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Applied Microbiology and Biotechnology, Volume 60, Number 5, January 2003, pp. 564-570

Original Paper

Food-grade host / vector expression system  for Lactobacillus casei based  on complementation  of plasmid-associated phospho-β-galactosidase  gene lacG

T. M. Takala¹, P. E. J. Saris^1,  and S. S. H. Tynkkynen²

(1) Department of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, Viikinkaari 9, P.O. Box 56, 00014 Helsinki, Finland
(2) Research and Development, Valio Ltd, 00370 Helsinki, Finland

Received: 4 July 2002 / Revised: 19 September 2002 / Accepted: 20 September 2002 / Published online: 7 November 2002

ABSTRACT

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-β-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-β-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

INTRODUCTION

The safe use of genetically modified lactic acid bacteria (LAB) in the dairy industry requires cloning systems that are composed solely of DNA from food-grade organisms and do not contain antibiotic resistance genes for selection. Several potentially useful food-grade cloning systems have been developed for LAB. Different selection marker systems, both complementation (e.g. lactose or pyrimidine metabolism) and dominant (e.g. bacteriocin resistance) markers have been published during the past decade (de Vos 1999; Sørensen et al. 2000). In addition to plasmid vectors carrying food-grade selection markers, several integrative food-grade expression systems have been developed (de Vos 1999; Gosalbes et al. 2000; Martín et al. 2000).

Lactobacillus casei is a LAB found in many food products, such as fermented vegetables, meat, and milk. The important role of Lb. casei as a non-starter LAB (NSLAB) in cheese has been reported (Jordan and Cogan 1993; McSweeney et al. 1993; Swearingen et al. 2001). Broome et al. (1990) showed that Lb. casei as adjunct starter in cheese increased the rate of peptidolytic activity, while flavor intensity increased during ripening. Furthermore, NSLAB, such as Lb. casei may have an important indirect contribution to cheese flavor by inhibiting undesirable adventitious bacteria responsible for off-flavor production (Swearingen et al. 2001).

An integrative food-grade expression system for Lb. casei has recently been published (Gosalbes et al. 2000). However, since the gene to be expressed in this system is integrated into the lactose operon, the cells cannot utilize lactose efficiently, resulting in weak growth at low concentrations of lactose, a condition which is typical in cheese after a few weeks ripening (Olson 1990; Turner and Thomas 1980). A host/vector system enabling growth at low lactose concentration and high expression level of foreign genes has not yet been established for Lb. casei.

Lactobacillar proteases and peptidases have an important role in composing the combination of peptides and free amino acids in cheese, thus contributing to the ripening process, texture of cheese, and flavor formation (Christensenet al. 1999; Kunji et al. 1996). Bitter flavors in cheese have been proposed to be a consequence of partial proteolytic digestion of the proline-rich milk protein, β-casein (Sullivan and Jago 1972). Proline-containing oligopeptides are suggested to cause a bitter taste in cheese, while proline as free amino acid has a sweet taste (Ishibashi et al. 1988; Langler et al. 1967). The liberation of proline from oligopeptides requires proline-specific peptidases (Christensen et al. 1999).

In this study, we present a new food-grade host/vector system for a non-starter Lb. casei strain E isolated from high-quality Edam cheese. The plasmid-associated phospho-β-galactosidase (P-β-Gal) gene lacG was inactivated in the host by gene replacement, and a food-grade plasmid vector expressing the lacG gene was transformed into the host with lactose selection. The ability of the food-grade recombinant strain to grow at low lactose concentrations and its capacity for expression of heterologous proline iminopeptidase were shown.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions

The bacterial strains and plasmids used in this study are presented in Table 1. Escherichia coli strains were grown in LB medium at 37 °C. Lactococcus lactis strains were grown in M17 medium (Oxoid) containing 0.5% glucose (GM17) at 30 °C. Lactobacilli were grown in MRS medium (Difco) at 37 °C. NSLAB strains were isolated by plating homogenized Edam-type cheese onto MRS agar (pH 5.6). The colonies were identified to species level by API 50CHL (bioMerieux, France), 16S rRNA sequencing, and species-specific PCR (Alander et al. 1999; Tilsala-Timisjärvi and Alatossava 1997). After electrotransformation, Lc. lactis cells were plated onto GM17 agar containing 0.5 M sucrose and Lb. casei cells were plated onto modified MRS agar containing 2% lactose. Modified MRS lacks carbohydrates and meat extract. Lactose indicator plates contained modified MRS supplemented with 2% lactose and 50 µg bromcresolpurple/ml. When needed, erythromycin was used at a final concentration of 200 µg/ml for E. coli and 2.5 µg/ml for Lb. casei and Lc. lactis. Food-grade transformant strains of Lb. casei were routinely grown in modified MRS supplemented with 0.5% lactose. Examining the effect of lactose concentration on growth, 3 µl of overnight Lb. casei cultures were added into 300 µl of modified MRS containing different concentration of carbohydrates (0.1-1.0%) in Bioscreen microtiter plates (100 wells). The plates were grown in the Bioscreen C apparatus (Labsystems, Helsinki, Finland) at 37 °C for 48 h. Every 30 min, the plates were mixed by moderate shaking for 10 s and the optical density was then measured with a wideband filter (420-580 nm).

Table 1. Bacterial strains and plasmids

DNA isolations and manipulations

E. coli plasmid DNA was isolated with the Wizard DNA miniprep purification kit (Promega, Madison, Wis.). Plasmid DNA from Lc. lactis was isolated as described by O'Sullivan and Klaenhammer (1993). Lb. casei DNA was isolated according to the method described by Anderson and McKay (1983), except that lysozyme and mutanolysin were used at concentrations of 20 mg/ml and 50 units/ml, respectively, for 1 h at 37 °C. Standard molecular cloning techniques were performed as described by Ausubel et al. (1987). DNA transfer from agarose gel to Nylon membrane was performed by vacuum blotting. DNA-probes were labeled and colorimetrically detected with the digoxigenin DNA-labeling and detection kit (Boehringer Mannheim, Germany). In the lactose operon sequence (5,219 bp, accession number Z80834) the lacG gene is located at positions 3,257-4,681. The 1,425-bp lacG-probe was amplified by PCR. Lc. lactis, Lb. casei, and E. coli were transformed by electroporation, as described by Holo and Nes (1989), Varmanen et al. (1998), and Zabarovsky and Winberg (1990). PCR was performed with the Mastercycler apparatus (Eppendorf), using standard procedures in reaction conditions recommended by the manufacturer of Dynazyme DNA polymerase (Finnzymes, Espoo, Finland).

Segregational and structural stability

The stable maintenance of the food-grade plasmid pLEB600 in Lb. casei under non-selective conditions was determined. Serial subcultures were performed by diluting overnight cultures to 10^-6 in fresh MRS broth. Subcultures were plated onto MRS agar and both the total amount of cells and the number of generations were calculated. Colonies were then picked onto lactose indicator plates and the percentage of lactose-negative (Lac^-) colonies was calculated. A total of 770 colonies from nine serial overnight subcultures were analyzed. Twenty streaks showing the weakest acid production on lactose indicator plates were chosen for determination of structural stability by plasmid isolation and restriction analysis.

Enzyme activity assays

For the determination of P-β-Gal and PepI activities, the cell suspensions were lysed, as described by Varmanen et al. (1996), except that potassium phosphate buffer (pH 7.0) was used instead of Hepes. PepI activity was determined by measuring spectrophotometrically at 410 nm the liberation of para-nitroaniline from the substrate L-proline-para-nitroanilide (Pro-p-NA) (Sigma) in 50 mM potassium phosphate bufferm pH 7.0 (El Soda and Desmazeaud 1982). P-β-Gal activity was determined by measuring spectrophotometrically at 420 nm the degradation of the substrate ortho-nitrophenyl-β-D-galactopyranoside-phosphate (ONPG-P; Sigma) in 50 mM sodium phosphate buffer (pH 7.0). Cuvettes containing 21 µg of protein extract and 0.55 mM Pro-p-NA or 0.17 mM ONPG-P were incubated for 20 min at 37 °C before measurement.

RESULTS

Localization of lactose operon in Lb. casei strain E

In screening for lactose-fermenting non-starter lactobacilli from a high-quality Edam-type cheese, a strain designated "E" was isolated and identified as Lb. casei (partial rRNA sequence accession number AJ507644). This strain utilized lactose via a phosphoenolpyruvate-dependent phosphotransferase system (data not shown). The organization of the Lb. casei lactose operon according to Alpert and Siebers (1997) is shown in Fig. 1.

Fig. 1. Structure of the lactose operon of Lactobacillus casei, according to Alpert and Siebers (1997). The corresponding proteins for genes lacTEGF are: regulatory antiterminator protein (lacT), lactose specific phosphotransferase proteins (lacE, lacF), and phospho-β-galactosidase (lacG). The black triangle and hairpin loops represent promoter and transcription terminators, respectively. The length of the lacG gene is 1,425 bp. Restriction sites SphI (S) and ClaI (C) are located at positions 584 and 724, respectively, in the lacG gene

For localization of the lactose operon, total DNA of Lb. casei E was isolated and separated in agarose gel. DNA was transferred to a Nylon membrane and hybridized with the lacG gene as a probe. Figure 2 shows the hybridization of the lacG probe with a large plasmid and the chromosomal DNA. Large plasmids are sensitive to degradation by shearing during plasmid isolation. Large plasmid DNA fragments (>20 kb) have the same mobility in agarose gel electrophoresis as chromosomal DNA, which upon isolation is always fragmented. Thus, the chromosomal band may contain also degraded plasmid DNA. Due to the strong hybridization of the lacG-probe to the large plasmid, we concluded that the lactose operon in Lb. casei E is located in a plasmid. The size of the lactose plasmid was estimated to be at least 30 kb (data not shown).

Fig. 2. Localization of the lactose operon in Lb. casei E. Left Plasmid profile of Lb. casei E in agarose gel. Right Southern blot of the same gel hybridized with a digoxigenin (DIG)-labeled lacG probe. Lane 1 DIG-labeled DNA digested with HindIII, lane 2 DNA digested with HindIII, lane 3 Lb. casei E plasmid profile. The lacG probe hybridized with a large plasmid (upper band in Southern blot lane 3), and with the chromosomal DNA (Chr, lower band in Southern blot lane 3). The chromosomal DNA band (Chr) may also contain degraded plasmid DNA. The molecular sizes of linear DNA markers (kb) are shown on the left

Construction of the lacG deletion by gene replacement

In order to construct a lactose-deficient food-grade cloning host from Lb. casei E, a deletion in the plasmid-encoded lacG gene was created via two single cross-over events, resulting first from insertion followed by excision of an integrative plasmid.

The integration plasmid was constructed in E. coli from the vector pLS19, containing a pUC19 replicon unable to replicate in lactobacilli and the erythromycin resistance gene ermC. The 1,425-bp lacG gene was cloned as a blunt-ended PCR-product into the blunt-ended SphI-site of the pLS19, resulting in plasmid pVS87. Then, a 141-bp SphI-ClaI fragment in the middle of the cloned lacG was restricted from pVS87. The locations of the SphI and ClaI sites in the lacG gene are presented in Fig. 1. In the lactose operon sequence (5,219 bp, accession number Z80834), SphI and ClaI are located at positions 3,841 and 3,981, respectively. The SphI-ClaI ends of the plasmid were made blunt-ended with T4 DNA-polymerase and self-ligated, resulting in the integration plasmid pVS88. The lacG gene containing the internal deletion was subsequently designated lacG. Plasmid pVS88 was transformed into Lb. casei E. Single cross-over recombination was obtained by selecting for erythromycin resistant (Erm^r) transformants. Correct integration in the lactose plasmid was verified by PCR and Southern hybridization, using the ermC gene as a probe. Since the cells of the integrant clone contained multiple copies of the lactose plasmid, the recombinant copy of the plasmid had to be enriched. Therefore, the cells were grown for approximately 100 generations in a medium containing erythromycin. After enrichment, the lactose utilization of the integrant was determined. The integrant showed no acid production from lactose on lactose-indicator plate, which indicated that there were no copies of the original lactose plasmid left in the cells. The integrated pVS88 in the lactose operon possibly hampered the transcription of the lactose operon, causing a Lac^- phenotype.

Next, the integrant was grown another 100 generations in non-selective medium to enable the second crossing-over between the homologies of the lacG and lacG genes, leading to gene replacement. Then, cells were plated onto non-selective MRS agar and screened for Erm^s, Lac^- colonies. The 141-bp SphI-ClaI deletion in the lacG gene of the Erm^s, Lac^- colonies was confirmed by PCR, restriction analysis, and Southern blotting with a lacG probe.

The lacG gene replaced in the plasmid-associated lactose operon encoded an inactive P-β-Gal enzyme, resulting in a lactose-negative strain. Next, we tested whether this lacG-strain could be used as a food-grade cloning host with lactose selection.

Complementation of the lacG mutation and construction of the food-grade vector

A lacG complementation plasmid had to be constructed for testing of the Lac^- lacG strain. This plasmid was constructed out of three DNA fragments from different food-grade organisms: the lacG gene from Lb. casei, the pSH71-based replicon from Lc. lactis, and the constitutive pepR-promoter (P_pepR) for lacG expression from Lb. rhamnosus. The lacG gene and the P_pepR were cloned as PCR fragments into the plasmid vector pLEB124 in Lc. lactis MG1614, resulting in plasmid pLEB591. The 1.6-kb P_pepR-lacG fragment from pLEB591 was cut as an EcoRI-BamHI fragment, ligated with the EcoRI-BamHI-digested pSH71 replicon from lactococcal plasmid vector pLEB590, and the ligation mixture was transformed into Lb. casei ElacG. Colonies obtained on lactose selection plates were picked and the acid production from lactose was confirmed on lactose indicator plate. One streak exhibiting a strong Lac⁺ phenotype was chosen for further analysis. According to plasmid isolation and restriction analysis, the transformant was shown to contain the expected plasmid construction. The resulting food-grade plasmid vector was designated pLEB600. Construction of pLEB600 is shown in Fig. 3. The plasmid is 3.6 kb in size, it is constructed entirely of DNA from food-grade organisms, and it is thus regarded as a food-grade plasmid.

Fig. 3. The food grade expression vector pLEB600 and the PepI expression plasmid pLEB604. The black dots represent transcription terminators

Segregational and structural stability

The segregational and structural stability of pLEB600 was then evaluated. Lb. casei ElacG (pLEB600) was grown in non-selective broth (MRS) for 170 generations. During the 170 generations in non-selective medium, no plasmid loss was observed. Analysis of the plasmid content of the 20 weakest Lac⁺ colonies showed no structural rearrangements. Thus, pLEB600 was shown to retain stable in Lb. casei ElacG without selection pressure.

Plasmid incompatibility

The wild-type Lb. casei strain E contains several cryptic plasmids of different sizes (Fig. 2, lane 3 in agarose gel). The deletion in the plasmid-encoded lacG gene caused no apparent changes in the plasmid content. However, transformation of pLEB600 displaced the smallest (approximately 2 kb) cryptic plasmid. This suggests that the smallest cryptic plasmid of Lb. casei E belongs to the same compatibility group as the food-grade plasmid. No other changes were observed.

Expression of proline iminopeptidase PepI in Lb. casei ElacG

To investigate whether new properties could be transferred into Lb. casei E using pLEB600 as a vehicle, the proline iminopeptidase gene (pepI) from Lb. helveticus was transcriptionally fused with the lacG gene in pLEB600, resulting in plasmid pLEB604 (Fig. 3). The pepI gene was cloned as a 1-kb BamHI-XhoI PCR fragment. Plasmid pLEB604 was transformed into Lb. casei ElacG and selected on lactose plates. PepI enzyme activities were determined from the transformant and compared with the wild-type Lb. casei and Lb. helveticus. The wild-type Lb. casei E was almost deficient in PepI. Expression of pepI from pLEB604 resulted in 20-fold higher, and 3- to 4-fold higher PepI activity, compared with the wild-type Lb. casei and Lb. helveticus, respectively. The lacG gene in pLEB600 and pLEB604 lacks a transcription terminator, allowing read-through expression of the promoterless pepI gene. Thus, pLEB600 can be regarded as a food-grade expression vector for Lb. casei ElacG.

The effect of lactose concentration on the growth of Lb. casei E variants

To examine the growth on different lactose concentrations simulating the conditions during cheese-making, Lb. casei strains E, lacG, and lacG (pLEB604) were grown for 48 h in modified MRS broth supplemented with 0.5% glucose, or 0.1-1.0% lactose. Figure 4 shows the growth of the strains during the first 20 h, after which the growth curves remained unchanged. Glucose supported growth of all three strains. The food-grade PepI expression strain and the wild-type strain grew equally well on every lactose concentration examined. The lacG strain could hardly grow on lactose-containing broth, as already seen in previous experiments.

Fig. 4. Growth curves of three Lb. casei E variants on medium supplemented with lactose (lac) or glucose (glc). Black circles Wild-type Lb. casei E, white triangles the Lac^- strain Lb. casei ElacG, white circles the Lac^- strain Lb. casei ElacG transformed with the PepI expression plasmid pLEB604. OD Optical density

The P-β-Gal activities of Lb. casei E variants were also measured. The P-β-Gal activity of the food-grade PepI expression strain was approximately 80% of the P-β-Gal activity of the wild-type Lb. casei E. However, the lower activity did not seem to affect growth on lactose, as shown in Fig. 4. As supposed, the Lac^- strain lacG showed no P-β-Gal activity.

DISCUSSION

Lb. casei strains have been shown to ferment lactose via PEP-dependent lactose phosphotransferase system located either in the chromosome or in large plasmids (Chassy and Alpert 1989). Lactose utilization of Lb. casei strain E used in this study was shown to be plasmid-associated. For the construction of a lactose-deficient mutant, a 141-bp in-frame fragment in the middle of the lacG gene encoding P-β-Gal was deleted by gene replacement. Mutated lacG gene encoded an inactive P-β-Gal lacking 47 amino acids, resulting in a stable Lac^- phenotype. Knock-out mutations in plasmid-encoded genes by gene replacement has previously been constructed only in the large (100 kb) low-copy E. coli R-factor plasmid, R100-1 (Nieto et al. 1998). Construction of a deletion by gene replacement in a multi-copy plasmid has several difficulties, since no selection method exists for the second homologous recombination and the desired phenotype is not achieved if any copy of the original plasmid is retained in the cell. However, this study shows that gene replacement is a possible method for the construction of knock-out mutations in multi-copy plasmids.

Food-grade systems based on replicable plasmids have been proposed to suffer from instability (Martín et al. 2000). However, we showed that pLEB600 is a stable plasmid, which remained in Lb. casei for more than 170 generations under conditions where the selective agent (lactose) was absent. The variation in the strength of the Lac⁺ phenotypes in the lactose-indicator plates was probably a consequence of variation in the plasmid copy number or expression level of the lacG gene. After a few hours growth in selective medium, no variation in the strength of the lactose utilization could be seen. Plasmid incompatibility is a common phenomenon with wild-type dairy strains, which might complicate the transfer of heterologous plasmid DNA. Lb. casei E contains multiple indigenous plasmids, of which the smallest (approximately 2 kb) was displaced when the food-grade plasmids pLEB600 or pLEB604 were transformed. Under lactose selection pressure, the transformed plasmids were more competitive and the small plasmid was cured. No phenotypic changes could be seen. The small plasmid probably contained only the genes needed for its own replication.

The food-grade system constructed in this study was used for transferring PepI activity into Lb. casei E. The pepI gene cloned from a Swiss-type cheese starter strain of Lb. helveticus and expressed in Lb casei E resulted in 20-fold higher PepI activity, compared with the level of the wild-type Lb. casei E. A high proline-specific peptidase activity decreases the concentration of bitter-tasting proline-containing oligopeptides and increases the amount of free proline amino acid. This has a favorable effect on the flavor formation and possibly accelerates the ripening process in cheese. Lactobacillar peptidases, including PepI, have previously been successfully expressed only in laboratory strains of Lc. lactis (Luoma et al. 2001; Wegmann et al. 1999). This paper presents the first example of food-grade expression of the peptidase gene in a wild-type Lactobacillus strain.

The pepI gene cloned into the food-grade vector pLEB600 did not hamper the ability to complement the P-β-Gal activity absent in the lacG mutant strain. The utilization of lactose was fully restored to the wild-type level. The PepI-producing recombinant Lb. casei grew well on lactose at low concentrations (0.1%), simulating the real situation in cheese after a few weeks of ripening. Depending on the conditions in a cheese curd, e.g. salt concentration, lactose is either exhausted in approximately 1 week by starter bacteria, or low concentration (0.1-0.5%) of lactose remain in the curd and is degraded later by NSLAB (Turner and Thomas 1980). The main energy source used by NSLAB for growth has not been clearly defined (Beresford et al. 2001). However, we believe that efficient utilization of lactose increases the competitiveness of the adjunct NSLAB and thus intensifies their contribution to the ripening process. Thus, the ability to grow at low lactose concentration provides a clear advantage, compared with the previously published food-grade system for Lb. casei, which was unable to do so (Gosalbes et al. 2000).

These results demonstrate that the food-grade system reported in this paper can be used for transferring new properties to Lb. casei in order to construct improved starter bacteria for cheese-making.

ACKNOWLEDGEMENT

This work was supported by the Academy of Finland (project number 177321).

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