敲除msn2对米曲霉生长和发酵产曲酸的影响

Effects of msn2 Knock-out on the Growth and Kojic Acid Production of Aspergillus oryzae

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 H_(2)O_(2)。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。

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/LH_(2)O_(2).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.