Cloning of the Bacillus pumilus β-xylosidase gene (xynB) and its expression in Saccharomyces cerevisiae

A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the β-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2(P)) and terminator (ADH2(T)) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone α-factor (MFα1(S)) before insertion between the ADH2(P) and ADH2(T) sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2(P)-MFα1(S)xynB-ADH2(T) was designated XLO1. Both plasmids pDLG11 and pDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional β-xylosidase. The total β-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45-50°C and pH 6.6 respectively. The enzyme was thermostable at 45°C; however, at 60°C most of the Xlo1 was inactive after 5 min.Articl

Optimization of Combinatorial Mutagenesis

Abstract. Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (stability, activity, immunogenicity, etc.). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by oneand two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, i

Grape and wine biotechnology: challenges, opportunities and potential benefits

The image of wine as a harmonious blend of nature, art and science invites tension between tradition and innovation, and no tension in the business of making wine is greater than that brought into play by the potential afforded by 21st century grape and wine biotechnology. The challenge is to realise the potential of technological innovation without stripping the ancient art of grapegrowing and winemaking of its charm, mysticism and romanticism. Equally challenging is the multitude of complex and interconnected agronomic, business, regulatory and social obstacles currently blocking commercial availability of transgenic grapes, wine yeast and malolactic bacterial starter strains. While the need to assess rigorously the potential negative impacts of new technologies is self-evident, over the long term, failure to overcome these hurdles will disadvantage the international wine sector and consumers alike. This contention is illustrated with reference to recent examples of genetically improved grapevine, yeast and bacterial prototypes showing potential for enhanced, cost-effective production of wine with minimised resource inputs, improved quality and low environmental impact