基于时间序列转录组筛选谷氨酸棒杆菌内源高效组成型启动子

Screening efficient constitutive promoters in Corynebacterium glutamicum based on time-series transcriptome analysis

摘要:

启动子是重要的转录调控元件，广泛用于工业菌株的代谢工程改造。谷氨酸棒杆菌Corynebacterium glutamicum 是重要的氨基酸生产菌株，但已报道的组成型强启动子较少。对谷氨酸高产菌Corynebacterium glutamicum SL4发酵过程的10个时间点样品进行转录组测序，筛选在发酵过程中稳定转录并且转录水平最高的10个基因；分别克隆其启动子序列至红色荧光蛋白 (RFP) 报告系统，通过荧光强度表征启动子在SL4菌株中的强度，再在野生型C. glutamicum ATCC 13869和ATCC 13032中验证部分启动子的通用性；并采用LacZ蛋白进一步评价强启动子的表达效果。结果显示，成功筛选到3个可以通用的组成型启动子PcysK、PgapA和PfumC。其中PcysK的表达强度最高，与诱导型强启动子Ptac对比，在SL4和13869菌株中均达到其2倍 (RFP) 和4倍 (LacZ) 以上；在ATCC 13032菌株中，PcysK的表达强度为Ptac的0.3?0.4倍。PcysK首次被报道为强启动子，可用于谷氨酸棒杆菌强化合成途径的代谢工程改造。

Abstract:

Promoter, an essential regulatory element, is widely used for metabolic engineering of industrial strains. Corynebacterium glutamicum is an important industrial workhorse to produce various amino acids. However, strong constitutive promoters that are applicable to C. glutamicum are rarely reported. In this study, we first performed a time-series transcriptome analysis of a glutamate hyper-producing strain C. glutamicum SL4 by using RNA-Seq. Overall, we picked 10 samples at different time during the fermentation process. By analyzing the time-series transcriptome data, we selected 10 candidate genes with the highest transcriptional level. These genes were all transcribed stably during the fermentation process. We subsequently cloned the promoter sequences and evaluated the promoters’ strength in strain SL4 using a red fluorescent protein reporter system. To evaluate the universality of the promoters in different C. glutamicum strains, we further tested the performance of some promoters in wild type C. glutamicum strains, including ATCC 13869 and ATCC 13032. The strongest promoter was further characterized using LacZ as a reporter in all the three C. glutamicum strains. Finally, we successfully obtained three constitutive promoters with universality, PcysK, PgapA and PfumC. PcysK is the most efficient promoter among the three C. glutamicum strains. In strains SL4 and ATCC 13869, the strength of PcysK is 2-fold of the strong inducible promoter Ptac using the red fluorescent protein as a reporter and 4-fold of Ptac using LacZ as a reporter. Moreover, the strength of PcysK reaches 30%?40% of Ptac in strain ATCC 13032. The promoter PcysK is identified as a strong promoter for the first time, which can be used as an efficient biobrick for metabolic engineering of synthesis pathways in C. glutamicum.