Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 99, Issue 13, Pages 5475-5485Publisher
SPRINGER
DOI: 10.1007/s00253-014-6355-6
Keywords
Rumen; Lipolysis; Fatty acid; Lipase; Esterase; Bacteria; Functional metagenomic
Categories
Funding
- Biotechnology and Biological Sciences Research Council (UK)
- Department of Environment Food and Rural Affairs
- English Beef and Lamb Executive
- Hybu Cig Cymru
- Quality Meat Scotland
- European Union ProSafeBeef [FOOD-CT-2006-36241]
- BBSRC [BBS/E/W/10964A01B] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BBS/E/W/10964A01B] Funding Source: researchfish
- The British Council [172629373] Funding Source: researchfish
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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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