Molecular cloning and biochemical characterization of isoprene synthases from the tropical trees Ficus virgata, Ficus septica, and Casuarina equisetifolia

Abstract

Three isoprene synthase (IspS) cDNA clones have been isolated from tropical trees (Ficus septica, F. virgata, and Casuarina equisetifolia), and their enzyme properties have been compared with those of Populus alba IspS. Phylogenetic analysis of the deduced amino acid sequences with known monoterpene synthase resolved IspS from F. septica and F. virgata and other IspSs in a clade together with TPS-b clade I, whereas IspS from C. equisetifolia was within another clade, sister to TPS-b clade II. The optimum reaction temperature was 40 °C for the IspSs isolated from the tropical trees, and 45 °C for P. alba IspS. The optimum pH of the IspSs from the tropical trees peaked between pH 8 and pH10 contrasting with the rather broad optimum pH (7.5-10.5) of P. alba IspS. IspSs from F. septica and F. virgata were activated solely by Mg(2+), whereas IspS from C. equisetifolia was dependent more on Mn(2+) than on Mg(2+). Michaelis constant (Km) values of IspSs from tropical trees were lower than that of P. alba IspS. Analysis of inter fragment interaction energy of IspS-substrate complex model and crystal structure of bornyl diphosphate synthase (1N20) found that the coordination geometry of amino acids with higher attraction force is similar at the active site of C. equisetifolia IspS and bornyl diphosphate synthase. These observations suggest the occurrence of another group of IspSs in TPS-b subfamily and extend the knowledge on biochemical regulatory mechanism of isoprene emission from tropical trees.

Functional Genetic Elements for Controlling Gene Expression in Cupriavidus necator H16

“…This was somewhat surprising, since the gene expression data using fluorescence reporter showed that the increase in strength of RBS translated to higher yields of protein. The observed limitation in the isoprene production can be explained by several lines of reasoning: (i) if the overproduction of isoprene synthase is toxic, cells can potentially reduce this effect by either turning down expression or increasing degradation of IspS protein; (ii) due to the inherently high K m of IspS, the reduced availability of substrate can become a limiting factor in the biosynthesis of isoprene ( 49 , 59 ); (iii) since isoprene synthase competes with geranyl pyrophosphate synthase and farnesyl pyrophosphate synthase for DMAPP, the overexpression of IspS can cause a depletion of prenyl phosphates (e.g., undecaprenyl phosphate) required for biosynthesis of various cell wall polymers ( 60 ); (iv) as it has been previously reported that isoprene synthase forms inclusion bodies when overexpressed ( 49 ), protein aggregation can inhibit the enzymatic activity of IspS.…”

Temperature controls on the basal emission rate of isoprene in a tropical tree Ficus septica: exploring molecular regulatory mechanisms

“…Two specific reverse primers (FsIspS‐pro1 and FsIspS‐pro2) were designed close to the 5′‐end of the FsIspS coding region (Oku et al . ). Sequences of primers used in this study are found in Supporting Information Table S3.…”

“…Briefly, genomic DNA was digested with four blunt-end restriction enzymes (Dra 1, Eco RV, Pvu 11 and Stu 1) to yield four libraries that were then ligated to the GenomeWalker adaptor. Two specific reverse primers (FsIspS-pro1 and FsIspS-pro2) were designed close to the 5′-end of the FsIspS coding region (Oku et al 2015). Sequences of primers used in this study are found in Supporting Information Table S3.…”

In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes

“…Sequences of the gene(s) encoding for isoprene synthase (IspS) and/or the isoprene synthase protein have been isolated from a variety of emitter species. These include poplars ( Populus alba , Populus × canescens , Populus tremuloides , Populus trichocarpa , Populus nigra ) [ 93 , 94 , 95 , 96 ], kudzu ( Pueraria montana ) [ 95 ], willow ( Salix glabra ) [Accession JQ943918.1], figs ( Ficus virgata , Ficus septica ), the Australian pine tree ( Casuarina equisetifolia ) [ 97 ], black locust ( Robinia pseudoacacia ), Wisteria sp. [ 63 ], and most recently Arundo donax , a herbaceous species [ 98 ].…”