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.
辅助活性蛋白家族(auxiliary activity family，AA family)中的裂解多糖单加氧酶(lytic polysaccharide monooxygenase, LPMO)能催化纤维素、几丁质和淀粉等多种难降解碳水化合物的氧化解聚。尽管目前对LPMO的酶学研究较多，但对LPMO基因失活的研究却鲜有报道。本研究利用同源重组方法定点敲除丝状真菌Podospora anserina中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)，分别构建了单突变体ΔPaLPMO11A (ΔA)、ΔPaLPMO11B (ΔB)、ΔPaLPMO11C (ΔC)、ΔPaLPMO11D (ΔD)和ΔPaLPMO11E (ΔE)，然后通过遗传杂交构建所有多基因突变体。通过在不同碳源培养基上的表型分析、DAB和NBT染色以及纤维素酶活测定分析野生型菌株与突变型菌株在生长速率、有性生殖、氧化应激和纤维素降解能力等方面的差异，揭示LPMO11基因在P. anserina菌株的生长发育和木质纤维素降解过程中的作用。实验结果表明，在不同纤维素碳源上，ΔBΔCΔE、ΔAΔBΔCΔE、ΔAΔCΔDΔE和ΔAΔBΔCΔDΔE突变型菌株的有性生殖能力降低，其余突变型菌株的孢子萌发效率、生长速率和生殖能力几乎没有差异。PaLPMO11家族5个基因的同时缺失，会导致菌株利用各种碳源的能力明显降低、生长速率降低、孢子萌发率降低、子实体数减少、部分子实体发育异常、寿命缩短和降解纤维素的能力显著下降，但仍有野生型45%以上的总纤维素酶活力。上述结果表明，LPMO11基因可能参与P. anserina的生长发育、有性生殖、衰老和纤维素降解过程。本研究为系统阐述丝状真菌P. anserina中木质纤维素降解的调控机制提供参考。.
Keywords: LPMO11; Podospora anserina; filamentous fungi; gene knockout; lignocellulose degradation.