First isolation of an isoprene synthase gene from poplar and successful expression of the gene in Escherichia coli

Abstract

For the first time, the complete functional gene for isoprene synthase has been isolated from poplar (Populus alba x Populus tremula). The gene was quite similar to known limonene and other monoterpene synthases, but was found to specifically catalyze the formation of isoprene from the precursor dimethylallyl diphosphate with only a marginal activity for the formation of the monoterpene limonene from geranyl diphosphate as compared with limonene synthases. Omitting the part of the gene that putatively encoded the signal peptide necessary for transport into the chloroplast led to an enhanced rate of isoprene formation by the recombinant protein.

Functional Characterization of Nine Norway Spruce TPS Genes and Evolution of Gymnosperm Terpene Synthases of the TPS-d Subfamily

“…In some cases, it was necessary to try several different vectors to optimize expression of active protein. We used a set of four pET-derived vectors for expression of spruce TPS cDNAs including the pSBET vector that codes for additional Arg tRNAs and was previously shown to enhance expression of active TPS enzymes in E. coli , and the pET100/D, pET101/ D, and pQE50 vectors, which were previously shown to work effectively with TPS expression (Miller et al, 2001;Fäldt et al, 2003b). Terpenoid products formed by the recombinant TPS enzymes (Table III) were analyzed using gas chromatography (GC) and mass spectrometry (MS) and/or GC-flame ionization detection (GC-FID).…”

“…Within the TPS-d1 subfamily, the newly characterized (2)-linalool synthase from Norway spruce also clusters closely to these farnesene synthases from Norway spruce and loblolly pine. Similarly, in the TPS-b subfamily this cluster is comprised of E,E-a-farnesene synthase from Malus 3 domestica (AAO22848), the hemiterpene synthase isoprene synthase from Populus alba 3 Populus tremula (Miller et al, 2001), and geraniol synthase from Cinnamomum tenuipilum (CAD29734), and likely the geraniol synthase from Ocimum basilicum (Iijima et al, 2004; not shown). The presence of sesqui-TPS in these mono-TPS subfamilies (and a hemi-TPS in TPS-b) was supported by high bootstrap values and demonstrates a similar evolution of these TPS within both angiosperms and gymnosperms.…”

Rapid Regulation of the Methylerythritol 4-Phosphate Pathway during Isoprene Synthesis

“…Isoprene emission has been hypothesized to provide leaves protection from heat damage (Sharkey and Singsaas, 1995), especially during short heatflecks , and to protect against ozone and other active oxygen species (Loreto and Velikova, 2001;Affek and Yakir, 2002). Isoprene is made in chloroplasts from dimethylallyl diphosphate (DMAPP) by isoprene synthase Fall, 1991, 1995;Schnitzler et al, 1996), an enzyme related to monoterpene synthases (Miller et al, 2001). Monoterpene synthases have a high affinity for their substrate, geranyl diphosphate (K m in the micromolar range), but isoprene synthases examined to date have a much lower affinity (K m s in the millimolar range; Wildermuth and Fall, 1996;Lehning et al, 1999).…”

How well can renewable resources mimic commodity monomers and polymers?

“…Nonetheless, unlike NR biosynthesis, which involves propagation of additional IPP units, a plant-based isoprene synthase converts the DMAPP to isoprene. 82 This process produces 2 g/L/h of isoprene.…”