[Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora].

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

Molecular cloning and functional identification of a high-efficiency (+)-borneol dehydrogenase from Cinnamomum camphora (L.) Presl.

Cinnamomum camphora (L.) Presl, rich in terpenoids, is an important commercial plant. The monoterpenes borneol and camphor are highly desired compounds that have been widely and diversely used in medicine and spices since ancient times. However, the key enzymes in the biosynthetic pathway of borneol and camphor in C. camphora remains unknown, which limits access to these natural products. Here, the chirality of borneol and camphor were identified in C. camphora leaves. Besides the main (+)-borneol and (+)-camphor, C. camphora also contains small amounts of (-)-borneol and (-)-camphor. Then, CcBDH3 - an efficient (+)-borneol dehydrogenase (BDH) - was identified that catalyzed (+)-borneol into (+)-camphor in the presence of NAD+. The Km value was 25.1 µM with a kcat value of 5.4 × 10-3 s-1 at pH 8.5 and 30 °C. CcBDH3, which also yields (-)-camphor from (-)-borneol as a substrate, had a Km value of 36.9 µM with a kcat of 2.1 × 10-3 s-1, and pH of 8.0 and temperature of 32 °C. We further compared the conformational specificity of two other reported BDHs, ZSD1 and ADH2, and found that ZSD1 had the highest conversion rate with (-)-borneol. These findings provide a new way for the production of camphor with various optical activities by metabolic engineering, and the identified camphor biosynthesis pathway provides the foundation for using genetic engineering to improve the production and purity of (+)-borneol in planta.

The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase.

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, ß-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.

[Cloning and expression analysis of 5-phosphomevalonate kinase gene (CcPMK) in Cinnamomum camphora].

The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.

[Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (CcDXR1) in Cinnamomum camphora L.) Presl].

1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the second rate-limiting enzyme of terpenoid biosynthesis in the methylerythritol-4-phosphate pathway. According to the transcriptome database of Cinnamomum camphora, the DXR cDNA was cloned by rapid amplification of cDNA ends (RACE) from C. camphora, and was named CcDXR1(GenBank number: KU886266). The ORF of CcDXR1 is composed of 1 413 bp, and it encodes 470 amino acids. The bioinformatics analysis suggests that the molecular weight of the encoded protein is 51.1 kD and the theoretically isoelectric point is 6.62, and there is no signal peptide and transmembrane structure in putative protein. The analysis of sequence alignment and phylogenetic tree showed that the CcDXR1 belonged to the DXR family. The results of the realtime PCR indicated that expression level of CcDXR1 in mature leaves was higher than tender leaves, which in roots was similar to leaves and the lowest in branches. The camphor is divided into five chemotypes, according to the main chemical compounds in C. camphora. It also showed that the expression level of CcDXR1 in borneol C. camphora was highest than that in cineol, iso-nerolidol, camphor and linalool. Our results revealed that the expression level of CcDXR1 exhibits diversity among plant tissues, growth periods and five chemical types, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.

[Cloning and expression analysis of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene in Cinnamomum camphora].

The 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase was the fourth key enzymes in plant terpenoid biosynthesis pathway of methyl erythritol phosphate pathway(MEP). According to the study of Cinnamomum camphora transcriptome data,we abtained the 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene using RT-PCR,and named CcCMK1,then deposited it in GeneBank(Accession number： Ku376098).Bioinformatics analysis showed the open reading frame (ORF) of the CcCMK1 was 1 212 bp.The putative protein encoded 403 amino acids,and its molecular weight was 44.46 kDa and theoretically isoelectric point was 4.99.Transmembrane structure analysis showed that there was no transmembrane structure. Signal peptide analysis showed that it was a non secretory protein, and there was no signal peptide. The subcellular localization showed that the chloroplast was located in the chloroplast.Analysis of the expression of CcCMK1 gene in five chemotypes of C. camphora using Real-time PCR showed its expression level was highest in C. longepaniculatum, and the lowest in Borneol camphor.This research provided a basis for characterizing the key enzyme genes of terpenoid biosynthetic pathway in C. camphora.

Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora.

A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.

Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree.

An iron-superoxide dismutase (SOD) was purified and characterized from the mature seeds of camphor tree (Cinnamomum camphora). The ultraviolet and visible absorption spectra of camphor Fe-SOD showed patterns typical of cambialistic Fe-SODs. The inductively coupled plasma assay indicated that there was 0.5-1 atom of Fe(2+) per camphor Fe-SOD subunit. The cDNA of camphor Fe-SOD, including the coding region and the 3' noncoding region, was obtained by reverse transcription polymerase chain reaction using the total RNA from immature seeds of C. camphora as template and then sequenced. The complete amino acid sequence of camphor Fe-SOD was deduced from the cDNA sequence. The correctness of the amino acid sequence was confirmed by directly sequencing five peptide fragments of the enzyme. The molecular mass calculated for the camphor Fe-SOD subunit from its 204 amino acid residues was 22,930.6 Da, The cDNA of camphor Fe-SOD was cloned into the expression vector PMFT7-5 and then expressed in Escherichia coli strain BL21. The reconstructed Fe- or Mn-SOD was purified to homogeneity through column chromatography. Activity of the Fe- or Mn-SOD was found to be almost equal to that of natural camphor Fe-SOD, which is the first cambialistic SOD isolated from eukaryotic cells.