Diversity, abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation

Abstract

A novel class of cysteine phytase showing ability to degrade phytate has recently been isolated from rumen bacteria. To expand our knowledge of this enzyme class, a total of 101 distinct cysteine phytase gene fragments were identified from the ruminal genomic DNA of Bore goats and Holstein cows, and most of them shared low identities (< 50%) with known sequences. By phylogenetic analysis, these sequences were separated into three clusters that showed substantial diversity. The two most abundant cysteine phytase genes of goat rumens were cloned and their protein products were characterized. Four findings were revealed based on our results. (i) Compared with soil and water environment, where β-propeller phytase is the most important phytate-degrading enzyme, cysteine phytase is the major phytate-degrading enzyme in the anaerobic ruminal environment. (ii) Cysteine phytase fragments in the rumen contents of goat and cow have the same diversity profile, although most of the sequences and their abundance differ in the two species. (iii) Each species has their respective high-abundance genes, which may play major roles for phytate degradation. (iv) Compared with previously reported cysteine phytases that have pH optimum at 4.5, the pH optima of the two most abundant secreted goat cysteine phytases are 6.5 and 6.0, which are within the pH range found in the rumens. This study provides valuable information about the diversity, abundance and enzymatic properties of the ruminal cysteine phytases and emphasizes the important role(s) of these cysteine phytases probably in the terrestrial cycle of phosphorus.

Comparative Quantitative Analysis of Gene Expression Profiles of Glycoside Hydrolase Family 10 Xylanases in the Sheep Rumen during a Feeding Cycle

“…The GFP gene harbored in recombinant P. pastoris was confirmed to have no cross-reaction with the DNA or cDNA of the rumen sample and was therefore suitable for use for normalizing for technical variations between rumen samples. Copy numbers for each gene matched the gene's abundance (based on sequencing data), thus confirming that there was no primer-associated bias; this is in agreement with the report by Huang et al (25). Quantitative analysis at the transcriptional level of the six representative xylanase genes at four time points showed that all six genes had lowered transcription at 4 h, and then four of the genes (including those having the highest and lowest abundance) were gradually upregulated.…”

“…We retrieved 44 distinct GH 10 xylanase gene fragments (Ͻ95% identity) from the rumen using a culture-independent approach. These fragments had shared identities with known xylanases in GenBank as low as 39%, which further confirms that there are large numbers of unidentified genes in the rumen ecosystem (25,26). Most of the retrieved GH 10 xylanase gene sequences showed significant expression variation at the genomic and transcriptional levels.…”

“…All six sequences showed identities of 55 to 92% to known xylanases from P. ruminicola, Prevotella dentalis, and Verrucomicrobiae bacterium. Full-length xynB and xynC were cloned using a modified TAIL-PCR procedure (25). xynB (1,053 bp) encodes 351 amino acids and includes a putative signal peptide of 24 residues (predicted by SignalP).…”

“…Using the combined methods of degenerate PCR, quantitative real-time PCR (qPCR), and modified thermal asymmetric interlaced PCR (TAIL-PCR) (25), the diversity and abundance of ruminal phytase in the rumen of Bore goats and Holstein cows have been evaluated. The two most abundant ruminal genes were identified, and it was shown that cysteine phytase, instead of the widely distributed ␤-propeller phytase, was the sole functional phytase in the rumen.…”

Rumen microbial (meta)genomics and its application to ruminant production

“…Rumen metagenome libraries have also been used to screen for other bioactivities, including novel lipases (Liu et al, 2009a;Bayer et al, 2010), polyphenol oxidase (Beloqui et al, 2006) and an enzyme capable of degrading 3,5,6-trichloro-2-pyridinol, a degradation product of the organophosphorus insecticide chlorpyrifos (Math et al, 2010). In addition, cysteine phytases have been isolated by screening ruminal genomic DNA from cows and goats using degenerate primer sets (Huang et al, 2010). Functional metagenomics has the potential to uncover new enzymes and metabolic pathways in the rumen if innovative strategies for screening are developed.…”