Abstract
With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.
中文概要
概 要
该综述较为全面地概述了当前针对大肠杆菌和谷 氨酸棒杆菌基因组遗传改造的各个方法的具体 流程、应用范围、注意事项以及其新颖之处, 比 较了针对基因定点突变、基因失活和基因过表达 的各个方法所存在的优缺点, 同时简单地介绍了 利用质粒介导基因过表达所存在的问题。此外, 还介绍了四种引物设计软件, 并简单分析了它们 的应用范围。为拟计划开展分子生物学实验的新 手对关于细菌基因组遗传改造方法做了可靠的 介绍, 同时也为已进行相关实验的实验员提供关 于基因定点突变、基因失活和基因过表达的最新 信息。
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1 Introduction
It is now well established that bacteria, such as Escherichia coli, Corynebacterium glutamicum, and yeast, exhibit excellent characteristics in producing amino acids, organic acids, and vitamins, etc. (Hou et al., 2012; Xu et al., 2013; 2014a; 2014b; 2015). Although the high-producing strains were obtained via repeated physical and/or chemical mutagenesis, there are many disadvantages to the processes, such as slow-growth, low sugar consumption rating, and low stress tolerance (Xu et al., 2013). Fortunately, the development of techniques for directed DNA manipulation and the availability of whole genome sequences make real the possibility of constructing high-producing strains using genetic engineering techniques (Kalinowski et al., 2003; Baba et al., 2006). These strategies have been successfully used in modifying E. coli or C. glutamicum strains for producing, e.g., amino acids (Georgi et al., 2005; Becker et al., 2011), organic acids (Inui et al., 2007), and vitamins (Martens et al., 2002). The strategies of genetic engineering breeding for constructing high-producing strains are as follows: (1) up-regulation of the key enzyme gene involved in the target product biosynthetic pathways; (2) relieving the inhibition and/or repression of the key enzyme; (3) interruption of the pathways for synthesizing by-products. The implementation of the above-mentioned strategies involves a wide variety of DNA manipulations, including site-directed mutagenesis (SDM), gene inactivation and over-expression. In contrast to conventional breeding via repeated physical and/or chemical mutagenesis, genetic engineering breeding gives more practicable options for subsequently isolating mutant strains because it can avoid poor physiological characteristics and unknown inherited characteristics. Therefore, develo** a new method to achieve faultless DNA manipulations is one of the most popular research subjects.
Here we review the common strategies used for DNA manipulations. We focus on the strategies for DNA manipulations via genetically modifying the E. coli and C. glutamicum genomes. In addition, the potential problems of multi-layered DNA manipulations are considered. However, this review avoids any discussion of plasmid-mediated gene over-expression. It intends to be an easy introduction for novices and a source of new experimental information for experienced investigators.
2 Strategies for SDM
SDM, also called “rational mutagenesis” (Mandaci, 2011), is commonly used to introduce mutations at definite sites of a target DNA fragment, including the genome and plasmid, via polymerase chain reaction (PCR) or restriction endonuclease reaction (RER). It plays a great role in understanding the regulatory motifs of operon and the relationship of protein structure to function (Ling and Robinson, 1997; Seyfang and **, 2004; Wu et al., 2013). Depending on the numbers of mutational sites, SDM can be divided into two types: single site-directed mutagenesis (Single-SDM) and multiple site-directed mutagenesis (Multi-SDM) (Liang et al., 2012).
2.1 Strategies of Single-SDM
Single-SDM is mainly based on the amplification of double-stranded DNA (dsDNA) plasmid using complementary primer pairs, which contain 20–30 oligonucleotides and the desired mutation (Ling and Robinson, 1997; Holland et al., 2015). Because of its simplicity, time-saving capability, and relatively high efficiency, this approach has become a common strategy for introducing mutation into E. coli and C. glutamicum genomes (Ling and Robinson, 1997; Muyrers et al., 2001; Xu et al., 2014b). Although most of the methods used for Single-SDM have been reviewed by researchers around 15–20 years ago (Chatellier et al., 1995; Ling and Robinson, 1997; Muyrers et al., 2001), many new methods are springing up along with the development of genetic engineering technique.
2.1.1 Single-SDM by enzyme digestion and homologous recombination (ED-HR)
Commercial kits are simple and easy to use for SDM (Imai et al., 1991; Martin et al., 1995; Chiu et al., 2004; Stoynova et al., 2004), but some defects usually limit their application in creating larger deletions (Li et al., 2008). In order to overcome the limitations of commercial kits, the methods used for larger deletion have been developed based on DNA ligation and hybridization in vitro (Imai et al., 1991; Chiu et al., 2004; Stoynova et al., 2004) or on homologous recombination in vivo (Martin et al., 1995). The improved homologous recombination technique has greatly simplified the procedure of mutagenesis (Li et al., 2008), with only two steps: PCR amplification and transformation (Martin et al., 1995). However, the efficiency is not invariable because the plasmid used as a template in PCR amplification is also transformed into a competent cell (Li et al., 2008). Although many efforts have been made to overcome this obstacle, such as gel purification of PCR products, they are time-consuming and do not work well for purifying the few products resulting from fewer PCR cycles and/or lower amplification efficiency (Li et al., 2008).
It is generally known that most bacteria possess DNA restriction-modification (R-M) systems that are used to protect the cell from intrusion by foreign DNA. In most cases, “self” DNA is marked by methylation, whereas in “non-self” DNA it is absence of methylation which marks it (Siwek et al., 2012). The DpnI restriction system is one of the R-M systems (Johnston et al., 2013). DpnI endonuclease cleaves only the double-stranded (ds)-methylated 5′-GmATC-3′ sequence in DNA (Lacks and Greenberg, 1977). Interestingly, the plasmid extracted from bacteria contains methyladenosine at the DpnI restriction site, which makes the plasmid susceptible to DpnI (Lu et al., 2002), whereas PCR-amplified DNA does not contain methyladenosine (Li et al., 2008). Based on this principle, PCR using plasmid DNA as a template is a very useful way to obtain replication and mutagenesis (Lu et al., 2002). PCR products digested by DpnI have been widely used in mutagenesis, and the protocols are listed in Fig. 1a. In general, the PCR products are digested in vitro by DpnI (Weiner et al., 1994; Martin et al., 1995; Qi and Scholthof, 2008). Li et al. (2008) pointed out a simple and rapid strategy to digest PCR products in vivo by DpnI when carrying out SDM. The crucial aspects of this strategy are inactivation of dam gene in chromosome and heterologous expression of dpnC gene (encoding DpnI) in the host cell. Since DpnI was expressed in vivo, there is no need to purchase DpnI and to manipulate PCR products.
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Project supported by the Natural Science Foundation of Jiangsu Province (No. BK20150149), the Fundamental Research Funds for the Central Universities (No. JUSRP51504), and the Youth Foundation of Jiangnan University (No. JUSRP115A19), China
ORCID: Jian-zhong XU, http://orcid.org/0000-0001-6555-6059
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Xu, Jz., Zhang, Wg. Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression. J. Zhejiang Univ. Sci. B 17, 83–99 (2016). https://doi.org/10.1631/jzus.B1500187
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DOI: https://doi.org/10.1631/jzus.B1500187
Keywords
- Escherichia coli
- Corynebacterium glutamicum
- DNA manipulation
- Site-directed mutagenesis
- Gene inactivation
- Gene over-expression