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

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

AiriPalva

AgriculturalResearch Centreof^{Finland,FoodResearch, FIN-31600 Jokioinen,}Finland. Current address:Facultyof

VeterinaryMedicine,Hämeentie57,FIN-00014 UniversityofHelsinki, Finland,e-mail:airi.palva@helsinki.fi

Lactic acid bacteria (LAB)areextensively used inthe manufacture ofawide varietyof fermented dairy, meat,vegetable, bakery and wineproducts inthe food and wineindustryas wellasin making silage for animal feed. SomeLAB strains also have ^anincreasingly importantroleas health-promot- ing probiotics. Moleculargeneticresearch ofLAB,focusedmainly onthe basic characterisation of traits essential for the industrial utilisation of thesebacteria,formsasolid scientific basis for stabili- sation, modificationand improvement ofthese characteristics. Theemphasisofthisreview is onthe moleculargenetic work done at the research laboratoryof the author. Our research team isengaged on, two mainprojects: moleculargenetic and biochemical characterisation of theproteolytic systems of industrialthermophiliclactobacilli and surfacelayer proteinstudies todevelop protein production systems for food, feed, vaccine anddiagnosticpurposes.

Key words:aminopeptidase N, B-glucuronidase,cheeseripening, E. coli6-lactamase, in vivo expres- sion ofslpA,luciferase, oligopeptide transport system,peptidases, protein secretion

ntroduction

Lactic acid bacteria (LAB) are adiverse group of micro-organisms inhabiting various ecologi- calniches, from plant surfaces tothe gastroin- testinal,genital and respiratorytractsofmanand animals (De Vuyst and Vandamme 1994, Wood 1992). LAB are also widely usedas starters in the manufacture of fermentedfoods, beverages, pickled vegetables and silage(DeVuyst and Van- damme 1994).Infermentation,several metabolic properties of LAB serve special functions with

adirectorindirect impactonfood 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 etal. 1995).Some milk products fer- mented with probiotic LAB may also have health and additional nutritionalbenefits,and theseare increasingly being subjected _to R+D (Jensen 1995, Wood 1992).The proposed key _targetsof the health-promoting effects of probiotic ^LAB are the prevention of intestinal infections,diar-

©Agriculturaland Food Science in Finland Manuscriptreceived December 1997

Voi7⁽¹⁹⁹⁸ V 267-282.

rhoeal diseases and upper gastrointestinaltract diseases, the prevention ofcancer and the hy- percholesterolemia, the improvement of lactose utilisation and the stabilisation of the _{gut mu-} cosal barrier (Kailasapathy and Rybka 1997, Wood 1992, Salminenetal. 1996).

The loosely defined group of LAB includes Gram-positiverods and cocci with low G+Ccon- tent^<50 mol% _{(Pot et}al. 1994). LAB arecata- lase negative, non-sporulating (except Sporol- actobacillus), acid- and aerotolerant anaerobic bacteria that produce lactic acid as a major or sole end-product of obligative fermentativeme- tabolism(Pot etal. 1994, Wood and Holzapfel

1995).Taxonomically LAB have been included in the generaLactobacillus, Lactococcus, Leu- conostoc, Pediococcus, Carnobacterium, Sporo- lactobacillus, Streptococcus,Enterococcus,Ae- rococcus, Vagococcus, Tetragenococcus and Atopium (Pot etal. 1994, Wood and Holzapfel 1995); This classificationis, however,notfinal.

Many characteristics typical of genuine LAB are

also shared by the genusBifidobacterium, ^which consists of increasingly important health-pro- moting intestinal bacteria (Kailasapathy and Rybka 1997).

Bifidobacterium

^{was considered}

tobe _oneof theLAB,but duetotheir high G+C content(55-67 mol%) and _on the basis of 16S rRNAdata, it is nowclear that the bifidobacte- ria belongto the actinomyces branch in the phy- logenetictreesof Gram-positive bacteria (Schlei- fer and Ludwig 1995).

Owingto the great diversity ofLAB, espe- cially amongoneof themostwidely used groups, i.e.Lactobacillus, molecular genetic studies of LAB arevery demanding. However,since being of such importance in the food industry, LAB, particularly thestarters used in the dairy prod- ucts, have been subjected ^toextensive molecu- lar genetic research for many years. The objec- tives of that research have been^tocharacterise, stabilise and improve traits essential for food processes (for recentreviews, see de Vos etal.

1993, Venema et al. 1996). Much is already known about the molecular biology of ^starter lactococci but during the last few years,a con- siderableamount of information has also been

gatheredonthe various lactobacillar species. In additiontothe development of genetictools, the main research targetshave 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 preser- vation,bacteriophages and bacteriophage resist- ance,and its gene expression and control (de Vos etal. 1993, Gasson and de Vos 1994, Venemaet al. 1996).We aregradually accumulating avery solid and promising basis for the further devel- opmentof better and safer food products, possi- bly 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 asoral vaccine carriers hasgot offtoapromis- ing start (Wells etal. 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 biotechnologycanhave on the health and nutrition of humans and ani- mals.

This review summarises, in the context of relevant research, some of the contributions made by ourLAB research team to effortsto establishabasic knowledge base for future food and health-promoting applications. In the first part of the review I shall examine thestatusof our work on the molecular genetic characteri- sation of lactobacillar proteolytic _systems.The proteolytic systems of LAB are considered^to have anessential impactoncheese ripening and the formation of flavour and texture in cheese.

The new informationnow available oncheese ripening and the exactrole of proteolytic and peptidolytic enzymes in the process will aid the development of low fat cheeses with a better health-affectingstatus.Furthermore, it will be possible tousethe characterised enzymes of the proteolytic systems in the targeted production of bioactive peptides, e.g. from milk proteins.

In the secondpart 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 pro- tein productionsystemsdeveloped for LAB are still rather unsophisticated, and the factors af- fecting protein production, secretion in partic- ular,poorly characterised compared with those established for other pro- and eukaryotic organ- isms. Furthermore, the utility of the S-protein itselfas acarrier of vaccine and diagnostic an- tigens is akey targetofcurrent and forthcom- ing research.

Molecular genetic characterisation of ^components of the proteolytic

systems ^of Lactobacilli

General background

Many LAB isolated from milk productsaremul- tiple amino acid auxotrophs (Chopin 1993).In milk, the abundances of free amino acids and short peptides are very low. To growin milkLAB are therefore highly dependenton theirproteo- lyticsystem.The biochemical and genetic char- acteristics of the proteolyticsystemin lactococ- ci have been extensively studiedtoascertain 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 pro- teinase (PrtP), which degrades casein into oli- gopeptides; ii) amino acid and peptidetransport

systems, which supply the degraded organic ni- trogen source ^to the cell by translocating the breakdown products of casein across the cyto- plasmic membrane; and iii) many different in- tracellularexo- and endopeptidases, which fur-

ther cleave oligopeptides into shorter peptides and amino acids (Kunji etal. 1996, Fig. 1).Mo- lecular characterisation of lactobacillarproteo- lyticsystems, mainly ofLactobacillus delbrueck- ii ssp. lactis,Lactobacillus helveticus and Lacto- bacillus paracasei,eventhough it_gotunderway

several years after that oflactococci, has made rapid progress. We now have large knowledge base on these bacteria too, and it has become clear thataclose overall similarity exits between the lactobacillar proteolytic systemsand the lac- tococci (Kunji et al. 1996). Due to their high peptidolytic activity, lactobacilli,L. helveticus in particular, have been used in various forms toreduce bitterness, improve flavour develop- mentand accelerate the ripening of varioustypes of cheese in processes conventionally based on the useof other starterstrains (Ardö and Lars- son 1989, Bartels etal. 1987a,_{b,El Soda} 1993).

Thus,genetic characterisation of thecomponents of the lactobacillar proteolytic systemswill en- able usto develop more precise modifications of starterbacteria 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 lac- tis subsp. cremoris strains, twoLactobacillus casei, oneLactobacillus delbrueckii subsp bul- garicus and three L. helveticus strains, and the substrate specificities of lactococcal ^Prts have been described in detail (Kunjietal. 1996).Ami- noacid and peptidetransport systemshave been studied mainly inlactococci,for which 10 ami- no acidtransport systems have been found but not yet cloned. The lactococcal di/tripeptide transporters and the oligopeptide transportsys- tems have been extensively analysed and even genetically modified (Kunji et al. 1996). The

mostextensive molecular characterisation work has, however,been doneonpeptidases. Thusfar, dozens of peptidases have been analysed, from Lactococcus lactis subsp. cremoris strains, L.

delbrueckii subsp. lactis,L. helveticus and oth- er LAB, including atleast 12 peptidases with distinct substrate specificities (see Kunji et al.

1996).

Vol. 7(1998):267-282.

The characterisation of lactobacillar proteolytic systems

Our laboratory has sought^tocharacterise the key

components ofandeventsin the proteolytic sys-

temof thermophilic industriallactobacilli,with special emphasis onL. 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 pepti- dolytic characteristics. During thepastfive years we have completed the molecular genetic char- acterisation of six L. helveticus andtwoL. del- brueckii subsp. bulgaricus peptidases. We are currently engaged in cloning and sequencinga novel _type of PrtP and _a tripeptidase from L.

helveticus(data not shown).

Characterisation of these components has allowedusto: i) initiate construction of modi- fied lactococcal_starterstrains(anEU-BIOTECH II Starlab project);ii)analyse the regulation and interactions ofthe 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 withan industrial_partner; and iv)test the overproduction of individual pepti- dases.

Cloning the oligopeptide transport system from L. delbrueckii subsp. bulgaricus will allow ustocompare thespecificity and capacity of the transport systemsof different LAB.

To control cell lysis, and thustorelease pepti- dases, wehave isolated and characterisedastress (e.g. NaCl) induced _promoter from L. helveti- cus(Smedset al.,unpublished). In the^next sec- tions I shall look atthe results wehave already published _orsubmitted for publication.

General arninopeptidases ^N, C ^{and D} of L helveticus 53/7

Arninopeptidasesarecapable of cleaving N-ter- minal amino acids from a wide variety of pep- tides differing in both size and composition. The general arninopeptidases _wehave characterised from L. helveticus 53/7 include PepN and PepC, which hydrolyse longer peptides, andadipepti- dase (PepD).

Fig. 1.^Schematicpresentationof theproteolyticsysteminlactic acid bacteria. (DrawnbyKirsiSavijoki).

We (Varmanen etal. 1994) cloned the ami- nopeptidase N gene using nucleic acid hybridi- sation_to detect the pepN gene from aL. helveti- cus53/7 genomic library, withafragment of the putative pepN gene from L. helveticus CNRZ32 (Nowakowski etal. 1993)asthe probe. A 3.7 kb hybridisation positive insert directing ami- nopeptidase activity in E. coli_was sequenced and found tocontain only one open reading frame (ORF) of2532 bp. This ORF hada coding ca- pacity fora95.8 kDa protein that corresponded to the size of the lactococcal (Tan et al. 1992) and L. delbrueckii subsp. lactis PepNs (Kleinet al. 1993).The deduced amino acid sequence_was 49% homologous ^toLactococcus lactis PepN, and showed 99% identity to the L. helveticus CNRZ32 pepN gene, sequenced ^later, (Chris- tensen etal. 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 withtwo transcriptionstart sites. Thesetwosites verified the existence of^twoputative overlapping pro-

moterregions in the DNA sequence. Our stud- ies onpepN expression as a function of growth in abioreactor showed that pepN transcriptsre- mained at a high level during the stationary growth phase,too, incontrast tothe steady-state levels of all other peptidase mRNAs wehave analysed. Similarly, the level of total aminopepti- dase activity remained constantthroughout the stationary growth phase. The inhibition profiles of PepN showed that it is indeedazinc-depend-

ent metallopeptidase, being completely inhibit- ed by ethylenediaminetetra-acetic acid (EDTA) whereas serine protease inhibitor phenyl- methylsulphonylfluoride (PMSF) and a thiol- blocking reagent, p-hydroxymercuribenzoate (pHMB), had only _aslight effect_onenzymeac- tivity (Varmanen etal. 1994).Thus, the charac- teristics of L. helveticus PepNarein accordance with PepNs from other LAB (Kunji etal. 1996).

The second aminopeptidasewecharacterised was the PepC (Vesanto etal. 1994).The isola-

tion procedure for the pepC genewas similar_to that for pepN. In a hybridisation positive clone showing aminopeptidase activity the pepC gene could be localised intoa3 kb fragment by dele- tion mapping. Sequencing of this fragment re- vealed two openreading frames (ORFI and ORF2)of1347 and 840 bp. ORFI waspreceded byatypical prokaryotic_promoterregion, andan

invertedrepeat structure with delta G of-49.0 kJmol'¹wasfound downstream of the codingre- gion. The deduced amino acid sequence of ORFI, with_anencoding capacity of_a51.4 kDa protein, shared 48.3% and 98% identity with the PepCproteins from Lactococcus lactis (Chapot- Chartieretal. 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 growthwhereas, atthesta- tionary growth phase, pepC transcripts derived from the pepCpromoter werebelow the detec- tion limit and ORF2 was expressed by its own promoter(Vesanto ^etal. 1994). The 5’ end map- ping of the pepC transcripts revealedatranscrip- tion start different from that suggested for the pepCin the L. helveticus CNRZ32 pepC gene (Fernandezetal. 1994). We also studied expres- sion of pepC in L. helveticusas a function of growth in_abioreactorcultivation,and found that transcription of pepC was typical of the expo- nential growth phase expression. The level of total thiol- aminopeptidase activity, however, remained nearlyconstantthroughout 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 athiol- dependent aminopeptidase belonging to the cysteine proteinase family. This allowed_arough estimation of the contributions of PepN and PepC tothe total aminopeptidase activity in L. helve- ticus. Addition of EDTA orEDTA andpHMB to L. helveticus cell lysates in the presence ofLys- p-nitroanilide (pNA) substrate reduced the ac- Vol.7(1998): 267-282.

tivity to 10% and tobelow the detectionlimit, respectively, indicating that the aminopeptidase activity in L. helveticus is mainly due to the metalloaminopeptidase (Varmanenetal. 1994).

Comparison of the level ofpepN and pepCtran- scripts _supportsthe conclusion that PepN is the main aminopeptidase in L. helveticus ^(datanot shown). Our further studieson ORF2 have re- vealed that it codes foratransmembrane protein homologoustoanunknown Bacillus protein that is expressed during sporulation (Vesanto and Palva, unpublished results).

We isolated L. helveticus 53/7 dipeptidase gene(pepD⁾in thesame wayaspepN and pepC (Vesanto et al. 1996).An open reading frame (ORF)of1422 bp wasidentified from_apositive 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 kDaprotein sharedno sig- nificant 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 ofprokaryotictran- scriptsweoptimisedamethod basedontheuse ofan automated DNA sequencer^{(Vesanto et}al.

1996).The 5’ end mapping of the 1.6 kb ^pepD transcript by the newand conventional primer extension methods gave consistent results. Ex- pression studies showed that pepDwasmaximal- ly expressedatlate exponential growth. We also overexpressed the pepD gene in E. coli and pu- rifiedPepD to homogeneity in three chromato- graphicsteps. Studies of the PepD substrate spe- cificity showed that PepDwasable tohydrolyse a number of dipeptides with the exception of those containing proline residues. Optimal PepD activity_wasobtainedatpH 6.0 and 55 °C. Inhi- bition studies of PepD revealed that ^theenzyme could be inhibitedbypHMB and reactivated by dithiothreitol; EDTA, incontrasthad _noinhibi-

toryeffect(Vesanto et al. 1996). In addition to the lack of a homologous counterpart among LAB, the enzymatic properties suggested that thetwo 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 _asweetfla- vour^tocheese(Biede and Hammond 1979, Fox

1989). Proline-specific peptidases thereforeplay animportant role in cheese ripening by degrad- ing proline-containing peptides, whichare some- timesbitter, 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 dipep- tidyl aminopeptidase (PepX) (Vesanto et al.

1995),prolinase (pepß) (Varmanen etal.

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 hy- drolysing the peptide bond atthe carboxyl side of the proline residue when this imino acid is the penultimate N-terminal residue. The pepX gene cloned andsequenced from strain 53/7was shownto be 2379 bp in size with acoding ca- pacity for a 90.6 kDa protein (Vesanto et al.

1995).ThepepXgenewasidentifiedas a mono- cistronic transcription unit flanked by atypical 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 etal. 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 mRNAwerein_agreement with the DNA sequence data. Similarly^totheotherL. helveti- cuspeptidases analysed, expression ofpepXwas typical of exponential growth. The pepX gene was also overexpressed in pKK223-3 in E. coli followed by purification of PepX tohomogene- ity by ion-exchange and hydrophobic interaction

chromatography. The enzymewas foundtobea dimer (165 kDa) with optimum activity atpH 6.5 and 45 °C.Furthermore, PepXwasshownto beametal-independent serine peptidase, having functional sulphhydryl groupsator nearthe ac- tive site(Vesanto etal. 1995).Thus, the charac- teristics of the L. helveticus PepX closelyresem- bled those of other PepXs.

Prolinases cleave dipeptides withaproline residueatthe N-terminal amino acid. We cloned aprolinase (pepß) from strain 53/7 withagene probe (Nowakowski etal. 1993) specific for a peptidase showntohave activity against di- and tripeptides(Varmanen etal.

1996

a). The hybrid- isation positive clonesobtained, however,didnot show any enzyme activity against di-ortripep- tides. Further analysis ofoneof the clones with a 5.5 kb insert revealed two ORFs, of912 and 1602 bp, ORF2, located upstreamof and oppo- site in orientationtoORFI, hada promoter re- gion overlapping that of ORFI. ORFI hada co- dine capacity fora 35 kDa protein. This protein was showntobe capable of hydrolysing dipep- tides when ORFI _was amplified by PCR with its control regions and expressed ⁱⁿ E. coli.

Among the dipeptides^tested,the highest activi- ty wasobtained with_aPro-Leu substrate where- as among the tripeptides ^tested, only Leu-Leu- Leuwas marginally degraded.Thus, the35 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 sequencedata,North- ern and primer-extension analyses of ^pepß showed a 1.25 kb transcript and two adjacent transcription startsites,respectively. Pepß pro- tein was found^to be99.6% identical^tothere- cently 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. Inter- estingly, analysis of theupstreamORF2 revealed that the deduced 59.5 kDa protein encoded by this gene showed significant homologytosever- al members of the family of ABC transporters.

Deletion constructsof ORF2 also clearly dem- onstrated that this upstream operon adversely affected Pepß activity in E. coli^, explaining the enzymatic inactivity of the original clones (Var- manen etal. 1996^a).

Proline iminopeptidases (Pepl) areable 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 etal.

1996

^{b), was foundto}be organised inanoperon-likestructureof three ORFs. ORFI waspreceded byatypical prokaryotic promoter

region, and a putative transcription terminator identifiedas the pepl gene was present down- stream of ORF3. Primer extension analyses on each ORF revealed only onetranscription start site, upstream ofORFI, indicating _an operon structure.The level of operon derived transcripts was solow thatwecould^not determine thetran- script size by Northern blot; nevertheless the RT-PCR clearly supported the operonstructure

of these three genes. The coding capacities of ORFI,ORF2 and ORF3 _werefor 50.7,24.5 and 33.8 kDa proteins, respectively. The ORF3-en- coded Pepl protein showed 65% identity ^tothe Pepl proteins from L. delbrueckii subsp. hulgari- cus(Allan_etal. 1994)and L. delbrueckii subsp.

lactis (Klein etal. 1994). The pepl gene was overexpressed in E. coli and purified tohomo- geneity in_twochromatographic_steps(Varmanen etal. 1996^{b).Peplwasshowntobeadimer with} optimum activity ^atpH7.5 and ⁴⁰ °C. Like the L. delbrueckii Pepls, the L. helveticus Pepl was foundtobeametal-independent serine peptidase with thiol group ator nearthe active site. Kinet- ic studies with PropNAassubstrate revealed K_m and V_max values of O.BmM and350 mmol min 1 mg ^', respectively and a very high turnover number, 135 000s'.The substrate specificities of the three Pepls identified differed from each other to some extent (Varmanen etal. 1996^b), but wedo notknow whether these differences were dueto assay conditions ortheywere true differences in the enzymatic properties of these Pepls.

The ORFI and ORF2 encoded proteinswere found to share homology with the members of Vol. 7(1998): 267-282.

the ABC(ATPbinding casette)transporterfam- ily but to represent an unusual _type (ORF1) among the bacterial ABCtransporters(Varmanen etal. 1996^b).

Proline specific peptidases of L delbrueckii subsp. bulgaricus

Due tothe importance of proline-specific pepti- dases we have also characterised two genes, pepQ andorfZ,^encodingaprolidase (PepQ) and aPep

Q-like

protein from L. delbrueckii subsp.

bulgaricus(Rantanenand Palva 1997).ThepepQ and

orfZ

genes showed 98% and 60%identity, respectively to the L. delbrueckii subsp. lactis pepQ; both pepQ and

orfZ

^werepreceded by _a putative _promoter region. The size of pepQ mRNA could be identified(1.1 kb) but, under the growth conditionsused,wecould only iden- tify the expression of

orfZ

by RT-PCR. Both geneswereshown^tobe monocistronic transcrip- tional 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 andL.helveticus(Rantanenand Palva 1997).The role of the cryptic

orfZ

gene and itsputative gene product remains tobe established.

Oligopeptide transport system of L delbrueckii subsp. bulgaricus

As wellasreforming peptidase analyses,wehave started the characterisation ofothercomponents of the proteolytic ^systemsofthermophilic lacto- bacilli. We have isolated the operon of the oli- gopeptidetransport system from _alambda gtlO based genomic library of L. delbrueckii subsp.

bulgaricus (Peltoniemi etal. 1998 unpublished results).This operon is 6.1 kb in size andcon-

sists of five genes encoding the peptide binding protein (OppA),twointegral membrane proteins (Oppß and OppC) andtwoATP-binding proteins (OppD and OppF). In L. delbrueckii subsp. hul- garicus, the opp genes in the operon areorgan- ized in much the same way as in Lactococcus lactis (Tynkkynen etal. 1993),i.e. oppDFBCA.

Interestingly, _an additional

opp A

-like gene is adjacent_tothe oppA of the operon. The identity of the oppDFBCA tothat of L. lactiswas shown to be 50%, 65%, 55%, 40% and 40%, respec- tively (Peltoniemi etal. 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 theover300 S-layer harbouringeu- 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-layersare^most-

ly unknown, but apparently thisstructure is es- sential since all attempts toinactivate sip genes have failed(Boot 1996).TheL. crispatusS-pro- tein has been showntomediate adhesion totype

IV collagen (Toba et al. 1995).

Foranaverage sizecell, 5 x 10⁵S-layer sub- units have tobe synthesised per cell generation in ordertocover the entire cell surface with the

S-layerproteins(Messnerand Sleytr 1992).The expression ofa sip gene and the secretionma- chinery ofan S-layerharbouring cell may thus be expected to be very efficient. These proper- ties_areobviousassetsin the utilisation of S-pro- teins in biotechnological applications. We chose

tostudy heterologous protein production in lac- tic acid bacteria, with the L. brevis slpA and ^to testthe possibility of using SlpA as acarrier for foreign antigenic epitopes. L. brevis isahetero- 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 brieflyatchar- 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 analy-

sis, which revealed only one majorband, with an apparentmolecular weight of46 kDa (Vidgrén etal. 1992).When the cellsweretreated withan antiserum raised against the isolated 46 kDa pro- tein and analysed by immuno-gold electron mi- croscopy,_post embedding immunoelectron mi- croscopyclearly showed that the 46 kDa protein was heavily concentrated in the outermostpart of the cell wall of L. brevis cells (Vidgrénatal.

1992).The slpA genewas PCR cloned accord- ing to the N-terminal sequence information of the intact S-protein and internal tryptic peptides (Vidgrénetal. 1992).

The L. brevis slpA gene is 1395 bp in size witha coding capacity for aprotein of 48 159 daltons. The first90 nucleotides of thestructur- al gene encode atypical Gram-positivetypesig- nal peptide of 30 amino acid residues (Vidgrén etal. 1992).The slpA gene is preceded by awell

conserved ribosome binding site^(RBS)andtwo promoter regions, PI and

P 2 with

^{the -35 and}

-10 regions resembling the conserved prokary- otic-35 and -10_consensus sequences(vonHei- jne 1987).Astrong transcription terminatorse- quence is present downstream of thetwo trans- lationstopcodons of slpA.

At the time the basiccharacterisation of the L. brevis S-protein and slpA waspublished, data bases contained nogenuinely related SlpA se- quences (Vidgrénet al. 1992). The predicted amino acid sequences of the recently described L. acidophilus (Bootetal. 1993) and L. Helveti- ans (EMBL Nucleotide Data Library: X91199 andX⁹²⁷⁵²⁾slpA genes,however, show 35.7%

(17)and28.8% similaritytothat of the L. brevis S-protein, respectively. Furthermore, L. brevis slpA probes hybridisestothe chromosomal DNA ofL. buchneri^{(Palva et}al. 1992).Two sip genes with phase variation have been found in L. aci- dophilus, and_onefunctional sip gene andatrun- cated sip 3’end in L. helveticus(Boot 1996).In L.brevis, incontrastonly_onesip gene ispresent (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 withtwo5’

ends located immediately downstream of thetwo -10 regions deduced from the DNA sequence, thus confirming that slpA isamonocistronictran- scriptional unit and possesses two functional promoters(PI,

P

2) (Vidgrénetal. 1992). Deter- mination of the stability of the slpA mRNA showed that the half- life of the slpA transcripts was 14 minutes (Kahala etal. 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 shownto be very stable(Boot etal. 1996, Chu et al. 1993). The long half-lives of these three S-layer mRNAs from three different spe- cies may indicate that_ahigh stability of mRNA is ageneral feature of S-layer mRNAs. As slpA Voi7^(1998): 267-282.

mRNA mediates the synthesis ofamajor_struc- turalcomponentof thecell,this high stability is not unexpected.

Study of the usage of thetwoL. brevis slpA promoters ^(PI,

P

^{2) at different stages of the}

growth revealed that the

P 2

^{promoter,whichwas}

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 bothpromoters are present throughout the entire growth phase and that the level oftranscripts derived frompromot- er

P 2 is

^ten times higher than that derived from PI (Kahala etal. 1997).

S-layer synthesis

In vivo expression studies of L. brevis showed that the kinetics of the accumulations of slpA mRNAand protein correlates well up^to theon- setof the stationary phase, when there isasharp decrease in the level of slpA mRNA. The^rateof mRNA decaywas,however,slower thanexpect- ed from the half-life of slpA transcripts, sug- gesting that residual transcription continueseven though the total amount of S-protein does not further increase atthe stationary phase(Kahala etal. 1997).The L. brevis S- layer protein is not released into thesupernatantfractions atany of the growth phases studied, suggesting tight reg- ulation of the S-layer synthesis and assembly (Vidgrénetal. 1992, Kahalaetal. 1997). Breit- wieseretal. (1992)demonstrated the presence of substantialamountsof S-layer subunits_onthe inner surfaceor 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 noL. brevis S-layer subunitsac- cumulated inside the peptidoglycan layer before translocationto the^outer surface.

Heterologous protein secretion with

the sIpA signals

To^constructasecretionvectorbased_on the slpA signals,_weused_aderivative (pKTH2O9S) of the shuttle vector pGKI2 (Kok et al. 1984) as the carrier of the secretioncassette. The modelse- cretion 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)upstreamof slpA. The reporter gene was the B-lactamase (bla) of pUCI9. The secretion ^vector (pKTH2I2I, see Fig. 2)wasconstructed stepwise by PCR (Savi-

jokietal. 1997).

To analyse the expression and secretion of Bla with the slpAcassette, wefirst transformed L. lactis(MG1614)with pKTH2I2I. The trans- formants kept the vectorstabile and efficiently secreted B-lactamase into the culture medium.

The utility of thecassettein 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 atthe early stationary phase. The highest production level of Bla in the earlysta- tionary phase L. brevis cells and in the exponen- tial phase L. plantarum cells was60% and 30%, respectively, of that in L. lactis (Savijoki etal.

1997).Therate of Bla production wasroughly equal in L. lactis and L. plantarum, whereas that of L. breviswas somewhat slower. In all strains studied, weobserved degradation of 6-lactamase due toproteolysis. With L. plantarum, L. gas-

seri and L. casei the Bla activity was already much lower atthe early stationary phase, sug- gesting higherprotease activity in these strains.

Comparison of theactivity and amount of Bla protein by Western blots revealeda goodcorre- lation and lack of cell associated 6-lactamase.

The size of Bla secretedtothe culture medium was equal to that of thematureBla 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 appearedtobe recognised ata lower level and in L. casei the level of transcripts was below the detection limit (Savijoki et al.

1997).Furthermore, high integrity and thecor- rectsize of bla mRNA weredemonstrated in all these species exceptL. casei.

Fig. 2.^Secretionand expressionvectors based ontheaslpA expressionand secretionsignals. A.TheL.brevis promoter- signalsequence (P_{sl A}-SS

dA)region, the transcription terminators (r and t_shA⁾and theE. collB-lactamase (bla) genewere isolatedbyPCRamplificationsandjoined stepwiseto form the final t-P_shA-SS_slA-bla-t_slAcassette, whichwasthen ligated withpKTH2O9S toresult inpKTH2I2I.The nucleotide and thecorrespondingamino acid sequences of the^PrfA-SS.A-bla joint regionof the secretion constructareshown,and thesignal peptide cleavagesite is indicatedbyavertical arrow.The reporter generegion is underlined.B.The expressioncassetteconsistingof theL. brevisslpA promoterregion and the reporter gene (gus, luc and pepN) with the nucleotide and thecorresponding amino acid sequences of thejoint region.The genes, whichwereisolatedusing PCR, containedasequence codingfor the mature part of thepolypeptide. The reporter generegion is underlined and the amino acid residues derived from therespectiverestriction enzymerecognition sitesare marked with dotted lines.

Vol.7(1998): 267-282.

To improve the stability and production of B-lactamase, wegrewL. lactis inabioreactorat constantpH and glucose feeding (Fig. 3). Under such conditions the yield of Bla could be in- creased up to 80 mg/ 1 (Savijoki ^et al. 1997).

After maximum activity was reached (10 after the cell density A₆₀₀=l) the level of 6-lactamase waskept stabileatthe 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 ahybrid expression-secretion unit consist- ing ofa strongcytoplasmic lactococcalpromot- erandanindigenous lactococcal signal sequence (Koivula^etal. 1991,Sibakov^etal. 1991)result- ed in only 14% of the Bla activity obtained with

the slpA-based secretion system. This finding supports the high efficiency of the slpA expres- sion and secretion signals. The kinetics of 6- lactamase accumulation shows that the Bla-pro- duction is basically restrictedto the exponential phase of growth. Sixty-five per^centof the_max- imum Bla activitywasreached within2 h of the cell density A_6oo^=l(Fig. 3),implying averyhigh rate of secretion witha calculated value of5 x 10⁵ molecules/cell/h. This value is comparable toorexceeds the best exoenzyme-producing lab- oratory strains of Bacillus (Simonen and Palva

1993), and thussuggests utility of lactococci as production hosts. However, lengthening the ef- fective duration of the production phasetoraise 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,weconstructed three reporter gene cassettes, 6-glucuronidase (gusA^), luciferase ⁽luc⁾ and aminopeptidase N (pepN^),in the pKTH2O9S based vectorunder the slpA PI and

P 2

^promoters(Fig. 2b) (Kahala and Palva 1998). The expression ofreporters was studied in three different lactic acid bacteria hosts,L. lactis^,L. plantarum and L. gasseri, as afunction of growth. The slpApromoters were identified in each strain but significant variations in GusA,Luc and PepN activities atbothtran- scriptional and product yield levels_wereevident in the different strains. The highest levels of GusA and Luc activitieswereobtained 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, wasachieved in L. plantarum (Kahalaand Palva 1998).The slpApromoters thus have significant potential for futureuse in protein production in different LAB. The utility of the slpA signals, is, however, strain and production gene depend- entand mustbe tested separately for each gene and host of interest.

Fig. 3.^Secretionof 6-lactamase underpHcontrolin L.lac- tis MG1614 carrying the pKTK2I2I secretion vector.

Closedand open circles show the Bla activities and cell densitiesasafunction of time. Cellsweregrownindouble strengthMl 7mediacontaining 2%glucose(2xM 17G) and in MRSbroth. Propagation was at30°Cinabioreactor (Biostat®B,Medical Brown) withgentle⁽¹⁰⁰rpm) stirring and without aeration. Glucosewas added inamountsre- quiredto maintain the final concentration of2%,and the pHof the culturewasadjustedto5.5 by 1 N NH3Samples weretaken at different times up to22 h,and the superna- tantfractionswereanalysed.

In the light ofpresentknowledge, we can expectthat lactobacillar S-layers will be further developed for different biotechnological appli- cations. One obvions application of the L. brevis S-proteincurrently being studied is as acarrier vehicle of foreign antigenic epitopes. Further elucidation of the adhesive properties of the L.

brevis S-protein may also allow and extend its utilisationas acarrier 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 im- prove fermentation processes.

Acknowledgements. The author isgratefultoM.Sc. Kirsi Savijokiand M.Sc. Minna Kahala for drawing the figures.

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