膨大弯颈霉中与Bmt生物合成相关的PKS基因功能研究  

Functional study of the PKS gene related to Bmt biosynthesis in Tolypocladium inflatum

作　　者：杨秀青 李丝竹 徐康 刘美洁 郭立忠[1] 于浩[1] YANG Xiuqing;LI Sizhu;XU Kang;LIU Meijie;GUO Lizhong;YU Hao(Shandong Provincial Key Laboratory of Applied Mycology,School of Life Sciences,Qingdao Agricultural University,Qingdao 266109,Shandong,China)

机构地区：[1]青岛农业大学生命科学学院、山东省应用真菌重点实验室,山东青岛266109

出　　处：《菌物学报》2023年第8期1691-1700,共10页Mycosystema

基　　金：国家自然科学基金(32000041);山东省自然科学基金(ZR2020QC005)。

摘　　要：环孢霉素A是一种主要由膨大弯颈霉Tolypocladium inflatum产生的环肽类次级代谢产物,临床上被广泛用作免疫抑制剂,其合成需要特殊底物(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine(Bmt),但目前相关Bmt的研究很少。基因敲除实验证实Bmt的生物合成需要一个聚酮合酶(polyketosynthase,PKS)基因(simG)参与,并推测其功能为合成Bmt的前体化合物羧酸分子3(R)-hydroxyl-4(R)-methyl6(E)-octenoic acid(B1)。本研究通过在同为虫生真菌的球孢白僵菌Beauveria bassiana中异源表达simG,以证明该基因的功能。通过克隆获得了较大基因simG,利用根癌农杆菌Agrobacterium tumefaciens介导方法将该基因转化到球孢白僵菌中,通过基因检测和半定量RT-PCR筛选出目标基因表达量高的菌株,获得4株simG高表达菌株。将其发酵培养后,利用LC-MS检测发酵产物,发现在simG高表达菌株中存在与B1分子量相同的产物峰。本研究进一步证明了simG负责化合物B1的合成,是Bmt合成的关键基因。本研究进一步加深了对环孢霉素的生物合成机理的认识,为构建Bmt高产的工程菌株提供理论支撑,有望从解决底物来源角度促进环孢霉素生产效率的提高。Cyclosporine A is a cyclic peptide secondary metabolite mainly produced by Tolypocladium inflatum and is widely used as an immunosuppressive agent in clinical practice.Its synthesis requires a special substrate(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine(Bmt).However,there are few studies on Bmt at present.Simulating gene knockouts confirmed that the biosynthesis of Bmt required the participation of a polyketosynthase(PKS)gene(simG)which was assumed as the precursor compound of Bmt synthesis,and named as 3(R)-hydroxyl-4(R)-methyl-6(E)-octenoic acid(B1).In this study,the simG was heterologously expressed in Beauveria bassiana to testify its function.The simG was cloned and transformed into B.bassiana through the mediation of Agrobacterium tumefaciens.Strains with a high expression of the simG were screened by gene detection and semi-quantitative RT-PCR.Four strains with high simG expression have been obtained.After fermentation and culture,the products of the strains were detected by LC-MS.It was shown that there was a product peak with the same molecular weight as B1 in the strain with high simG expression.This study further proves that the simG is probably responsible for the synthesis of compound B1 and is a key gene in the synthesis process of Bmt.Our results further deepened the understanding of the biosynthesis mechanism of cyclosporine,providing theoretical support for the construction of engineering strains with a high yield of Bmt.It was expected that the production efficiency of cyclosporine could be improved by solving the source of the substrate.

关 键 词：环孢霉素 BMT 膨大弯颈霉 生物合成 异源表达 

分 类 号：R914.5[医药卫生—药物化学]