2023, Vol. 50
表1(Table 1)
乳酸菌的基因组编辑技术研究进展
扩展功能
文章信息
- 赵难难, 贾爱卿, 王永强, 林影, 王健
- ZHAO Nannan, JIA Aiqing, WANG Yongqiang, LIN Ying, WANG Jian
- 乳酸菌的基因组编辑技术研究进展
- Genome editing technology of lactic acid bacteria
- 微生物学通报, 2023, 50(7): 3088-3103
- Microbiology China, 2023, 50(7): 3088-3103
- DOI: 10.13344/j.microbiol.china.221003
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文章历史
- 收稿日期: 2022-10-13
- 接受日期: 2022-11-23
- 网络首发日期: 2022-12-28
赵难难, 贾爱卿, 王永强, 林影, 王健. 乳酸菌的基因组编辑技术研究进展[J]. 微生物学通报, 2023, 50(7): 3088-3103.
ZHAO Nannan, JIA Aiqing, WANG Yongqiang, LIN Ying, WANG Jian. Genome editing technology of lactic acid bacteria[J]. Microbiology China, 2023, 50(7): 3088-3103.
乳酸菌的基因组编辑技术研究进展
1. 广东海大集团股份有限公司畜牧水产研究中心, 广东 广州 511400;
2. 广东海大集团股份有限公司, 广东 广州 511400;
3. 华南理工大学生物科学与工程学院, 广东 广州 511400
2. 广东海大集团股份有限公司, 广东 广州 511400;
3. 华南理工大学生物科学与工程学院, 广东 广州 511400
摘要: 乳球菌(Lactococcus sp.)和乳杆菌(Lactobacillus sp.)是工业上常用的乳酸菌(lactic acid bacteria, LAB)，长期应用于食品和饮料的发酵。近年来，随着分子操作及遗传改造技术的不断完善，推动了乳球菌和乳杆菌的基础和应用研究，其作为功能菌株和工业微生物细胞工厂的重要潜能也不断突显出来。本文综述了工业常用乳酸菌的基因组编辑技术研究进展，着重介绍了基于整合质粒的敲除、敲入，基于基因组重组工程的精细修饰和敲除、敲入，以及基于成簇的规律性间隔的短回文重复序列及其相关蛋白9 [(clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease9 (Cas9), CRISPR/Cas9]系统的基因组编辑技术。
关键词:
乳球菌 乳杆菌 整合质粒 基因组重组工程 CRIPSR/Cas9系统
Genome editing technology of lactic acid bacteria
1. Animal Husbandry and Fisheries Research Center of Guangdong Haid Group Limited Company by Shares, Guangzhou 511400, Guangdong, China;
2. Guangdong Haid Group Limited Company by Shares, Guangzhou 511400, Guangdong, China;
3. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 511400, Guangdong, China
2. Guangdong Haid Group Limited Company by Shares, Guangzhou 511400, Guangdong, China;
3. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 511400, Guangdong, China
Abstract: Lactococcus sp. and Lactobacillus sp. are lactic acid bacteria commonly used in industry and have long been used in food and beverage fermentation. In recent years, with the continuous improvement of molecular manipulation and genetic modification techniques, the basic research and application of Lactococcus and Lactobacillus have been promoted, and their important potential as functional strains as well as industrial microbial cell factories has been highlighted. In this paper, the research progress of genome editing technology of lactic acid bacteria commonly used in industry was reviewed, and the knockout knock-in based on integrative plasmid, fine modification and knockout knock-in based on genome recombination engineering, and gene editing of CRIPSR/Cas9 system based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease9 (Cas9) were emphatically introduced.
Keywords:
Lactococcus sp. Lactobacillus sp. integrative plasmids genome recombination engineering CRIPSR/Cas9 system
乳酸菌(lactic acid bacteria, LAB)是一组无芽孢、兼性厌氧、碳水化合物代谢产物主要以乳酸为主的一类细菌。LAB种类繁多，在遗传组成和生理代谢方面具有高度多样性[1]，其中乳球菌属和乳杆菌属在遗传代谢和基因工程领域研究较多[2-5]。作为公认安全级(generally recognized as safe, GRAS)菌株，发酵食品是乳酸菌最重要的经济应用之一，商业化使用时间最长，预计未来5年市场增长7.2%[6]。近年来，随着基因工程和合成生物学技术的发展，提升乳酸菌潜在的生产性能、安全性及稳定性并构建乳酸菌细胞工厂受到了工业界和医学界的广泛关注[7-8]。
由于乳酸菌的重要应用价值及潜力，越来越多的研究致力于构建适用于该菌株的遗传改造工具和方法。20世纪80年代，基于异源转座子和原噬菌体整合酶等方法在乳酸菌中实现了染色体整合和基因失活[9-11]。21世纪初，第1个乳酸菌基因组序列(Lc. lactis subsp. lactis IL1403)被公布[12]，为实现乳酸菌遗传背景解析和定向基因组改造奠定了基础。随着基因组学和分子生物学的快速发展，研究者对乳酸菌的遗传操作系统如质粒复制系统、异源表达的启动子系统、选择系统、表达系统、转化技术等不断完善，促进了乳酸菌基因组工程技术的开发和优化，进一步推动了乳酸菌应用和基础研究[13-14]。目前，乳酸菌作为工业重要的微生物细胞工厂已成功用于高附加值化合物、抗菌剂、甜味剂和重要工业酶的生产[6, 15]。除此之外，乳酸菌还被开发作为药物和疫苗的载体用于生物治疗、预防和诊断方面的医疗应用[16-18]。本文总结了针对工业上常用的乳球菌和乳杆菌开发的基于整合质粒、基因组重组工程及基于成簇的规律性间隔的短回文重复序列及其相关蛋白9 [(clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease9 (Cas9), CRISPR/Cas9]系统的基因组编辑技术(表 1)，并概述了其在构建乳酸菌细胞工厂中的应用。
表 1 乳球菌和乳杆菌中可用的基因组编辑技术
Table 1 Genome editing techniques available in Lactococcus and Lactobacillus
| Tools | Host range | Characteristics | References |
| Integrative plasmid | Lc. Lactis, Lb. plantarum, Lb. rhamnosus, Lb. acidophilus, Lb. gasseri, Lb. casei | Based on two rare single crossing-over, low integration efficiency and time consuming; No resistance markers; dependent on counter-screening markers | [19-21] |
| ssDNA recombination | Lb. reuteri, Lc. lactis, Lb. plantarum, Lb. gasseri | Recombinase mediated, suitable for genome fine modification, low recombination efficiency; Requires high concentration of oligonucleotides and mismatch repair (MMR) system; No resistance markers | [2-3] |
| dsDNA recombination | Lb. plantarum, Lb. casei, Lb. paracasei | Recombinase mediated, suitable for gene deletion and insertion; Combined with Cre/loxP system to cause lox72 residue; No resistance marker | [22-23] |
| CRIPSR/Cas9 | Lb. reuteri, Lc. lactis, Lb. plantarum, Lb. brevis | Combined with ssDNA or dsDNA recombination, recombination before cleavage, providing efficient counter-screening tools; Recombinase mediated, cleavage before recombination, enabling gene deletion insertion and fine modification; No resistance markers | [24-28] |
| CRISPR/Cas9D10A | Lb. casei | Suitable for deletion and insertion of genes; No resistance marker; Cas9D10A causes genomic single-strand DNA breaks, which is easier to repair and can effectively avoid high mortality caused by double-strand breaks (DSBs) and is suitable for strains with low recombination capacity | [29] |
| CRIPSR/dCas9 | Lc. lactis, Lb. plantarum | Specific targeting of single or multiple target genes using dCas9 without endonuclease activity for downregulation at the mRNA level; No resistance marker | [30-32] |
| CRISPR/DBE | Lc. lactis | CRISPR-deaminase-assisted base editor, including CRISPR/cDBE and CRISPR/aDBE; Specifically targeted by sgRNA, can efficiently achieve base editing; No resistance marker | [33] |
目前，常用的乳酸菌基因组编辑技术主要是依赖细胞内源重组蛋白RecA介导的整合型质粒与基因组发生的同源双交换事件。由于RecA依赖性的双交换频率低，阳性克隆子筛选周期长，研究者开发了一系列非复制型[19, 34]和条件复制型[21, 35]整合质粒结合反向筛选标记[36-38]，进一步提高了该系统的整合和筛选效率。
1.1 整合质粒整合质粒在很多微生物的基因组编辑技术发展历程中都发挥着不可或缺的作用，载有筛选标记基因和基因组靶标基因上、下游同源臂的整合质粒，在细胞自身RecA作用下实现与基因组同源区域的两轮重组交换[20, 39]。如图 1所示，在第一轮交换中，质粒上的上游同源臂与基因组同源区进行交换，将整个质粒整合到基因组上，实现载体基因的复制，同时赋予重组菌株抗性标记。第二轮交换中，下游同源臂与基因组同源区进行交换，导致靶标基因、筛选标记基因及整合质粒上其他元件从基因组上去除。最终整合质粒上携带的上、下游同源臂取代靶标基因及其上、下游同源序列，实现基因的无痕敲除。目前乳酸菌中常用的非复制型整合质粒主要由pACYC184[40]、pUC[34]和pBluescript SK−[41]等衍生而来。例如，宿主范围比较广的突变载体pNZ5319 (衍生于pACYC184)被证明适用于乳酸菌的自杀突变，目前已广泛用于乳酸乳球菌(Lactococcus lactis, Lc. Lactis)和植物乳杆菌(Lactobacillus plantarum, Lb. plantarum)中基因的敲除和敲入[19-20]。张雅婷[42]通过对基于突变载体pNZ5319的基因组编辑方法进行优化，以Lc. lactis NZ9000-3为出发菌株构建了一系列基因组敲除菌株，为乳球菌合成生物学底盘的构建奠定了基础。由于非复制型整合质粒介导的基因整合对宿主的转化效率要求比较高，随后研究者开发了条件型复制质粒，如热敏性pG+宿主系统[35]和RepA依赖性pORI系统[21, 43] (衍生于pWV01)等来提高第1轮的交换效率。传统的整合载体筛选标记还是基于双抗性基因分别在2次单交换中进行抗性筛选，然后再结合Cre/loxP位点特异性重组系统对双交换菌株基因组上的抗性标记进行切除，实现基因无标记敲除[19-20]，但会存在lox72位点残留问题。
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| 图 1 基于整合质粒的同源重组示意图 Figure 1 Schematic diagram of homologous recombination based on integrative plasmids. HA: Homologous arm. Emr: Erythromycin resistance cassette. |
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为了加快第2轮单交换事件中双交换菌株的筛选及质粒去除，反向筛选系统应运而生。例如，广泛应用于谷氨酸棒杆菌(Corynebacterium glutamicum)基于自杀质粒pK18mobsacB/ pK19mobsacB的基因无痕敲除系统[39, 44-45]，其利用果聚糖酶SacB作为反向筛选标记，SacB可以水解蔗糖并合成果聚糖，导致谷氨酸棒杆菌对蔗糖敏感。许黎明等[46]在1株高产l-乳酸的鼠李糖乳杆菌(Lactobacillus rhamnosu, Lb. rhamnosus) JCM1553中引入了基于pK18mobsacB的反向筛选系统，成功获得了ldhD基因缺失的突变菌株，使得发酵液中l-乳酸含量达到99.92%。研究表明，编码尿嘧啶磷酸核糖转移酶的upp基因参与尿嘧啶代谢的替代途径，upp基因的缺失可以赋予突变菌株对5-氟尿嘧啶的耐药性且不会显著影响宿主的生物学特性[47]。目前upp基因已作为一种反选择标记在嗜酸乳杆菌(Lactobacillus acidophilus, Lb. acidophilus)[48]、格氏乳酸杆菌(Lactobacillus gasseri, Lb. gasseri)[49]、干酪乳杆菌(Lactobacillus casei, Lb. casei)[50]和Lc. lactis[47]中使用。由于upp基因在几乎所有的生物体中被发现，因此，该筛选系统的应用需要先构建upp基因缺失菌株。Solem等[37]构建了一个含有乳清酸转运蛋白基因(oroP)的新载体pCS1966，用于Lc. lactis在红霉素或乳清酸作用下进行染色体重组及在5-氟尿酸盐作用下进行反选择，但是oroP基因目前并未得到广泛应用。编码苯丙氨酸tRNA合成酶α亚基的基因pheS已被证明可以在很多细菌中作为反向筛选标记[51-54]。基于此，Xin等[38]在Lc. lactis NZ9000中鉴定了1个PheS蛋白，证明细胞中PheS蛋白发生突变A312G (PheS*)会使Lc. lactis NZ9000和Lb. casei BL23对p-Cl-Phe (对氯苯丙氨酸)敏感。Zhang等[55]通过在万古霉素耐药乳酸菌中异源表达二肽连接酶Ddl赋予乳酸菌对万古霉素的敏感性，随后将二肽连接酶基因整合到自杀载体上，开发了一种用于万古霉素耐药乳酸菌的反选择标记，从而扩大了目前大多数菌株可利用的基因组编辑工具箱。反向选择标记只允许失去自杀载体的细胞生长，但不能杀死恢复突变(野生型)细胞，因此理论筛选效率只有50%。
2 基于基因组重组工程的精细修饰和敲除、敲入基于整合质粒-反筛选系统的基因组整合技术虽然应用广泛，但是整个双交换菌株筛选过程操作烦琐，耗时且低效[20, 56]。随着分子生物学和合成生物学技术的发展，一些新兴的基因组改造系统，如基于原噬菌体重组酶的单链DNA (single-stranded DNA, ssDNA)重组系统及双链DNA (double-stranded DNA, dsDNA)重组系统成为操作细菌基因组的有力工具[25, 57-60]。
2.1 基因组重组工程自1998年以来，通过噬菌体重组酶介导的同源重组使细菌遗传学发生了革命性的变化[61]。其中来源于大肠杆菌λ噬菌体的Red系统和Rac噬菌体的RecET系统是最经典的重组系统[62-63]。Red重组系统由3个噬菌体编码蛋白Redα、Redβ和Redγ组成[64]，RecET系统仅由RecE和RecT这2个噬菌体编码蛋白组成[63]。蛋白Redα和RecE作为dsDNA依赖性的5′→3′核酸外切酶，可切除线性dsDNA暴露的3′端ssDNA；ssDNA-退火蛋白Redβ和RecT是单链DNA退火蛋白(single-stranded DNA annealing protein, SSAP)家族的成员，又称重组酶，可以和ssDNA末端结合，保护其不被宿主核酸酶降解的同时促进互补DNA链的退火、交换和侵入，这一过程被称为dsDNA重组工程[62-63] (图 2A)。然而在ssDNA重组工程中只有SSAP对促进单链寡核苷酸的重组是必需的[65] (图 2B)。除大肠杆菌外，Red/Rec系统仅在少数的细菌可以直接使用，可能是由于宿主遗传操作系统及未知重组机制的差异导致[59, 66-67]。因此，挖掘内源噬菌体重组酶成为提高重组效率、扩大基于重组工程的基因组编辑技术的有效策略[22, 60, 68]。
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| 图 2 基因组重组工程原理示意图 Figure 2 Schematic diagram of the principle of genome recombination engineering. A: dsDNA. B: ssDNA. HA: Homologous arm. Beta/RecT: 5ʹ→3ʹ exonuclease. Exo/RecE: Single-stranded DNA annealing protein. Cre: Cyclization recombination enzyme. |
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乳酸菌中ssDNA重组技术的应用最早是源于van Pijkeren等[3]在罗伊氏乳杆菌(Lactobacillus reuteri, Lb. reuteri)中鉴定得到了ssDNA结合蛋白RecT同系物，在无选择压力下，实现了Lb. reuteri和Lc. lactis基因组精准的碱基突变，但是效率仅达到0.4%−19%；对重组Lb. reuteri和Lc. lactis菌株的全基因组序列分析表明，ssDNA重组具有特异性，不具有高致突变性。随后该课题组在乳杆菌和Lc. Lactis中又鉴定得到一系列ssDNA结合蛋白同系物，同时探索了ssDNA重组系统在Lb. reuteri和Lc. lactis应用中的优化参数，结果表明，重组酶的来源及表达水平、寡核苷酸的设计和浓度、转化效率及错配修复(mismatch repair, MMR)系统地避免是该系统在乳酸菌中应用的关键因素[2, 68]。Guo等[69]通过筛选不同来源的重组酶，研究表明粪肠球菌来源的重组酶RecT在Lc. lactis具有最高活性，利用利福平筛选时重组效率可达到100%。ssDNA重组系统的限制因素是在无筛选压力下的效率比较低，导致无表型突变菌株的筛选成为挑战。为了解决这一问题，研究者将ssDNA重组技术耦合高效的酿脓链球菌来源Ⅱ型CRISPR/Cas9反选择系统，先利用前者使基因组靶标基因和前间隔序列邻近基序(protospacer adjacent motif, PAM)序列发生突变，然后利用Cas9的核酸内切酶活性对未发生突变的细胞基因组进行切割，造成未突变或回复突变菌株的死亡，进一步提高了突变菌株的筛选效率[24, 26, 69]。
2.3 Red/Rec系统介导的dsDNA重组Red/Rec系统介导的dsDNA重组大大促进了大肠杆菌及其他几种生物的快速、精确的基因组编辑和功能基因分析[59, 67, 70-71]。此外，研究表明宿主核酸酶抑制剂Redγ与全长的RecE/RecT或Redα/Redβ表达可有效提高dsDNA和基因组同源区的重组效率[64, 72]。基于van Pijkeren等[3]在Lb. plantarum WCFS1鉴定的RecT/Redβ同系物Lp_0641，Yang等[23]在Lb. plantarum WCFS1原噬菌体P1中鉴定得到潜在的Redγ和Redα蛋白同系物Lp_0640和Lp_0642；随后操纵子Lp_0640-41-42被证明能有效地进行dsDNA底物和宿主基因组DNA之间的同源重组，结合Cre/loxP系统，成功在Lb. plantarum中开发了一种无标记的基因组操作方法。由于Lp_0640-41-42存在宿主特异性，从而限制了该系统在其他乳酸菌中的应用。Xin等[22]通过生物信息学分析从Lb. casei BL23原噬菌体PLE3中鉴定得到1个λRed类似的重组酶操纵子LCABL_13040-50-60，并对它们的功能进行了鉴定；结合Cre/loxP系统，在Lb. casei BL23中实现了基因的敲除、敲入和精确点突变；此外，在Redγ的辅助下该系统被证明在副干酪乳杆菌(Lactobacillus paracasei, Lb. paracasei)和Lb. plantarum WCFS1中也有重组活性。利用该系统，研究者对Lb. casei BL23基因组进行大片段的连续敲除，获得了基因组减少1.68%的突变菌株[73]。dsDNA重组结合Cre/loxP系统为乳酸菌作为细胞工厂提供了简单高效的基因组操作工具，但是由于存在lox72位点的残留，并不属于无痕敲除。随后，研究者将CRISPR/Cas9反选择系统应用到dsDNA重组系统中实现了目的基因的无痕敲除，并将该无痕基因组操作方法应用到N-乙酰葡萄糖胺代谢途径的改造中[74]。
3 基于CRIPSR/Cas9系统的基因组编辑技术乳酸菌的遗传多样性导致目前已开发的基因表达元件及基因组操作工具都存在一定的宿主特异性，因此亟须开发一套通用性更强、编辑效率更高、操作更简便的基因组编辑技术。作为细菌和古菌的适应性免疫系统，CRISPR及其相关蛋白(Cas)可以有效地切割进入细胞的外源DNA，如噬菌体DNA或质粒。2013年，Cong等[75]利用CRISPR/Cas9系统在哺乳动物细胞中实现了多基因编辑，使CRISPR技术在生物基因编辑中的应用得到了推广。截至目前，CRISPR/Cas9系统已被广泛用于大肠杆菌[76-77]、枯草芽孢杆菌[77-78]、谷氨酸棒杆菌[79-80]、酿酒酵母[81-82]等工业重要细胞工厂中的基因组高效、快速的编辑及代谢调控，但是在乳酸菌中仍处于探索阶段。
3.1 CRIPSR/Cas9系统作用机制作为天然防御机制，CRISPR/Cas系统为微生物提供了序列特异性的保护，以抵御外来核酸的入侵[83]。CRISPR/Cas系统在自然界中极其多样化，并根据其相关的Cas蛋白进行分类。其中，Ⅱ型CRISPR/Cas系统(尤其是CRISPR/Cas9系统)由于具有可编程、精确、便携和高效的Cas9特征核酸酶的基因组编辑能力，是截至目前研究最广泛的系统[84]。
天然CRISPR/Cas9系统由CRISPR RNA (crRNA)、反式激活crRNA (trans-activating crRNA, tracrRNA)和Cas9核酸酶组成，Cas9核酸酶与由crRNA和tracrRNA组成的双RNA复合体形成核糖核蛋白(ribonucleoprotein, RNP)复合体，并以crRNA的间隔序列识别外源DNA的原间隔序列，将RNP复合体定位到PAM和原间隔序列区域，在PAM上游3个碱基处诱导双链断裂(double-strand break, DSB)[85-86]。然后，在宿主修复途径的帮助下，可以将感兴趣的基因修饰引入突变位点；失去原间隔子或PAM序列的突变体可能在Cas9/tracrRNA-crRNA复合物的攻击下存活下来[3, 24]。后来，研究者将CRIPSR/Cas9系统设计成由Cas9核酸酶和单向导RNA (single-guide RNA, sgRNA)组成的双组分系统，sgRNA是一种人工设计的、更简便的crRNA-tracrRNA双工形式，包含原有的gRNA骨架和可编程的20 nt的引导序列，使复合物与引导序列互补到DNA靶点[75, 87]。因此，基于CRISPR/Cas9的基因组编辑可以很容易地通过替换间隔序列来重新编程。
3.2 CRIPSR/Cas9系统在乳酸菌中的应用 3.2.1 基于CRIPSR/Cas9系统的反向筛选工具由于大多数细菌不存在非同源末端重组(non-homologous end joining, NHEJ)修复机制，当细菌染色体中被引入DSBs，细胞就会死亡，因此，CRISPR/Cas9系统可视为一种强大的反向筛选工具来实现预期突变菌株的快速筛选[2, 88] (图 3A)。在乳酸菌中，CRIPSR/Cas9的反向筛选通常是通过耦合基于寡核苷酸的重组工程来实现的，寡核苷酸使sgRNA靶向的基因组部位(包含PAM序列)突变，Cas9的序列特异性和致死性只针对未经历重组的细胞[2]。在Lb. reuteri中，为了提高重组菌株的筛选效率，研究者将CRIPSR/Cas9基因组编辑结合ssDNA重组，使得精确点突变菌株的筛选效率可以达到90%−100%[24]。在Lc. lactis中，研究者通过选择合适的重组酶和提高CRIPSR/Cas9的致死率，可以在72 h内实现精确点突变、片段敲除和敲入，筛选效率达到75%以上[69]。在Lb. plantarum中，Zhou等[26]通过建立CRIPSR/Cas9辅助的dsDNA和ssDNA重组技术，实现了对基因组的无缝编辑，包括基因敲除、敲入和点突变；随后，他们将这些策略应用于N-乙酰氨基葡萄糖的代谢工程改造，在不引入外源基因和质粒的情况下重组菌株可产生797.3 mg/L的N-乙酰氨基葡萄糖。此外，CRIPSR/Cas9系统还可以与整合质粒结合，用于Lc. lactis中单交换重组菌株的反选择，加快双交换重组菌株的构建和选择[89]。
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| 图 3 基于CRIPSR/Cas9系统的基因组编辑示意图 Figure 3 Schematic diagram of genome editing based on CRIPSR/Cas9 system. A: Counterselection in precision genome engineering. B: Gene knockout and knock-in of the genome. |
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除了作为反向筛选工具，CRIPSR/Cas9系统还被进一步开发应用于乳酸菌基因组和噬菌体基因的编辑技术。Lc. lactis作为一种应用广泛的食品和饮料发酵剂，噬菌体感染一直是食品行业持续存在并引起广泛关注的问题。研究者通过对CRIPSR/Cas9系统进行优化，成功实现了侵染Lc. lactis的强毒性噬菌体p2的基因组修饰，为研究噬菌体和宿主互作及防御噬菌体感染提供了有效的工具[27]。由于Lb. casei LC2W较低的同源重组修复(homology directed repair, HDR)潜力，CRIPSR/Cas9诱导的DSBs对LC2W是高致死的，而CRIPSR/Cas9D10A (切口酶)诱导的染色体损伤是一种不太严重的触发修复的方法，因此有助于LC2W的基因组编辑[90]。Song等[29]通过将Cas9D10A取代Cas9，并对Cas9D10A和sgRNA的表达水平进行优化，在Lb. casei LC2W中实现了染色体基因的敲除和敲入，效率可以达到25%−62%；与CRIPSR/Cas9介导的DSBs相比，切口酶Cas9D10A诱导的单链DNA断裂(切口)可能更容易修复，从而导致更多的野生型细胞逃逸。随后，他们将内源噬菌体重组酶和CRIPSR/Cas9系统相结合，实现了在Lb. plantarum和短乳杆菌(Lactobacillus brevis, Lb. brevis)中的基因组编辑，其中单基因敲除在7 d内即可完成，敲除效率可达50%−100%；染色体基因替换效率可达到35.7%[25] (图 3B)。该研究表明，表达噬菌体重组酶并引入dsDNA修复模板可以有效地提高重组效率，避免Cas9/sgRNA诱导的DSBs产生的高致死率。这种策略在其他微生物中也已得到验证[91-92]。
3.2.3 基于CRIPSR/dCas9系统的基因表达调控和碱基编辑基因表达调控在研究未知蛋白功能及代谢途径优化方面起到非常重要的作用。除在基因组靶基因处引入点突变、缺失和敲入外，CRISPR系统还可以通过CRISPR干扰(CRISPR interference, CRISPRi)来调控基因表达[93]。研究者通过构建含dCas9和gRNA的单质粒系统，在Lc. lactis中用于upp基因mRNA转录本下调，获得的重组菌株能够在含5-氟尿嘧啶的半培养基上存活，证实了upp基因沉默[30]。随后，Xiong等[31]在Lc. lactis NZ9000中又构建了用于基因转录抑制的双质粒CRISPR/dCas9系统，分别用诱导型启动子Pnisin和组成型启动子P44驱动SpdCas9和gRNA的表达；结果表明，该系统可以介导单个或多个靶基因沉默，基因表达降低率高达99%；此外，研究者利用该系统鉴定出假定的青霉素酰化酶LLNZ_07335为胆盐水解酶，其对NZ9000的胆盐抗性起作用。除了Lc. lactis，研究者也证明了CRISPR/dCas9干扰系统可以用于研究Lb. plantarum中多个重要基因功能[32]，表明CRISPRi系统是快速筛选必需基因和非必需基因表型的理想工具。近期，Tian等[33]将dCas9融合胞苷和腺苷脱氨酶分别构建了适用于Lc. lactis的CRISPR/cDBE和CRISPR/aDBE碱基编辑器，在无供体DNA模板和引入DSB的情况下，CRISPR/DBE可以有效地编辑Lc. lactis的目标碱基；而且CRISPR/cDBE被证明可以在Lc. lactis NZ9000中同时实现多个基因的失活，其中2个基因的失活效率可达100%。
4 总结与展望近些年，乳酸菌的安全性和益生作用引起了研究学者的广泛关注[94-95]。此外，许多乳酸菌具有稳健的生理特性，能够耐受乙醇和胃酸等恶劣条件，这使得它们成为工业生产菌株和生物疗法开发的优选载体[15, 96]。然而，对于具有工业和医学重要性的微生物菌株，其基因组能否被高效编辑是关键。虽然针对乳酸菌的基因组操作已开发了很多工具和方法，但是普遍存在编辑效率低、耗时长等问题，而且由于乳酸菌的系统发育多样性，多数基因编辑技术存在宿主特异性。因此有必要开发一种简单、高效、通用性更强的基因组编辑技术来促进乳酸菌代谢网络的重构和修饰。
作为新兴的基因组编辑技术，CRIPSR/Cas9系统仅在少数乳酸菌中实现了基因组编辑，并且Cas9核酸酶识别位于靶序列3′端的富含胞嘧啶的PAM序列(5′-NGG-3′)，这也限制了Cas9在低GC含量乳酸菌中有效的可编辑位点。不同来源或经过改造的Cas9突变体具有不同的PAM偏好性，根据靶位点选择合适的Cas9蛋白，可以拓展该系统在乳酸菌中的可编辑范围[97-98]。V型CRISPR/Cas系统CRISPR/Cas12 (Cpf1)识别靶序列5′端富含胸腺嘧啶(T)的PAM序列，来源于新凶手弗朗西斯菌的FnCpf1已实现了在多种微生物中的基因编辑，而且研究表明该系统脱靶效率更低，更适合多基因编辑[99-101]。该系统能否适用于乳酸菌基因组编辑有待进一步的开发验证。原核生物argonaute (pAgo)蛋白是一种新型DNA核酸内切酶，可以在gRNA/gDNA的引导下实现靶标RNA/DNA的切割，目前该系统已经实现了在禽多杀性巴氏杆菌、大肠杆菌中和枯草芽孢杆菌的基因组编辑[102-104]。该系统和CRISPR/Cas系统相比无PAM序列依赖性，因此有希望成为新一代基因组编辑技术。
由于菌株遗传背景和生长特性的差异，异源基因编辑技术的开发和应用往往存在转化效率低、酶活性低和细胞毒性的问题。例如，Cas9核酸酶的表达对很多微生物造成不可避免的细胞毒性，需要通过密码子优化及严格调控Cas9蛋白的表达来缓解细胞毒性[79-80]。因此，进一步挖掘或者设计宿主通用强的表达载体和调控元件对实现乳酸菌基因编辑技术的开发至关重要。此外，许多乳酸菌的基因组中含有不同亚型的CRISPR/Cas系统，也为基因工程提供了原生的CRISPR核酸酶[105-106]。
REFERENCES
| [1] |
KHALID K. An overview of lactic acid bacteria[J]. International Journal of Biosciences, 2011, 1: 1-13. |
| [2] |
van PIJKEREN JP, BRITTON RA. Precision genome engineering in lactic acid bacteria[J]. Microbial Cell Factories, 2014, 13(Suppl 1): S10. DOI:10.1186/1475-2859-13-S1-S10 |
| [3] |
van PIJKEREN JP, BRITTON RA. High efficiency recombineering in lactic acid bacteria[J]. Nucleic Acids Research, 2012, 40(10): e76. DOI:10.1093/nar/gks147 |
| [4] |
LIANG Y, CUI X, WANG Z, WANG T, XU ZS. Research and application of food-grade expression vectors of lactic acid bacteria[J]. Microbiology China, 2021, 48(3): 906-915. (in Chinese) 梁琰, 崔欣, 王哲, 王婷, 徐振上. 乳酸菌食品级表达载体的研究与应用[J]. 微生物学通报, 2021, 48(3): 906-915. DOI:10.13344/j.microbiol.china.200430 |
| [5] |
GUAN CR, YUAN Y, MA Y, WANG X, ZHANG CC, LU ML, GU RX, CHEN DW. Development of a novel expression system in lactic acid bacteria controlled by a broad-host-range promoter PsrfA[J]. Microbial Cell Factories, 2022, 21(1): 23. DOI:10.1186/s12934-022-01754-z |
| [6] |
BÖRNER RA, KANDASAMY V, AXELSEN AM, NIELSEN AT, BOSMA EF. Genome editing of lactic acid bacteria: opportunities for food, feed, pharma and biotech[J]. FEMS Microbiology Letters, 2019, 366(1): fny291. |
| [7] |
van TILBURG AY, CAO H, van DER MEULEN SB, SOLOPOVA A, KUIPERS OP. Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories[J]. Current Opinion in Biotechnology, 2019, 59: 1-7. DOI:10.1016/j.copbio.2019.01.007 |
| [8] |
HIDALGO-CANTABRANA C, O՚Flaherty S, Barrangou R. CRISPR-based engineering of next-generation lactic acid bacteria[J]. Current Opinion in Microbiology, 2017, 37: 79-87. DOI:10.1016/j.mib.2017.05.015 |
| [9] |
FITZGERALD GF, GASSON MJ. In vivo gene transfer systems and transposons[J]. Biochimie, 1988, 70(4): 489-502. DOI:10.1016/0300-9084(88)90085-5 |
| [10] |
LEENHOUTS KJ, KOK J, VENEMA G. Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis[J]. Applied and Environmental Microbiology, 1989, 55(2): 394-400. DOI:10.1128/aem.55.2.394-400.1989 |
| [11] |
CHOPIN MC, CHOPIN A, ROUAULT A, GALLERON N. Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome[J]. Applied and Environmental Microbiology, 1989, 55(7): 1769-1774. DOI:10.1128/aem.55.7.1769-1774.1989 |
| [12] |
BOLOTIN A, WINCKER P, MAUGER S, JAILLON O, MALARME K, WEISSENBACH J, EHRLICH SD, SOROKIN A. The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403[J]. Genome Research, 2001, 11(5): 731-753. DOI:10.1101/gr.169701 |
| [13] |
WU JP, XIN YP, KONG J, GUO TT. Genetic tools for the development of recombinant lactic acid bacteria[J]. Microbial Cell Factories, 2021, 20(1): 118. DOI:10.1186/s12934-021-01607-1 |
| [14] |
HATTI-KAUL R, CHEN L, DISHISHA T, ENSHASY HE. Lactic acid bacteria: from starter cultures to producers of chemicals[J]. FEMS Microbiology Letters, 2018, 365(20): fny213. |
| [15] |
SHARMA A, GUPTA G, AHMAD T, KAUR B, HAKEEM KR. Tailoring cellular metabolism in lactic acid bacteria through metabolic engineering[J]. Journal of Microbiological Methods, 2020, 170: 105862. DOI:10.1016/j.mimet.2020.105862 |
| [16] |
MAYS ZJ, NAIR NU. Synthetic biology in probiotic lactic acid bacteria: at the frontier of living therapeutics[J]. Current Opinion in Biotechnology, 2018, 53: 224-231. DOI:10.1016/j.copbio.2018.01.028 |
| [17] |
WANG M, GAO ZQ, ZHANG YG, PAN L. Lactic acid bacteria as mucosal delivery vehicles: a realistic therapeutic option[J]. Applied Microbiology and Biotechnology, 2016, 100(13): 5691-5701. DOI:10.1007/s00253-016-7557-x |
| [18] |
VÅGESJÖ E, ÖHNSTEDT E, MORTIER A, LOFTON H, HUSS F, PROOST P, ROOS S, PHILLIPSON M. Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria[J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(8): 1895-1900. |
| [19] |
LAMBERT JM, BONGERS RS, KLEEREBEZEM M. Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum[J]. Applied and Environmental Microbiology, 2007, 73(4): 1126-1135. DOI:10.1128/AEM.01473-06 |
| [20] |
ZHU DL, ZHAO K, XU HJ, ZHANG XM, BAI YL, SARIS PEJ, QIAO MQ. Construction of thyA deficient Lactococcus lactis using the Cre-loxP recombination system[J]. Annals of Microbiology, 2015, 65(3): 1659-1665. DOI:10.1007/s13213-014-1005-x |
| [21] |
RUSSELL WM, KLAENHAMMER TR. Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination[J]. Applied and Environmental Microbiology, 2001, 67(9): 4361-4364. DOI:10.1128/AEM.67.9.4361-4364.2001 |
| [22] |
XIN YP, GUO TT, MU YL, KONG J. Identification and functional analysis of potential prophage-derived recombinases for genome editing in Lactobacillus casei[J]. FEMS Microbiology Letters, 2017, 364(24): fnx243. |
| [23] |
YANG P, WANG J, QI QS. Prophage recombinases- mediated genome engineering in Lactobacillus plantarum[J]. Microbial Cell Factories, 2015, 14(1): 154. DOI:10.1186/s12934-015-0344-z |
| [24] |
OH JH, VAN PIJKEREN JP. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri[J]. Nucleic Acids Research, 2014, 42(17): e131. DOI:10.1093/nar/gku623 |
| [25] |
HUANG H, SONG X, YANG S. Development of a RecE/T-assisted CRISPR-Cas9 toolbox for Lactobacillus[J]. Biotechnology Journal, 2019, 14(7): 1800690. DOI:10.1002/biot.201800690 |
| [26] |
ZHOU D, JIANG ZN, PANG QX, ZHU Y, WANG Q, QI QS. CRISPR/Cas9-assisted seamless genome editing in Lactobacillus plantarum and its application in N-acetylglucosamine production[J]. Applied and Environmental Microbiology, 2019, 85(21): e01367-19. |
| [27] |
LEMAY ML, TREMBLAY DM, MOINEAU S. Genome engineering of virulent lactococcal phages using CRISPR-Cas9[J]. ACS Synthetic Biology, 2017, 6(7): 1351-1358. DOI:10.1021/acssynbio.6b00388 |
| [28] |
LEENAY RT, VENTO JM, SHAH M, MARTINO ME, LEULIER F, BEISEL CL. Streamlined, recombinase-free genome editing with CRISPR-Cas9 in Lactobacillus plantarum reveals barriers to efficient editing[J]. bioRxiv, 2018. DOI:10.1101/352039 |
| [29] |
SONG X, HUANG H, XIONG ZQ, AI LZ, YANG S. CRISPR-Cas9D10A nickase-assisted genome editing in Lactobacillus casei[J]. Applied and Environmental Microbiology, 2017, 83(22): e01259-e01217. |
| [30] |
BERLEC A, ŠKRLEC K, KOCJAN J, OLENIC M, ŠTRUKELJ B. Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis[J]. Scientific Reports, 2018, 8: 1009. DOI:10.1038/s41598-018-19402-1 |
| [31] |
XIONG ZQ, WEI YY, KONG LH, SONG X, YI HX, AI LZ. Short communication: an inducible CRISPR/dCas9 gene repression system in Lactococcus lactis[J]. Journal of Dairy Science, 2020, 103(1): 161-165. DOI:10.3168/jds.2019-17346 |
| [32] |
MYRBRÅTEN IS, WIULL K, SALEHIAN Z, HÅVARSTEIN LS, STRAUME D, MATHIESEN G, KJOS M. CRISPR interference for rapid knockdown of essential cell cycle genes in Lactobacillus plantarum[J]. mSphere, 2019, 4(2): e00007-e00019. |
| [33] |
TIAN KR, HONG X, GUO MM, LI YN, WU H, CAIYIN Q, QIAO JJ. Development of base editors for simultaneously editing multiple loci in Lactococcus lactis[J]. ACS Synthetic Biology, 2022, 11(11): 3644-3656. DOI:10.1021/acssynbio.1c00561 |
| [34] |
GROOT MN, KLAASSENS E, de VOS WM, DELCOUR J, HOLS P, KLEEREBEZEM M. Genome-based in silico detection of putative manganese transport systems in Lactobacillus plantarum and their genetic analysis[J]. Microbiology (Reading, England), 2005, 151(Pt 4): 1229-1238. |
| [35] |
MAGUIN E, DUWAT P, HEGE T, EHRLICH D, GRUSS A. New thermosensitive plasmid for gram-positive bacteria[J]. Journal of Bacteriology, 1992, 174(17): 5633-5638. DOI:10.1128/jb.174.17.5633-5638.1992 |
| [36] |
MARTINUSSEN J, HAMMER K. Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis[J]. Journal of Bacteriology, 1994, 176(21): 6457-6463. DOI:10.1128/jb.176.21.6457-6463.1994 |
| [37] |
SOLEM C, DEFOOR E, JENSEN PR, MARTINUSSEN J. Plasmid pCS1966, a new selection/counterselection tool for lactic acid bacterium strain construction based on the oroP gene, encoding an orotate transporter from Lactococcus lactis[J]. Applied and Environmental Microbiology, 2008, 74(15): 4772-4775. DOI:10.1128/AEM.00134-08 |
| [38] |
XIN YP, GUO TT, MU YL, KONG J. Development of a counterselectable seamless mutagenesis system in lactic acid bacteria[J]. Microbial Cell Factories, 2017, 16(1): 116. DOI:10.1186/s12934-017-0731-8 |
| [39] |
SCHÄFER A, TAUCH A, JÄGER W, KALINOWSKI J, THIERBACH G, PÜHLER A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum[J]. Gene, 1994, 145(1): 69-73. DOI:10.1016/0378-1119(94)90324-7 |
| [40] |
ROSE RE. The nucleotide sequence of pACYC184[J]. Nucleic Acids Research, 1988, 16(1): 355. DOI:10.1093/nar/16.1.355 |
| [41] |
LELOUP L, EHRLICH SD, ZAGOREC M, MOREl-DEVILLE F. Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes[J]. Applied and Environmental Microbiology, 1997, 63(6): 2117-2123. DOI:10.1128/aem.63.6.2117-2123.1997 |
| [42] |
ZHANG YT. Genome reduction and phenotypic identification of Lactococcus lactis[D]. Tianjin: Master's Thesis of Nankai University, 2021 (in Chinese). 张雅婷. 乳酸乳球菌基因组删减底盘构建与表型研究[D]. 天津: 南开大学硕士学位论文, 2021. |
| [43] |
LAW J, BUIST G, HAANDRIKMAN A, KOK J, VENEMA G, LEENHOUTS K. A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes[J]. Journal of Bacteriology, 1995, 177(24): 7011-7018. DOI:10.1128/jb.177.24.7011-7018.1995 |
| [44] |
HWANG GH, CHO JY. Enhancement of l-ornithine production by disruption of three genes encoding putative oxidoreductases in Corynebacterium glutamicum[J]. Journal of Industrial Microbiology & Biotechnology, 2014, 41(3): 573-578. |
| [45] |
PARK SH, KIM HU, KIM TY, PARK JS, KIM SS, LEE SY. Metabolic engineering of Corynebacterium glutamicum for l-arginine production[J]. Nature Communications, 2014, 5: 4618. DOI:10.1038/ncomms5618 |
| [46] |
XU LM, CHENG CY, LV J. Knockout of d-lactate dehydrogenase gene ldhD in Lactobacillus rhamnosus[J]. Genomics and Applied Biology, 2016, 35(6): 1421-1427. (in Chinese) 许黎明, 成春燕, 吕军. 鼠李糖乳杆菌d-乳酸脱氢酶基因ldhD的敲除[J]. 基因组学与应用生物学, 2016, 35(6): 1421-1427. |
| [47] |
SONG L, CUI HY, TANG LJ, QIAO XY, LIU M, JIANG YP, CUI W, LI YJ. Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid[J]. Journal of Microbiological Methods, 2014, 102: 37-44. DOI:10.1016/j.mimet.2014.04.011 |
| [48] |
DOUGLAS GL, GOH YJ, KLAENHAMMER TR. Integrative food grade expression system for lactic acid bacteria[M]//Methods in Molecular Biology. Totowa, NJ: Humana Press, 2011: 373-387.
|
| [49] |
SELLE K, GOH YJ, O՚FLAHERTY S, KLAENHAMMER TR. Development of an integration mutagenesis system in Lactobacillus gasseri[J]. Gut Microbes, 2014, 5(3): 326-525. DOI:10.4161/gmic.29101 |
| [50] |
SONG BF, JU LZ, LI YJ, TANG LJ. Chromosomal insertions in the Lactobacillus casei upp gene that are useful for vaccine expression[J]. Applied and Environmental Microbiology, 2014, 80(11): 3321-3326. DOI:10.1128/AEM.00175-14 |
| [51] |
KAST P. Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations[J]. Journal of Molecular Biology, 1991, 222(1): 99-124. DOI:10.1016/0022-2836(91)90740-W |
| [52] |
XIE ZJ, OKINAGA T, QI FX, ZHANG ZJ, MERRITT J. Cloning-independent and counterselectable markerless mutagenesis system in Streptococcus mutans[J]. Applied and Environmental Microbiology, 2011, 77(22): 8025-8033. DOI:10.1128/AEM.06362-11 |
| [53] |
CARR JF, DANZIGER ME, HUANG AL, DAHLBERG AE, GREGORY ST. Engineering the genome of Thermus thermophilus using a counterselectable marker[J]. Journal of Bacteriology, 2015, 197(6): 1135-1144. DOI:10.1128/JB.02384-14 |
| [54] |
KINO Y, NAKAYAMA-IMAOHJI H, FUJITA M, TADA A, YONEDA S, MURAKAMI K, HASHIMOTO M, HAYASHI T, OKAZAKI K, KUWAHARA T. Counterselection employing mutated pheS for markerless genetic deletion in Bacteroides species[J]. Anaerobe, 2016, 42: 81-88. DOI:10.1016/j.anaerobe.2016.09.004 |
| [55] |
ZHANG SW, OH JH, ALEXANDER LM, ÖZÇAM M, van PIJKEREN JP. d-alanyl-d-alanine ligase as a broad-host-range counterselection marker in vancomycin-resistant lactic acid bacteria[J]. Journal of Bacteriology, 2018, 200(13): e00607-e00617. |
| [56] |
LIU FL, ZHANG YT, QIAO WJ, ZHU DL, XU HJ, SARIS PEJ, QIAO MQ. Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system[J]. Microbial Cell Factories, 2019, 18(1): 198. DOI:10.1186/s12934-019-1249-z |
| [57] |
DATTA S, COSTANTINO N, ZHOU XM, COURT DL. Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(5): 1626-1631. |
| [58] |
LAJOIE MJ, GREGG CJ, MOSBERG JA, WASHINGTON GC, CHURCH GM. Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering[J]. Nucleic Acids Research, 2012, 40(22): e170. DOI:10.1093/nar/gks751 |
| [59] |
HUANG YY, LI L, XIE S, ZHAO NN, HAN SY, LIN Y, ZHENG SP. Recombineering using RecET in Corynebacterium glutamicum ATCC14067 via a self-excisable cassette[J]. Scientific Reports, 2017, 7: 7916. DOI:10.1038/s41598-017-08352-9 |
| [60] |
SWINGLE B, BAO ZM, MARKEL E, CHAMBERS A, CARTINHOUR S. Recombineering using RecTE from Pseudomonas syringae[J]. Applied and Environmental Microbiology, 2010, 76(15): 4960-4968. DOI:10.1128/AEM.00911-10 |
| [61] |
MURPHY KC. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli[J]. Journal of Bacteriology, 1998, 180(8): 2063-2071. DOI:10.1128/JB.180.8.2063-2071.1998 |
| [62] |
GOTTESMAN MM, GOTTESMAN ME, GOTTESMAN S, GELLERT M. Characterization of bacteriophage lambda reverse as an Escherichia coli phage carrying a unique set of host-derived recombination functions[J]. Journal of Molecular Biology, 1974, 88(2): 471-487. DOI:10.1016/0022-2836(74)90496-3 |
| [63] |
ZHANG YM, BUCHHOLZ F, MUYRERS JPP, STEWART AF. A new logic for DNA engineering using recombination in Escherichia coli[J]. Nature Genetics, 1998, 20(2): 123-128. DOI:10.1038/2417 |
| [64] |
YU D, ELLIS HM, LEE EC, JENKINS NA, COPELAND NG, COURT DL. An efficient recombination system for chromosome engineering in Escherichia coli[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(11): 5978-5983. |
| [65] |
SAWITZKE JA, THOMASON LC, BUBUNENKO M, LI XT, COSTANTINO N, COURT DL. Recombineering: highly efficient in vivo genetic engineering using single-strand oligos[J]. Methods in Enzymology, 2013, 533: 157-177. |
| [66] |
HU SB, FU J, HUANG F, DING XZ, STEWART AF, XIA LQ, ZHANG YM. Genome engineering of Agrobacterium tumefaciens using the lambda Red recombination system[J]. Applied Microbiology and Biotechnology, 2014, 98(5): 2165-2172. DOI:10.1007/s00253-013-5412-x |
| [67] |
LUO X, YANG YW, LING W, ZHUANG H, LI Q, SHANG GD. Pseudomonas putida KT2440 markerless gene deletion using a combination of λ Red recombineering and Cre/loxP site-specific recombination[J]. FEMS Microbiology Letters, 2016, 363(4): fnw014. DOI:10.1093/femsle/fnw014 |
| [68] |
van PIJKEREN JP, NEOH KM, SIRIAS D, FINDLEY AS, BRITTON RA. Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri[J]. Bioengineered, 2012, 3(4): 209-217. DOI:10.4161/bioe.21049 |
| [69] |
GUO TT, XIN YP, ZHANG Y, GU XY, KONG J. A rapid and versatile tool for genomic engineering in Lactococcus lactis[J]. Microbial Cell Factories, 2019, 18(1): 22. DOI:10.1186/s12934-019-1075-3 |
| [70] |
ZHAO NN, LI L, LUO GJ, XIE S, LIN Y, HAN SY, HUANG YY, ZHENG SP. Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum[J]. Journal of Industrial Microbiology & Biotechnology, 2020, 47(8): 599-608. |
| [71] |
BUNNY K, LIU J, ROTH J. Phenotypes of lexA mutations in Salmonella enterica: evidence for a lethal lexA null phenotype due to the Fels-2 prophage[J]. Journal of Bacteriology, 2002, 184(22): 6235-6249. DOI:10.1128/JB.184.22.6235-6249.2002 |
| [72] |
FU J, BIAN XY, HU S, WANG HL, HUANG F, SEIBERT PM, PLAZA A, XIA LQ, MÜLLER R, STEWART AF, ZHANG YM. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting[J]. Nature Biotechnology, 2012, 30(5): 440-446. DOI:10.1038/nbt.2183 |
| [73] |
XIN YP, GUO TT, MU YL, KONG J. Coupling the recombineering to Cre-lox system enables simplified large-scale genome deletion in Lactobacillus casei[J]. Microbial Cell Factories, 2018, 17(1): 21. DOI:10.1186/s12934-018-0872-4 |
| [74] |
ZHOU D. Development of genome editing method and modification of N-acetylglucosamine metabolic pathway in Lactobacillus plantarum[D]. Jinan: Master's Thesis of Shandong University, 2019 (in Chinese). 周丁. 植物乳杆菌基因组编辑方法开发及N-乙酰葡萄糖胺的代谢途径改造[D]. 济南: 山东大学硕士学位论文, 2019. |
| [75] |
CONG L, RAN FA, COX D, LIN SL, BARRETTO R, HABIB N, HSU PD, WU XB, JIANG WY, MARRAFFINI LA, ZHANG F. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121): 819-823. DOI:10.1126/science.1231143 |
| [76] |
PYNE ME, MOO-YOUNG M, CHUNG DA, CHOU CP. Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified chromosomal gene replacement in Escherichia coli[J]. Applied and Environmental Microbiology, 2015, 81(15): 5103-5114. DOI:10.1128/AEM.01248-15 |
| [77] |
LI YJ, YAN FQ, WU HY, LI GL, HAN YK, MA Q, FAN XG, ZHANG CL, XU QY, XIE XX, CHEN N. Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli[J]. Journal of Industrial Microbiology & Biotechnology, 2019, 46(1): 81-90. |
| [78] |
LIU DY, HUANG C, GUO JX, ZHANG PJ, CHEN T, WANG ZW, ZHAO XM. Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for Bacillus subtilis[J]. Biotechnology for Biofuels, 2019, 12: 197. DOI:10.1186/s13068-019-1537-1 |
| [79] |
CHO JS, CHOI KR, PRABOWO CPS, SHIN JH, YANG D, JANG J, LEE SY. CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum[J]. Metabolic Engineering, 2017, 42: 157-167. DOI:10.1016/j.ymben.2017.06.010 |
| [80] |
LIU J, WANG Y, LU YJ, ZHENG P, SUN JB, MA YH. Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum[J]. Microbial Cell Factories, 2017, 16(1): 205. DOI:10.1186/s12934-017-0815-5 |
| [81] |
STOVICEK V, BORODINA I, FORSTER J. CRISPR-Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains[J]. Metabolic Engineering Communications, 2015, 2: 13-22. DOI:10.1016/j.meteno.2015.03.001 |
| [82] |
WIJSMAN M, ŚWIAT MA, MARQUES WL, HETTINGA JK, van den BROEK M, de la TORRE CORTÉS P, MANS R, PRONK JT, DARAN JM, DARAN-LAPUJADE P. A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains[J]. FEMS Yeast Research, 2019, 19(1): foy107. |
| [83] |
HORVATH P, BARRANGOU R. CRISPR/Cas, the immune system of bacteria and archaea[J]. Science, 2010, 327(5962): 167-170. DOI:10.1126/science.1179555 |
| [84] |
JINEK M, CHYLINSKI K, FONFARA I, HAUER M, DOUDNA JA, CHARPENTIER E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096): 816-821. DOI:10.1126/science.1225829 |
| [85] |
JIANG WY, BIKARD D, COX D, ZHANG F, MARRAFFINI LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nature Biotechnology, 2013, 31(3): 233-239. DOI:10.1038/nbt.2508 |
| [86] |
KARVELIS T, GASIUNAS G, MIKSYS A, BARRANGOU R, HORVATH P, SIKSNYS V. crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus[J]. RNA Biology, 2013, 10(5): 841-851. DOI:10.4161/rna.24203 |
| [87] |
SANDER JD, JOUNG JK. CRISPR-Cas systems for editing, regulating and targeting genomes[J]. Nature Biotechnology, 2014, 32(4): 347-355. DOI:10.1038/nbt.2842 |
| [88] |
LIU J, WANG Y, ZHENG P, SUN JB. CRISPR/Cas9- mediated ssDNA recombineering in Corynebacterium glutamicum[J]. Bio-protocol, 2018, 8(19): e3038. |
| [89] |
van DER ELS S, JAMES JK, KLEEREBEZEM M, BRON PA. Versatile Cas9-driven subpopulation selection toolbox for Lactococcus lactis[J]. Applied and Environmental Microbiology, 2018, 84(8): e02752-e02717. |
| [90] |
ATHUKORALAGE JS, McMAHON SA, ZHANG CY, GRÜSCHOW S, GRAHAM S, KRUPOVIC M, WHITAKER RJ, GLOSTER TM, WHITE MF. An anti-CRISPR viral ring nuclease subverts type Ⅲ CRISPR immunity[J]. Nature, 2020, 577(7791): 572-575. DOI:10.1038/s41586-019-1909-5 |
| [91] |
JIANG Y, CHEN B, DUAN CL, SUN BB, YANG JJ, YANG S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system[J]. Applied and Environmental Microbiology, 2015, 81(7): 2506-2514. DOI:10.1128/AEM.04023-14 |
| [92] |
WANG B, HU QT, ZHANG Y, SHI RL, CHAI X, LIU Z, SHANG XL, ZHANG Y, WEN TY. A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum[J]. Microbial Cell Factories, 2018, 17(1): 1-16. DOI:10.1186/s12934-017-0850-2 |
| [93] |
QI LS, LARSON MH, GILBERT LA, DOUDNA JA, WEISSMAN JS, ARKIN AP, LIM WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression[J]. Cell, 2013, 152(5): 1173-1183. DOI:10.1016/j.cell.2013.02.022 |
| [94] |
CLARKE G, CRYAN JF, DINAN TG, QUIGLEY EM. Review article: probiotics for the treatment of irritable bowel syndrome-focus on lactic acid bacteria[J]. Alimentary Pharmacology & Therapeutics, 2012, 35(4): 403-413. |
| [95] |
LIU JM, CHAN SHJ, CHEN J, SOLEM C, JENSEN PR. Systems biology-a guide for understanding and developing improved strains of lactic acid bacteria[J]. Frontiers in Microbiology, 2019, 10: 876. DOI:10.3389/fmicb.2019.00876 |
| [96] |
BRON PA, KLEEREBEZEM M. Engineering lactic acid bacteria for increased industrial functionality[J]. Bioengineered Bugs, 2011, 2(2): 80-87. DOI:10.4161/bbug.2.2.13910 |
| [97] |
HOU ZG, ZHANG Y, PROPSON NE, HOWDEN SE, CHU LF, SONTHEIMER EJ, THOMSON JA. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(39): 15644-15649. |
| [98] |
HIRANO H, GOOTENBERG JS, HORII T, ABUDAYYEH OO, KIMURA M, HSU PD, NAKANE T, ISHITANI R, HATADA I, ZHANG F, NISHIMASU H, NUREKI O. Structure and engineering of Francisella novicida Cas9[J]. Cell, 2016, 164(5): 950-961. DOI:10.1016/j.cell.2016.01.039 |
| [99] |
ZETSCHE B, GOOTENBERG JS, ABUDAYYEH OO, SLAYMAKER IM, MAKAROVA KS, ESSLETZBICHLER P, VOLZ SE, JOUNG J, van der OOST J, REGEV A, KOONIN EV, ZHANG F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 2015, 163(3): 759-771. DOI:10.1016/j.cell.2015.09.038 |
| [100] |
JIANG Y, QIAN FH, YANG JJ, LIU YM, DONG F, XU CM, SUN BB, CHEN B, XU XS, LI Y, WANG RX, YANG S. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum[J]. Nature Communications, 2017, 8: 15179. DOI:10.1038/ncomms15179 |
| [101] |
ZHANG XY, GU SJ, ZHENG XY, PENG SQ, LI YR, LIN Y, LIANG SL. A novel and efficient genome editing tool assisted by CRISPR-Cas12a/Cpf1 for Pichia pastoris[J]. ACS Synthetic Biology, 2021, 10(11): 2927-2937. DOI:10.1021/acssynbio.1c00172 |
| [102] |
FU L, XIE CY, JIN ZH, TU ZZ, HAN L, JIN ML, XIANG YZ, ZHANG AD. The prokaryotic argonaute proteins enhance homology sequence-directed recombination in bacteria[J]. Nucleic Acids Research, 2019, 47(7): 3568-3579. DOI:10.1093/nar/gkz040 |
| [103] |
LEE KZ, MECHIKOFF MA, KIKLA A, LIU A, PANDOLFI P, FITZGERALD K, GIMBLE FS, SOLOMON KV. NgAgo possesses guided DNA nicking activity[J]. Nucleic Acids Research, 2021, 49(17): 9926-9937. DOI:10.1093/nar/gkab757 |
| [104] |
HUANG S, WANG KH, MAYO SL. Guide-directed DNA cleavage by a prokaryotic Argonaute protein induces chromosome recombination in Escherichia coli[J]. bioRxiv, 2022. DOI:10.1101/2021.11.17.469050 |
| [105] |
ROBERTS A, BARRANGOU R. Applications of CRISPR-Cas systems in lactic acid bacteria[J]. FEMS Microbiology Reviews, 2020, 44(5): 523-537. DOI:10.1093/femsre/fuaa016 |
| [106] |
LIU L, YANG DL, ZHANG ZY, LIU T, HU GQ, HE MX, ZHAO SM, PENG N. High-efficiency genome editing based on endogenous CRISPR-cas system enhances cell growth and lactic acid production in Pediococcus acidilactici[J]. Applied and Environmental Microbiology, 2021, 87(20): e0094821. DOI:10.1128/AEM.00948-21 |