The rumen microbial metagenome associated with high methane production in cattle

Acknowledgements The Rowett Institute of Nutrition and Health and SRUC are funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government. The project was supported by Defra and the DA funded Agricultural Greenhouse Gas Inventory Research Platform, the Technology Strategy Board (Project No: TP 5903–40240) and the Biotechnology and Biological Sciences Research Council (BBSRC; BB/J004243/1, BB/J004235/1). Our thanks are due to the excellent support staff at the SRUC Beef and Sheep Research Centre, Edinburgh, and to Silvia Ramos Garcia for help in interrogating the data. MW and RR contributed equally to the paper and should be considered as joint last authors.Peer reviewedPublisher PD

Supplementary Material for: Accessing Carboxylesterase Diversity from Termite Hindgut Symbionts through Metagenomics

A shotgun metagenomic library was constructed from termite hindgut symbionts and subsequently screened for esterase activities. A total of 68 recombinant clones conferring esterolytic phenotypes were identified, of which the 14 most active were subcloned and sequenced. The nucleotide lengths of the esterase-encoding open reading frames (ORFs) ranged from 783 to 2,592 bp and encoded proteins with predicted molecular masses of between 28.8 and 97.5 kDa. The highest identity scores in the GenBank database, from a global amino acid alignment ranged from 39 to 83%. The identified ORFs revealed the presence of the G-X-S-X-D, G-D-S-X, and S-X-X-K sequence motifs that have been reported to harbour a catalytic serine residue in other previously reported esterase primary structures. Five of the ORFs (EstT5, EstT7, EstT9, EstT10, and EstT12) could not be classified into any of the original eight esterase families. One of the ORFs (EstT9) showed a unique primary structure consisting of an amidohydrolase-esterase fusion. Six of the 14 esterase-encoding genes were recombinantly expressed in <i>Escherichia coli</i> and the purified enzymes exhibited temperature optima of between 40–50°C. Substrate-profiling studies revealed that the characterised enzymes were ‘true’ carboxylesterases based on their preferences for short to medium chain length <i>p</i>-nitrophenyl ester substrates. This study has demonstrated a successful application of a metagenomic approach in accessing novel esterase-encoding genes from the gut of termites that could otherwise have been missed by classical culture enrichment approaches

Sustainability assessment of selected biowastes as feedstocks for biofuel and biomaterial production by emergy evaluation in five African countries

11siAfrica is a continent with enormous natural resources in the form of biomass and innovative ways are needed to exploit those ones available from agricultural processes and other production systems. This paper aims to assess the sustainability of a set of potential feedstocks for the production of biofuels and other value added products in Egypt, Ghana, Kenya, Morocco and South Africa. These feedstocks are residues from agricultural and industrial food processing systems that we assess by emergy evaluation for insights into their sustainability. The feedstocks are grouped into sugar-rich (corn stover, cassava peels, pineapple peels, olive oil pomace and rejected bananas) and nutrient-rich (cocoa pods, discarded cabbage leaves, cattle manure and soybean processing residues). Where possible, comparison is made between traditional and commercial production of the same good. Despite higher environmental impacts, commercial systems were found to perform better in exploiting natural resources. Finally, sugar-and nutrient-rich feedstocks were compared on the basis of glucan and ash content, respectively. Cassava peels and cattle manure gave the best performance from an emergy point of view. This approach enabled emergy evaluation of feedstocks that also considered their potential for the production of useful bioproductsreservedmixedSaladini, Fabrizio; Vuai, Said A.; Langat, Benard K.; Gustavsson, Mathias; Bayitse, Richard; Gidamis, Andrew B.; Belmakki, Mohammed; Owis, Amal S.; Rashamuse, Konanani; Sila, Daniel N.; Bastianoni, SimoneSaladini, Fabrizio; Vuai, Said A.; Langat, Benard K.; Gustavsson, Mathias; Bayitse, Richard; Gidamis, Andrew B.; Belmakki, Mohammed; Owis, Amal S.; Rashamuse, Konanani; Sila, Daniel N.; Bastianoni, Simon

Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics▿†

It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various β-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C β-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities