RRC ID 50675
Author Kudoh K, Kubota G, Fujii R, Kawano Y, Ihara M.
Title Exploration of the 1-deoxy-d-xylulose 5-phosphate synthases suitable for the creation of a robust isoprenoid biosynthesis system.
Journal J. Biosci. Bioeng.
Abstract 1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for production of two precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). Previously, we attempted the overexpression of endogenous DXS in Synechocystis sp. PCC6803, and revealed that although the mRNA level was 4-fold higher, the DXS protein level was only 1.5-fold higher compared with those of the original strain, suggesting the lability of endogenous DXS protein. Therefore, for the creation of a robust isoprenoid synthesis system, it is necessary to build a novel MEP pathway by combining stable enzymes. In this study, we expressed 11 dxs genes from 9 organisms in Escherichia coli and analyzed their protein solubility. Furthermore, we purified the recombinant DXSes and evaluated their specific activities and protease tolerance, thermostability, and feedback inhibition tolerance. Among DXSes we examined in this study, the highest protein solubility was observed in Paracoccus aminophilus DXS (PaDXS). The DXS with the highest activity was one from Rhodobacter capsulatus (RcDXSA). The highest protease tolerance, thermostability, and tolerance of feedback inhibition were found in Bacillus subtilis DXS (BsDXS), RcDXSA, PaDXS, BsDXS, respectively. These DXSes can be potentially used for the design of robust isoprenoid synthesis system.
Volume 123(3)
Pages 300-307
Published 2017-3
DOI 10.1016/j.jbiosc.2016.10.005
PII S1389-1723(16)30375-9
PMID 27856234
MeSH Bacillus subtilis / enzymology Bacillus subtilis / genetics Enzyme Stability Erythritol / analogs & derivatives Erythritol / biosynthesis Escherichia coli / genetics* Escherichia coli / metabolism* Hemiterpenes / biosynthesis Hemiterpenes / metabolism Organophosphorus Compounds / metabolism Paracoccus / enzymology Paracoccus / genetics Pentosephosphates / biosynthesis Peptide Hydrolases / metabolism Rhodobacter capsulatus / enzymology Rhodobacter capsulatus / genetics Solubility Sugar Phosphates / biosynthesis Synechocystis / genetics Synechocystis / metabolism Terpenes / metabolism* Transferases / chemistry Transferases / genetics* Transferases / metabolism*
IF 2.015
Resource
General Microbes JCM11003