Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

<Emphasis Type="Italic">Sfi</Emphasis>NX: a method for assembly of protein coding sequences with high success rates

Objective

Concatenation of two NdeI–XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli.

Results

A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid.

Conclusion

This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.

     

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.

     

Secreted expression of truncated capsid protein from porcine circovirus type 2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To achieve secreted expression of the truncated capsid protein from porcine circovirus type 2 (PCV2) in Pichia pastoris.

Results

A truncated cap gene (tcap) with a deleted N-terminal nuclear localization signal was optimized and synthesized. Effective secreted expression was achieved in P. pastoris GS115. The high-productive recombinant strain for tCap was grown in a 5 l bioreactor and the productivity of tCap in supernatant reached 250 μg/ml. Furthermore, serum antibody test demonstrated that adjuvant-assisting tCap induced a significant increase of specific PCV2-Cap antibody over time in mice and a similar antibody level in pigs compared with a commercial Cap-based subunit vaccine.

Conclusion

This work establishes a secreted expression strategy in P. pastoris for the production of PCV2 Cap with superior bioactivity, and this strategy might provide potential uses in developing Cap-based subunit vaccine in the future.

     

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.

     

<Emphasis Type="Italic">massPix</Emphasis>: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

     

Enhancement of crystallinity of cellulose produced by <Emphasis Type="Italic">Escherichia coli</Emphasis> through heterologous expression of <Emphasis Type="Italic">bcsD</Emphasis> gene from <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>

Objectives

To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001.

Results

The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD.

Conclusion

The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

     

Development and application of a T7 RNA polymerase-dependent expression system for antibiotic production improvement in <Emphasis Type="Italic">Streptomyces</Emphasis>

Objective

To develop a reliable and easy to use expression system for antibiotic production improvement of Streptomyces.

Results

A two-compound T7 RNA polymerase-dependent gene expression system was developed to fulfill this demand. In this system, the T7 RNA polymerase coding sequence was optimized based on the codon usage of Streptomyces coelicolor. To evaluate the functionality of this system, we constructed an activator gene overexpression strain for enhancement of actinorhodin production. By overexpression of the positive regulator actII-ORF4 with this system, the maximum actinorhodin yield of engineered strain was 15-fold higher and the fermentation time was decreased by 48 h.

Conclusion

The modified two-compound T7 expression system improves both antibiotic production and accelerates the fermentation process in Streptomyces. This provides a general and useful strategy for strain improvement of important antibiotic producing Streptomyces strains.

     

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.

     

Metabolic changes of genetically engineered grapes (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) studied by <Superscript>1</Superscript>H-NMR,metabolite heatmaps and <Emphasis Type="Italic">i</Emphasis>PLS

Introduction

The Deficiens Homologue 9-iaaM (DefH9-iaaM) gene is an ovule-specific auxin-synthesizing gene which is expressed specifically in placenta/ovules and promotes auxin-synthesis. It was introduced into the genome of two grape cultivars Thompson Seedless and Silcora and both transgenic cultivars had an increased number of berries per bunch.

Objectives

This study investigates the down-stream metabolic changes of Silcora and Thompson seedless grape cultivars when genetically modified through the insertion of the DefH9-iaaM gene into their genome.

Methods

The effects of the genetic modification upon the grape metabolome were evaluated through ¹H-NMR and exploratory data analysis. Chemometric tools such as Interval Partial Least Squares regression and metabolite heatmaps were employed for scrutinizing the changes in the transgenic metabolome as compared to the wild type one.

Results

The results show that the pleiotropic effect on the grape metabolome as a function of the gene modifications is relatively low, although the insertion of the transgene caused a decrement in malic acid and proline and an increment in p-coumaric acid content. In addition, the concentration of malic acid was successfully correlated with the number of inserted copies of transgene in the Silcora cultivar, proving that the increased production of berries, promoted by the inserted gene, is achieved at the expense of a decrement in malic acid concentration.

Conclusion

NMR together with chemometrics is able to identify specific metabolites that were up- or down regulated in the genetically engineered plants allowing highlighting alterations in the down-stream metabolic pathways due to the up-stream genetic modifications.

     

Expression on roots and contribution to maize phytostimulation of 1-aminocyclopropane-1-decarboxylate deaminase gene <Emphasis Type="Italic">acdS</Emphasis> in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> F113

Aims

The plant-beneficial bacterium Pseudomonas fluorescens F113 harbours an acdS gene, which enables deamination of 1-aminocyclopropane-1-carboxylate. The impact of abiotic and biotic factors on the expression of this gene was assessed, as well as the plant-beneficial properties of F113 under different soil moistures.

Methods

An acdS-egfp biosensor was constructed in F113, validated in vitro and used to analyse, by microscopy, its expression on roots of Zea mays comparatively to Beta vulgaris. An acdS mutant was constructed and compared with the wild-type to characterize plant-beneficial effects of F113 on maize lines EP1 and FV2, under well-watered and water deficit conditions.

Results

Different patterns of root colonization and acdS expression were observed according to plant genotype. acdS rhizoplane expression was higher on Beta vulgaris, and on maize line FV2 and hybrid PR37Y15 than on maize line EP1 and teosinte. Strain F113 but not its acdS mutant promoted root growth of EP1 under well-watered conditions and germination of FV2 under water deficit conditions.

Conclusions

Maize lines differed in their ability to induce acdS expression and to respond to P. fluorescens F113. The maize line leading to higher acdS expression, FV2, was the one benefiting from inoculation under water deficit.

     

Expression and characterisation of neopullulanase from <Emphasis Type="Italic">Lactobacillus mucosae</Emphasis>

Objectives

To clone and express a neopullulanase gene from Lactobacillus mucosae LM1 in Escherichia coli and characterise the resulting recombinant neopullulanase.

Results

An ORF in L. mucosae corresponding to a neopullulanase was cloned and expressed in E. coli. The predicted amino acid sequence of the neopullulanase contained catalytic sites and conserved motifs that are present in members of the neopullulanase subfamily. The resulting recombinant neopullulanase was efficiently purified by Ni–NTA affinity chromatography. The purified enzyme optimally hydrolyses pullulan at 37 °C and pH 6.0, producing panose as the major reaction product.

Conclusions

To the best of our knowledge, this is the first report of the cloning, expression and characterisation of a neopullulanase gene from a lactic acid bacterium.

     

Role of <Emphasis Type="Italic">PUF60</Emphasis> gene in Verheij syndrome: a case report of the first Chinese Han patient with a de novo pathogenic variant and review of the literature

Background

Verheij syndrome is a rare microdeletion syndrome of chromosome 8q24.3 that harbors PUF60, SCRIB, and NRBP2 genes. Subsequently, loss of function mutations in PUF60 have been found in children with clinical features significantly overlapping with Verheij.

Case presentation

Here we present the first Chinese Han patient with a de novo nonsense variant (c.1357C?>?T, p.Gln453*) in PUF60 by clinical whole exome sequencing. The 5-year-old boy presents with dysmorphic facial features, intellectual disability, and growth retardation but without apparent cardiac, renal, ocular, and spinal anomalies.

Conclusions

Our finding contributes to the understanding of the genotype and phenotype in PUF60 related disorder.

     

Disruption of <Emphasis Type="Italic">YLR162W</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> results in increased tolerance to organic solvents

Objective

To identify a novel gene responsible for organic solvent-tolerance by screening a transposon-mediated deletion mutant library based on Saccharomyces cerevisiae L3262.

Results

One strain tolerant of up to 0.5 % (v/v) n-hexane and cyclohexane was isolated. The determination of transposon insertion site identified one gene, YLR162W, and revealed disruption of the ORF of this gene, indicating that organic solvent tolerance can be conferred. Such a tolerant phenotype reverted to the sensitive phenotype on the autologous or overexpression of this gene. This transposon mutant grew faster than the control strain when cultured at 30 °C in YPD medium containing 0.5 % (v/v) n-hexane and cyclohexane respectively.

Conclusion

Disruption of YLR162W in S. cerevisiae results in increased tolerance to organic solvents.

     

The kinase MSK1 is required for induction of c-<Emphasis Type="Italic">fos</Emphasis> by lysophosphatidic acid in mouse embryonic stem cells

Background

The regulation of the immediate-early gene c-fos serves as a paradigm for signal-activated gene induction. Lysophosphatidic acid is a potent serum-borne mitogen able to induce c-fos.

Results

Analysing the signalling events following stimulation of mouse embryonic stem cells with serum and lysophosphatidic acid, we show that the extracellular signal-regulated kinase (ERK) pathway is involved in mediating c-fos induction. We demonstrate that the ERK-activated kinase MSK1 is required for full c-fos promoter activation, as well as for the phosphorylation of cAMP-responsive element (CRE) binding proteins. We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins.

Conclusion

These results show that MSK1 is an important ERK-activated mediator of mitogen-stimulated c-fos induction. In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation. Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos.

     

Two acetyl-CoA synthetase isoenzymes are encoded by distinct genes in marine yeast <Emphasis Type="Italic">Rhodosporidium diobovatum</Emphasis>

Objectives

Two genes encoding two acetyl-CoA synthetase (ACS) isoenzymes have been identified in the marine yeast Rhodosporidium diobovatum MCCC 2A00023.

Results

ACS1 encoded a polypeptide with a sequence of 578 amino acid residues, a predicted molecular weight of 63.73 kDa, and pI of 8.14, while the ACS2 encoded a polypeptide containing 676 amino acid residues with a deduced molecular mass of 75.61 kDa and a pI of 5.95. Biological activity of Acs1p and Acs2p was confirmed by heterologous expression in Escherichia coli. A 1.5-kb DNA fragment of the ACS1 gene and a 2.7-kb DNA fragment of the ACS2 gene were deleted using the RNA guide CRISPR-Cas9 system. The strain lacking ACS1 was unable to grow on acetate and ethanol media, while the ACS2 deletant was unable to grow on glucose medium. ACS1-ACS2 double mutants of R. diobovatum were non-viable.

Conclusions

ACS isoenzymes are essential to the yeast metabolism, and other sources of ACSs cannot compensate for the lack of ACSs encoded by the two genes.

     

A versatile <Emphasis Type="Italic">Trichoderma reesei</Emphasis> expression system for the production of heterologous proteins

Objectives

To develop a versatile Trichoderma reesei (teleomorph Hypocrea jecorina) expression system for the high-purity production of heterologous proteins.

Results

The versatile T. reesei expression system is based on xyn1 and xyn2 promoters, A824V transition in XYRI, and a bicomponent carbon source strategy. Red fluorescent protein gene rfp and alkaline endoglucanase EGV gene egv3 from Humicola insolens were used as reporter genes to test our versatile expression system

Conclusions

The versatile T. reesei expression system can be applied to produce heterologous proteins with high purity and high yield.