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J Biosci Bioeng. 2017 Mar;123(3):300-307. doi: 10.1016/j.jbiosc.2016.10.005. Epub 2016 Nov 14.

Exploration of the 1-deoxy-d-xylulose 5-phosphate synthases suitable for the creation of a robust isoprenoid biosynthesis system.

Author information

1
Department of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
2
Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
3
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
4
Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan; JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: m_ihara@shinshu-u.ac.jp.

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.

KEYWORDS:

1-Deoxyxylulose 5-phosphate synthase; Activity analysis; Feedback inhibition; Isoprenoid; Protein solubility

PMID:
27856234
DOI:
10.1016/j.jbiosc.2016.10.005
[Indexed for MEDLINE]

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