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

3. Challenges in Vector Engineering

When designing a vector, researchers must address several bottlenecks that determine the efficiency of a gene expression system, as follows:

 

3.1. Copy Number

Plasmid-based vectors are self-replicating DNA molecules and maintain a specific number of copies in the host cell [43]. It is believed that a high copy number of a vector supports plasmid segregation into daughter cells during cell division and that an additional burden on the partition system is not required to increase the stability of plasmid segregation; however, it places a metabolic burden on the host cell, which in turn affects the cell’s metabolism [33]. On the contrary, low-copy-number plasmid vectors, while not subjected to host cell metabolic stress, have a high risk of segregationally being unstable [60]. In order to facilitate the process of plasmid segregation, partition systems are often present in low-copy plasmids [53]; however, the presence of partition systems in the vectors is not recommended as it increases the size of the vector, which in turn decreases the vector’s carrying capacity. In addition, the replicon type also plays a crucial role in the copy number and segregational stability, since theta-replicating plasmids mostly have a low to medium copy number and are segregationally stable while rolling circle plasmids have a high copy number and are unstable due to their single-strand intermediate [56]. In fact, gene expression in low-copy vectors has been found to have improved functionality and stability compared to multi-copy plasmids [61]. Therefore, special care should be taken in selecting appropriate replicons for gene expression system design.

 

3.2. Plasmid Incompatibility

In recent years, the use of multiple gene expression systems to express multiple genes simultaneously in a single host in a modular manner has attracted much interest from researchers. However, bacterial plasmids exhibit an idiosyncratic property of incompatibility, meaning that plasmids with similar replication machinery cannot be stably maintained simultaneously in the same host [44,62]. Therefore, it is important to consider plasmid incompatibility while designing new genetic tools for heterologous protein production.

 

3.3. Plasmid Structural and Segregation Stability

Plasmid instability is one of the main problems in research; it also affects pilot-scale fermentation in industry. Researchers have repeatedly reported that plasmids are simultaneously lost during cell division (segregational instability), or DNA segments are rearranged and lost from the plasmid backbone (structural stability) [32,60,63,64]. Segregational instability occurs mainly due to defects in the partition machinery and depends mainly on the plasmid replicon. Therefore, proper selection of the replicon is critical for vector construction. Whereas, when excess sequences homologous to the genome are present in the plasmid backbone, recombination and structural instability of the vector can result. When designing new gene expression constructs, attention must, therefore, be paid to the stability of the vector.

 

3.4. Vector Size and Carrying Capacity

The carrying capacity of a vector is defined as the size of an insert that can be cloned into a gene expression system without affecting the stability and functionality of the plasmid. The vector size plays a crucial role in determining the carrying capacity of the vector since a replicon of a plasmid possesses the ability to carry out the replication of a certain length of DNA; if this limit is exceeded, severe instability is observed as a result of a failure to reproduce. Therefore, before constructing a plasmid-based vector, it is important to characterize the components well (as indicated above) and minimize the junk DNA from the vector backbone to limit the vector size so that a large amount of genetic load can be stably inserted.

 

3.5. Host Range

The host range is one of the most important determinants of vector fitness. A vector’s ability to be stable across a broad host range increases its applicability in modern research, as narrow-host-range platforms are highly specific, only addressing the specific requirements of a particular host, and thereby limiting their use to heterologous hosts [65]. While broad host systems allow for the cloning and expression of genes in a variety of heterologous hosts, researchers can switch to other models without changing the circuitry. The vector replicon primarily determines the host range of the system; therefore, it is important to select a replicon with a broad host range for the development of the gene expression system. Considering the need for an efficient gene expression system for LAB to widen the spectrum of applications of LAB from the laboratory to the land, the well-characterized components, namely the origin of replication, from food-grade LAB plasmids should be the first choice to construct a vector. Since the origin of replication plays a crucial role in controlling the copy number, stability, host range, incompatibility, and carrying capacity, special care should be taken in the appropriate selection of ori for the construction of a stable gene expression system for LAB. Furthermore, as previously mentioned, constitutive expression in LAB is preferred for steady-state overproduction of recombinant proteins of medical and industrial relevance; however, constitutive expression often results in a metabolic burden on the host, which in turn leads to a plasmid instability, followed by plasmid loss. Therefore, a stable theta-type replicon is preferred over a rolling circle replicon for LAB (vide supra).

 

4. Vectors for Lactic Acid Bacteria

There are mainly two types of plasmid vectors: cloning and expression vectors. Cloning vectors are generally used to construct a library to obtain specific DNA fragments of interest for various purposes in molecular biology, such as probe generation, restriction mapping, sequencing, etc., while the expression vectors with their strong transcriptional and translational signal sequences are used to produce various homologous and heterologous proteins in large quantities. Over the past two decades, researchers have been involved in the growth of an array of cloning and expression vectors for the engineering of various LAB species [66]. The vectors developed for LAB or other Gram-positive bacteria that are currently in use are constructed largely based on the previously characterized plasmids of LAB (Table 1) and can be divided into two major classes based on the replicon used, which are as follows:

 

 

Class 1 vectors: These vectors are represented by the large theta-replicating, segregational and structurally stable conjugative plasmids pIP501 and pAMβ1 and their derivatives, which are resistant to macrolides, lincosamides and spectogramin B (MLS resistance), and other antibiotics [53,67–69]. These plasmids have a broad host range and can replicate in various LAB such as Lactococcus spp., Lactobacillus spp., and Pediococcus spp. [70–72]. However, pIP501 and pAMβ1 derived from streptococci and enterococci cannot be considered food-grade since these organisms do not have GRAS status, and are, therefore, not suitable for food and pharmaceutical applications. Class 2 vectors: These vectors are derived from small cryptic plasmids (pSH71, pD125, pCL2.1, pWC1, pBM02, and pWV01) of several lactococcal species. Being rolling circle plasmids, these vectors are generally segregationally and structurally unstable and have a very low carrying capacity; they are only useful for cloning smaller genes. At the same time, however, pSH71 and pWV01 replicons and their derivatives have a broad host range [73,74].

In addition to these classes of vectors, research is driven towards the development of stable gene expression systems derived from plasmids of food-grade LAB [66]. Expression systems for LAB can be broadly classified into two major types:

 

4.1. Constitutive Gene Expression Systems

As mentioned above, constitutive gene expression systems for LAB have a wide range of biotechnological applications in the development of next-generation health-promoting bio-functional foods and LAB-based therapeutics. To date, a variety of constitutive gene expression systems for LAB have been developed, particularly for Lactococcus sp. and Lactobacillus sp., using the promoters from housekeeping genes [31,75]. A constitutive gene expression system pUBU was engineered using the theta-type replicon of pUCL287 from Tetragenococcus halophilus, the promoter of the L-lactate dehydrogenase (ldhL) gene from Lactobacillus plantarum, and the cadmium resistance (Cdr ) gene from pND918. The host range of this vector protracted up to Lactobacillus, Lactococcus, Pediococcus, Enterococcus, Leuconostoc, and Tetragenococcus and was able to remain stable for at least 100 generations under non-selective pressure [76]. A constitutive expression system comprising a constitutive L-lactate dehydrogenase promoter (PldhL) from L. sakei and a gene downstream of the promoter encoding green fluorescent protein (GFP) has been reported [77]. A constitutive gene expression cassette, pTRK892, was recently developed for Lactobacillus sp. The vector was generated using the Lactobacillus acidophilus phosphoglycerate mutase promoter (Ppgm) and the pWV01 replicon and the gene cloned into this vector was found to be constitutively expressed at a level that was more than 10-fold higher than that observed in inducible vectors [78]. Recently, pPBT-GFP, a constitutive gene expression system based on the erythromycin (ery) gene promoter, was developed. The low copy number shuttle vector pPBT-GFP consists of pCP289 and pLES003 replicons and is capable of stably replicating in host cells in multiple hosts and can be used as an ideal tool for improving LAB strains of commercial value using genetic engineering [79]. Despite the immense importance of constitutive gene expression systems for lactic acid bacteria in industrial settings, their development has been highly retarded due to the scarcity of constitutive promoters characterized in LAB [59,80]. To date, a handful of constitutive promoters of LAB have been characterized, including P8: Phosphopentomutase promoter; P5: Hypothetical protein L1010 promoter; P6: Hypothetical protein L141485 promoter; etc. [59]. However, these were mostly conditionally constitutive, i.e., they are constitutively active in the presence of certain sugars, and the expression of these promoters was generally weak. The commercially available constitutive vectors pNZ2103 and pNZ2122 from Mobitec are constructed using the lacA promoter from Lactococcus lactis [81]. The lacA promoter of L. lactis is believed to be regulated by lacR (repressor) and in the presence of glucose, the promoter is inactivated. The expression of the lacA promoter was reported to be constitutive in particular hosts. In L. lactis MG1363, the activity of the promoter was quite high in the presence of glucose. Whereas, in another host L. Lactis MG5267 (lacR+ ), the expression of the lacA promoter was greatly reduced in glucose and the promoter was induced in the presence of lactose [81]. Therefore, the L. lactis lacA promoter is not a constitutive promoter in the strict sense, and thus, the constitutive expression of this vector is host specific.

 

4.2. Inducible Expression Systems

Inducible systems for LAB have received considerable attention worldwide for the overproduction of various proteins in lactic acid bacteria. To date, many inducible gene expression systems exist and are employed extensively in laboratory and industrial settings. Recently, a series of sugar-inducible expression systems pTRK888 (Pfos), pTRK889 (Plac), and pTRK890 (Ptre) were constructed for use in lactobacilli, based on the broad host range replicon of pWV01 and the use of promoters from FOS (Pfos), lac (Plac), and tre (Ptre) operons [78]. In addition, thermo-inducible expression systems and stress-inducible expression systems have also been developed, which Landette discussed extensively in 2017 [20]. Recently, pMY01 was developed using the PsrfA promoter to fine-tune gene expression in a broad range of LAB hosts, namely L. casei 5257, L. plantarum 97, L. fermentum 087, and Weissella confusa 10, yielding the recombinant strain L. casei 5257-01, L. plantarum 97-01, L. fermentum 087-01, and Weissella confusa 10-0. In addition, the promoter PsrfA was used to construct an autoinducible expression system in B. subtilis and E. coli [82]. Among the inducible systems, the two-component induction system of the nisin operon has gained relatively more attention due to its stringency of induction and expression and thus a myriads of gene expression systems, the nisin-controlled expression system (NICE), have been developed based on the nisin promoter (PnisA) from Lactococcus lactis. Nisin is a bacteriocin generated by certain strains of Lactococcus lactis. It is known that a cluster of eleven genes transcriptionally organized into four operons is involved in the biosynthesis of mature nisin following post-translational modifications. Molecular characterization of the nisA promoter revealed that the promoter is self-managed by a two-component regulatory system, comprising the sensor kinase NisK and the response regulator NisR, that responds to extracellular nisin [83–85]. Since the nisin-mediated autoinduction of PnisA occurs only in the nisin-producing L. lactis via NisR and NisK, a host-vector system was developed by exploiting the auto-induction mechanism of the nisin operon through the chromosomal insertion of the genes involved in signal transduction, nisK and nisR into L. lactis subsp. cremoris MG1363 (nisin-negative), creating the strain NZ9000 [84,85]. Likewise, several genetically engineered hosts have been developed to utilize this auto-induction mechanism to produce heterologous proteins in LAB (Table 2). In addition, plasmids have been constructed to facilitate translational and transcriptional fusions and intracellular fabrication or secretion of the gene product.

The vector pNZ8048 is mostly employed for translational fusions, where a gene of interest can be inserted at the canonical NcoI site around the ATG start codon for expression. At the same time, two further variants of this plasmid were constructed: pNZ8148 and pNZ8150, wherein pNZ8148, a 60-bp remnant DNA-fragment of Bacillus subtilis, was deleted, and in plasmid pNZ8150, the NcoI site was swapped for a ScaI site that is present just upstream of the ATG start codon. The upgraded pNZ8148 eschews the drawback created by the mandatory usage of the NcoI site, which sometimes mandates changing the first base of the second codon in the gene being pursued. In pNZ8150, the pursued gene may be amplified, beginning at the ATG start codon and straightforwardly fused to the vector, resulting in an accurate nisA translational fusion [85]. Although several nisininducible systems have been developed for efficient gene expression in the engineered host, this host specificity has limited the applicability of these vectors for the genetic engineering of industrially important LAB. Therefore, other strategies were used for a wide range of applications, and a vector pMSP3535 was developed that carries both the nisRK genes and the nisA promoter on one plasmid, facilitating the induction of gene expression by exogenous addition of nisin [86]. A food-grade-inducible gene expression system has also been created for the expression of heterologous proteins in L. lactis [87]. This system consists of a food-grade cytoplasmic-inducible expression vector pRNA48 containing the α-galactosidase gene, theta replicon from pRAF800, and PnisA-MCS-TpepN from pNZ8048; and a cell-wallanchored expression vector pRNV48 containing α-aga, theta replicon, and PnisA-SPUsp45- nucA-CWAM6-t1t2 from pRNA48 and pVE5524 have been described. The OprF/H fusion derived from Pseudomonas aeruginosa was cloned into plasmids pRNA48 and pRNV48 to construct the pRNA48-OprF/H and pRNV48-OprF/H for the expression of OprF/H using a nisin-inducible system, resulting in 9.6% intracellular soluble protein and 9.8% cell-wallanchored protein in L. lactis NZ9000, respectively [87]. Along with nisin-inducible systems, a series of vectors, the pSIP series, was developed using a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P, and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The pSIP series of vectors has been used for tightly controlled and efficient expression of β-glucuronidase in both L. sakei and L. plantarum [88]. A Lactobacillus/Escherichia coli shuttle vector, pKRV3, was constructed, including the sakacin signal transducing system. The gusA gene fused to the PsapA promoter cloned into this vector allowed for inducible beta-glucuronidase expression in L. sakei and L. plantarum after induction with sakacin A [89]. A bile-responsive E. coli-Lactobacillus shuttle expression vector, pULP3-PLDH, for Lactobacillus spp. was generated by fusing pUC19 with the L. plantarum plasmid pLP27, which has been found to persist in L. plantarum for up to 80 generations without selection pressure [90].