细菌启动子识别及应用研究进展

细菌启动子是细菌中基因表达的必需调控元件,决定了细菌基因表达的强度和时机。通过启动子的插入或缺失,可以改变细菌基因的表达,实现对菌体生长发育以及代谢调控的研究。启动子也是构建各种表达系统、实现异源基因表达的基础。启动子的识别和应用研究,对于实现异源基因的可控表达、有效获得目的产物、促进生物催化和代谢工程研究具有重要的意义。以下对细菌启动子进行了简单的介绍,总结了细菌启动子的识别方法,并对细菌启动子的研究进展和具体应用进行了概述。     

Dynamic change in promoter activation during lysine biosynthesis in Escherichia coli cells

We investigated the expression dynamics of genes involved in lysine biosynthesis in Escherichia coli cells to obtain a quantitative understanding of the gene regulatory system. By constructing reporter strains expressing the green fluorescence protein (gfp) gene under the control of the promoter regions of those genes associated with lysine biosynthesis, time-dependent changes in gene expression in response to changes in lysine concentration in the medium were monitored by flow cytometry. Five promoters involved in lysine biosynthesis respond to the changes in lysine concentration in the medium. For these five promoters, time-dependent gene expression data were fitted to a simple dynamical model of gene expression to estimate the parameters of the gene regulatory system. According to the fitting parameters, dapD shows a significantly larger coefficient of repression than the other genes in the lysine synthesis pathway, which indicates the weak binding activity of the repressor to the dapD promoter region. Moreover, there is a trend that the closer an enzyme is to the start of the lysine biosynthesis pathway, the smaller its maximal promoter activity is. The results provide a better quantitative understanding of the expression dynamics in the lysine biosynthesis pathway.     

Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 microg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p(R) promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.     

A gene fusion at a homeobox locus: alterations in leaf shape and implications for morphological evolution.

Compound leaves are seen in many angiosperm genera and are thought to be either fundamentally different from simple leaves or elaborations of simple leaves. The knotted1-like homeobox (knox) genes are known to regulate plant development. When overexpressed in homologous or heterologous species, this family of genes can cause changes in leaf morphology, including excessive leaf compounding in tomato. We describe here an instance of a spontaneously arisen fusion between a gene encoding a metabolic enzyme and a homeodomain protein. We show that the fusion results in overexpression of the homeodomain protein and a change in morphology that approximates the changes caused by overexpression of the same gene under the control of the cauliflower mosaic virus 35S promoter in transgenic plants. Exon-shuffling events can account for the modularity of proteins. If the shuffled exons are associated with altered promoters, changes in gene expression patterns can result. Our results show that gene fusions of this nature can cause changes in expression patterns that lead to altered morphology. We suggest that such phenomena may have played a role in the evolution of form.     

Cloning of an ovule specific promoter from Arabidopsis thaliana and expression of beta-glucuronidase

Tissue specific expression of transgenes in plant species has several advantages over constitutive expression. Identification of ovule specific promoters would be useful in genetic engineering of plants with a variety of desirable traits such as genetically engineered parthenocarpy, female sterile plants or seedless fruits. Relative inaccessibility and difficulty in harvesting adequate amounts of tissue at known developmental stages has impeded the progress in cloning of promoters involved in ovule development. In the present study an ovule specific promoter was cloned from Arabidopsis AGL11 gene and used to express GUS (beta-glucuronidase) gene in transgenic Arabidopsis. Histochemical staining of GUS appeared in the center of young ovary (ovules), but no detectable GUS activity was observed in vegetative plant tissues, sepals, petals and androecium. AGL11 gene promoter can be useful to modify the developmental path of plants by expressing either plant hormones or lethal genes for agronomic purpose.     

Improving lycopene production in Escherichia coli by engineering metabolic control

Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.     

谷氨酸棒杆菌代谢工程高效生产L-缬氨酸北大核心CSCD

L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。     

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.     

Metabolic Engineering of Carotenoid Biosynthesis in Escherichia coli by Ordered Gene Assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 μg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p_R promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.     

启动子PpetE与Pcpc560对集胞藻PCC 6803生物合成乙醇的影响

为了增加工程集胞藻PCC 6803的乙醇合成产量,通过选用强启动子Pcpc560 驱动并提高外源乙醇合成基因(pdc,yqhD)的表达,从而促进乙醇的生产。具体方法利用同源双交换引入来源于运动型发酵单胞菌的丙酮酸脱羧酶基因(pdc)与来源于大肠杆菌的NADPH依赖型醛还原酶基因(yqhD)并选用不同的启动子来驱动其表达。通过逆转录定量PCR分析,比较在不同启动子驱动的情况下,外源乙醇合成基因(pdc,yqhD)的表达情况并检测相应突变株的乙醇产量。结果显示相较于中等启动子,铜离子诱导启动子PpetE,来源于集胞藻PCC 6803的光强启动子Pcpc560显著促进了外源乙醇合成基因(pdc,yqhD)的表达,并增加了工程菌株乙醇合成的产量。超强启动子Pcpc560搭配pdc,yqhD的组合表达,显著提高了工程菌株的乙醇合成产量。