Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China.
School of Materials and New Energy, South China Normal University, Shanwei 516622, China.
Yi Chuan. 2023 Dec 20;45(12):1128-1146. doi: 10.16288/j.yczz.23-223.
The lytic polysaccharide monooxygenase (LPMO) in the auxiliary active protein family (AA family) catalyzes the oxidative depolymerization of various refractory carbohydrates including cellulose, chitin and starch. While accumulating studies investigate the enzymology of LPMO, the research on the inactivation of LPMO genes has been rarely explored. In this study, five LPMO genes PaLPMO11A (Pa_4_4790), PaLPMO11B (Pa_1_5310), PaLPMO11C (Pa_2_7840), PaLPMO11D (Pa_2_8610) and PaLPMO11E (Pa_3_9420) of the AA11 family in the filamentous fungus Podospora anserina were knocked out by homologous recombination. Single mutants ΔPaLPMO11A (ΔA), ΔPaLPMO11B (ΔB), ΔPaLPMO11C (ΔC), ΔPaLPMO11D (ΔD) and ΔPaLPMO11E (ΔE) were constructed, and then all polygenic mutants were constructed via genetic crosses. The differences in the growth rate and sexual reproduction between wild type and mutant strains were observed on different carbon source media. The alteration of oxidative stress and cellulose degradation ability were found on DAB and NBT staining and cellulase activity determination. These results implicated that LPMO11 genes play a key role in the growth, development, and lignocellulose degradation of P. anserina. The results showed that the spore germination efficiency, growth rate and reproductive capacity of mutant strains including ΔBΔCΔE, ΔAΔBΔCΔE, ΔAΔCΔDΔE and ΔAΔBΔCΔDΔE was significantly decreased on different cellulose carbon sources and the remaining strains have no difference. The reduced utilization of various carbon sources, the growth rate, the spore germination rate, the number of fruiting bodies, the normal fruiting bodies, the shortened life span and the ability to degrade cellulose were found in strains which all five genes in the PaLPMO11 family were deleted. However, the strain still had 45% cellulase activity compared to wild type. These results suggest that LPMO11 genes may be involved in the growth and development, sexual reproduction, senescence and cellulose degradation of P. anserina. This study provides information for systematically elucidating the regulatory mechanism of lignocellulose degradation in filamentous fungus P. anserina.
在辅助活性蛋白家族（AA 家族）中的溶细胞多糖单加氧酶（LPMO）催化各种难降解碳水化合物的氧化解聚，包括纤维素、壳聚糖和淀粉。虽然越来越多的研究调查了 LPMO 的酶学特性，但对 LPMO 基因失活的研究却很少被探索。在这项研究中，丝状真菌 Podospora anserina 中的 AA11 家族的 5 个 LPMO 基因 PaLPMO11A（Pa_4_4790）、PaLPMO11B（Pa_1_5310）、PaLPMO11C（Pa_2_7840）、PaLPMO11D（Pa_2_8610）和 PaLPMO11E（Pa_3_9420）通过同源重组被敲除。构建了单突变体 ΔPaLPMO11A（ΔA）、ΔPaLPMO11B（ΔB）、ΔPaLPMO11C（ΔC）、ΔPaLPMO11D（ΔD）和 ΔPaLPMO11E（ΔE），然后通过遗传杂交构建了所有多基因突变体。观察野生型和突变菌株在不同碳源培养基上的生长速度和有性生殖的差异。通过 DAB 和 NBT 染色以及纤维素酶活性测定发现氧化应激和纤维素降解能力的改变。这些结果表明 LPMO11 基因在 P. anserina 的生长、发育和木质纤维素降解中起关键作用。结果表明，突变株（包括ΔBΔCΔE、ΔAΔBΔCΔE、ΔAΔCΔDΔE 和ΔAΔBΔCΔDΔE）在不同的纤维素碳源上的孢子萌发效率、生长速度和繁殖能力显著降低，而其余菌株则没有差异。在所有 5 个 PaLPMO11 家族基因缺失的菌株中，发现对各种碳源的利用率降低、生长速度、孢子萌发率、产孢体数量、正常产孢体、寿命缩短和纤维素降解能力降低。然而，与野生型相比，该菌株仍具有 45%的纤维素酶活性。这些结果表明，LPMO11 基因可能参与 P. anserina 的生长发育、有性生殖、衰老和纤维素降解。本研究为系统阐明丝状真菌 P. anserina 木质纤维素降解的调控机制提供了信息。
