Evolvement of nutraceutical onion plants engineered for resveratrol biosynthetic pathway.

KEY MESSAGE: Genetically engineered onion expressing codon-optimized VvSTS1 gene accumulated stilbenes and extended life span in yeast and can serve as potential nutraceutical. Resveratrol (RV) is a natural polyphenolic compound found in certain plant species including grapes. RV is well known for its nutraceutical properties and to assuage several disease conditions. Onion is the second most consumed vegetable worldwide and contains large quantities of precursor molecules, malonyl-CoA and para-coumaroyl-CoA that are needed for RV biosynthesis. The present study reports the development of nutraceutical onion by engineering RV biosynthetic pathway. A codon-optimized grapevine synthetic stilbene synthase gene (VvSTS1) was synthesized using native grapevine sequence. Six-week-old healthy yellowish compact nodular calli were co-cultivated with Agrobacterium tumefaciens harbouring pCAMBIA1300-hpt II-CaMV35S-VvSTS1-nos. PCR analysis revealed the presence of VvSTS1 and hpt II genes in putative transgenics. Southern blot analysis confirmed the integration of VvSTS1 gene and independent nature of transformants. LC-ESI-HRMS analysis revealed the accumulation of variable quantities of RV (24.98-50.18 µg/g FW) and its glycosylated form polydatin (33.6-67.15 µg/g FW) in both leaves and bulbs, respectively, indicating the successful engineering of RV biosynthetic pathway into onion. The transgenic onion bulb extracts extended the life span in haploid yeast. The transgenic onion accumulating RV and polydatin, developed for the first of its kind, may serve as a potential nutraceutical resource.

Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties.

KEY MESSAGE: We have developed a unique male-sterility and fertility-restoration system in rice by combining Brassica napus cysteine-protease gene (BnCysP1) with anther-specific P12 promoter of rice for facilitating production of hybrid varieties. In diverse crop plants, male-sterility has been exploited as a useful approach for production of hybrid varieties to harness the benefits of hybrid vigour. The promoter region of Os12bglu38 gene of rice has been isolated from the developing panicles and was designated as P12. The promoter was fused with gusA reporter gene and was expressed in Arabidopsis and rice systems. Transgenic plants exhibited GUS activity in tapetal cells and pollen of the developing anthers indicating anther/pollen-specific expression of the promoter. For engineering nuclear male sterility, the coding region of Brassica napus cysteine protease1 (BnCysP1) was isolated from developing seeds and fused to P12 promoter. Transgenic rice plants obtained with P12-BnCysP1 failed to produce functional pollen grains. The F1 seeds obtained from BnCysP1 male-sterile plants and untransformed controls showed 1:1 (tolerant:sensitive) ratio when germinated on the MS medium supplemented with phosphinothricin (5 mg/l), confirming that the male sterility has been successfully engineered in rice. For male fertility restoration, transgenic rice plants carrying BnCysP1Si silencing system were developed. The pollination of BnCysP1 male-sterile (female-fertile) plants with BnCysP1Si pollen resulted in normal grain filling. The F1 seeds of BnCysP1 × BnCysP1Si when germinated on the MS basal medium containing PPT (5 mg/l) and hygromycin (70 mg/l) exhibited 1:1 (tolerant:sensitive) ratio and the tolerant plants invariably showed normal grain filling. The overall results clearly suggest that the customized male-sterility & fertility-restoration system can be exploited for quality hybrid seed production in various crops.

Engineered Pichia pastoris for enhanced production of S-adenosylmethionine.

A genetically engineered strain of Pichia pastoris expressing S-adenosylmethionine synthetase gene from Saccharomyces cerevisiae under the control of AOX 1 promoter was developed. Induction of recombinant strain with 1% methanol resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band. Further, the recombinant strain showed 17-fold higher enzyme activity over control. Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L. The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine. In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase. The recombinant P pastoris seems promising as potential source for industrial production of S-adenosylmethionine.

Expression of pigeonpea hybrid-proline-rich protein encoding gene (CcHyPRP) in yeast and Arabidopsis affords multiple abiotic stress tolerance.

A hybrid-proline-rich protein encoding gene (CcHyPRP) has been isolated and characterized, for the first time, from the subtracted cDNA library of pigeonpea (Cajanus cajan L.) plants subjected to drought stress. Functionality of CcHyPRP has been validated for abiotic stress tolerance using the heterologous yeast and Arabidopsis systems. The CcHyPRP contained a repetitive proline-rich (PR) N-terminal domain and a conserved eight cysteine motif (8CM) at the C-terminus. Southern analysis disclosed single-copy nature of CcHyPRP gene in the pigeonpea genome. Northern analysis revealed higher levels of CcHyPRP transcripts in PEG, NaCl, heat (42 degrees C), cold and ABA-treated plants compared with the weak signals observed in the untreated plants, suggesting stress-responsive nature of the CcHyPRP gene. In yeast, expression of CcHyPRP imparted marked tolerance against abiotic stresses exerted by PEG, high temperature, NaCl and LiCl. Transgenic Arabidopsis lines, expressing CcHyPRP under the control of CaMV35S and rd29A promoters, when subjected to PEG, mannitol, NaCl, LiCl and heat (42 degrees C) stress, developed into healthy plants with profuse root system and increased biomass in contrast to the weak-stunted wild-type plants. The CcHyPRP-transgenics driven by stress-inducible rd29A exhibited similar stress-tolerance as that of CaMV35S-lines without any negative effects on plant morphology, implying that stress-inducible promoters are preferable for production of stress tolerant transgenics. The overall results amply demonstrate the profound effect of CcHyPRP in bestowing multiple abiotic stress tolerance at cellular and whole plant levels. Accordingly, the multipotent CcHyPRP seems promising as a prime candidate gene to fortify crop plants with abiotic stress tolerance.

Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetase expressed in Escherichia coli.

S-adenosylmethionine (SAM) synthetase (EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine using l-methionine and ATP as substrates. SAM synthetase gene (metE) from Bacillus subtilis was cloned and over-expressed, for the first time, in the heterologus host Escherichia coli as an active enzyme. Size-exclusion chromatography (SEC) revealed a molecular weight of ~180 kDa, suggesting that the enzyme is a homotetramer stabilized by non-covalent interactions. SAM synthetase exhibited optimal activity at pH 8.0 and 45 degrees C with the requirement of divalent cation Mg(2+), and stimulated by the monovalent cation K(+). The enzyme followed sequential mechanism with a V(max) of 0.362 micromol/min/mg, and a K(m) of 920 microM and 260 microM for ATP and l-methionine, respectively. The urea-induced unfolding equilibrium of the recombinant enzyme revealed a multistate process, comprising partially unfolded tetramer, structural dimer, structural monomer and completely unfolded monomer, as evidenced by intrinsic and extrinsic fluorescence, circular dichroism (CD) and SEC. Absence of trimer in the SEC implicates that the enzyme is a dimer of dimer. Concordance between results of SEC and enzyme activity in the presence of urea amply establishes that tetramer alone with intersubunit active site(s) exhibits enzyme activity.