生物技术通报 ›› 2023, Vol. 39 ›› Issue (12): 311-319.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0702

• 研究报告 • 上一篇    下一篇

米曲霉磷酸甲羟戊酸激酶功能研究

尚怡彤1,2(), 闫欢欢1,2, 王丽红1,2, 田学琴1,2, 薛萍红1,2, 罗涛1, 胡志宏1,2()   

  1. 1.江西科技师范大学生命科学学院,南昌 330013
    2.江西科技师范大学江西省生物加工过程重点实验室,南昌 330013
  • 收稿日期:2023-07-21 出版日期:2023-12-26 发布日期:2024-01-11
  • 通讯作者: 胡志宏,男,博士,副教授,研究方向:微生物分子生物学;E-mail: huzhihong426@163.com
  • 作者简介:尚怡彤,女,硕士研究生,研究方向:微生物分子生物学;E-mail: syt15735905986@163.com
  • 基金资助:
    江西省科技厅自然科学基金面上项目(20212BAB205001);江西省科技厅自然科学基金重点项目(20192ACBL20012);国家自然科学基金项目(32260009)

Study on the Function of Phosphomevalonate Kinase in Aspergillus oryzae

SHANG Yi-tong1,2(), YAN Huan-huan1,2, WANG Li-hong1,2, TIAN Xue-qin1,2, XUE Ping-hong1,2, LUO Tao1, HU Zhi-hong1,2()   

  1. 1. College of Life Science, Jiangxi Science and Technology Normal University, Nanchang 330013
    2. Jiangxi Key Laboratory of Bioprocess Engineering, Jiangxi Science and Technology Normal University, Nanchang 330013
  • Received:2023-07-21 Published:2023-12-26 Online:2024-01-11

摘要:

磷酸甲羟戊酸激酶(PMK)是甲羟戊酸(MVA)途径的关键酶。在真菌中,MVA途径是麦角甾醇生物合成的上游,因此PMK也被称为Erg8。为了研究磷酸甲羟戊酸激酶在米曲霉(Aspergillus oryzae)麦角甾醇合成通路中的作用,对米曲霉AoErg8基因功能进行研究。采用生物信息学方法鉴定米曲霉中的该基因,通过系统发育树和酵母异源互补分析其是否保守,利用实时荧光定量PCR(RT-qPCR)检测其表达模式,同时通过荧光蛋白标记对其亚细胞定位进行分析,最后测定AoErg8基因过表达对米曲霉生长和麦角甾醇含量的影响。结果表明,AoErg8进化保守,其表达量在不同生长时间和不同非生物胁迫下均发生了改变;AoErg8能恢复酿酒酵母erg8突变体的温度敏感表型;AoErg8定位于细胞质中;米曲霉中AoErg8过表达导致麦角甾醇含量降低,并且影响米曲霉生长和孢子形成。因此,米曲霉AoErg8的功能相对保守,其过表达可以降低麦角甾醇含量并影响菌落生长和孢子形成。该研究进一步揭示丝状真菌米曲霉麦角甾醇生物合成和调控机理,为米曲霉或其他真菌脂质代谢的基因工程奠定基础。

关键词: 米曲霉, 磷酸甲羟戊酸激酶, 麦角甾醇, 亚细胞定位, 孢子形成

Abstract:

Phosphomevalonate kinase(PMK)is a key enzyme in the mevalonate(MVA)pathway. In fungi, the MVA pathway is the upstream of ergosterol biosynthesis and therefore PMK is also known as Erg8. In order to study the role of phosphomevalonate kinase in ergosterol synthesis pathway of Aspergillus oryzae, the function of AoErg8 gene in A. oryzae was preliminarily studied. Bioinformatics method was used to identify AoErg8. Phylogenetic tree and yeast allogeneic complementation were used to analyze whether this gene was conserved. Then the subcellular location was determined via fluorescence protein labelling and gene expression pattern were analyzed via RT-qPCR. Finally, the effects of overexpression of this gene on the growth and ergosterol content of A. oryzae were determined. AoErg8 was evolutionarily conservative, and its expression was different under different growth times and different abiotic stress. AoErg8 restored the temperature sensitive phenotype of Saccharomyces cerevisiae erg8 mutant. AoErg8 was located in the cytoplasm. The overexpression of AoErg8 led to the decrease of ergosterol content, and affected growth and spore formation. Therefore, the function of AoErg8 in A. oryzae was conservative and its overexpression reduceed the ergosterol content, also affected the growth of colonies and sporulation. This study further reveals the biosynthesis and regulatory mechanisms of ergosterol in this important filamentous fungus A. oryzae and lays the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi.

Key words: Aspergillus oryzae, phosphomevalonate kinase, ergosterol, subcellular localization, sporulation