Improved monoterpene biotransformation with <Emphasis Type="Italic">Penicillium</Emphasis> sp. by use of a closed gas loop bioreactor

A closed gas loop bioprocess was developed to improve fungal biotransformation of monoterpenes. By circulating monoterpene-saturated process gas, the evaporative loss of the volatile precursor from the medium during the biotransformation was avoided. Penicillium solitum, isolated from kiwi, turned out to be highly tolerant towards monoterpenes and to convert α-pinene to a range of products including verbenone, a valuable aroma compound. The gas loop was mandatory to reproduce the production of 35 mg L⁻¹ verbenone obtained in shake flasks and also in the bioreactor. Penicillium digitatum DSM 62840 regioselectively converted (+)-limonene to the aroma compound α-terpineol, but shake flask cultures revealed a pronounced growth inhibition when initial concentrations exceeded 1.9 mM. In the bioreactor, toxic effects on P. digitatum during biotransformation were alleviated by starting a sequential feeding of non-toxic limonene portions after a preceding growth phase. Closing the precursor-saturated gas loop during the biotransformation allowed for an additional replenishment of limonene via the gas phase. The gas loop system led to a maximum α-terpineol concentration of 1,009 mg L⁻¹ and an average productivity of 8–9 mg L⁻¹ h⁻¹ which represents a doubling of the respective values previously reported. Furthermore, a molar conversion yield of up to 63% was achieved. M. Pescheck and M. A. Mirata have contributed equally to this work.     

Chemical composition and antibacterial potential of Artemisia arborescens L. essential oil

This study was undertaken to characterize the essential oil (EO) of Artemisia arborescens growing wild in Sicily. EO, extracted by steam distillation, was examined for its chemical composition and for its capability to inhibit some food-borne pathogen bacteria. A total of 43 compounds (13 monoterpene hydrocarbons, 14 oxygenated monoterpenes, 10 sesquiterpene hydrocarbons, three oxygenated sesquiterpenes and less amount of other three compounds), which account 93.73% of the total oil, were identified by gas chromatography and gas chromatography–mass spectrometry. Oxygenated monoterpenes (57.32%) constituted the main fraction, with β-thujone as the main compound (45.04%), followed by the sesquiterpene hydrocarbon chamazulene (22.71%). Undiluted EO showed a large inhibition spectrum against strains of Listeria monocytogenes (34 out of 44), whilst it was ineffective against enterobacteria and salmonellas. The minimum inhibition concentration (MIC) was evaluated for the two most sensitive strains (L. monocytogenes 186 and 7BO) at two cellular concentrations (10⁶ and 10⁷ CFU ml⁻¹). The lowest MIC (0.625 μl ml⁻¹, dilution of oil with acetone) was found for strain L. monocytogenes 186 at 10⁶ CFU ml⁻¹.     

Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems

Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO₂ production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μ_max) was 0.02 h⁻¹ and 0.06 h⁻¹ and the half-velocity constant (K _s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO₂ production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g⁻¹ h⁻¹ to 2.1 μg g⁻¹ h⁻¹. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays. Received: 10 September 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Essential‐Oil Variability of Juniperus deltoides R.P.Adams along the East Adriatic Coast – How Many Chemotypes Are There?

The composition of the essential oils isolated from twigs of ten Juniperus deltoides R.P . Adams populations from the east Adriatic coast was determined by GC‐FID and GC/MS analyses. Altogether, 169 compounds were identified, representing 95.6–98.4% of the total oil composition. The oils were dominated by monoterpenes (average content of 61.6%), which are characteristic oil components of species of the Juniperus section. Two monoterpenes, α‐pinene and limonene, were the dominant constituents, comprising on average 46.78% of the essential oils. Statistical methods were deployed to determine the diversity of the terpene classes and the common terpenes between the investigated populations. These statistical analyses revealed the existence of three chemotypes within all populations, i.e., a α‐pinene, limonene, and limonene/α‐pinene type.     

Intraspecific Variation in Oil Components ofBoronia megastigmaNees. (Rutaceae) Flowers

Intraspecific variation in the oil composition ofBoronia megastigmaNees.(Rutaceae) was examined. Boronia absolute is extracted fromblossom primarily for use as a food additive. A major componentis ß-ionone andB. megastigmais one of the commercial,natural sources of this compound. Genotypes superior in productionof ß-ionone and low in monoterpene hydrocarbons weresought from natural populations in the south west of WesternAustralia as part of a breeding programme. Flowers were collectedfrom 25 plants in each of 29 different populations. Blossomwas extracted with ethanol and analysed using a gas liquid chromatographfitted with ionisation detectors. The contents of ß-ionone,dodecyl acetate, -pinene, ß-pinene and limonene inthe oil extract were compared. Intra-population variation wasas great as inter-population variation and no distinct chemotypeswere found. Considerable variation existed in the content ofcomponents. The highest ß-ionone content was 1787mg g^-1f. wt. Some genotypes contained all five components analysed,others lacked one or more of the monoterpenes: -pinene, ß-pineneor limonene. Principle components analysis indicated that contentsof ß-ionone and dodecyl acetate were associated andthat they were distinct from the content of the monoterpenes,which were associated with each other. Natural shading was associatedwith lower levels of monoterpenes but other oils were unaffected.Young plants contained less pinenes than older plants and oldplants contained the most dodecyl acetate. Vigorous plants producedmore pinenes. Red flowers contained the least ß-iononeand dodecyl acetate.Copyright 1999 Annals of Botany Company Boronia megastigma,boronia, Rutaceae, oil, -pinene, ß-pinene, limonene, ß-ionone, dodecyl acetate, monoterpenes, chemotypes.     

Influence of plant growth regulators on volatiles produced by in vitro grown shoots of Agastache rugosa (Fischer & C.A.Meyer) O. Kuntze

The composition of volatile organic compounds emitted by in vitro shoots of Agastache rugosa (Fischer & C.A. Meyer) O. Kuntze (Lamiaceae) was studied using headspace solid-phase microextraction–gas chromatography–mass spectrometry and compared to the those emitted by adult plants and in vitro-germinated seedlings. Shoot-tip explants were cultured on a solid MS medium supplemented with either 4.4 μM 6-benzyladenine (BA), 9.3 μM kinetin, or 0.45 μM thidiazuron and with either 0.57 μM indole-3-acetic acid (IAA) or 0.41 μM picloram. Shoot proliferation was observed in all these treatments. The presence of these plant growth regulators in the culture medium significantly influenced the composition of volatiles as well as morphogenetic responses observed. The number and quality of regenerating shoots and frequency of axillary bud break were highest in medium containing the BA + IAA combination. Sixty-five compounds were identified in the headspace of the in vitro-produced material and plants cultivated in the field. The in vitro shoots emitted both hydrocarbon (limonene, α-pinene) and oxidized (menthone, isomenthone, pulegone) monoterpenes. The composition of monoterpenes differed depending on the type of auxin—rather than cytokinin—in the medium. The emission of phenylallyl compounds, such as estragole, a major compound in field-grown plants, was markedly lower in shoot cultures.     

Several cis-elements including a palindrome involved in pollen-specific activity of SBgLR promoter

SBgLR (Solanum tuberosum genomic lysine-rich) is a pollen-specific gene cloned from potato (Solanum tuberosum L.). The region from −269 to −9 (The A of translation start site “ATG” as +1) of the SBgLR promoter was identified as critical for gene specific expression in pollen grains. Sequence analysis indicates a palindromic sequence “TTTCTATTATAATAGAAA” in the −227 to −209 region, in which two pollen-specific motifs TTTCT and AGAAA surround a unique putative TATA box. Moreover, nine putative pollen-specific motifs are located in the region between the TATA box and ATG. We placed the −227 to −9 region (reserving the palindrome) and the −222 to −9 region (breaking the palindrome) downstream of the CaMV35S enhancer, respectively, to construct two fusion promoters. Histochemical assays in transgenic plants demonstrated that the region from −222 to −9 is necessary and sufficient for pollen-specific expression of the uidA gene. However, the region of −227 to −9 is incapable of driving GUS expression in pollen grains and parts of vegetative tissues. A series of 5′ deletions from −269 to −9 of SBgLR promoter were constructed. A transient expression assay indicated that the region from the −227 to −9 suppressed gfp gene expression in pollen, and a positive regulatory element was present in the region of −253 to −227. The function of the palindromic sequence as a repressor inhibiting gene expression in pollen was further confirmed by the mutated promoter, breaking the palindrome by substituting its 3′-flanking five base pairs, which resumes the reporter gene expression in mature pollen.     

Variability of the Needle Essential Oils of Juniperus deltoides R.P.Adams from Different Populations in Serbia and Croatia

The essential‐oil compositions of one Croatian and three Serbian populations of Juniperus deltoides R.P.Adams have been determined by GC/MS analysis. In total, 147 compounds were identified, representing 97.3–98.3% of the oil composition. The oils were dominated by monoterpenes, which are characteristic components for the species of the section Juniperus. Two monoterpenes, α‐pinene and limonene, were the dominant constituents, with a summed‐up average content of 49.45%. Statistical methods were used to determine the diversity of the terpene classes and the common terpenes between the newly described J. deltoides populations from Serbia and Croatia. Only reports on several specimens from this species have been reported so far, and there are no studies that treat population diversity. Cluster analysis of the oil contents of 21 terpenes showed high correlation with the geographical distribution of the populations, separating the Croatian from the Serbian populations. The comparison of the essential‐oil compositions obtained in the present study with literature data, showed the separation of J. deltoides and J. oxycedrus ssp. oxycedrus from the western Mediterranean region.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

Induced monoterpene and lignin production in mechanically stressed and fungal elicited cultured Cupressus lusitanica cells

Cultured Cupressus lusitanica cells induced by various stresses are thought to produce different complexes of defense chemicals to optimize defense. To compare the induced products of two stimulations, we investigated the emission of monoterpenes, biosynthesis of β-thujaplicin, and accumulation of lignin in mechanically stressed and fungal elicited cultured C. lusitanica cells. Both mechanical stress and fungal elicitor caused emission of qualitatively similar monoterpene blends indicating de novo biosynthesis of these compounds after stimulation, while mechanical stress alone is sufficient to induce fungal elicitor-related monoterpene emission. Sabinene and limonene were the dominant compounds over the time course in both volatile blends. Although the emitted volatile blends were qualitatively similar, the time course and the relative ratios of the constituents of the volatile blends differed with the type of stimulation. While fungal elicited cells produced significant amounts of β-thujaplicin over the 5-day time course, no β-thujaplicin was observed in the mechanically stressed cells. The production of β-thujaplicin was the main dissimilarity of the induced products of these two treatments, suggesting that synthesis of β-thujaplicin is not a general response to all types of stresses, but is a specific response and serves as a strong toxic compound against already invaded fungus. Significantly higher amounts of lignin accumulations were observed in the fungal elicited and mechanically stressed cells on the 5th day after induction. Based on these results, we suggest the composition of induced products was dependent on the method of stimulation.     

Validation of <Emphasis Type="Italic">DGAT1</Emphasis>-<Emphasis Type="Italic">2</Emphasis> polymorphisms associated with oil content and development of functional markers for molecular breeding of high-oil maize

The gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1-2) is a key quantitative trait locus that controls oil content and oleic acid composition in maize kernels. Here we re-sequenced the DGAT1-2 region responsible for oil variation in a maize landrace set and in 155 inbred lines (35 high-oil and 120 normal lines). The high-oil DGAT1-2 allele was present in most Northern Flint and Southern Dent populations but was absent in five of eight Corn Belt Dent open-pollinated populations and in most of the earlier inbred lines. Loss of the high-oil DGAT1-2 allele possibly resulted from genetic drift in the early twentieth century when a few Corn Belt Dent populations were selected for the development of high-grain-yield inbred lines. Association analysis detected significant effects of two PCR-based functional markers (HO06 and DGAT04; developed based on DGAT1-2 polymorphisms) on kernel oil content and oleic acid composition using the 155 inbred lines. Zheng58 and Chang7-2, the parent inbred lines of elite hybrid Zhengdan958, were used to transfer the favorable allele from the high-oil line By804 using marker-assisted backcrossing with the two functional markers. In BC5F2:3 populations, oil content of the three genotypes (−/−, +/−, and +/+) was, respectively, 3.37, 4.20, and 4.61% (Zheng58 recipient line) and 4.14, 4.67, and 5.25% (Chang7-2 recipient line). Oil content of homozygous kernels containing the high-oil DGAT1-2 allele increased by 27–37% compared with recurrent parents. Hence, these functional markers can be used to re-introduce the high-oil DGAT1-2 allele into modern inbred lines for increased oil content through marker-assisted backcrossing.     

A novel wound-responsive cis-element, VWRE, of the vascular system-specific expression of a tobacco peroxidase gene, tpoxN1

The wound-induced expression of tpoxN1, encoding a tobacco peroxidase, is unique because of its vascular system-specific expression and insensitivity to known wound-signal compounds such as jasmonic acid, ethylene, and plant hormones [Sasaki et al. (2002) Plant Cell Physiol 43:108–117]. To study the mechanism of expression, the 2-kbp tpoxN1 promoter region and successive 5′-deletion of the promoter were introduced as GUS fusion genes into tobacco plants. Analysis of GUS activity in transgenic plants indicated that a vascular system-specific and wound-responsive cis-element (VWRE) is present at the −239/−200 region of the promoter. Gel mobility shift assays suggested that a nuclear factor(s) prepared from wounded tobacco stems binds a 14-bp sequence (−229/−215) in the −239/−200 region in a sequence-specific manner. A mutation in this 14-bp region of the −239 promoter fragment resulted in a considerable decrease in wound-responsive GUS activity in transgenic plants. An 11-bp sequence, which completely overlaps with the 14-bp sequence, was found in the 5′ distal region (−420/−410) and is thought to contribute to the wound-induced expression together with the 14-bp. The −114-bp core promoter of the tpoxN1 gene was indispensable for wound-induced expression, indicating that the 14-bp region is a novel wound-responsive cis-element VWRE, which may work cooperatively with other factors in the promoter.     

Induction of multiple shoots in a monopodial orchid hybrid (Aerides vandarum Reichb.f × Vanda stangeana Reichb.f) using thidiazuron and analysis of their genetic stability

The effect of thidiazuron (TDZ) on direct multiple shoot induction in axenic seedlings of a monopodial orchid hybrid Aerides vandarum × Vanda stangeana, using a dual phase culture medium was studied. The culture system consisted of a basal agar solidified half-strength Murashige and Skoog medium overlaid by a liquid fraction of the same composition. Highest regeneration of multiple shoots (15.8 shoots per seedling) was obtained in the medium containing 2% sucrose (w/v) supplemented with 2 mgl⁻¹ TDZ. Genetic stability of the regenerated shoots was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), and restriction fragment length polymorphism of the PCR amplified (PCR-RFLP) nrITS region, as well as those of the coding (matK) and non-coding (trnL-F) regions of the cpDNA. Across the randomly selected mother plant and nine of its regenerated shoots, 2,680 bands were generated by 19 RAPD and 12 ISSR primers, exhibiting monomorphic banding profiles. Homogenous PCR-RFLP profiles were generated for nrITS using four restriction enzymes (REs), matK using five REs, and trnL-F using six REs. These molecular analyses showed no genomic alterations in all regenerated shoots obtained on medium containing 2 mgl⁻¹ TDZ.     

Specific biodegradation of polychlorinated biphenyls (PCBs) facilitated by plant terpenoids

The aim of this study was to examine how plant terpenoids, as natural growth substrates or inducers, would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. Degradation activities of the PCB congeners, 4,4′-dichlorobiphenyl (4,4′-DCBp) and 2,2′-dichlorobiphenyl (2,2′-DCBp), were initially monitored through a resting cell assay technique that could detect their degradation products. The PCB degraders,Pseudomonas sp. P166 andRhodococcus sp. T104, were found to grow on both biphenyl and terpenoids ((S)-(−) limonene,p-cymene and α-terpinene) whereasArthrobacter sp. B1B could not grow on the terpenoids as a sole carbon source. The B1B strain grown on biphenyl exhibited good degradation activity for 4,4′-DCBp and 2,2′-DCBp, while the activity of strains P166 and T104 was about 25% that of the B1B strain, respectively. Concomitant GC analysis, however, demonstrated that strain T104, grown on (S)-(−) limonene,p-cymene and α-terpinene, could degrade 4,4′-DCBp up to 30%, equivalent to 50% of the biphenyl induction level. Moreover, strain T104 grown on (S)-(−) limonene, could also degrade 2,2′-DCBp up to 30%. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate(s) for PCB biodegradation in the environment.     

Effects of ‘Candidatus Phytoplasma asteris’ on the Volatile Chemical Content and Composition of Grindelia robusta Nutt.

Grindelia robusta, a perennial herb, contains an essential oil that is used as an antitussive, sedative, and analgesic agent. During the spring of 2007, ‘Candidatus Phytoplasma asteris’‐related phytoplasmas were identified in plants showing virescence and phyllody symptoms. The qualitative and quantitative composition of the oil of healthy and infected plants was compared by gas chromatography/mass spectrometry. Samples from six symptomatic and five asymptomatic plants tested by nested PCR followed by RFLP analyses confirmed the presence of ‘Ca. P. asteris’ in all symptomatic samples. The oils from healthy and infected plants, obtained by steam distillation, contained 42 components; that of healthy plants contained a higher concentration of monoterpenes, especially limonene and bornyl acetate, which were nearly 50% higher.     

Single-stranded conformation polymorphism (SSCP)-driven indirect sequencing in detection of short deletion

To seek for novel rare and/or sporadic mutations within genomic neighborhood of common −344 C>T (rs2427827) FCER1A proximal promoter polymorphism, which by its prevalence could have masked the presence of less frequent genetic variants in our previous single-stranded conformational polymorphism (SSCP) mutational search study, SSCP screening was repeated using primers fixing −344 C>T variant. The genomic region surrounding −344 C>T polymorphism was confirmed to be fairly conservative and only a single sporadic mutation (present in 1 out of 196 chromosomes), a 6-bp deletion −262 to 257 del CTAGAC, was found. From the methodological point of view, we demonstrated a successful detection of a short-length polymorphism using SSCP screening in a population, in which the same genomic region contained frequent single-nucleotide polymorphic variant. In conjunction with subsequent cloning of aberrantly migrating PCR products and SSCP-driven indirect sequencing of the clones, this method offers a superior (to direct sequencing of PCR products) detection of rare mutations. The cost-effective method applied by us enables a reliable characterization of infrequent (thus present only in heterozygotic form) short-length variance of the sequence which are difficult to interpret by direct sequencing.     

Detection of divergent genes in microbial aCGH experiments

Background  

Array-based comparative genome hybridization (aCGH) is a tool for rapid comparison of genomes from different bacterial strains. The purpose of such analysis is to detect highly divergent or absent genes in a sample strain compared to an index strain. Development of methods for analyzing aCGH data has primarily focused on copy number abberations in cancer research. In microbial aCGH analyses, genes are typically ranked by log-ratios, and classification into divergent or present is done by choosing a cutoff log-ratio, either manually or by statistics calculated from the log-ratio distribution. As experimental settings vary considerably, it is not possible to develop a classical discriminant or statistical learning approach.     

Discrimination of <Emphasis Type="Italic">Oribotritia</Emphasis> species by oil gland chemistry (Acari,Oribatida)

The chemical composition of secretions from opisthonotal (oil) glands in four species of the oribatid mite genus Oribotritia (Mixonomata, Euphthiracaroidea, Oribotritiidae) was compared by means of gas chromatography—mass spectrometry. The secretions of all, O. banksi (from North America) and three Austrian oribotritiids (O. berlesei, O. hermanni, O. storkani), are shown to be based on certain unusual compounds, the iridoid monoterpenes chrysomelidial and epi-chrysomelidial and the diterpene β-springene. These components probably represent general chemical characteristics of oribotriid oil glands. Their relative abundance in the secretions along with further components (mainly saturated and unsaturated C₁₃-, C₁₅-, C₁₇-hydrocarbons, and the tentatively identified octadecadienal) led to well-distinguishable, species-specific oil gland secretions profiles. In addition a reduced set of “Astigmata compounds” (sensu Sakata and Norton in Int J Acarol 27:281–291, 2001)—namely the two monoterpenes neral and geranial—could be detected in extracts of O. banksi nevertheless indicating the classification of euphthiracaroids within the (monophyletic) group of “Astigmata compounds-bearing”-Oribatida. These compounds are considered to be apomorphically reduced in all Austrian species. Our findings emphasize the potential of chemosystematics using oil gland secretion profiles in the discrimination of morphologically very similar, syntopically living or even cryptic oribatid species.