谷氨酸棒杆菌人工合成启动子文库的构建及应用

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...     

人工合成启动子文库研究进展

随着合成生物学的发展,人工合成功能元件在代谢工程领域显示出巨大应用潜力。启动子是调控基因转录水平的重要元件,可以利用人工合成启动子文库对代谢途径进行精细调控,使基因适量表达,实现代谢途径的组合优化。文章综述了人工合成启动子文库的研究进展,评述了不同文库的构建策略,讨论了人工合成启动子文库在代谢工程领域的应用,并展望了人工合成启动子文库的发展方向。     

Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression

Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering.     

Synthetic promoter libraries for Corynebacterium glutamicum

The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7–59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.     

Screening of promoters from <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. CGMCC 3584 using a green fluorescent protein reporter system

Available molecular and genetic tools for the genetic manipulation of Arthrobacter species are limited until now. In gene engineering, a continuous set of promoters with various strengths are of importance for fine-tuning gene expression in metabolic optimization and control analysis. Here, for the first time, we constructed a promoter trap system using green fluorescence protein (GFP) as a reporter, for screening and characterizing functional Arthrobacter promoters. Twenty-three Arthrobacter transformants of various GFP fluorescence strengths were isolated and characterized through the analysis of DNA sequences. Among the 23 putative promoters, 2 were selected for deletion analysis of promoter elements. As a result, the deletion of the upstream of the putative promoter P8 and P13 caused a 43.8% decrease and a 29.1% increase in the fluorescence signals, respectively. Finally, we obtained the strongest promoter P13-3 which was 4.4 times more potent than the promoter of 6–hydroxyl–d–nicotine oxidase gene which was previously reported in Arthrobacter nicotinovorans, and the obtained promoter was used to improve the production of cyclic adenosine monophosphate in Arthrobacter sp. CGMCC 3584. The screening strategy together with obtained promoters in this study would contribute to the future engineering of Arthrobacter species.     

Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli

Due to their induction characteristics stationary-phase promoters have a great potential in biotechnological processes for the production of heterologous proteins on a large-scale. In order to broaden the utility of stationary-phase promoters in bacterial expression systems and to create novel promoters induced by metabolic conditions, a library of synthetic stationary-phase/stress promoters for Escherichia coli was constructed. For designing the promoters the known -10 consensus sequence as well as the extended -10 region and an A/T-rich region downstream of the -10 region were kept constant, while sequences from -37 to -14 were partially or completely randomized. For detection and selection of stationary-phase promoters GFP with enhanced fluorescence was used. The expression pattern of the GFP reporter system was compared with that of the LacZ reporter system. To screen and characterize colonies containing stationary-phase/stress promoters a bioinformatic approach was developed. In total, 33 promoters were selected which cover a broad range of promoter activities and induction times indicating that the strength of promoters can be modulated by partially randomizing the sequence upstream of the -10 region. The induction ratio of synthetic promoters at the transition from exponential to stationary-phase was from 4 to over 6000 and the induction time relative to the entrance into stationary-phase from -1.4 to 2.7 h. Ninety-one percentage of the promoters had no or only low background activity during exponential growth. The broad variability of the promoters offers good possibilities for fine-tuning of gene expression and for applications in industrial bioprocesses.     

Red/ET重组及其在生物医学中的应用

通过应用Rac噬菌体的RecE RecT和λ噬菌体的RedαRedβ系统而建立的DNA工程平台———Red ET重组,是一种不依赖于限制性内切酶的分子克隆新技术。运用该技术能够介导PCR产物或寡核苷酸对目标基因进行剪切、插入、融合及突变等多种操作,在生物医学领域里具有广阔的应用前景,尤其在基因组功能研究中对BACs、PACs和细菌染色体的打靶修饰以及基因敲除动物DNA靶分子的快速构建等方面最有效。随着Red ET重组的推广与应用,该技术本身也在不断被改进,在工作效率得到显著提高的同时,其操作也变得更加简单、省时、省力。结合自身的一些研究结果,对Red ET重组的技术特点、发展现状和在生物医学中的应用进行了详细阐述。     

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.     

Optimization of phage λ promoter strength for synthetic small regulatory RNA-based metabolic engineering

Synthetic small regulatory RNAs (sRNAs) are gene-silencing tools that can be used to tune gene expression in prokaryotes. A recent study by our group proposed rational design principles, introduced a regulatory system that may be used to implement synthetic sRNAs, and showed their utility in metabolic engineering. The regulatory system employed the strong phage λ P_R promoter to tightly control synthetic sRNA production. Here, we fine-tuned the strength of the P_R promoter via mutagenesis in order to optimize the level of synthetic sRNAs while maintaining the ability of the promoter to be regulated by CI proteins. Five mutant promoters of different strengths, ranging from 24 to 87% of that of the wild-type P_R promoter, were identified and confirmed to be repressed by CI proteins. A mutated promoter with only 40% of the original strength still produced enough synthetic sRNAs to inhibit the translation of the target mRNA to ~10% of the original level. As a practical application, we tested our promoters as drivers for a synthetic anti-murE sRNA, which was used to adjust the production of cadaverine. As the promoter strength decreased, the cadaverine titer first increased and then dropped. A mutated promoter with 39% of the original strength achieved the improved cadaverine titer of 2.15 g/L. The mutant promoters developed in this study should prove useful for tuning the expression levels of synthetic sRNAs for metabolic engineering.     

Constitutive expression of a native human interferon-alpha 1 gene in Escherichia coli

1. A plasmid for constitutive expression of the human interferon-alpha 1 (hIFN-alpha 1) gene in Escherichia coli is constructed on the basis of the cloning plasmid pBR322 using a strong synthetic promoter, synthetic ribosome binding site and a native hIFN-alpha 1 gene excised from a chromosomal clone. 2. The yield of recombinant hIFN-alpha 1 from E. coli LE392 cells transformed with the expression plasmid pJP1R9-hIFN-alpha 1 is evaluated to be 2-6 x 10(7) U/l bacterial culture for metabolic shaker and 6-8 x 10(7) U/l for fermentor.