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

Abstract

Background: 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. 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.

Results: To 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.

Conclusions: Increasing 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.

Figure 1

Overview of trans-polyisoprene biosynthesis pathway in plants. In the isoprenoid biosynthetic pathway, the basic five-carbon unit IPP is synthesized in the cytoplasm via the MVA pathway or in plastids via the DXP pathway. Then, IPP is interconverted to its highly electrophilic isomer, DMAPP, by the isomerase IPI at the first step. DMAPP then loses its inorganic pyrophosphate to form isoprene, which sequentially condenses with IPP to generate the short-chain isoprenoid precursors GPP, FPP, and GGPP. These precursors are further metabolized for the biosynthesis of distinct sets of isoprenoids, such as monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and polyisoprenes (C>5000) by various isoprenyl diphosphate synthases.

Figure 2

Nucleotide and deduced amino acid sequences of EuIPI. EuIPI (Accession No. AB041629) consists of 1028 bp with a 675-bp ORF encoding a protein with 224 amino acid residues. Numbers of nucleotide sequence and amino acid sequence are indicated on left and right, respectively. Start codon (ATG) is underlined; stop codon (TAG) is marked with *. Broken lines indicate four highly conserved regions of IPIs. Residues critical for catalytic activity of the enzyme are highlighted in red bold font. Boxes show the conserved C (cysteine) motif and the conserved E (glutamic acid) motif.

Figure 3

Phylogenetic analysis based on comparison of deduced amino acid sequence of EuIPI with those of other IPIs from different organisms including plants, bacteria, fungi, and animals (a) or with plant two type I IPIs (b). EuIPI shares a common evolutionary origin with other plant IPIs indicating that EuIPI belongs to the plant IPI group and has high homology with C. acuminata IPI2 (90.9% identity). Alignments were performed with Clustal W and visualized with TreeView. Distances between sequences are expressed as 0.1 changes per amino acid residue. Accession numbers and abbreviations: An, Aspergillus nidulans (AF479816); Ap, Acyrthosiphon pisum (FJ824667); At1, Arabidopsis thaliana (U47324); At2, Arabidopsis thaliana (U49259); Bc, Bupleurum chinense clone (GQ433719); Bm, Bombyx mori (AB274994), Ca1, Camptotheca acuminata (AF031079); Ca2, Camptotheca acuminata (AF031080); Ec, Escherichia coli (EU896065); Ce, Caenorhabditis elegans (AY597336); Eu, Eucommia ulmoides (AB041629); Hb1, Hevea Brasiliensis (AB294696); Hb2, Hevea Brasiliensis (AB294697); Hs, Homo sapiens (AF271720); Ik, Ipomoea sp. Kenyan (AB499048); Nc, Neurospora crassa (AB299023); Ng, Natronobacterium gregoryi (AJ564483); Nt1, Nicotiana tabacum (AB049815); Nt2, Nicotiana tabacum (AB049816); Ps, Periploca sepium (AB091677); Pt, Pinus taeda (GQ476784); Sl, Solanum lycopersicum (EU253957); Sp, Schizosaccharomyces pombe (U21154); and Zm, Zea mays (AF330034).

Figure 4

¹H-NMR spectra of reaction products obtained by incubating IPP substrate with (a) and without (b) purified EuIPI protein in enzymatic activity assay. Red arrows indicate four signals characteristic of DMAPP (1.53, 1.58, 4.3, 5.3 ppm), which was produced from IPP catalyzed by EuIPI, and were absent from the reaction without EuIPI. Signals of IPP substrate (1.59, 2.3, 3.9, 4.7 ppm; blue arrows) were detected in the reaction either with or without purified EuIPI protein. These results indicated that the cDNA clone isolated from E. ulmoides encoded a functional IPP isomerase capable of catalyzing the conversion of IPP to DMAPP in vitro.

Figure 5

Comparison of EuIPI expression levels between transgenic E. ulmoides root lines and wild-type control. EuIPI was transformed into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines overexpressing EuIPI showed increased expression of EuIPI gene (19-fold higher expression in the pOEB5-6 line) compared with that in the wild-type control. Data represent means ± standard error, n=3; different letters indicate significant differences at P < 0.01 (ANOVA, Statistica, St. Tulsa, OK, USA).

Figure 6

Comparison of total trans-polyisoprenes contents between transgenic E. ulmoides root lines and wild-type control. EuIPI was transformed into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines overexpressing EuIPI showed 3- to 4-fold increases in contents of trans-polyisoprenes compared with that in the wild-type control. Data represent means ± standard error, n=3; different letters indicate significant differences at P < 0.05 (ANOVA, Statistica).

Figure 7

Comparison of molecular weight distribution of trans-polyisoprenes between transgenic E. ulmoides root lines and wild-type control. Compared with those in the wild-type control, the three representative transgenic lines overexpressing EuIPI showed significantly increased peak areas in both the low molar mass region from 1.5×10³ to 2.5×10⁴ and the high molar mass region from 2.5×10⁴ to 4.0×10⁶, especially the latter.

Figure 8

Schematic structure of T-DNA region of overexpression vector used for E. ulmoides transformation. cDNA of EuIPI was inserted into pMSIsGFP binary vector between 35S promoter and a NOS terminator. NPT II gene was used as a selective marker, and sGFP(S65T) gene was used to optimize conditions for transformation by monitoring the expression of green-fluorescent protein. An intron was fused within the N-terminal part of the sGFP(S65T) coding sequence to discriminate between Agrobacterium and plant expression (because bacteria can not splice the intron). RB, right border; LB, left border; NOS-P, nopaline synthase promoter; NOS-T, nopaline synthase terminator; 35S-P, cauliflower mosaic virus (CaMV) 35S promoter; 35S-Ω-P, 35S promoter with additional omega element translational enhancer; NPT II, neomycin phosphotransferase; sGFP(65T), synthetic green-fluorescent protein with S65T mutation; I: intron of castor bean catalase gene CAT-1.