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

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.

Keywords: Streptomyces albus; herbicide; heterologous production; thaxtomins; unnatural analog.

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).

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).

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).

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).