Monoterpene engineering in a woody plant Eucalyptus camaldulensis using a limonene synthase cDNA

Metabolic engineering aimed at monoterpene production has become an intensive research topic in recent years, although most studies have been limited to herbal plants including model plants such as Arabidopsis. The genus Eucalyptus includes commercially important woody plants in terms of essential oil production and the pulp industry. This study attempted to modify the production of monoterpenes, which are major components of Eucalyptus essential oil, by introducing two expression constructs containing Perilla frutescens limonene synthase ( PFLS ) cDNA, whose gene products were designed to be localized in either the plastid or cytosol, into Eucalyptus camaldulensis . The expression of the plastid-type and cytosol-type PFLS cDNA in transgenic E. camaldulensis was confirmed by real-time polymerase chain reaction (PCR). Gas chromatography with a flame ionization detector analyses of leaf extracts revealed that the plastidic and cytosolic expression of PFLS yielded 2.6- and 4.5-times more limonene than that accumulated in wild-type E. camaldulensis , respectively, while the ectopic expression of PFLS had only a small effect on the emission of limonene from the leaves of E. camaldulensis. Surprisingly, the high level of PFLS in Eucalyptus was accompanied by a synergistic increase in the production of 1,8-cineole and α-pinene, two major components of Eucalyptus monoterpenes. This genetic engineering of monoterpenes demonstrated a new potential for molecular breeding in woody plants.     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

Deoxyhypusine synthase from tobacco. cDNA isolation, characterization, and bacterial expression of an enzyme with extended substrate specificity.

Deoxyhypusine synthase catalyzes the formation of a deoxyhypusine residue in the translation eukaryotic initiation factor 5A (eIF5A) precursor protein by transferring an aminobutyl moiety from spermidine onto a conserved lysine residue within the eIF5A polypeptide chain. This reaction commences the activation of the initiation factor in fungi and vertebrates. A mechanistically identical reaction is known in the biosynthetic pathway leading to pyrrolizidine alkaloids in plants. Deoxyhypusine synthase from tobacco was cloned and expressed in active form in Escherichia coli. It catalyzes the formation of a deoxyhypusine residue in the tobacco eIF5A substrate as shown by gas chromatography coupled with a mass spectrometer. The enzyme also accepts free putrescine as the aminobutyl acceptor, instead of lysine bound in the eIF5A polypeptide chain, yielding homospermidine. Conversely, it accepts homospermidine instead of spermidine as the aminobutyl donor, whereby the reactions with putrescine and homospermidine proceed at the same rate as those involving the authentic substrates. The conversion of deoxyhypusine synthase-catalyzed eIF5A deoxyhypusinylation pinpoints a function for spermidine in plant metabolism. Furthermore, and quite unexpectedly, the substrate spectrum of deoxyhypusine synthase hints at a biochemical basis behind the sparse and skew occurrence of both homospermidine and its pyrrolizidine derivatives across distantly related plant taxa.     

Expression of tobacco cDNA encoding phytochelatin synthase promotes tolerance to and accumulation of Cd and As inSaccharomyces cerevisiae

The first tobacco cDNA encoding phytochelatin synthase (NtPCS1) has been cloned by complementing the YCF1 (vacuolar ABC type transporter)-depleting yeast mutant DTY167 with an expression library fromNicotiana tabacum. When NtPCSI was over-expressed in DTY165 (WT) and DTY167 (mutant), tolerance to and the accumulation of cadmium (Cd) were enhanced. Interestingly, its expression promoted these responses as well to arsenic (As), but only in DTY167. We conclude thatNtPCS1 plays a role in tolerance to and the accumulation of both toxic metals inSaccharomyces cerevisiae. These authors contributed equally to the work.     

Molecular analysis of resveratrol synthase. cDNA, genomic clones and relationship with chalcone synthase

Resveratrol synthase (RS), a key enzyme in biosynthesis of stilbene-type phytoalexins, catalyzes the formation of resveratrol from coumaroyl-CoA and malonyl-CoA. Two cDNA clones, pGSC1 and pGSC2, have been isolated from cDNA libraries established with poly(A)-rich RNA from peanut (Arachis hypogaea) cell cultures specifically induced for RS. These cDNAs were used to identify two genomic clones (pGSG10 and pGSG11). Sequence analysis shows that the two clones overlap in a large stretch of nearly identical sequences, and that pGSG10 contains the 5' and pGSG11 the 3' end of RS genes. The sequences reveal a single intron, and the size of the predicted protein is 42.7 kDa, in close agreement with that observed in polyacrylamide gels (43 kDa). Chalcone synthase (CHS), a key enzyme of flavonoid biosynthesis, utilizes the same substrates as RS, but the product is different (naringenin chalcone). Comparison of RS with CHS consensus sequences shows that the two genes are related. Homology extends throughout the coding region, and the intron in RS is at the same position as a conserved intron in CHS. However, RS reveals a substantial number of amino acid differences to CHS in positions highly conserved in all CHS enzymes. It is proposed that the two proteins possess a common scaffold necessary for binding of the substrates and the type of enzyme reaction, and that the differences are responsible for the formation of different products.     

Sucrose synthase isoforms in cultured tobacco cells.

The plant enzyme sucrose synthase (SuSy; EC 2.4.1.13) catalyzes the reversible conversion of sucrose and UDP into UDP-glucose (UDP-Glc) and fructose. The enzyme exists in different isoforms and is both located in the cytosol, membrane-bound and associated to the actin cytoskeleton. We here investigate sucrose synthase from tobacco (Nicotiana tabacum L.) BY-2 heterotrophic cell suspensions. Two different isoforms of sucrose synthase SuSy1 and SuSy2, could be purified from cytosolic extracts of these cells using a combination of poly(ethylene glycol) (PEG) precipitation, gel filtration, ion-exchange chromatography and affinity chromatography. They were clearly distinct, both with regard to the binding to the ion-exchange column and with regard to their kinetic and regulatory properties. SuSy1, the more abundant species, showed lower V(max) and K(m) for sucrose and UDP compared to the less abundant SuSy2. The activity of SuSy2 in the breakdown direction was stimulated by 60% by actin, in contrast to that of SuSy1, which showed a 17% inhibition. An indication of interaction between SuSy1 and actin was obtained by partitioning in aqueous Dextran-PEG two-phase systems. Furthermore, fructose 2,6-bisphosphate (F26BP) at micromolar concentrations stimulated SuSy2 in the presence of actin while SuSy1 was strongly inhibited by fructose. Possible roles of these two isoforms in the sucrose turnover in BY-2 cells are discussed.     

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400 mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100 mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.     

Cyclitol production in transgenic tobacco

High levels of cyclic sugar alcohols (cyclitols) correlate with tolerance to osmotic stress in a number of plant species. A gene encoding a cyclitol biosynthesis enzyme from a halophyte, Mesembryanthemum crystallinum has been introduced into tobacco. The gene, lmt1 , encodes a myo -inositol O -methyl transferase that, in M. crystallinum , catalyzes the first step in the stress-induced accumulation of the cyclitol pinitol. Tobacco transformed with the lmt1 cDNA under the control of the CaMV 35S promoter appeared phenotypically normal and exhibited IMT1 enzyme activity. Transformants accumulated a carbohydrate product not detectable in non-transformed control plants. This product was identified by HPLC and NMR as ononitol (1- d -4- O -methyl myo -inositol). Ononitol was a major carbohydrate constituent in leaf tissue of plants expressing the lmt1 gene, accumulating to up to 25% the level of sucrose in transformant seedlings. The identification of ononitol as the IMT1 product and the specific accumulation of this compound in transformed tobacco support a role for ononitol as a stable intermediate in pinitol biosynthesis and indicate that an epimerization activity lacking in tobacco is responsible for the conversion of ononitol to pinitol in M. crystallinum . The production of ononitol in tobacco indicates that plant carbohydrate metabolism is flexible and can accommodate the synthesis and accumulation of non-endogenous metabolites. The transgenic system described here will serve as a useful model to test the ability of cyclitols such as ononitol to confer tolerance to environmental stress in a normally glycophytic plant.     

Direct evidence for anthocyanidin synthase as a 2-oxoglutarate-dependent oxygenase: molecular cloning and functional expression of cDNA from a red forma of Perilla frutescens

Anthocyanidin synthase (ANS), an enzyme of the biosynthetic pathway to anthocyanin, has been postulated to catalyze the reaction(s) from the colorless leucoanthocyanidins to the colored anthocyanidins. Although cDNAs have been isolated that encode putative ANS, which exhibits significant similarities in amino acid sequence with members of a family of 2-oxoglutarate-dependent oxygenases, no biochemical evidence has been presented which identifies the actual reaction that is catalyzed by ANS. Here we show that anthocyanidins are formed in vitro through 2-oxoglutarate-dependent oxidation of leucoanthocyanidins catalyzed by the recombinant ANS and subsequent acid treatment. A cDNA encoding ANS was isolated from red and green formas of Perilla frutescens by differential display of mRNA. Recombinant ANS tagged with maltose-binding-protein (MBP) was purified, and the formation of anthocyanidins from leucoanthocyanidins was detected by the ANS-catalyzed reaction in the presence of ferrous ion, 2-oxoglutarate and ascorbate, being followed by acidification with HCI. Equimolar stoichiometry was confirmed for anthocyanidin formation and liberation of CO2 from 2-oxoglutarate. The presumptive two-copy gene of ANS was expressed in leaves and stems of the red forma of P. frutescens but not in the green forma plant. This corresponds to the accumulation pattern of anthocyanin. The mechanism of the reaction catalyzed by ANS is discussed in relation to the molecular evolution of a family of 2-oxoglutarate-dependent oxygenases.     

A flower-specific cDNA encoding a novel thionin in tobacco

Summary A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42° C. A B. subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E. coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins. The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon. No similarity to csaA was found among DNA or protein sequences deposited in databases. In contrast to other homologous or heterologous suppressors of the E. coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E. coli sec Y24(Ts) or lep9(Ts) mutations. However, it restored the ability of a SecB-deficient mutant to grow on complex medium. It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.     

PSTV infection in tobacco (Nicotiana tabacum L.) transformed with PSTV cDNA

Tobacco cv. White Burley was transformed with disarmed expression vector pCB1314 containing dimeric cDNA of potato spindle tuber viroid (PSTV, severe strain) in plus orientation regulated from the mannopine promoter. Amount of PSTV specific (?) and (+) sequences and PSTV circular forms was measured in transformed tobacco stock and compared with PSTV content in untransformed tomato and tobacco grafts. It follows from the results that lower rate of accumulation of PSTV in tobacco as compared with tomato is due to less intensive viroid transportation through the cytoplasm and/or cell to cell moverment, whereas both, viroid replication and processing showed comparable characteristics in tomato and tobacco with respect to accumulation of minus and plus strands and circular forms in infected tissues. Despite of accumulation of viroid in comparable amount in both transformed tobacco and infected tomato, no expression of any morphological symptom of disease was observed in transgenic tobacco.     

Role of tryptophanyl residues in tobacco acetolactate synthase.

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of three classes of herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines. Five mutants (W266F, W439F, W490F, W503F, and W573F) of the ALS gene from Nicotiana tabacum were constructed and expressed in Escherichia coli, and the enzymes were purified. The W490F mutation abolished the binding affinity for cofactor FAD and inactivated the enzyme. The replacement of Trp573 by Phe yielded a mutant ALS resistant to the three classes of herbicides. The other three mutations, W266F, W439F, and W503F, did not significantly affect the enzymatic properties and the sensitivity to the herbicides. These results indicate that the Trp490 residue is essential for the binding of FAD and that Trp573 is located at the herbicide binding site. The data also suggest that the three classes of herbicides bind ALS competitively.     

A methionine synthase homolog is associated with secretory vesicles in tobacco pollen tubes

Seven isoforms of 85 kDa polypeptides (p85) were identified as methionine synthase (MetE) homologs by partial aminoacid sequencing in tobacco pollen tube extracts. Immunocytochemistry data showed a localization of the antigen on the surface of tip-focussed post-Golgi secretory vesicles (SVs), that appear to be partially associated with microtubules (Mts). The chemical dissection of pollen tube high speed supernatant (HSS) showed that two distinct pools of MetE are present in pollen tubes, one being the more acidic isoforms sedimenting at 15S and the remaining at 4S after zonal centrifugation through a sucrose density gradient. The identification of the MetE within the pollen tube and its possible participation as methyl donor in a wide range of metabolic reactions, makes it a good subject for studies on pollen tube growth regulation.     

Roles of conserved methionine residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The conserved methionine residues of ALS from plants were identified by multiple sequence alignment using ClustalW. The alignment of 17 ALS sequences from plants revealed 149 identical residues, seven of which were methionine residues. The roles of three well-conserved methionine residues (M350, M512, and M569) in tobacco ALS were determined using site-directed mutagenesis. The mutation of M350V, M512V, and M569V inactivated the enzyme and abolished the binding affinity for cofactor FAD. Nevertheless, the secondary structure of each of the mutants determined by CD spectrum was not affected significantly by the mutation. Both M350C and M569C mutants were strongly resistant to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine), while M512C mutant did not show a significant resistance to the herbicides. The mutant M350C was more sensitive to pH change, while the mutant M569C showed a profile for pH dependence activity similar to that of wild type. These results suggest that M512 residue is likely located at or near the active site, and that M350 and M569 residues are probably located at the overlapping region between the active site and a common herbicide binding site.