One-Pot Biocombinatorial Synthesis of Herbicidal Thaxtomins

• Published in: ACS catalysis 2018-11, Vol.8 (11), p.10761-10768
• Main Authors: Jiang, Guangde, Zuo, Ran, Zhang, Yi, Powell, Magan M, Zhang, Peilan, Hylton, Sarah M, Loria, Rosemary, Ding, Yousong
• Format: Article
• Language: English
• ISSN: 2155-5435; 2155-5435
• DOI: 10.1021/acscatal.8b03317

Thaxtomins are a group of phytotoxic diketopiperazines produced by tens of plant pathogenic Streptomyces strains and have received considerable attention as bioherbicide. To synthesize thaxtomin analogue libraries for herbicide development, we here develop an in vitro biocombinatorial approach. We first prepared two recombinant single-module multifunctional nonribosomal peptide synthetases (NRPSs), TxtA and TxtB. Biochemical studies revealed that TxtA and TxtB tolerated small substituents on the aromatic moiety of l-Phe and l-Trp, respectively. Intriguingly, TxtB showed a control of the substrate scopes of TxtA and a previously characterized, pathway-specific nitration promoting P450 TxtE. We further demonstrated that the methyltransferase (MT) domain of TxtA and TxtB posed a minimal influence on the assembly of the diketopiperazine core by other domains of these two enzymes, providing a way for structural diversification. The pathway-specific bifunctional P450 TxtC was then recombinantly produced in Escherichia coli after being fused with the reductase domain of P450BM3. Biochemical and kinetic studies indicated that this self-sufficient biocatalyst promoted two hydroxylation reactions first on the aliphatic C14 and then the aromatic C20 of thaxtomin D to sequentially produce thaxtomin B and A. Using these enzymes, a one-pot biocatalytic reaction was developed to synthesize 124 thaxtomin analogues, whose structures were validated in high-resolution MS, tandem MS, and sometimes 1D and 2D NMR analysis. Select thaxtomin analogues showed potent herbicidal activities in radish seedling assay. This work demonstrated the feasibility of biocombinatorial synthesis in the creation of natural product-like libraries and provided useful insights into the model of diketopiperazine structural diversity generation in nature, aiding the development of bioactive peptidic compounds in general.