Diversification of barley beta-D-glucan endohydrolases in Neurospora crassa.

Abstract

Similarities in the primary sequences and three dimensional structures of barley (1-3)-beta-D-glucan endohydrolases and (1-3,1-4)-beta-D-glucan endohydrolases suggest they are closely related in evolutionary terms, yet they perform completely different functions (Stewart et al., 2001). While (1-3)-beta-D-glucanases capable of hydrolyzing the linear, substituted and branched (1-3)-beta-D-glucans often found in fungal cell walls appear to be involved in plant protection, the (1-3,1-4)-beta-D-glucanases are responsible for digestion of the starchy endosperm cell wall during germination of barley grains (Hrmova and Fincher, 2001). We are using an in vivo gene diversification technique developed in Neurospora crassa (Catcheside et al., 2003) to investigate the molecular basis of their divergence and to generate a more thermostable (1-3,1-4)-beta-D-glucanase intended for industrial use. Gene diversification in Neurospora takes advantage of the meiotic recombination hotspot cogL,shuffling exogenous genes inserted between cogL and the nearby his-3 locus during meiosis. Functional genes encoding the barley beta-D-glucanases will be inserted between cogL and his-3 by transplacement while non-functional duplicates will be incorporated at random. Variant alleles generated by pre-meiotic Repeat-Induced Point mutation (RIP) will be shuffled by recombination, increasing variation and providing a potential rescue for deleterious mutations. Progeny will be screened using a colorimetric plate assay to identify novel variants.