多基因同步调控结合高通量筛选构建高产L-苯丙氨酸的谷氨酸棒杆菌工程菌株  

Construction of L-phenylalanine High-producing Corynebacterium glutamicum Engineered Strains via Multi-gene Simultaneous Regulation Combined with High-throughput Screening

作　　者：薛宁[1,2,3] 王瑾 李世新 刘叶 程海娇 张玥 毛雨丰 王猛 XUE Ning;WANG Jin;LI Shi-xin;LIU Ye;CHENG Hai-jiao;ZHANG Yue;MAO Yu-feng;WANG Meng(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308;Key Laboratory of Engineering Biology for Low-Carbon Manufacturing,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308 [3]中国科学院天津工业生物技术研究所低碳合成工程生物学重点实验室,天津300308

出　　处：《生物技术通报》2023年第9期268-280,共13页Biotechnology Bulletin

基　　金：国家重点研发计划(2018YFA0902900);国家自然科学基金项目(32101186);天津市合成生物技术创新能力提升行动项目(TSBICIP-PTJS-003)。

摘　　要：L-苯丙氨酸(L-Phe)是重要的食品及医药中间体,但由于其生物合成途径较长且调控机制复杂,仅依靠质粒或者周期漫长的基因组逐轮改造,难以实现各个模块之间的通量平衡,在一定程度上限制了其产量的进一步提升。本研究将L-Phe生物合成途径中的7个携带组成型启动子PF11及核糖体结合位点(ribosome binding site,RBS)为GGGGGGGG的关键基因ppsA、tktA、aroF^(fbr)、aroE、aroL、pheA^(fbr)和tyrB,整合到谷氨酸棒杆菌(Corynebacterium glutamicum)的基因组中;利用课题组前期研发的基于CRISPR/Cas系统和胞苷脱氨酶的无模板基因表达调控技术(base editor-targeted and template-free expression regulation,BETTER),对每个关键基因的RBS进行同步编辑,生成富含G/A的RBS突变文库;利用前期开发的荧光蛋白型L-Phe生物传感器结合液滴微流控系统(droplet microfluidics)对RBS突变文库进行高通量筛选。最终,从大约7万个RBS突变文库中筛选到4株L-Phe产量提升不同程度的突变菌株,其72 h摇瓶发酵产量分别为2.06、1.30、4.42和7.44 mmol/L,相比对照菌株C.g-2(0.6 mmol/L)分别提高了2.43、1.17、6.37、11.40倍。利用碱基编辑器同时调节多基因的表达水平并筛选组合文库,可以为L-Phe工程育种提供一种可行策略。L-phenylalanine(L-Phe)is an important intermediate in the food and pharmaceutical industries.However,due to the complex regulation mechanisms and the long its biosynthetic pathway,it is difficult to achieve the flux balance between each module by relying only on plasmids or long-cycle iterations of genome editing,thus limiting its production.In this study,seven key genes(ppsA,tktA,aroF^(fbr),aroE,aroL,pheA^(fbr),and tyrB)carrying the constitutive promoter PF11 and a ribosome binding site(RBS)with the sequence GGGGGGGG in the L-Phe biosynthesis pathway were integrated into the genome of Corynebacterium glutamicum.Using the Base Editor-Targeted and Templatefree Expression Regulation(BETTER)technology in our labbased on the CRISPR/Cas system and cytidine deaminase,the RBSs for each key gene were simultaneously edited to generate a RBS mutant library enriched with G/A.The library was subjected to high-throughput screening using a fluorescence protein-based L-Phe biosensor combined with a droplet microfluidics system.Finally,four mutant strains were selected from about 70000 RBS mutants with varied degrees of L-Phe production improvement.The L-Phe titers in these strains at 72 h during shake-flask fermentation were 2.06,1.30,4.42,and 7.44 mmol/L,respectively,which were 2.43,1.17,6.37 and 11.40 times higher than the control strain C.g-2(0.6 mmol/L).The simultaneous regulation of multigene expression levels and screening of combinatorial libraries using base editing techniques may provide a feasible strategy for L-Phe engineering breeding.

关 键 词：多基因同步调控 碱基编辑技术 L-苯丙氨酸 谷氨酸棒杆菌 代谢工程改造 高通量筛选 液滴微流控 生物传感器 

分 类 号：Q78[生物学—分子生物学]