A second cinnamoyl ester hydrolase (CEH) encoding gene (cinB) has been characterized from the ruminal bacterium Butyrivibrio fibrisolvens E14. CinB is more similar to CinA (previously named Cinl) (28% amino acid identity), the first CEH described from B. fibrisolvens E14, than either of the enzymes are to any other member of the family of hydrolases to which they belong. Upstream of cinB, and in the opposite orientation, is a gene (cinR) encoding a protein with substantial similarity to members of the MarR family of negative regulators of bacterial gene expression. By alignment of these sequences, a possible helix-turn-helix DNA-binding domain has been identified. CinR was expressed at a high level in Escherichia coli using the lac promoter. In E. coli CinR repressed the expression of CinB, but had no effect on the expression of CinA. In gel mobility-shift assays, CinR bound specifically to the cinR-cinB intergenic region. Two identical 16 nucleotide inverted repeats adjacent to the putative PcinR and PcinB promoters are likely binding sites for CinR. The addition of FAXX (O-[5-O-(trans-feruloyl)-α-L-arabinofuranosyl]-(1,3)-O-ß-D-xylopyranosyl-(1,4)-D-xylopyranose) and Fara [5-O-(trans-feruloyl)-arabinofuranose], but not xylobiose, ferulic acid and a number of other soluble components of hemicellulose, inhibited the binding of CinR to DNA.

Aims:  To obtain reliable transformation of a range of Butyrivibrio fibrisolvens strains and to express a Neocallimastix patriciarum xylanase gene in the recipients. Methods and Results:  Eight strains (H17c, E14, LP1309, LP1028, AR11a, OB156, LP210B and LP461A) of Bu. fibrisolvens were transformed by the Gram‐positive vector pUB110. A xylanase expression/secretion cassette containing Bu. fibrisolvens promoter and signal peptide elements fused to catalytic domain II of the N. patriciarum xylanase A cDNA (xynANp) was inserted into pUB110 to create the plasmid pUBxynA. pUBxynA was used to transform seven of the Bu. fibrisolvens strains transformed by pUB110. In strain H17c pUBxynA, which produced native xylanase, 2·46 U mg−1 total xylanase activity was produced with 45% extracellular xylanase. In strain H17c pUMSX, 0·74 U mg−1 total xylanase activity was produced with 98% extracellular xylanase. H17c pUBxynA exhibited increased (28·7%) degradation of neutral detergent fibre compared with unmodified H17c; however, progressive loss of pUBxynA was observed in long‐term cultivation. Conclusions:  A stable transformation system was developed that was applicable for a range of Bu. fibrisolvens strains and high levels of expression of a recombinant xylanase were obtained in H17c which lead to increased fibre digestion. Significance and Impact of the Study:  This stable transformation system with the accompanying recombinant plasmids will be a useful tool for further investigation aimed at improving ruminal fibre digestion.

A gene (cinI) encoding a cinnamoyl ester hydrolase (CEH) has been isolated from the ruminal bacterium, Butyrivibrio fibrisolvens E14, using a model substrate, MUTMAC [4-methylumbelliferoyl (p-trimethylammonium cinnamate chloride)]. CinI has significant amino-acid similarities with members of a large and diverse family of hydrolases with a serine/aspartic acid/histidine catalytic triad. Our analyses identified two previously unclassified amino acid sequences, the amino-terminal domain of unknown function in XynZ from Clostridium thermocellum and XynC, an acetylxylan esterase from Caldicellulosiruptor saccharolyticus, as members of the same family of hydrolases. A previously described esterase with CEH activity, XylD from Pseudomonas fluorescens ssp. cellulosa, is not similar to CinI. CinI was expressed at high levels in the periplasmic fraction of E. coli TOPP2 and released ferulic acid from Fara [5-O-(trans-feruloyl)-arabinofuranose] prepared from wheat bran.

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynBfamily, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.