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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.

Promoters and signal sequences for expression and secretion of a fungal xylanase encoded by a modified Neocallimastix patriciarum xynA cDNA in the rumen bacterium, Butyrivibrio fibrisolvens OB156, were investigated. Successful expression of the fungal xylanase in OB156 was obtained using the putative xylanase promoter from B. fibrisolvens strain 49. Replacing the putative -35 region sequence (TTGCAC) of the xylanase promoter with the sequence TTGACA by mutagenesis reduced the fungal xylanase expression level 4-fold in OB156, indicating that this B. fibrisolvens strain did not efficiently recognise the E. coli consensus -35 sequence. Reduction of the spacer length between the -35 and -10 regions of the xylanase promoter from 18 to 17 base-pairs (bp) considerably increased the expression levels of the fungal enzyme in both E. coli and OB156. Insertion of a pUB110 mob promoter upstream of the xylanase promoter also significantly improved the fungal xylanase expression. Secretion of the fungal xylanase mediated by the alpha-amylase signal peptide from B. fibrisolvens strain H17c was efficient in E. coli, but very poor in OB156. An increase in the hydrophobicity of the signal sequence resulted in a 4-fold increase in the extracellular portion of the fungal xylanase in OB156, indicating marked improvement in xylanase secretion efficiency. The recombinant plasmids and xylanase expression/secretion cassettes were found to be stable in OB156 after prolonged cultivation (100 generations) in the absence of antibiotic selection. These results suggest that the rumen bacterium B. fibrisolvens can be manipulated to produce and secrete a eukaryotic extracellular protein with stable maintenance of the expression cassette in plasmid form.

Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.

A Neocallimastix patriciarum xylanase cDNA with the core coding sequence essentially identical to xynA was isolated and modified for high-level expression in Escherichia coli. The xylanase cDNA was truncated into individual catalytic domains, which were modified at the N-terminus. These modified xylanases were synthesised as non-fusion proteins under the control of the tac promoter. High-level expression was obtained with the modified domain II construct, accounting for approx. 25% of total cellular protein. However, with the same vector and expression cassette, expression levels of constructs containing domain I or domains I and II fused in tandem were very low. RNA analysis revealed that the striking difference in expression levels of these three constructs was not due to transcription efficiency, but was mainly related to transcript stability. Further analysis of the domain II construct revealed that the high-level expression of the domain II xylanase was largely attributed to the presence of a favourable N-terminal coding sequence, as mutation at the N-terminus of the domain II dramatically reduced the expression level. The modified domain II xylanase produced in E. coli had a specific activity of 1229 U mg-1 protein at pH 7 and 50 degrees C without purification. The availability of a recombinant fungal xylanase with high specific activity and in high yield offers a potentially attractive source of xylanase for industrial applications.