Overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat) increases the growth rate, Ca2+ concentration and cellulase production in Ganoderma lucidum

Abstract

Identifying new and economical means to utilize diverse lignocellulosic biomass is an urgent task. Ganoderma lucidum is a well-known edible and medicinal basidiomycete with an excellent ability to degrade a wide range of cellulosic biomass, and its nutrient use efficiency is closely related to the activity of extracellular cellulase. Intracellular nicotinamide adenine dinucleotide (NAD⁺) biosynthesis is controlled in response to nutritional status, and NAD⁺ is an essential metabolite involved in diverse cellular processes. Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a common enzyme in three NAD⁺ synthesis pathways. In this study, a homologous gene of nmnat was cloned from G. lucidum and two G. lucidum overexpression strains, OE::nmnat4 and OE::nmnat19, were constructed using an Agrobacterium tumefaciens-mediated transformation method. The G. lucidum nmnat overexpression strains showed obviously increased colony growth on different carbon sources, and intracellular Ca²⁺ concentrations in the G. lucidum OE::nmnat4 and OE::nmnat19 strains were increased by 2.04- and 2.30-fold, respectively, compared with those in the wild-type (WT) strains. In the G. lucidum OE::nmnat4 and OE::nmnat19 strains, endo-β-glucanase (CMCase) activity increased by approximately 2.8- and 3-fold, while β-glucosidase (pNPGase) activity increased by approximately 1.9- and 2.1-fold, respectively, compared with the activity in the WT strains. Furthermore, overexpression of NAD⁺ synthesis pathways was found to elicit cellulase production by increasing the intracellular Ca²⁺ concentration. In summary, this study is the first to demonstrate that increased intracellular NAD⁺ contents through overexpression of the nmnat gene of NAD⁺ synthesis pathways may increase cellulase production by increasing intracellular Ca²⁺ concentrations in G. lucidum. KEY POINTS: • The concentration of NAD⁺influences cellulase production in G. lucidum. • The concentration of NAD⁺influences the intracellular Ca²⁺concentration in G. lucidum. • The concentration of NAD⁺influences cellulase production by eliciting a change in intracellular Ca²⁺in G. lucidum.

Keywords: Ca2 +; Cellulase production; Ganoderma lucidum; NAD+.