乳酸菌食品级基因表达系统

酸菌是一类重要工业菌株。最近,乳酸菌遗传学和分子生物学的研究取得长足进步,导致发展了乳酸菌食品级基因表达系统。通过介绍乳酸菌食品级基因表达系统的基本要求、食品级选择性标记、食品级诱导物及该系统的研究进展,展示了乳酸菌食品级基因表达系统的建立对研究乳酸菌的基因表达调控和它的深层次的开发利用所具有的重要意义。     

食品级高效诱导表达系统-NICE系统

乳酸菌NICE系统是在乳链菌肽诱导下由nisA启动子控制目的基因表达的,含nisR和nisK的两组分调节系统的高效诱导表达系统。由于NICE系统的诱导剂、宿主菌和载体都是食品级的,其应用前景十分广阔。     

食品级乳酸菌表达系统研究进展

乳酸菌表达系统是近几年发展起来的食品级高效表达系统。乳酸菌具有益生菌特征,因此该表达系统与其他细菌表达系统相比有很多优点。介绍了糖诱导表达系统、噬菌体Φ31爆发式诱导的表达系统、乳链球菌素调控表达系统、温控表达系统等的研究进展,以及这些系统的应用前景。     

乳酸菌蛋白质分泌表达研究进展

食品级乳酸菌不仅是食品或消化道中传递异源蛋白质的合适的候选菌,在工业发酵中还可用于生产蛋白质。在过去20多年中,人们设计了许多乳酸菌蛋白质表达和标记系统,这些系统已用在乳酸菌工程菌的细胞内或细胞外生产各种细菌、病毒和真核生物来源的蛋白质。在目的蛋白生产和发酵中,分泌表达由于可持续培养和简化纯化步骤并使目的蛋白与其靶位相互作用而优于细胞质表达。目前只有少数研究报道了目的蛋白在乳酸菌细胞内或分泌表达产量的比较,研究表明分泌表达比细胞质表达更优越。     

产Ⅱ类细菌素乳酸菌群体感应及其应用

群体感应(quorum sensing,QS)是微生物通过感知与细胞密度相关的信号分子的浓度来调控基因表达的一种行为。许多产Ⅱ类细菌素乳酸菌通过自诱导肽介导的QS系统调控其细菌素的合成。本文综述了乳酸菌Ⅱ类细菌素合成的QS调控现象、调控机制、QS系统组分以及QS的应用。产Ⅱ类细菌素乳酸菌QS的研究,必将为揭示发酵调控机理、调控发酵过程提供新的研究平台,为食品级基因表达系统的开发提供新的选择。     

乳酸菌食品级载体选择标记

食品级载体不依靠抗生素抗性基因作为选择标记,因而更为安全。近年来,人们已构建了多种乳酸菌的食品级克隆载体。这些食品级克隆载体是在显性选择标记和互补选择标记的基础上建立的。最近提出了第三种新的方法。该就这三种食品级载体选择标记的功能、分子机制及应用前景作了简明的介绍。     

乳酸菌载体pMG36e的应用现状

乳酸乳球菌通用表达质粒pMG36e是一个经典的人工构建的组成型表达载体,是以乳酸乳球菌乳脂亚种蛋白酶基因的转录和翻译信号为基础构建而成的。它包含一个强启动子,能够在多种细菌中表达外源蛋白。已用于研究细菌素作用机制,乳酸菌基因工程菌株的改造以及口服疫苗的开发等,应用领域十分广泛,已成为乳酸菌基因工程研究的重要工具质粒之一。本文主要从载体构成、基因表达与食品级载体改造等三方面的应用对其进行综述,旨在为该质粒今后研究提供资料。     

乳酸菌食品级表达载体的研究与应用

乳酸菌是能够发酵糖类产生大量有机酸的革兰氏阳性菌的通称,在发酵食品中有着悠久的应用历史.乳酸菌通常被认为是安全菌株,这些微生物的基因工程操作在食品、医学等方面具有广阔的应用前景.表达载体是基因工程中常用的工具之一,大多数乳酸菌的表达载体通常以抗生素抗性基因作为选择标记,然而抗性基因具有潜在的转移性,因此需要开发食品级表...     

乳酸乳球菌食品级表达载体的研究进展

乳酸乳球菌（L.lactis）是乳球菌属中最重要和最典型的一个种,在食品工业中应用广泛,被公认为安全的（generally regards as safe,GRAS）食品级微生物。以乳酸乳球菌作为宿主菌,构建表达载体用来表达异源蛋白和酶,逐渐成为食品工业、生物制药和疫苗研究的热点。近年来,乳酸乳球菌的分子微生物学研究取得了重大进展,这为表达载体的构建奠定了基础,一些具有不同用途的乳酸乳球菌基因表达载体已经构建,用来表达抗原蛋白、细胞因子和生物酶等。其中,以来源于食品级微生物的DNA片段构建的食品级表达载体引起人们的关注。     

细菌异源表达抗菌肽的研究进展

抗菌肽是一类从动植物、微生物体内分离得到的阳离子小分子量肽,具有天然的抗菌活性。它作用迅速,广谱,不易产生耐药性,具有重要的应用价值,近年来成为研究热点。普遍认为异源表达是生产大量抗菌肽的最有效方法。大肠杆菌作为经典的表达宿主,具有生长速度快、遗传背景清晰、有大量可利用的商业表达载体、易操作等优势,现已成为抗菌肽表达的首选宿主。乳酸菌作为世界公认安全的食品级微生物,近年来广泛用于抗菌肽的异源表达。着重阐述了抗菌肽在大肠杆菌、乳酸菌中重组表达的研究进展。     

Food-grade gene expression in lactic acid bacteria

In the 1990s, significant efforts were invested in the research and development of food-grade expression systems in lactic acid bacteria (LAB). At this time, Lactococcus lactis in particular was demonstrated to be an ideal cell factory for the food-grade production of recombinant proteins. Steady progress has since been made in research on LAB, including Lactococcus, Lactobacillus and Streptococcus, in the areas of recombinant enzyme production, industrial food fermentation, and gene and metabolic pathway regulation. Over the past decade, this work has also led to new approaches on chromosomal integration vectors and host/vector systems. These newly constructed food-grade gene expression systems were designed with specific attention to self-cloning strategies, food-grade selection markers, plasmid replication and chromosomal gene replacements. In this review, we discuss some well-characterized chromosomal integration and food-grade host/vector systems used in LAB, with a special focus on sustainability, stability and overall safety, and give some attractive examples of protein expression that are based on these systems.     

Incorporation of nisI-mediated nisin immunity improves vector-based nisin-controlled gene expression in lactic acid bacteria

Lactic acid bacteria (LAB) have been used successfully to express a wide variety of recombinant proteins, ranging from flavor-active proteins to antibiotic peptides and oral vaccines. The nisin-controlled expression (NICE) system is the most prevalent of the systems for production of heterologous proteins in LAB. Previous optimization of the NICE system has revealed a strong limit on the concentration of the inducer nisin that can be tolerated by the culture of host cells. In this work, the nisin immunity gene, nisI, has been inserted into the recently reported pMSP3535H2 vector that contains the complete NICE system on a high-copy Escherichia coli-LAB shuttle vector. Fed-batch fermentation data show that Lactococcus lactis IL1403 cells transformed with the new vector, pMSP3535H3, tolerate a 5-fold increase in the concentration of the inducer nisin, and, at this elevated concentration, produce a 1.8-fold increased level of green fluorescent protein (GFP), a model recombinant protein. Therefore, the incorporation of nisI in the pMSP3535H3 NICE system described here unveils new ranges of induction parameters to be studied in the course of optimizing recombinant protein expression in LAB.     

10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis

Lactococcus lactis is a Gram-positive lactic acid bacterium that, in addition to its traditional use in food fermentations, is increasingly used in modern biotechnological applications. In the last 25 years great progress has been made in the development of genetic engineering tools and the molecular characterization of this species. A new versatile and tightly controlled gene expression system, based on the auto-regulation mechanism of the bacteriocin nisin, was developed 10 years ago—the NIsin Controlled gene Expression system, called NICE. This system has become one of the most successful and widely used tools for regulated gene expression in Gram-positive bacteria. The review describes, after a brief introduction of the host bacterium L. lactis, the fundaments, components and function of the NICE system. Furthermore, an extensive overview is provided of the different applications in lactococci and other Gram-positive bacteria: (1) over-expression of homologous and heterologous genes for functional studies and to obtain large quantities of specific gene products, (2) metabolic engineering, (3) expression of prokaryotic and eukaryotic membrane proteins, (4) protein secretion and anchoring in the cell envelope, (5) expression of genes with toxic products and analysis of essential genes and (6) large-scale applications. Finally, an overview is given of growth and induction conditions for lab-scale and industrial-scale applications.     

A review of food-grade vectors in lactic acid bacteria: from the laboratory to their application

Lactic acid bacteria (LAB) have a long history of use in fermented foods and as probiotics. Genetic manipulation of these microorganisms has great potential for new applications in food safety, as well as in the development of improved food products and in health. While genetic engineering of LAB could have a major positive impact on the food and pharmaceutical industries, progress could be prevented by legal issues related to the controversy surrounding this technology. The safe use of genetically modified LAB requires the development of food-grade cloning systems containing only the DNA from homologous hosts or generally considered as safe organisms, and not dependent antibiotic markers. The rationale for the development of cloning vectors derived from cryptic LAB plasmids is the need for new genetic engineering tools, therefore a vision from cryptic plasmids to applications in food-grade vectors for LAB plasmids is shown in this review. Replicative and integrative vectors for the construction of food-grade vectors, and the relationship between resistance mechanism and expression systems, will be treated in depth in this paper. Finally, we will discuss the limited use of these vectors, and the problems arising from their use.     

Development of Vaccine Delivery Vehicles Based on Lactic Acid Bacteria

Live recombinant bacteria represent attractive antigen delivery systems able to induce both mucosal and systemic immune responses against heterologous antigens. The first live recombinant bacterial vectors developed were derived from attenuated pathogenic microorganisms. In addition to the difficulties often encountered in the construction of stable attenuated mutants of pathogenic organisms, attenuated pathogens may retain a residual virulence level that renders them unsuitable for the vaccination of partially immunocompetent individuals such as infants, the elderly or immunocompromised patients. As an alternative to this strategy, non-pathogenic food-grade lactic acid bacteria (LAB) maybe used as live antigen carriers. This article reviews LAB vaccines constructed using antigens other than tetanus toxin fragment C, against bacterial, viral, and parasitic infective agents, for which protection studies have been performed. The antigens utilized for the development of LAB vaccines are briefly described, along with the efficiency of these systems in protection studies. Moreover, the key factors affecting the performance of these systems are highlighted.     

乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

Structure/function relationship of homopolysaccharide producing glycansucrases and therapeutic potential of their synthesised glycans

The capability of lactic acid bacteria (LAB) to produce exopoly- and oligosaccharides was and is the subject of expanding research efforts. Due to their physicochemical properties and health-promoting potential, exopoly- and oligosaccharides from food-grade LAB can be used in the food and other industries and may have additional medical applications. In the last years, many LAB have been screened for their ability to produce exopoly- and oligosaccharides, and several glycosyltransferases involved in their biosynthesis have been characterised at biochemical and genetic levels. These research efforts aim to exploit the full potential of these organisms and to understand the structure/function relationship of glycosyltransferases. The latter knowledge is a prerequisite for the production of tailored exopoly- and oligosaccharides for the diverse applications. This review will survey the results of recent works on the structure/function relationship of homopolysaccharide producing glycosyltransferases and the therapeutic potential of their synthesised exopoly- and oligosaccharides.