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)