CHO细胞表达系统启动子

中国仓鼠卵巢（Chinese hamsters ovary,CHO）细胞是目前重组蛋白质生产的首选宿主细胞。利用CHO细胞生产重组蛋白质,启动子是启动转基因转录的关键。核心启动子是RNA聚合酶与转录起始复合物集合的部位,分为集中型和分散型两种类型。目前,CHO细胞常用的启动子为病毒启动子、异源启动子、内源性和诱导性启动子等。也可以利用合成生物学及相关的数据库,人工设计合成启动子及鉴定新型启动子。本文综述了CHO细胞常用的启动子以及人工设计的合成启动子在CHO细胞中重组蛋白质表达方面的进展,为哺乳动物细胞选择合适的启动子,保证蛋白质表达量最大化,并确保长时间表达稳定性提供参考。     

CHO细胞表达系统启动子

中国仓鼠卵巢（Chinese hamsters ovary,CHO）细胞是目前重组蛋白质生产的首选宿主细胞。利用CHO细胞生产重组蛋白质,启动子是启动转基因转录的关键。核心启动子是RNA聚合酶与转录起始复合物集合的部位,分为集中型和分散型两种类型。目前,CHO细胞常用的启动子为病毒启动子、异源启动子、内源性和诱导性启动子等。也可以利用合成生物学及相关的数据库,人工设计合成启动子及鉴定新型启动子。本文综述了CHO细胞常用的启动子以及人工设计的合成启动子在CHO细胞中重组蛋白质表达方面的进展,为哺乳动物细胞选择合适的启动子,保证蛋白质表达量最大化,并确保长时间表达稳定性提供参考。     

Model-based reconstruction of synthetic promoter library in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Objective

To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum.

Results

A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the ??10 and ??35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model. A new promoter library was further reconstructed with improved promoter strength and coverage based on the results of correlation analysis. Tandem promoter P70 was finally constructed with increased strength by 121% over the tac promoter. The promoter library developed in this study showed a great potential for applications in metabolic engineering and synthetic biology for the optimization of metabolic networks.

Conclusions

To the best of our knowledge, this is the first reconstruction of synthetic promoter library based on statistical analysis of C. glutamicum.

     

Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.     

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

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.     

瘤胃微生物元基因组来源的新的组成型启动子获取

【目的】自极端环境来源的微生物的基因组中筛选新型的可用于合成生物学底盘细胞设计的启动子元件。【方法】本研究以含有绿色荧光蛋白结构基因和核糖体结合位点的探针型质粒pUC18-GFP为载体,通过构建瘤胃微生物元基因组质粒文库,从文库中快速高效筛选具有启动子功能的DNA片段。并且通过基于神经网络的启动子预测分析,获得可能的启动子区域。以绿色荧光蛋白和施氏假单胞菌Pseudomonas stutzeri来源的麦芽四糖淀粉酶作为报告基因验证所获得的新启动子片段的功能。【结果】我们从约3750个转化子中筛选到22条具有组成型启动子功能的DNA片段。这些片段与NCBI数据库中已报道的基因序列同源性较低,启动效率高低不等。我们通过启动子预测和亚克隆的方法获得两条全新的启动子片段RFa1p2 (76 bp)和RFb4p (547 bp)。此新的组成型启动子可以在不添加任何诱导剂的情况下启动异源蛋白在大肠杆菌基因工程菌中高效表达。