大肠杆菌合成启动子的构建及在顺,顺-粘康酸生物合成中的应用

Construction of synthetic promoters for Escherichia coli and application in the biosynthesis of cis,cis-muconic acid

摘要:

启动子是基因表达调控的重要元件。在代谢工程和合成生物学研究中，经常需要利用不同强度的启动子对代谢途径进行精细调控，来实现代谢平衡，降低中间产物积累，提高目标产物合成。然而目前可获得的启动子难以满足以上要求，而且不同来源的启动子通用性差，缺乏标准化。针对这些问题，设计了1条88个碱基对的启动子，包含典型的?35区、?10区以及核糖体结合区。同时，在转录起始位点上游6个碱基、?35与?10区间隔区14个碱基对中引入简并序列，构建了合成启动子文库。利用合成启动子控制红色荧光蛋白mCherry的表达强度，经过两轮筛选，从5 000多个克隆中获得了720个不同强度的启动子。随机挑选35条不同强度的启动子进行测序分析，结果表明不同强度的启动子具有碱基偏好性。对于强启动子，?13位点嘌呤碱基出现频率高，转录起始区除?4位点外，嘧啶碱基出现的频率高于嘌呤碱基，而?10区与?35区间14个位点的嘌呤碱基与嘧啶碱基出现频率大致相当。最后选取5条不同强度启动子应用于顺,顺-粘康酸合成途径调控优化，结果显示不同强度的启动子可以调节目标产物顺,顺-粘康酸的合成和中间产物儿茶酚的积累。

Abstract:

Promoter is one of important elements for gene expression and regulation. In the construction of recombinants for metabolic engineering and synthetic biology, it is necessary to have the promoters with varying strengths for fine-tuning metabolic pathway to reach the metabolic balance, decrease the accumulation of intermediate and increase the production of target metabolite. However, the natural promoters available are not completely suitable for fine-tuning metabolic pathway due to discrete strength, lack of versatility and standardization. To deal with this problem, in this study, a new 88 bp synthetic promoter, which contains the typical ?35 box, ?10 box as well as ribosome bind site, was designed. Then, the promoter library was constructed by introducing some degenerate base pairs in the sequence of 6 bp in the upstream of the initial transcription site and 14 bp in spacer region between ?35 and ?10 box. 720 promoters with varying strengths were screened out from a library of more than 5 000 clones via the expression of red fluorescent protein mCherry under the control of the synthetic promoter. The sequence analysis based on 35 promoters with varying strengths showed the promoters with varying strengths are base preference. The purine bases in ?13 site and pyrimidine bases in the transcriptional initiation sequence are of high frequency; the purine and pyrimidine bases are of the similar frequency in the spacer sequence between ?35 and ?10 box in strong promoter. In the end, five characterized promoters with varying strengths were selected to tune the synthetic pathway of cis,cis-muconic acid in Escherichia coli. The results showed that the promoters with varying strengths can regulate the production of cis, cis-muconic acid and the accumulation of the intermediate catechol.