Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver

Ren Chen,Takeshi Bamba,Yoshihisa Nakazawa,Koichiro Gyokusen,Yoko Harada

doi:10.1186/1472-6750-12-78

Ren Chen, Takeshi Bamba + Show 3 more

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https://doi.org/10.1186/1472-6750-12-78

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Journal: BMC Biotechnology	Publication Date: Oct 30, 2012
Citations: 62	License type: cc-by

Affiliation: Osaka University, Kyushu University

Abstract

BackgroundNatural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree) produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production.ResultsTo develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629) encoding isopentenyl diphosphate isomerase (IPI) was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type) and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line) control.ConclusionsIncreasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our results demonstrated that regulation of IPI expression is a key target for efficient production of trans-polyisoprene in E. ulmoides.

Highlights

Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer
Similar to other eukaryotic isopentenyl diphosphate isomerase (IPI) reported previously, EuIPI contained a number of highly conserved regions that are common to IPIs from higher plants and humans to yeasts [11,13], and two residues, C and E, in the TNTCCSHPL motif and the WGEHEXDY motif (Figure 2), respectively, which are critical for catalytic activity of the enzyme [13,14,15,16]
The spectra of isopentenyl diphosphate (IPP) and DMAPP were nearly identical to those published previously [17]. These results unambiguously indicated that the digested EuIPI protein was able to catalyze the conversion of IPP to DMAPP in vitro, confirming that the cDNA clone isolated from E. ulmoides encoded a functional IPP isomerase

Summary

Introduction

Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. Hevea brasiliensis (rubber tree) produces cis-polyisoprene, and is the key source of commercial rubber. Trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. Plant polyisoprenes can be classified into two types; cis- or (Z)-polyisoprene and trans- or (E)-polyisoprene, depending on the type of polymerization of the isoprene unit (Figure 1) [1,2]. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene [3]. OPP Dimethylallyl diphosphate (DMAPP, C5) GPP synthase. OPP Geranyl diphosphate (GPP, C10) IPP FPP synthase. Mevalonic acid (MVA) pathway 1-Deoxy-D-xylulose-5-phospahte (DXP) pathway IPP isomerase

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