Cytoplasmic expression of a thermostable invertase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.

Results

The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.

Conclusions

Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

     

New molecular evidence of wine yeast-bacteria interaction unraveled by non-targeted exometabolomic profiling

Introduction

Bacterial malolactic fermentation (MLF) has a considerable impact on wine quality. The yeast strain used for primary fermentation can systematically stimulate (MLF+ phenotype) or inhibit (MLF?) bacteria and the MLF process as a function of numerous winemaking practices, but the underlying molecular evidence still remains a mystery.

Objectives

The goal of the study was to elucidate such evidence by the direct comparison of extracellular metabolic profiles of MLF+ and MLF? phenotypes.

Methods

We have applied a non-targeted metabolomic approach combining ultrahigh-resolution FT-ICR-MS analysis, powerful statistical tools and a comprehensive wine metabolite database.

Results

We discovered around 2500 unknown masses and 800 putative biomarkers involved in phenotypic distinction. For the putative biomarkers, we also developed a biomarker identification workflow and elucidated the exact structure (by UPLC-Q-ToF–MS²) and/or exact physiological impact (by in vitro tests) of several novel biomarkers, such as D-gluconic acid, citric acid, trehalose and tripeptide Pro-Phe-Val. In addition to valid biomarkers, molecular evidence was reflected by unprecedented chemical diversity (around 3000 discriminant masses) that characterized both the yeast phenotypes. While distinct chemical families such as phenolic compounds, carbohydrates, amino acids and peptides characterize the extracellular metabolic profiles of the MLF+ phenotype, the MLF? phenotype is associated with sulphur-containing peptides.

Conclusion

The non-targeted approach used in this study played an important role in finding new and unexpected molecular evidence.

     

Identification and characterization of a novel antioxidant peptide from feather keratin hydrolysate

Objectives

To improve the potential value of feather, which is a valuable protein resource, we have separated and identified antioxidant peptide(s) from feather hydrolysate.

Results

Feather hydrolysate was prepared by fermentation with Bacillus subtilis S1–4. Antioxidative peptides were separated by sequential acid precipitation, cation exchange, and reversed-phase fast performance liquid chromatography. Finally, a peptide with antioxidative activity was identified as Ser-Asn-Leu-Cys-Arg-Pro-Cys-Gly by MALDI time-of-flight (TOF)/TOF analysis, and determined to represent a portion of feather keratin near its N-terminal. A synthesized peptide with the same sequence was used to characterize its antioxidative properties, including scavenging free radicals, reducing power, and Fe²⁺ chelation. In terms of the peptide’s amino acid composition, the antioxidative activity might be mainly attributed to Cys and other amino acid residues.

Conclusion

Feather keratin is a good source for the quantitative preparation of antioxidative peptides.

     

Molecular cloning and characterization of <Emphasis Type="Italic">α</Emphasis>-amylase/subtilisin inhibitor from rhizome of <Emphasis Type="Italic">Ligusticum chuanxiong</Emphasis>

Objectives

To clone and characterize a novel bi-functional α-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine.

Results

The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the α-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only α-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin. In addition, the expression level of LASI in rhizome was higher than that in leaf and LASI expression was enhanced by salt, chilling and drought treatment.

Conclusions

This is the first member of the Kunitz-protease inhibitor family identified in traditional Chinese medicine and it might be involved in the plant defense responses against lepidopterous pests, microorganisms and abiotic stresses.

     

Interactions of in vitro selected fluorogenic peptide aptamers with calmodulin

Objectives

We examined the importance of aptamer usage under the same condition as the selection process by employing the previously selected aptamers for calmodulin (CaM) which includes a non-natural fluorogenic amino acid, 7-nitro-2,1,3-benzoxadiazole.

Results

We added five amino acids at the N-terminus which was employed for the selection and then we tested the affinity and selectivity for CaM binding. Surface plasmon resonance and fluorescence measurements showed that the additional amino acids for one of the aptamers drastically improved binding affinity to CaM, indicating the importance of aptamer use under the same conditions as the selection process. Such drastic improvement in affinity was not observed for the sequence which had been reported previously. Nuclear magnetic resonance data identified that the primary binding site is located in a C-terminal of CaM and the additional residues enhance interactions with CaM.

Conclusions

We found that the addition of the common sequence, which was employed for ribosome display, makes the affinity of a selected peptide as strong as the previously reported peptide.

     

The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Objective

α-Galactosidases are widely used in many fields. It is necessary to improve the production of enzymes through microbiological processes. The aim of this study was to construct recombinant Aspergillus niger strains with high α-galactosidase production.

Results

Two recombinant A. niger strains were constructed: AB and AGB. The recombinant AB strain contained the α-galactosidase aglB gene from A. niger with its native AglB signal peptide regulated by the glucoamylase promoter. In the AGB recombinant strain, the AglB signal peptide was replaced with the glucoamylase (GlaA) signal peptide. The extracellular maximum α-galactosidase activity of the AGB strain was 215.7 U/ml and that of the AB strain was 9.8 U/mL. The optimal conditions for α-galactosidase were pH 3.5 and 35 °C.

Conclusions

The GlaA signal peptide substantially increased the yield of secreted α-galactosidase in A. niger. This recombinant strain holds great potential for industrial applications.

     

Intramuscular injection of mechano growth factor E domain peptide regulated expression of memory-related <Emphasis Type="Italic">sod</Emphasis>, miR-134 and miR-125b-3p in rat hippocampus under simulated weightlessness

Objective

To investigate the expression of memory-related antioxidant genes and miRNAs under simulated weightlessness and the regulation of mechano growth factor (MGF) E domain, the peptide preventing nerve damage.

Results

Igf-iea and mgf mRNA levels, expression of antioxidant genes sod1 and sod2 and levels of miR-134 and miR-125b-3p increased in rat hippocampus after 14 days tail suspension to simulate weightlessness which was inhibited with intramuscular injection of E domain peptide. Therefore, administration of MGF E domain peptide could reverse increased expressions of memory-related igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p in rat hippocampus under simulated weightlessness.

Conclusions

MGF may regulate the redox state and miRNA-targeted NR-CREB signaling, and intramuscular injection may be the alternative administration because of its safety, convenience and ability to pass through the blood brain barrier.

     

Differential metabolomic responses of PAMP-triggered immunity and effector-triggered immunity in Arabidopsis suspension cells

Introduction

The rhizobacterial tomato pathogen Pseudomonas syringae pv. tomato str. DC3000 (PstDC3000), like many plant pathogenic bacteria, can elicit hypersensitive response in non-host plant cells. PstDC3000 uses a type III protein secretion system (T3SS) to deliver effector proteins.

Objectives

We compared metabolomic responses of Arabidopsis suspension cells to a wild-type PstDC3000, a T3SS deletion mutant PstDC3000D28E, and a pathogen associated molecular pattern (PAMP) flagellin’s N-terminal domain’s 22-aa peptide (flg22) to obtain metabolomics insights into the plant cell PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI).

Methods

Using targeted HPLC-MRM-MS and untargeted GC-MS approaches, we monitored qualitative and quantitative changes of 312 metabolites in central and specialized metabolic pathways in a time-course study.

Results

The overall metabolomic changes induced by the three treatments included phenylpropanoid, flavonoid, and phytohormone biosynthetic pathways, as well as primary metabolism in amino acid and sugar biosynthesis. In addition to shared metabolites, flg22, PstDC3000D28E and PstDC3000 each caused unique metabolite changes in the course of the development of PTI and ETI.

Conclusion

PstDC3000D28E triggered PTI responses were different from those of flg22. This study has not only revealed the discernible metabolomics features associated with the flg22, PstDC3000D28E and PstDC3000 treatments, but also laid a foundation toward further understanding of metabolic regulation and responses underlying plant PTI and ETI.

     

Secretory expression of a novel human spermatozoa antigen in <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis> and its application to a protein chip

Objective

To produce a recombinant spermatozoa antigen peptide using the E. coli: PhoA system on a protein chip for screening anti-sperm antibodies (ASA).

Results

The purity of the recombinant spermatozoa antigen exceeded 95% after two-step purification, as assessed using SDS-PAGE and HPLC. The diagnostic performance of a protein chip coated with the recombinant antigen peptide was evaluated by examining ASA in 51 infertile patients in comparison with a commercial ELISA kit. The area under the receiver operating characteristic curve (AUC) was 0.944, which indicated that the protein chip coated with recombinant spermatozoa antigen peptide was consistent with ELISA for ASA detection.

Conclusion

A recombinant spermatozoa antigen was expressed in the E. coli PhoA secretory expression system and its potential application for clinical ASA detection was validated.

     

Screening,tandem expression and immune activity appraisal of <Emphasis Type="Italic">Deinagkistrodon acutus</Emphasis> (pit viper) venom mimotopes from a phage display 12-mer peptide library

Objective

To design a specific polyclonal antibody against Deinagkistrodon acutus venom (DA-pAb) by immunizating New Zealand white rabbits.

Results

The IgG fraction was purified by affinity chromatography, and specific antibodies were purified by immunoaffinity chromatography. Polyclonal antibodies were subjected to ELISA and western blotting to evaluate their immune reactivity. We identified the mimotopes by screening a phage display 12-mer peptide library against D. acutus venom. After three rounds of biopanning with DA-pAb, 30 positive clones were identified. Eighteen phage clones were sequenced, and their corresponding amino acid sequences were deduced. Additional immunoassays with the peptides and DA-pAb identified five sequences as possible epitopes. Recombinant antigens synthesized with the five epitopes were used for the immunization of BALB/c mice.

Conclusion

The antibodies induced by these peptides recognized the recombinant antigen and D. acutus venom and protected mice against the hemorrhagic effects of the venom.

     

Characterization of an inhibitor-resistant endo-1,4-β-mannanase from the gut microflora metagenome of <Emphasis Type="Italic">Hermetia illucens</Emphasis>

Objective

Hermetia illucens is a voracious insect scavenger that efficiently decomposes food waste. To exploit novel hydrolytic enzymes from this insect, we constructed a fosmid metagenome library using unculturable H. illucens intestinal microorganisms.

Results

Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone with a product displaying hydrolytic activity. Fosmid sequence analysis revealed a novel mannan-degrading gene (ManEM17) composed of 1371 base pairs, encoding 456 amino acids with a deduced 54 amino acid N-terminal signal peptide sequence. Conceptual translation and domain analysis revealed that sequence homology was highest (46%) with endo-1,4-β-mannosidase of Anaerophaga thermohalophila. Phylogenetic and domain analysis indicated that ManEM17 belongs to a novel β-mannanase containing a glycoside hydrolase family 26 domain. The recombinant protein (rManEM17) was expressed in Escherichia coli, exhibiting the highest activity at 55 °C and pH 6.5. The protein hydrolyzed substrates with β-1,4-glycosidic mannoses; maximum specific activity (5467 U mg^?1) occurred toward locust bean gum galactomannan. However, rManEM17 did not hydrolyze p-Nitrophenyl-β-pyranosides, demonstrating endo-form mannanase activity. Furthermore, rManEM17 was highly stable under stringent conditions, including polar organic solvents as well as chemical reducing and denaturing reagents.

Conclusions

ManEM17 is an attractive candidate for mannan degradation under the high-organic-solvent and protein-denaturing processes in food and feed industries.

     

Profiling,isolation and structure elucidation of specialized acylsucrose metabolites accumulating in trichomes of <Emphasis Type="Italic">Petunia</Emphasis> species

Introduction

Acylsugar specialized metabolites function as defenses against insect herbivores, and are the most abundant specialized metabolites produced in Solanaceous trichomes. Metabolite profiling provides the foundation for determining the genetic basis of specialized metabolism and its evolution.

Objectives

To profile and identify acylsugar specialized metabolites in three Petunia species: P. axillaris, P. integrifolia and P. exserta.

Methods

Metabolites were profiled using ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOF MS). Metabolites were purified using solid phase extraction and HPLC, and structures were established using NMR spectroscopy.

Results

Twenty-eight distinct acylsucrose formulas, representing a sampling of more than 100 different detected chemical forms, were purified from three Petunia species and structures have been proposed based on one- and two-dimensional NMR data. 15 of the 28 purified acylsugars were sucrose pentaesters that possess a malonyl group on the fructose ring. These malonate esters can be readily distinguished from other acylsugars based on distinct masses of pseudomolecular ions and fragment ions generated using multiplexed collision-induced dissociation. Chemical diversity of acylsugars was observed between Petunia species, particularly with respect to the lengths of acyl chains and specific acylation positions.

Conclusions

These findings suggest substrate selectivity of various acyltransferases in Petunia species.

     

Production of an antioxidative peptide from hairy basil seed waste by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To circumvent the time-consuming and costly problems associated with natural product extraction, a potential antioxidative peptide selected from hairy basil waste after oil extraction was produced by recombinant DNA technology.

Results

Because the target peptide is short, the recombinant peptide containing seven repeats of the target sequence, QTFQYSRGWTN, and the DNA fragment coding this sequence was cloned into the pQE-30 Xa expression vector and transformed into Escherichia coli. After 6 h of recombinant peptide expression in E. coli, the target peptide was purified by Ni²⁺ affinity chromatography and gel extraction. The expected 15 kDa recombinant target peptide construct was verified by modified dot blot analysis. Compared with the chemically synthesized peptide, the recombinant peptide revealed significantly higher antioxidant activities (p < 0.05), as determined by DPPH and ABTS radical scavenging assays, and in vitro DNA damage induced by hydroxyl radicals.

Conclusion

This approach provides an alternative to produce an antioxidative peptide that provides a potential scaffold for the further development of antioxidative peptides for industrial applications.

     

Developing a flippase-mediated maker recycling protocol for the oleaginous yeast <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis>

Objectives

To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides.

Results

Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites. When FLP was fused with a native nuclear localization signal (NLS) peptide, the system was found functional. Moreover, R. toruloides recombinant strains expressing the NLS-fused FLP under the control of PADH2, an promoter of alcohol dehydrogenase 2 gene (RHTO_03062), were obtained to realize homologous recombination upon growing in glucose-deficient medium.

Conclusions

We have devised a homologous recombination method for R. toruloides based on the FLP/FRT system, which may facilitate further metabolic engineering and designing advanced cell factories for value-added chemicals.

     

Development of a novel engineered antibody targeting <Emphasis Type="Italic">Neisseria</Emphasis> species

Objectives

A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).

Results

Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.

Conclusions

The recombinant scFv could detect Neisseria strains at 10⁶ CFU/ml.

     

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.

     

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.

     

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.