- Open Access
Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis
© The Author(s) 2017
- Received: 19 June 2017
- Accepted: 9 September 2017
- Published: 18 September 2017
Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000.
Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB.
By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.
- Lactococcus lactis
- Knocked-in heterologous gene
- Knock-in reporter system
Lactococcus lactis, a food-grade Gram-positive lactic acid bacterium, is commonly used to manufacture fermented dairy products, such as (soft) cheese, buttermilk, and sour cream [1, 2]. Since 1980s, extensive research on L. lactis has revealed considerable information on the biological, genetic, and immunological characteristics of this species [3, 4]. L. lactis has been broadly used as an “efficient cell factory” for recombinant protein production  because of the following properties: (i) As a generally regarded as safe (GRAS) microorganism , L. lactis elicits weak immune responses against itself and does not colonize the gut of humans and animals . Thus, L. lactis can be directly used in the digestive tract [7, 8]. (ii) L. lactis is genetically easy to manipulate, because of its completely sequenced genome [9–11] and many available genetic tools [3, 5, 12]. (iii) The downstream purification processes of secreted recombinant proteins are simple because L. lactis secretes only one major protein, namely, Usp45 . Several kinds of heterologous proteins, such as enzymes [13–15], therapeutic proteins [16–18], growth factors [19–21], and antigens [3, 6, 12, 22], have been expressed in L. lactis. Therefore, L. lactis is a suitable host for heterologous gene expression and becomes the focus of food industry, biopharmaceuticals, and vaccine research.
Heterologous proteins can be expressed in L. lactis by encoding their genes harbored in vectors, such as pNZ8148 [3, 23], pMG36e [15, 24], pAMJ399 [19–21], and pLEB590 [25, 26]. However, this approach is limited by several disadvantages. (i) In these vectors, antibiotic-resistant genes, which are banned for use in humans, are commonly employed as selective markers. (ii) Food-grade selective markers, such as nisin resistance gene (nsr), have been applied in L. lactis. However, most of the food-grade selective markers cannot be used in Escherichia coli. Therefore, plasmids containing these food-grade selective markers can only be constructed in L. lactis, but the efficiency of constructing plasmids in L. lactis is much lower than that in E. coli. (iii) Plasmids in L. lactis are unstable in human and animal digestive tracts in the abundance of selective pressure. As an efficient alternative approach, the knock-in of target genes into L. lactis chromosome through double-crossover recombination is performed to stably express heterologous proteins without antibiotic-selective markers.
Temperature-sensitive (Ts) plasmids are usually utilized to integrate heterologous genes into the L. lactis genome. The entire process is accomplished in two steps . First, a Ts plasmid harboring a target heterologous gene is transformed into L. lactis. Single-crossover recombinants are then obtained by culturing the transformants with antibiotics at a nonpermissive temperature. Second, plasmid-integrated clones are grown at a permissive temperature in an antibiotic-free medium. The integrated vector can be excised from the genome at a low frequency through a second recombination and consequently produce wild-type or heterologous gene knocked-in (HGK) strain without antibiotic resistance. To screen non-resistant clones, we individually examine the antibiotic resistance provided by integrated plasmids in colonies. HGK clones are subsequently checked through PCR. However, screening is laborious and time consuming, that is, this process requires several days to weeks. Therefore, a rapid screening method for HGK clones is desirable.
In this study, a heterologous gene knock-in reporter system was established for L. lactis NZ9000 (β-galactosidase negative strain) through visual selection. The proposed system comprised a Ts pJW plasmid and a recombinant L. lactis NZB strain. pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. Afterward, the lacZ (β-galactosidase) gene was knocked-in the chromosome of NZ9000 by pJW. The resulting mutant strain, named NZB, formed blue colonies on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plate. To knock a target gene into L. lactis chromosome through the knock-in reporter system, the heterologous gene was firstly inserted in pJW. Then, the pJW vector harboring the target gene was transformed into NZB. When the heterologous gene was knocked into the NZB chromosome and replaced the lacZ gene by double-crossover recombination, the HGK clones formed white colonies on X-Gal plate and were distinguished from the other blue colonies. The HGK clones were then screened by selecting the white colonies from blue colonies rather than by checking colonies individually. By utilizing our knock-in reporter system, the HGK clones were produced simply and efficiently.
Construction of the Ts plasmid pJW
Construction of NZB strain
We first integrated a lacZ gene with a promoter into the His locus of the chromosome of NZ9000 because β-galactosidase gene is absent in the NZ9000 chromosome. The PZLT fragment containing a nisin promoter P nisZ from L. lactis N8 , lacZ from Lactobacillus acidophilus , and a terminator from the pMG36e plasmid [15, 35] was cloned into pJW to create pJW-PZLT (Additional file 1: Figure S2). P nisZ , which can be induced in the presence of nisin in NZ9000, was artificially synthesized on the basis of the reference sequence .
Evaluating the efficiency of heterologous gene knock-in reporter system in L. lactis
The “efficient heterologous gene knock-in reporter system in L. lactis” is composed of the pJW plasmid and the NZB strain. To integrate a heterologous gene into the NZB chromosome, we used the built-in lacZ gene as a target counter-selection marker. The NZB strain loses its ability to produce β-galactosidase and forms white colonies that are easily distinguished from the blue parental colonies on an X-Gal plate when the built-in lacZ gene is replaced by the gene of interest via double-crossover recombination.
White colony occurrence and accuracy rate in the evaluation of the knock-in reporter system
HDF length (bp)
White colony occurrence rate (‰)
Accuracy rate (%)
In conclusion, our knock-in reporter system can effectively knock-in the HDFs ranging from 1.3 to 14.6 kb into the L. lactis chromosome. Furthermore, the white colonies were HGK colonies, and the accuracy rate was 100%.
In this work, we developed an efficient heterologous gene knock-in reporter system in L. lactis. The knock-in reporter system contains a Ts plasmid pJW and a NZB strain (a derivative of NZ9000). By using the knock-in reporter system, the HDF knocked-in NZB clones can be selected directly by colony phenotype (color) change (blue to white). Our assay largely reduced the required labor and time, and thus improved the efficiency of knock-in assay for L. lactis.
In the original assay, the knocked-in strains were screened by examining the loss of antibiotic resistance, hundreds and thousands of colonies must be examined individually to obtain antibiotic-sensitive clones. Some of these colonies were HDF knocked-in clones, and others were parental clones. In our assay, screening was simplified through visual observation of phenotypic colony changes, and the screening time was reduced from several weeks to several days.
The parental strain used in this study was NZ9000 (L. lactis subsp. cremoris) , a derivative of MG1363, and is the most commonly used host of the NIsin Controlled gene Expression (NICE) system. The NICE system is a highly successful and widely used tool for regulating of gene expression in L. lactis [11, 23, 36, 37]. When a gene of interest is placed at the downstream of the inducible promoter P nisA , P nisF , or P nisZ in NZ9000, industrial-scale gene expression can be induced by adding nisin (a food-grade antimicrobial peptide produced by some strains of L. lactis) to the culture medium [36–38]. Besides nisin inducible promoters, other promoters, like constitutive promoters P 32  and P 45 , and pH inducible promoter P 170 , can also work in NZ9000. Therefore, NZ9000 was chosen to develop the host strain of the knock-in reporter system instead of other L. lactis strains.
Two kinds of plasmids namely, non-replicative and Ts plasmids, are employed in gene knockout and knock-in. Non-replicative plasmids can be maintained by the target host only by specific recombination with the host genome, and the resulted clones are selected using antibiotics. Obtaining plasmid-integrated clones is difficult in certain hosts, such as L. lactis, because of low transformation efficiency. Owing to its large size, Ts plasmids are difficult to use in transforming target hosts. However, this kind of plasmids can be replicated in the target host at a permissive temperature. Transformants are easily achieved, and only one transformant is needed for subsequent operations. Therefore, we chose Ts plasmid as a vehicle of the gene of interest in our knock-in reporter system.
In the present study, the His locus, corresponding to a nonessential gene involved in histidine biosynthesis in L. lactis , was employed as the integration site, which was also described in a previous study by Simões-Barbosa et al. . Their results demonstrated that the disruption of the His locus did not significantly affect growth of the heterologous gene integrated strain , which was confirmed by our results. Besides the His locus, thyA, a thymidylate synthase-encoding gene, was also used as integration site for the construction of delivery system in vivo [17, 41]. Replacement of thyA by heterologous genes creates thyA-deficient strains, which are self-limited and die rapidly in the absence of thymidine or thymine. Thus, the thyA-deficient bacteria can survive in vivo, but cannot accumulate in the environment .
This system can be only applied in β-galactosidase negative and nisRK-positive strains, such as NZ9000, because the nisin inducible promoter P nisZ can only work in nisRK-positive strains. However, we believe that, the strategy can also be applied in other hosts. For β-galactosidase and nisRK genes negative strains, the nisin inducible promoter P nisZ in lacZ expression cassette should be replaced by constitutive promoters or other kind of inducible promoters (depend on host strains) . For β-galactosidase positive strains, the β-galactosidase gene should be used as counter-selective marker.
In conclusion, the “efficient heterologous gene knock-in reporter system in L. lactis” is a convenient and practical tool to knock heterologous genes into L. lactis NZB strain (a derivative of NZ9000) efficiently, and enhances downstream research works.
Bacteria, plasmids, and culture conditions
Bacterial strains and plasmids used in this study
E. coli DH5α
Cloning host for maintaining recombinant plasmids
L. lactis NZ9000
Derivative of MG1363; pepN::nisRK
, Lab collection
L. lactis NZB
Derivative of NZ9000; P nisZ ::lacZ::terminator
Cloning vector; Ap r
Derivative of pUC18; containing His fragment from L. lactis
An improved version of the pCrePA  plasmid containing Ts replicon; Ap r , Em r
Kindly gifted by Stephen H. Leppla
L. lactis integration vector; derivative of pUC-H; containing Ts replicon from pCrePA2; Ap r , Em r
Wide-host-range vector; Em r
, Lab collection
Derivative of the expression vector pQE31; containing lacZ from Lactobacillus acidophilus; Ap r
, Lab collection
derivative of pMG36e; containing P nisZ ::lacZ; template of P nisZ ::lacZ::terminator; Em r
L. lactis integration vector; derivative of pJW; containing P nisZ ::lacZ::terminator; Ap r , Em r
DNA manipulations and sequencing
Primers used in this study
Construction of Ts plasmid pJW
The His fragment (2472 bp), an internal sequence of the histidine operon, was amplified from the genome of NZ9000 by PCR using PrimerStar DNA polymerase (high-fidelity DNA polymerase, Takara, China) with primers HisF and HisR from NZ9000 genome. The His fragment was then digested with EcoRI, and inserted in the SmaI/EcoRI sites of pUC18. The resulting vector was named pUC-H. The “Ts-replicon::Em r ” fragment amplified from pCrePA2 vector with primers TsF and TsR was digested and inserted in the BamHI/PstI sites of pUC-H. The constructed vector was named pJW (Additional file 1: Figure S1).
Construction of NZB strain
The nisin promoter P nisZ  was artificially synthesized as template, and was amplified with the primers PZF and PZR. The lacZ gene (from L. acidophilus) was amplified from pQE31-LacZ  with the primers LF and LR. P nisZ and lacZ gene were combined by overlap PCR. The PCR product was digested by EcoRI/XbaI and cloned into the same sites of pMG36e  to yield the plasmid pMG-PZL. The PZLT fragment (P nisZ ::lacZ::terminator) was then amplified from pMG-PZL with primers PZRec and TerRec and inserted into the AscI site of pJW by seamless cloning (Additional file 1: Figure S2).
The vector pJW-PZLT was introduced into L. lactis NZ9000 by electroporation, and the transformants were selected at 30 °C on M17GS-NX (M17GS agar containing X-Gal and nisin) medium containing erythromycin (Additional file 1: Figure S3A). One blue colony was streaked onto the same medium and incubated at 30 °C. In the next step, a blue colony was inoculated in M17GS broth with erythromycin and incubated at 30 °C for 8 h and then diluted 1000-fold in the same medium and grown at nonpermissive temperature (38.5 °C) overnight to select the chromosomal-plasmid-integrated strain (Additional file 1: Figure S3B). Next, the cultures were then diluted 1:106 in M17GS medium without antibiotic and grown overnight at permissive temperature (25 °C) to stimulate a second recombination event , and the plasmid was excised from chromosome (Additional file 1: Figure S3C). Dilutions of the overnight cultures were plated on M17GS-XN and incubated at 37 °C to eliminate the excised Ts plasmid. Single blue colonies were screened by replica plating on M17GS-XN plates versus M17GS-XN plates containing erythromycin. The erythromycin-sensitive blue colonies represented the strains with a plasmid region excised in a double-crossover event and the PZLT fragment inserted in the NZ9000 genome. The resultant stain was named NZB (Additional file 1: Figure S3).
Evaluating the efficient heterologous gene knock-in reporter system in L. lactis
To evaluate the knock-in reporter system, we knocked five different lengths (1.3–14.6 kb) of DNA fragments into the L. lactis NZB genome as heterologous genes. The five fragments were amplified from the vectors of our laboratory’s collection with the primer pair P32Rec–TerRec or PZRec–TerRec and cloned into the vector pJW through its AscI site by seamless cloning. The constructed vectors were named pJW-1.3, pJW-2.2, pJW-3.8, pJW-7.3, and pJW-14.6 (Additional file 1: Table S2.). The five vectors were transformed into NZB, and the transformants were selected at 30 °C on M17GS-NX medium containing erythromycin (Fig. 3a). The plasmids harboring NZB were inoculated in M17GS broth with erythromycin and incubated at 30 °C for 8 h, and were then diluted at 1:103 in the same medium and incubated at 38.5 °C to select plasmid-integrated clones (Fig. 3b). The overnight cultures were then diluted at 1:106 in M17GS medium without antibiotic and grown overnight at 25 °C (Fig. 3c). The overnight cultures were diluted and plated on M17GS-XN at 37 °C until most colonies turned blue. The white colonies were picked for further identification.
YFL, HXY, JZD, ZGH, XRJ, YLY and QWH designed and carried out the experiments, analyzed the data and drafted the manuscript. FQH and JW contributed to editing and revising the manuscript. JW draft the basic idea and supervised the study. All authors read and approved the final manuscript.
We would like to thank Prof. Stephen H. Leppla for kindly providing plasmid pCrePA2.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this manuscript.
This study was funded by National Natural Science Foundation of China (NSFC, Grant No. 31201341), and Third Military Medical University Youth Science Foundation (Grant No. 2010XQN06).
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