Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Microbiology Department, College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, People's Republic of China.
Appl Microbiol Biotechnol. 2020 Aug;104(16):7079-7091. doi: 10.1007/s00253-020-10763-0. Epub 2020 Jul 7.
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 Ca 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 Ca 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 Ca concentrations in G. lucidum. KEY POINTS: • The concentration of NADinfluences cellulase production in G. lucidum. • The concentration of NADinfluences the intracellular Caconcentration in G. lucidum. • The concentration of NADinfluences cellulase production by eliciting a change in intracellular Cain G. lucidum.
鉴定利用各种木质纤维素生物质的新的和经济的方法是一项紧迫的任务。灵芝是一种著名的可食用和药用担子菌，具有降解广泛的纤维素生物质的优异能力，其营养利用效率与细胞外纤维素酶的活性密切相关。细胞内烟酰胺腺嘌呤二核苷酸（NAD）的生物合成受到营养状况的控制，而 NAD 是一种参与多种细胞过程的必需代谢物。烟酰胺单核苷酸腺苷转移酶（NMNAT）是三种 NAD 合成途径中的常见酶。在这项研究中，从灵芝中克隆了同源的 nmnat 基因，并利用农杆菌介导的转化方法构建了两个灵芝过表达菌株 OE::nmnat4 和 OE::nmnat19。灵芝 nmnat 过表达菌株在不同碳源上的菌落生长明显增加，灵芝 OE::nmnat4 和 OE::nmnat19 菌株的细胞内 Ca 浓度分别比野生型（WT）菌株增加了 2.04 倍和 2.30 倍。在灵芝 OE::nmnat4 和 OE::nmnat19 菌株中，内切-β-葡聚糖酶（CMCase）活性分别增加了约 2.8 倍和 3 倍，而β-葡萄糖苷酶（pNPGase）活性分别增加了约 1.9 倍和 2.1 倍。此外，通过增加细胞内 Ca 浓度，发现 NAD 合成途径的过表达会引起纤维素酶的产生。总之，这项研究首次表明，通过过表达 NAD 合成途径的 nmnat 基因增加细胞内 NAD 含量，可能通过增加灵芝细胞内 Ca 浓度来增加纤维素酶的产生。关键点：· NAD 浓度影响灵芝中的纤维素酶生产。· NAD 浓度影响灵芝细胞内 Ca 浓度。· NAD 浓度通过诱导灵芝细胞内 Ca 的变化来影响纤维素酶的产生。
