One-Pot Biocombinatorial Synthesis of Herbicidal Thaxtomins

Abstract

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 singlemodule 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 highresolution 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.

A Promiscuous Cytochrome P450 Hydroxylates Aliphatic and Aromatic C−H Bonds of Aromatic 2,5‐Diketopiperazines

“…Recently, we biochemically confirmed the dual functions of recombinant TxtC fused with the reductase domain of P450BM3 (BM3R) through a 14‐amino acid linker, named as TCB14 . In addition, we revealed substantial substrate promiscuity of TCB14 toward nitro‐containing thaxtomins in aliphatic and aromatic hydroxylation as the biocatalytic production of 58 hydroxylated thaxtomin analogues from 3 l ‐Trp and 12 l ‐Phe analogues . This work provides the first biochemical evidence for the existence of natural P450s for both aliphatic and aromatic C−H hydroxylation, which is further supported by a recent report on the structural characterization of TxtC .…”

“…Among these fusion enzymes, TB14 demonstrates the highest activity in nitrating l ‐Trp, whereas TB3, TB6, and TB9 are not properly folded and inactive. Recently, we used the same design to create self‐sufficient TCB14, which was highly soluble and catalytically active toward 32 analogues of thaxtomin D ( 3 ) . To identify highly active TxtC biocatalysts for DKP hydroxylation, we sought to examine the potential effect of the linker length on the catalytic performance of chimeric TxtC–BM3R fusion enzymes.…”

“…All variants were expressed as soluble proteins in Escherichia coli and possessed featured Soret bands at λ =450 nm in their differential CO‐reduced spectra (Figures S1 and S2 in the Supporting Information). We then incubated 1.5 μ m recombinant enzymes and the glucose dehydrogenase (GDH)‐based NADPH regeneration system with 0.1 m m 3 as a substrate for 2 h . HPLC analysis revealed the production of 2 in all reactions, and TCB14 was the most active enzyme, followed by TCB11 and TCB17 (73.5 and 55.7 % of the activity of TCB14, respectively; Figure B).…”

“…The N12− and N15−CH 3 groups are installed by the MT domain of NRPS TxtB and TxtA, respectively, whereas TxtC hydroxylates the phenyl group as one of its two reactions (Scheme B). Our recent in vitro study revealed that the N‐methylation of thaxtomins was controlled by the availability of SAM in TxtA and TxtB reactions . If [SAM] is limiting, one thaxtomin analogue without any N−CH 3 group ( 5 ) is produced, along with small quantities of 3 and 4 carrying the N12−CH 3 group (Scheme B) .…”

“…Genetic studies of thaxtomin biosynthesis imply that the second pathway‐specific P450 TxtC sequentially hydroxylates the aliphatic tertiary C14 and aromatic C20 of 3 to produce thaxtomins B ( 2 ) and A ( 1 ; Scheme B) . Recently, we biochemically confirmed the dual functions of recombinant TxtC fused with the reductase domain of P450BM3 (BM3R) through a 14‐amino acid linker, named as TCB14 . In addition, we revealed substantial substrate promiscuity of TCB14 toward nitro‐containing thaxtomins in aliphatic and aromatic hydroxylation as the biocatalytic production of 58 hydroxylated thaxtomin analogues from 3 l ‐Trp and 12 l ‐Phe analogues .…”