Transcriptome changes in adaptive evolution of xylose-fermenting industrial Saccharomyces cerevisiae strains with δ-integration of different xylA genes.

It is of utmost importance to construct industrial xylose-fermenting Saccharomyces cerevisiae strains for lignocellulosic bioethanol production. In this study, two xylose isomerase-based industrial S. cerevisiae strains, O7 and P5, were constructed by δ-integration of the xylose isomerase (XI) gene xylA from the fungus Orpinomyces sp. and from the bacterium Prevotella ruminicola, respectively. The xylose consumption of the strains O7 and P5 at 48-h fermentation was 17.71 and 26.10 g/L, respectively, in synthetic medium with xylose as the sole sugar source. Adaptive evolution further improved the xylose fermentation capacity of the two strains to 51.0 and 28.9% in average, respectively. The transcriptomes of these two strains before and after evolution were analyzed using RNA-Seq. The expression levels of the genes involved in cell integrity, non-optimal sugar utilization, and stress response to environment were significantly up-regulated after evolution and did not depend on the origin of xylA; the expression levels of the genes involved in transmembrane transport, rRNA processing, cytoplasmic translation, and other processes were down-regulated. The expression of genes involved in central carbon metabolism was fine-tuned after the evolution. The analysis of transcription factors (TFs) indicated that most of the genes with significant differential expression were regulated by the TFs related to cell division, DNA damage response, or non-optimal carbon source utilization. The results of this study could provide valuable references for the construction of efficient xylose-fermenting XI strains.

Functional expression of xylose isomerase in flocculating industrial Saccharomyces cerevisiae strain for bioethanol production.

Saccharomyces cerevisiae strains with xylose isomerase (XI) pathway were constructed using a flocculating industrial strain (YC-8) as the host. Both strains expressing wild-type xylA (coding XI) from the fungus Orpinomyces sp. and the bacterium Prevotella ruminicola, respectively, showed better growth ability and fermentation capacity when using xylose as the sole sugar than most of the reported strains expressing XI. Codon optimization of both XIs did not improve the xylose fermentation ability of the strains. Adaption significantly increased XI activity resulting in improved growth and fermentation. The strains expressing codon-optimized XI showed a higher increase in xylose consumption and ethanol production compared to strains expressing wild XI. Among all strains, the adapted strain YCPA2E expressing XI from P. ruminicola showed the best performance in the fermentation of xylose to ethanol. After 48 h of fermentation, YCPA2E assimilated 16.95 g/L xylose and produced 6.98 g/L ethanol. These results indicate that YC-8 is a suitable host strain for XI expression, especially for the codon-optimized XI originating from P. ruminicola.

Functional Characterization Reveals Novel Putative Coding Sequences in Prevotella ruminicola Genome Extracted from Rumen Metagenomic Studies.

AIM: To reassemble Prevotella ruminicola genome from rumen metagenomic data of cattle and buffalo and compare with the published reference genome. METHOD: Rumen microbial communities from Mehsani buffaloes (n = 8) and Kankrej cattle (n = 8), each adapted to different proportions of a dry or green roughage diet, were subjected to metagenomic sequencing by Ion Torrent PGM, and subsequent reads were analyzed by MG-RAST. Using reference-guided assembly of the sequences against the published P. ruminicola strain 23, draft genomes of 2.56 and 2.46 Mb were reconstructed from Mehsani buffalo and Kankrej cows, respectively. The genomes were annotated using the RAST Server and carbohydrate active enzyme (CAZyme) analysis. RESULTS: Taxonomic analysis by MG-RAST revealed P. ruminicola to be the most abundant species present among the rumen microflora. Functional annotation of reconstructed genomes using the RAST Server depicted the maximum assignment of coding sequences involved in the subsystems amino acid and derivatives and carbohydrate metabolism. CAZyme profiling revealed the glycoside hydrolases (GH) family to be the most abundant. GH family subclassification revealed that the extracted genomes had more sequence hits for GH2, GH3, GH92 and GH97 as compared to the reference. CONCLUSION: The results reflect the metabolic significance of rumen-adapted P. ruminicola in utilizing a coarse diet for animals based on acquisition of novel genetic elements.

Purification, characterization, and expression of multiple glutamine synthetases from Prevotella ruminicola 23.

The Prevotella ruminicola 23 genome encodes three different glutamine synthetase (GS) enzymes: glutamine synthetase I (GSI) (ORF02151), GSIII-1 (ORF01459), and GSIII-2 (ORF02034). GSI, GSIII-1, and GSIII-2 have each been heterologously expressed in and purified from Escherichia coli. The subunit molecular mass of GSI was 56 kDa, while GSIII-1 and GSIII-2 were both 83 kDa. Optimal conditions for γ-glutamyl transferase activity were found to be 35°C at pH 5.6 with 0.25 mM Mn(2+) ions (GSI) or 37°C at pH 6.0 (GSIII-1 and GSIII-2) with 0.50 to 1.00 mM Mn(2+) ions. GSIII biosynthetic activity was found to be optimal at 50 to 60°C and pH 6.8 to 7.0 with 10 mM Mn(2+) ions, while GSI displayed no GS biosynthetic activity. Kinetic analysis revealed K(m) values for glutamate and ammonium as well as for hydrolysis of ATP to be 8.58, 0.48, and 1.91 mM, respectively, for GSIII-1 and 1.72, 0.43, and 2.65 mM, respectively, for GSIII-2. A quantitative reverse transcriptase PCR assay (qRT-PCR) revealed GSIII-2 to be significantly induced by high concentrations of ammonia, and this corresponded with increases in measured GS activity. Collectively, these results show that both GSIII enzymes in P. ruminicola 23 are functional and indicate that GSIII-2, flanked by GOGAT (gltB and gltD genes), plays an important role in the acquisition and metabolism of ammonia, particularly under nonlimiting ammonia growth conditions.

Biochemical characterization and relative expression levels of multiple carbohydrate esterases of the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate.

We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOS(FA,Ac)) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOS(FA,Ac), a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose.

Comparative genome analysis of Prevotella ruminicola and Prevotella bryantii: insights into their environmental niche.

The Prevotellas comprise a diverse group of bacteria that has received surprisingly limited attention at the whole genome-sequencing level. In this communication, we present the comparative analysis of the genomes of Prevotella ruminicola 23 (GenBank: CP002006) and Prevotella bryantii B(1)4 (GenBank: ADWO00000000), two gastrointestinal isolates. Both P. ruminicola and P. bryantii have acquired an extensive repertoire of glycoside hydrolases that are targeted towards non-cellulosic polysaccharides, especially GH43 bifunctional enzymes. Our analysis demonstrates the diversity of this genus. The results from these analyses highlight their role in the gastrointestinal tract, and provide a template for additional work on genetic characterization of these species.

Biochemical analysis of a beta-D-xylosidase and a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in Prevotella ruminicola 23.

Prevotella ruminicola 23 is an obligate anaerobic bacterium in the phylum Bacteroidetes that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) and expressed the recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-beta-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 beta-D-glucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of xyn10D-fae1A. Biochemical analyses of the purified recombinant protein revealed that the putative beta-D-glucosidase has activity for pNP-beta-D-xylopyranoside, pNP-alpha-L-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated xyl3A. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 beta-D-xylosidase.

Utility of enzymes from Fibrobacter succinogenes and Prevotella ruminicola as detergent additives.

In this study, we investigated the application of cellulase and protease purified from rumen bacteria as detergent additives. Cellulase and protease were purified from the rumen cellulytic bacteria Fibrobacter succinogenes S85, and Prevotella ruminicola 23, respectively. An inhibitor test indicated that the purified protease belongs to the category of serine proteases and metalloproteases. Both the enzymes were effective at a high temperature (50 degrees C) and neutral pH (pH 7-8), but the protease activity increased with the increase in temperature and pH. The purified protease was treated with ten types of surfactants/detergents; it was found to retain over 60% of its activity in the presence of anionic and nonionic detergents. The cellulose plus protease combination was still effective after treatment with Triton X-100 and Tween 80, but the residual activity was low after treatment with Tween 20 than that after treatment with other nonionic detergents. Washing tests indicated that enzyme addition produced no significant improvement in the removal of grass stains, but individual enzyme addition in surfactants/detergents, especially in nonionic detergents, could improve the washing performance of the detergents by improving its ability to remove blood stains. This suggested that the surfactant/detergent class, enzyme properties, and the mixing ratio of ingredients should be considered simultaneously to enhance the washing performance.