High-Yield Production of Herbicidal Thaxtomins and Thaxtomin Analogs in a Nonpathogenic Streptomyces Strain
High-Yield Production of Herbicidal Thaxtomins and Thaxtomin Analogs in a Nonpathogenic Streptomyces Strain
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DOI：
10.1128/aem.00164-18
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发表时间：
2018-06-01
影响因子：
4.4
通讯作者：
Ding, Yousong
中科院分区：
文献类型：
--
作者：
Jiang, Guangde;Zhang, Yucheng;Ding, Yousong
泰素是大多数植物病原链霉菌的毒力因子。由于其强大的除草活性、诱人的环境兼容性和固有的生物降解性，泰素是美国环境保护局批准的生物除草剂的关键活性成分。然而，国内链霉菌产量低，限制了其广泛的农业应用。在这里，我们描述了在异源宿主中高产生产他红霉素的方法。利用染色体整合技术，克隆了恶臭链霉菌87.22的thaxtomin基因簇，并在白色链霉菌J1074中进行了表达。采用液质联用(LC-MS)分析方法，观察雷公藤红素和硝基色氨酸类似物的产生。当工程菌J1074在添加1%纤维二糖(TDMc)的Thx定义的最小培养基中培养时，紫杉素A的产量是赤霉菌87.22的10倍，优化的培养条件使紫杉素类似物的产量最高，约为222 mg/L。通过基因缺失进一步改造他红霉素生物合成基因簇，导致产生对新类似物的化学合成至关重要的多种生物合成中间体。此外，利用白花蛇舌草J1074中紫杉素生物合成系统的多功能性，合成了一种非天然的氟代类似物5-氟紫杉素A(5-F-thaxtomin A)，其结构经MS、一维(1D)和二维(2D)核磁共振分析确定。天然和非天然红景天素在萝卜幼苗试验中表现出较强的除草活性。这些结果表明，白花蛇舌草J1074具有高产生产紫杉素及其类似物的潜力，促进了其农业应用。在白链霉菌J1074中通过异源表达获得了迄今报道的最高产量，标志着其在农业上的广泛应用取得了重大飞跃。此外，目前合成紫杉素及其类似物的路线很长，本工作中以高产量生产的两个紫杉素生物合成中间体可以为先进的合成路线提供前体和构建块。重要的是，在白链霉菌J1074中生产5-F-他索托菌素A证明了在合成新的泰索托菌素类似物中替代化学方法的可行方法。此外，我们的工作提出了一种有吸引力的合成生物学策略来改善除草剂泰素的供应，可能会在许多其他生物活性天然产品的发现和生产中找到广泛的应用。
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.