Contribution of modern biotechnology of lactic acid bacteria to development of health-promoting foods

Lactic acid bacteria (LAB) are extensively used in the manufacture of a wide variety of fermented dairy, meat, vegetable, bakery and wine products in the food and wine industry as well as in making silage for animal feed. Some LAB strains also have an increasingly important role as health-promoting probiotics. Molecular genetic research of LAB, focused mainly on the basic characterisation of traits essential for the industrial utilisation of these bacteria, forms a solid scientific basis for stabilisation, modification and improvement of these characteristics. The emphasis of this review is on the molecular genetic work done at the research laboratory of the author. Our research team is engaged on, two main projects: molecular genetic and biochemical characterisation of the proteolytic systems of industrial thermophilic lactobacilli and surface layer protein studies to develop protein production systems for food, feed, vaccine and diagnostic purposes.

ntroduction Lactic acid bacteria (LAB) are a diverse group of micro-organisms inhabiting various ecological niches, from plant surfaces to the gastrointestinal, genital and respiratory tracts of man and animals (De Vuyst and Vandamme 1994, Wood  1992).LAB are also widely used as starters in the manufacture of fermented foods, beverages, pickled vegetables and silage (De Vuyst and Van- damme 1994).In fermentation, several metabolic properties of LAB serve special functions with a direct or indirect impact on food processes.Fer- mentation allows the preservation of food and affects the development of flavour and texture, whilst, starter cultures bring about a variety of beneficial metabolic and sensory changes in food (Lindgren and Dobrogosz 1990, Olson 1990,  Holzapfel et al. 1995).Some milk products fer- mented with probiotic LAB may also have health and additional nutritional benefits, and these are increasingly being subjected to R+D (Jensen  1995, Wood 1992).The proposed key targets of the health-promoting effects of probiotic LAB are the prevention of intestinal infections, diar- rhoeal diseases and upper gastrointestinal tract diseases, the prevention of cancer and the hypercholesterolemia, the improvement of lactose utilisation and the stabilisation of the gut mu- cosal barrier (Kailasapathy and Rybka 1997,  Wood 1992, Salminen et al. 1996).
The loosely defined group of LAB includes Gram-positiverods and cocci with low G+C content < 50 mol% (Pot et al. 1994).LAB are cata- lase negative, non-sporulating (except Sporolactobacillus), acid-and aerotolerant anaerobic bacteria that produce lactic acid as a major or sole end-product of obligative fermentative me- tabolism (Pot et al. 1994, Wood and Holzapfel  1995).Taxonomically LAB have been included in the genera Lactobacillus, Lactococcus, Leu- conostoc, Pediococcus, Carnobacterium, Sporolactobacillus, Streptococcus, Enterococcus, Aerococcus, Vagococcus, Tetragenococcus and Atopium (Pot et al. 1994, Wood and Holzapfel  1995); This classification is, however, not final.Many characteristics typical of genuine LAB are also shared by the genus Bifidobacterium, which consists of increasingly important health-promoting intestinal bacteria (Kailasapathy and   Rybka 1997).Bifidobacterium was considered to be one of the LAB, but due to their high G+C content (55-67 mol%) and on the basis of 16S rRNA data, it is now clear that the bifidobacte- ria belong to the actinomyces branch in the phylogenetic trees of Gram-positive bacteria (Schleifer and Ludwig 1995).
Owing to the great diversity of LAB, especially among one of the most widely used groups, i.e.Lactobacillus, molecular genetic studies of LAB are very demanding.However, since being of such importance in the food industry, LAB, particularly the starters used in the dairy products, have been subjected to extensive molecu- lar genetic research for many years.The objectives of that research have been to characterise, stabilise and improve traits essential for food processes (for recent reviews, see de Vos et al.  1993, Venema et al. 1996).Much is already known about the molecular biology of starter lactococci but during the last few years, a con- siderable amount of information has also been gathered on the various lactobacillar species.In addition to the development of genetic tools, the main research targets have been characterisation of the key metabolic genes (e.g.lactose and cit- rate utilisation, proteolytic enzymes) and meta- bolic engineering, isolation, characterisation and modification of antimicrobials for food preservation, bacteriophages and bacteriophage resist- ance, and its gene expression and control (de Vos et al. 1993, Gasson and de Vos 1994, Venema et al. 1996).We are gradually accumulating a very solid and promising basis for the further devel- opment of better and safer food products, possibly even with novel characteristics.Recently, work has begun on elucidating the different health-promoting effects and validating the claimed benefits of probiotics with the aid of molecular biology (Klaenhammer 1995,Tannock  1995).Furthermore, the development of LAB as oral vaccine carriers has got off to a promising start (Wells et al. 1996).These new fields have opened fascinating perspectives on novel utilisations of certain LAB strains.Eventually such applications may be seen as evidence of the major impact modern biotechnology can have on the health and nutrition of humans and animals.
This review summarises, in the context of relevant research, some of the contributions made by our LAB research team to efforts to establish a basic knowledge base for future food and health-promoting applications.In the first part of the review I shall examine the status of our work on the molecular genetic characteri- sation of lactobacillar proteolytic systems.The proteolytic systems of LAB are considered to have an essential impact on cheese ripening and the formation of flavour and texture in cheese.The new information now available on cheese ripening and the exact role of proteolytic and peptidolytic enzymes in the process will aid the development of low fat cheeses with a better health-affecting status.Furthermore, it will be possible to use the characterised enzymes of the proteolytic systems in the targeted production of bioactive peptides, e.g. from milk proteins.In the second part of the review I shall look at the molecular characterisation of the surface layer protein (S-protein, SlpA) and gene ( slpA ) ofLactobacillus brevis and the development of a protein production system based on the ex- pression and secretion signals ofslpA.The protein production systems developed for LAB are still rather unsophisticated, and the factors af- fecting protein production, secretion in particular, poorly characterised compared with those established for other pro-and eukaryotic organisms.Furthermore, the utility of the S-protein itself as a carrier of vaccine and diagnostic an- tigens is a key target of current and forthcom- ing research.
Molecular genetic characterisation of components of the proteolytic systems of Lactobacilli

General background
Many LAB isolated from milk products are mul- tiple amino acid auxotrophs (Chopin 1993).In milk, the abundances of free amino acids and short peptides are very low.To grow in milk LAB are therefore highly dependent on their proteolytic system.The biochemical and genetic char- acteristics of the proteolytic system in lactococ- ci have been extensively studied to ascertain the influence of the components of the system on the degradation of milk proteins, and thus on cheese ripening (for review, see Kunji et al.  1996).Briefly, the proteolytic system in lacto- cocci consists of: i) the cell wall-associated proteinase (PrtP), which degrades casein into oli- gopeptides; ii) amino acid and peptide transport systems, which supply the degraded organic nitrogen source to the cell by translocating the breakdown products of casein across the cytoplasmic membrane; and iii) many different intracellular exo-and endopeptidases, which fur- ther cleave oligopeptides into shorter peptides and amino acids (Kunji et al. 1996, Fig. 1).Mo- lecular characterisation of lactobacillar proteo- lytic systems, mainly ofLactobacillus delbrueck- ii ssp.lactis, Lactobacillus helveticus and Lacto- bacillus paracasei, even though it got under way several years after that of lactococci, has made rapid progress.We now have large knowledge base on these bacteria too, and it has become clear that a close overall similarity exits between the lactobacillar proteolytic systems and the lac- tococci (Kunji et al. 1996).Due to their high peptidolytic activity, lactobacilli, L. helveticus in particular, have been used in various forms to reduce bitterness, improve flavour development and accelerate the ripening of various types of cheese in processes conventionally based on the use of other starter strains (Ardö and Larsson 1989, Bartels et al. 1987 a, b, El Soda 1993).
Thus, genetic characterisation of the components of the lactobacillar proteolytic systems will en- able us to develop more precise modifications of starter bacteria for improvements to lowfat cheeses and many other processes in the food industry.
To date, the cell wall-bound proteinase has been characterised from several Lactococcus lactis subsp.cremoris strains, two Lactobacillus casei, one Lactobacillus delbrueckii subsp bul- garicus and three L. helveticus strains, and the substrate specificities of lactococcal Prts have been described in detail (Kunji et al. 1996).Amino acid and peptide transport systems have been studied mainly in lactococci, for which 10 amino acid transport systems have been found but not yet cloned.The lactococcal di/tripeptide transporters and the oligopeptide transport systems have been extensively analysed and even genetically modified (Kunji et al. 1996).The most extensive molecular characterisation work has, however, been done on peptidases.Thus far, dozens of peptidases have been analysed, from Lactococcus lactis subsp.cremoris strains, L. delbrueckii subsp.lactis, L. helveticus and oth- er LAB, including at least 12 peptidases with distinct substrate specificities (see Kunji et al.   1996).

The characterisation of lactobacillar proteolytic systems
Our laboratory has sought to characterise the key components ofand events in the proteolytic system of thermophilic industrial lactobacilli, with special emphasis on L. helveticus and L. del- brueckii subsp.bulgaricus.The L. helveticus we have used in our studies is an industrially uti- lised strain, 53/7, with highly favourable peptidolytic characteristics.During the past five years we have completed the molecular genetic char- acterisation of six L. helveticus and two L. del- brueckii subsp.bulgaricus peptidases.We are currently engaged in cloning and sequencing a novel type of PrtP and a tripeptidase from L. helveticus (data not shown).
Characterisation of these components has allowed us to: i) initiate construction of modi- fied lactococcal starter strains (an EU-BIOTECH II Starlab project); ii) analyse the regulation and interactions of the peptidase genes in L. helveti- cus (in collaboration with the Department of Food Science, University of Wisconsin-Madi- son), iii) test modifiedL.helveticus strains in a cheese process with an industrial partner; and iv) test the overproduction of individual peptidases.
Cloning the oligopeptide transport system from L. delbrueckii subsp.bulgaricus will allow us to compare the specificity and capacity of the transport systems of different LAB.
To control cell lysis, and thus to release peptidases, we have isolated and characterised a stress (e.g.NaCl) induced promoter from L. helveti- cus (Smeds et al., unpublished).In the next sec- tions I shall look at the results we have already published or submitted for publication.
General arninopeptidases N, C and D of L helveticus 53/7 Arninopeptidases are capable of cleaving N-ter- minal amino acids from a wide variety of peptides differing in both size and composition.The general arninopeptidases we have characterised from L. helveticus 53/7 include PepN and PepC, which hydrolyse longer peptides, and a dipeptidase (PepD).We (Varmanen et al. 1994) cloned the ami- nopeptidase N gene using nucleic acid hybridisation to detect the pepN gene from a L. helveti- cus 53/7 genomic library, with a fragment of the putative pepN gene from L. helveticus CNRZ32 (Nowakowski et al. 1993) as the probe.A 3.7 kb hybridisation positive insert directing ami- nopeptidase activity in E. coli was sequenced and found to contain only one open reading frame (ORF) of 2532 bp.This ORF had a coding ca- pacity for a 95.8 kDa protein that corresponded to the size of the lactococcal (Tan et al. 1992) and L. delbrueckii subsp.lactis PepNs (Klein et  al. 1993).The deduced amino acid sequence was 49% homologous to Lactococcus lactis PepN, and showed 99% identity to the L. helveticus CNRZ32 pepN gene, sequenced later, (Christensen et al. 1995).Furthermore, the conserved catalytic and zinc-binding sites of the neutral zinc metallo-peptidase family were identified from the PepN sequence, confirming that the ORF cloned and sequenced did indeed encode the aminopeptidase N activity.The pepN mRNA analyses revealed 2.75 kb transcripts with two transcription start sites.These two sites verified the existence of two putative overlapping pro- moter regions in the DNA sequence.Our stud- ies on pepN expression as a function of growth in a bioreactor showed that pepN transcripts re- mained at a high level during the stationary growth phase, too, in contrast to the steady-state levels of all other peptidase mRNAs we have analysed.Similarly, the level of total aminopeptidase activity remained constant throughout the stationary growth phase.The inhibition profiles of PepN showed that it is indeed a zinc-dependent metallopeptidase, being completely inhibit- ed by ethylenediaminetetra-acetic acid (EDTA) whereas serine protease inhibitor phenylmethylsulphonylfluoride (PMSF) and a thiol- blocking reagent, p-hydroxymercuribenzoate (pHMB), had only a slight effect on enzyme ac- tivity (Varmanen et al. 1994).Thus, the charac- teristics of L. helveticus PepN are in accordance with PepNs from other LAB (Kunji et al. 1996).
The second aminopeptidase we characterised was the PepC (Vesanto et al. 1994).The isola-tion procedure for the pepC gene was similar to that for pepN.In a hybridisation positive clone showing aminopeptidase activity the pepC gene could be localised into a 3 kb fragment by dele- tion mapping.Sequencing of this fragment re- vealed two open reading frames (ORFI and ORF2) of 1347 and 840 bp.ORFI was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -49.0 kJmol' 1 was found downstream of the coding re- gion.The deduced amino acid sequence of ORFI, with an encoding capacity of a 51.4 kDa protein, shared 48.3% and 98% identity with the PepC proteins from Lactococcus lactis (Chapot-Chartier et al. 1993) and L. helveticus CNRZ32 (Fernändez et al. 1994), thus confirming that ORFI codes for an aminopeptidase C. mRNA size analyses revealed 1.7 and 2.7 kb transcripts.Further analysis with the pepC and ORF 2 spe- cific probes showed that the downstream ORF2 was co-transcribed with the pepC gene at the exponential phase of growth whereas, at the sta- tionary growth phase, pepC transcripts derived from the pepC promoter were below the detection limit and ORF2 was expressed by its own promoter (Vesanto et al. 1994).The 5' end mapping of the pepC transcripts revealed a transcription start different from that suggested for the pepC in the L. helveticus CNRZ32 pepC gene (Fernandez et al. 1994).We also studied expression of pepC in L. helveticus as a function of growth in a bioreactor cultivation, and found that transcription of pepC was typical of the exponential growth phase expression.The level of total thiolaminopeptidase activity, however, remained nearly constant throughout the station- ary growth phase.Strain 53/7 PepC expressed in E. coli could be completely inhibited by pHMB and partially by PMSF whereas EDTA had only a minor effect.Thus, PepC is a thiol- dependent aminopeptidase belonging to the cysteine proteinase family.This allowed a rough estimation of the contributions of PepN and PepC to the total aminopeptidase activity in L. helve- ticus.Addition of EDTA or EDTA and pHMB to L. helveticus cell lysates in the presence ofLys- p-nitroanilide (pNA) substrate reduced the ac- tivity to 10% and to below the detection limit, respectively, indicating that the aminopeptidase activity in L. helveticus is mainly due to the metalloaminopeptidase (Varmanen et al. 1994).
Comparison of the level of pepN and pepC tran- scripts supports the conclusion that PepN is the main aminopeptidase in L. helveticus (data not shown).Our further studies on ORF2 have re- vealed that it codes for a transmembrane protein homologous to an unknown Bacillus protein that is expressed during sporulation (Vesanto and  Palva, unpublished results).
We isolated L. helveticus 53/7 dipeptidase gene (pepD ) in the same way as pepN and pepC (Vesanto et al. 1996).An open reading frame (ORF) of 1422 bp was identified from a positive clone with a coding capacity of 53.5 kDa.The promoter and transcription terminator regions were also identified.The deduced amino acid sequence of the 53.5 kDa protein shared no significant similarity with the sequences in the data bases but showed 99.8% overall similarity to PepDA from L. helveticus CNRZ32 (Dudley et  al. 1996).For s'end mapping ofprokaryotic tran- scripts we optimised a method based on the use of an automated DNA sequencer (Vesanto et al.  1996).The 5' end mapping of the 1.6 kb pepD transcript by the new and conventional primer extension methods gave consistent results.Expression studies showed that pepD was maximal- ly expressed at late exponential growth.We also overexpressed the pepD gene in E. coli and pu- rified PepD to homogeneity in three chromato- graphic steps.Studies of the PepD substrate specificity showed that PepD was able to hydrolyse a number of dipeptides with the exception of those containing proline residues.Optimal PepD activity was obtained at pH 6.0 and 55 °C.Inhibition studies of PepD revealed that the enzyme could be inhibitedby pHMB and reactivated by dithiothreitol; EDTA, in contrast had no inhibi- tory effect (Vesanto et al. 1996).In addition to the lack of a homologous counterpart among LAB, the enzymatic properties suggested that the two PepDs isolated from L. helveticus 53/7 and CNRZ32 represented a novel dipeptidase type.
Proline specific peptidases of L helveticus 53/7 Milk proteins contain a large amount of proline residues, which, in free form impact a sweet fla- vour to cheese (Biede and Hammond 1979, Fox  1989).Proline-specific peptidases thereforeplay an important role in cheese ripening by degrading proline-containing peptides, which are some- times bitter, and by making peptides accessible to further degradation by other peptidases through the removal of proline residues (Baank- reis and Exterkate 1991).
We have performed the molecular genetic and biochemical characterisation of three different proline specific peptidases, i.e.X-prolyl dipeptidyl aminopeptidase (PepX) (Vesanto et al.   1995), prolinase (pepß) (Varmanen et al. 1996 a)   and iminopeptidase (Pepi) (Varmanen et al.   1996 b), from strain 53/7.The X-prolyl dipeptidyl aminopeptidases cleave dipeptidyl residues from peptides by hydrolysing the peptide bond at the carboxyl side of the proline residue when this imino acid is the penultimate N-terminal residue.The pepX gene cloned and sequenced from strain 53/7 was shown to be 2379 bp in size with a coding ca- pacity for a 90.6 kDa protein (Vesanto et al.  1995).The pepX gene was identified as a monocistronic transcription unit flanked by a typical prokaryotic promoter region and transcription terminator(delta G -84.1 kJ mol' 1 ).The deduced amino acid sequence of the 90.6 kDa protein shared 49.3, 49.4 and 77.7% overall similarity with the PepX proteins from Lactococcus lactis subsp.lactis (Mayo et al. 1991), Lactococcus lactis subsp.cremoris (Nardi et al. 1991) and L. delbrueckii subsp.lactis (Meyer-Barton et al.  1993), respectively.The size (2.6 kb) and s'end ofpepX mRNA were in agreement with the DNA sequence data.Similarly to the other L. helveti- cus peptidases analysed, expression ofpepX was typical of exponential growth.The pepX gene was also overexpressed in pKK223-3 in E. coli followed by purification of PepX to homogeneity by ion-exchange and hydrophobic interaction chromatography.The enzyme was found to be a dimer (165 kDa) with optimum activity at pH 6.5 and 45 °C.Furthermore, PepX was shown to be a metal-independent serine peptidase, having functional sulphhydryl groups at or near the ac- tive site (Vesanto et al. 1995).Thus, the characteristics of the L. helveticus PepX closely resem- bled those of other PepXs.
Prolinases cleave dipeptides with a proline residue at the N-terminal amino acid.We cloned a prolinase (pepß) from strain 53/7 with a gene probe (Nowakowski et al. 1993) specific for a peptidase shown to have activity against di-and tripeptides (Varmanen et al. 1996 a).The hybrid- isation positive clones obtained, however, did not show any enzyme activity against di-or tripeptides.Further analysis of one of the clones with a 5.5 kb insert revealed two ORFs, of 912 and 1602 bp, ORF2, located upstream of and opposite in orientation to ORFI, had a promoter re- gion overlapping that of ORFI.ORFI had a co- dine capacity for a 35 kDa protein.This protein was shown to be capable of hydrolysing dipeptides when ORFI was amplified by PCR with its control regions and expressed in E. coli.Among the dipeptides tested, the highest activi- ty was obtained with a Pro-Leu substrate whereas among the tripeptides tested, only Leu-Leu- Leu was marginally degraded.Thus, the 35 kDa protein was identified as a prolinase (Pepß) from the substrate-specificity profile and protein ho- mologies (Varmanen et al. 1996 a).The activity of the cloned Pepß was inhibited by pHMB.In accordance with the DNA sequence data, North- ern and primer-extension analyses of pepß showed a 1.25 kb transcript and two adjacent transcription start sites, respectively.Pepß protein was found to be 99.6% identical to the recently sequenced prolinase (PepPN) from L. hel- veticus CNRZ32 (Dudley and Steele 1994), thus further confirming the close similarity of the peptidases of these two L. helveticus strains, which otherwise differ from each other.Interestingly, analysis of the upstream ORF2 revealed that the deduced 59.5 kDa protein encoded by this gene showed significant homology to sever- al members of the family of ABC transporters.
Deletion constructs of ORF2 also clearly dem- onstrated that this upstream operon adversely affected Pepß activity in E. coli, explaining the enzymatic inactivity of the original clones (Varmanen et al. 1996 a).
Proline iminopeptidases (Pepl) are able to liberate the N-terminal proline residue from di- and tripeptides.The L. helveticus 53/7 proline iminopeptidase gene (pepl) cloned by us (Var- manen et al. 1996 b), was found to be organised in an operon-like structure of three ORFs.ORFI was preceded by a typical prokaryotic promoter region, and a putative transcription terminator identified as the pepl gene was present down- stream of ORF3.Primer extension analyses on each ORF revealed only one transcription start site, upstream of ORFI, indicating an operon structure.The level of operon derived transcripts was so low that we could not determine the transcript size by Northern blot; nevertheless the RT-PCR clearly supported the operon structure of these three genes.The coding capacities of ORFI, ORF2 and ORF3 were for 50.7, 24.5 and 33.8 kDa proteins, respectively.The ORF3-en- coded Pepl protein showed 65% identity to the Pepl proteins from L. delbrueckii subsp.hulgaricus (Allan et al. 1994) and L. delbrueckii subsp.lactis (Klein et al. 1994).The pepl gene was overexpressed in E. coli and purified to homo- geneity in two chromatographic steps (Varmanen   et al. 1996 b).Pepl was shown to be a dimer with optimum activity at pH7.5 and 40 °C.Like the L. delbrueckii Pepls, the L. helveticus Pepl was found to be a metal-independent serine peptidase with thiol group at or near the active site.Kinetic studies with PropNA as substrate revealed K m and V values of O.BmM and 350 mmol min 1 max mg ', respectively and a very high turnover number, 135 000 s'.The substrate specificities of the three Pepls identified differed from each other to some extent (Varmanen et al. 1996 b), but we do not know whether these differences were due to assay conditions or they were true differences in the enzymatic properties of these Pepls.
The ORFI and ORF2 encoded proteins were found to share homology with the members of the ABC (ATP binding casette) transporter fam- ily but to represent an unusual type (ORF1)  among the bacterial ABC transporters (Varmanen   et al. 1996 b).
Proline specific peptidases of L delbrueckii subsp.bulgaricus Due to the importance of proline-specific peptidases we have also characterised two genes, pepQ and orfZ, encoding a prolidase (PepQ) and a Pep Q-like protein from L. delbrueckii subsp.bulgaricus (Rantanen and Palva 1997).ThepepQ and orfZ genes showed 98% and 60% identity, respectively to the L. delbrueckii subsp.lactis pepQ; both pepQ and orfZ were preceded by a putative promoter region.The size of pepQ mRNA could be identified (1.1 kb) but, under the growth conditions used,we could only iden- tify the expression of orfZ by RT-PCR.Both genes were shown to be monocistronic transcriptional units.The OrfZ protein, overexpressed in E. coll, revealed no enzymatic activity against the peptide substrates tested whereas the L. del- brueckii subsp.bulgaricus PepQ hydrolysed X-Pro substrates similarly to other prolidases.Note that homologues of the L. delbrueckii sub- sp.bulgaricus orfZ and pepQ genes appeared to be present in both L. delbrueckii subsp.lactis and L. helveticus (Rantanen and Palva 1997).The role of the cryptic orfZ gene and its putative gene product remains to be established.

Oligopeptide transport system of L delbrueckii subsp. bulgaricus
As well as reforming peptidase analyses, we have started the characterisation ofother components of the proteolytic systems ofthermophilic lacto- bacilli.We have isolated the operon of the oligopeptide transport system from a lambda gtlO based genomic library of L. delbrueckii subsp.bulgaricus (Peltoniemi et al. 1998 unpublished  results).This operon is 6.1 kb in size and con- sists of five genes encoding the peptide binding protein (OppA), two integral membrane proteins (Oppß and OppC) and two ATP-binding proteins (OppD and OppF).In L. delbrueckii subsp.hul- garicus, the opp genes in the operon are organ- ized in much the same way as in Lactococcus lactis (Tynkkynen et al. 1993), i. e. oppDFBCA.
Interestingly, an additional opp A -like gene is adjacent to the oppA of the operon.The identity of the oppDFBCA to that of L. lactis was shown to be 50%, 65%, 55%, 40% and 40%, respec- tively (Peltoniemi et al. 1998, unpublished).The operon structure deduced from the DNA se- quence of oppDFBCA has also been confirmed by mRNA analyses.
Characterisation of surface layer protein and gene ( sIpA) from Lactobacillus brevis and use of sIpA signals for heterologous protein production

General background of prokaryotic S-layer structures
There are also many Lactobaci/lus-species among the over 300 S-layer harbouring eu-and archaebacterial species (Messner and Sleytr  1992).The DNA sequence of the lactobacillar S-protein gene (sip) has been published for L. brevis , L. acidophilus and L. helveticus.The functions of the lactobacillar S-layers are most- ly unknown, but apparently this structure is es- sential since all attempts to inactivate sip genes have failed (Boot 1996).The L. crispatus S-protein has been shown to mediate adhesion to type IV collagen (Toba et al. 1995).
For an average size cell, 5 x 10 5 S-layer subunits have to be synthesised per cell generation in order to cover the entire cell surface with the S-layer proteins (Messner and Sleytr 1992).The expression of a sip gene and the secretion ma- chinery of an S-layer harbouring cell may thus be expected to be very efficient.These proper- ties are obvious assets in the utilisation of S-pro- teins in biotechnological applications.We chose to study heterologous protein production in lac- tic acid bacteria, with the L. brevis slpA and to test the possibility of using SlpA as a carrier for foreign antigenic epitopes.L. brevis is a hetero- fermentative lactic acid bacterium commonly found in vegetable fermentations, sour dough, silage and the intestine of humans and animals (Wood 1992).Here, I shall look briefly at char- acterisation of the L. brevis S-protein, gene, mRNA and in vivo expression and discuss the demonstration of heterologous protein secretion and intracellular production with of aid the slpA signals.

Characteristics of the L brevis S-protein
and the slpA gene We demonstrated the presence of the S-protein in intact L. brevis ATCC 8287 cells, boiled in Laemmli sample buffer, by SDS-PAGE analysis, which revealed only one major band, with an apparent molecular weight of46 kDa (Vidgrén  et al. 1992).When the cells were treated with an antiserum raised against the isolated 46 kDa pro- tein and analysed by immuno-gold electron microscopy, post embedding immunoelectron microscopy clearly showed that the 46 kDa protein was heavily concentrated in the outermost part of the cell wall of L. brevis cells (Vidgrén at al.  1992).The slpA gene was PCR cloned accord- ing to the N-terminal sequence information of the intact S-protein and internal tryptic peptides (Vidgrén et al. 1992).The L. brevis slpA gene is 1395 bp in size with a coding capacity for a protein of 48 159 daltons.The first 90 nucleotides of the structural gene encode a typical Gram-positive type signal peptide of 30 amino acid residues (Vidgrén  et al. 1992).The slpA gene is preceded by a well conserved ribosome binding site (RBS) and two promoter regions, PI and P 2 with the -35 and -10 regions resembling the conserved prokaryotic -35 and -10 consensus sequences (von Heijne 1987).A strong transcription terminator se- quence is present downstream of the two translation stop codons of slpA.
At the time the basic characterisation of the L. brevis S-protein and slpA was published, data bases contained no genuinely related SlpA se- quences (Vidgrén et al. 1992).The predicted amino acid sequences of the recently described L. acidophilus (Boot et al. 1993) and L. Helveti- ans (EMBL Nucleotide Data Library: X91199 and X 92752) slpA genes, however, show 35.7% (17) and 28.8% similarity to that of the L. brevis S-protein, respectively.Furthermore, L. brevis slpA probes hybridises to the chromosomal DNA ofL.buchneri (Palva et al. 1992).Two sip genes with phase variation have been found in L. aci- dophilus, and one functional sip gene and a trun- cated sip 3'end in L. helveticus (Boot 1996).In L. brevis, in contrast only one sip gene is present (Palva et al. 1992).
In vivo expression of the slpA gene Determination of the size and s'ends of the slpA transcripts revealed 1.5 kb mRNAs with two 5' ends located immediately downstream of the two -10 regions deduced from the DNA sequence, thus confirming that slpA is a monocistronic tran- scriptional unit and possesses two functional promoters (PI, P 2) (Vidgrén et al. 1992).Deter- mination of the stability of the slpA mRNA showed that the half-life of the slpA transcripts was 14 minutes (Kahala et al. 1997), indicating exceptional stability when compared with the typical half-lives of prokaryotic mRNAs.Recent- ly, the half-lives of the L. acidophilus slpA and Aeromonas salmonicida vapA mRNAs have also been shown to be very stable (Boot et al. 1996,  Chu et al. 1993).The long half-lives of these three S-layer mRNAs from three different species may indicate that a high stability of mRNA is a general feature of S-layer mRNAs.As slpA mRNA mediates the synthesis of a major struc- tural component of the cell, this high stability is not unexpected.
Study of the usage of the two L. brevis slpA promoters (PI, P 2) at different stages of the growth revealed that the P 2 promoter, which was located closer to the start codon, is efficiently used during both the logarithmic and early sta- tionary phases, whereas slpA mRNA derived from PI was only weakly detectable (Kahala et  al. 1997).Further quantitative analyses showed that transcripts derived from both promoters are present throughout the entire growth phase and that the level oftranscripts derived from promot- er P 2 is ten times higher than that derived from PI (Kahala et al. 1997).

S-layer synthesis
In vivo expression studies of L. brevis showed that the kinetics of the accumulations of slpA mRNA and protein correlates well up to the on- set of the stationary phase, when there is a sharp decrease in the level of slpA mRNA.The rate of mRNA decay was, however, slower than expected from the half-life of slpA transcripts, suggesting that residual transcription continues even though the total amount of S-protein does not further increase at the stationary phase (Kahala  et al. 1997).The L. brevis S-layer protein is not released into the supernatant fractions at any of the growth phases studied, suggesting tight regulation of the S-layer synthesis and assembly (Vidgrén et al. 1992, Kahala et al. 1997).Breit- wieser et al. (1992) demonstrated the presence of substantial amounts of S-layer subunits on the inner surface or within the peptidoglycan layer of Bacillus stearothermophilus, suggesting an intermediate phase between the synthesis and final location of the S-layer protein.This has also frequently been observed in S-layers of other gram-positive eubacteria (Breitwieser et al.  1992).Flowever, in L. brevis over 95% of the S-layer subunits could be released with the SDS-PAGE sample buffer from intact cells, as indi- cated by Western blot analyses ofintact and dis- rupted cells (unpublished data).It appears, then, that basically no L. brevis S-layer subunits ac- cumulated inside the peptidoglycan layer before translocation to the outer surface.

Heterologous protein secretion with
the sIpA signals To construct a secretion vector based on the slpA signals, we used a derivative (pKTH2O9S) of the shuttle vector pGKI2 (Kok et al. 1984) as the carrier of the secretion cassette.The model secretion cassette contained the two promoters (PI,P2), the signal sequence (SS ) and the tran- scription terminator (,t slpA ) of the L. brevis slpA and another terminator (t) upstream of slpA.The reporter gene was the B-lactamase (bla) of pUCI9.The secretion vector (pKTH2I2I, see Fig. 2) was constructed stepwise by PCR (Savijoki et al. 1997).
To analyse the expression and secretion of Bla with the slpA cassette, we first transformed L. lactis (MG 1614) with pKTH2I2I.The trans- formants kept the vector stabile and efficiently secreted B-lactamase into the culture medium.The utility of the cassette in other LAB was con- firmed by transferring pKTH2I2I into L. brevis (ATCCB2B7), L. plantarum (NCDO 1193), L.  gasseri (NCK 334) and L. casei (ATCC 393) hosts and studying the expression and secretion of 6-lactamase as a function of growth in flask cultivations.In each strain carrying pKTH2I2I, all detectable Bla activity was in the growth medium.The highest yield (10240 U/ml; 50 mg Bla/1) in the culture supernatants was obtained with L. lactis at the early stationary phase.The highest production level of Bla in the early sta- tionary phase L. brevis cells and in the exponen- tial phase L. plantarum cells was 60% and 30%, respectively, of that in L. lactis (Savijoki et al.  1997).The rate of Bla production was roughly equal in L. lactis and L. plantarum, whereas that of L. brevis was somewhat slower.In all strains studied, we observed degradation of 6-lactamase due to proteolysis.With L. plantarum, L. gas- seri and L. casei the Bla activity was already much lower at the early stationary phase, suggesting higher protease activity in these strains.Comparison of the activity and amount of Bla protein by Western blots revealed a good corre- lation and lack of cell associated 6-lactamase.The size of Bla secreted to the culture medium was equal to that of the mature Bla of E. coli, suggesting that the enzyme was correctly proc- essed.The L. brevis slpA promoters were very efficiently recognized in L. lactis, L. brevis and L. plantarum, whereas in L. gasseri, the slpA promoter region appeared to be recognised at a lower level and in L. casei the level of transcripts was below the detection limit (Savijoki et al.  1997).Furthermore, high integrity and the cor- rect size of bla mRNA were demonstrated in all these species except L. casei.  region, the transcription terminators (r and t shA ) and the E. coll B-lactamase (bla) gene were isolated by PCR amplifications and joined stepwise to form the final t-P shA -SS sl A -bla-t sl A cassette, which was then ligated with pKTH2O9S to result in pKTH2I2I.The nucleotide and the corresponding amino acid sequences of the P rf A -SS.A -bla joint region of the secretion construct are shown, and the signal peptide cleavage site is indicated by a vertical arrow.The reporter gene region is underlined.B. The expression cassette consisting of the L. brevis slpA promoter region and the reporter gene (gus, luc and pepN) with the nucleotide and the corresponding amino acid sequences of the joint region.The genes, which were isolated using PCR, contained a sequence coding for the mature part of the polypeptide.The reporter gene region is underlined and the amino acid residues derived from the respective restriction enzyme recognition sites are marked with dotted lines.
To improve the stability and production of B-lactamase, we grew L. lactis in a bioreactor at constant pH and glucose feeding (Fig. 3).Under such conditions the yield of Bla could be increased up to 80 mg/ 1 (Savijoki et al. 1997).After maximum activity was reached (10 after the cell density A 600 =l) the level of 6-lactamase was kept stabile at the stationary phase of growth, indicating stabilisation of Bla activity with the pH control and glucose.Comparison of yields ofB-lactamase production in lactococci revealed that a hybrid expression-secretion unit consist- ing of a strong cytoplasmic lactococcal promoter and an indigenous lactococcal signal sequence (Koivula et al. 1991,Sibakov et al. 1991) resulted in only 14% of the Bla activity obtained with the slpA-based secretion system.This finding supports the high efficiency of the slpA expression and secretion signals.The kinetics of 6lactamase accumulation shows that the Bla-production is basically restricted to the exponential phase of growth.Sixty-five per cent of the max- imum Bla activity was reached within 2 h of the cell density A 6oo =l (Fig. 3), implying a very high rate of secretion with a calculated value of 5 x 10 5 molecules/cell/h.This value is comparable to or exceeds the best exoenzyme-producing lab- oratory strains of Bacillus (Simonen and Palva  1993), and thus suggests utility of lactococci as production hosts.However, lengthening the ef- fective duration of the production phase to raise product yields requires either optimization of growth or use of immobilized cell systems.

Intracellular protein production with the slpA signals
To analyse the utility of the slpA promoters for intracellular protein production, we constructed three reporter gene cassettes, 6-glucuronidase (gusA ), luciferase ( luc ) and aminopeptidase N (pepN ), in the pKTH2O9S based vector under the slpA PI and P 2 promoters (Fig. 2b) (Kahala and   Palva 1998).The expression of reporters was studied in three different lactic acid bacteria hosts, L. lactis, L. plantarum and L. gasseri, as a function of growth.The slpA promoters were identified in each strain but significant variations in GusA, Luc and PepN activities at both tran- scriptional and product yield levels were evident in the different strains.The highest levels of GusA and Luc activities were obtained in L. lac- tis, which produced, for example, GusA up to 15% of total cellular proteins.The highest level of PepN activity, 28% of total cellular proteins, was achieved in L. plantarum (Kahala and Palva  1998).The slpA promoters thus have significant potential for future use in protein production in different LAB.The utility of the slpA signals, is, however, strain and production gene dependent and must be tested separately for each gene and host of interest.Closed and open circles show the Bla activities and cell densities as a function of time.Cells were grown in double strength M l 7 media containing 2% glucose (2xM 17G) and in MRS broth.Propagation was at 30°C in a bioreactor (Biostat®B, Medical Brown) with gentle (100 rpm) stirring and without aeration.Glucose was added in amounts re- quired to maintain the final concentration of 2%, and the pH of the culture was adjusted to 5.5 by 1 N NH 3 Samples were taken at different times up to 22 h, and the superna- tant fractions were analysed.
In the light of present knowledge, we can expect that lactobacillar S-layers will be further developed for different biotechnological applications.One obvions application of the L. brevis S-protein currently being studied is as a carrier vehicle of foreign antigenic epitopes.Further elucidation of the adhesive properties of the L. brevis S-protein may also allow and extend its utilisation as a carrier of oral vaccines and other substances for animal and human use.Further developments will include the extension of the use of the L. brevis slpA signal for both secre- tion and intracellular protein production to improve fermentation processes.

Fig. 2 .
Fig. 2. Secretion and expression vectors based on the a slpA expression and secretion signals.A. The L. brevis promoter- signal sequence (P sl A -SS d A