敲除msn2对米曲霉生长和发酵产曲酸的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-1492

摘要/Abstract

摘要：

Msn2作为C2H2型锌指结构转录激活因子,参与调控真菌生长速率、分生孢子发育及多种应激反应等生物学过程。本论文以产曲酸米曲霉3.042为出发菌株,构建pyrG营养缺陷型菌株,通过msn2基因的上下游序列为同源重组臂和回补pyrG基因为筛选标记,最终获得一株敲除msn2且回补pyrG基因的转化子g-5号菌株。较原始菌株米曲霉3.042相比,g-5菌株生长受到抑制且孢子生成量显著降低60%,在含10%及以上葡萄糖环境中不产孢,但可耐受30 mmol/L H₂O₂。g-5菌株摇瓶发酵曲酸产量达到21.55 g/L,较米曲霉3.042的11.86 g/L,提升了81%。经RT-qPCR分析,与米曲霉3.042相比,g-5菌株的msn2基因不表达;在发酵6 d时米曲霉调节曲酸合成相关基因kojR和LaeA基因表达量分别提高4.13倍和21倍;g-5菌株菌丝生长及产孢相关基因brlA与tps1表达量较3.042下调63倍和128倍,相反ageB基因表达量大幅上调59倍。

关键词: 米曲霉, msn2, 基因敲除, 曲酸

Abstract:

Msn2,as a C2H2-type zinc finger structure transcriptional activator,is involved in regulating biological processes such as fungal growth rate,conidial development and various stress responses. In this thesis,Aspergillus oryzae 3.042 was used as the starting strain to construct the pyrG nutrition-deficient strain. The upstream and downstream sequences of msn2 gene were as homologous recombination arms and the complementary pyrG gene was used as the screening marker. Finally,a mutant strain g-5 with msn2 knockout and the complementary pyrG gene was obtained. Compared with the original strain A. oryzae 3.042,the growth of strain g-5 was inhibited and the spore production significantly reduced by 60%. Strain g-5 could not sporulate in the environment containing 10% or more glucose,but tolerated 30 mmol/LH₂O₂. g-5 achieved 21.55 g/L kojic acid in shake flasks,which was 81% higher than that of A. oryzae 3.042（11.86 g/L）. By RT-qPCR analysis,the msn2 gene was not expressed in strain g-5 compared with A. oryzae 3.042. After 6 d of fermentation,the expressions of kojR and LaeA genes related to kojic acid synthesis increased by 4.13 and 21 times,respectively. The expressions of mycelial growth and sporulation related genes brlA and tps1 in g-5 strain were down-regulated by 63 and 128 folds compared with A. oryzae 3.042,while the expressions of ageB gene were up-regulated by 59 folds.

Key words: Aspergillus oryzae, msn2, gene knock-out, kojic acid

史亚楠, 王德培, 王一川, 周昊, 薛鲜丽. 敲除msn2对米曲霉生长和发酵产曲酸的影响[J]. 生物技术通报, 2022, 38(8): 188-197.

SHI Ya-nan, WANG De-pei, WANG Yi-chuan, ZHOU Hao, XUE Xian-li. Effects of msn2 Knock-out on the Growth and Kojic Acid Production of Aspergillus oryzae[J]. Biotechnology Bulletin, 2022, 38(8): 188-197.

图/表 8

表1 RT-qPCR 的引物

Table 1 RT-qPCR primers

目的基因Target gene	引物序列Sequence of primer（5'-3'）
brlA	TTATCACAAGGCGGAGAAGG
brlA	CGAACAGTCAGTATGGGTTGC
tps1	GAATACGGGACTGGCCCT
tps1	GGGCTCGAAACATATCTGTGA
ageB	CACCATCTCCAACCACTCG
ageB	CAGCGTAGGTGGGAGACC
LaeA	TCCATTCATAGCAAGAGTTCTGAA
LaeA	TTGCTCCTGCTCATCGC
kojR	CCGCAGACGTTAGCGTAAAT
kojR	GCGATCGGCCATATTCATAC
his2A	GCCTGGTGGAAAGGGAAAG
his2A	CTTTGCGCTGTGGGACTT
msn2	AAGCGAGGCTGTGACTTTG
msn2	AGGCGCTGTCTCTTTTCG

图1 质粒pk1的构建及△msn2菌株的筛选 A：pk1质粒示意图;B：msn2敲除示意图;C：转化子的平板筛选;D：△msn2菌株的PCR验证。M：DL5000;1：以3.042基因组为模板作为阴性对照（2 965 bp）;2：质粒pk1为模板作为阳性对照（3 706 bp）;3-5：转化子,其中3为质粒随机插入,4为质粒未插入,5为阳性转化子,6为以5号转化子基因组为PCR扩增msn2L上游到-pyrG中间（2 602 bp）

Fig.1 Construction of plasmid pk1 and screening of △msn2 strain A：pk1 plasmid diagram. B：msn2 knock-out diagram. C：Screening of transformants on plate. D：PCR verification of △msn2 strains. M：DL5000;1：negative control with genome template in 3.042（2 965 bp）;2：positive control with pk1 template（3 706 bp）;3-5：transformants,3 refers to that pk1 was randomly inserted in it,4 refers to that pk1 was not inserted in it,5 is positive transformant,and 6 refers to msn2L up stream-pyrG middle（2 602 bp）was validation by PCR with transformant 5 genome as template

表2 米曲霉3.042和g-5菌株生长形态观察

Table 2 Growth morphology of Aspergillus oryzae 3.042 and g-5 strain

菌株Strain	24 h	36 h	48 h
3.042
g-5

表3 米曲霉3.042和g-5菌株的H2O2耐受性

Table 3 H2O2 tolerance of A. oryzae 3.042 and g-5 strain

时间 Time/h	菌株 Strain	0 mmol·L^-1	15 mmol·L^-1	25 mmol·L^-1	30 mmol·L^-1
36	3.042
36	g-5
48	3.042
48	g-5

表4 米曲霉3.042和g-5菌株高糖耐受性

Table 4 High sugar tolerance of A. oryzae 3.042 and g-5 strains

时间 Time/h	菌株Strain	2%	10%	15%
36	3.042
36	g-5
48	3.042
48	g-5

图2 米曲霉3.042和g-5号菌株发酵产曲酸分析及菌丝球形态观察 A：米曲霉3.042和g-5号菌株发酵产曲酸分析;B：3.042发酵7 d菌丝球形态;C：g-5菌株发酵7d菌丝球形态

Fig. 2 Fermentation results of A. oryzae 3.042 and strain g-5 and observation of mycelial pellet morphology A：Kojic acid yield of A. oryzae 3.042 and g-5 strain. B：Mycelial pellet morphology of A. oryzae 3.042 in at 7 d in fermentation. C：Mycelial pellet morphology of g-5 strain at 7 d of fermentation

图3 米曲霉3.042和g-5菌株brlA、tps1和ageB相对表达量

Fig. 3 Relative expressions of A. oryzae 3.042 and g-5 strain brlA,tps1 and ageB

图4 米曲霉3.042和g-5菌株kojR、msn2和LaeA相对表达量

Fig. 4 Relative expressions of A. oryzae 3.042 and g-5 strain kojR,msn2 and LaeA

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