Cell-Free Expression of a Plant Membrane Protein BrPT2 From Boesenbergia Rotunda.

Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.

Determination of Quercetin and Flavonol Synthase in Boesenbergia rotunda Rhizome.

BACKGROUND AND OBJECTIVE: Flavonols in plants are catalyzed by flavonol synthase (FLS) enzyme. FLS was reported expressed in flowers and fruits, i.e., Dianthus caryophyllus L. (Caryophyllaceae), Petunia hybrida Hort. (Solanaceae), Arabidopsis thaliana L. (Brassicaceae), Citrus unshiu Marc. (Rutaceae). However, none reported about FLS in medicinal plants, particularly those which possess anti-inflammatory activity. This study was aimed to extract and identify FLS in the rhizome of Boesenbergia rotunda (Zingiberaceae) and to determine quercetin in the ethanol extract of the rhizome. MATERIALS AND METHODS: The protein extraction of the rhizome was carried out by employing Laing and Christeller's (2004) and Wang's (2014) methods. The extracted-proteins were separated by using SDS-PAGE, followed by the measurement of FLS intensity by using Gel Analyzer. The FLS-1 of recombinant A. thaliana was employed as the standard. The determination of quercetin in the rhizome was carried out using LC-MS. RESULTS: The FLS occurred as a thick band at 38 kDa with intensity 116-158. The LC chromatogram of the extract indicated a small peak at 7.94 min similar to that of quercetin standard. The MS spectra at 7.94 min indicated that quercetin is present in the B. rotunda rhizome (m/z = 303.0549). The concentration of quercetin in the extract is 0.022% w/v. CONCLUSION: The FLS, an enzyme which plays an important role in producing quercetin, was detected in B. rotunda rhizome planted in Indonesia. As a consequence, quercetin in a small amount, was also quantified in the rhizome of this plant. This report will add a scientific insight of B. rotunda for biological sciences.

A Hedychium coronarium short chain alcohol dehydrogenase is a player in allo-ocimene biosynthesis.

KEY MESSAGE: An enzyme is crucial for the formation of Hedychium coronarium scent and defense responses, which may be responsible for the biosynthesis of allo-ocimene in H. coronarium. Hedychium coronarium can emit a strong scent as its main scent constituents are monoterpenes and their derivatives. Among these derivatives, allo-ocimene is not only a very important volatile substance in flower aroma, but is also crucial to plant defense. However, the molecular mechanism of allo-ocimene biosynthesis has not been characterized in plants. In this study, a new alcohol dehydrogenase gene, HcADH, was cloned. The amino acid sequences encoded by HcADH contained the most conserved motifs of short chain alcohol dehydrogenase/reductases (SDRs), which included NAD+ binding domain, TGxxx[AG]xG and active site YxxxK. Real-time PCR analyses showed that the HcADH was highly expressed in the outer labellum but was almost undetectable in vegetative organs. The change in its expression level in petals was positively correlated with the emission pattern of allo-ocimene during flower development. HcADH expression coincides also the release level of allo-ocimene among different Hedychium species. Although HcADH is not expressed in the leaves, HcADH expression and allo-ocimene release in leaves can be induced by mechanical wounding or methyl jasmonate (MeJA) treatment. In addition, the expression of HcADH induced by mechanical wounding can be prevented by acetylsalicylic acid, a jasmonic acid biosynthesis inhibitor, suggesting that jasmonic acid might participate in the transmission of wounding signals. Using the Barley stripe mosaic virus (BSMV)-VIGS method, it was found that BSMV:HcADH335 inoculation was able to down-regulate HcADH expression, decreasing only the release of allo-ocimene in flowers while the content of other volatile substances did not decrese. In vitro characterization showed that recombinant HcADH can catalyze geraniol into citral, and citral is an intermediate of allo-ocimene biosynthesis. HcADH may be responsible for the biosynthesis of allo-ocimene in H. coronarium, which is crucial for the formation of H. coronarium scent and defense function.

Production of sesquiterpenoid zerumbone from metabolic engineered Saccharomyces cerevisiae.

Zerumbone, the predominant sesquiterpenoid component of Zingiber zerumbet, exhibits diverse pharmacological properties. In this study, de novo production of zerumbone was achieved in a metabolically engineered yeast cell factory by introducing α-humulene synthase (ZSS1), α-humulene 8-hydroxylase (CYP71BA1) and zerumbone synthase variant (ZSD1S114A) from Z. zerumbet, together with AtCPR1 from Arabidopsis thaliana into the yeast strain. Multistep metabolic engineering strategies were applied, including the over-expression of the mevalonate (MVA) pathway rate-limiting enzymes tHMG1 and ERG20, regulation of ERG9 by an inducible promoter and competitive pathway deletion to redirect metabolic flux toward the desired product. In the engineered strain, α-humulene production increased by 18-fold, to 92 mg/L compared to that in the original strain. Five cytochrome P450 reductases (CPRs) from different sources were selected for CYP71BA1 adaptability tests, and AtCPR1 from A. thaliana was found to be the optimal, producing 113.16 µg/L of 8-hydroxy-α-humulene. Multicopy integration of CYP71BA1, AtCPR1, ZSS1 and ICE2 (type III membrane protein) genes resulting in strain LW14 increased the production of 8-hydroxy-α-humulene by 134-fold to 15.2 mg/L. Expressing ZSD1S114A in the ura3 site of strain LW14 resulted in the production of 7 mg/L zerumbone. Multicopy integration of ZSD1S114A increased the production of zerumbone to 20.6 mg/L. The high zerumbone-producing strain was used for batch and fed-batch fermentation in a 5-L bioreactor and zerumbone degradation by yeast was observed; the production of zerumbone finally reached 40 mg/L by fed-batch fermentation in a 5-L bioreactor.

Purification, biochemical characterization and antioxidant property of ZCPG, a cysteine protease from Zingiber montanum rhizome.

Zingiber montanum cysteine protease glycoprotein (ZCPG) was purified to homogeneity by DEAE- cellulose and Sephadex G50 resulting in sixteen fold purification and total activity of 39.4U/mg. ZCPG presented a prominent single peak in HPLC chromatogram with an estimated molecular weight of 48kDa on native PAGE. SDS-PAGE gave two subunits of ∼24.3 and ∼24.6kDa showing its heterodimeric form. Protein sequencing was studied by MALDI-TOF MS/MS. Isoelectrofocusing exhibited two isoforms with pI values of 4.8 and 5.1. Analysis of the total carbohydrate by GC-MS/MS showed the presence of glucose, mannose, fucose and xylose. The pH and temperature optimum were 9 and 60°C respectively while Km and Vmax values were 0.5±0.03µg and 13.73±2.07U/ml respectively. ZCPG was strongly inhibited by NEM indicating the cysteine-type. Substrates such as casein, azocasein, gelatin, BSA and haemoglobin showed high relative activity. Metal ions of CuCl2, CoCl2, HgCl2 and ZnCl2 showed partial inhibition at 1mM concentration. Furthermore, ZCPG exhibited promising antioxidant activity in biochemical systems as well as THP-1 cells. These findings suggested, ZCPG with significant antioxidant activity might have potential applications in therapeutic and food industry.

Fluorescence quenching, structural and unfolding studies of a purified cysteine protease, ZCPG from Zingiber montanum rhizome.

A first attempt was made to study the fluorescence quenching, structure and unfolding nature of the purified Zingiber montanum (J.Koenig) Link ex A.Dietr. cysteine protease glycoprotein (ZCPG). ATR-IR spectra showed the presences of amide groups along with carbohydrate stretch indicating the glycoprotein nature. UV-vis spectra determined the presences of peptide groups and aromatic sidechains of tyrosine, tryptophan and phenylalanine. Far UV-Circular Dichroism spectrum revealed that the secondary structure consists of 47.6% α-helix, 14.1% ß-sheet, 16.1% ß-turn, and 22.2% random coil. CD signals revealed pronounced structural stability until 70°C followed by a significant variation in the secondary structure content in the transition temperature between 80-90°C. ZCPG retained most of its secondary structure in the pH range of 3.0-10.0. The extrinsic study shows that at pH 2.0, ZCPG revealed characteristics of a molten globule-like state exhibiting strong ANS binding. The effect of GdnHCl on ZCPG evaluated by far-CD emission maximum and fluorescence emission revealed that the unfolding was incomplete determining the stability of the protein. The microenvironment of the tryptophan residues indicated the presence of relatively exposed single tryptophan residue (per monomer) with positively charged side chains.

Type III polyketide synthase repertoire in Zingiberaceae: computational insights into the sequence, structure and evolution.

Zingiberaceae or 'ginger family' is the largest family in the order 'Zingiberales' with more than 1300 species in 52 genera, which are mostly distributed throughout Asia, tropical Africa and the native regions of America with their maximum diversity in Southeast Asia. Many of the members are important spice, medicinal or ornamental plants including ginger, turmeric, cardamom and kaempferia. These plants are distinguished for the highly valuable metabolic products, which are synthesised through phenylpropanoid pathway, where type III polyketide synthase is the key enzyme. In our present study, we used sequence, structural and evolutionary approaches to scrutinise the type III polyketide synthase (PKS) repertoire encoded in the Zingiberaceae family. Highly conserved amino acid residues in the sequence alignment and phylogram suggested strong relationships between the type III PKS members of Zingiberaceae. Sequence and structural level investigation of type III PKSs showed a small number of variations in the substrate binding pocket, leading to functional divergence among these PKS members. Molecular evolutionary studies indicate that type III PKSs within Zingiberaceae evolved under strong purifying selection pressure, and positive selections were rarely detected in the family. Structural modelling and protein-small molecule interaction studies on Zingiber officinale PKS 'a representative from Zingiberaceae' suggested that the protein is comparatively stable without much disorder and exhibited wide substrate acceptance.

Characterization of two monoterpene synthases involved in floral scent formation in Hedychium coronarium.

Hedychium coronarium, a perennial herb belonging to the family Zingiberaceae, is cultivated as a garden plant or cut flower as well as for medicine and aromatic oil. Its flowers emit a fresh and inviting scent, which is mainly because of monoterpenes present in the profile of the floral volatiles. However, fragrance produced as a result of monoterpenes has not been well studied. In the present study, two novel terpene synthase (TPS) genes (HcTPS7 and HcTPS8) were isolated to study the biosynthesis of monoterpenes in H. coronarium. In vitro characterization showed that the recombinant HcTPS7 was capable of generating sabinene as its main product, in addition to nine sub-products from geranyl diphosphate (GPP). Recombinant HcTPS8 almost specifically catalyzed the formation of linalool from GPP, while it converted farnesyl diphosphate (FPP) to α-bergamotene, cis-α-bisabolene, ß-farnesene and other ten sesquiterpenes. Subcellular localization experiments revealed that HcTPS7 and HcTPS8 were located in plastids. Real-time PCR analyses showed that HcTPS7 and HcTPS8 genes were highly expressed in petals and sepals, but were almost undetectable in vegetative organs. The changes of their expression levels in petals were positively correlated with the emission patterns of sabinene and linalool, respectively, during flower development. The results indicated that HcTPS7 and HcTPS8 were involved in the biosynthesis of sabinene and linalool in H. coronarium flowers. Results on these two TPSs first characterized from H. coronarium provide new insights into molecular mechanisms of terpene biosynthesis in this species and also lay the basis for biotechnological modification of floral scent profile in Hedychium.

Studies on antioxidant enzymes in Canna indica plant under copper stress.

Bright red-flowered Canna indica L. plants were subjected to grow in nutrient solution supplemented with five different concentrations (0, 5, 10, 30 and 50 microM) of CuCl2 to study antioxidant defense responses of the plant. Accumulation of Cu was dose-dependent and much higher in the roots (108-191 microg g(-1) d. wt.) than in the leaves (23.36-40.43 microg g d.wt.). Total ascorbate content did not changed in both tissues, but ascorbate redox state decreased (0.570-0.640) in Cu-treated Canna roots. In contrast, both total and reduced glutathione contents increased (387-591.9 nmol g(-1) f. wt.) considerably in roots, accompanied with enhanced activities of dehydroascorbate reductase (153.3-160 nmol mg(-1) protein) and glutathione reductase (67-87.5 nmol mg(-1) protein). No significant change, however, was observed for monodehydroascorbate reductase activity in both tissues of the treated plant. The efficient scavenging of hydrogen peroxide was performed by normal (control level) activities of both ascorbate peroxidase and catalase in leaf and increased activity of only catalase in root, preventing its accumulation at toxic concentrations (despite high superoxide dismutase activity) and subsequent damage of membrane lipids by peroxidation. Together, these ensured normal dry weight of leaves and roots, indicating tolerance of Canna indica plant to Cu-induced oxidative stress.

Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids.

Farnesyl pyrophosphate synthase (FPPS EC 2.5.1.10) catalyzes the production of farnesyl pyrophosphate (FPP), which is a key precursor for many sesquiterpenoids such as floral scent and defense volatiles against herbivore attack. Here we report a new full-length cDNA encoding farnesyl diphosphate synthase from Hedychium coronarium. The open reading frame for full-length HcFPPS encodes a protein of 356 amino acids, which is 1068 nucleotides long with calculated molecular mass of 40.7 kDa. Phylogenetic tree analysis indicates that HcFPPS belongs to the plant FPPS super-family and has strong relationship with FPPS from Musa acuminata. Expression of the HcFPPS gene in Escherichia coli yielded FPPS activity. Tissue-specific and developmental analyses of the HcFPPS mRNA and corresponding volatile sesquiterpenoid levels in H. coronarium flowers revealed that the HcFPPS might play a regulatory role in floral volatile sesquiterpenoid biosynthesis. The emission of the FPP-derived volatile terpenoid correlates with strong expression of HcFPPS induced by mechanical wounding and Udaspes folus-damage in leaves, which suggests that HcFPPS may have an important ecological function in H. coronarium vegetative organ.

A short-chain dehydrogenase involved in terpene metabolism from Zingiber zerumbet.

The rhizome oil of Zingiber zerumbet Smith contains an exceptionally high content of sesquiterpenoids with zerumbone, a predominating potential multi-anticancer agent. Biosynthetic pathways of zerumbone have been proposed, and two genes ZSS1 and CYP71BA1 that encode the enzymes catalyzing the first two steps have been cloned. In this paper, we isolated a cDNA clone (ZSD1) that encodes an alcohol dehydrogenase capable of catalyzing the final step of zerumbone biosynthesis. ZSD1 has an open reading frame of 804 bp that encodes a 267-residue enzyme with a calculated molecular mass of 28.7 kDa. After expression in Escherichia coli, the recombinant enzyme was found to catalyze 8-hydroxy-α-humulene into zerumbone. ZSD1 is a member of the short-chain dehydrogenase/reductase superfamily (SDR) and shares high identities with other plant SDRs involved in secondary metabolism, stress responses and phytosteroid biosynthesis. In contrast to the transcripts of ZSS1 and CYP71BA1, which are almost exclusively expressed in rhizomes, ZSD1 transcripts are detected in leaves, stems and rhizomes, suggesting that ZSD1 may also be involved in other biological processes. Consistent with its proposed flexible substrate-binding pocket, ZSD1 also converts borneol to camphor with K(m) and k(cat) values of 22.8 µm and 4.1 s(-1) , displaying its bisubstrate feature.

Isolation and functional characterization of a beta-eudesmol synthase, a new sesquiterpene synthase from Zingiber zerumbet Smith.

In this paper, we have identified a new sesquiterpene synthase gene (ZSS2) from Zingiber zerumbet Smith. Functional expression of ZSS2 in Escherichia coli and in vitro enzyme assay showed that the encoded enzyme catalyzed the formation of beta-eudesmol and five additional by-products. Quantitative RT-PCR analysis revealed that ZSS2 transcript accumulation in rhizomes has strong seasonal variations. To further confirm the enzyme activity of ZSS2 and to assess the potential for metabolic engineering of beta-eudesmol production, we introduced a gene cluster encoding six enzymes of the mevalonate pathway into E. coli and coexpressed it with ZSS2. When supplemented with mevalonate, the engineered E. coli produced a similar sesquiterpene profile to that produced in the in vitro enzyme assay, and the yield of beta-eudesmol reached 100 mg/L.