依赖于AcPpm1的甲硫氨酸参与的头孢菌素生物合成的分子调控

Cephalosporins are the most important anti-bacterial drugs. Methionine dramatically stimulates cephalosporin production in Acremonium chrysogenum and has been widely used in industrial cephalosporin production, but its regulatory mechanism is poorly understood. Recently, the improvement of the fungal transformation system and the genomic sequencing of A. chrysogenum provide favorable conditions for discovering the regulatory mechanism of methionine on cephalosporin biosynthesis. Our group previously constructed an Acppm1 disruption mutant of A. chrysogenum, the mutant produced more cephalosporin C when decreasing the concentration of methionine in the fermentation medium. Based on the investigation of Acppm1 disruption mutant, we will discover the biological function of Acppm1 in A. chrysogenum and reveal the molecular mechanism of AcPpm1-dependent methionine regulation in cephalosporin biosynthesis through transcriptome analysis, gene disruptions and genetic complementation, enzyme activity analysis and so on. We will further improve the regulatory pathway through genetic engineering, obtain the methionine-independent cephalosporin high-producing strain and lay the foundation for upgrading of antibiotic industry.

头孢类抗生素是临床上使用最为广泛的抗感染药物。甲硫氨酸作为头孢菌素合成的重要促进剂广泛应用于工业生产中，但其参与头孢菌素合成调控的机制一直不清楚。近期，头孢菌素产生菌-顶头孢霉遗传操作系统的逐渐完善以及基因组序列的完成都为阐明甲硫氨酸参与的头孢菌素生物合成调控机制提供了有利条件。本课题前期构建了一株S-腺苷甲硫氨酸（SAM）依赖的甲基转移酶编码基因Acppm1缺失突变株，该突变株中头孢菌素的产生明显不再依赖于发酵培养基中添加的甲硫氨酸。我们拟在此基础上，通过转录组分析、基因敲除与互补、体外酶学活性测定等，系统阐明Acppm1的功能，揭示依赖于AcPpm1的甲硫氨酸参与的头孢菌素生物合成调控的分子机制，并实现甲硫氨酸参与的头孢菌素生物合成调控的定向遗传改造，获得不依赖于甲硫氨酸诱导的头孢菌素高产菌株，降低生产成本，为传统抗生素工业的升级换代奠定基础。

甲硫氨酸作为头孢菌素合成的重要促进剂广泛应用于工业生产中，但其机制一直不清楚。我们研究发现，甲硫氨酸是通过形成S-腺苷甲硫氨酸（SAM）促进头孢菌素C(CPC)的合成，提高胞内SAM的含量可以显著提高CPC的产量。通过高表达AcsamS和mecB、筛选并敲除S-腺苷甲硫氨酸依赖的甲基转移酶基因Acppm1，在顶头孢霉中获得了不依赖甲硫氨酸刺激却能提高CPC产量的菌株，该菌株在不添加甲硫氨酸的发酵培养基中CPC产量是同样培养条件下出发菌株的5.5倍。同时发现在发酵培养基中添加甲硫氨酸或SAM都可以显著提高CPC合成相关基因pcbAB、pcbC、cefEF、cefG以及参与甲硫氨酸循环的相关基因AccysD、胱硫醚-β-合成酶编码基因AcmecA和mecB的转录水平，说明SAM通过增加CPC合成前体物半胱氨酸而促进CPC的产生。此外，发现外源添加甲硫氨酸显著抑制顶头孢霉分生孢子产生。外源添加S-腺苷甲硫氨酸或者在体内高表达AcsamS时也导致分生孢子产量明显下降。外源添加的甲硫氨酸也同时抑制了顶头孢霉中自噬的发生。进而发现甲硫氨酸是通过自噬控制Acgph1所编码的糖原磷酸酶的量来影响顶头孢霉产孢。为进一步揭示自噬在CPC产生中的作用，我们对自噬基因Acatg8、Acatg11和Acatg12开展了研究。这些结果系统揭示了甲硫氨酸在CPC合成中的作用机制。