Soil management exerts profound impact on the structure and function of the microbiome.Organic practice is believed to pos. affect disease suppressiveness compared to conventional systems.While Pseudomonas has been established as a major player in conferring suppressiveness, the impact of farming practices on its diversity at species level has not been conclusively understood.Also, translation of such knowledge into application requires a mechanistic understanding of the mechanism of action of potent biocontrol strains of Pseudomonas.And results: We compared fields under conventional and organic farming, under long-term (∼19 years) management.Profiling of Pseudomonas community structure using rpoD-based amplicon sequencing revealed a higher diversity of known antagonistic Pseudomonas spp. in soils sampled from organic fields compared to conventional field.Cultivation and genome sequencing of selected Pseudomonas isolates from the organic field identified species with multiple secondary metabolite biosynthetic gene clusters and antagonistic activity towards phytopathogens.The selected Pseudomonas isolates showed colonization of wheat root system as confirmed by SEM.In vitro tube assay using wheat also revealed the suppressive activity of potent Pseudomonas isolates against Fusarium oxysporum and Verticillium dahliae.Production of metabolites with antimicrobial activity was verified using untargeted metabolomics (LC/MS).Overall, the mechanistics involved in the contribution of Pseudomonas spp. to disease suppressiveness in the field under organic farming could be established.This study reports that microbiome from organic field exhibits enhanced diversity of antagonistic Pseudomonas spp. effective against phytopathogens, compared to the conventional practice, and has the potential to promote sustainable agriculture and disease management.

2024-08-01·Journal of Biological Chemistry

The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain

Article

作者: Townsend, Craig A ; Li, Rongfeng ; Lichstrahl, Michael S ; Gulick, Andrew M ; Patel, Ketan D ; Oliver, Ryan A

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation, and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual β-lactam-forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for β-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the β-lactone-forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized β-lactam-forming biosynthetic gene clusters by genome mining.

2024-07-19·ACS Catalysis

Deciphering the Key Loop: Enhancing l-Threonine Transaldolase’s Catalytic Potential

作者: Rao, Jingxin ; Zhang, Xinyi ; Xi, Zhiwen ; Xu, Yan ; Zhang, Wenchi ; Zhang, Rongzhen ; Li, Lihong ; Liu, Zhiyong ; Zheng, Mingyue

L-Threonine transaldolase (LTTA) is an attractive biocatalyst because of its potential diastereoselectivity in the synthesis of β-hydroxy-α-amino acids (βHAAs).However, prospective development of LTTA has been hampered by its low activity.Here, a combination of techniques involving structural comparison, computational anal., Loop deletion, and alanine scanning was used to identify a key Loop region (Loop 1) regulating the catalytic ability of Chitiniphilus shinanonensis LTTA (CsLTTA).Saturation mutagenesis and iterative saturation mutagenesis at the hot spots in Loop 1 were performed, and the best variant containing an F70T/C57Q/Y69T (TQT) triple mutation was screened.The diastereoisomer excess (de) produced by the TQT variant (95.4%syn) was greater than that produced by the wild-type (WT) enzyme (75.2%syn), and the catalytic efficiency (kcat/Km) of the TQT variant was four times higher than that of the wild-type enzyme.Mol. dynamics simulations, metadynamics simulations, and CAVER anal. revealed the critical role of the Loop 1 structure in regulating the hydrogen bond network and thus reshaping the active-site pocket to control the syn-tunnel direction.Further engineering of Loop 1 in ObiH, an LTTA responsible for obafluorin biosynthesis, resulted in the development of the F70T-C57Q-H69T (ObiH-TQT) variant producing a de of 97%syn.Using the ObiH-TQT variant for kilogram-scale synthesis of L-syn-p-methylsulfonylphenylserine, coupled with acetaldehyde elimination, resulted in space-time yields of up to 12.7 g L-1 h-1.The method achieved 98.3% substrate conversion and 99.2%synde within 6 h, marking the highest reported levels to date.The above findings will contribute to the industrial production of β-hydroxy-α-amino acids, offer insights into the mechanism of Loop regions regulating the catalytic function of LTTAs, and provide ideas for engineering other enzymes.

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适应症	最高研发状态	国家/地区	公司	日期
革兰氏阴性菌感染	临床前	英国	Norwich Research Partners LLP	2019-11-01
革兰氏阳性细菌感染	临床前	英国	Norwich Research Partners LLP	2019-11-01