Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum
Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum
复制标题
DOI：
10.3390/jof5040096
复制
发表时间：
2019-10
期刊：
影响因子：
4.7
通讯作者：
Yasaman Mahmoudjanlou;Birgit Hoff;U. Kück
中科院分区：
文献类型：
--
作者：
Yasaman Mahmoudjanlou;Birgit Hoff;U. Kück
短密青霉菌是一种丝状子囊菌，用于制药工业生产麦考酚酸，一种免疫抑制剂。为了扩展这种真菌的基因工程选择，我们测试了两种以前未应用于短密青霉的抗性标记。虽然普遍可用的腐草霉素抗性标记（ble）成功地用于DNA介导的转化实验，我们不能使用一个普遍适用的诺尔丝菌素抗性盒（nat 1）。为了避免这一失败，我们构建了一个新的nat基因，考虑到密码子偏好的短密青霉。然后，我们在随后的转化实验中使用这种修饰的nat基因，靶向破坏两个核基因，MAT 1 -2-1和flbA。对于MAT 1 -2-1，我们获得了频率约为10%的缺失菌株。在flbA的情况下，频率为约4%，并且该破坏菌株也显示出减少的分生孢子形成。为了确认缺失，我们使用ble重新引入野生型基因。该步骤恢复了flbA缺失菌株中的野生型表型，其具有孢子形成缺陷。这里描述的成功的转化系统大大扩展了对生物技术相关真菌短密青霉进行遗传操作的选择。
Penicillium brevicompactum is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to P. brevicompactum. Although a generally available phleomycin resistance marker (ble) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette (nat1). To circumvent this failure, we constructed a new nat gene, considering the codon bias for P. brevicompactum. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, MAT1-2-1 and flbA. For MAT1-2-1, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used ble to reintroduce the wild-type genes. This step restored the wild-type phenotype in the flbA deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus P. brevicompactum.