ABSTRACT
The key and crucial step of metabolic engineering during quinic acid biosynthesize using shikimic acid pathway is high expression of quinate 5-dehydrogenase. The gene qa-3 which code quinate 5-dehydrogenase from Neurospora crassa doesn't express in Escherichia coli. By contrast with codon usage in Escherichia coli, there are 27 rare codons in qa-3, including eight AGG/AGA (Arg) and nine GGG (Gly). Two AGG are joined together (called box R) and some GGG codons are relative concentrate (called box G). Along with the secondary structure of mRNA analysed in computer, the free energy of mRNA changes a lot from -374.3 kJ/mol to least -80.5 kJ/mol when some bases in the end of qa-3 were transformed, and moreover, the change of free energy is quite small when only some bases in the box G and box R transformed. After the change of rare codon and optimization of some bases in the end, qa-3 was expression in E. coli and also the enzyme activity of quinate 5-dehydrogenase can be surveyed accurately. All the work above benefit the further research on producing quinic acid engineering bacterium.
Subject(s)
Alcohol Oxidoreductases , Genetics , Base Sequence , Codon , Chemistry , Genetics , Escherichia coli , Genetics , Metabolism , Hydro-Lyases , Genetics , Molecular Sequence Data , Neurospora crassa , Genetics , RNA, Messenger , Chemistry , Genetics , Recombinant Proteins , Genetics , Shikimic Acid , Metabolism , Transformation, BacterialABSTRACT
<p><b>AIM</b>To investigate effect and mechanism of vasonatrin peptide (VNP) on Ca2+ activated K+ channels (K(Ca)) of vascular smooth muscle cells (VSMCs) isolated from rat mesentery arteries.</p><p><b>METHODS</b>Changes of K(Ca) induced by VNP were measured by the means of whole cell recording mode of patch clamp, furthermore effects of HS-142-1(0.3 g/L), 8-Br-cGMP and methylene blue (MB) were observed.</p><p><b>RESULTS</b>K(Ca) was significantly enhanced by VNP (10(-6) mol/L), which was mimicked by 8-Br-cGMP(10(-3) mol/L) and blocked completely by HS-142-1 or MB (2 x 10(-5) mol/L).</p><p><b>CONCLUSION</b>VNP increases K(Ca) of VSMCs isolated from rat mesenteric arteries, by binding with natriuretic peptide guanylate cyclase-coupled receptors and increasing the intracellular level of cGMP in VSMCs.</p>