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 Ca ²⁺ concentration in G. lucidum.

• The concentration of NAD ⁺ influences cellulase production by eliciting a change in intracellular Ca ²⁺ in G. lucidum.

中文翻译：

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烟酰胺单核苷酸腺苷酸转移酶（nmnat）的过表达增加了灵芝中的生长速率，Ca2 +浓度和纤维素酶的产生。

摘要

确定利用多样化的木质纤维素生物质的新的经济方法是当务之急。灵芝是一种著名的食用和药用担子菌，具有出色的降解多种纤维素生物质的能力，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 ⁺）的生物合成受营养状况的影响而受到控制，而NAD ⁺是参与多种细胞过程的必需代谢产物。烟酰胺单核苷酸腺苷酸转移酶（NMNAT）是三种NAD ⁺合成途径中的常见酶。在这项研究中，nmnat的同源基因从使用根癌农杆菌介导的转化方法构建了灵芝和两个灵芝过表达菌株OE :: nmnat4和OE :: nmnat19 。所述灵芝NMNAT过表达菌株显示明显增加在不同碳源菌落生长，和细胞内Ca ²⁺浓度的灵芝OE :: nmnat4和OE :: nmnat19菌株分别增加了2.04- 2.30倍，分别与野生型（WT）菌株相比。在灵芝OE :: nmnat4和OE :: nmnat19菌株中，内切β-葡聚糖酶（CMCase）活性增加了约2.8和3倍，而β与WT菌株中的活性相比，β-葡萄糖苷酶（p NPGase）活性分别增加了约1.9-和2.1-倍。此外，发现NAD ⁺合成途径的过表达通过增加细胞内Ca ^2+的浓度来引发纤维素酶的产生。总之，这项研究是第一个证明通过过表达NAD ⁺合成途径的nmnat基因而增加细胞内NAD ⁺含量的方法，可以通过提高灵芝中细胞内Ca ^2+的浓度来增加纤维素酶的产量。

关键点

•NAD ⁺ 的浓度会影响灵芝中纤维素酶的产生。

•NAD ⁺ 的浓度会影响 灵芝中细胞内Ca ^2+的 浓度。

•NAD ⁺ 的浓度通过引起 灵芝中细胞内Ca ²⁺ 的变化来影响纤维素酶的产生。