PNNL

Abstract

Genome resequencing is an efficient strategy for associating mutant phenotypes with physical genomic loci (Baker 2009). A pilot study of this approach demonstrated that the Neurospora crassa genetic map was critical in narrowing the possible candidate mutations in a strain to a small number in a limited, defined region of the genome (McCluskey, et al. 2011). In this study, we utilize a resequencing strategy to identify the mutations underlying the gluc-1 and gluc-2 genes in N. crassa. Fungal CAZymes are of special interest for biofuel production because they can deconstruct plant derived biomass into fermentable sugars (Ottum, et al. 2021). Neurospora crassa has been a good model to learn about the biochemistry and regulation of cellulose deconstructing enzymes for many decades (Coradetti, et al. 2013; Eberhart, et al. 1977; Gabriel, et al. 2021; Schmoll, et al. 2012; Tian, et al. 2009; Yazdi, et al. 1990a; Yazdi, et al. 1990b; Znameroski, et al. 2012). In cellulose saccharification, beta glucosidases release glucose from cellobiose (Ketudat Cairns and Esen 2010). The N. crassa, beta-glucosidase system has been well characterized and exploited for advances in biotechnology related to sugar utilization (Eberhart, et al. 1964; Eberhart and Beck 1970, 1973; Galazka, et al. 2010; Ha, et al. 2011; Karkehabadi, et al. 2018; Mahadevan and Eberhart 1964a). Numerous studies of biomass deconstruction continue to be facilitated by the generation and wide distribution of N. crassa gene knockout library (Colot, et al. 2006; Seibert, et al. 2016; Znameroski and Glass 2013). By screening gene knockout strains many of the regulatory circuits and enzymes that drive N. crassa biomass deconstruction have been characterized (Coradetti et al. 2013; Gabriel et al. 2021; Reilly, et al. 2015; Schmoll et al. 2012; Sun, et al. 2012). While these advances have had significant impact, in many cases, connections to prior literature and classical or forward genetic mutant strains have not been made. Fractionation studies indicated N. crassa had at least two beta-glucosidases one with active that was thermostable (Eberhart et al. 1964). The induction and enzymatic properties of beta-glucosidases were characterized, in part, utilizing two mutations, gluc-1 and gluc-2 which reduce measured thermostable beta glucosidase activity by ~90% and over 99% respectively. gluc-1 and gluc-2 map to Linkage Group III and were proposed to be allelic (Eberhart and Beck 1970, 1973; Mahadevan and Eberhart 1964b; Mahadevan and Eberhart 1962). To bridge current beta glucosidase studies with prior literature, we sequenced the genomes of two N. crassa strains, one with gluc-1 (FGSC 1224) and the other with gluc-1 and gluc-2 (FGSC 1227). Combined with prior genetic mapping of the mutant to LG III, our sequencing analysis led us to discover that gluc-1 and gluc-2 are both encoded by mutations in NCU08755 (Table 1). Sequence homology demonstrates that NCU08755 is a member of the glycoside hydrolase family 3 (GH3) beta-glucosidase. The gluc-1 mutation is a leucine to proline at amino acid residue 425 while gluc-2 encodes a tryptophan to STOP mutation at amino acid residue 46, presumably resulting in a truncated and nonfunctional peptide. In summary, we have identified mutations in the gene NCU08755 that underly the classical mutations, gluc-1 and gluc-2, connecting a robust biochemistry and genetics literature with current systems biology studies enabled by the N. crassa gene knockout strain library as well as by the diverse tools available for research into fundamental and applied areas of fungal biology and biotechnology.