Plasmid-Based Gene Expression Systems for Lactic Acid Bacteria: A Review

 

 

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1. Introduction

Lactic acid bacteria represent a family of non-pathogenic, non-sporulating, microaerophilic, Gram-positive bacteria that produce lactic acid as the major end resultant during carbohydrate fermentation and have been employed in food supplements, medicines, and cosmetics for ages to promote modern human civilization [1,2].

 

non-pathogenic 비병원성

sporulating 포자형성

microaerophilic 미호기성

resultant 그 결과로 생긴

 fermentation 발효

 

LAB are generally recognized as safe (GRAS) and due to this status, these food-grade Gram-positive bacteria have been used as starter cultures for food fermentation and as cell factories for the production of various macromolecules, enzymes, and relevant metabolites in the food, pharmaceutical, and dietary supplement industries [3–7].

 

These microbes not only support the fermentation process but also improve the organoleptic and rheological properties of food products [8]. Furthermore, due to their ability to produce various bacteriocins, they also help to extend the shelf life of various foods [4,9–12].

 

LAB are used as probiotics to promote human health as they maintain and modulate the intestinal flora; prevent hypersensitivity reactions and stimulate the immune response; and help protect against pathogens by producing antibacterial peptides, such as bacteriocin, in the gut [13].

 

hypersensitivity reactions 과민반응

 

Probiotic lactic acid bacteria are also used to treat several diseases such as diarrhea, inflammatory bowel diseases, and autoimmune diseases [14–17]. Because of their ability to efficiently produce recombinant proteins, LAB are considered as emerging candidates for the expression of proteins of medical, industrial, and biotechnological relevance [18–20].

 

diarrhea 설사

 inflammatory bowel diseases(IBD) 염증성 장질환

autoimmune diseases 자가면혁질환

 

Recently, Pediococcus sp. has progressively become an attractive and promising host for the development of next-generation probiotics and microbial cell factory for potential applications as live delivery vectors for use as therapeutics [13].

 

progressively 계속해서

 

In the last decade, scientific interest has focused on the application of this group of food-grade bacteria as effective vehicles for mucosal delivery and for the delivery of recombinant prophylactic and therapeutic proteins, neutralizing antibodies, and the variable domain of heavy-chain-only antibodies from camelids—known as VHH antibodies or nanobodies—into the human gut, to prevent and treat inflammatory bowel diseases (IBDs), autoimmune disorders, infectious and non-infectious gastrointestinal diseases, and infections by pathogenic microorganisms from mucosal surfaces [18,21–26].

 

 

As lactic acid bacteria (LAB) is a cell factory and delivery vehicle for the delivery of therapeutic proteins, an LAB strain (Lactococcus lactis) was used as a vector to deliver HuNoV (human noroviruses) antigen and it demonstrated that this LAB-based HuNoV vaccine induced protective immunity in gnotobiotic piglets [27].

 

In modern times, advances in genetic engineering and the development of several sophisticated genome engineering tools, such as CRISPR/Cas, RecT-mediated recombineering, etc., have created opportunities to further expand the biotechnological and industrial application of LAB. For the expression and delivery of recombinant proteins and RNA for successful genome engineering using these tools, plasmid-based vectors are essential [28].

 

Plasmids are extrachromosomal, covalently closed, circular, and self-replicating DNA molecules that are important for the rapid adaptation of bacterial populations to different environmental conditions, protection from phages, niche development, and community structure shifts [11]. Because many LABs have played critical roles in human well-being, from food production to drug delivery, plasmids have been extensively studied to expand the uses of LAB through genetic modification, and hence research continues to develop gene expression systems [20]. This review focuses on the plasmid-based vectors developed for LAB to date, and their functionality, efficacy, and applications. The need for a new, stable, and effective gene expression platform for LAB has also been outlined.

 

 

2. Components of Plasmid-Based Vectors

Discovered in the early 1950s, plasmids were first conceived as vectors for gene cloning in 1972 when a small group of scientists consisting of Stanley Cohen, Herbert Boyer, and Charles Brinton used the enzyme EcoRI to linearize the tetracycline-resistant plasmid pSC101 and cloned a kanamycin-resistant gene to develop double-resistant pSC102. The success of this study not only established pSC101 as the first plasmid-based cloning vector but also revolutionized the concept and development of molecular biology [29,30].

 

A fundamental precondition for efficient genetic manipulation of organisms is the availability of appropriate vectors that guarantee duplication and maintenance of both the vector DNA and the implanted foreign DNA. For a plasmid to be an efficient expression system, it should have several distinct features: a functional and stable replicon to maintain the recombinant plasmid in the host system; a selectable marker, such as an antibiotic resistance gene or a bacteriocin immunity gene, etc., for the selection of positive recombinants; a strong promoter with an associated ribosome-binding site (RBS) along with other regulatory sequences for an effective inducible or constitutive production of the protein of interest; and a multiple cloning site adjacent to the promoter region for successful in-frame cloning of the desired gene to achieve expression. However, if the vector is constructed for food-grade application, all DNA components must be acquired from food-grade organisms with a GRAS and/or Qualified Safe Acceptance Status (QPS) and no antimicrobial resistance will be entertained as mandated by the US Food and Drug Administration and by the European Food Safety Authority [20,31].

 

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2.1. Replicon and Mode of Replication

A replicon, the most important region in the plasmid-based expression system, consists of the origin of replication (ori) and all its control elements. The replicon is the site of DNA replication initiation that allows a plasmid to reproduce itself and survive within the host cell.

 

reproduce itself 자가복제

 

It is generally an A-T-rich sequence where the DNA melts, allowing the replication machinery to step in and actively participate in the making of copies.

 

The host range of a vector depends mostly on the plasmid’s replicon. Not only the host range but also the segregation stability, i.e., the ability of the vector to segregate into daughter cells during cell division, is replicon dependent [32,33]. There are two types of plasmid replication mechanisms, namely the rolling circle or sigma mode of replication (RCR) and theta mode of replication [34,35].

 

segregation 분리

 

2.1.1. Rolling Circle Replicating Plasmids

Rolling circle replicating (RCR) plasmids [34,36] are omnipresent in both Grampositive and Gram-negative bacteria and even in archaea [37]. The usual elements of RCR plasmids are the replication initiator (Rep) protein, the double-stranded origin (dso), and the single-stranded origin (sso).

 

 omnipresent 어디에나 있는

 

 

The unidirectional and disengaged synthesis of the primary strand and the lagging strand are the two key phases of the sigma replication mode [34,38–40].

 

 

Plasmid-encoded Rep protein binds to the cognate dso, which consist of either an inverted repeat or a set of two to three direct repeats either adjacent to the nick site or separated by up to 100 bp [34,38,40], and initiates plasmid replication with the introduction of a single-strand nick at the specific nick site. This consists of an inverted repeat, on the parent plus strand, leaving a free 30 -OH, which in turn serves as a primer for leading strand synthesis. Leading strand synthesis generates a single-stranded DNA (ssDNA) replication intermediate [37,40].

 

 

Conversion of the displaced ssDNA intermediate to double-stranded DNA (dsDNA) occurs through lagging strand synthesis beginning at the sso [34,38–40]. There are several families of these plasmids which can be classified as the pT181, pE194/pMV158, pC194, and pSN2 family. In general, RCR-type plasmids from Gram-positive bacteria exhibit a broad host range and high copy number as host dependency is non-existent; hence, they are ideal for vector construction. However, having an ssDNA replication intermediate, they exhibit low structurally and segregationally stability, making them unsuitable for the construction of expression systems for starter cultures and drug delivery systems [32,34,37,40]. RCR plasmids are highly disseminated, so they possess the threat of spreading the desired trait or resistance to the undesired organisms, which could affect food production or vaccine delivery.

 

2.1.2. Theta-Type-Replicating Plasmids

The marginal replicon of a theta-replicating plasmid consists of the ori region and a gene encoding a replication initiator protein. The ori region is an AT-rich region usually followed by iterons (three and a half 22 bp direct repeats) and two tiny, inverted repeats that overlap the -35 element of the rep promoter and the RBS of the rep gene. Theta plasmid replication is initiated with the softening of the parental strands, followed by the continuous and discontinuous production of the leading and lagging strands, respectively [34].

The iterons of the ori site are not only crucial for the initiation of replication [34,41] but also important for the control of plasmid replication [34,41–43]. To date, six classes of theta replicons [41,44] have been identified, class A-F: Class A includes plasmids that have a Rep protein with a related origin of replication and replicate independently of DNA Pol I. Most LAB theta-type-replicating plasmids belong to class A. Theta replicons of plasmids, such as pCI305 [45], pUCL22 [46], pWVO2 [47], pW563 [48], pCD4 [44], pSM409 [49], etc., from lactic acid bacteria are representatives of class A theta replicons. Class B replicons lack oriA and do not encode a Rep protein. Their replication is initiated by processing a transcript synthesized by the host RNA polymerase. Interestingly, class C replicons do not have an oriA-like structure but encode a replication protein and require DNA Pol I for replication. pColE2 and pColE3 are the most known class C replicons. Class D replicons, although dependent on the plasmid-encoded replication protein (Rep), is independent of the DNA structure typical of the origin of replication of most Rep-dependent plasmids, and is initiated by DNA polymerase I. Some LAB theta-type-replicating plasmids (pAMβl, pEF1, pIP501, and pSM19035) belongs to this class. Class E replicons are yet to be researched and characterized and are represented by plasmid pLS20 from Bacillus subtilis. As observed, the structural organization of the minimal replicon of pLS20 differs from the typical plasmids of Gram-positive bacteria. Class F replicons possess a replication initiation protein (RepN) but lack the characteristic AT-rich region at the replication initiation site. The origin of replication differs structurally from those of class A and is identified by the presence of multiple iterons on the coding sequence of RepN, but replication is independent of DNA polymerase I. Plasmids of diverse origins, such as pLJ1 of Lactobacillus sp. [50], pAD1 [51], pCF10 [52], and pPD1 [53] from Enterococcus sp., and the plasmids pNP40 [54] and pLS32 from B. natto and pCI2000 [55] from Lactococcus sp. belong to Class F. The creation of theta plasmids can be uni- or bi-directional and can initiate from multiple origins, and produces double-stranded DNA replication intermediates compared to the RCR plasmid; therefore, theta-replicating plasmids show structural and segregational stability compared to RCR plasmids and can incorporate and maintain large DNA fragments, making them useful as cloning and expression vectors [32,56].

2.2. Selectable Marker

The selectable marker aids in the identification and elimination of non-transformants and selectively allows transformants to grow in the presence of appropriate selective pressure. A selectable marker for a vector is, therefore, mandatory for efficient screening and easy handling [20,33,56]. In general, antibiotic resistance genes are considered to be the most suitable marker for the vector, since they can specifically eliminate the untransformed cells and the transformants can only survive in the presence of suitable antibiotics. Apart from antibiotics, bacteriocin production and immunity, phage resistance, or heavy metal resistance have been identified as efficient and straightforward mechanisms to separate the transformants from the non-transformed ones and are, therefore, also used by researchers as markers for vector construction. Since the use of antibiotic resistance genes as selection markers for food-grade vectors has been restricted by regulatory bodies, the sugar utilization gene, bacteriophage resistance, stress tolerance, bacteriocin resistance, and immunity, etc. have been used as selection markers for the development of food gene expression systems for LAB [57].

 

2.3. Promoter

The efficiency of an expression system is often determined by the strength of its promoter and the way it works since the expression of the heterologous proteins is controlled solely by the promoter system [20,56,58,59]. Although several gene expression systems have been developed in LAB, the choice of an appropriate promoter still remains a very important consideration when constructing expression cassettes. For laboratory protein production, the type of promoter (inducible or constitutive) is not a major concern, but when expression for therapeutic applications is desired, several factors come into play [59]. The strong inducible promoters are used to overproduce recombinant proteins of medical and industrial relevance in LAB, whereas the constitutive promoter is preferred for in vivo delivery and steady-state production of therapeutic proteins in the human gut since constitutive promoters do not require external induction, unlike the inducible promoter.