Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome

Applied Microbiology and Biotechnology, Jan 2015

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

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Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome

Florence Priv C Jamie Newbold Naheed N. Kaderbhai Susan G. Girdwood Olga V. Golyshina Peter N. Golyshin Nigel D. Scollan Sharon A. Huws 0 ) Institute of Biological, Environmental and Rural Sciences, Aberystwyth University , Aberystwyth SY23 3DA , UK Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses. - Rumen lipid metabolism plays a significant role in regulating the overall lipid composition of microbial cells and also of meat and milk produced by ruminants (Harfoot and Hazlewood 1997; Scollan et al. 2006; Loureno et al. 2010; Shingfield et al. 2013). The lipid content of forage ingested by ruminants ranges from 2 to 10 % of the total dry weight (Harfoot and Hazlewood 1997), which represent 1.5 kg of ingested lipids through forage per day by dairy cattle (Harfoot 1978). Dietary lipids enter the rumen either as triglycerides (neutral lipids) in concentrate-based feeds or as glycolipids or phospholipids (polar lipids) in forages (Harfoot and Hazlewood 1997; Bauman et al. 2003). Other polar lipids, like sulpholipids, are also present as minor components in forage (<5 %) (Harfoot and Hazlewood 1997). Fresh forage is typically composed of approx. 50 % 18:3 n-3, 15 % 18:2 n-6 and 15 % 16:0 with the rest being minor contributions from other fatty acids (Huws et al. 2009; Huws et al. 2012). Nonetheless, the fatty acid content of meat and milk does not directly correspond to that in their diets, with ruminant products being relatively high in saturated fatty acids. This is due to lipolysis and subsequent biohydrogenation of dietary lipids within the rumen. On entering the rumen, lipids are hydrolyzed by lipases/esterases, which results in the liberation of glycerol and unsaturated and saturated fatty acids. These fatty acids go through microbial biohydrogenation and are transformed to more saturated end products. Indeed, approximately 92 % 18:3 n-3 and 86 % 18:2 n-6 ingested are biohydrogenated in the rumen (Loureno et al. 2010; Huws et al. 2010; Huws et al. 2011; Shingfield et al. 2013; Huws et al. 2014). Research on lipid metabolism in the rumen has largely focused on biohydrogenation of polyunsaturated fatty acids; however, there is a dearth of data on microbial lipolysis, the first step in lipid metabolism in the rumen. Lipolysis is a crucial step in rumen lipid metabolism, and its control could subsequently alter the degree of ruminal biohydrogenation. It is known that dietary lipids are predominantly hydrolyzed in the rumen by obligate anaerobic bacteria (Jenkins et al. 2008), and there is little convincing evidence that rumen protozoa and/or fungi are significantly involved in ruminal lipolysis (Harfoot and Hazlewood 1997; Loureno et al. 2010, Jenkins et al. 2008). However, to date, only six pure cultures of obligately anaerobic, lipolytic bacteria have been isolated from the rumen, including Anaerovibrio lipolytica isolated in the 1960s (Hobson and Mann 1961; Henderson 1970; Henderson 1971; Prins et al. 1975; Priv et al. 2013) and other bacteria belonging to the genera Butyrivibrio, Clostridium and Propionibacterium (Jarvis and Moore 2010). Nonetheless, the major hurdle of being able to culture many of the rumen bacteria means that we are potentially missing a wealth of information on rumen bacterial lipolysis. Since the first published paper detailing functional metagenomic-based techniques for enhanced gene discovery in whole populations, irrespective of the ability to culture (Handelsman 1994), there has been an explosion in its use resulting in the discovery of many novel enzymes. Indeed, many new families of lipase (...truncated)


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Florence Privé, C Jamie Newbold, Naheed N. Kaderbhai, Susan G. Girdwood, Olga V. Golyshina, Peter N. Golyshin, Nigel D. Scollan, Sharon A. Huws. Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome, Applied Microbiology and Biotechnology, 2015, pp. 5475-5485, Volume 99, Issue 13, DOI: 10.1007/s00253-014-6355-6