Molecular and biochemical characterization of a novel isoprene synthase from Metrosideros polymorpha
Isoprene is a five-carbon chemical that is an important starting material for the synthesis of rubber, elastomers, and medicines. Although many plants produce huge amounts of isoprene, it is very difficult to obtain isoprene directly from plants because of its high volatility and increasing environmental regulations. Over the last decade, microorganisms have emerged as a promising alternative host for efficient and sustainable bioisoprene production. Isoprene synthase (IspS) has received much attention for the conversion of isoprene from dimethylallyl diphosphate (DMAPP). Herein, we isolated a highly expressible novel IspS gene from Metrosideros polymorpha (MpIspS), which was cloned and expressed in Escherichia coli, using a plant cDNA library and characterized its molecular and biochemical properties.
The signal sequence deleted MpIspS was cloned and expressed in E. coli as a 65-kDa monomer. The maximal activity of the purified MpIspS was observed at pH 6.0 and 55 °C in the presence of 5 mM Mn2+. The Km, kcat, and kcat/Km for DMAPP as a substrate were 8.11 mM, 21 min− 1, and 2.59 mM− 1 min− 1, respectively. MpIspS was expressed along with the exogenous mevalonate pathway to produce isoprene in E. coli. The engineered cells produced isoprene concentrations of up to 23.3 mg/L using glycerol as the main carbon source.
MpIspS was expressed in large amounts in E. coli, which led to increased enzymatic activity and resulted in isoprene production in vivo. These results demonstrate a new IspS enzyme that is useful as a key biocatalyst for bioisoprene production in engineered microbes.
KeywordsIsoprene synthase DMAPP Escherichia coli Mevalonate pathway
Isopropyl β- d − 1-thiogalactopyranoside
Isoprene synthase from Metrosideros polymorpha
Isoprene (2-methyl-1,3-butadiene) is a volatile five-carbon terpene that is an important platform chemical in the synthetic chemistry industry for the synthesis of rubber, pharmaceuticals, flavors, and potential biofuels [1, 2, 3, 4, 5]. Almost all isoprene has been produced from petrochemical sources, mainly by direct isolation from C5 cracking fractions or through the dehydrogenation of C5 isoalkanes and isoalkenes [6, 7]. However, the C5 supply has been dependent upon the petroleum industry and the chemical production process is relatively energy-intensive and environment-unfriendly, and the yields may be insufficient for future demands .
To overcome these disadvantages, bioisoprene has become an attractive alternative. Bioisoprene is synthesized by isoprene synthase (IspS; EC 188.8.131.52) from dimethylallyl diphosphate (DMAPP). IspSs have been isolated and characterized from several plants, such as kudzu , poplars , aspen [11, 12], velvet bean , willows , and oaks . Some IspSs have also been also employed to produce isoprene in Escherichia coli [15, 16, 17, 18, 19], Saccharomyces cerevisiae , Synechocystis , and Bacillus subtilis . Although isoprene has been produced from these engineered microorganisms, their production level remains insufficient to satisfy the supply needed by industry . As IspS is the key enzyme for the isoprene biosynthetic pathway needs to be established. Identification of new IspSs may provide enzymes with improved kinetics that will benefit the constructed microbial cell factories for efficient isoprene production.
In this study, our goal was to isolate a new IspS from a plant cDNA library and functionally characterize the IspS in E. coli. We mined transcriptome datasets of various plants to identify an IspS homologue. Among seven plants, a putative IspS was discovered from Metrosideros polymorpha, which emits volatile terpene . However, emission of isoprene or IspS from M. polymorpha has not been reported. The gene encoding the putative IspS from M. polymorpha (MpIspS) was cloned and expressed in E. coli. The biochemical properties of the purified MpIspS, such as the effects of metal ions, pH, temperature, and kinetics, were investigated. Finally, the characterized MpIspS successfully produced isoprene in E. coli harboring an exogenous mevalonate (MVA) pathway. MpIspS could be used as a potential enzyme for the production of isoprene in an E. coli system.
Results and discussion
Discovery of new IspS
Gene cloning, purification, and molecular mass determination
Effects of metals on the activity of MpIspS
Effects of pH and temperature on the activity of MpIspS
Kinetic parameters of MpIspS
Isoprene production by E. coli expressing MpIspS
In this work, we discovered a new IspS by sequence homology searches and identified the gene potentially encoding IspS M. polymorpha. The gene encoding the putative IspS from M. polymorpha was cloned, expressed in E. coli, purified, and characterized. Recombinant MpIspS demonstrated IspS activity with a preference for Mn2+ as a cofactor, and its optimum pH and temperature were 6.0 and 50 °C, respectively. The MpIspS was successfully used to produce isoprene from glycerol in E. coli expressing the heterologous MVA pathway genes. The MpIspS enzyme could be expressed in large amounts in E. coli, which led to increased enzymatic activity and resulted in high isoprene production in the E. coli. Based on our results, further protein engineering of MpIspS toward improved enzyme activity will pave the way for the high-level production of isoprene. Furthermore, engineered MpIspS in combination with the exogenous MVA pathway will be a platform for the enhanced production of isoprene in microorganisms. Overall, this study will provide a new IspS enzyme that is a useful key biocatalyst for isoprene production in engineered microbes.
Chemicals and materials
All chemical reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oligonucleotides and gene synthesis were provided by Macrogen (Seoul, Korea). Restriction endonucleases, polymerases, and DNA cloning kits were purchased from New England Biolabs (Ipswich, MA, USA). DNA preparation and manipulation techniques were carried out according to standard protocols for molecular biology. The kits of PCR product purification, gel extraction, and plasmid preparation were purchased from Promega (Madison, WI, USA). Profinia™ purification kits and all materials for SDS-PAGE were purchased from Bio-Rad (Hercules, CA, USA).
Gene-mining of novel IspS
To mine the novel IspS from the transcriptome datasets of plants belonging the family Fabaceae, Salicaceae, and Myrtaceae, approximately 113 GB of unassembled reads from the transcriptome projects (PRJDB3212, PRJNA275266, PRJNA296824, PRJNA232948, PRJEB10287, PRJNA203514, and PRJEB11301) were downloaded from the Sequence Read Archive website of GenBank. BLASTX searches of individual reads (E-value ≤1e− 10) were performed against an in-house database consisting of IspSs and other terpene synthases retrieved from the GenBank database . Reads assigned to IspS and other terpene synthases were extracted and sorted into sub-datasets of IspS and the other group. Reads in the IspS sub-datasets from each organism were assembled into six contigs using a sequence assembler. Open reading frames from each scaffold were identified using Prodigal v2.6.1 . The predicted open reading frames were BLAST-searched against the UniProt databases . Candidate isoprene synthase sequences were aligned using MUSCLE algorithm in the MEGA6 package program v.6.06 .
Gene synthesis and cloning of MpIspS
The MpIspS (IspS from Metrosideros polymorpha) gene identified from gene mining was codon-optimized for E. coli using the by IDT Codon Optimization Tool (Integrated DNA Technologies Inc., Coralville, IA, USA; http://sg.idtdna.com/CodonOpt) and synthesized by Macrogen co. (Seoul, South Korea) (Additional file 3: Table S1). E. coli C2566 (New England Biolabs) and the pET-28a(+) plasmid (Novagen, Merck KGaA, Darmstadt, Germany) were used as host cells and expression vectors, respectively. The gene encoding MpIspS was amplified by PCR using the synthetic DNA as the template. The MpIspS coding region was cloned between the T7 promoter and terminator in the pET-28a(+) plasmid containing an N-terminal His6 tag. Forward (5′-CGGCAGCCATATGTGTAGTG-3′) and reverse (5′-GGTGGTGCTCGAGTTAACGCGG-3′) primers were designed for the introduction of the NdeI and XhoI restriction sites (underlined), respectively. The PCR product was subcloned into the pET-28a(+) plasmid digested with the same restriction enzymes and then transformed into E. coli C2566. For isoprene production in E. coli, we constructed an MpIspS-expressing plasmid (pTSN-MpIspS). Forward (5′-ACACAGGAGGTTAAACCATGTGTAGTGCTTCCACACAAGT-3′) and reverse (5′-CATGCCTGCAGGTCGACTCTAGATTAACGC-3′) primers were designed and the MpIspS coding region was amplified by PCR using the synthetic DNA as the template. Vector backbone was prepared after digestion of the pTSN plasmid  with NcoI and XbaI, and then the PCR product was ligated into the digested pTSN plasmid using the Gibson Assembly Master Mix (New England Biolabs). For MVA pathway expression, we constructed the pSEVA231-MVA plasmid. The first fragment containing the lacI gene was amplified using forward (5′-TCACACAGGACGAAGCGGCATGCATTTACG-3′) and reverse (5′-GCGTTCGAACGGCAGAATTGCAGCTCATTTCAGAATATTT-3′) primers from the pSNA-MrBBS plasmid containing MVA pathway genes , the second fragment containing MVA pathway genes was amplified using forward (5′-CAATTCTGCCGTTCGAACGCTAATCTAGAGCGCAACGCAA-3′) and reverse (5′-CAGTCACGACAAGAGTTTGTAGAAACGCAA-3′) primers from the pSNA-MrBBS plasmid as template, and the third fragment as vector backbone was amplified using forward (5′-ACAAACTCTTGTCGTGACTGGGAAAACCCT-3′) and reverse (5′- TGCCGCTTCGTCCTGTGTGAAATTGTTATC-3′) primers from the pSEVA231 plasmid . The three fragments were assembled using the Gibson Assembly Master Mix (New England Biolabs). The correctness of the constructed plasmid was confirmed by Sanger sequencing (Macrogen).
The MpIspS expressing cells were harvested from culture broth and disrupted on ice using ultrasonication (Thermo Fisher Scientific, Waltham, MA, USA) in buffer A (50 mM sodium monophosphate, 300 mM NaCl, 10 mM imidazole, and 0.1 mM phenylmethylsulfonyl fluoride as a protease inhibitor). Unbroken cells and cell debris were removed by centrifugation at 14,000 rpm for 10 min at 4 °C, and the supernatants were filtered through a 0.45 μm filter and applied to an IMAC column (Bio-Rad) equilibrated with buffer A. Supernatants collected from lysates were loaded into the Profinia™ Purification System (Bio-Rad). Supernatants were loaded onto a 1-mL IMAC cartridge and washed twice with 5 and 10 mM imidazole buffer A. Proteins were eluted with 250 mM imidazole in buffer A. Imidazole and other salts were removed and changed with 50 mM MOPS buffer (pH 6.0) using a desalting cartridge. The resulting solution was used as the purified MpIspS enzyme. The protein concentration was quantified by the standard Bradford method . The purified proteins were confirmed by SDS-PAGE.
Molecular mass determination of MpIspS
The subunit molecular mass of MpIspS was examined by SDS-PAGE under denaturing conditions, using the proteins of a pre-stained ladder (Bio-Rad) as reference proteins. All protein bands were stained with Coomassie Blue for visualization. The native molecular mass of the enzyme was determined by gel-filtration chromatography on a Superose 12 10/300 GL column (GE Healthcare, Buckinghamshire, UK). The purified enzyme was applied to the column and eluted with 25 mM Tris-HCl (pH 7.4) buffer containing 200 mM NaCl at a flow rate of 1 mL/min. The column was calibrated with apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (13.7 kDa), and carbonic anhydrase (29 kDa) as reference proteins (Sigma-Aldrich), and the native molecular mass of the enzyme was calculated by comparing with the migration length of reference proteins.
Effects of metal ions, pH, and temperature
To evaluate the effect of metal ions on enzyme activity, an enzyme assay was conducted after the treatment with 1 mM ethylenediaminetetraacetic acid at 4 °C for 1 h or after addition a 1 mM concentration of each metal ion (Mn2+, Zn2+, Cu2+, Ni2+, Co2+, Mg2+, Ca2+, or Fe2+). The reactions were performed in 50 mM MOPS buffer (pH 6.0) containing each metal ion at 50 °C. To examine the effect of pH on the activity of MpIspS, the pH was varied between 4.5 and 7.5 using 50 mM sodium citrate (pH 4.5–6.0) and 50 mM MOPS buffer (3-(N-morpholino)propanesulfonic acid; pH 6–7.5) containing 5 mM Mn2+ or 1 mM Mg2+. To investigate the effect of temperature on MpIspS enzyme activity, the temperature was varied from 30 to 60 °C. One unit (U) of MpIspS activity was defined as the amount of enzyme required to produce 1 μM of isoprene per min at 50 °C and pH 6.0.
To measure isoprene concentration as described previously , 50 μL headspace of the sealed serum bottle used for the enzyme reaction or the cultivation of the engineered E. coli was directly injected into the GC system equipped with a flame ionization detector (FID) and an HP-5 column (30 m × 0.320 mm × 0.25 μm) at a flow rate of 1 mL/min. The starting temperature of the oven was maintained at 40 °C for 3 min, then was increased by 10 °C/min to 100 °C, held at 100 °C for 3 min, increased at the rate of 30 °C/min to 200 °C, and then again held at 200 °C for 1 min. Commercial isoprene (Sigma-Aldrich) was used as an external standard for the quantification of isoprene. The retention time (R.T.) of the standard isoprene was 2.8 min.
Various concentrations of DMAPP (0–10 mM) were used to determine the kinetic parameters of the MpIspS enzyme. The reaction was conducted in 50 mM MOPS buffer (pH 6.0) containing 5 mM Mn2+ at 50 °C for 10 min. The amounts of isoprene in the headspace were detected by GC-FID. The enzyme kinetic parameters, K m and kcat values for substrates, were determined by fitting the data to the Michaelis–Menten equation.
Bioisoprene production by E. coli expression with MpIspS
For isoprene production in E. coli, pTSN-MpIspS, and pSEVA231-MVA were co-transformed into E. coli DH5α. Transformants were selected on LB (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) agar plates containing 100 μg/mL ampicillin and 25 μg/mL kanamycin and incubated at 30 °C for 16 h. To prepare the seed culture, a single colony was cultivated in LB media containing 100 μg/mL ampicillin and 25 μg/mL kanamycin at 30 °C for 16 h. Culture for isoprene production was conducted in a sealed bottle containing 500 μL TB medium (24 g/L yeast extract, 12 g/L tryptone, 9.2 g/L K2HPO4, and 2.2 g/L KH2PO4) containing 0.025 mM IPTG, 100 μg/mL ampicillin, 25 μg/mL kanamycin, and 3.5% (w/v) glycerol as the main carbon source at 30 °C for 3 days. After cultivation, isoprene in the headspace was analyzed by GC-FID.
We appreciate the assistance of Dr. Byung Kwon Kim (OmicsPia, Inc., Daejeon, Korea) for bioinformatics analyses. And also, the authors would like to thank Dr. Victor D. Lorenzo for the kind donation of the pSEVA plasmids.
This research was supported by C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2018M3D3A1A01055732), the Intelligent Synthetic Biology Center of the Global Frontier Project (2011–0031944) and the KRIBB Research Initiative Program.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its Additional files.
SY and MK designed and performed experiments and wrote the manuscript. SKK, KKK, and HL performed experiments for isoprene production and revised the manuscript. DK, DL, HK, and SL revised the manuscript and prepared it for submission. All authors reviewed the results and approved the manuscript.
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