Swimming exercise to control precocious maturation in male seabass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>)

Background

Male European seabass, already predominant (~?70%) in cultured stocks, show a high incidence (20–30%) of precocious sexual maturation under current aquaculture practices, leading to important economic losses for the industry. In view of the known modulation of reproductive development by swimming exercise in other teleost species, we aimed at investigating the effects of sustained swimming on reproductive development in seabass males during the first year of life in order to determine if swimming could potentially reduce precocious sexual maturation.

Methods

Pre-pubertal seabass (3.91?±?0.22 g of body weight (BW)) were subjected to a 10 week swimming regime at their optimal swimming speed (U_opt) in an oval-shaped Brett-type flume or kept at rest during this period. Using Blazka-type swim tunnels, U_opt was determined three times during the course of the experiment: 0.66 m s^??1 at 19?±?1 g BW, 10.2?±?0.2 cm of standard length (SL) (week 1); 0.69 m s^??1 at 38?±?3 g BW, 12.7?±?0.3 cm SL (week 5), and also 0.69 m s^??1 at 77?±?7 g BW, 15.7?±?0.5 cm SL (week 9). Every 2 weeks, size and gonadal weight were monitored in the exercised (N?=?15) and non-exercised fish (N?=?15). After 10 weeks, exercised and non-exercised males were sampled to determine plasma 11-ketotestosterone levels, testicular mRNA expression levels of genes involved in steroidogenesis and gametogenesis by qPCR, as well as the relative abundance of germ cells representing the different spermatogenic stages by histological examination.

Results

Our results indicate that sustained swimming exercise at U_opt delays testicular development in male European seabass as evidenced by decreased gonado-somatic index, slower progression of testicular development and by reduced mRNA expression levels of follicle stimulating hormone receptor (fshR), 3-beta-hydroxysteroid dehydrogenase (3βhsd), 11-beta hydroxysteroid dehydrogenase (11βhsd), estrogen receptor-beta (erβ2), anti-mullerian hormone (amh), structural maintenance of chromosomes protein 1B (smc1β), inhibin beta A (inhba) and gonado-somal derived factor 1 (gsdf1) in exercised males as compared with the non-exercised males.

Conclusions

Swimming exercise may represent a natural and non-invasive tool to reduce the incidence of sexually precocious males in seabass aquaculture.

     

Discovery of novel feruloyl esterase activity of BioH in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)

Objectives

To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3.

Results

The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K _m values of 0.48, 6.3, and 1.9 mM, respectively, and k _cat /K _m values of 9.3, 3.8, and 3.8 mM^?1 s^?1, respectively.

Conclusions

Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.

     

4-Vinylphenol production from glucose using recombinant <Emphasis Type="Italic">Streptomyces mobaraense</Emphasis> expressing a tyrosine ammonia lyase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Objectives

To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.

Results

The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s k_cat/K_m value was 0.54 mM ^?1 s^?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l^?1 from 10 g glucose l^?1.

Conclusion

A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

A novel malic enzyme gene, <Emphasis Type="Italic">Mime2</Emphasis>, from <Emphasis Type="Italic">Mortierella isabellina</Emphasis> M6-22 contributes to lipid accumulation

Objective

This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation.

Methods

Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as K_m and V_max for NADP⁺ were determined. The effects of EDTA or metal ions (Mn²⁺, Mg²⁺, Co²⁺, Cu²⁺, Ca²⁺, or Zn²⁺) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively.

Results

The act ivity of MIME2 was significantly increased by Mg²⁺, Ca²⁺, or Mn²⁺ at 0.5 mM but inhibited by Cu²⁺ or Zn²⁺ (p?<?0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the K_m and V_max for NADP⁺ are 0.703 mM and 156.25 μg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15?±?0.24 vs. 2.17?±?0.31 g/L, p?<?0.01).

Conclusions

The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

     

Increased production of plumbagin in <Emphasis Type="Italic">Plumbago indica</Emphasis> root cultures by biotic and abiotic elicitors

Objectives

To evaluate the effects of 12 biotic and abiotic elicitors for increasing the production of plumbagin in Plumbago indica root cultures.

Results

Most elicitors showed minimal effects on the root dry weight, except for 250 mg chitosan l^?1 and 10 mM l-alanine that markedly decreased root biomass by about 40 % compared to the untreated root cultures (5 g l^?1). Treatments with 100 µM AgNO₃ significantly increased intracellular plumbagin production by up to 7.6 mg g^?1 DW that was 4-fold more than the untreated root cultures (1.9 mg g^?1 DW). In contrast, treatments with 150 mg chitosan l^?1, 5 mM l-alanine, and 50 µM 1-naphthol significantly enhanced the extracellular secretion of plumbagin by up to 10.6, 6.9, and 5.7 mg g^?1 DW, respectively, and increased the overall production of plumbagin by up to 12.5, 12.5, and 9.4 mg g^?1 DW, respectively.

Conclusions

Chitosan (150 mg l^?1), l-alanine (5 mM), and 1-naphthol (50 µM) were the best elicitors to enhance plumbagin production in P. indica root cultures.

     

Genome-wide association study of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">Paratuberculosis</Emphasis> infection in Chinese Holstein

Background

Paratuberculosis is a contagious, chronic and enteric disease in ruminants, which is caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection, resulting in enormous economic losses worldwide. There is currently no effective cure for MAP infection or a vaccine, it is thus important to explore the genetic variants that contribute to host susceptibility to infection by MAP, which may provide a better understanding of the mechanisms of paratuberculosis and benefit animal genetic improvement. Herein we performed a genome-wide association study (GWAS) to identify genomic regions and candidate genes associated with susceptibility to MAP infection in dairy cattle.

Results

Using Illumina Bovine 50?K (54,609 SNPs) and GeneSeek HD (138,893 SNPs) chips, two analytical approaches were performed, GRAMMAR-GC and ROADTRIPS in 937 Chinese Holstein cows, among which individuals genotyped by the 50?K chip were imputed to HD SNPs with Beagle software. Consequently, 15 and 11 significant SNPs (P?<?5?×?10^??5) were identified with GRAMMAR-GC and ROADTDRIPS, respectively. A total of 10 functional genes were in proximity to (i.e., within 1?Mb) these SNPs, including IL4, IL5, IL13, IRF1, MyD88, PACSIN1, DEF6, TDP2, ZAP70 and CSF2. Functional enrichment analysis showed that these genes were involved in immune related pathways, such as interleukin, T cell receptor signaling pathways and inflammatory bowel disease (IBD), implying their potential associations with susceptibility to MAP infection. In addition, by examining the publicly available cattle QTLdb, a previous QTL for MAP was found to be overlapped with one of regions detected currently at 32.5?Mb on BTA23, where the TDP2 gene was anchored.

Conclusions

In conclusion, we identified 26 SNPs located on 15 chromosomes in the Chinese Holstein population using two GWAS strategies with high density SNPs. Integrated analysis of GWAS, biological functions and the reported QTL information helps to detect positional candidate genes and the identification of regions associated with susceptibility to MAP traits in dairy cattle.

     

Long-term H<Subscript>2</Subscript> photoproduction from starch by co-culture of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in a repeated batch process

Objectives

To prove the possibility of efficient starch photofermentation in co-culture of heterotrophic and phototrophic bacteria over prolonged period.

Results

Repeated batch photofermentation of starch was demonstrated in co-culture Clostridium butyricum and Rhodobacter sphaeroides under microaerobic conditions. It continued 15 months without addition of new inoculum or pH regulation when using 4–5 g starch l^?1 and 0.04 g yeast extract l^?1. The complete degradation of starch without volatile fatty acids accumulation was shown in this co-culture. The average H₂ yield of 5.2 mol/mol glucose was much higher than that in Clostridium monoculture. The species composition of co-culture was studied by q-PCR assay. The concentration of Clostridium cells in prolonged co-culture was lower than in monoculture and even in a single batch co-culture. This means that Clostridia growth was significantly limited whereas starch hydrolysis still took place.

Conclusion

The prolonged repeated batch photofermentation of starch by co-culture C. butyricum and R. sphaeroides provided efficient H₂ production without accumulation of organic acids under conditions of Clostridia limitation.

     

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlAB–rhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlAB–rhlC increased rhamnolipids production (0.64 ± 0.02 g l^?1) than that in strain only expressing rhlAB–rhlC (0.446 ± 0.009 g l^?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C₁₀–C₁₀.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.

     

Recombinant Expression and Bioactivity Comparison of Four Typical Fungal Immunomodulatory Proteins from Three Main <Emphasis Type="Italic">Ganoderma</Emphasis> Species

Background

More than a dozen of fungal immunomodulatory proteins (FIPs) have been identified to date, most of which are from Ganoderma species. However, little is known about the similarities and differences between different Ganoderma FIPs’ bioactivities. In the current study, two FIP genes termed FIP-gap1 and FIP-gap2 from G. applanatum, along with LZ-8 and FIP-gsi, another two representative Ganoderma FIP genes from G. lucidum and G. sinense were functionally expressed in Pichia. Subsequently, bioactivities of four recombinant Ganoderma FIPs were demonstrated and compared.

Results

All the four Ganoderma FIP genes could be effectively expressed in P. pastoris GS115 at expression levels ranging from 197.5 to 264.3?mg?L^??1 and simply purified by one step chromatography using HisTrap? FF prepack columns. Amino acid sequence analysis showed that they all possessed the FIP conserved fragments. The homologies of different Ganoderma FIPs were from 72.6 to 86.4%. In vitro haemagglutination exhibited that FIP-gap1, FIP-gsi and LZ-8 could agglutinate human, sheep and mouse red blood cells but FIP-gap2 agglutinated none. Besides, the immunomodulation activities of these Ganoderma FIPs were as: rFIP-gap2?>?rFIP-gap1?>?rLZ-8 and rFIP-gsi in terms of proliferation stimulation and cytokine induction on murine splenocytes. Additionally, the cytotoxic activity of different FIPs was: rFIP-gap1?>?rLZ-8?>?rFIP-gsi?>?rFIP-gap2, examined by their inhibition of three human carcinomas A549, Hela and MCF-7.

Conclusions

Taken together, four typical Ganoderma FIP genes could be functionally expressed in P. pastoris, which might supply as feasible efficient resources for further study and application. Both similarities and differences were indeed observed between Ganoderma FIPs in their amino acid sequences and bioactivities. Comprehensively, rFIP-gaps from G. applanatum proved to be more effective in immunomodulation and cytotoxic assays in vitro than rLZ-8 (G. lucidum) and rFIP-gsi (G. sinense).

     

Decreased formation of branched-chain short fatty acids in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> by metabolic engineering

Objectives

To reduce the unpleasant odor during 1-deoxynojirimycin (DNJ) production, the genes of leucine dehydrogenase (bcd) and phosphate butryltransferase (ptb) were deleted from Bacillus amyloliquefaciens HZ-12, and the concentrations of branched-chain short fatty acids (BCFAs) and DNJ were compared.

Results

By knockout of the ptb gene, 1.01 g BCFAs kg^?1 was produced from fermented soybean by HZ-12Δptb. This was a 56% decrease compared with that of HZ-12 (2.27 g BCFAs kg^?1). Moreover, no significant difference was found in the DNJ concentration (0.7 g kg^?1). After further deletion of the bcd gene from HZ-12Δptb, no BCFAs was detected in fermented soybeans with HZ-12ΔptbΔbcd, while the DNJ yield decreased by 26% compared with HZ-12.

Conclusions

HZ-12Δptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell.

     

Effects of chocolate containing <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> strain NTM048 on immune function: a randomized,double-blind,placebo-controlled trial

Background

Previous reports showed that oral administration of Leuconostoc mesenteroides strain NTM048 increases IgA levels and CD4+ T cell population in feces and mice, respectively, as revealed by flow cytometric analysis of splenocytes. This study aimed to evaluate the effect of chocolate supplemented with L. mesenteroides strain NTM048 (>?1.00?×?10⁹?CFU/day, NTM048) on the immune parameters of healthy subjects, using a randomized, placebo-controlled, double-blinded study design.

Methods

Participants (mean age: 46.3?years) ingested 28?g of test food daily, at a time of their own choice, for 4?weeks. The immunological parameters of all participants were evaluated two times (pre- and post- ingestion). At the end of the study, various immunological parameters of the participants were measured and scoring of immunological vigor (SIV) was performed using a comprehensive algorithm.

Results

Ingestion of NTM048-supplemented chocolate significantly improved SIV in the NTM048 group (18.6?±?1.6) compared to that in the placebo group (17.8?±?2.0) after 4?weeks (p?=?0.049). Several immunological parameters (CD8⁺T cells, CD8⁺CD28⁺ T cells, and memory T cells) were significantly elevated in the NTM048 group as compared to the placebo group (all p?<?0.05). In addition, T cell proliferation index at post-ingestion significantly increased compared with that at pre-ingestion in the NTM048 (p?=?0.017) and placebo groups (p?=?0.037), although no differences were observed between the two groups.

Conclusion

Our results suggest that ingestion of chocolate supplemented with NTM048 is effective against the age-related decline in T cell-related immune functions.

Trial registration

UMIN Clinical Trials Registry UMIN000021989. Registered 19 April 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000025321

     

The effects of condensed tannins derived from senescing <Emphasis Type="Italic">Rhizophora mangle</Emphasis> leaves on carbon,nitrogen and phosphorus mineralization in a <Emphasis Type="Italic">Distichlis spicata</Emphasis> salt marsh soil

Background and aims

Low nitrogen negatively affects soil fertility and plant productivity. Glucose-6-phosphate dehydrogenase (G6PDH) and Epichloë gansuensis endophytes are two factors that are associated with tolerance of Achnatherum inebrians to abiotic stress. However, the possibility that E. gansuensis interacts with G6PDH in enhancing low nitrogen tolerance of host grasses has not been examined.

Methods

A. inebrians plants with (E+) and without E. gansuensis (E?) were subjected to different nitrogen concentration treatments (0.1, 1, and 7.5 mM). After 90 days, physiological studies were carried out to investigate the participation of G6PDH in the adaption of host plants to low nitrogen availability.

Results

Low nitrogen retarded the growth of A. inebrians. E+ plants had higher total dry weight, chlorophyll a and b contents, net photosynthesis rate, G6PDH activity, and GSH content, while having lower plasma membrane (PM) NADPH oxidase activity, NADPH/NADP⁺ ratios, and MDA and H₂O₂ than in E? A. inebrians plants under low nitrogen concentration.

Conclusions

The presence of E. gansuensis played a key role in maintaining the growth of the A. inebrians plants under low nitrogen concentration by regulating G6PDH activity and the NADPH/NADP⁺ ratio and improving net photosynthesis rate.

     

Green synthesis of gold nanoparticles by a newly isolated strain <Emphasis Type="Italic">Trichosporon</Emphasis><Emphasis Type="Italic">montevideense</Emphasis> for catalytic hydrogenation of nitroaromatics

Objective

To investigate green synthesis of gold nanoparticles (AuNPs) by Trichosporon montevideense, and to study their reduction of nitroaromatics.

Results

AuNPs had a characteristic absorption maximum at 535 nm. Scanning electron microscopy images revealed that the biosynthesized nanoparticles were attached on the cell surface. X-ray diffraction analysis indicated that the particles formed as face-centered cubic (111)-oriented crystals. The average size of AuNPs decreased from 53 to 12 nm with increasing biomass concentration. The catalytic reduction of 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, o-nitrophenylamine and m-nitrophenylamine (0.1 mM) by NaBH₄ had reaction rate constants of 0.32, 0.44, 0.09, 0.24 and 0.39 min^?1 with addition of 1.45 × 10^?2 mM AuNPs.

Conclusions

An eco-friendly approach for synthesis of AuNPs by T. montevideense is reported for the first time. The biogenic AuNPs could serve as efficient catalysts for hydrogenation of various nitroaromatics.

     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

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.