Studies on the formation and synthetic mechanism of related substance G in potassium clavulanate production

Abstract

The objective of this study was to investigate the formation and synthetic mechanism of related substance G in potassium clavulanate production. The impurity in the potassium clavulanate final product, with a retention time of 13.5 min, was confirmed as related substance G by high performance liquid chromatography-mass spectrometry/mass spectrometry. Related substance G analysis during the production of clavulanic acid showed that this impurity could be synthesized during fermentation, and the amount increased with the fermentation time. Studies on its synthetic mechanism showed that L-tyrosine and succinic acid were the precursors for biosynthesis of related substance G in vivo. The reaction was deduced to be catalyzed by an enzyme. The enzyme was a type of extracellular enzyme present in the fermentation supernatant.Uniterms: Potassium clavulanate/production. Clavulanic acid/production. Related substance G./structure identification. Related substance G./synthetic mechanism. High Performance Liquid ChromatographyMass Spectrometry-Mass Spectrometry/analysis O objetivo deste estudo foi investigar a formação e o mecanismo sintético da substância G relacionada na produção de clavulanato de potássio. A impureza do produto final clavulanato de potássio, com tempo de retenção de 13,5 min, foi confirmada como substância G relacionada por cromatografia líquida de alta eficiência-espectrometria de massas/espectrometria de massas. A análise da substância G relacionada durante a produção do ácido clavulânico mostrou que essa impureza poderia ser sintetizada durante a fermentação e que a quantidade aumenta com o tempo de fermentação. Estudos do seu mecanismo sintético mostraram que a L-tirosina e o ácido succínico foram os precursores in vivo para a biossíntese da substância G relacionada. Deduziu-se que a reação foi catalisada por uma enzima. A enzima foi do tipo extracelular, presente no sobrenadante da fermentação. Unitermos: Clavulanato de potássio/produção. Ácido clavulânico/produção. Substância G relacionada/ identificação estrutural. Substância G relacionada/mecanismo sintético. Cromatografia Líquida de Alta Eficiência-Espectrometria de massas-Espectrometria de massas/análise

The CagRS Two-Component System Regulates Clavulanic Acid Metabolism via Multiple Pathways in Streptomyces clavuligerus F613-1

“…All strains and plasmids used in this study are listed in Supplementary Table S1. S. clavuligerus F613-1 is an industrial strain (Jin et al, 2015) and was used as the parental strain in this study. Cloning procedures were performed in Escherichia coli DH5a, protein expression was performed using E. coli BL21(DE3), and E. coli ET12567/pUZ8002 was used for intergeneric conjugative transfer of plasmid DNA into S. clavuligerus (Kieser et al, 2000).…”

“…The diameter of the inhibition zone was gauged after overnight culturing at 37°C. The concentration of CA during the liquid-state fermentation was detected by HPLC with an Inertsil ODS-3 4.6 mm × 150 mm, 5 μm column (Jin et al, 2015; Qin et al, 2017), using clavulanate lithium (provided by Lunan Pharmaceutical Co.) as the standard for quantification. For bioassay and HPLC analysis of CA production, the experiments were conducted in triplicate.…”

Clavulanic Acid Production by Streptomyces clavuligerus: Insights from Systems Biology, Strain Engineering, and Downstream Processing

“…Degradation of CA is considered consequence of susceptibility to nucleophilic attacks at specific points. This acts as a hydrolytic mechanism in two steps: an equilibrium reaction leading to an active intermediate and an irreversible reaction of this intermediate with another CA molecule, thus leading to the degradation product [67,176,177]. As consequence, CA increases its own degradation rate when increasing concentration in the fermentation broth [68,176].…”

The two-component system CepRS regulates the cephamycin C biosynthesis in Streptomyces clavuligerus F613-1

“…CA concentrations were analyzed by HPLC analysis as previously described (Fu et al 2019; Jin et al 2015; Qin et al 2017). Briefly, the concentration of CA in the SCF fermentation medium was detected by HPLC using an Inertsil ODS-3 4.6 mm × 150 mm, 5 μm column, and using clavulanate lithium as the standard for quantification.…”

“…S. clavuligerus F613-1 is used industrially to produce the β-lactamase inhibitor CA. Cephamycin C, which is also present in the fermentation broth, is considered undesirable when CA is produced (Jin et al 2015; Qin et al 2017).…”