ABSTRACT
Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.
Subject(s)
Stilbenes/metabolism , Escherichia coli/metabolism , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/genetics , Biological Products , Coenzyme A Ligases , Fatty Acids , Metabolic EngineeringABSTRACT
Esta lesión poco común, se presenta con gran frecuencia como muerte súbita, por lo que su diagnóstico muchas veces se realiza posmortem. Gracias al desarrollo de las técnicas de resucitación y reanimación en los centros especializados en politrauma, han sobrevivido algunos pacientes con dislocación occipito-atloidea, tema de este trabajo. Se presentan 7 casos atendidos en el Servicio de Neurocirugía del Hospital Universitario General Calixto García(AU)
This uncommon injury is frequently presented as sudden death. That`s why its diagnosis is many times made after death. Thanks to the development of resuscitation and reanimation techniques in the centers specialized in polytrauma, some patients with atlanto-occipital dislocation have survived, and this is the topic we deal with in this paper. 7 cases that received attention at the Neurosurgery Service of General Calixto García Teaching Hospital are presented here(AU)