Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

A novel method for single-grain-based metabolic profiling of Arabidopsis seed

Introduction

In plant metabolomics, metabolite contents are often normalized by sample weight. However, accurate weighing of very small samples, such as individual Arabidopsis thaliana seeds (approximately 20 µg), is difficult, which may lead to irreproducible results.

Objectives

We aimed to establish alternative normalization methods for seed-grain-based comparative metabolomics of A. thaliana.

Methods

Arabidopsis thaliana seeds were assumed to have a prolate spheroid shape. Using a microscope image of each seed, the lengths of major and minor axes were measured by fitting a projected 2-dimensional shape of each seed as an ellipse. Metabolic profiles of individual diploid or tetraploid A. thaliana seeds were measured by our highly sensitive protocol (“widely targeted metabolomics”) that uses liquid chromatography coupled with tandem quadrupole mass spectrometry. Mass spectrometric analysis of 1 µL of solution extract identified more than 100 metabolites. The data were normalized by various seed-size measures, including seed volume (single-grain-based analysis). For comparison, metabolites were extracted from 4 mg of diploid and tetraploid A. thaliana seeds and their metabolic profiles were analyzed by normalization of weight (weight-based analysis).

Results

A small number of metabolites showed statistically significant differences in the single-grain-based analysis compared to weight-based analysis. A total of 17 metabolites showed statistically different accumulation between ploidy types with similar fold changes in both analyses.

Conclusion

Seed-size measures obtained by microscopic imaging were useful for data normalization. Single-grain-based analysis enables evaluation of metabolism of each seed and elucidates the metabolic profiles of precious bioresources by using small amounts of samples.

     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

Plant growth-promoting bacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> FR1 secrets a novel type of extracellular polyhydroxybutyrate polymerase involved in abiotic stress response in plants

Objectives

Identification of novel microbial factors contributing to plant protection against abiotic stress.

Results

The genome of plant growth-promoting bacterium Pseudomonas fluorescens FR1 contains a short mobile element encoding a novel type of extracellular polyhydroxybutyrate (PHB) polymerase (PhbC) associated with a type I secretion system. Genetic analysis using a phbC mutant strain and plants showed that this novel extracellular enzyme is related to the PHB production in planta and suggests that PHB could be a beneficial microbial compound synthesized during plant adaptation to cold stress.

Conclusion

Extracellular PhbC can be used as a new tool for improve crop production under abiotic stress.

     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Degradation of carbazole,dibenzothiophene and polyaromatic hydrocarbons by recombinant <Emphasis Type="Italic">Rhodococcus</Emphasis> sp.

Objectives

With the view of designing a single biocatalyst for biorefining, carbazole dioxygenase was cloned from Pseudomonas sp. and expressed in Rhodococcus sp.

Results

The recombinant, IGTS8, degraded both carbazole and dibenzothiophene at 400 mg/l in 24 h. Maximum carbazole degradation was in 1:1 (v/v) hexadecane/aqueous phase. Anthracene, phenanthrene, pyrene, fluoranthene and fluorine were also degraded without affecting the aliphatic component.

Conclusions

Recombinant Rhodococcus sp. IGTS8 can function as a single biocatalyst for removing major contaminants of fossil fuels viz. dibenzothiophene, carbazole and polyaromatic compounds.

     

Synthesis of rosmarinic acid analogues in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS).

Results

Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues.

Conclusion

This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.

     

<Emphasis Type="Italic">Candida auris</Emphasis>: Antifungal Multi-Resistant Emerging Yeast

Purpose of Review

The purpose of this review is to contribute to the knowledge about the existence of Candida auris as an emerging pathogenic fungus, multi-resistant to antifungal, and causing health care-associated infections (HCAI).

Recent Findings

C. auris emerges as yeast with clonal transmission resistance to three families of commonly used antifungals, mainly azoles (fluconazole and voriconazole), diminishing therapeutic options for the treatment of fungal infections. In 2009, C. auris was isolated for the first time in Japan and by the time of this review, it has been reported in different countries in Africa, America, Asia, and Europe.

Summary

It is important to identify yeasts of the Candida genus up to species, to perform susceptibility tests and to implement surveillance, prevention, and control measures, to minimize the global spread of this fungus, due to its impact on public health.

     

Monoterpene biotransformation by <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Objective

To investigate the biocatalytic potential of Colletotrichum acutatum and Colletotrichum nymphaeae for monoterpene biotransformation.

Results

C. acutatum and C. nymphaeae used limonene, α-pinene, β-pinene, farnesene, citronellol, linalool, geraniol, perillyl alcohol, and carveol as sole carbon and energy sources. Both species biotransformed limonene and linalool, accumulating limonene-1,2-diol and linalool oxides, respectively. α-Pinene was only biotransformed by C. nymphaeae producing campholenic aldehyde, pinanone and verbenone. The biotransformation of limonene by C. nymphaeae yielded 3.34–4.01 g limonene-1,2-diol l^?1, depending on the substrate (R-(+)-limonene, S-(?)-limonene or citrus terpene (an agro-industrial by-product). This is among the highest concentrations already reported for this product.

Conclusions

This is the first report on the biotransformation of these terpenes by Colletotrichum spp. and the biotransformation of limonene to limonene-1,2-diol possibly involves enzymes similar to those found in Grosmannia clavigera.

     

<Emphasis Type="Italic">Enterococcus faecalis</Emphasis> promotes osteoclast differentiation within an osteoblast/osteoclast co-culture system

Objective

To investigate the effects of heat-killed Enterococcus faecalis ATCC 29212 and P25RC clinical strain (derived from an obturated root canal with apical periodontitis) on osteoclast differentiation within an osteoblast/osteoclast co-culture system.

Results

Heat-killed E. faecalis significantly increased the proportion of multinucleated osteoclastic cells (MNCs) within the co-culture system. The IL-6 level was significantly increased upon exposure to heat-killed E. faecalis. Gene expression levels of NFATc1 and cathepsin K were significantly up-regulated compared to the untreated control. EphrinB2 and EphB4 expressions at both the mRNA and protein levels were also significantly upregulated compared to the untreated control.

Conclusions

Heat-killed E. faecalis can induce osteoclast differentiation within the osteoblast/osteoclast co-culture system in vitro, possibly through ephrinB2-EphB4 bidirectional signaling.

     

Asymmetric reduction of ketopantolactone using a strictly (<Emphasis Type="Italic">R</Emphasis>)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy

Objectives

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].

Results

The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.

Conclusions

Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

     

Carbon catabolite repression in <Emphasis Type="Italic">Thermoanaerobacterium saccharolyticum</Emphasis>

Background

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.

Results

We report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.

Conclusion

Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

     

Metabolic engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for linalool production

Objectives

To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool.

Results

Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l^?1 in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l^?1.

Conclusions

Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

     

A metabolomic approach to characterize the acid-tolerance response in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

Introduction

Sinorhizobium meliloti establishes a symbiosis with Medicago species where the bacterium fixes atmospheric nitrogen for plant nutrition. To achieve a successful symbiosis, however, both partners need to withstand biotic and abiotic stresses within the soil, especially that of excess acid, to which the Medicago-Sinorhizobium symbiotic system is widely recognized as being highly sensitive.

Objective

To cope with low pH, S. meliloti can undergo an acid-tolerance response (ATR(+)) that not only enables a better survival but also constitutes a more competitive phenotype for Medicago sativa nodulation under acid and neutral conditions. To characterize this phenotype, we employed metabolomics to investigate the biochemical changes operating in ATR(+) cells.

Methods

A gas chromatography/mass spectrometry approach was used on S. meliloti 2011 cultures showing ATR(+) and ATR(?) phenotypes. After an univariate and multivariate statistical analysis, enzymatic activities and/or reserve carbohydrates characterizing ATR(+) phenotypes were determined.

Results

Two distinctive populations were clearly defined in cultures grown in acid and neutral pH based on the metabolites present. A shift occurred in the carbon-catabolic pathways, potentially supplying NAD(P)H equivalents for use in other metabolic reactions and/or for maintaining intracellular-pH homeostasis. Furthermore, among the mechanisms related to acid resistance, the ATR(+) phenotype was also characterized by lactate production, envelope modification, and carbon-overflow metabolism.

Conclusions

Acid-challenged S. meliloti exhibited several changes in different metabolic pathways that, in specific instances, could be identified and related to responses observed in other bacteria under various abiotic stresses. Some of the observed changes included modifications in the pentose-phosphate pathway (PPP), the exopolysaccharide biosynthesis, and in the myo-inositol degradation intermediates. Such modifications are part of a metabolic adaptation in the rhizobia that, as previously reported, is associated to improved phenotypes of acid tolerance and nodulation competitiveness.

     

Differences in water-use strategies along an aridity gradient between two coexisting desert shrubs (<Emphasis Type="Italic">Reaumuria soongorica</Emphasis> and <Emphasis Type="Italic">Nitraria sphaerocarpa</Emphasis>): isotopic approaches with physiological evidence

Background and aims

Understanding the responses of different plant species to changes in available water sources is critical for accurately modeling and predicting species dynamics. Our study aimed to explore whether there were differences in water-use strategies between the two coexisting shrubs (Reaumuria soongorica and Nitraria sphaerocarpa) in response to different amounts of summer precipitation.

Methods

We conducted 3 years of field observations at three sites along an aridity gradient from the middle to lower reaches of the Heihe River basin, northwestern China. Stable oxygen composition (δ¹⁸O) in plant xylem water, soil water and groundwater were analyzed concurrently with ecophysiological measurements at monthly intervals during the growing seasons.

Results

Water source for coexisting R. soongorica and N. sphaerocarpa did not differ at the sites with high precipitation, but significantly differed in more arid locations. The N. sphaerocarpa was more sensitive to summer precipitation than R. soongorica in terms of predawn water potential, stomatal conductance and foliage carbon-isotope discrimination.

Conclusions

The plants relying on groundwater maintained consistent water use strategies, but not plants that took up precipitation-derived water. We also found that the difference in water source uptake between the coexisting species was more apparent in more arid locations.

     

<Emphasis Type="Italic">Sedum</Emphasis> root foraging in layered green roof substrates

Background and aims

Layered profiles of designed soils may provide long-term benefits for green roofs, provided the vegetation can exploit resources in the different layers. We aimed to quantify Sedum root foraging for water and nutrients in designed soils of different texture and layering.

Methods

In a controlled pot experiment we quantified the root foraging ability of the species Sedum album (L.) and S. rupestre (L.) in response to substrate structure (fine, coarse, layered or mixed), vertical fertiliser placement (top or bottom half of pot) and watering (5, 10 or 20 mm week^?1).

Results

Water availability was the main driver of plant growth, followed by substrate structure, while fertiliser placement only had marginal effects on plant growth. Root foraging ability was low to moderate, as also reflected in the low proportion of biomass allocated to roots (5–13%). Increased watering reduced the proportion of root length and root biomass in deeper layers.

Conclusions

Both S. album and S. rupestre had a low ability to exploit water and nutrients by precise root foraging in substrates of different texture and layering. Allocation of biomass to roots was low and showed limited flexibility even under water-deficient conditions.