 
               
               
                   
               
               
                 Attachment site (att) sequence for  S. acidiscabies  (SEQ ID NO: 63) 
               
               
                 TTGAACCGGAAC 
               
               
                   
               
               
                 Third attachment site (att) sequence for  S . sp. 96-12 (SEQ ID NO: 64) 
               
               
                 TTGAACCTGAAC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 List of primers used in Example 1 
               
            
           
           
               
               
               
            
               
                 Primer 
                 Sequence 5′-3′ (SEQ ID NO (SEQ): 20-35) 
                 Use 
               
               
                   
               
               
                 a 
                 TCCACCTCCTGACCACCAAG (SEQ: 20) 
                 Detect the site-specific 
               
               
                 b 
                 AAGATCCCCGAACCGACCT (SEQ: 21) 
                 integration of TR in aviX1 
               
               
                   
               
               
                 c 
                 GTAGCGAAGGCGAGAGTCTCACTG (SEQ: 22) 
                 PCR amplification of the 
               
               
                 d 
                 GAGCCGACGAACAAGTACTACCCG (SEQ: 23) 
                 integrase gene (SCAB_31871) 
               
               
                   
                   
                 located in the TR2 region 
               
               
                   
               
               
                 e 
                 CGAAGATCGAGAACGTCAGGAAGG (SEQ: 24) 
                 Detect the site-integration of 
               
               
                 f 
                 GACCGACGAGGACTTCAAGAACGA (SEQ: 25) 
                 TR2 or the whole TR in aviX1 
               
               
                   
               
               
                 g 
                 TACGAGACCATCGGCAGGGA (SEQ: 26) 
                 PCR amplification of txtH 
               
               
                 h 
                 ACATCCTCACCGAGCCGGAA (SEQ: 27) 
                 (SCAB_31771) located in the 
               
               
                   
                   
                 TR1 region 
               
               
                   
               
               
                 DRB201 
                 TGCCGGGCCCTCTTTGCCGACTAGGAGAAATTCACCGTG 
                 txtH (SCAB_31771) redirect 
               
               
                   
                 TGTAGGCTGGAGCTGCTT (SEQ: 28) 
                 deletion cassette 
               
               
                 DRB202 
                 GGCGACCCGTGGCCCCGCTCGATGTTATTGGCCGGGTCA 
                   
               
               
                   
                 ATTCCGGGGATCCGTCGACC (SEQ: 29) 
                   
               
               
                   
               
               
                 DRB431 
                 ATGAAGAACTTCGAAGCCGCGACCACTCAGGTCGATGTG 
                 lanA (SCAB_32021) and lanB 
               
               
                   
                 TGTAGGCTGGAGCTGCTTC (SEQ: 30) 
                 (SCAB_32031) redirect 
               
               
                 DRB432 
                 TCACGGCGTCCTCCAGTGTTCGCGGGCGCTCTGGCGCAG 
                 deletion cassette 
               
               
                   
                 ATTCCGGGGATCCGTCGACC (SEQ: 31) 
                   
               
               
                   
               
               
                 US-F 
                 GCGCGCTAGCGATTCACGGCAAACTGC (SEQ: 32) 
                 PCR amplification of 1 Kb 
               
               
                 US-R 
                 GCACCTGATTTCGCGATCCGTTAACAGGTCGTCGAAACC 
                 upstream of thaxtomin 
               
               
                   
                 CAGATCG (SEQ: 33) 
                 biosynthetic gene cluster 
               
               
                   
               
               
                 DS-F 
                 CGATCTGGGTTTCGACGACCTGTTAACGGATCGCGAAAT 
                 PCR amplification of 1 Kb 
               
               
                   
                 CAGGTGC (SEQ: 34) 
                 downstream of thaxtomin 
               
               
                 DS-R 
                 GCGCGCATGCCTCAAAGGCCAGGTTGTAGG 
                 biosynthetic gene cluster 
               
               
                   
                 (SEQ: 35) 
               
               
                   
               
               
                 US-F/R; upstream forward/reverse primer; DS-F/R: downstream forward/reverse primer 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 List of primers used in Example 2 
               
            
           
           
               
               
            
               
                 Primers 
                 Sequence (5′ −&gt; 3′) (SEQ ID NO (SEQ): 32-61) 
               
               
                   
               
            
           
           
               
            
               
                 Plasmid construction (pTARa-thx) (SEQ ID Nos: 32-35 as in Table 7 above) 
               
            
           
           
               
               
            
               
                 Upstream-F 
                 GCGCGCTAGCGATTCACGGCAAACTGC (SEQ: 32) 
               
               
                 Upstream-R 
                 GCACCTGATTTCGCGATCCGTTAACAGGTCGTCGAAACCCAGATCG (SEQ: 33) 
               
               
                 Downstream-F 
                 CGATCTGGGTTTCGACGACCTGTTAACGGATCGCGAAATCAGGTGC (SEQ: 34) 
               
               
                 Downstream-R 
                 GCGCGCATGCCTCAAAGGCCAGGTTGTAGG (SEQ: 35) 
               
               
                   
               
            
           
           
               
            
               
                 Plasmid construction (pLST9828-thx) 
               
            
           
           
               
               
            
               
                 9828-Vector-F 
                 TCTAGAGTCGACCTGCAGCCCA (SEQ: 36) 
               
               
                 9828-Vector-R 
                 GTAATCATGTCATAGCTGTTTC (SEQ: 37) 
               
               
                 Thx-fragment-1-F 
                 GAAACAGCTATGACATGATTACACGTATCGGCGACCTGCTCCTG (SEQ: 38) 
               
               
                 Thx-fragment-1-R 
                 TTCACCAACAGGCCGGCGTTCG (SEQ: 39) 
               
               
                 Thx-fragment-2-F 
                 AAGAGAGGCCATCGTCTGGGA (SEQ: 40) 
               
               
                 Thx-fragment-2-R 
                 TGGGCTGCAGGTCGACTCTAGAGACGAGTACCTGGCGGACTA (SEQ: 41) 
               
               
                   
               
            
           
           
               
            
               
                 Plasmid construction (pLST9828-thx-ΔC) 
               
            
           
           
               
               
            
               
                 9828AC-Vector-F 
                 CATGTTCGAGCGACCGAACGAC (SEQ: 42) 
               
               
                 9828AC-Vector-R 
                 GTAATCATGTCATAGCTGTTTC (SEQ: 43) 
               
               
                 ThxΔC-fragment-1-F 
                 GAAACAGCTATGACATGATTACGGTGTCGTTTCCTTTCCAAGAC (SEQ: 44) 
               
               
                 ThxΔC-fragment-1-R 
                 GTGATCCAGTACTTTCCCTCAG (SEQ: 45) 
               
               
                 ThxΔC-fragment-2-F 
                 CAGTAGGTCGAACAGGTAATCG (SEQ: 46) 
               
               
                 ThxΔC-fragment-2-R 
                 GTCGAGGTCGAAGTCGTTCGGTC (SEQ: 47) 
               
               
                   
               
            
           
           
               
            
               
                 Plasmid construction (pLST9828-thx-ΔABHC) 
               
            
           
           
               
               
            
               
                 9828ΔABCH-Vector-F 
                 TCTAGAGTCGACCTGCAGCCCA (SEQ: 48) 
               
               
                 9828ΔABCH-Vector-R 
                 GTAATCATGTCATAGCTGTTTC (SEQ: 49) 
               
               
                 ThxΔABCH-F 
                 GAAACAGCTATGACATGATTACAGGTATCCGTTCCTCTCTGTC (SEQ: 50) 
               
               
                 ThxΔABCH-R 
                 TGGGCTGCAGGTCGACTCTAGAGACGAGTACCTGGCGGACTAC (SEQ: 51) 
               
               
                   
               
            
           
           
               
            
               
                 Colony PCR screening 
               
            
           
           
               
               
            
               
                 TxtA-F 
                 CTTCTCGTCCCCGAGTTTCGAC (SEQ: 52) 
               
               
                 TxtA-R 
                 GATCGCTCATGGTGGCGCAGAC (SEQ: 53) 
               
               
                 TxtB-F 
                 GTCATCTACACCTCGGGTTC (SEQ: 54) 
               
               
                 TxtB-R 
                 CCAACGGTCCGCTCATGGTC (SEQ: 55) 
               
               
                 TxtC-F 
                 TGTTCTCCGGGTATGAGGATG (SEQ: 56) 
               
               
                 TxtC-R 
                 GTTCGGGAAGACACTCTCGTC (SEQ: 57) 
               
               
                 TxtD-F 
                 GCAACTCCAGTCGCTGCATC (SEQ: 58) 
               
               
                 TxtD-R 
                 CGAAGAACCGGGGTTTGTCGTC (SEQ: 59) 
               
               
                 TxtE-F 
                 CAAGAACGTGGTGATGTACAC (SEQ: 60) 
               
               
                 TxtE-R 
                 GAGCTTTTCCAGGATTATCTC (SEQ: 61) 
               
               
                   
               
            
           
         
       
     
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