Recombinant Protein Production in Bacillus Species, Isolation and Methionine Production in Methionine-producing Microorganisms

Linoleic acid isomerase (LAI) is the enzyme that converts linoleic acid to conjugated linoleic acids (CLAs) which appear to be capable of lowering cancer risk and enhancing immunity. There is an interest in developing commercial processes for the production of single isomers of CLA by biotransformation of LA using microbial cells and enzymes. However, the evaluation of these strains suggest that growth and LA isomerase production levels by these anaerobes are insufficient to support economic commercial production of single CLA isomers. A better alternative is to clone the isomerase gene and to generate new production strains using recombinant technology. Bifidobacterium LAI genes were cloned into pET24a(+) as an expression vector and transferred into E. coli BL21 (DE3) as the expression host while Propionibacterium acnes LAI were cloned into Bacillus species as the expression hosts. Unfortunately, the expressed Bifodobacterium LAI formed inclusion bodies and did not exhibit detectable enzymatic activities. Propeptide Staphylococcus hyicus lipase fused with P. acnes LAI expressed in B. megaterium YYBm1 was secreted into the surrounding medium. Based on MALDI-TOF MS results showed that this propeptide S. hyicus lipase still attached with the secreted LAI and might inhibit activity. However, other propeptides (B. subtilis nprE, B. subtilis amyE, B. megaterium nprM) did not protect LAI from proteolytic degradation. Methionine is the first limiting amino acid in poultry feed and must be supplemented to the poultry feed mixture. Many studies attempted to isolate methionine producing microoragnisms from environments and genetically modified by using chemical mutagens. However, genetically-modified organisms are considered unacceptable for use in organic food production. Therefore, wild type strains with methionine-producing ability are necessary for the organic poultry industry. In this study, numerous bacteria were isolated from natural environments that appeared to produce high methionine; however, all of them were identified as pathogens. In addition, bacteria excreted methionine into the surrounding medium and it was concluded that this could be impractical for large scale recovery of methionine. Conversely, isolated yeast strain K1 yielded high methionine content, compared to other yeasts. Based on large subunit rRNA sequences, these isolated strains were identified as Pichia kudriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is recognized as a generally recognized as safe (GRAS) organism. Yeast strain K1 may be suitable as a source of methionine for dietary supplements in organic poultry feed

Establishment, identification, quantification of methanogenic archaea in chicken ceca and methanogenesis inhibition in in vitro chicken ceca by using nitrocompounds

In the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks

High Prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae Detected in the Human Gut Using an Improved DNA Detection Protocol

Background: The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. Methodology/Principal Findings: Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens ( 15 for M. smithii and 15 for M. stadtmanae) yielded a sequence similarity of 99-100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. Conclusions/Significance: In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome

Broadband, site selective and time resolved photoluminescence spectroscopic studies of finely size-modulated Y2O3:Eu3+ phosphors synthesized by a complex based precursor solution method

Undoped and Eu3+-doped cubic yttria (Y2O3) nanophosphors of good crystallinity, with selective particle sizes ranging between 6 and 37 nm and showing narrow size distributions, have been synthesized by a complex-based precursor solution method. The systematic size tuning has been evidenced by transmission electron microscopy, X-ray diffraction, and Raman scattering measurements. Furthermore, size-modulated properties of Eu3+ ions have been correlated with the local structure of Eu3+ ion in different sized Y2O3:Eu3+ nanophosphors by means of steady-state and time-resolved site-selective laser spectroscopies. Time-resolved site-selective excitation measurements performed in the 7F0 ¿ 5D0 peaks of the Eu3+ ions at C2 sites have allowed us to conclude that Eu3+ ions close to the nanocrystal surface experience a larger crystal field than those in the nanocrystal core. Under the site-selective excitation in the 7F0 ¿ 5D0 peaks, energy transfer between the sites has also been observed.Authors are grateful to Ministerio de Ciencia e Innovacion of Spain (MICINN) under The National Program of Materials (MAT2010-21270-C04-02/03/04), the Consolider-Ingenio 2010 Program (MALTA CSD2007-0045), Generalitat Valenciana (GVA-ACOMP-2013-012), and to the EU-FEDER Funds for their financial support. F.J.M. and O.G. are grateful to the Vicerrectorado de Investigacion y Desarrollo of the Universitat Politecnica de Valencia (UPV2011-0914 PAID-05-11 and UPV2011-0966 PAID-06-11). S.F.L-L. wishes to thank MICCIN for an FPI grant (BES-2008-003353). Finally, S. R. wishes to thank Universitat Politecnica de Valencia and Universidad de La Laguna for the financial support during her research stays.Ray, S.; León-Luis, SF.; Manjón Herrera, FJ.; Mollar García, MA.; Gomis Hilario, O.; Rodríguez-Mendoza, UR.; Agouram, S.... (2014). Broadband, site selective and time resolved photoluminescence spectroscopic studies of finely size-modulated Y2O3:Eu3+ phosphors synthesized by a complex based precursor solution method. Current Applied Physics. 14(1):72-81. https://doi.org/10.1016/j.cap.2013.07.027S728114

Extensive microbial and functional diversity within the chicken cecal microbiome

Chickens are major source of food and protein worldwide. Feed conversion and the health of chickens relies on the largely unexplored complex microbial community that inhabits the chicken gut, including the ceca. We have carried out deep microbial community profiling of the microbiota in twenty cecal samples via 16S rRNA gene sequences and an in-depth metagenomics analysis of a single cecal microbiota. We recovered 699 phylotypes, over half of which appear to represent previously unknown species. We obtained 648,251 environmental gene tags (EGTs), the majority of which represent new species. These were binned into over two-dozen draft genomes, which included Campylobacter jejuni and Helicobacter pullorum. We found numerous polysaccharide- and oligosaccharide-degrading enzymes encoding within the metagenome, some of which appeared to be part of polysaccharide utilization systems with genetic evidence for the co-ordination of polysaccharide degradation with sugar transport and utilization. The cecal metagenome encodes several fermentation pathways leading to the production of short-chain fatty acids, including some with novel features. We found a dozen uptake hydrogenases encoded in the metagenome and speculate that these provide major hydrogen sinks within this microbial community and might explain the high abundance of several genera within this microbiome, including Campylobacter, Helicobacter and Megamonas

Group 4 Metallocene and Half-sandwich Derivatives of Spherosilicate: Preparation fromSi8O20(SnMe3)8 and Their Olefin Polymerization Activity

Three metallocene and half-sandwich Group 4 complexes of cuboctameric spherosilicate (Si8O20(TiCp2Cl)8, Si8O20(ZrCp2Cl)8, and Si8O20(TiCp*Cl2)8) were successfully prepared by the exchange reaction of Group 4 metal chlorides with the spherosilicate starting material, Si8O20(SnMe3)8. These complexes were found to be catalytically active in olefin polymerization. Their preparative reactions clearly demonstrate a synthetically useful route towards many other derivatives of spherosilicate through the Si8O20(SnMe3)8 starting material. A new synthesis of the key precursor, octa(trimethyltin) cuboctameric spherosilicate Si8O20(SnMe3)8, was developed. It was found that most of the waters of hydration of the initial hydrate ([NMe4][Si8O20] @ n H2O, = 60! 70) could be removed by heating to 90 ! 100 °C. This “dehydrated” material proved to be synthetically useful and could be exhaustively silylated and stannylated in good to fair yields. The structures of Si8O20(SnMe3)8, Si8O20(SiMe3)8, and Si8O20(TiCp2Cl)8 are reported

Identification and Quantification of Methanogenic Archaea in Adult Chicken Ceca

By using molecular methods for the identification and quantification of methanogenic archaea in adult chicken ceca, 16S rRNA genes of 11 different phylotypes, 10 of which were 99% similar to Methanobrevibacter woesei, were found. Methanogen populations, as assessed by cultivation, and the 16S rRNA copy number were between 6.38 and 8.23 cells/g (wet weight) and 5.50 and 7.19 log(10)/g (wet weight), respectively