Biotechnology

High-Yield Production of Herbicidal Thaxtomins and Thaxtomin Analogs in a Nonpathogenic Streptomyces Strain

Guangde Jiang, Yucheng Zhang, Magan M. Powell, Peilan Zhang, Ran Zuo, Yi Zhang, Dimitris Kallifidas, Albert M. Tieu, Hendrik Luesch, Rosemary Loria, Yousong Ding
Rebecca E. Parales, Editor
DOI: 10.1128/AEM.00164-18

ABSTRACT

Thaxtomins are virulence factors of most plant-pathogenic Streptomyces strains. Due to their potent herbicidal activity, attractive environmental compatibility, and inherent biodegradability, thaxtomins are key active ingredients of bioherbicides approved by the U.S. Environmental Protection Agency. However, the low yield of thaxtomins in native Streptomyces producers limits their wide agricultural applications. Here, we describe the high-yield production of thaxtomins in a heterologous host. The thaxtomin gene cluster from S. scabiei 87.22 was cloned and expressed in S. albus J1074 after chromosomal integration. The production of thaxtomins and nitrotryptophan analogs was observed using liquid chromatography-mass spectrometry (LC-MS) analysis. When the engineered S. albus J1074 was cultured in the minimal medium Thx defined medium supplemented with 1% cellobiose (TDMc), the yield of the most abundant and herbicidal analog, thaxtomin A, was 10 times higher than that in S. scabiei 87.22, and optimization of the medium resulted in the highest yield of thaxtomin analogs at about 222 mg/liter. Further engineering of the thaxtomin biosynthetic gene cluster through gene deletion led to the production of multiple biosynthetic intermediates important to the chemical synthesis of new analogs. Additionally, the versatility of the thaxtomin biosynthetic system in S. albus J1074 was capitalized on to produce one unnatural fluorinated analog, 5-fluoro-thaxtomin A (5-F-thaxtomin A), whose structure was elucidated by a combination of MS and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) analyses. Natural and unnatural thaxtomins demonstrated potent herbicidal activity in radish seedling assays. These results indicated that S. albus J1074 has the potential to produce thaxtomins and analogs thereof with high yield, fostering their agricultural applications.

IMPORTANCE Thaxtomins are agriculturally valuable herbicidal natural products, but the productivity of native producers is limiting. Heterologous expression of the thaxtomin gene cluster in S. albus J1074 resulted in the highest yield of thaxtomins ever reported, representing a significant leap forward in its wide agricultural use. Furthermore, current synthetic routes to thaxtomins and analogs are lengthy, and two thaxtomin biosynthetic intermediates produced at high yields in this work can provide precursors and building blocks to advanced synthetic routes. Importantly, the production of 5-F-thaxtomin A in engineered S. albus J1074 demonstrated a viable alternative to chemical methods in the synthesis of new thaxtomin analogs. Moreover, our work presents an attractive synthetic biology strategy to improve the supply of herbicidal thaxtomins, likely finding general applications in the discovery and production of many other bioactive natural products.

INTRODUCTION

Herbicides play a crucial role in agricultural production all over the world, but their extensive and broad uses have resulted in herbicide resistance in weeds (1–5). The situation is further exaggerated by the fact that no new class of herbicides has been commercialized in the past decades. Known as virulence factors 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 (Fig. 1) (6–13). Thaxtomin A (compound 1) inhibits cellulose biosynthesis in the nanomolar range, a unique mechanism of plant pathogenicity (6, 14). As weeds have a significantly higher demand of cellulose for rapid growth, a bioherbicide with compound 1 as the main active ingredient has been approved by the U.S. Environmental Protection Agency for pre- and postemergence weed control on various crops at ppm concentrations (14–22). Economically and environmentally acceptable production of thaxtomins is thus critical to foster and sustain their agricultural applications. Accordingly, several synthetic routes have been developed, but they generally generate environmentally damaging wastes, produce racemic products, and have a low overall yield, therefore being impractical for industrial production (7, 23–26). On the other hand, the isolation yield of compound 1 from its native producers (e.g., S. scabiei and S. acidiscabiei) is lower than 10 mg/liter, rendering a costly production process (27, 28).

FIG 1
FIG 1

(A) Schematic representation of an 18-kb thaxtomin gene cluster. (B) The biosynthetic pathway of thaxtomin leading to the production of multiple analogs (compounds 1 to 5) and nitrotryptophan analogs (compounds 6 to 8).

The gene cluster of compound 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. 1A) (27–29). TxtD generates nitric oxide (NO) from l-arginine, which is then used by TxtE to nitrate C-4 of l-tryptophan, resulting in 4-NO2-l-tryptophan (compound 6). Compound 6 is then cyclized with l-phenylalanine by TxtA and TxtB to form a diketopiperazine thaxtomin D (compound 5), which is further hydroxylated twice to produce thaxtomin A, likely by TxtC (Fig. 1B). The expression of the thaxtomin gene cluster is regulated by TxtR, which is known to be activated by cellobiose (27). The relative brevity of thaxtomin biosynthesis compared to that of other more complicated natural products provides opportunities to address the supply issue; i.e., the cluster can be expressed in capable hosts to achieve overproduction. Multiple Streptomyces strains, including S. coelicolor, S. lividans, S. avermitilis, and S. albus J1074 are commonly used to express active and silent natural product gene clusters. Compared with others, S. albus J1074 has the smallest genome within the genus Streptomyces (30), indicating fewer competing pathways for efficient heterologous expression. Indeed, S. albus J1074 has been used to produce a number of structurally diverse natural products of actinomycete and nonstreptomycete origins (31–37) and can demonstrate a higher productivity than other hosts (38–41). Very recently, we expressed 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 ATCC 12309, and S. lividans 1326 (42). Strikingly, S. albus J1074 expressing the 177-kb PAI from S. scabiei produced the highest level of thaxtomins among all 10 engineered strains and two native producers when cultured in Thx defined medium supplemented with 1% cellobiose (TDMc). This result indicates the significant influences of the genetic backgrounds of expression hosts on gene cluster expression and clearly 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 limitations to developing industrial processes, e.g., the mobility nature of PAI, potentially leading to the genetic instability of the production strain, technical difficulties in engineering the biosynthetic gene cluster for further improving the production yield, and cost and safety concerns regarding the use of two antibiotics in the fermentation media (42).

In this work, we describe the high-yield production of thaxtomins in S. albus J1074 after chromosomal integration of only its biosynthetic gene cluster. The yield of thaxtomins (Fig. 1B) was improved by about 12 times in comparison with that by 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-fluoro-l-trytophan (5-F-l-trytophan) into the culture medium led to the generation of one unnatural analog, 5-F-thaxtomin A. The natural and unnatural thaxtomins demonstrated potent herbicidal activity and possessed weak cytotoxicity.

RESULTS AND DISCUSSION

Thaxtomin production in S. albus.We used the transformation-associated recombination approach (43, 44) 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 (see Fig. S1 in the supplemental material). We cultured this strain along with the untransformed S. albus J1074 as a control in the minimal medium TDMc. TDMc contains 1% cellobiose, which induced the production of thaxtomin in native producers in our previous studies (45, 46), presumably by its binding to the pathway-specific regulator TxtR and likely one global regulator, CebR. After a 6-day fermentation, S. albus thx1 produced thaxtomin A (compound 1), four thaxtomin analogs as minor metabolites (compounds 2 to 5), N-acetyl-4-nitrotryptophan (compound 7), and a trace amount of N-methyl-4-nitrotryptophan (compound 8) as shown in a high-pressure liquid chromatography (HPLC) trace (Fig. 2A). The identities of these compounds were revealed by high resolution-mass spectrometry (HR-MS) analysis including the fragmentation of target ions (see Fig. S2 in the supplemental material). 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 (compound 1), reached about 23 mg/liter after only 24 h, highlighting a rapid and highly efficient production. Compounds 7 and 8 are the derivatives of 4-NO2-l-tryptophan and have been isolated from the culture medium of S. scabiei 87.22 at low titers (47). Nitrotryptophan analogs can be valuable building blocks for the synthesis of bioactive chemicals such as the protein kinase C activator indolactam V (48) and new analogs of numerous tryptophan-containing compounds (49–51). Chemical synthesis of optically pure 4-NO2-l-tryptophan (compound 6), however, is complicated (24). S. albus thx1 produced a large amount of N-acetyl-nitrotryptophan (compound 7) (>50.0 mg/liter) and can provide a green route to nitrotryptophan analogs.

FIG 2
FIG 2

(A) Representative HPLC traces of culture extracts of S. albus J1074, S. scabiei 87.22, S. albus thx1, and S. albus thx2. The two engineered S. albus strains have the same product profiles with multiple thaxtomin metabolites. (B) S. albus thx1 and S. albus thx2 produce about 20 times more thaxtomin A (compound 1) per gram of dry weight of biomass than S. scabiei 87.22. Data represent means ± standard deviations (SD) (n ≥ 3). Significant differences between S. scabiei 87.22 and two engineered S. albus strains were determined by Student's t test analysis and are shown (***, P < 0.001).

The integrative bacterial artificial chromosome (BAC) vector pTARa is specific for assembling natural product gene clusters and is over 15 kb in size (43, 44). To assess the potential size effect of integrative vectors on the production of thaxtomins and to ease the cluster engineering as detailed below, we cloned the assembled thaxtomin gene cluster from pTARa-thx into a smaller integrative vector, pLST9828 (∼5.7 kb), to generate pLST9828-thx via a Gibson assembly approach (see Fig. S3 in the supplemental material) (52, 53). We then used this new construct to create S. albus thx2. The metabolite profile of S. albus thx2 cultured in TDMc was identical to that of S. albus thx1 (Fig. 2A), including the increasing accumulation of compounds 7 and 8 during the fermentation (see Fig. S4 in the supplemental material). 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 compounds 1, 2, and 7 (Fig. 2A). Of note, the engineered S. albus strains produced about 10 times more thaxtomin A (compound 1) than S. scabiei 87.22 (91.2 ± 6.8 mg/liter versus 9.1 ± 0.4 mg/liter) (Table 1) after a 6-day fermentation in TDMc. We further determined the productivity of thaxtomin A (compound 1) per biomass unit, and two S. albus strains demonstrated even greater improvement (Fig. 2B). S. albus thx2 showed a slightly, statistically insignificantly higher yield of thaxtomin A (compound 1) than S. albus thx1 (142 ± 4 mg/g versus 137 ± 3 mg/g of dry weight [DW]) (Fig. 2B), both of which were about 20 times higher than that of the native producer (7.2 ± 0.7 mg/g of DW). Collectively, these results indicate the superior production of thaxtomins in S. albus.

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TABLE 1

Yields of thaxtomins (compounds 1 to 5) and nitrotryptophan derivatives (compounds 7 and 8) from S. albus thx2 in different media and from S. scabiei 87.22 in TMDc

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 (27–29, 54), while Trypticase soy broth (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, we prepared it in water or artificial seawater (PDBS) for screening because it contains ingredients of plant origin, as does OBB. We selected S. albus thx2 (Fig. 2B) 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 (compound 1) at a low level (about 0.5 mg/liter) (Table 1). This result presumably suggested that cellobiose remains indispensable for the induction of thaxtomin biosynthesis in S. albus. Consequently, we supplemented these media (except TDMc) with 1% cellobiose, which is indicated by a lowercase c (e.g., ISP4c). Remarkably, ISP4c, R5c, and PDBSc supported the production of ∼150 to ∼170 mg/liter of thaxtomin A (compound 1) by S. albus thx2 after a 6-day fermentation, 1.7 to 1.9 times higher than that supported by TDMc (Table 1). Furthermore, when PDBSc was prepared directly from raw potato materials, S. albus thx2 was able to produce ∼142 mg/liter of thaxtomin A (compound 1), providing a highly economical option for the production of thaxtomins. In these cellobiose-containing media, S. albus thx2 also produced compounds 2 to 5, whose total productivity was about one-third of that for thaxtomin A (compound 1), 50 to 80 mg/liter of N-acetyl-4-nitrotryptophan (compound 7), and a trace amount of compound 8 (Table 1).

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., semisynthesis) to produce thaxtomins and analogs (7, 23–26). Thaxtomin D (compound 5) is an advanced intermediate carrying a featured diketopiperazine core (Fig. 1B), but only a trace amount was produced by both the native producers and two engineered S. albus strains (Fig. 2A). To overproduce this compound, we deleted the txtC gene from the thaxtomin cluster cloned in pLST9828 and then created S. albus thx2-ΔC (see Fig. S5 in the supplemental material). This new strain was cultured in the thaxtomin production media TDMc, ISP4c, R5c, and PDBSc for 6 days. All selected media supported the production of thaxtomin D (compound 5), and the highest yield, of 76 mg/liter, was achieved in ISP4c (Table 2). This strain also produced a low concentration of thaxtomin C (compound 4) in all media (see Fig. S6 in the supplemental material) and 55 to 65 mg/liter of compounds 7 and 8 in TDMc, R5c, and ISP4c (Table 2). However, only about 11 mg/liter of nitrotryptophans was produced when the stain was cultured in PDBSc (Table 2), strikingly different from the case for S. albus thx2, which produced ∼80 mg/liter of compounds 7 and 8 in PDBSc (Table 1). It remains unclear how the removal of txtC influences the production of nitrotryptophan analogs in some media.

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TABLE 2

Yields of thaxtomin biosynthetic intermediates (compounds 5, 7, and 8) from two engineered strains in different media

We next deleted the txtABCH genes from the thaxtomin gene cluster (Fig. S5), and created S. albus thx2-ΔABCH, which can potentially overproduce nitrotryptophans. Indeed, about 50 mg/liter of compound 7 was produced by S. albus thx2-ΔABCH when cultured in TDMc and ISP4c for 6 days (Table 2). R5c and PDBSc were less effective production media (producing about less than 18 mg/liter). Interestingly, no compound 8 was detected from any extract of S. albus thx2-ΔABCH (see Fig. S7 in the supplemental material). Future studies can investigate the potential role of the methyltransferase domain of TxtB/TxtA in the formation of compound 8. S. albus thx2-ΔABCH is not able to produce thaxtomin analogs and therefore has no competing pathway using 4-NO2-l-tryptophan (compound 6) as the substrate (Fig. 1B). The comparable amounts of compound 7 with S. albus thx2 and S. albus thx2-ΔC indicated that the cellular availability of 4-NO2-l-tryptophan (compound 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.We previously demonstrated the impressive substrate promiscuity of TxtE in in vitro studies, and 5-F-l-tryptophan was identified as the best enzyme substrate (55, 56). To further probe the feasibility of the thaxtomin biosynthetic system as an alternative to chemical methods in synthesizing thaxtomin analogs, we sought to produce unnatural thaxtomin analogs by feeding tryptophan analogs. Specifically, serial concentrations (0 to 50 μM) of 5-F-l-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 (see Fig. S8A in the supplemental material). The peak content showed a molecular ion of 457.1497 in HR-MS analysis (Fig. S8B), matching the value of 5-F-thaxtomin A (compound 9) (calculated, 457.1523). We further isolated this compound (1.8 mg/liter) by semipreparative HPLC analysis and determined its structure as 5-F-thaxtomin A (compound 9) by multiple one-dimensional (1D) (1H, 19F, and 13C) and 2D (correlation spectroscopy [COSY], heteronuclear multiple bond correlation [HMBC], heteronuclear single quantum coherence [HSQC], and nuclear Overhauser effect spectroscopy [NOESY]) nuclear magnetic resonance (NMR) analyses (Fig. 3; see Fig. S9 in the supplemental material). 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 19F NMR analysis, confirming the aromatic C-5–F substitution (Fig. S9C). Compared with reported NMR spectra of thaxtomin A (compound 1) (11), this new analog lost a proton signal at its C-5 and possessed an increased chemical shift of its C-5 (δ = 151.7 ppm, versus 119.2 ppm in thaxtomin A) caused by the deshielding of the C-5–F substitution (see Table S1 in the supplemental material). These results illustrated the remarkable plasticity of thaxtomin biosynthetic system and the use of S. albus as a host to produce new thaxtomin analogs. In addition, fluorine substitution is a common and effective strategy to improve properties (e.g., metabolism) of bioactive compounds (57).

FIG 3
FIG 3

Selected key COSY (bold bonds, black), HMBC (→, blue), and NOESY (↔ , red) correlations of the isolated compound by 2D NMR analyses, leading to the elucidation of its structure as 5-F-thaxtomin A (compound 9).

Bioactivities of natural and unnatural thaxtomin analogs.Thaxtomins have potent herbicidal activity, and their analogs can possess additional bioactivities, including antifungal and antiviral activities (24–26). We measured the herbicidal activity of 5-F-thaxtomin A (compound 9) in a radish seedling assay (Fig. 4) (58). Its 50% inhibitory value (I50), at 0.56 ± 0.03 μM, was statistically insignificantly weaker than that of thaxtomin A (compound 1) (I50 = 0.45 ± 0.05 μM, P = 0.04) but significantly stronger than that of ortho-thaxtomin A (compound 2) (I50 = 0.81 ± 0.04 μM, P = 0.0005). We further examined the cytotoxicity of compound 9 along with compounds 1 to 3 and observed weak growth inhibition toward the human T-cell leukemia Jurkat and prostate cancer PC-3 cell lines in a dose-dependent manner (see Fig. S10 in the supplemental material). The 30% inhibitory concentrations (IC30 values) 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. 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. The IC30 values of compounds 2 and 3 were significantly different from the IC30 of compound 1, with P values of 0.019 and 0.013, respectively, while the difference in IC30 between compound 9 and compound 1 was statistically insignificant (P = 0.112).

FIG 4
FIG 4

Characterization of herbicidal activity of thaxtomin analogs. Thaxtomin A (compound 1), ortho-thaxtomin A (compound 2), and 5-F-thaxtomin A (compound 9) demonstrated potent growth inhibition activity toward radish seedling. DMSO was used as the negative control for normalizing the herbicidal activities of thaxtomins. Data represent mean ± SD (n = 18).

In conclusion, despite the promising agricultural applications of thaxtomins, there is a lack of cost-effective production approaches. Here, we describe 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/liter. This significant achievement can lay a solid basis toward the wide agricultural use of this novel class of bioherbicide. Furthermore, we engineered the thaxtomin biosynthetic gene cluster to produce two valuable biosynthetic intermediates at high yields, both of which can act as precursors and building blocks for the development of advanced strategies for synthesis of chemicals. This work also demonstrated the versatility and feasibility of the thaxtomin biosynthetic system by the production of 5-F-thaxtomin A (compound 9), which showed potent herbicidal activity similar to those of thaxtomin A (compound 1) and ortho-thaxtomin A (compound 2). Overall, our work 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.

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. Escherichia coli EPI300 competent cells were purchased from Epicenter. DNA sequencing was performed at Eurofins. The plasmids and strains used in this study are listed in Table 3. Streptomyces strains were cultivated on soybean flour-mannitol agar plates and ISP4 agar plates (BD Biosciences, San Jose, CA, USA) for sporulation and conjugation, respectively. Spores were collected, suspended in 20% (vol/vol) 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 by 50 mm) and coupled with a photodiode array (PDA) detector was used for HPLC analysis. An Agilent Zorbax SB-C18, (5 μm, 9.4 by 250 mm) or YMC-Pack Ph (5 μm, 4.6 by 250 mm) column was used for semipreparative HPLC analysis to isolate the metabolite. 1D and 2D NMR spectra were recorded in CDOD3 on a Bruker 400-MHz or Bruker 500-MHz instrument at the University of Florida, Gainesville, FL, USA. Spectroscopy data were collected using Topspin 3.5 software. HR-MS data were obtained using a Thermo Fisher Q Exactive Focus mass spectrometer equipped with an electrospray probe on a Universal Ion Max API source. Unless otherwise specified, all the samples were analyzed at the end of a 6-day fermentation.

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TABLE 3

Microbial strains and plasmids used in this study

Preparation of thaxtomin-producing S. albus strains.We followed the transformation-associated recombination (TAR) approach (43, 44) to clone the thaxtomin gene cluster into pTARa vector as shown in Fig. S1 in the supplemental material. The primers used are listed in Table 4. 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 cosmid 1989 as the 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 EPI300. 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.

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TABLE 4

Primers used in this study

Isolation of thaxtomins and nitrotryptophan 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-liter flask was inoculated with 15 ml of mycelial suspension solutions and then incubated at 30°C and 250 rpm for 6 days. Clean supernatants were collected after centrifugation at 5,000 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% methanol (MeOH) while thaxtomins were eluted with 100% MeOH. The eluted solvents containing targeted compounds were further dried in in vacuo evaporation. A 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 semipreparative HPLC. After drying corresponding fractions in in vacuo evaporation, the weights of thaxtomins were measured with the microscale balance. Alternatively, the concentrations of thaxtomins were calculated on the basis of their areas under peaks 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 nitrotryptophans were analyzed in HR-MS and tandem MS (MS/MS) studies.

HPLC analysis.The HPLC program included 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 to 50% solvent B over 8 min, followed by another linear gradient of 50 to 99% solvent B over 5 min. The column was further cleaned with 99% solvent B for 3 min and then reequilibrated 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 semipreparative 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 to 50% solvent B for 8 min, followed by a linear gradient of 50 to 99% solvent B for 5 min. The column was then cleaned by 99% solvent B for 1 min and reequilibrated 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 ultraperformance liquid chromatography (UPLC) system fitted with an Agilent Poroshell 120 EC-C18 column (2.7 μm, 4.6 by 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 turbospray conditions were as follows: curtain gas, 30 lb/in2; ion spray voltage, 5,500 V; temperature, 600°C; ion source gas 1, 50 lb/in2; and ion source gas 2, 60 lb/in2. 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) and water (A) containing 0.1% FA were used as mobile phases with a linear gradient program (10 to 90% solvent B over 15 min) to separate chemicals at a flow rate of 0.3 ml/min. A prewash phase of 15 min with 10% solvent B was added at the beginning of each run, in which the eluate was diverted to the waste by a diverting valve. MS1 spectra were acquired under the full-scan mode of Orbitrap, in which a mass range of m/z 150 to 2,000 was covered and data were collected in the positive-ion mode. Fragmentation was introduced by the higher-energy collisional dissociation (HCD) technique with optimized collision energy ranging from 6 to 15 eV. Other settings for the Orbitrap scan were as follow: resolution, 15,000; AGC target, 5 × 105. Full-scan mass spectra and targeted MS/MS spectra for each of the preselected parental ions were extracted from the raw files of the HPLC-MS/MS Experiment II using Xcalibur 2.1 (Thermo Scientific).

Feeding experiment with 5-F-l-trptophan.S. albus thx2 was cultured in TDMc under the same conditions described above for 2 days, and filter sterilized 5-F-l-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 an equal volume of ethyl acetate three times. The combined organic layers were washed, dried over sodium sulfate, and then evaporated to dryness in vacuo. The residue was suspended in methanol for HPLC analysis as described above. To purify compound 9 for structural determination, the mixture was separated on one C18 column (Agilent Zorbax SB-C18; 5 μm, 9.4 by 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 to 99% solvent B for 0.5 min. After eluting in 99% solvent B for 0.5 min, a liner gradient of 99 to 10% solvent B for 0.5 min was used. The column was further reequilibrated 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. 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 was eluted at 30°C with 50% solvent B (methanol with 0.1% FA) for 13.5 min and then with a linear gradient of 50 to 99% solvent B for 0.5 min, followed by another linear gradient of 50 to 99% solvent B for 0.5 min. After eluting in 99% solvent B for 0.5 min, a liner gradient of 99 to 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. Compound 9 was eluted and collected for NMR analysis.

5-F-thaxtomin A (compound 9): yellow solid; [α]20D +148.3 (c 0.0022, MeOH); HRMS (ESI-TOF) m/z 457.1497 [M + H]+ (calculated for C22H21FN4O6, 457.1523); 19F NMR (500 MHz, CD3OD) δ −137.41 (dd, J = 10.9 Hz, 4.5 Hz, 1F). 1H and 13C NMR data are described in Table S1 in the supplemental material.

Herbicidal activity assay of thaxtomins.Serial concentrations (0 to 4 μM) of thaxtomins in DMSO were added to 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 disinfected, pregerminated, and selected when the radicle was 1 ± 2 mm and just emerged from the seed coat. Six radish seedlings were located equally on the surface of each plate with the root ends all pointed in the same direction. The agar plates were covered and 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. The 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 (59), and I50 values were estimated from these curves.

MTT assay to characterize cytotoxicity of thaxtomins.PC-3 and Jurkat cells were cultured in Dulbecco modified Eagle medium (DMEM) or RPMI 1640 medium containing 10% fetal bovine serum and 100 U/ml penicillin and streptomycin and were maintained at 37°C in a humidified incubator under 5% CO2. The cells (1 × 104 for Jurkat, and 5,000 for PC-3 in 100 μl) were seeded into 96-well plates and incubated overnight. Various 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 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg/ml) in PBS was added and incubated for 4 h, followed by the aspiration of the medium. DMSO (100 μl) was added to each well to dissolve the MTT in the wells, and the plate was agitated for 1 h for recording the absorbance at 570 nm using a UV/visible microplate spectrophotometer (BioTek). Three to six replications were performed per treatment of each sample.

Statistical analysis.Statistical significance among multiple groups was analyzed by one-way analysis of variance (ANOVA) followed by Student's t test for comparison of the results between two groups using Prism 5 (GraphPad Software, Inc.). A P value of <0.05 is considered to be statistically significant.

ACKNOWLEDGMENTS

We thank Steven D. Bruner and Isolde M. Francis for insightful discussions.

NMR spectra were obtained at the Advanced Magnetic Resonance Imaging and Spectroscopy facility at the University of Florida, supported by grant ML-Ding-001. This study was partially supported by the University of Florida Opportunity Fund (R.L. and Y.D.) and by University of Florida Emerging Scholar and University Scholar programs (M.M.P.). Y.D. is an Air Force Office of Scientific Research Young Investigator.

FOOTNOTES

    • Received 20 January 2018.
    • Accepted 27 March 2018.
    • Accepted manuscript posted online 30 March 2018.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00164-18.

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    . 1976. Restriction of a bacteriophage of Streptomyces albus G involving endonuclease SalI. J Bacteriol 128:644–650.
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KEYWORDS

Streptomyces albus
herbicide
heterologous production
thaxtomins
unnatural analog

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