谷氨酸棒杆菌人工合成启动子文库的构建及应用  

Construction and application of a synthetic promoter library for Corynebacterium glutamicum

作　　者：刘莫识 刘娇[2,3] 孙冠男 路福平 王钰[2,3] 郑平[2,3] 孙际宾 LIU Moshi;LIU Jiao;SUN Guannan;LU Fuping;WANG Yu;ZHENG Ping;SUN Jibin(School of Biological Engineering,Tianjin University of Science and Technology,Tianjin 300457,China;Key Laboratory of Systems Microbial Biotechnology,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;National Technology Innovation Center of Synthetic Biology,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院系统微生物工程重点实验室天津工业生物技术研究所,天津300308 [3]国家合成生物技术创新中心,天津300308

出　　处：《生物工程学报》2022年第2期831-842,共12页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2019YFA0904900);国家自然科学基金(31800038);天津市自然科学基金(18JCQNJC10300)。

摘　　要：启动子是实现基因精细表达调控的重要工具,广泛应用于微生物的代谢工程改造。谷氨酸棒杆菌是重要的工业底盘,已报道的启动子文库较少且主要是基于完全人工设计的突变序列构建获得。本研究对谷氨酸棒杆菌odhA基因天然启动子的–10区及附近序列进行随机突变,借助rfp报告基因和荧光成像系统进行高通量筛选,构建了包含57个相对强度为2.4–16.7倍的人工启动子文库,最高强度可达强诱导型启动子P_(trc)的2.3倍。分析文库的突变序列,发现55个启动子突变体的–10区保守序列“TANNNT”均向3′端移动1–4个碱基,其中移动4个碱基的突变体占68%;同时发现强、中、弱启动子突变体在不同位置还呈现T或G保守碱基。选择5个不同强度的启动子应用于L-脯氨酸合成途径γ-谷氨酰激酶(ProB)的表达调控,结果显示,L-脯氨酸产量随着启动子强度增强逐渐提高,相对强度为9.8倍的启动子达到最高产量(6.4 g/L),更高强度的启动子将不再提高L-脯氨酸的产量。本研究基于谷氨酸棒杆菌天然启动子P_(odhA)构建文库,成功获得1个强度增强显著、分布均匀的新型启动子文库,可用于谷氨酸棒杆菌的系统代谢工程改造,还可为更多启动子文库的构建提供思路和方法借鉴。Promoter is an important genetic tool for fine-tuning of gene expression and has been widely used for metabolic engineering. Corynebacterium glutamicum is an important chassis for industrial biotechnology. However, promoter libraries that are applicable to C. glutamicum have been rarely reported, except for a few developed based on synthetic sequences containing random mutations. In this study, we constructed a promoter library based on the native promoter of odhA gene by mutating the -10 region and the bystanders. Using a red fluorescent protein (RFP) as the reporter, 57 promoter mutants were screened by fluorescence imaging technology in a high-throughput manner. These mutants spanned a strength range between 2.4-fold and 19.6-fold improvements of the wild-type promoter. The strongest mutant exhibited a 2.3-fold higher strength than the widely used strong inducible promoter P_(trc). Sequencing of all 57 mutants revealed that 55 mutants share a 1-4 bases shift (4 bases shift for 68% mutants) of the conserved -10 motif “TANNNT” to the 3′ end of the promoter, compared to the wild-type promoter. Conserved T or G bases at different positions were observed for strong, moderate, and weak promoter mutants. Finally, five promoter mutants with different strength were employed to fine-tune the expression of γ-glutamyl kinase (ProB) for L-proline biosynthesis. Increased promoter strength led to enhanced L-proline production and the highest L-proline titer of 6.4 g/L was obtained when a promoter mutant with a 9.8-fold higher strength compared to the wild-type promoter was used for ProB expression. The use of stronger promoter variants did not further improve L-proline production. In conclusion, a promoter library was constructed based on a native C. glutamicum promoter P_(odhA). The new promoter library should be useful for systems metabolic engineering of C. glutamicum. The strategy of mutating native promoter may also guide the construction of promoter libraries for other microorganisms.

关 键 词：谷氨酸棒杆菌 人工合成启动子文库 P_(odhA)启动子 L-脯氨酸 

分 类 号：Q78[生物学—分子生物学]