P42: Heterologous expression of wood degrading enzymes laccases, manganese peroxidase, and pectinases from Shiitake mushroom Lentinula edodes

The shitake mushroom, Lentinula edodes, one of the most cultivated edible mushrooms, is a white rot basidiomycetes capable of degrading wood efficiently. The fungus secrets two ligninolytic enzymes, laccases and manganese peroxidases, assisted by hydrolytic enzymes such as pectinase. Laccases also take part in fungal morphogenesis, melanin synthesis and pigmentation. Ligninolytic enzymes are used in biofuel production, waste-water treatment, paper bleaching and detoxification of byproduct from food processing. We determined the genome sequence of L. edodes and predicted fourteen laccases, five manganese peroxidases, and 14 pectinases.  Comparative sequence analysis revealed that 6 laccases have the four signature sequences of fungal laccases with perfect matches, while 1 laccase missed two signature sequences. Four manganese peroxidases have highly conserved domains for ion binding and enzyme activity. They are group I peroxidases. Pectinases include 5 endo-galacturonases, 2 exo-rhamnogalacturonases, 2 polygalacturonases and 1 endo-xylogalacturonases.

The expressions of ligninolytic and pectin degrading genes was investigated at various growth stages and grown in different media. Different ligninolytic genes had a wide array of expression patterns, suggesting that they play different roles. Ten laccases, 1 manganese peroxidase and 4 pectinases genes were cloned in the yeast Pichia pastoris. Four laccases were expressed, characterised, and compared in kinetics, stability, and degradation of dyes and polyaromatic hydrocarbons (PAHs). One pectinase gene encoding a pectin depolymerase was also expressed. These heterologous expressed recombinant enzymes in yeast make them modifiable by genetic means. They are useful resources for enzymatic pre-treatment of woods for biofuel, bio-remediation, and other biotechnological applications.