双向启动子(bidirectional promoters, BDPs)是指在启动子转录起始位点左右两端7~25 bp内存在一个相反的转录位点的启动子,BDPs可以作为基因元件协调不同基因的表达,并且在单边元件受损的情况下会导致两边基因共同失活,从而实现共调控。然而,如何在原核生物中迅速筛选出高活性BDPs缺乏高效快速的手段。该研究构建了一种在原核生物中迅速筛选出高活性BDPs的方法。根据实验室前期工作,构建了一种BDPs活性探针载体p19BDP,用于完成平板上BDPs快速筛选,最终筛选出2个来自大肠杆菌以及1个来自谷氨酸棒杆菌的-10或-35区单区杂合的高活性BDPs。随后设计了7种对称性文库筛选出8个在谷氨酸棒杆菌中具有双边活性的高活性BDPs,荧光检测发现这8个启动子在大肠杆菌中也具有双向性。最后将筛选出的BDPs应用到大肠杆菌甲羟戊酸(mevalonate,MVA)生产中,与添加0.5 mmol/L异丙基硫代半乳糖苷的串联表达的T7启动子相比,双边分别表达单个基因的BDP-W6在24 h的MVA产量与对照菌产量相似,为19.8 mg/L,48 h的MVA产量为对照菌72.6％的产量,为32.2 mg/L。该研究提供了一种大规模筛选BDPs的研究方案,同时验证了BDPs的有效性,与T7启动子的诱导表达相比,BDPs的双向高活性表达更具有经济效益。
Bidirectional promoters (BDPs) are defined as promoters possessing an opposing transcription site within 7-25 bp of both the right and left ends of the transcription start site of the promoter.BDPs serve as gene elements that coordinate the expression of different genes, and can lead to the inactivation of both genes if a single side of the element is compromised, thereby facilitating co-regulation.However, there is a lack of efficient and rapid methods for screening highly active BDPs in prokaryotes.In this study, a method was developed to rapidly screen for highly active BDPs in prokaryotes.Based on previous laboratory work, the active probe vector p19BDP for rapid BDP screening on plates was developed.Finally, two highly active BDPs from Escherichia coli and one from Corynebacterium glutamicum, each with heterozygous -10 or -35 regions, were identified.Subsequently, seven symmetrical libraries were developed to identify eight highly active BDPs exhibiting bilateral activity in C.glutamicum.Fluorescence detection confirmed that these eight promoters also functioned bidirectionally in E.coli.The selected BDPs were utilized for the production of mevalonate (MVA) in E.coli.Compared to the T7 promoter induced with 0.5 mmol/L isopropyl-beta-D-thiogalactopyranoside, BDP-W6 expressing genes on both sides achieved a similar MVA yield to the control strain at 24 hours (19.8 mg/L).At 48 hours, the MVA yield reached 72.6% of the control strain’s yield, totalling 32.2 mg/L.This study outlines a research methodology for the large-scale screening of BDPs and confirms their effectiveness.Compared with the induced expression of the T7 promoter, the bidirectional high-activity expression of BDPs is more economical.
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