丝状真菌Podospora anserina AA11家族裂解多糖单加氧酶基因的鉴定和功能研究

doi:10.16288/j.yczz.23-223

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Hereditas(Beijing) ›› 2023, Vol. 45 ›› Issue (12): 1128-1146.doi: 10.16288/j.yczz.23-223

• Research Article • Previous Articles Next Articles

Identification and functional study of AA11 family polysaccharide monooxygenase genes in filamentous fungus Podospora anserina

Wenzhen Du¹(), Yuanjing Li², Jialing Wu¹, Siyu Chen¹, Liang Jiang¹, Gang Liu¹, Ning Xie¹()

1. Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China
2. School of Materials and New Energy, South China Normal University, Shanwei 516622, China

Received:2023-08-21 Revised:2023-10-28 Online:2023-12-20 Published:2023-11-10
Contact: Ning Xie E-mail:2100251038@email.szu.edu.cn;ning.xie@szu.edu.cn
Supported by:
National Key R&D Program of China(2021YFA0910800);Basic and Applied Basic Research Fund of Guangdong Province(2021A1515012166);Basic and Applied Basic Research Fund of Guangdong Province(2021A1515012118);National Natural Science Foundation of China(31601014);National Natural Science Foundation of China(22078199)

Abstract

Abstract:

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.

Key words: filamentous fungi, Podospora anserina, LPMO11, gene knockout, lignocellulose degradation

Wenzhen Du, Yuanjing Li, Jialing Wu, Siyu Chen, Liang Jiang, Gang Liu, Ning Xie. Identification and functional study of AA11 family polysaccharide monooxygenase genes in filamentous fungus Podospora anserina[J]. Hereditas(Beijing), 2023, 45(12): 1128-1146.

Figures/Tables 11

Fig. 1

Table 1

Primers used in this study"

引物名称	序列 (5′→3′)	用途
PaLPMO11A_1F	CATTGACGCTCTCACCTTGG	敲除PaLPMO11A基因
PaLPMO11A_2R	CTATTTAACGACCCTGCCCTGAACCGATGCCCGACCTTCTGGTCCT
PaLPMO11A_MKF	AGGACCAGAAGGTCGGGCATCGGTTCAGGGCAGGGTCGTTAAATAG
PaLPMO11A_MKR	AGGTGACTACGATCCAGGCGCATCGAACTGGATCTCAACAGCGGTAAG
PaLPMO11A_3F	CTTACCGCTGTTGAGATCCAGTTCGATGCGCCTGGATCGTAGTCACCT
PaLPMO11A_4R	CCAGCCAACTTCCTCGTCAT
PaLPMO11B_1F	CCCAGACAATGACCACACGAGT	敲除PaLPMO11B基因
PaLPMO11B_2R	CTATTTAACGACCCTGCCCTGAACCGCTGCTGCATGTCACTGAAGG
PaLPMO11B_MkF	CCTTCAGTGACATGCAGCAGCGGTTCAGGGCAGGGTCGTTAAATAG
PaLPMO11B_MkR	CAAGGGTAGCTGATGAGGCACATCGAACTGGATCTCAACAGCGGTAAG
PaLPMO11B_3F	CTTACCGCTGTTGAGATCCAGTTCGATGTGCCTCATCAGCTACCCTTG
PaLPMO11B_4R	CCGCGGTGAAGCTTGACACT
PaLPMO11C_1F	ACAGGGATCATCGGTACAACTG	敲除PaLPMO11C基因
PaLPMO11C_2R	CTATTTAACGACCCTGCCCTGAACCGCTGGTTAGGTTCGAAGTCGAGT
PaLPMO11C_MKF	ACTCGACTTCGAACCTAACCAGCGGTTCAGGGCAGGGTCGTTAAATAG
PaLPMO11C_MKR	CTGGCTTCCAGTCGTCGATGCATCGAACTGGATCTCAACAGCGGTAAG
PaLPMO11C_3F	CTTACCGCTGTTGAGATCCAGTTCGATGCATCGACGACTGGAAGCCAG
PaLPMO11C_4R	CATCAGAACGACTCGCAGAT
PaLPMO11D_1F	AGAACATCCAACGTGCGTGG	敲除PaLPMO11D基因
PaLPMO11D_2R	CTATTTAACGACCCTGCCCTGAACCGAATGACAGGACAAGTGCGGT
PaLPMO11D_MKF	ACCGCACTTGTCCTGTCATTCGGTTCAGGGCAGGGTCGTTAAATAG
PaLPMO11D_MKR	CCTGACAATGCTGAAACGACTGCATCGAACTGGATCTCAACAGCGGTAAG
PaLPMO11D_3F	CTTACCGCTGTTGAGATCCAGTTCGATGCAGTCGTTTCAGCATTGTCAGG
PaLPMO11D_4R	GCATCTCCATCAGCGCAGTG
PaLPMO11E_1F	ACTAGAACGGCTCAGGCACACT	敲除PaLPMO11E基因
PaLPMO11E_2R	CTATTTAACGACCCTGCCCTGAACCGCGGATAGGCGAGTGATCGAT
PaLPMO11E_MKF	ATCGATCACTCGCCTATCCGCGGTTCAGGGCAGGGTCGTTAAATAG
PaLPMO11E_MKR	AGCTGCAGAAGTCCATCTCCCATCGAACTGGATCTCAACAGCGGTAAG
PaLPMO11E_3F	CTTACCGCTGTTGAGATCCAGTTCGATGGGAGATGGACTTCTGCAGCT
PaLPMO11E_4R	GCATGATATTCCCTCCACAGGT
5 Test	TGAGAAGCACACGGTCAC	检测目的基因的敲除
3 Test	TCGGGGCGAAAACTCTC	检测目的基因的敲除
verify_PaLPMO11A_1F	AGGCCGGATACATACCGTTG	验证PaLPMO11A基因敲除突变体
verify_PaLPMO11A_2R	CACAATGTCCTCCAACACCG	验证PaLPMO11A基因敲除突变体
verify_PaLPMO11B_1F	ACCTTGCGAGAAGGTTGGTG	验证PaLPMO11B基因敲除突变体
verify_PaLPMO11B_2R	GAGGGAGGTGGCCTCTGAT	验证PaLPMO11B基因敲除突变体
verify_PaLPMO11C_1F	GAGCAAGTCCAGCCCTGACT	验证PaLPMO11C基因敲除突变体
verify_PaLPMO11C_2R	GCAGCGCAATCGTCGTGT	验证PaLPMO11C基因敲除突变体
verify_PaLPMO11D_1F	CAGCTGAGGCTAATGATCCG	验证PaLPMO11D基因敲除突变体
verify_PaLPMO11D_2R	CTCCTCGGCAGGTCACAAGT	验证PaLPMO11D基因敲除突变体
verify_PaLPMO11E_1F	AACCAGGTTCCCCAGGATCT	验证PaLPMO11E基因敲除突变体
verify_PaLPMO11E_2R	ATAACAGGCAGCTTGGCCTT	验证PaLPMO11E基因敲除突变体

Table 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

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