Ramzan Rabia, Safiullah Virk Muhammad, Muhammad Zafarullah, Ahmed Amani Mohedein Mohammed, Yuan Xi, Chen Fusheng
Hubei International Scientific and Technological Cooperation Base of Traditional Fermented Foods, Huazhong Agricultural University, Wuhan 430070, China.
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Microorganisms. 2019 Sep 24;7(10):390. doi: 10.3390/microorganisms7100390.
The biosynthesis of penicillin G (PG) is compartmentalized, which forces penicillin and its intermediates to cross the membrane barriers. Although many aspects around the penicillin intermediates traffic system remain unclosed, the transmembrane transporter protein involvement has been only predicted. In the present work, detection of PG and isopenicillin N (IPN) in M7 was performed and functions of gene as a transporter were investigated by the combination of gene deletion (Δ) complementation (Δ::) and overexpression (M7::PtrpC-). While, the feeding of PG pathway precursor side chain and amino acids, i.e., phenylacetic acid, D-valine, and L-cysteine was performed for the interpretation of gene role as an intermediate transporter. The results showed that, the feeding of phenylacetic acid, D-valine, and L-cysteine possessed a significant effect on morphologies, secondary metabolites (SMs) production of all above-mentioned strains including M7. The results of UPLC-MS/MS revealed that, Δ interrupt the penicillin G (PG) production in M7 by blocking the IPN transportation, while PG and IPN produced by the Δ:: have been recovered the similar levels to those of M7. Conclusively, these findings suggest that the M7 is, not only a PG producer, but also, indicate that the gene is supposed to play a key role in IPN intermediate compound transportation during the PG production in M7.
青霉素G(PG)的生物合成是分区进行的,这使得青霉素及其中间体必须穿过膜屏障。尽管围绕青霉素中间体转运系统的许多方面仍未明确,但仅预测了跨膜转运蛋白的参与情况。在本研究中,对M7中的PG和异青霉素N(IPN)进行了检测,并通过基因缺失(Δ)、互补(Δ::)和过表达(M7::PtrpC-)相结合的方法研究了基因作为转运蛋白的功能。同时,通过添加PG途径前体侧链和氨基酸,即苯乙酸、D-缬氨酸和L-半胱氨酸,来阐释该基因作为中间体转运蛋白的作用。结果表明,添加苯乙酸、D-缬氨酸和L-半胱氨酸对包括M7在内的所有上述菌株的形态和次级代谢产物(SMs)产量都有显著影响。超高效液相色谱-串联质谱(UPLC-MS/MS)结果显示,Δ通过阻断IPN转运中断了M7中青霉素G(PG)的产生,而Δ::产生的PG和IPN已恢复到与M7相似的水平。总之,这些发现表明M7不仅是PG的生产者,而且表明该基因在M7中PG产生过程中IPN中间化合物的转运中可能起关键作用。