Metabolically engineered lactic acid bacteria and means for providing same

FIELD OF INVENTION

[0001] The present invention pertains to the field of lactic acid bacterial starter cultures which are useful in the production of food products, animal feed or aroma compounds, and specifically there is provided means for metabolically engineering such lactic acid bacteria which are thereby modified in their production of metabolic end products including aroma or flavour compounds and/or compounds having antimicrobial effects.

TECHNICAL BACKGROUND AND PRIOR ART

[0002] Lactic acid bacteria are used extensively as starter cultures in the food industry in the manufacture of fermented products including milk products such as e.g. yoghurt and cheese, meat products, bakery products, wine and vegetable products. Lactococcus lactis is one of the most commonly used lactic acid bacteria in dairy starter cultures. However, several other lactic acid bacteria such as Leuconostoc species, Lactobacillus species and Streptococcus species are also commonly used in food starter cultures. In the art, species of the obligate anaerobic bacteria belonging to Bifidobacterium which are taxonomically different from the group of bacteria generally referred to as lactic acid bacteria, are frequently included in the group of lactic acid bacteria due to their application as dairy starter cultures. Lactic acid bacteria are also commonly used as inoculants in feedstuffs of plant and animal origin, i.a. for preservation purposes.

[0003] When a lactic acid bacterial starter culture is added to a substrate including milk or any other food or feed product starting material under appropriate conditions, the bacteria grow rapidly with concomitant conversion of lactose or other sugars to lactic acid/lactate and minor amount of acetate resulting in a pH decrease. In addition, several other metabolites are produced during the growth of lactic acid bacteria. Among these metabolites, diacetyl is one essential flavour compound which is formed during fermentation of the citrate-utilizing species of e.g. Lactococcus, Leuconostoc, and Lactobacillus. Diacetyl is formed by an oxidative decarboxylation (R1, FIG. 1) of α-acetolactate which is formed from two molecules of pyruvate by the action of α-acetolactate synthase (R2, FIG. 1).

[0004] Pyruvate is a key intermediate of several lactic acid bacterial metabolic pathways including the citrate metabolism and the degradation of lactose or glucose to lactate. The pool of pyruvate in the cells is critical for the flux through the pathway leading to diacetyl, acetoin and 2,3 butylene glycol due to α-acetolactate synthase affinity for pyruvate. Overproduction of α-acetolactate synthase in Lactococcus lactis as an approach for increased production of diacetyl has been disclosed by Platteuw et al. 1995.

[0005] An alternative metabolic engineering approach to providing an increased pool of pyruvate in lactic acid bacteria is to block one or several pyruvate degrading pathways. As an example hereof, a Lactococcus lactis mutant defective in the lactate dehydrogenase (R3, FIG. 1) has been disclosed by Gasson et al. (ref. 8, unpublished data, in Platteuw et al. 1995). Under aerobic conditions pyruvate is accumulated in this mutant leading to the formation of increased levels of acetoin and 2,3 butylene glycol. However, formate and ethanol were the major metabolic end products obtained under anaerobic conditions, but the formation of the latter end products in high amounts is generally undesired in fermented dairy products typically being produced under anaerobic conditions.

[0006] The reaction whereby pyruvate is converted to formate and acetyl coenzyme A (acetyl CoA) (R4, FIG. 1) by the action of pyruvate formate-lyase (Pfl) takes place only under anaerobic conditions (Frey et al. 1994). An alternative pathway for the formation of acetyl CoA from pyruvate (R5, FIG. 1) in a lactic acid bacterium is by the activity of the pyruvate dehydrogenase complex (PDC). In contrast to Pfl, the activity of PDC appears to be optimal under aerobic conditions (Snoep et al. 1992). Consequently, the pyruvate pool assumingly will be increased under anaerobic conditions by partially or completely blocking the Pfl activity. As mentioned above, an increased pyruvate pool may in turn lead to an increased flux from pyruvate towards acetoin and diacetyl via the intermediate α-acetolactate. Fermented foods or feed products produced by using a starter culture with reduced Pfl activity therefore may contain an increased amount of diacetyl or other products derived from conversion of α-acetolactate. In contrast, starter cultures with increased Pfl activity should result in enhanced production of the antimicrobially active metabolite formate and the use of such cultures in the production of feed or food products having increased shelf life can therefore be contemplated.

[0007] The pfl gene has been isolated from several microorganisms including Escherichia coli, Haemophilus influenzae, Clostridium pasteurianum and Streptococcus mutans. The Pfl enzyme is post-translationally activated by the Pfl activase via formation of an organic free radical into a glycine residue located at the C-terminal of Pfl (Frey et al. 1994). This modification of Pfl occurs only in the absence of oxygen. Although the activation gene, act encoding the Pfl activase flanks the pfl gene in E. coli, H. influenzae and C. pasteurianum, the act gene is transcribed from its own promoter, and the expression is essentially constitutive (Weidner et al. 1996). In contrast, the pfl expression is induced 12 to 15 fold by anaerobiosis (Sauter and Sawers 1990). The free radical enzyme, i.e. the activated Pfl, is destroyed by oxygen with concomitant fragmentation of the polypeptide chain (ref. 2 in Kessler 1992). However, in E. coli a Pfl deactivase activity has been found which under anaerobic conditions reverts the active radical form to the native non-radical form of Pfl (Kessler et al. 1992). By this activity, Pfl deactivase protects Pfl against being irreversibly destroyed by oxygen.

[0008] The AdhE protein of E. coli has acetaldehyde dehydrogenase activity, catalyzing the conversion of acetyl CoA to acetaldehyde (R6, FIG. 1), and ethanol dehydrogenase activity, catalyzing the conversion of acetaldehyde to ethanol (R7, FIG. 1). Additionally, the E. coli AdhE protein is responsible for the Pfl deactivase activity.

[0009] In the strict anaerobe, Clostridium acetobutylicum an adhE analogue, aad, has been cloned and characterized. However, the presence of Pfl deactivase activity could not be verified for the Aad protein, since no evidence exists for the presence of Pfl in C. acetobutylicum (Nair et al. 1994).

[0010] Lactic acid bacteria including Lactococcus lactis species are facultatively anaerobic organisms like E. coli, indicating that the occurrence of Pfl activase and deactivase activities in these organisms is to be expected. Analysis of the expression of adhE in E. coli has shown an eight fold increase under anaerobic growth (Chen and Lin 1991). The facts that the regulation of expression of pfl and adhE under anaerobic conditions is similar and that expression of act in E. coli is constitutive suggest that an equilibrium is formed between activated and deactivated Pfl under anaerobic conditions. If the deactivase activity of the AdhE protein is partially or completely blocked in lactic acid bacteria, an increased Pfl activity is expected to occur while, on the other hand, a reduced Pfl activity is expected to occur if the deactivase activity is overexpressed. If the Pfl activase is blocked, a decreased Pfl activity is contemplated.

[0011] The acetaldehyde dehydrogenase and the ethanol dehydrogenase activities of the AdhE protein are also potential targets for metabolic engineering in lactic acid bacterial food starter cultures and cultures used in feed production or as cultures for the production of aroma compounds or antimicrobially active compounds. Thus, it can be contemplated that a block or modification of the ethanol dehydrogenase activity of such cultures may result in the overproduction of acetaldehyde which is an important flavour compound in yoghurt. Alternatively, a block of the acetaldehyde dehydrogenase activity could give rise to an increased production of acetate which in turn may result in improved preservation of fermented foods or feed products in whose production such modified cultures are used. Additionally, it is contemplated that such modifications of starter cultures would increase the pyruvate pool and consequently, the formation of diacetyl or other compounds derived from the conversion of α-acetolactate. Increasing one or both dehydrogenase activities will most likely direct the conversion of acetyl CoA from acetate to acetaldehyde or ethanol.

[0012] Based on the above analysis of the potential means of regulating the size of the pyruvate pool in lactic acid bacteria and the intracellular fluxes from this metabolic intermediate pool towards desirable end products, a novel approach has been developed for metabolically engineering lactic acid bacteria allowing the provision of useful lactic acid bacterial starter cultures either having an enhanced production of desirable flavour compounds or an increased production of antimicrobially active compounds which can be used to increase the shelf life of food or feed products.

[0013] In particular, the starting point for the invention is the achievement of the isolation and sequencing of the entire adhE and pfl genes of Lactococcus lactis. Based on these findings, it has become possible, by appropriate modifications of the genes and their expression and/or activity of one or more of the enzyme activities encoded by these genes, to provide in a goal-directed manner lactic acid bacterial starter cultures having the above desirable characteristics, including cultures of strains having reduced or enhanced production of particular metabolites.

SUMMARY OF THE INVENTION

[0014] Accordingly, the present invention provides novel means for metabolically engineering lactic acid bacteria, and lactic acid bacteria being modified by such means. Specifically, the invention relates in a first aspect to an isolated DNA sequence comprising a sequence derived from a lactic acid bacterium, said sequence coding for a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

[0015] In further aspects, the invention pertains to a recombinant replicon comprising the above DNA sequence and to a recombinant lactic acid bacterial cell comprising such a replicon.

[0016] In still further aspects, there is provided an-isolated DNA sequence comprising a sequence derived from a lactic acid bacterium, said sequence coding for a polypeptide having pyruvate formate-lyase activity, subject to the limitation that the sequence is not derived from oral Streptococcus species, a recombinant replicon comprising such a DNA sequence and a recombinant lactic acid bacterial cell comprising such a replicon.

[0017] In another aspect, the invention relates to a method of producing a lactic acid bacterial metabolite, the method comprising cultivating a lactic acid bacterium comprising a DNA sequence as defined above which is modified so as to inactivate or reduce or enhance the expression of at least one of the enzymatic activities selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity, or a lactic acid bacterium comprising a DNA sequence which is modified whereby its production of pyruvate formate-lyase is reduced or inhibited, or whereby the enzyme is expressed in a modified form having a reduced pyruvate formate-lyase activity, or wherein the DNA sequence is modified whereby the expression of pyruvate formate-lyase is enhanced or whereby the enzyme is expressed in a modified form having an increased pyruvate formate-lyase activity, and isolating the metabolite from the culture.

[0018] The invention also pertains to methods of producing a food product or an animal feed, the method comprising the step of admixing to the food product or feed starting materials a starter culture of a lactic acid bacterium according to the invention and keeping the mixture under conditions allowing the starter culture to be metabolically active.

[0019] There is also provided an isolated DNA sequence derived from a lactic acid bacterium, said sequence coding for a product having a formate transporter activity.

DETAILED DISCLOSURE OF THE INVENTION

[0020] The facultative anaerobe Escherichia coli is capable of carrying out mixed-acid fermentation during anaerobic growth in the absence of exogenous electron acceptors. In this connection, a major fermentation product is ethanol which is synthesized from acetyl CoA by two consecutive NADH-dependent reductions catalyzed by a single polypeptide, AdhE, with an acetaldehyde dehydrogenase (ACDH) domain and alcohol dehydrogenase (ADH) domain. It has also been found that this polypeptide is responsible for pyruvate formate-lyase deactivase activity.

[0021] It has now been found that a DNA sequence showing significant homology to the E. coli gene, adhE which codes for a polypeptide showing substantial similarity with the above multi-functional E. coli AdhE polypeptide is present in lactic acid bacteria which are also facultative anaerobes, such as in Lactococcus lactis. It was therefore hypothesized that the gene product of the thus identified and isolated lactic acid bacterial DNA sequence might have similar enzymatic activities as the corresponding E. coli gene. This was found to be the case.

[0022] Accordingly, the present invention provides, as mentioned above, in its first aspect an isolated DNA sequence which comprises a sequence derived from a lactic acid bacterium, which sequence codes for a multi-functional polypeptide having at least one of the following enzymatic activities: (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity. The coding sequence for the multifunctional polypeptide is also referred to herein as the adhE gene, and the polypeptide encoded by the gene as the AdhE polypeptide.

[0023] In accordance with the invention, the DNA sequence coding for the multi-functional polypeptide may be derived from any lactic acid bacterium. In the present context, the term “lactic acid bacterium” designates gram-positive, microaerophilic or facultatively anaerobic bacteria which ferment sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species and Pediococcus species. Additionally, the strict anaerobic Bifidobacterium species, which are commonly used in the manufacture of dairy products, are included in the group of lactic acid bacteria. The group of lactic acid bacteria comprises so-called mesophilic species which have optimum growth temperatures in the range of 15-30° C. and which in many cases do not grow at temperatures exceeding 35-40° C. Other groups of lactic acid bacteria have higher growth temperatures, in particular species for which humans and/or animals are the natural habitat, e.g. Enterococcus species, oral streptococci and pathogenic streptococci.

[0024] In certain preferred embodiments, the above DNA sequence is derived from Lactococcus lactis including Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies diacetylactis (also frequently referred to as Lactococcus lactis subspecies lactis biovar diacetylactis) and Lactococcus lactis subspecies cremoris.

[0025] In useful embodiments of the invention, the lactic acid bacterium-derived DNA sequence codes for a multifunctional polypeptide that is at least 30% identical with the gene products of the adhE gene of E. coli (FASTA, GCG Wisconsin accession No. P17547) or the aad gene of Clostridium acetobutylicum (FASTA, GCG Wisconsin accession No. P33744) or the gene product of the sequence of Table 1.4 herein (SEQ ID NO:3). In other useful embodiments, the identity to such other gene products is at least 40%, such as at least 50%, such as at least 60% identity or even at least 70% identity. The homology between the .above gene products may also be expressed in terms of amino acid similarity in which case the similarity suitably is at least 60%, such as at least 70%, e.g. at least 80% similarity. In this context, the expression “amino acid similarity” indicates that a particular amino acid in a polypeptide sequence can be replaced by another amino acid having similar physical/chemical characteristics such as charge or polarity characteristics.

[0026] The sequence according to the invention which codes for the AdhE protein also includes such a coding sequence of lactic acid bacterial origin which hybridizes to the adhE coding sequence from L. lactis strain DB1341 under the following conditions: hybridization overnight at 65° C. followed by washing the filters twice in 5×SSC at room temperature for 30 minutes and subsequently once in 3×SSC; 0.1% SDS at 65° C. for 30 minutes.

[0027] In one specific embodiment, the DNA sequence according to the invention comprises the sequence as shown herein in Table 1.4 (SEQ ID NO:3) or the sequence designated adhemg1363 as shown in the below Table 1.8 (SEQ ID NO:12) or the sequence shown in Table 1.9 (SEQ ID NOS:28/30), or a mutant or variant hereof which codes at least in part for a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

[0028] In the present context, the above term “mutant or variant” is used to designate any naturally occurring or constructed nucleotide modification of the above DNA sequence which still allows a polypeptide having at least one of the defined activities to be expressed by the thus modified sequence. Accordingly, the modification may consist in one or more nucleotide substitutions in one or more codons, resulting in the translation of the same or different amino acid(s), or the modification may be in the form of the insertion or deletion of one or more nucleotides/codons. The modifications can be provided by any conventional method including, where appropriate, modifications hereof, such as e.g. the use of restriction enzymes or random or site-directed mutagenesis, e.g. by means of transposable elements. It will be understood that the above DNA sequence according to the invention may also be provided-as a synthetically produced sequence or it may be a hybrid sequence comprising in part a native sequence and in part a synthetically prepared sequence. Additionally, the above term “mutant and variant” includes any mutein of the sequence.

[0029] The above lactic acid bacterial DNA sequence whether in its native form or in a modified mutant or variant form may further comprise one or more sequences that regulate the expression of the coding sequence. Such regulatory sequences may be located upstream and/or downstream of the coding sequence or they can be placed on a different replicon, i.e. in trans. The regulatory sequences may be sequences which are natively associated with the coding sequence or they may be inserted or modified promoter sequences not natively associated with the coding sequence, which can be operably linked to the coding sequence. Such sequences which are not natively associated with the coding sequence may be derived from the bacterial strain which is the source of the coding sequence or from a different organism. In this context, a regulatory sequence includes a promoter/operator sequence, a ribosome binding site, a sequence coding for a gene product which either enhances or inhibits the expression the coding sequence, such as a repressor or activator substance including e.g. a RNA sequence including an antisense RNA, a terminator sequence or a leader sequence regulating the excretion of the above multifunctional enzyme product. A promoter which is derived from a different organism or from the same organism may, depending on the desired characteristics of the resulting bacterial cell, have a stronger or a weaker promoter activity than the promoter with which the coding sequence is natively associated.

[0030] In a useful embodiment, the coding sequence is under the control of a regulatable promoter. As used herein, the term “regulatable promoter” is used to describe a promoter sequence, the activity of which is dependent on physical or chemical factors present in the medium where organisms comprising the above coding sequence and its regulatory sequences are cultivated. Such factors include the cultivation temperature, the pH and/or the arginine content of the medium, a temperature shift eliciting the expression of heat shock genes, the composition of the growth medium including the ionic strength/NaCl content and the growth phase/growth rate of the host cell and stringent response.

[0031] A promoter sequence as defined above may further comprise sequences whereby the activity of the promoter becomes regulated. Thus, in lactic acid bacterial cultures for which it is advantageous to have a gradually decreasing activity of the coding sequence under control of the promoter sequence such further sequences may provide a regulation by a stochastic event and may e.g. be sequences, the presence of which results in a recombinational excision of the promoter or of genes coding for substances which are positively needed for the promoter function.

[0032] It has been found that in e.g. Lactococcus lactis there may be, upstream of the sequence coding for the above multifunctional polypeptide, DNA sequences coding for one or more open reading frames. Thus, such open reading frames were identified in both L. lactis strain DB1341 and strain MG1363. These open reading frames were designated orfB.

[0033] In a further aspect, the invention relates, as it is mentioned above, to a recombinant replicon comprising the above DNA sequence coding for the multifunctional polypeptide. As used herein, the term “replicon” designates a DNA sequence which is capable of autonomous replication in a lactic acid bacterium. Such a replicon can be selected from a plasmid capable of replicating in a lactic acid bacterium, a lactic acid bacterial chromosome and a bacteriophage derived from a lactic acid bacterium.

[0034] The replicon may comprise further sequences including marker sequences and linker sequences for the insertion of genes coding for desirable gene products. Thus, in useful embodiments, the replicon may comprise a gene coding for a lipase, a peptidase, a gene coding for a gene product involved in carbohydrate or citrate metabolism, a gene coding for a gene product involved in bacteriophage resistance or a gene coding for a lytic enzyme or a gene coding for a bacteriocin such as e.g. nisin or pediocin. The gene may also be one which codes for a gene product conferring resistance to an antibiotic.

[0035] The gene coding for a desired gene product may be a homologous gene, i.e. a gene isolated from the same species as the host cell for the replicon, or a heterologous gene including a gene isolated from a lactic acid bacterial species which is of a species different from the host cell.

[0036] The invention also provides a recombinant lactic acid bacterial cell comprising the above replicon. Such a host cell may be derived from any species of lactic acid bacteria as defined herein, such as a Lactococcus species, a Lactobacillus species, a Streptococcus species, a Pediococcus species, a Bifidobacterium species and a Leuconostoc species.

[0037] The above lactic acid bacterial cell is useful in starter culture compositions for the manufacturing of food products including dairy products, meat products, wine, vegetables and bakery products, or in the preservation of animal feed. In the latter context, the present recombinant lactic acid bacterial cells are particularly useful as inoculants in field crops which are to be ensiled or as preserving agents in feedstuff components of animal origin such as waste products from the slaughtering and fish processing industries.

[0038] When the cells are to be used for these purposes they are conveniently provided in the form of freeze-dried or frozen concentrates typically containing 108 to 1012 colony forming units (CFUs) per g of concentrate. Such concentrates may be provided as starter culture compositions comprising further suitable components such as e.g. preserving agents, stabilizing agent, cryoprotectants, nutrients, bacterial growth factors or further active components including enzymes.

[0039] An interesting use of the above lactic acid bacterial cell is in the manufacturing of a probiotically active composition. In the present context, the term “probiotically active” indicates that the bacteria selected for this purpose have characteristics which enable them to colonize in the gastrointestinal tract and hereby exert a beneficial regulatory effect on the microbial flora in this habitat. Such an effect may be recognizable as an improved food or feed conversion in humans or animals to which the cells are administered, or as an increased resistance against invading pathogenic microorganisms.

[0040] The above lactic acid bacterial cell can also be provided in the form of a culture for the production of an aroma or antimicrobially active compound.

[0041] In a particularly useful embodiment, the above lactic acid bacterial cell is one wherein the DNA sequence comprising the sequence coding for the multifunctional polypeptide is modified so as to inactivate or reduce the production of or the activity of at least one of the enzymatic activities selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

[0042] Such a modification can be made by methods which are known per se in the art. Thus, as typical examples, a DNA modification can be in the form of deletion, insertion or substitution of one or more nucleotides in the coding sequence possibly leading to the translation of a polypeptide having a modified amino acid composition. Such a modified polypeptide may have lost one or more of the above enzymatic activities or it/they may be reduced. An inactivation of the coding sequence may also be obtained by random or site-directed mutagenesis, e.g. using a transposable element which is integratable in the replicon comprising the coding sequence. Another useful means of providing inactivated mutants is Campbell-like homologous integration as it is described in the below examples.

[0043] The level of production of the multi-functional polypeptide can also be reduced by modifying or regulating regulatory sequences controlling the expression of the gene coding for the polypeptide. Thus, as one example, a native constitutive promoter can be replaced by a regulatable promoter, the function of which can be reduced or inhibited under appropriate conditions such as those physical and chemical promoter regulating factors as mentioned above. Alternatively, a native promoter which is in itself regulatable by certain factors may be replaced by another regulatable promoter which is negatively regulatable by other factors present in the cultivation medium for the recombinant cell.

[0044] Generally, the term “metabolic engineering” in relation to lactic acid bacteria covers manipulations of the bacteria themselves or of the conditions under which they are cultivated whereby the production of metabolites from the fermentation of sugars or citrate is modulated quantitatively or qualitatively. Accordingly, a lactic acid bacterial cell which is modified as described above in one or more of its glycolytic pathways can be characterized as a metabolically engineered cell. Dependent on the type and the site of the DNA modification such a cell will be at least partially blocked in one or more of the above pathways catalyzed by the multi-functional polypeptide (R6/R7 in FIG. 1) and/or the pyruvate formate-lyase deactivase activity will be reduced or blocked. Accordingly, such a metabolically engineered cell may as a result of these modifications produce increased amounts of i.a. acetaldehyde, ethanol and/or acetate.

[0045] In a further useful embodiment, the above lactic acid bacterial cell is one wherein the DNA sequence comprising the sequence coding for the multi-functional polypeptide is modified so as to enhance the production of or the activity of at least one of its native enzymatic activities as defined above. It is contemplated that such a modification can be provided by appropriate modifications of the coding sequence itself which result in an enhanced production level of the polypeptide and/or the production of a modified polypeptide having an enhanced activity of at least one of its native activities. Such modification can be made by substitution, deletion or insertion of one or more nucleotides using any conventional methods for such DNA modifications, including random or site-directed mutagenesis followed by selection of the desired mutants.

[0046] Alternatively, a lactic acid bacterial cell having enhanced production of and/or enhanced activity of at least one of its native enzymatic activities can be provided by suitable modifications of sequences regulating the production and/or the activity of the multifunctional polypeptide. One suitable manner whereby this can be obtained is by operably linking the coding sequence to a promoter sequence having a stronger promoter activity than the native promoter for the coding sequence.

[0047] In suitable embodiments such an inserted promoter is regulatable by a factor as mentioned above and the expression of the polypeptide can then be enhanced by cultivating the cell in the presence of a factor which mediates a strong promoter activity. It is contemplated that an enhanced production of the AdhE polypeptide in a host cell can be obtained by using a replicon which occurs in a high copy number in that host cell.

[0048] It is aimed at that such a metabolically engineered lactic acid bacterial cell having enhanced production of and/or enhanced activity of at least one of its native enzymatic activities will result in that the cell produces increased amounts of at least one metabolite selected from the group consisting of acetaldehyde, ethanol, formate, acetate, acetoin, diacetyl and 2,3 butylene glycol. Thus, in preferred embodiments, such metabolically engineered have a production of one or more of these metabolites which, in comparison with a wild type strain, is at least 2-fold higher such as at last 5-fold higher, e.g. at least 10-fold higher or even at least 20-fold higher.

[0049] The present invention relates in a still further aspect to an isolated lactic acid bacterial DNA sequence that comprises a sequence coding for a polypeptide having pyruvate formate-lyase activity, i.e. a pfl gene. In useful embodiments, such a DNA sequence further comprises at least one regulatory sequence operably linked to the coding sequence and regulating the production of the pyruvate formate-lyase polypeptide or coding for a gene product regulating the pyruvate formate-lyase activity of the polypeptide. In the following, the gene product of pfl will also be referred to as a Pfl polypeptide.

[0050] Such regulatory sequences may be located upstream and/or downstream of the coding sequence. The regulatory sequences may be sequences which are natively associated with the coding sequence or they may be inserted or modified promoter sequences not natively associated with the coding sequence, but which can be operably linked to the coding sequence. Such sequences which are not natively associated with the coding sequence may be derived from the bacterial strain which is the source of the coding sequence or from a different organism. In this context, regulatory sequences include a promoter sequence, a ribosome binding site, a sequence coding for a gene product which either enhances or inhibits the expression of the coding sequence, such as a repressor or activator substance including e.g an antisense RNA, a transcription terminator sequence or a leader sequence directing the excretion of the Pfl polypeptide. In a useful embodiment, the coding sequence is under the control of a regulatable promoter as defined hereinbefore and being regulatable as also described above.

[0051] The activity of the pyruvate formate-lyase enzyme can be regulated or modulated under anaerobic conditions by the presence or absence of an activase and a deactivase, respectively. Accordingly, the DNA sequence comprising the sequence coding for the Pfl polypeptide preferably comprises sequences coding for a pyruvate formate-lyase activase (act gene) and/or a pyruvate formate-lyase deactivase. In preferred embodiments, such a deactivase is a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity as defined hereinbefore.

[0052] In accordance with the invention, the Pfl-encoding DNA sequence can be derived from any lactic acid bacterium including a Lactobacillus species, a Streptococcus species, a Pediococcus species a Bifidobacterium species, a Leuconostoc species and a Lactococcus species such as Lactococcus lactis including Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies lactis biovar diacetylactis and Lactococcus lactis subspecies cremoris.

[0053] It has been found that the Pfl polypeptide as encoded by the pfl gene of Lactococcus lactis subspecies lactis biovar diacetylactis strain DB1341 comprises 787 amino acids (Table 3.2 below) (SEQ ID NO:15) and has a deduced molecular weight of 89.1 kDa. This polypeptide shows considerable identity with known pfl gene products (Table 3.1). Furthermore, it has been found that the corresponding pfl gene in Lactococcus lactis subspecies lactis MG1363 differs from the DB1341 gene in only about 50 of the nucleotides.

[0054] In specific embodiments, the DNA sequence comprising a Pfl encoding sequence comprises the coding sequence as shown in Table 3.2 below (SEQ ID NO:15), the sequence designated mg1363-pfl as shown in Table 3.6 (SEQ ID NO:22) and the sequence shown in Table 5.3 (SEQ ID NOS:36 and 38), or a DNA sequence which is a mutant or variant hereof which codes for a polypeptide having pyruvate formate-lyase activity, the term “mutant or variant” being used in the same manner as defined hereinbefore.

[0055] In accordance with the invention, a pfl gene as defined herein encompasses any of the specific sequences as exemplified in the following and a lactic acid bacterial sequence coding for a polypeptide having the enzymatic activity of the gene products of such isolated sequences which has a DNA homology of at least 50% with the coding sequence of the plf of L. lactis strains DB1341 or MG1363 such as at least 60% homology including at least 70% homology or at least 80% homology, e.g. at least 90% homology.

[0056] In useful embodiments of the invention, the lactic acid bacterium-derived DNA sequence codes for a Pfl protein that is at least 30% identical with the gene products of the pfl gene of Streptococcus mutans (FASTA, GCG Wisconsin, Accession No. D50491) or the pfl gene of Hemophilus influenzae (FASTA, GCG Wisconsin, Accession Nos. U32812 and L42023) or the gene product of the sequence of Table 3.2 herein (SEQ ID NO:15). In other useful embodiments, the identity to such gene products is at least 40%, such as at least 50%, such as at least 60% identity or even at least 70% identity. The homology between the above gene products may also be expressed in terms of amino acid similarity in which case the similarity suitably is at least 60%, such as at least 70%, e.g. at least 80% similarity.

[0057] In accordance with the invention, the DNA sequence coding for the Pfl polypeptide may also be a coding sequence of lactic acid bacterial origin that hybridizes to the pfl encoding sequence isolated from L. lactis strain MG1363, under the following conditions: hybridization overnight at 65° C. followed by washing the filter twice in 5×SSC at room temperature for 30 minutes and subsequently once in 3×SSC; 0.1% SDS at 65° C. for 30 minutes.

[0058] It was found that e.g. in L. lactis open reading frames may be identified upstream of the coding region for the Pfl polypeptide. Such open reading frames were designated orfA and it was found that the gene products hereof has a function in transport across cell membranes of formate. Thus, it was found that a mutant strain of L. lactis wherein the open reading had been disrupted showed an increased tolerance to the toxic formate analogue, hypophosphite.

[0059] In accordance with the invention there is also provided herein a recombinant replicon comprising the above Pfl-encoding DNA sequence. Such a replicon can be derived from a plasmid, a lactic acid bacterial bacteriophage or a lactic acid bacterial chromosome.

[0060] In one aspect the invention relates to a recombinant lactic acid bacterial host cell comprising such a replicon. The cell can be selected from the group consisting of a Lactococcus species, a Lactobacillus species, a Streptococcus species, a Pediococcus species a Bifidobacterium species and a Leuconostoc species.

[0061] The lactic acid bacterial cell may conveniently be provided in the form of a starter culture composition for use in the manufacturing of food products as described above. It is also contemplated that the above cells may be used as probiotically active cultures or as inoculants in animal feed preservation. In this connection, a particular use is as inoculants in field crops or animal waste materials which are subjected to an ensiling process.

[0062] In particularly useful embodiments, the above lactic acid bacterial cell is one wherein the DNA sequence coding for pyruvate formate-lyase activity is modified whereby the production of the pyruvate formate-lyase is reduced or eliminated or whereby the enzyme is produced in a modified form having a reduced pyruvate formate-lyase activity.

[0063] Such a modification can, as it has been described above for a cell comprising a sequence coding for the AdhE polypeptide, be made by methods which are known per se in the art. Thus, as typical examples, a DNA modification can e.g. be made by deletion, insertion or substitution of one or more nucleotides in the coding sequence possibly leading to the expression of a polypeptide having a modified amino acid composition. An inactivation of the coding sequence can also be obtained by random or site-directed mutagenesis, e.g. by using a transposable element which is integratable in the replicon comprising the coding sequence. Another possible means of providing Pfl-inactivated (pfl−)mutants is Campbell-like homologous integration.

[0064] The level of expression of the Pfl polypeptide can also be reduced by modifying or regulating regulatory sequences controlling the production of the polypeptide. Thus, as one example, a native constitutive promoter can be replaced by a regulatable promoter, the function of which can be reduced or inhibited under appropriate conditions such as those physical and chemical promoter regulating factors as mentioned hereinbefore. Alternatively, a native promoter which is in itself regulatable by certain factors may be replaced by another regulatable promoter which is negatively regulatable by other factors present in the cultivation medium for the recombinant cell.

[0065] A cell being modified in this manner will be a metabolically engineered cell, since under conditions where the pyruvate formate-lyase is normally metabolically active as shown in FIG. 1 such a modified cell will lack one of the major pathways whereby the pyruvate pool in normally consumed. This will result in a modification of the metabolic pathways based on pyruvate including an enhanced flux towards α-acetolactate which is a precursor substance for diacetyl, acetoin and 2,3 butylene glycol. Such a cell is particularly useful in dairy starter cultures where such flavour compounds are generally desirable.

[0066] In further useful embodiments, the lactic acid bacterial cell according to the invention is a cell wherein the DNA sequence comprising the sequence coding for pyruvate formate-lyase is modified so that the production of the pyruvate formate-lyase is enhanced or so that the enzyme is produced in a modified form having an increased pyruvate formate-lyase activity.

[0067] Analogously with what is described above with respect to the modifications leading to an enhanced expression or activity of the AdhE polypeptide, it is contemplated that such a modification can be provided by appropriate modifications of the coding sequence itself which result in an enhanced production level of the Pfl polypeptide and/or the production of a modified polypeptide having an enhanced activity of at least one of its native activities. Such modifications can be made by substitution, deletion or insertion of one or more nucleotides using any conventional methods for such DNA modifications, including random or site-directed mutagenesis followed by selection of the desired mutants.

[0068] Alternatively, a lactic acid bacterial cell having enhanced production of and/or enhanced activity of pyruvate formate-lyase can be provided by suitable modifications of sequences regulating the expression of the pfl gene and/or the activity of the enzyme. One suitable manner whereby this can be obtained is by operably linking the coding sequence to a promoter sequence having a stronger promoter activity than the native promoter for the coding sequence. In suitable embodiments such an inserted promoter is regulatable by a factor as mentioned above and the production of the polypeptide can then be enhanced by cultivating the cell in the presence of a factor which confers a strong promoter activity. It is contemplated that a thus modified lactic acid bacterial cell produces increased amounts of formate and/or acetate. Enhanced production of the Pfl polypeptide may also be obtained in a host by using a replicon which occurs in a high copy number in that host cell or by chromosomal amplification.

[0069] In accordance with the invention, there is also provided a recombinant lactic acid bacterial cell comprising both the DNA sequence comprising the above sequence coding for an AdhE polypeptide, and the above sequence comprising a sequence coding for pyruvate formate-lyase, in both instances including sequences regulating the production and/or the activity of the enzyme activities. As used herein, the term “recombinant” implies that at least one of the coding sequences or regulatory sequences is not a naturally occurring sequence. The sequences may be located on the same replicon or they may be on separate replicons.

[0070] Preferably, at least one of the sequences of the above cell is modified so as to modify the production of the pyruvate formate-lyase or the activity hereof, or the distribution of the amounts of end products resulting from the lactose and/or citrate metabolism of the cell.

[0071] It will be understood that a lactic acid bacterium which is metabolically engineered in accordance with the invention so that it has an enhanced production of one or more metabolites is useful in a method of producing such a metabolite or such metabolites. In general, such a the method comprises cultivating a lactic acid bacterium which is metabolically engineered in accordance with the invention under conditions where the metabolite is produced, and isolating the metabolite from the culture. The isolation of the metabolite may be carried out according to any conventional methods of recovering the particular substance, such as e.g. distillation.

[0072] As it is also mentioned above, the lactic acid bacterial cells according to the invention are useful as food starter cultures. In accordance herewith, the invention also provides a method of producing a food product, the method comprising the step of admixing to the food product starting materials a starter culture of a lactic acid bacterium as defined above and keeping the mixture under conditions allowing the starter culture to be metabolically active. Such a method where a starter culture which is metabolically engineered in accordance with the invention is used will, dependent on the type of metabolite modifications, result in a food product having an improved flavour and/or a product which has an improved shelf life due to an enhanced production of antimicrobially active metabolites by the starter culture.

[0073] The invention will now be further illustrated in the below examples and the drawing wherein:

[0074]
FIG. 1 illustrates selected metabolic pathways in citrate fermenting lactic acid bacteria;

[0075]
FIG. 2 shows an overview of the cloned L. lactis DB1341 adhE gene (open arrow), the sequence strategy for clone 1 (box in middle) and the regions covered by the λZAP clones adhE1 and adhE3 (bottom). The nucleotide position of relevant restriction sites is shown (top). The position of PCR and sequencing primers is shown as small open arrows. A putative transcription terminator present downstream of the stop codon is shown as a circle. The rbs box shows the position of a consensus lactococcal ribosome binding site. Arrows show the sequencing strategy for clone 1 (middle);

[0076]
FIG. 3 shows an overview of the cloned L. lactis DB1341 adhE gene fragment (open arrow). The nucleotide position of relevant restriction sites is shown (top). The position of PCR and sequencing primers is shown as small open arrows. A putative transcription terminator present downstream of the stop codon is shown as a circle. The rbs box shows the position of a consensus lactococcal ribosome binding site. The cloned PCR fragments of the L. lactis MG1363 adhE gene are shown as lines (MGadhESTART and MGadhESTOP). The PCR fragments used to clone into pSMA500 for gene inactivation in strain DB1341 are shown as open boxes (pSMAKAS4 and pSMAKAS5);

[0077]
FIG. 4 is an overview of the cloned Lactococcus lactis DB1341 strain (L. lactis subspecies lactis biovar diacetylactis) pfl gene (open arrow box). The nucleotide positions of relevant restriction sites are shown (top). The position of PCR and sequencing primers is shown as small open arrows. A putative ribosome binding site (rbs box) and a transcription terminator present downstream of the stop codon is shown as a circle. The plf1 (open box) shows the fragment of the λZAP clone of the DB1341 genomic library containing a pfl gene fragment. The cloned PCR fragment of the L. lactis subspecies lactis MG1363 pfl fragment is shown as a line (MGpfl1). A Sau3AI fragment used for gene inactivation in strain DB1341 is shown as an open box (pSMAKAS7). The pfl region included in the fragment as obtained by inverse PCR from DB1341 using EcoRI digestion and primers pfl1-250 and pfl1-390 is shown as a dotted box (pflup-1);

[0078]
FIG. 5 is a genetic map of the L. lactis MG1363 adhE locus including the orfB open reading frame. In the upper part are indicated primer sequences;

[0079]
FIG. 6 illustrates the structure of the L. lactis OrfA protein. The shadowed box at the terminal region of OrfA depicts the area covered by the internal orfA fragment used for gene inactivation. The two transmembrane regions were identified using the PredictProtein server at the EMBL, Heidelberg, Germany;

[0080]
FIG. 7 illustrates expression of orfA in L. lactis. A: genetic map of orfA showing the region covered by the probe (thick line below orfA) used in expression studies and in the construction of a null mutant strain. B: Northern blot analysis. RNA isolated from MG1363 was hybridized to the orfA probe. Lane 1: exponential culture in GM17 aerobic; lane 2: same, anaerobic; lane 3: stationary culture in GM17, aerobic; lane 4: same, anaerobic; lane 5: exponential culture i GalM17, aerobic; lane 6: same, anaerobic. The transcript size is shown in kb to the left. The autoradiogram was exposed for 14 days;

[0081]
FIG. 8 illustrates inhibition of growth by hypophosphite in strains of L. lactis. Strains were grown anaerobically overnight in GM17 supplemented with different concentrations of hypophosphite. At the end of the incubation period (about 18 hours), OD600 was measured. Symbols: (♦) MG1363; (▴) MG1363Δo-rfA; (▪) MG1363 pAK80::orfA;

[0082]
FIG. 9 shows a genetic map of the L. lactis MG1363 pfl gene, showing the region used as a probe in the identification of pfl homologues in other lactic acid bacteria, including the position of EcoR1 sites;

[0083]
FIG. 10 shows autoradiograms from Southern hybridization of genomic DNA from non-Lactococcus lactic acid bacteria to a L. lactis pfl probe; Lane 1: L. lactis MG1363; lane 2: Streptococcus thermophilus; lane 3: Leuconostoc mesenteroides; lane 4 Lactobacillus acidophilus. Bands are shown in kb. Filters were exposed 2 h (A) or overnight (B);

[0084]
FIG. 11 illustrates two Sau3AI fragments including most of the L. lactis strain DB1341 adhE coding sequence used in Southern hybridization experiments with EcoRI-digested genomic DNA from non-Lactococcus lactic acid bacteria;

[0085]
FIG. 12 illustrates detection of adhE homologues in other lactic acid bacteria by Southern hybridization experiments with EcoRI-digested genomic DNA from non-Lactococcus lactic acid bacteria. Lane 1: L. lactis MG1363; lane 2: S. thermophilus; lane 3: L. mesenteroides; lane 4 L. acidophilus. Bands are shown in kb. Filters were exposed overnight;

EXAMPLE 1

Cloning of the L. lactis adhE Gene

[0086] 1. Construction of a L. lactis ssp. lactis Biovar diacetylactis DB1341 Genomic Library for Genetic Complementation

[0087] A genomic library was constructed by cloning partially Sau3AI-digested chromosomal DNA from strain DB1341 into BamHI-digested pSMA500 (Madsen et al. 1996) and transforming into E. coli MC1000 by electroporation (Sambrook et al., 1989). Strain DB1341 was kindly provided by Chr. Hansen A/S, Hørsholm, Denmark. The genomic library consisted of about 10,000 independent recombinant clones with an average insert size of 4 kb. A mixed culture, containing all clones obtained, was grown in LB+erythromycin term, 50 μg/ml) and plasmid DNA was isolated for genetic complementation.

[0088] 2. Genetic Complementation in E. coli NZN111 Using the pSMA500 Library

[0089]

E. coli
strain NZN111 (pfl−; ldb::Tn5; kanR) is unable to grow in the absence of O2 due to the accumulation of NADH derived from the lack of fermentative enzyme activities encoded by the pfl and ldh genes (Mat-Jan et al., 1989).

[0090] Genetic complementation was attempted by transformation of NZN111 using 200 ng plasmid DNA from the library (see above). Transformation mixtures were plated on LB+erm (50 μg/ml)+Kanamycin (kan; 50 μg/ml) and incubated at 37° C. in anaerobic jars. As a control, pSMA500-transformed strain NZN111 was used. After two days, transformation plates were incubated aerobically for another two days to allow weak complementing clones to grow. A clone was identified (clone 1) in the library-transformed plates, and no growth was observed in the pSMA500 control.

[0091] In a preliminary screening, protein extracts of clone 1 were used in a modified “Ldh” assay (Crow and Pritchard 1977), where the pyruvate-dependent conversion of NADH to NAD is monitored, to ensure that complementation of the fermentative defects in strain NZN111 had occurred. Protein extraction was carried out adding 100 μl 100 mM MOPS buffer (pH 6.5); 2% Triton X-100 to the cell pellet from 1.5 ml stationary cultures grown in LB+erm (50 μg/ml) which had been washed in fresh ice cold LB, and frozen at −80° C. for 15 min. Pellets were dissolved and transferred to Eppendorf tubes. Lysozyme (5 mg) was added and samples were incubated on ice for 30 min. Subsequently, glass beads (100 μM, Sigma; 100 μl) were added and samples were vortexed for 30 sec and kept on ice for 30 sec. This step was repeated 10-15 times, and samples were centrifuged at maximum speed for 2 min. Supernatants were transferred to a new Eppendorf tube and kept at −80° C. until assayed. To measure NADH oxidation, the following components were mixed in a quartz cuvette: 700 μl 100 mM MOPS, pH 6.5; 100 μl 120 mM Na-Pyruvate; 50 μl 2.56 mM NADH and 50 μl H2O. The decrease in OD340 as a result of the oxidation of NADH to NAD was monitored after the addition of 100 μl sample. As control reaction, pyruvate was omitted. No significant decrease in OD was observed in the control. A relatively high conversion rate (approximately 2-fold as compared to the NZN111::pSMA500 control) was observed in clone 1.

[0092] Plasmid DNA was isolated from clone 1 and used to retransform E. coli NZN111. Duplicate LB+erm plates were incubated (i) aerobically for 4 days or (ii) anaerobically for 2 days and then 2 days aerobically at 37° C. A similar number of transformants was obtained in both procedures (see Table 1.1 below) Thus, clone 1 did not result from artifact cloning and can indeed complement the defect in strain NZN111.

1TABLE 1.1Retransformation of clone 1 into E. coil NZN111No. of colonies per 10 ng DNAPlasmidanaerobic growthaerobic growthclone 1600800pSMA50001000

[0093] NZN111 competent cells were electroporated with the corresponding plasmid, and one half of the cell mixture was plated onto LB+kan+erm and incubated without O2 (anaerobic growth), and the other half was plated onto the same medium and incubated with O2 (aerobic growth). Transformants were scored after 4 days (see main text).

[0094] A sample of clone 1 in E. coli was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession No. DSM 11093.

[0095] 3. Sequence Analysis of Clone 1 and Identification of an adhE Fragment

[0096] Clone 1 was further characterized by restriction enzyme analysis and included a 2.2 kb insert. Sequence analysis determined that it included a 1.7 kb fragment of an open reading frame (ORF) showing homology to the E. coli adhE gene disclosed by Goodlove et al., 1989. The sequence of the 2.2 kb insert is shown in Table 1.2 below (SEQ ID NO:1).

2TABLE 1.2Sequence of the insert in clone 1Sau3AI1GATCTGTCCTTAGTACGAGAGGACCGGGATGGACTTACCGCTGGTGTACC(SEQ ID NO:1)51AGTTGTTCCGCCAGAGCACGGCTGGATAGCTATGTAGGGAAGGGATAAGC101GCTGAAAGCATCTAAGTGCGAAGCCACCTCAAGATGAGATTACCCATTCG Sau3AI151AGAATTAAGAGCCCAGAGAGATGATCAAGATGTCAATAATTTGCAAAAAA201TCTTCTTTCAGCAAAACGGGATTTGAGTTTTTGCTCGATTTGTGGGAATT Sau3AI251TAACAGAAAGTGATCTGTTGAAATCGCAAGCCCTCTCGGTGTACTTGCTG301GTATCGTTCCAACGACTAATCCAACATCAACAGCAATCTTTAAATCTTTA351TTGACTGCAAAAACACGTAATGCTATTGTTTTCGCTTTCCACCCTCAAGC401TCAAAAATGTTCAAGCCATGCAGCAAAAATTGTTTACGATGCTGCAATTG451AAGCTGGTGCACCGGAAGACTTTATTCAATGGATTGAAGTACCAAGCCTT501GACATGACTACCGCCTTGATTCAAAACCGTGGACTTGCAACAATCCTTGC551AACTGGTGGCCCAGGAATGGTAAACGCCGCACTCAAATCTGGTAACCCTT601CACTCGGTGTTGGAGCTGGTAATGGTGCTGTTTATGTTGATGCAACTGCA651AATATTGAACGTGCCGTTGAAGACCTTTTGCTTTCAAAACGTTTTGATAA −35701TGGGATGATTTGTGCCACTGAAAATTCAGCTGTTATTGATGCTTCAGTTT −10 SD →751ATGATGAATTTATTGCTAAAATGCAAGAACAAGGCGCTTATATGGTTCCT {overscore (M )}V P3(SEQ ID NO:2)801AAAAAAGACTACAAAGCTATTGAAAGTTTCGTTTTTGTTGAACGTGCTGGK K D Y K A I E S F V F V E R A G20851TGAAGGTTTTGGAGTAACTGGTCCTGTTGCCGGTCGTTCTGGTCAATGGA E G F G V T G P V A G R S G Q W I37901TTGCTGAACAAGCTGGTGTCAAAGTTCCTAAAGATAAAGATGTCCTTCTT A E Q A G V K V P K D K D V L L53951TTTGAACTTGATAAGAAAAATATTGGTGAAGCACTTTCTTCTGAAAAACTF E L D K K N I G E A L S S E K L701001TTCTCCTTTGCTTTCAATCTACAAAGCTGAAACACGTGAAGAAGGAATTG S P L L S I Y K A E T R E E G I E871051AGATTGTACGTAGCTTACTTGCTTATCAAGGTGCTGGACATAATGCTGCA I V R S L L A Y Q G A G H N A A103 Sau3AI1101ATTCAAATCGGTGCAATGGATGATCCATTCGTTAAAGAATATGGCGAAAAI Q I G A M D D P F V K E Y G E K1201151AGTTGAAGCTTCTCGTATCCTCGTTAACCAACCAGATTCTATTGGTGGGG V E A S R I L V N Q P D S I G G V1371201TCGGAGATATCTATACTGATGCAATGCGTCCATCACTTACACTTGGAACT G D I Y T D A M R P S L T L G T153 Sau3AI1251GGTTCATGGGGGAAAAATTCACTTTCACACAATTTGAGTACATACGATCTG S W G K N S L S H N L S T Y D L1701301ATTGAATGTTAAAACAGTGGCTAAACGTCGTAATCGCCCACAATGGGTTC L N V K T V A K R R N R P Q W V R1871351GTTTGCCAAAAGAAATTTACTACGAAAAAAATGCAATTTCTTACTTACAA L P K E I Y Y E K N A I S Y L Q2031401GAATTGCCACACGTCCACAAAGCTTTCATCGTTGCTGACCCTGGTATGGTE L P H V H K A F I V A D P G M V2201451TAAATTTGGTTTCGTTGATAAAGTTTTGGAACAACTTGCTATCCGCCCAA K F G F V D K V L E Q L A I R P T2371501 CTCAAGTTGAAACAAGCATTTATGGCTCTGTTCAACCTGACCCAACTTTG Q V E T S I Y G S V Q P D P T L2531551AGCGAAGCAATTGCAATCGCTCGTCAAATGAAACAATTTGAACCTGACACS E A I A I A R Q M K Q F E P D T2701601TGTCATCTGTCTTGGTGGTGGTTCTGCTCTCGATGCCGGTAAGATTGGTC V I C L G G G S A L D A G K I G R2871651GTTTGATTTATGAATATGATGCTCGTGGTGAAGCTGACCTTTCTGATGAT L I Y E Y D A R G E A D L S D D3031701GCAAGTTTGAAAGAACTTTTCCAAGAATTAGCTCAAAAATTTGTCGATATA S L K E L F Q E L A Q K F V D I3201751TCGTAAACGTATTATTAAATTCTACCATCCACATAAAGCACAAATGGTTG R K R I I K F Y F Y H P H K A Q M V A3371801CAATTCCTACTACTTCTGGTACTGGTTCTGAAGTGACTCCATTTGCAGTT I P T T S G T G S E V T P F A V3531851ATCACTGATGATGAAACTCATGTTAAGTACCCACTTGCTGACTACCAATTI T D D E T H V K Y P L A D Y Q L3701901AACACCACAAGTTGCCATTGTTGACCCTGAGTTTGTTATGACTGTACCAA T P Q V A I V D P E F V M T V P K3871951AACGTACTGTTTCTTGGTCTGGTATTGATGCGATGTCACACGCGCTTGAA R T V S W S G I D A M S H A L E4032001TCTTACGTTTCTGTTATGTCTTCTGACTATACAAAACCAATTTCACTTCAS Y V S V M S S D Y T K P I S L Q420 Sau3AI2051AGCGATCCCGGGTCTAGATTAGGGTAACTTTGAAAGGA A I P G L D *

[0097] Sau3AI recognition sites are indicated above the sequence. DNA homology to the E. coli adhE starts at nucleotide position 262 (data not shown). A Sau3AI fragment with 100% homology to the 23S rRNA of L. lactis is shown doubly underlined at the top (positions 1-173). Putative expression signals functional in E. coli are shown: −35, −10 promoter regions (underlined); Shine Dalgarno (SD, doubly underlined) and putative start codon (bold, discontinuous underline). The amino acid sequence of the open reading frame is given in one-letter-code. The open reading frame ends in the multiple cloning site of vector pSMA500 (doubly underlined at bottom) (Madsen et al., 1996).

[0098]

E. coli
AdhE is a multi-functional protein consisting of 890 amino acids that catalyzes the conversion of acetyl CoA into ethanol and has acetaldehyde-DHase (ACDH) and alcohol-DHase (ADH) activities. Additionally, AdhE shows Pfl deactivase activity involved in the inactivation of pyruvate-formate lyase, a key enzyme in anaerobic metabolism (Knappe et al. 1991).

[0099] As shown in the above Table 1.2 and Table 1.3 below, clone 1 includes the ADH domain of a L. lactis AdhE homologue, and it contains expression signals necessary for expression in E. coli (Shine Dalgarno and −35 and −10 regions). The putative gene product of 427 amino acids is highly homologous to a number of other iron-dependent ADHs. Comparison at the protein level showed a 41.4% identity (78% similarity) with E. coli AdhE, in addition to significant homology to other ADHs of both eukaryotic and prokaryotic origin (Table 1.3).

3TABLE 1.3Homology search (FASTA, GCG Wisconsin package version 8,Genetics Computer Group) using the 427 amino acid putativeprotein encoded by clone 1 (see also TABLE 1.2)The region of homology to AdhE corresponds to the centralregion, where the ADH domain is possibly located. Only homologyto the best score is shown.(Peptide) FASTA of: clone1.pep from: 1 to: 427TRANSLATE of: clone1.seq check: 2521 from: 792 to: 2072The best scores are:init1initnopt..sw:adhe_ecoli P17547 escherichia coli. alcohol dehydroge.276736768sw:adhe_cloab P33744 clostridium acetobutylicum. alcoh..256600703sw:adh1_cloab P13604 clostridium acetobutylicum. nadph..256357279sw:medh_bacmt P31005 bacillus methanolicus. nad-depend..169224173sw:adh4_yeast P10127 saccharomyces cerevisiae (baker's..146224165sw:adhf_schpo Q09669 schizosaccharomyces pombe (fission.146219162sw:yiay_ecoli P37686 escherichia coli. hypothetical 40..158218187sw:sucd_clok1 P38947 clostriclium kluyveri. succinate-s..132186179sw:adh2_zymmo P06758 zymomonas mobilis. alcohol dehydr..129180169sw:fuco_ecoli P11549 escherichia coli. lactaldehyde re..141175147sw:adha_cloab Q04944 clostridium acetobutylicum. nadh-..136153145clone1.pepsw:adhe_ecoliID ADHE_ECOLI STANDARD; PRT; 890 AA.AC P17547;DE ALCOHOL DEHYDROGENASE (EC 1.1.1.1) (ADH)/ACETALDEHYDE DEHYDROGENASE . . . SCORES Init1: 276 Initn: 736 Opt: 76841.4% identity in 430 aa overlap 10 20 30clone 1 MVPKKDYKAIESFVFVERAGEGFGVTGPVA(SEQ ID NO:2) ::: |: ||::: :: ::| :::::::adhe_eGVICASEQSVVVVDSVYDAVRERFATHGGYLLQGKELKAVQDVIL--KNG---ALNAAIV(corresponding to a.a. residues 43-762 of SEQ ID NO:6) 250 260 270 280 290 40 50 60 70 80 90clone 1GRSGQWIAEQAGVKVPKDKDVLLFELDKKNIGEALSSEKLSPLLSIYKAETREEGIEIVR|::: ||| || :||:::::|: |:: : :|::: ||||| |::|:|:: |:::| :adhe_eGQPAYKIAELAGFSVPENTKILIGEVTVVDESEPFAHEKLSPTLAMYRAKDFEDAVEKAE 300 310 320 330 340 350 100 110 120 130 140 149clone1SLLAYQGAGHNAAIQIGAMDDP-FVKEYGEKVEASRILVNQPDSIGGVGDIYTDAMRPSL:|:| | ||:: : ::: ::| |: :|:|::::|||:| |:| ||:||:|: : |||adhe_eKLVAMGGIGHTSCLYTDQDNQPARVSYFGQKMKTARILINTPASQGGIGDLYNFKLAPSL 360 370 380 390 400 410150 160 170 180 190 200clone1TLGTGSWGKNSLSHNLSTYDLLNVKTVAKRRNRPQWVRLPKEIYYEKNAISY-LQE-LPH||| |||| ||:|:|::: :|:| |||||| :: | :|||:||: ::::: |:| :::adhe_eTLGCGSWGGNSISENVGPKHLINKKTVAKRAENMLWHKLPKSIYFRRGSLPIALDEVITD 420 430 440 450 460 470210 220 230 240 250 260clone1VHK-AFIVADPGMVKFGFVDKVLEQLAIRPTQVETSIYGSVQPDPTLSEAIAIARQMKQF || |:||:|: : : |::|:: : | ::: |||::: :|::||||| : | : |adhe_eGHKRALIVTDRFLFNNGYADQITSVL--KAAGVETEVFFEVEADPTLSIVRKGAELANSF 480 490 500 510 520 530 270 280 290 300 310 320clone1EPDTVICLGGGSALDAGKIGRLIYEYDARGEADLSDDASLKELFQELAQKFVDIRKRIIK:||::| |||||::||:|| :::|| : |:: |:||| :|:|||||| |adhe_eKPDVIIALGGGSPMDAAKIMWVMYE---HPETH----------FEELALRFMDIRKRIYK 540 550 560 570 580 330 340 350 360 370 380clone1FYH-PHKAQMVAIPTTSGTGSEVTPFAVITDDETHVKYPLADYQLTPQVAIVDPEFVMTV| : ||:|:|::||||||||||||||:|||:| |||||||:|||::||||:::||::adhe_eFPKMGVKAKMIAVTTTSGTGSEVTPFAVVTDDATGQKYPLADYALTPDMAIVDANLVMDM 590 600 610 620 630 640 390 400 410 420clone1PKRTVSWSGIDAMSHALESYVSVMSSDYTKPISLQAIPGLD||: :::|:||::||:|:||||::|:::: :|||: |:adhe_ePKSLCAFGGLDAVTHAMEAYVSVLASEFSDGQALQALKLLKEYLPASYHEGSKNPVARER 650 660 670 680 690 700adhe_eVHSAATIAGIAFANAFLGVCHSMAHKLGSQFHIPHGLANALLICNVIRYNANDNPTKQTA 710 720 730 740 750 760

[0100] 4. DNA Hybridization of the DB1341 λZAP Library Using an adhE Fragment

[0101] Sequence comparison of clone 1 with the previously cloned adhE gene indicated that the first 500 bp and the last 600 bp of the putative L. lactis adhE homologue were not present in clone 1. Therefore, a λZAP genomic library of strain DB1341 was constructed according to manufacturer's instructions (Stratagene). The average insert size was estimated to be approx. 3 kb, with 80% recombinant clones. Approximately 2×105 pfu were screened using a 0.8 kb Sau3AI fragment (position 1296-2054 in Table 1.2) and 10 positive clones (named adhE-1 to 10 were selected for characterization.

[0102] 5. Sequencing of Positive λZAP adhE Clones

[0103] Following ‘in vivo’ excision of the pBK plasmid version (Stratagene) of the clones, restriction mapping and sequencing of clones adhE-1 and adhE-3 was carried out as shown in FIG. 2. Clone adhE-1 included a 1.7 kb insert that was identical to the adhE fragment of clone 1 (position 262-2054 in Table 1.2). Clone adhE-3 contained a 4 kb insert spanning from the Sau3AI site at position 1296 in Table 1.2. This fragment could harbour the 3′-end of the L. lactis adhE gene. Sequence analysis of this clone confirmed that it included the 3′-end of the L. lactis adhE gene, which ends with a double stop codon (TAATAA, position 2854-2859 in Table 1.4 below). Downstream from this position, a possible transcription terminator was found (position 2883-2905 in Table 1.4).

[0104] A sample of clones adhE-1 and adhE-3, respectively in E. coli was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 25 Jul. 1996 under the accession Nos DSM 11101 and DSM 11102, respectively.

4TABLE 1.4Sequence of the L. lactis DB1341 adhE gene (SEQ ID NO:3)1AAGCTTGTTACAAAACCGTTTTCTAAACTTTTGATGAGTGTTTTTGTAAA(SEQ ID NO:3)1---------+---------+---------+---------+---------+50AACTATCACAATATTGCTTGACATCTATAAAAAACTTTGTTAAACTATTC51---------+---------+---------+---------+---------+100ACGTAAAAGAAAGTGAATGAAGTCACAAAGGAGAACCTACAAATATGGCA101---------+---------+---------+---------+---------+150 MetAla—(SEQ ID NO:4)ACTAAAAAAGCCGCTCCAGCTGCAAAGAAAGTTTTAAGCGCTGAAGAAAA151---------+---------+---------+---------+---------+200ThrLysLysAlaAlaProAlaAlaLysLysValLeuSerAlaGluGluLys—AGCCGCAAAATTCCAAGAAGCTGTTGCTTATACTGACAAATTAGTCAAAA201---------+---------+---------+---------+---------+250 AlaAlaLysPheGlnGluAlaValAlaTyrThrAspLysLeuValLysLys—AAGCACAAGCTGCTGTTCTTAAATTTGAAGGATATACACAAACTCAAGTC251---------+---------+---------+---------+---------+300 AlaGlnAlaAlaValLeuLysPheGluGlyTyrThrGlnThrGlnVal—GATACTATTGTCGCTGCAATGGCTCTTGCAGCAAGCAAACATTCTCTAGA301---------+---------+---------+---------+---------+350AspThrIleValAlaAlaMetAlaLeuAlaAlaSerLysHisSerLeuGlu—ACTCGCTCATGAAGCCGTTAACGAAACTGGTCGTGGTGTTGTCGAAGACA351---------+---------+---------+---------+---------+400 LeuAlaHisGluAlaValAsnGluThrGlyArgGlyValValGluAspLys—AATGACAAAACTGTTGGTGTCATTTCTGAAAACAAGGTTGCTGGATCTGT401---------+---------+---------+---------+---------+450 AspThrLysAsnHisPheAlaSerGluSerValTyrAsnAlaIleLys—AATGACAAAACTGTTGGTGTCATTTCTGAAAACAAGGTTGCTGGATCTGT451---------+---------+---------+---------+---------+500AsnAspLysThrValGlyValIleSerGluAsnLysValAlaGlySerVal—TGAAATCGCAAGCCCTCTCGGTGTACTTGCTGGTATCGTTCCAACGACTA501---------+---------+---------+---------+---------+550 GluIleAlaSerProLeuGlyValLeuAlaGlyIleValProThrThrAsn—ATCCAACATCAACAGCAATCTTTAAATCTTTATTGACTGCAAAAACACGT551---------+---------+---------+---------+---------+600 ProThrSerThrAlaIlePheLysSerLeuLeuThrAlaLysThrArg—AATGCTATTGTTTTCGCTTTCCACCCTCAAGCTCAAAAATGTTCAAGCCA601---------+---------+---------+---------+---------+650AsnAlaIleValPheAlaPheHisProGlnAlaGlnLysCysSerSerHis—TGCAGCAAAAATTGTTTACGATGCTGCAATTGAAGCTGGTGCACCGGAAG651---------+---------+---------+---------+---------+700 AlaAlaLysIleValTyrAspAlaAlaIleGluAlaGlyAlaProGluAsp—ACTTTATTCAATGGATTGAAGTACCAAGCCTTGACATGACTACCGCCTTG701---------+---------+---------+---------+---------+750 PheIleGlnTrpIleGluValProSerLeuAspMetThrThrAlaLeu—ATTCAAAACCGTGGACTTGCAACAATCCTTGCAACTGGTGGCCCAGGAAT751---------+---------+---------+---------+---------+800IleGlnAsnArgGlyLeuAlaThrIleLeuAlaThrGlyGlyProGlyMet —GGTAAACGCCGCACTCAAATCTGGTAACCCTTCACTCGGTGTTGGAGCTG801---------+---------+---------+---------+---------+850 ValAsnAlaAlaLeuLysSerGlyAsnProSerLeuGlyValGlyAlaGly—GTAATGGTGCTGTTTATGTTGATGCAACTGCAAATATTGAACGTGCCGTT851---------+---------+---------+---------+---------+900 AsnGlyAlaValTyrValAspAlaThrAlaAsnIleGluArgAlaVal—GAAGACCTTTTGCTTTCAAAACGTTTTGATAATGGGATGATTTGTGCCAC901---------+---------+---------+---------+---------+950GluAspLeuLeuLeuserLysArgPheAspAsnGlyMetIleCysAlaThr—TGAAAATTCAGCTGTTATTGATGCTTCAGTTTATGATGAATTTATTGCTA951---------+---------+---------+---------+---------+1000 GluAsnSerAlaValIleAspAlaSerValTyrAspGluPheIleAlaLys—AAATGCAAGAACAAGGCGCTTATATGGTTCCTAAAAAAGACTACAAAGCT1001---------+---------+---------+---------+---------+1050 MetGlnGluGlnGlyAlaTyrMetValProLysLysAspTyrLysAla—ATTGAAAGTTTCGTTTTTGTTGAACGTGCTGGTGAAGGTTTTGGAGTAAC1051---------+---------+---------+---------+---------+1100IleGluSerPheValPheValGluArgAlaGlyGluGlyPheGlyValThr—TGGTCCTGTTGCCGGTCGTTCTGGTCAATGGATTGCTGAACAAGCTGGTG1101---------+---------+---------+---------+---------+1150 GlyProValAlaGlyArgSerGlyGlnTrpIleAlaGluGluAlaGlyVal—TCAAAGTTCCTAAAGATAAAGATGTCCTTCTTTTTGAACTTGATAAGAAA1151---------+---------+---------+---------+---------+1200 LysValProLysAspLysAspValLeuLeuPheGluLeuAspLysLys—AATATTGGTGAAGCACTTTCTTCTGAAAAACTTTCTCCTTTGCTTTCAAT1201---------+---------+---------+---------+---------+1250AsnIleGlyGluAlaLeuSerSerGluLysLeuSerProLeuLeuSerIle—CTACAAAGCTGAAACACGTGAAGAAGGAATTGAGATTGTACGTAGCTTAC1251---------+---------+---------+---------+---------+1300 TyrLysAlaGluThrArgGluGluGlyIleGluIleValArgSerLeuLeu—TTGCTTATCAAGGTGCTGGACATAATGCTGCAATTCAAATCGGTGCAATG1301---------+---------+---------+---------+---------+1350 AlaTyrGlnGlyAlaGlyHisAsnAlaAlaIleGlnIleGlyAlaMet—GATGATCCATTCGTTAAAGAATATGGCGAAAAAGTTGAAGCTTCTCGTAT1351---------+---------+---------+---------+---------+1400AspAspProPheValLysGluTyrGlyGluLysValGluAlaSerArgIle—CCTCGTTAACCAACCAGATTCTATTGGTGGGGTCGGAGATATCTATACTG1401---------+---------+---------+---------+---------+1450 LeuValAsnGlnProAspSerIleGlyGlyValGlyAspIleTyrThrAsp—ATGCAATGCGTCCATCACTTACACTTGGAACTGGTTCATGGGGGAAAAAT1451---------+---------+---------+---------+---------+1500 AlaMetArgProSerLeuThrLeuGlyThrGlySerTrpGlyLysAsn—TCACTTTCACACAATTTGAGTACATACGATCTATTGAATGTTAAAACAGT1501---------+---------+---------+---------+---------+1550SerLeuSerHisAsnLeuSerThrTyrAspLeuLeuAsnValLysThrVal—GGCTAAACGTCGTAATCGCCCACAATGGGTTCGTTTGCCAAAAGAAATTT1551---------+---------+---------+---------+---------+1600 AlaLysArgArgAsnArgProGlnTrpValArgLeuProLysGluIleTyr—ACTACGAAAAAAATGCAATTTCTTACTTACAAGAATTGCCACACGTCCAC1601---------+---------+---------+---------+---------+1650 TyrGluLysAsnAlaIleSerTyrLeuGlnGluLeuProHisValHis—AAAGCTTTCATCGTTGCTGACCCTGGTATGGTTAAATTTGGTTTCGTTGA1651---------+---------+---------+---------+---------+1700LysAlaPheIleValAlaAspProGlyMetValLysPheGlyPheValAsp—TAAAGTTTTGGAACAACTTGCTATCCGCCCAACTCAAGTTGAAACAAGCA1701---------+---------+---------+---------+---------+1750 LysValLeuGluGlnLeuAlaIleArgProThrGlnValGluThrSerIle—TTTATGGCTCTGTTCAACCTGACCCAACTTTGAGCGAAGCAATTGCAATC1751---------+---------+---------+---------+---------+1800 TyrGlySerValGlnProAspProThrLeuSerGluAlaIleAlaIle—GCTCGTCAAATGAAACAATTTGAACCTGACACTGTCATCTGTCTTGGTGG1801---------+---------+---------+---------+---------+1850AlaArgGlnMetLysGlnPheGluProAspThrValIleCysLeuGlyGly—TGGTTCTGCTCTCGATGCCGGTAAGATTGGTCGTTTGATTTATGAATATG1851---------+---------+---------+---------+---------+1900 GlySerAlaLeuAspAlaGlyLysIleGlyArgLeuIleTyrGluTyrAsp—ATGCTCGTGGTGAAGCTGACCTTTCTGATGATGCAGGTTTGAAAGAACTT1901---------+---------+---------+---------+---------+1950 AlaArgGlyGluAlaAspLeuSerAspAspAlaSerLeuLysGluLeu—TTCCAAGAATTAGCTCAAAAATTTGTCGATATTCGTAAACGTATTATTAA1951---------+---------+---------+---------+---------+2000PheGlnGluLeuAlaGlnLysPheValAspIleArgLysArgIleIleLys—ATTCTACCATCCACATAAAGCACAAATGGTTGCAATTCCTACTACTTCTG2001---------+---------+---------+---------+---------+2050 PheTyrHisProHisLysAlaGlnMetValAlaIleProThrThrSerGly—GTACTGGTTCTGAAGTGACTCCATTTGCAGTTATCACTGATGATGAAACT2051---------+---------+---------+---------+---------+2100 ThrGlySerGluValThrProPheAlaValIleThrAspAspGluThr—CATGTTAAGTACCCACTTGCTGACTACCAATTAACACCACAAGTTGCCAT2101---------+---------+---------+---------+---------+2150HisValLysTyrProLeuAlaAspTyrGlnLeuThrProGlnValAlaIle—TGTTGACCCTGAGTTTGTTATGACTGTACCAAAACGTACTGTTTCTTGGT2151---------+---------+---------+---------+---------+2200 ValAspProGluPheValMetThrValProLysArgThrValSerTrpSer—CTGGTATTGATGCGATGTCACACGCGCTTGAATCTTACGTTTCTGTTATG2201---------+---------+---------+---------+---------+2250 GlyIleAspAlaMetSerHisAlaLeuGluSerTyrValSerValMet—TCTTCTGACTATACAAAACCAATTTCACTTCAAGCGATCAAACTTATCTT2251---------+---------+---------+---------+---------+2300SerSerAspTyrThrLysProIleSerLeuGlnAlaIleLysLeuIlePhe—TGAAAACTTGACTGAGTCTTATCATTATGACCCAGCGCATCCAACTAAAG2301---------+---------+---------+---------+---------+2350 GluAsnLeuThrGluSerTyrHisTyrAspProAlaHisProThrLysGlu—AAGGACAAAAAGCCCGCGAAAACATGCACAATGCTGCAACACTCGCTGGT2351---------+---------+---------+---------+---------+2400 GlyGlnLysAlaArgGluAsnMetHisAsnAlaAlaThrLeuAlaGly—ATGGCCTTCGCTAATGCTTTCCTTGGAATTAACCACTCACTTGCTCATAA2401---------+---------+---------+---------+---------+2450MetAlaPheAlaAsnAlaPheLeuGlyIleAsnHisSerLeuAlaHisLys—AATTGGTGGTGAATTTGGACTTCCTCATGGTCTTGCCATTGCCATCGCTA2451---------+---------+---------+---------+---------+2500 IleGlyGlyGluPheGlyLeuProHisGlyLeuAlaIleAlaIleAlaMet—TGCCACATGTCATTAAATTTAACGCTGTAACAGGAAACGTTAAACGTACC2501---------+---------+---------+---------+---------+2550 ProHisValIleLysPheAsnAlaValThrGlyAsnValLysArgThr—CCTTACCCACGTTATGAAACATATCGTGCTCAAGAGGACTACGCTGAAAT2551---------+---------+---------+---------+---------+2600ProTyrProArgTyrGluThrTyrArgAlaGlnGluAspTyrAlaGluIle—TTCACGCTTCATGGGATTTGCTGGTAAAGATGATTCAGATGAAAAAGCTG2601---------+---------+---------+---------+---------+2650 SerArgPheMetGlyPheAlaGlyLysAspAspSerAspGluLysAlaVal—TGCAAGCTCTGGTTGCTGAACTTAAGAAACTGACTGATAGCATTGATATT2651---------+---------+---------+---------+---------+2700 GlnAlaLeuValAlaGluLeuLysLysLeuThrAspSerIleAspIle—AATATCACCCTTTCAGGAAATGGTATCGATAAAGCTCACCTTGAACGTGA2701---------+---------+---------+---------+---------+2750AsnIleThrLeuSerGlyAsnGlyIleAspLysAlaHisLeuGluArgGlu—ACTTGATAAATTGGCTGACCTTGTTTATGATGATCAATGTACTCCTGCTA2751---------+---------+---------+---------+---------+2800 LeuAspLysLeuAlaAspLeuValTyrAspAspGlnCysThrProAlaAsn—ATCCTCGTCAACCAAGAATTGATGAGATTAAACAGTTGTTGTTAGATCAA2801---------+---------+---------+---------+---------+2850 ProArgGlnProArgIleAspGluIleLysGlnLeuLeuLeuAspGln—TACTAATAATCTGTTGATAAAATTATTAAAACGCTCTGATGAATTCGTCA2851---------+---------+---------+---------+---------+2900TyrEndEndGAGCATTTTTTATTATAGCTTATACAACTATCAAAAGGTATAAATCAATT2901---------+---------+---------+---------+---------+2950TCGATATAGGCTCTTTTCACTCCATTGATTTATGCATTTCTATAAAAATC2951---------+---------+---------+---------+---------+3000AATAATTAATTAGCGATAGAAGTCGAGTTCATGCATGCTAATAATGAAAT3001---------+---------+---------+---------+---------+3050TGTTTTAAATTCTGGTTTTTCTTTATGTTCTTTGCGAACATCTTTCACAG3051---------+---------+---------+---------+---------+3100TTTCTTTGTTCATGAAAATTCCTCCTTATTATGGTACTATTTTGAGCCCA3101---------+---------+---------+---------+---------+3150AATAGTTATATAAGAATCCTAAACTTCGGATATCTTATCAAAG3151---------+---------+---------+---------+--- 3193In this Table a putative ribosome binding site is shown in bold (position 127-133), 12 bp upstream the putative start codon (position 145-147), deduced from homology comparisons (FIGS. 2 and 3). Two adjacent stop codons, located at position 2854-2859) are shown (double underline). A putative rho-independent transcription terminator (de Vos and Simons, 1994) is also shown downstream of the stop codons at position 2883-2904 (single and dotted underline show stem and loop sequences, respectively).

[0105] The L. lactis adhE gene of strain DB1341 encodes a 903 amino acid long protein, as deduced from the DNA sequence (Table 1.5), with an estimated molecular weight of 98.2 KDa. A putative ribosome binding site (AAAGGAG, position 127-133 in Table 1.4 is found 11 bp upstream of the start codon (de Vos and Simmons 1994).

[0106] Homology comparisons have shown a 44% identity (81% similarity) of the L. lactis AdhE to the E. coli protein and 42.4% identity (80% similarity) to the Clostridium acetobutylicum Aad protein throughout an approx. 750 amino acids fragment (Tables 1.4 and 1.5). A significantly lower homology is observed at the C-terminal region of these three proteins.

5TABLE 1.5Protein homology search (FASTA. GCG Wisconsin package version 8. Genetics Computer Group) using the deducedsequence of the AdhE Protein encoded by the L. lactis DB1341adhE geneIn this Table only alignment of the best two scores (E. coliAdhE and C. acetobutylicum Aad) is shown.(Peptide) FASTA of: adhedb1341.pep from: 1 to: 904TRANSLATE of: adhedb246.seq check: 3519 from: 145 to: 2856The best scores are:init1initnoptsw:adhe_ecoli P17547 escherichia coli. alcohol dehydr....70818191507sw:adhe_cloab P33744 clostridium acetobutylicum. alcoh...40412971053sw:adh1_cloab P13604 clostridiuxn acetobutylicum. nadph...283581434sw:sucd_clok1 P38947 clostridium kluyveri. succinate-s...290460621sw:medh_bacmt P31005 bacillus methanolicus. nad-depend...187389298sw:adh2_zymmo P06758 zymomonas mobilis. alcohol dehydr...170376299sw:adh4_yeast P10127 saccharomyces cerevisiae (baker's ..173368295sw:dhat_citfr P45513 citrobacter freundii. 1,3-Propan....163329295sw:eute_salty P41793 salmonella typhimurium. ethanolam...150309372adhedb1341.pepsw:adhe_ecoliID ADHE_ECOLI STANDARD; PRT; 890 AA.AC P17547;DT 01 AUG. 1990 (REL. 15, CREATED)DT 01 AUG. 1990 (REL. 15, LAST SEQUENCE UPDATE)DT 01 NOV. 1995 (REL. 32, LAST ANNOTATION UPDATE)DE ALCOHOL DEHYDROGENASE (EC 1.1.1.1) (ADH)/ACETALDEHYDE DEHYDROGENASE . . . SCORES Init1: 708 Initn: 1819 Opt: 150744.3% identity in 757 aa overlap 10 20 30 40 50 60adhe24 MATKKAAPAAKKVLSAEEKAAKFQEAVAYTDKLVKKAQAAVLKFEGYTQTQVDTIVAAMA : ||:::: | :::::||:|||:| | |adhe_e AVTNVAELNALVERVKKAQREYASFTQEQVDKIFRAAA 10 20 30 70 80 90 100 110 120adhe24 LAASKHSLELAHEAVNETGRGVVEDKDTKNHFASESVYNAIKNDKTVGVISENKVAGSVE |||:: :: ||: ||:|:|:|:|||| :||||||| :||| |::|| ||:||::: |:::adhe_e LAAADARIPLAKMAVAESGMGIVEDKVIKNHFASEYIYNAYKDEKTCGVLSEDDTFGTIT 40 50 60 70 80 90 130 140 150 160 170 180adhe24 IASPLGVLAGIVPTTNPTSTAIFKSLLTAKTRNAIVFAFHPQAQKCSSHAAKIVYDAAIE ||:|:|:: |||||||||||||||||:: ||||||:|: ||:|:: :::||:|| :||| adhe_e IAEPIGIICGIVPTTNPTSTAIFKSLISLKTRNAIIFSPHPRAKDATNKAADIVLQAAIA 100 110 120 130 140 150 190 200 210 220 230 240adhe24 AGAPEDFIQWIEVPSLDMTTALIQNRGLATILATGGPGMVNAALKSGNPSLGVGAGNGAV ||||:|:| ||: ||:::::||::::::: ||||||||||:|| :||:|::||||||::|adhe_e AGAPKDLIGWIDQPSVELSNALMHHPDINLILATGGPGMVKAAYSSGKPAIGVGAGNTPV 160 170 180 190 200 210 250 260 270 280 290 300adhe24 YVDATANIERAVEDLLLSKRFDNGMICATENSAVIDASVYDEFIAKMQEQGAYMVPKKDY :|:||:|:|||:::|:|| ||||:|||:|:|:|: :||||: ::::::|:|::: |:adhe_e VIDETADIKRAVASVLMSKTFDNGVICASEQSVVVVDSVYDAVRERFATHGGYLLQGKEL 220 230 240 250 260 270 310 320 330 340 350 360adhe24 KAIESFVFVERAGEGFGVTGPVAGRSGQWIAEQAGVKVPKDKDVLLFELDKKNIGEALSS ||::: :: ::| :::::::|::: ||| || :||:::::|: |:: : :|:::adhe_e KAVQDVIL--KNG---ALNAAIVGQPAYKIAELAGFSVPENTKILIGEVTVVDESEPFAH 280 290 300 310 320 330 370 380 390 400 410 419adhe24 EKLSPLLSIYKAETREEGIEIVRSLLAYQGAGHNAAIQIGAMDDP-FVKEYGEKVEASRI ||||| |::|:|:: |:::| : :|:| | ||:: : ::: ::| |: :|:|::::||adhe_e EKLSPTLAMYRAKDFEDAVEKAEKLVAMGGIGHTSCLYTDQDNQPARVSYFGQKMKTARI 340 350 360 370 380 390420 430 440 450 460 470 479adhe24 LVNQPDSIGGVGDIYTDAMRPSLTLGTGSWGKNSLSHNLSTYDLLNVKTVAKRRNRPQWV |:| |:| ||:||:|: : |||||| |||| ||:|:|::: :|:| |||||| :: |adhe_e LINTPASQGGIGDLYNFKLAPSLTLGCGSWGGNSISENVGPKHLINKKTVAKRAENMLWH 400 410 420 430 440 450480 490 500 510 520 530adhe24 RLPKEIYYEKNAISY-LQE-LPHVHK-AFIVADPGMVKFGFVDKVLEQLAIRPTQVETSI :|||:||: ::::: |:| ::: || |:||:|: : : |::|:: : | ::: ||| adhe_e KLPKSIYFRRGSLPIALDEVITDGHKRALIVTDRFLFNNGYADQITSVL--KAAGVETEV 460 470 480 490 500 510 540 550 560 570 580 590adhe24 YGSVQPDPTLSEAIAIARQMKQFEPDTVICLGGGSALDAGKIGRLIYEYDARGEADLSDD : :|::||||| : | : |:||::| |||||::||:|| :::|| : |::adhe_e FFEVEADPTLSIVRKGAELANSFKPDVIIALGGGSPMDAAKIMWVMYE---HPETH---- 520 530 540 550 560 600 610 620 630 640 650adhe24 ASLKELFQELAQKFVDIRKRIIKFYH-PHKAQMVAIPTTSGTGSEVTPFAVITDDETHVK |:||| :|:|||||| || : ||:|:|::||||||||||||||:|||:| |adhe_e ------FEELALRFMDIRKRIYKFPKMGVKAKMIAVTTTSGTGSEVTPFAVVTDDATGQK 570 580 590 600 610 660 670 680 690 700 710adhe24 YPLADYQLTPQVAIVDPEFVMTVPKRTVSWSGIDAMSHALESYVSVMSSDYTKPISLQAI ||||||:|||::||||:::||::||: :::|:||::||:|:||||::|:::: :||| adhe_e YPLADYALTPDMAIVDANLVMDMPKSLCAFGGLDAVTHAMEAYVSVLASEFSDGQALQAL 620 630 640 650 660 670 720 730 740 750 760 770adhe24 KLIFENITESYHYDPAHPTKEGQKARENMHNAATLAGMAFANAFLGINHSLAHKIGGEFG ||: | |::||| :: :|: :: ::: :: ::|: || :: :::|:|: ||::::|adhe_e KLLKEYLPASYHEGSKNPVARERVHSAATIAGIAFAN-AFLGVCHSMAHKLGSQFHIPHG 680 690 700 710 720 730 780 790 800 810 820 830adhe24 LPHGLAIAIAMPHVIKFNAVTGNVKRTPYPRYETYRAQEDYAEISRFMGFAGKDDSDEKA |:::| |adhe_e LANALLICNVIRYNANDNPTKQTAFSQYDRPQARRRYAEIADHLGLSAPGDRTAAKIEKL 740 750 760 770 780 790adhe24: SEQ ID NO:5; adh_e: SEQ ID NO:6adhedb1341.pepsw:adhe_cloabID ADHE_CLOAB STANDARD; PRT; 862 AA.ACP33744;DT 01 FEB. 1994 (REL. 28, CREATED)DT 01 FEB. 1994 (REL. 28, LAST SEQUENCE UPDATE)DT 01 FEB. 1995 (REL. 31, LAST ANNOTATION UPDATE)DE ALCOHOL DEHYDROGENASE (EC 1.1.1.1) (ADH)/ACETALDEHYDE DEHYDROGENASESCORES Init1: 404 Initn: 1297 Opt: 105338.6% identity in 568 aa overlap 10 20 30 40 50 60adhe24 MATKKAAPAAKKVLSAEEKAAKFQEAVAYTDKLVKKAQAAVLKFEGYTQTQVDTIVAAMA |: :| ::| ||: |:|: ||:| : |adhe_c MKVTTVKELDEKLKVIKEAQKKFSCYSQEMVDEIFRNAA 10 20 30 70 80 90 100 110 120adhe24 LAASKHSLELAHEAVNETGRGVVEDKDTKNHFASESVYNAIKNDKTVGVISENKVAGSVE :|| : ::|||::|| |||:|:|||| :|||||:| :|| |::|| |:|: |: | ::adhe_c MAAIDARIELAKAAVLETGMGLVEDKVIKNHFAGEYIYNKYKDEKTCGIIERNEPYGITK40 50 60 70 80 90 130 140 150 160 170 180adhe24 IASPLGVLAGIVPTTNPTSTAIFKSLLTAKTRNAIVFAFHPQAQKCSSHAAKIVYDAAIE ||:|:||:|:|:|:||||||:|||||:: ||||:| |: ||:|:|:: |||:: |||::adhe_c IAEPIGVVAAIIPVTNPTSTTIFKSLISLKTRNGIFFSPHPRAXKSTILAAKTILDAAVK100 110 120 130 140 150 190 200 210 220 230 240adhe24 AGAPEDFIQWIEVPSLDMTTALIQNRGLATILATGGPGMVNAALKSGNPSLGVGAGNGAV :||||::| ||: ||:::| |:|: ::: ||||||::|::| :||:|::|||:||::|adhe_c SGAPENIIGWIDEPSIELTQYLMQKADIT--LATGGPSLVKSAYSSGKPAIGVGPGNTPV160 170 180 190 200 210 250 260 270 280 290 300adhe24 YVDATANIERAVEDLLLSKRFDNGMICATENSAVIDASVYDEFIAKMQEQGAYMVPKKDY :|::|:|::||::::||| :|||:|||:|:|::: |:|:: :::||:|||:: |::adhe_c IIDESAHIKMAVSSIILSKTYDNGVICASEQSVIVLKSIYNKVKDEFQERGAYIIKKNEL 220 230 240 250 260 270 310 320 330 340 350 360adhe24 KAIESFVFVERAGEGFGVTGPVAGRSGQWIAEQAGVKVPKDKDVLLFELDKKNIGEALSS : : : :| ::| :|: ::|:|: ||: ||:||||:: :|: |::: : :|:::adhe_c DKVREVIF--KDG---SVNPKIVGQSAYTIAAMAGIKVPKTTRILIGEVTSLGEEEPFAH 280 290 300 310 320 330 370 380 390 400 410 419adhe24 EKLSPLLSIYKAETREEGIEIVRSLLAYQGAGHNAAIQIGAMDDP-FVKEYGEKVEASRI |||||:|::|:|:: ::::: : :|:: | ||:::| :::::: :: ::: ::: |:adhe_c EKLSPVLAMYEADNFDDALKKAVTLINLGGLGHTSGIYADEIKARDKIDRFSSAMKTVRT 340 350 360 370 380 390420 430 440 450 460 470 479adhe24 LVNQPDSIGGVGDIYTDAMRPSLTLGTGSWGKNSLSHNLSTYDLLNVKTVAKRRNRPQWV :|| |:| |: ||:|: ::||:||| | || ||:|:|::: :|||:||||:||:: |adhe_c FVNIPTSQGASGDLYNFRIPPSFTLGCGFWGGNSVSENVGPKHLLNIKTVAERRENMLWF 400 410 420 430 440 450480 490 500 510 520 530adhe24 RLPKEIYYEKNAISY-LQELPHVHK--AFIVADPGMVKFGFVDKVLEQLAIRPTQVETSI |:|:::|:: : : : |::| :::| ||||:|:: ::::||:::: |: : ::: ::adhe_c RVPHKVYFKFGCLQFALKDLKDLKKKRAFIVTDSDPYNLNYVDSIIKILE--HLDIDFKV 460 470 480 490 500 510 540 550 560 570 580 590adhe24 YGSVQPDPTLSEAIAIARQMKQFEPDTVICLGGGSALDAGKIGRLIYEYDARGEADLSDD :::| ::::|:: : :|: | |||:| |||:::::::|: :::||: | :||:adhe_c FNKVGREADLKTIKKATEEMSSFMPDTIIALGGTPEMSSAKLMWVLYEHPEVKFEDLAIK 520 530 540 550 560 570 600 610 620 630 640 650adhe24 ASLKELFQELAQKFVDIRKRIIKFYHPHKAQMVAIPTTSGTGSEVTPFAVITDDETHVKYadhe_cFMDIRKRIYTFPKLGKKAMLVAITTSAGSGSEVTPFALVTDNNTGNKYMLADYEMTPNMA 580 590 600 610 620 630adhe24: Corresponding to amino acid residues 1-656 of SEQ ID NO:5adh_c: corresponding to amino acid residues 1-630 of SEQ ID NO:11

[0107] 6. Inverse PCR to Obtain Sequences Upstream of the L. lactis DB1341 adhE Coding Sequence and Cloning of PCR Fragments

[0108] Inverse PCR was used to obtain additional sequences from the upstream region of the L. lactis DB1341 adhE gene. HindIII-, HpaI- or PvuII-digested genomic DNA of strain DB1341 was ligated at low concentration and PCR was carried out using primers adhE-350 and adhE-700 (or adhE1300x) (see FIG. 2). Sequence analysis of the obtained PCR products, using primers adhE-240 (or adhE-1300x), allowed the identification of the upstream region of the adhE gene. A 0.6 kb PCR product obtained from HindIII inverse PCR amplification was subsequently cloned into pSMA500 resulting in E. coli DH5α strain adhEup-1.

[0109] A sample of adhEup-1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession No. DSM 11091.

[0110] Further inverse PCR was carried out using PstI-digested and religated chromosomal DNA of strain DB1341, using primers derived from the above sequence. An about 5 kb PCR product was obtained which in addition to the entire coding sequence of the adhE gene comprises about 1800 bp upstream of the coding sequence. This upstream sequence includes an open reading frame, designated orfB that encodes a putative 341 aa protein having no homology to in available databases.

6TABLE 1.6DNA sequence upstream of the coding sequence of theL. lactis DB1341 adhE gene PstI1CTGCAGCTTGTTTTTTAGTACCAACAAAAAGGACTACTGCACCTTCTTGT50(SEQ ID NO:26)51GAAGCGTTTTTTACATAGTTGTAAGCATCGTCAACAAGTTTTACAGTTTT100101TTGAAGGTCGATAACGTGGATACCATTACGTTCTGTGAAGATGTATGGTT150151TCATTTTTGGGTTCCAACGACGAGTTTGGTGACCGAAGTGAACACCAGCT200201TCAAGAAGTTGTTTCATTGAAATAACTGACATGTTAATGTCTCCTTTTAA250251AATAGTTTTTCCTCTTTCATCTGTCATCCGCAGCCGCAATACTTGCGTAC300301ACTACGACTTTGTCGAGACGAAATGCGAGATGGTTGCATAGCAACTCTAT350351CATTATACATTGTTTGACCTATTTTTGCAAGTATCTATTCATGCTTCTAT400401TGTTCAGTAAATCTATTTTTCTAACCACTCCTATTATCTGACAAATTTAA450451TTGTTAATTAGGCTCTATAATCACTAAAAGAGTAAGTTTTTTAAATTTTT500501TTCTAAGAAAAAAATTAATATTTTTGCTGAAACCGCTTTTTTTGTGATAA550551AATAATTATAGTAAATAAATTAGTTTGTGAGGAGAGAAATATGAAAGAAA600 orfB M K E K(SEQ ID NO:27)601AAATCCTTTTAGGCGGCTATACAAAACGTGTATCTAAAGGCGTATATAGT650 I L L G G Y T K R V S K G V Y S651GTTCTTTTGGACACTAAAGCTGCTGAATTATCATCATTAAATGAAGTCGC700V L L D T K A A E L S S L N E V A701TGCGGTTCAAAACCCTACTTATATCACTCTCGATGAAAAGGGACACCTCT750 A V Q N P T Y I T L D E K G H L Y751ATACTTGTGCAGCAGATAGTAATGGTGGAGGAATCGCCGCCTTTGATTTT800 T C A A D S N G G G I A A F D F801GATGGCGAAACTGCTACTCATCTCGGAAATGTCACAACCACGGGAGCTCC850D G E T A T H L G N V T T T G A P851ACTCTGCTATGTTGCCGTGGACGAAGCGCGACAATTAGTTTACGGAGCGA900 L C Y V A V D E A R Q L V Y G A N901ACTATCATCTTGGAGAAGTTCGTGTTTATAAGATTCAAGCTAATGGCTCA950 Y H L G E V R V Y K I Q A N G S951CTCCGATTAACGGATACAGTAAAACATACCGGTTCTGGACCACGTCCTGA1000L R L T D T V K H T G S G P R P E1001ACAAGCTAGCTCACACGTTCATTATTCTGATTTGACTCCTGACGGACGAC1050 Q A S S H V H Y S D L T P D G R L1051TTGTCACCTGTGATTTGGGAACAGATGAAGTCACTGTTTATGATGTCATT1100 V T C D L G T D E V T V Y D V I1101GGTGAAGGTAAACTCAATATTGCTACAATTTATCGGGCAGAAAAAGGAAT1150G E G K L N I A T I Y R A E K G M1151GGGTGCTCGTCATATTACTTTCCATCCAAATGGTAAAATCGCTTATTTGG1200 G A R H I T F H P N G K I A Y L V1201TTGGAGAGTTAAATTCAACAATTGAAGTTTTAAGTTACAATGAAGAAAAA1250 G E L N S T I E V L S Y N E E K-1251GGACGCTTTGCTCGTCTTCAAACAATTAGCACCCTACCTGAAGATTATCA1300G R F A R L Q T I S T L P E D Y H1301TGGAGCAAATGGTGTTGCTGCCATCCGTATTTCATCTGACGGTAAATTCC1350 G A N G V A A I R I S S D G K F L1351TCTATACTTCTAATCGTGGACATGATTCTTTGACAACTTACAAAGTAAGT1400 Y T S N R G H D S L T T Y K V S1401CCTCTTGGTACAAAACTTGAAACTATTGGCTGGACAAATACTGAAGGTCA1450P L G T K L E T I G W T N T E G H1451TATCCCTCGCGATTTTAATTTCAACAAAACTGAAGATTATATCATTGTCG1500 I P R D F N F N K T E D Y I I V A1501CTCATCAAGAATCTGATAATTTATCTCTTTTCTTGCGAGATAAAAAAACC1550 H Q E S D N L S L F L R D K K T1551GGTACTTTAACTTTGGAACAAAAAGATTTTTACGCTCCTGAAATCACTTG1600G T L T L E Q K D F Y A P E I T C1601TGTTTTACCACTATAAAAATTTATTTTTTCACAAAGTTTGACTGATAAAC1650 V L P L Stop1651TAAAAAAGATTGCTAATTTCTCTCAAAGAATTAGCAATCTTTTTTTCTTC17001701AGTAAAGCTTGTTACAAAACCGTTTTCTAAACTTTTGATGAGTGTTTTTG17501751TAAAAACTATCACAATATTGCTTGACATCTATAAAAAACTTTGTTAAACT18001801ATTCACGTAAAAGAAAGTGAATGAAGTCACAAAGGAGAACCTACAAAT

[0111] 7. Sequence of a Fragment of the L. lactis Strain MG1363 adhE Gene

[0112] PCR was used to characterize the adhE homologue of strain MG1363. Primers adhE-mg1 and adhE-1697 were used to amplify a 1.5 kb fragment from this strain, named MGadhESTART. Primers adhE-1300x and adhE-mg2 were used to amplify an overlapping 1.5 kb fragment, named MGadhESTOP (FIG. 3).

[0113] The above fragments were subsequently cloned into the plasmid pGEM and transformed into E. coli DH5α resulting in strains MGadhESTART and MGadhESTOP, respectively. Using the relevant primers a sequence was obtained that spans from position 1306-2775 shown in Table 1.2. An additional primer adhE-mg3 (5′-CTTCTTTGGTTGGATGAGC-3′) (SEQ ID NO:7), derived from the MG1363 adhE sequence and corresponding to position 2359-2335 of the DB1341 adhE sequence (Table 1.4) was used to fill a sequence gap. A limited sequence variation at the DNA level (84 base changes, no insertion/deletions in the 1470 bp MG1363 adhE fragment, corresponding to 5.7% variation; Table 1.7 below), resulting in only 8 amino acid substitutions (or 1.6% variation; Table 1.7).

[0114] A sample of E. coli DH5α strain MGadhESTART and strain MGadhESTOP, respectively were deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession Nos DSM 11089 and DSM 11090, respectively.

7TABLE 1.7Multialignment of the deduced L. lactis AdhE proteinfrom strain MG1363 (fragment, adhemg1363) and DB1341 (adhedb13-41) with the E. coli (adhe_ec) and C. acetobutylicum (aad_ca)AdhE homologuesThe Program lineup (GCG Wisconsin package version 8, GeneticsComputer Group) was used for the alignment. The consensussequence (bold type at bottom) shows only conserved residuesfor all proteins. The differences between the two L. lactisAdhE proteins are shown as bold, underlined in adhemg1363.1 50adhemg1363.......... .......... .......... .......... ..........adhedb1341MATKKAAPAA KKVLSAEEKA AKF.QEAVAY TDKLVKKAQA AVLK.FEGYTadhe_ecMAVTNVA... ..ELNALVER VKKAQREYAS FT......QE QVDKIFRA..aad_caMKVTTVK... ..ELDEKLKV IKEAQKKFSC YS......QE MVDEIFRN..consensusM......... ...L...... .K..Q..... ........Q. .V...F....51 100adhemg1363.......... .......... .......... .......... ..........adhedb1341QTQVDTIVAA MALAASKHSL ELAHEAVNET GRGVVEDKDT KNHFASESVYadhe_ec.......... AALAAADARI PLAKMAVAES GMGIVEDKVI KNHFASEYIYaad_ca.......... AAMAAIDARI ELAKAAVLET GMGLVEDKVI KNHFAGEYIYconsensus.......... .A.AA..... .LA..AV.E. G.G.VEDK.. KNHFA.E..Y101 150adhemg1363.......... .......... .......... .......... ..........adhedb1341NAIKNDKTVG VISENKVAGS VEIASPLGVL AGIVPTTNPT STAIFKSLLTadhe_ecNAYKDEKTCG VLSEDDTFGT ITIAEPIGII CGIVPTTNPT STAIFKSLISaad_caNKYKDEKTCG IIERNEPYGI TKIAEPIGVV AAIIPVTNPT STTIFKSLISconsensusN..K..KT.G ........G. ..IA.P.G.. ..I.P.TNPT ST.IFKSLI.151 200adhemg1363.......... .......... .......... .......... ..........adhedb1341AKTRNAIVFA FHPQAQKCSS HAAKIVYDAA IEAGAPEDFI QWIEVPSLDMadhe_ecLKTRNAIIFS PHPRAKDATN KAADIVLQAA IAAGAPKDLI GWIDQPSVELaad_caLKTRNGIFFS PHPRAKKSTI LAAKTILDAA VKSGAPENII GWIDEPSIELconsensus.KTRN..... .HP.A..... .AA.....AA ...GAP...I .WI..PS...201 250adhemg1363.......... .......... .......... .......... ..........adhedb1341TTALIQNRGL ATILATGGPG MVNAALKSGN PSLGVGAGNG AVYVDATANIadhe_ecSNALMHHPDI NLILATGGPG MVKAAYSSGK PAIGVGAGNT PVVIDETADIaad_caTQYLMQKADI T..LATGGPS LVKSAYSSGK PAIGVGPGNT PVIIDESAHIconsensus...L...... ...LATGGP. .VK.A..SG. P.IGVG.GN. .V..D..A.I251 300adhemg1363.......... .......... .......... .......... ..........adhedb1341ERAVEDLLLS KRFDNGMICA TENSAVIDAS VYDEFIAKMQ EQGAYMVPKKadhe_ecKRAVASVLMS KTFDNGVICA SEQSVVVVDS VYDAVRERFA THGGYLLQGKaad_caKMAVSSIILS KTYDNGVICA SEQSVIVLKS IYNKVKDEFQ ERGAYIIKKNconsensus..AV.....S ...DNG.ICA .E.S.....S .Y.......G ..G.Y.....301 350adhemg1363.......... .......... .......... .......... ..........adhedb1341DYKAIESFVF VERAGEGFGV TGPVAGRSGQ WIAEQAGVKV PKDKDVLLFEadhe_ecELKAVQDVIL ..KNG...AL NAAIVGQPAY KIAELAGFSV PENTKILIGEaad_caELDKVREVIF ..KDG...SV NPKIVGQSAY TIAAMAGIKV PKTTRILIGEconsensus.......... ....G..... .....G.... .IA..AG..V P.....LIGE351 400adhemg1363.......... .......... .......... .........Y QGAGHNAAIQadhedb1341LDKKNIGEAL SSEKLSPLLS IYKAETREEG IEIVRSLLAY QGAGHNAAIQadhe_ecVTVVDESEPF AHEKLSPTLA MYRAKDFEDA VEKAEKLVAM GGIGHTSCLYaad_caVTSLGEEEPF AHEKLSPVLA MYEADNFDDA LKKAVTLINL GGLGHTSGIYconsensus.......E.. ..EKLSP.L. .Y.A...... ......L... .G.GH.....401 450adhemg1363IGAMDDP.FV KEYGIKVEAS RILVNQPDSI GGVGDIYTDA MRPSLTLGTGadhedb1341IGAMDDP.FV KEYGEKVEAS RILVNQPDSI GGVGDIYTDA MRPSLTLGTGadhe_ecTDQDNQPARV SYFGQKMKTA RILINTPASQ GGIGDLYNFK LAPSLTLGCGaad_caADEIKARDKI DRFSSAMKTV RTFVNIPTSQ GASGDLYNFR IPPSFTLGCGconsensus.......... .......... R...N.P.S. G..GD.Y... ..PS.TLG.G451 500adhemg1363SWGKNSLSHN LSTYDLLNVK TVAKRRNRPQ WVRLPKEIYY EKNAISYLQEadhedb1341SWGKNSLSHN LSTYDLLNVK TVAKRRNRPQ WVRLPKEIYY EKNAISYLQEadhe_ecSWGGNSISEN VGPKHLINKK TVAKRAENML WHKLPKSIYF RRGSLPIALDaad_caFWGGNSVSEN VGPKHLLNIK TVAERRENML WFRVPHKVYF KFGCLQFALKconsensus.WG.NS.S.N .....L.N.K TVA.R..... W...P...Y. ..........501 550adhemg1363LPHVHK...A FIVADPGMVK FGFVDKVLEQ LAIRPTQVET SIYGSVQPDPadhedb1341LPHVHK...A FIVADPGMVK FGFVDKVLEQ LAIRPTQVET SIYGSVQPDPadhe_ecEVITDGHKRA LIVTDRFLFN NGYADQITSV L..KAAGVET EVFFEVEADPaad_caDLKDLKKKRA FIVTDSDPYN LNYVDSIIKI L..EHLDIDF KVFNKVGREAconsensus.......... .IV.D..... ....D..... L......... .....V....551 600adhemg1363TLSEAIAIAR QMNHFEPDTV ICLGGGSALD AGKIGRLIYE YDARGEADLSadhedb1341TLSEAIAIAR QMKQFEPDTV ICLGGGSALD AGKIGRLIYE YDARGEADLSadhe_ecTLSIVRKGAE LANSFKPDVI IALGGGSPMD AAKIMWVMYE ...HPETH..aad_caDLKTIKKATE EMSSFMPDTI IALGGTPEMS SAKLMWVLYE ...HPEVK..consensus.S........ ....F.PD.. I.LGG..... ..K.....YE .....E....601 650adhemg1363DDASLKEIFQ ELAQKFVDIR KRIIKFYH.P HKAQMVAIPT TSGTGSEVTPadhedb1341DDASLKELFQ ELAQKFVDIR KRIIKFYH.P HKAQMVAIPT TSGTGSEVTPadhe_ec........FE ELALRFMDIR KRIYKFPKMG VKAKMIAVTT TSGTGSEVTPaad_ca........FE DLAIKFMDIR KRIYTFPKLG KKAMLVAITT SAGSGSEVTPconsensus........F. .LA..F.DIR KRI..F.... .KA...A..T ....GSEVTP651 700adhemg1363FAVITDDETH VKYPLADYQL TPQVAIVDPE FVMTVPKRTV SWSGIDAMSHadhedb1341FAVITDDETH VKYPLADYQL TPQVAIVDPE FVMTVPKRTV SWSGIDAMSHadhe_ecFAVVTDDATG QKYPLADYAL TPDMAIVDAN LVMDMPKSLC AFGGLDAVTHaad_caFALVTDNNTG NKYMLADYEM TPNMAIVDAE LMMKMPKGLT AYSGIDALVNconsensusFA..TD..T. .KY.LADY.. TP..AIVD.. ..M..PK... ...G.DA...701 750adhemg1363ALESYVSVMS SDYTKPISLQ AIKLIFENLT ESYEYDPAHP TKEGQKARENadhedb1341ALESYVSVMS SDYTKPISLQ AIKLIFENLT ESYHYDPAHP TKEGQKARENadhe_ecAMEAYVSVLA SEFSDGQALQ ALKLLKEYLP ASYHEGSKNP .....VARERaad_caSIEAYTSVYA SEYTNGLALE AIRLIFKYLP EAYKNGRTNE .....KAREKconsensus..E.Y.SV.. S.......L. AI.L....L. ..Y....... ......ARE.751 800adhemg1363MHNAATLAGM AFANAFLGIN HSLAHKIAGE FGLPHGLAIA IAMPHVIKFNadhedb1341MHNAATLAGM AFANAFLGIN HSLAHKIGGE FGLPHGLAIA IAMPHVIKFNadhe_ecVHSAATIAGI AFANAFLGVC HSMAHKLGSQ FHIPHGLANA LLICNVIRYNaad_caMAHASTMAGM ASANAFLGLC HSMAIKLSSE HNIPSGIANA LLIEEVIKFNconsensus...A.T.AG. A.ANAFLG.. HSMA.K.... ...P.G.A.A .....VI..N801 850adhemg1363AVTGNVKTP YPRYETYRAQ EDYAEISRFM GFAGKEDSDE KAVKAFVAELadhedb1341AVTGNVKRTP YPRYETYRAQ EDYAEISRFM GFAGKDDSDE KAVQALVAELadhe_ecANDNPTKQTA FSQYDRPQAR RRYAEIADHL GLSAPGDRTA AKIEKLLAWLaad_caAVDNPVKQAP CPQYKYPNTI FRYARIADYI KLGGNTDEEK VDLLINKIHEconsensusA.....K... ...Y...... ..YA.I.... ......D... ..........851 900adhemg1363KKLTDSIDIN ITLSGN..GV DKAHLERELD KLADLVadhedb1341KKLTDSIDIN ITLSGN..GI DKAHLERELD KLADLVYDDQ CTPANPRQPRadhe_ecETLKA..ELG IPKSIREAGV QEADFLANVD KLSEDAFDDQ CTGANPRYPLaad_caLKKAL....N IPTSIKDAGV LEENFYSSLD RISELALDDQ CTGANPRFPLconsensus.......... I..S....G. .........D .......DDQ CT.ANPR.P.901 941adhedb1341IDEIKQLLLD QY*adhe_ecISELKQILLD TYYGPDYVEG ETAAKKEAAP AKAEKKAKKS Aaad_caTSEIKEMYIN CFKKQPconsensus..E.K..... .......... .......... .......... .adhemg1363: SEQ ID NO:8; adhedb1341: SEQ ID NO:9; adhe_ec: SEQ ID NO:10; aad_ca: SEQ ID NO:11

[0115]

8

TABLE 1.8

Alignment of the adhE sequences from L. lactis

DB1341 and MG1363

The complete sequence of the adhE gene of strain DB1341 is

compared to the sequence obtained via PCR amplification of

MG1363 adhE fragments (see FIG. 2).

1 50

adhemg1363
.......... .......... .......... .......... ..........
(SEQ ID NO:12)

adhedb1341
AAGCTTGTTA CAAAACCGTT TTCTAAACTT TTGATGAGTG TTTTTGTAAA
(SEQ ID NO:13)

consensus
.......... .......... .......... .......... ..........

51 100

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
AACTATCACA ATATTGCTTG ACATCTATAA AAAACTTTGT TAAACTATTC

consensus
.......... .......... .......... .......... ..........

101 150

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
ACGTAAAAGA AAGTGAATGA AGTCACAAAG GAGAACCTAC AAATATGGCA

consensus
.......... .......... .......... .......... ..........

151 200

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
ACTAAAAAAG CCGCTCCAGC TGCAAAGAAA GTTTTAAGCG CTGAAGAAAA

consensus
.......... .......... .......... .......... ..........

201 250

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
AGCCGCAAAA TTCCAAGAAG CTGTTGCTTA TACTGACAAA TTAGTCAAAA

consensus
.......... .......... .......... .......... ..........

251 300

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
AAGCACAAGC TGCTGTTCTT AAATTTGAAG GATATACACA AACTCAAGTC

consensus
.......... .......... .......... .......... ..........

301 350

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
GATACTATTG TCGCTGCAAT GGCTCTTGCA GCAAGCAAAC ATTCTCTAGA

consensus
.......... .......... .......... .......... ..........

351 400

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
ACTCGCTCAT GAAGCCGTTA ACGAAACTGG TCGTGGTGTT GTCGAAGACA

consensus
.......... .......... .......... .......... ..........

401 450

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
AAGATACCAA AAACCACTTT GCTTCTGAAT CTGTTTATAA CGCAATTAAA

consensus
.......... .......... .......... .......... ..........

451 500

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
AATGACAAAA CTGTTGGTGT CATTTCTGAA AACAAGGTTG CTGGATCTGT

consensus
.......... .......... .......... .......... ..........

501 550

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
TGAAATCGCA AGCCCTCTCG GTGTACTTGC TGGTATCGTT CCAACGACTA

consensus
.......... .......... .......... .......... ..........

551 600

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
ATCCAACATC AACAGCAATC TTTAAATCTT TATTGACTGC AAAAACACGT

consensus
.......... .......... .......... .......... ..........

601 650

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
AATGCTATTG TTTTCGCTTT CCACCCTCAA GCTCAAAAAT GTTCAAGCCA

consensus
.......... .......... .......... .......... ..........

651 700

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
TGCAGCAAAA ATTGTTTACG ATGCTGCAAT TGAAGCTGGT GCACCGGAAG

consensus
.......... .......... .......... .......... ..........

701 750

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
ACTTTATTCA ATGGATTGAA GTACCAAGCC TTGACATGAC TACCGCCTTG

consensus
.......... .......... .......... .......... ..........

751 800

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
ATTCAAAACC GTGGACTTGC AACAATCCTT GCAACTGGTG GCCCAGGAAT

consensus
.......... .......... .......... .......... ..........

801 850

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
GGTAAACGCC GCACTCAAAT CTGGTAACCC TTCACTCGGT GTTGGAGCTG

consensus
.......... .......... .......... .......... ..........

851 900

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
GTAATGGTGC TGTTTATGTT GATGCAACTG CAAATATTGA ACGTGCCGTT

consensus
.......... .......... .......... .......... ..........

901 950

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
GAAGACCTTT TGCTTTCAAA ACGTTTTGAT AATGGGATGA TTTGTGCCAC

consensus
.......... .......... .......... .......... ..........

951 1000

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
TGAAAATTCA GCTGTTATTG ATGCTTCAGT TTATGATGAA TTTATTGCTA

consensus
.......... .......... .......... .......... ..........

1001 1050

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
AAATGCAAGA ACAAGGCGCT TATATGGTTC CTAAAAAAGA CTACAAAGCT

consensus
.......... .......... .......... .......... ..........

1051 1100

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
ATTGAAAGTT TCGTTTTTGT TGAACGTGCT GGTGAAGGTT TTGGAGTAAC

consensus
.......... .......... .......... .......... ..........

1101 1150

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
TGGTCCTGTT GCCGGTCGTT CTGGTCAATG GATTGCTGAA CAAGCTGGTG

consensus
.......... .......... .......... .......... ..........

1151 1200

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
TCAAAGTTCC TAAAGATAAA GATGTCCTTC TTTTTGAACT TGATAAGAAA

consensus
.......... .......... .......... .......... ..........

1201 1250

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
AATATTGGTG AAGCACTTTC TTCTGAAAAA CTTTCTCCTT TGCTTTCAAT

consensus
.......... .......... .......... .......... ..........

1251 1300

adhemg1363
.......... .......... .......... .......... ..........

adhedb1341
CTACAAAGCT GAAACACGTG AAGAAGGAAT TGAGATTGTA CGTAGCTTAC

consensus
.......... .......... .......... .......... ..........

1301 1350

adhemg1363
.....TACCA AGGAGCTGGT CACAACGCTG CAATTCAAAT CGGTGCAATG

adhedb1341
TTGCTTATCA AGGTGCTGGA CATAATGCTG CAATTCAAAT CGGTGCAATG

consensus
.....TA.CA AGG.GCTGG. CA.AA.GCTG CAATTCAAAT CGGTGCAATG

1351 1400

adhemg1363
GACGACCCAT TTGTCAAAGA ATACGGAATT AAAGTCGAAG CTTCTCGTAT

adhedb1341
GATGATCCAT TCGTTAAAGA ATATGGCGAA AAAGTTGAAG CTTCTCGTAT

consensus
GA.GA.CCAT T.GT.AAAGA ATA.GG.... AAAGT.GAAG CTTCTCGTAT

1401 1450

adhemg1363
CCTCGTTAAC CAACCTGACT CTATCGGTGG GGTCGGAGAT ATTTATACTG

adhedb1341
CCTCGTTAAC CAACCAGATT CTATTGGTGG GGTCGGAGAT ATCTATACTG

consensus
CCTCGTTAAC CAACC.GA.T CTAT.GGTGG GGTCGGAGAT AT.TATACTG

1451 1500

adhemg1363
ATGCAATGCG TCCATCATTG ACGCTCGGAA CTGGTTCATG GGGGAAAAAT

adhedb1341
ATGCAATGCG TCCATCACTT ACACTTGGAA CTGGTTCATG GGGGAAAAAT

consensus
ATGCAATGCG TCCATCA.T. AC.CT.GGAA CTGGTTCATG GGGGAAAAAT

1501 1550

adhemg1363
TCACTTTCAC ACAATTTGAG TACATACGAT CTATTGAATG TTAAAACAGT

adhedb1341
TCACTTTCAC ACAATTTGAG TACATACGAT CTATTGAATG TTAAAACAGT

consensus
TCACTTTCAC ACAATTTGAG TACATACGAT CTATTGAATG TTAAAACAGT

1551 1600

adhemg1363
GGCTAAACGT CGTAATCGCC CTCAATGGGT TCGTTTGCCA AAAGAAATTT

adhedb1341
GGCTAAACGT CGTAATCGCC CACAATGGGT TCGTTTGCCA AAAGAAATTT

consensus
GGCTAAACGT CGTAATCGCC C.CAATGGGT TCGTTTGCCA AAAGAAATTT

1601 1650

adhemg1363
ACTACGAAAA AAATGCAATT TCTTACTTAC AAGAATTGCC ACACGTCCAC

adhedb1341
ACTACGAAAA AAATGCAATT TCTTACTTAC AAGAATTGCC ACACGTCCAC

consensus
ACTACGAAAA AAATGCAATT TCTTACTTAC AAGAATTGCC ACACGTCCAC

1651 1700

adhemg1363
AAAGCTTTCA TTGTTGCCGA CCCTGGTATG GTTAAATTCG GTTTCGTTGA

adhedb1341
AAAGCTTTCA TCGTTGCTGA CCCTGGTATG GTTAAATTTG GTTTCGTTGA

consensus
AAAGCTTTCA T.GTTGC.GA CCCTGGTATG GTTAAATT.G GTTTCGTTGA

1701 1750

adhemg1363
TAAAGTTTTG GAACAACTTG CTATCCGCCC AACTCAAGTT GAAACAAGCA

adhedb1341
TAAAGTTTTG GAACAACTTG CTATCCGCCC AACTCAAGTT GAAACAAGCA

consensus
TAAAGTTTTG GAACAACTTG CTATCCGCCC AACTCAAGTT GAAACAAGCA

1751 1800

adhemg1363
TTTATGGCTC AGTCCAACCT GACCCAACTT TGAGTGAAGC AATTGCAATC

adhedb1341
TTTATGGCTC TGTTCAACCT GACCCAACTT TGAGCGAAGC AATTGCAATC

consensus
TTTATGGCTC .GT.CAACCT GACCCAACTT TGAG.GAAGC AATTGCAATC

1801 1850

adhemg1363
GCTCGTCAAA TGAACCATTT TGAACCTGAC ACTGTCATCT GTCTTGGTGG

adhedb1341
GCTCGTCAAA TGAAACAATT TGAACCTGAC ACTGTCATCT GTCTTGGTGG

consensus
GCTCGTCAAA TGAA.CA.TT TGAACCTGAC ACTGTCATCT GTCTTGGTGG

1851 1900

adhemg1363
TGGTTCTGCT CTCGATGCTG GTAAGATTGG TCGTTTGATT TATGAATATG

adhedb1341
TGGTTCTGCT CTCGATGCCG GTAAGATTGG TCGTTTGATT TATGAATATG

consensus
TGGTTCTGCT CTCGATGC.G GTAAGATTGG TCGTTTGATT TATGAATATG

1901 1950

adhemg1363
ATGCTCGTGG TGAGGCTGAC CTTTCCGATG ACGCAAGTTT GAAAGAGATC

adhedb1341
ATGCTCGTGG TGAAGCTGAC CTTTCTGATG ATGCAAGTTT GAAAGAACTT

consensus
ATGCTCGTGG TGA.GCTGAC CTTTC.GATG A.GCAAGTTT GAAAGA..T.

1951 2000

adhemg1363
TTCCAAGAGT TAGCTCAAAA ATTTGITGAT ATTCGTAAAC GTATTATCAA

adhedb1341
TTCCAAGAAT TAGCTCAAAA ATTTGTCGAT ATTCGTAAAC GTATTATTAA

consensus
TTCCAAGA.T TAGCTCAAAA ATTTGT.GAT ATTCGTAAAC GTATTAT.AA

2001 2050

adhemg1363
ATTCTACCAC CCACACAAAG CACAAATGGT TGCTATCCCT ACTACTTCTG

adhedb1341
ATTCTACCAT CCACATAAAG CACAAATGGT TGCAATTCCT ACTACTTCTG

consensus
ATTCTACCA. CCACA.AAAG CACAAATGGT TGC.AT.CCT ACTACTTCTG

2051 2100

adhemg1363
GTACTGGTTC TGAAGTGACT CCATTTGCGG TTATCACTGA TGATGAAACT

adhedb1341
GTACTGGTTC TGAAGTGACT CCATTTGCAG TTATCACTGA TGATGAAACT

consensus
GTACTGGTTC TGAAGTGACT CCATTTGC.G TTATCACTGA TGATGAAACT

2101 2150

adhemg1363
CACGTTAAAT ATCCACTTGC TGACTATCAA TTGACACCTC AAGTTGCCAT

adhedb1341
CATGTTAAGT ACCCACTTGC TGACTACCAA TTAACACCAC AAGTTGCCAT

consensus
CA.GTTAA.T A.CCACTTGC TGACTA.CAA TT.ACACC.C AAGTTGCCAT

2151 2200

adhemg1363
TGTTGACCCT GAGTTTGTTA TGACTGTACC AAAACGTACT GTTTCTTGGT

adhedb1341
TGTTGACCCT GAGTTTGTTA TGACTGTACC AAAACGTACT GTTTCTTGGT

consensus
TGTTGACCCT GAGTTTGTTA TGACTGTACC AAAACGTACT GTTTCTTGGT

2201 2250

adhemg1363
CTGGGATTGA TGCTATGTCA CACGCGCTTG AATCTTATGT TTCTGTCATG

adhedb1341
CTGGTATTGA TGCGATGTCA CACGCGCTTG AATCTTACGT TTCTGTTATG

consensus
CTGG.ATTGA TGC.ATGTCA CACGCGCTTG AATCTTA.GT TTCTGT.ATG

2251 2300

adhemg1363
TCTTCTGACT ATACAAAACC AATTTCACTT CAAGCCATCA AACTCATCTT

adhedb1341
TCTTCTGACT ATACAAAACC AATTTCACTT CAAGCGATCA AACTTATCTT

consensus
TCTTCTGACT ATACAAAACC AATTTCACTT CAAGC.ATCA AACT.ATCTT

2301 2350

adhemg1363
TGAAAACTTG ACTGAGTCTT ATCATTATGA CCCAGCTCAT CCAACCAAAG

adhedb1341
TGAAAACTTG ACTGAGTCTT ATCATTATGA CCCAGCGCAT CCAACTAAAG

consensus
TGAAAACTTG ACTGAGTCTT ATCATTATGA CCCAGC.CAT CCAAC.AAAG

2351 2400

adhemg1363
AAGGTCAAAA AGCTCGCGAA AACATGCACA ATGCTGCAAC ACTCGCTGGT

adhedb1341
AAGGACAAAA AGCCCGCGAA AACATGCACA ATGCTGCAAC ACTCGCTGGT

consensus
AAGG.CAAAA AGC.CGCGAA AACATGCACA ATGCTGCAAC ACTCGCTGGT

2401 2450

adhemg1363
ATGGCCTTCG CCAATGCTTT CCTTGGAATT AACCACTCAC TTGCTCATAA

adhedb1341
ATGGCCTTCG CTAATGCTTT CCTTGGAATT AACCACTCAC TTGCTCATAA

consensus
ATGGCCTTCG C.AATGCTTT CCTTGGAATT AACCACTCAC TTGCTCATAA

2451 2500

adhemg1363
AATTGCTGGT GAATTTGGGC TTCCTCATGG TCTTGCCATT GCTATCGCTA

adhedb1341
AATTGGTGGT GAATTTGGAC TTCCTCATGG TCTTGCCATT GCCATCGCTA

consensus
AATTG.TGGT GAATTTGG.C TTCCTCATGG TCTTGCCATT GC.ATCGCTA

2501 2550

adhemg1363
TGCCACATGT CATTAAATTT AACGCTGTAA CAGGAAACGT TAAATTTACC

adhedb1341
TGCCACATGT CATTAAATTT AACGCTGTAA CAGGAAACGT TAAACGTACC

consensus
TGCCACATGT CATTAAATTT AACGCTGTAA CAGGAAACGT TAAA..TACC

2551 2600

adhemg1363
CCTTACCCAC GTTATGAAAC TTATCGTGCG CAAGAAGACT ACGCTGAAAT

adhedb1341
CCTTACCCAC GTTATGAAAC ATATCGTGCT CAAGAGGACT ACGCTGAAAT

consensus
CCTTACCCAC GTTATGAAAC .TATCGTGC. CAAGA.GACT ACGCTGAAAT

2601 2650

adhemg1363
TTCACGCTTC ATGGGATTTG CTGGCAAAGA AGATTCAGAT GAAAAAGCGG

adhedb1341
TTCACGCTTC ATGGGATTTG CTGGTAAAGA TGATTCAGAT GAAAAAGCTG

consensus
TTCACGCTTC ATGGGATTTG CTGG.AAAGA .GATTCAGAT GAAAAAGC.G

2651 2700

adhemg1363
TCAAAGCTTT TGTTGCTGAA CTTAAAAAAT TGACTGATAG TATTGATATT

adhedb1341
TGCAAGCTCT GGTTGCTGAA CTTAAGAAAC TGACTGATAG CATTGATATT

consensus
T..AAGCT.T .GTTGCTGAA CTTAA.AAA. TGACTGATAG .ATTGATATT

2701 2750

adhemg1363
AATATCACCC TTTCAGGAAA TGGTGTAGAT AAAGCTCACC TTGAACGTGA

adhedb1341
AATATCACCC TTTCAGGAAA TGGTATCGAT AAAGCTCACC TTGAACGTGA

consensus
AATATCACCC TTTCAGGAAA TGGT.T.GAT AAAGCTCACC TTGAACGTGA

2751 2800

adhemg1363
GCTTGATAAA TTGGCTGACC TTGTT

adhedb1341
ACTTGATAAA TTGGCTGACC TTGTTTATGA TGATCAATGT ACTCCTGCTA

consensus
.CTTGATAAA TTGGCTGACC TTGTT..... .......... ..........

2801 2850

adhedb1341
ATCCTCGTCA ACCAAGAATT GATGAGATTA AACAGTTGTT GTTAGATCAA

consensus
.......... .......... .......... .......... ..........

2851 2900

adhedb1341
TACTAATAAT CTGTTGATAA AATTATTAAA ACGCTCTGAT GAATTCGTCA

consensus
.......... .......... .......... .......... ..........

2901 2950

adhedb1341
GAGCATTTTT TATTATAGCT TATACAACTA TCAAAAGGTA TAAATCAATT

consensus
.......... .......... .......... .......... ..........

2951 3000

adhedb1341
TCGATATAGG CTCTTTTCAC TCCATTGATT TATGCATTTC TATAAAAATC

consensus
.......... .......... .......... .......... ..........

3001 3050

adhedb1341
AATAATTAAT TAGCGATAGA AGTCGAGTTC ATGCATGCTA ATAATGAAAT

consensus
.......... .......... .......... .......... ..........

3051 3100

adhedb1341
TGTTTTAAAT TCTGGTTTTT CTTTATGTTC TTTGCGAACA TCTTTCACAG

consensus
.......... .......... .......... .......... ..........

3101 3150

adhedb1341
TTTCTTTGTT CATGAAAATT CCTCCTTATT ATGGTACTAT TTTGAGCCCA

consensus
.......... .......... .......... .......... ..........

3151 3193

adhedb1341
AATAGTTATA TAAGAATCCT AAACTTCGGA TATCTTATCA AAG

consensus
.......... .......... .......... .......... ...

[0116] 8. Obtaining and Sequencing the Entire adhE Locus from L. lactis Strain MG1363

[0117] Inverse PCR was carried out on digested and religated chromosomal DNA of strain MG1363, using primers adhE-146 and adhE-MG5 (see FIG. 5). A PCR fragment was obtained which in addition to the above fragment of the MG1363 adhE sequence comprised an about 2.9 kb sequence upstream of that fragment including the 5′-end of the adhE coding sequence and and open reading frame, designated orfB showing a high homology with the corresponding open reading frame from strain DB1341.

[0118] The entire sequence of the adhE locus of Lactococcus lactis strain MG1363 is shown in Table 1.9 below.

9TABLE 1.9The adhE locus of strain MG1363 1 TTTGGTGACCGAAGTGAACACCAGCTTCAAGAAGTTGTTTCATTGAAATA 50(SEQ ID NOS:28/30) 51 ACTGACATGTTAATGTCTCCTTTTAAAATAGTTTTTCCTCTTTCATCTGT 100 101 CATCCGCAGCCGCAATACTTGCGTACACTACGACTTTGTCGAGACGAAAT 150 151 GCGAGATGGTTGCATAGCAACTCTCTCATTATACATTGTTTAAGCTACTT 200 201 TTGCAAGCATCTATTCATTTATTTCTTTTATCAATATGAGTAAATGAAAG 250 251 CTATCCTACCCCCCTTTCTTTTTATTCTGTTTTTTATATCTCAATGTTGT 300 301 CTGACAAATTTAACGAATATTTTTGCCTATATAATCCCCATAAGGGAGAT 350 351 TTTTACATTTTTTTCTAAGAATAAAATTAATATTTTTGCTGAAAACGCTT 400 401 TTTTTGTGATAAAATAATTATAGTAAATAAAATAGTTTGTGAGGAGAGAA 450 451 ATATGAAAGAAAAAATCCTTTTAGGCGGTTATACTAAACGTGTATCTAAA 500 orfB M K E K I L L G G Y T K R V S K(SEQ ID NO:29) 501 GGCGTTTACAGTGTTCTATTAGATAGCAAGAAAGCTGAATTGTCGGCTTT 550 G V Y S V L L D S K K A E L S A L Sau3AI 551 AACTGAAGTTGCAGCGGTTCAAAATCCAACTTATATCACTCTTGATCAAA 600 T E V A A V Q N P T Y I T L D Q K 601 AAGGGCACCTCTACACTTGTGCTGCTGATGGAAATGGTGGTGGAATTGCT 650 G H L Y T C A A D G N G G G I A 651 GCCTTTGATTTCGATGGTCAAAATACAACTCACCTAGGGAATGTAACGAG 700 A F D F D G Q N T T H L G N V T S 701 TACTGGAGCCCCTTTGTGTTATGTGGCTGTTGATGAAGCACGTCAACTCG 750 T G A P L C Y V A V D E A R Q L V 751 TTTATGGTGCCAACTATCACTTGGGTGAAGTTCGTGTGTACAAAATTCAA 800 Y G A N Y H L G E V R V Y K I Q 801 GCTGATGGTTCCCTTAGATTAACCGATACAGTTAAACATAATGGTTCTGG 850 A D G S L R L T D T V K H N G S G 851 CCCTCGACCTGAGCAAGCAAGTTCTCATGTCCATTACTCTGATTTAACTC 900 P R P E Q A S S H V H Y S D L T P 901 CAGATGGTCGTCTTGTTACTTGTGATTTAGGTACAGATGAAGTGACTGTT 950 D G R L V T C D L G T D E V T V 951 TACGATGTTATTGGTGAAGGTAAACTCAATATCGTTACGATTTATCGTGC 1000 Y D V I G E G K L N I V T I Y R A1001 CGAAAAAGGAATGGGAGCTCGTCACATCAGCTTCCATCCTAATGGAAAAA 1050 E K G M G A R H I S F H P N G K I1051 TTGCTTATCTCGTCGGAGAATTAAATTCAACTATTGAAGTTCTAAGCTAT 1100 A Y L V G E L N S T I E V L S Y1101 AATGAAGAAAAAGGACGATTCGCTCGTCTTCAAACAATCAGTACTTTACC 1150 N E E K G R F A R L Q T I S T L P1151 TGAAGACTATCACGGAGCCAATGGAGTAGCTGCTATTCGAATTTCTTCTG 1200 E D Y H G A N G V A A I R I S S D1201 ATGGTAAGTTCCTCTATGCTTCTAATCGTGGGCACGACTCTTTAGCAATT 1250 G K F L Y A S N R G H D S L A I1251 TACAAGGTAAGTCCTCTCGGAACAAAATTAGAATCTATTGGTTGGACAAA 1300 Y K V S P L G T K L E S I G W T K1301 GACTGAATATCATATTCCACGCGATTTTAATTTTAATAAAACCGAAGATT 1350 T E Y H I P R D F N F N K T E D Y1351 ATATCATTGTCGCTCATCAAGAATCTGATAATTTAACTCTTTTCTTGAGA 1400 I I V A H Q E S D N L T L F L R1401 GATAAAAATACAGGGTCATTAACGTTAGAACAAAAAGACTTTTACGCTCC 1450 D K N T G S L T L E Q K D F Y A P1451 TGAAATTACTTGTGTTTTACCTTTGTAAAAACTAAACTTTAGTAAATCTT 1500 E I T C V L P L Stop1501 GCTTTTGTTTTTTCACAAAGTTTTACTAAATCAGACAAAAAAATATTGCC 15501551 AAATCTTTAAAAGGATTGGCAATATTTTTTTGTCTGAAACCCTTGCTTAT 16001601 AAAGCGATTTCTAAAAGTTTGATGAGTTTTTTTGTAAATTTCATCACAAT 16501651 ATCGCTTGACTTCTTTAAAAAACTTTGTTAAACTATTCACGTAAAAGAAA 17171701 GTGAATGGAATCACAAAGGAGAACGTACACATATGGCAACTAAAAAAGCC 1750 adhE M A T K K A(SEQ ID NO:31)1751 GCTCCAGCTGCAAAGAAAGTTTTAAGCGCTGAAGAAAAAGCCGCAAAATT 1800 A P A A K K V L S A E E K A A K F Sau3AI1801 CCAAGGAAGTGTCGCTTATACTGATCAATTAGTCAAAAAAGCTCAAGCTG 1850 Q G S V A Y T D Q L V K K A Q A A1851 CAGTTCTTAAATTTGAAGGATACACACAAACTCAAGTTGATACTATTGTT 1900 V L K F E G Y T Q T Q V D T I V1901 GCTGCAATGGCTCTTGCAGCAAGCAAACATTCTCTGGAACTCGCTCACGA 1950 A A M A L A A S K H S L E L A H E1951 AGCCGTTAATGAAACTGGCCGTGGAGTTGTTGAGGACAAAGATACAAAAA 2000 A V N E T G R G V V E D K D T K N2001 ACCATTTTGCTTCTGAATCTGTTTATAATGCAATCAAAAATGATAAAACA 2050 H F A S E S V Y N A I K N D K T2051 GTTGGCGTTATCGCTGAAAACAAAGTTGCTGGTTCTGTTGAAATCGCAAG 2100 V G V I A E N K V A G S V E I A S2101 CCCCCTTGGAGTACTTGCTGGTATTGTCCCAACAACTAATCCAACATCAA 2150 P L G V L A G I V P T T N P T S T2151 CAGCCATCTTTAAATCATTATTAACTGCAAAGACACGTAATGCTATTGTC 2200 A I F K S L L T A K T R N A I V2201 TTTGCCTTTCACCCACAAGCACAAAAATGCTCAAGCCATGCGGCAAAAAT 2250 F A F H P Q A Q K C S S H A A K I2251 TGTTTATGATGCTGCGATTGAAGCTGGTGCACCTGAAGACTTTATTCAAT 2300 V Y D A A I E A G A P E D F I Q W2301 GGATTGAAGTACCCAGTCTTGATATGACGACTGCTTTGATTCAAAATAGA 2350 I E V P S L D M T T A L I Q N R2351 GGAATTGCTACAATTCTTGCAACTGGTGGTCCAGGTATGGTCAATGCCGC 2400 G I A T I L A T G G P G M V N A A2401 GCTTAAGTCTGGTAATCCTTCACTTGGTGTAGGTGCTGGTAATGGTGCAG 2450 L K S G N P S L G V G A G N G A V Sau3AI Sau3AI2451 TTTATGTTGATGCAACTGCAAATATCGATCGTGCTGTTGAAGATCTTTTG 2500 Y V D A T A N I D R A V E D L L2501 CTTTCAAAACGTTTTGATAACGGAATGATTTGTGCGACTGAAAACTCTGC 2550 L S K R F D N G M I C A T E N S A2551 AGTTATTGATGCATCAATCTATGATGAATTTGTCGCTAAAATGCCAACGC 2600 V I D A S I Y D E F V A K M P T Q2601 AAGGCGCTTATATGGTTCCTAAAAAAGATTACAAGGCAATTGAAAGTTTT 2650 G A Y M V P K K D Y K A I E S F2651 GTTTTCGTTGAACGTGCTGGTGAAGGTTTTGGTGTAACTGGTCCTGTTGC 2700 V F V E R A G E G F G V T G P V A2701 TGGTCGTTCTGGTCAATGGATTGCTGAACAAGCTGGTGTTAACGTCCCTA 2750 G R S G Q W I A E Q A G V N V P K2751 AAGATAAAGATGTTCTTCTTTTTGAACTTGATAAGAAAAATATTGGGGAA 2800 D K D V L L F E L D K K N I G E2801 GCTCTTTCTTCTGAAAAACTTTCTCCTTTGCTTTCAATCTACAAATCAGA 2850 A L S S E K L S P L L S I Y K S E2851 AACACGTGAAGAAGGAATTGAAATTGTACGTAGCTTACTTGCTTACCAAG 2900 T R E E G I E I V R S L L A Y Q G2901 GAGCTGGTCACAACGCTGCCATTCAAATCGGTGCAATGGACGACCCATTT 2950 A G H N A A I Q I G A M D D P F2951 GTCAAAGAATACGGAATTAAAGTCGAAGCTTCTCGTATCCTCGTTAACCA 3000 V K E Y G I K V E A S R I L V N Q3001 ACCTGACTCTATCGGTGGGGTCGGAGATATTTATACTGATGCAATGCGTC 3050 P D S I G G V G D I Y T D A M R P3051 CATCATTGACGCTCGGAACTGGTTCATGGGGGAAAAATTCACTTTCACAC 3100 S L T L G T G S W G K N S L S H Sau3AI3101 AATTTGAGTACATACGATCTATTGAATGTTAAAACAGTGGCTAAACGTCG 3150 N L S T Y D L L N V K T V A K R R3151 TAATCGCCCTCAATGGGTTCGTTTGCCAAAAGAAATTTACTACGAAAAAA 3200 N R P Q W V R L P K E I Y Y E K N3201 ATGCAATTTCTTACTTACAAGAATTGCCACACGTCCACAAAGCTTTCATT 3250 A I S Y L Q E L P H V H K A F I3251 GTTGCCGACCCTGGTATGGTTAAATTCGGTTTCGTTGATAAAGTTTTGGA 3300 V A D P G M V K F G F V D K V L E3301 ACAACTTGCTATCCGCCCAACTCAAGTTGAAACAAGCATTTATGGCTCAG 3350 Q L A I R P T Q V E T S I Y G S V3351 TCCAACCTGACCCAACTTTGAGTGAAGCAATTGCAATCGCTCGTCAAATG 3400 Q P D P T L S E A I A I A R Q M3401 AACCATTTTGAACCTGACACTGTCATCTGTCTTGGTGGTGGTTCTGCTCT 3450 N H F E P D T V I C L G G G S A L3451 CGATGCTGGTAAGATTGGTCGTTTGATTTATGAATATGATGCTCGTGGTG 3500 D A G K I G R L I Y E Y D A R G E Sau3AI3501 AGGCTGACCTTTCCGATGACGCAAGTTTGAAAGAGATCTTCCAAGAGTTA 3550 A D L S D D A S L K E I F Q E L3551 GCTCAAAAATTTGTTGATATTCGTAAACGTATTATCAAATTCTACCACCC 3600 A Q K F V D I R K R I I K F Y H P3601 ACACAAAGCACAAATGGTTGCTATCCCTACTACTTCTGGTACTGGTTCTG 3650 H K A Q M V A I P T T S G T G S E3651 AAGTGACTCCATTTGCGGTTATCACTGATGATGAAACTCACGTTAAATAT 3700 V T P F A V I T D D E T H V K Y3701 CCACTTGCTGACTATCAATTGACACCTCAAGTTGCCATTGTTGACCCTGA 3750 P L A D Y Q L T P Q V A I V D P E3751 GTTTGTTATGACTGTACCAAAACGTACTGTTTCTTGGTCTGGGATTGATG 3800 F V M T V P K R T V S W S G I D A3801 CTATGTCACACGCGCTTGAATCTTATGTTTCTGTCATGTCTTCTGACTAT 3850 M S H A L E S Y V S V M S S D Y3851 ACAAAACCAATTTCACTTCAAGCCATCAAACTCATCTTTGAAAACTTGAC 3900 T K P I S L Q A I K L I F E N L T3901 TGAGTCTTATCATTATGACCCAGCTCATCCAACCAAAGAAGGTCAAAAAG 3950 E S Y H Y D P A H P T K E G Q K A3951 CTCGCGAAAACATGCACAATGCTGCAACACTCGCTGGTATGGCCTTCGCC 4000 R E N M H N A A T L A G M A F A4001 AATGCTTTCCTTGGAATTAACCACTCACTTGCTCATAAAATTGCTGGTGA 4050 N A F L G I N H S L A H K I A G E4051 ATTTGGGCTTCCTCATGGTCTTGCCATTGCTATCGCTATGCCACATGTCA 4100 F G L P H G L A I A I A M P H V I4101 TTAAATTTAACGCTGTAACAGGAAACGTTAAATTTACCCCTTACCCACGT 4150 K F N A V T G N V K F T P Y P R4151 TATGAAACTTATCGTGCGCAAGAAGACTACGCTGAAATTTCACGCTTCAT 4200 Y E T Y R A Q E D Y A E I S R F M4201 GGGATTTGCTGGCAAAGAAGATTCAGATGAAAAAGCGGTCAAAGCTTTGG 4250 G F A G K E D S D E K A V K A L V4251 TTGCTGAACTTAAAAAATTGACTGATAGTATTGATATTAATATCACCCTT 4300 A E L K K L T D S I D I N I T L4301 TCAGGAAATGGTGTAGATAAAGCTCATCTTGAACGTGAGCTTGATAAATT 4350 S G N G V D K A H L E R E L D K L4351 GGCTGACCTTGTTTACGATGACCAATGTACACCTGCTAATCCACGTCAAC 4400 A D L V Y D D Q C T P A N P R Q P4401 CAAGAATTGATGAGATTAAACAACTCTTGTTAGACCAATATTAATATATT 4450 R I D E I K Q L L L D Q Y Stop4451 AATTATAGTATTTGGAACCGAACGATATCCATGCTCGCTAACCTGCTAAA 45004501 GCAGGAAGTCGCAATGGTACGTCAACCAAGAATTGATGAGATTAAACAAC 4550 Sau3AI4551 TCTTGTTAGATCAATACTAATAATCTGTTGATAAAAATAATTAAAACGCT 46004601 CTGATGAATTCGTCAGAGCGTTTTTTATTATAGCTTATACAACTATCAAA 46504651 AGGTATAAATCAATTTCGATATAGGCTCTTTTCACTCCATTGATTTATAT 4700 Sau3AI4701 TATATAAAAATCAATAATTAATTAGCGATAGAAGTGATCC 4741

EXAMPLE 2

[0119] 1. Construction of L. lactis DB1341 and MG1363 adhE Mutant Strains by Gene Inactivation

[0120] Inactivation of the adhE gene of strain DB1341 was carried out by Campbell-like integration (Leenthous et al., 1991) of pSMA-500 derivatives into the DB1341 chromosome. The adhE gene of strain DB1341 was inactivated at two different positions by cloning of PCR fragments (see FIG. 2) into the integration vector pSMA500 (Madsen et al., 1996). A 706 bp internal adhE fragment was amplified from the DB1341 chromosome using-primer adhP1 (position 1069-1088 in Table 1.4) and primer adhP2 (position 1775-1756 in Table 1.4). These primers contain a XhoI and a BamHI recognition site at the 5′ end. The PCR fragment was digested with XhoI and BamHI followed by cloning into pSMA500. The resulting plasmid, pSMAKAS4 (FIG. 3), was introduced into E. coli MC1000 by electroporation (Sambrook et al., 1989). Plasmid pSMAKAS4 was purified and subsequently introduced into strain DB1341 by electroporation (Holo and Nes 1989) and transformants were selected on SGM17 plates containing 1 μg/ml erythromycin and 80 μg/ml X-gal (Madsen et al., 1996). Homologous integration leads to an adhE gene which is interrupted after amino acid residue Asp543. About 100 blue transformants were obtained, indicating that a transcriptional fusion of the adhE gene to the lacLM reporter gene of pSMA500 had occurred. Eight blue transformants were restreaked and the integration point was verified by PCR analysis. One strain, DBKAS4, was selected for further studies.

[0121] Another integration further downstream in the adhE gene was constructed by a similar strategy. A 616 bp adhE fragment was amplified from the DB1341 chromosome using primer orf3P1 (position 2112-2138 in Table 1.4) and primer orf3P2 (position 2728-2708 in Table 1.4). The cloning of this fragment into pSMA500 resulted in plasmid pSMAKAS5 (FIG. 3). Introduction of pSMAKAS5 into DB1341 and subsequent integration into the adhE gene leads to an adhE gene, which is interrupted after amino acid residue Ile861. About 400 blue transformants were obtained, which again indicated that a transcriptional fusion of the adhE gene to the lacLM reporter gene of pSMA500 had occurred. Eight blue transformants were restreaked and the integration point was verified by PCR analysis. One strain, DBKAS5, was selected for further studies.

[0122] pSMAKAS4 and pSMAKAS5 were used also to inactivate the MG1363 adhE gene. One transformant from each transformation that turned blue on X-gal plates (MGKAS4 and MGKAS5), and therefore contained a translational fusion of the lacLM reporter gene of pSMA500 to the MG1363 adhE gene, was isolated for further studies.

[0123] A sample of Lactococcus lactis subspecies lactis biovar diacetylactis strains DBKAS4 and DBKAS5, respectively and of Lactococcus lactis subspecies lactis strains MGKAS4 and MGKAS5, respectively were deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession Nos DSM 11084, DSM 11085, DSM 11081 and DSM 11082, respectively.

[0124] A further adhE mutant strain was obtained by PCR using MG1363 DNA as template and primers adhP1-XhoI (sequence 5′-GGCCGCTCGAGGTTGAACGTGCTGGTGAAGG-3′; spanning position 2657-2676 in the MG1363 adhE sequence) (SEQ ID NO:32) and adhP2-BamHI (sequence 5′-TAGTAGGATCCGGGTCAGGTTGGACTGAGCC-3′; spanning position 3363-3344 in the MG1363 adhE sequence) (SEQ ID NO:33). A 700 bp fragment was digested with XhoI and BamHI, cloned into likewise digested pSMA500 and transformed into E. coli MC1000. The new construction, pSMAKAS14 was introduced into L. lactis MG1363 via electroporation. Integration led to disruption of the resident adhE gene and one transformant that turned blue on X-gal plates (integration results in transcriptional fusion to lacLM, a reporter gene) was selected for further analysis and was named MGKAS14. This integrant should express an AdhE protein truncated at position Asp543.

[0125] A sample of MGKAS14 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 10 Jul. 1997 under the accession No. DSM 11654.

[0126] 2. Physiological Characterization of MGKAS14

[0127] Physiological studies of MGKAS14 was carried by cultivating the strain in anaerobiosis in M17 medium supplemented with either glucose (GM17) or galactose (GalM17). The production under these conditions of the metabolites formate, acetaldehyde and pyruvate, respectively was measured and compared to corresponding measurement for the wild type strain, cultivated under similar conditions. In GM17 the production of formate in the mutant strain was reduced (4.86 in GM1363 vs. 1.67 in MGKAS14), the production of acetaldehyde was increased (0.52 in MG1363 vs. 0.67 in MGKAS14). No pyruvate was detected with any of the test strains. In the GalM17 medium, the production of formate was reduced substantially in the mutant strain (39.11 in GM1363 vs. 4.39 in MGKAS14) and that of acetaldehyde increased (0.67 in MG1363 vs. 1.12 in MGKAS14). None of the strains produced pyruvate.

EXAMPLE 3

Cloning of the L. lactis pfl Gene

[0128] The sequence of the pfl gene encoding pyruvate formate-lyase, a key enzyme in anaerobic metabolism, has only been reported in a few bacteria. DNA sequence homology between the different bacterial pfl genes is limited, making it difficult to clone this gene from other organisms (Table 3.1). Recently, this gene has been cloned in Streptococcus mutans (Yamamoto et al., 1996). The S. mutans pfl gene encodes a 775 amino acid protein as deduced from the published DNA sequence.

10TABLE 3.1Homology (DNA and protein level) of the L. lactispfl with other bacterial pfl genesHomology to theL. lactis Pfl proteinPfl proteinIdentitySimilarityDNA homologyaOrganism759 aa42.2%73%55.1%E. coli769 aa42.1%76%55.4%H. influenzae740 aaNANA52.6%C. pasteurianum775 aaNANA71.8%S. mutansaDNA homology through the L. lactis pfl sequence obtained. NA: not submitted to the databases; NF: not found in database searches.

[0129] 1. Construction of Lactococcus lactis λZAP Genomic Libraries

[0130] λZAP genomic libraries of L. lactis strains DB1341 and MG1363 were constructed according to the manufacturer's instructions (Stratagene) using partially Sau3AI-digested chromosomal DNA (average size about 5 kb) cloned into λ vector BamHI arms. Average insert size was estimated to be 3 kb.

[0131] 2. Screening of a λZAP Genomic Library of Strain DB1341 with a S. mutans pfl Probe

[0132] A 1 kb EcoRI fragment from the S. mutans pfl gene, encompassing positions 1190-2213 of the published S. mutans sequence (codons 298-639 of the pfl gene) was randomly labelled and used for screening the λZAP genomic library of strain DB1341 (approximately 2×105 pfu; Sambrook et al., 1989). Filters were washed at low stringency (2×30 min at room temperature in 5×SSC, then 1×30 min at 65° C. in 3×SSC; 0.1% SDS), and two positive clones, pfl1 and pfl2 were identified.

[0133] A sample of an E. coli strain transformed with clone pfl1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 25 Jul. 1996 under the accession Nos DSM 11103.

[0134] 3. Sequencing Positive λZAP Clones and Identification of Clones Containing a pfl Fragment

[0135] Following in vivo excision (Stratagene) and plasmid DNA isolation, sequence analysis (ALF sequenator, Pharmacia) was carried out for pfl1 using T7 and T3 primers (Stratagene). Approximately 2.1 kb was sequenced from one end of clone pfl1 (from position 1342 in Table 3.2 below), and a truncated, uninterrupted ORF spanning 1.1 kb was found that showed significant homology to other pfl genes, both at the DNA and protein level (Tables 3.3 and 3.4). A putative rho-independent transcription terminator (de Vos and Simons 1994) is located 26 bp downstream of the stop codon (positions 2468-2490 in Table 3.2).

11TABLE 3.2Sequence of the L. lactis DB1341 pf1 geneThe coding sequence starts at position 80 and ends at position 2443.A putative ribosome binding site is shown in bold, double underline(positions 65-71). A putative rho-independent transcriptional terminator(de Vos and Simons 1994) is found at positions 2468-2490 andis shown in bold, underline (stem) or dotted underline (loop).EcoRIGAATTCTGTTTGCTATTCTCAAACTGTATGATATAATGAAGTTGTAATTT(SEQ ID NO:15)1---------+---------+---------+---------+---------+50GAAACAGAAAGAACAAAGGAGATTTCAAAATGAAAACCGAAGTTACGGAA51---------+---------+---------+---------+---------+100 MetLysThrGluValThrGlu-(SEQ ID NO:16)AATATCTTTGAACAAGCTTGGGATGGTTTTAAAGGAACCAACTGGCGCGA1---------+---------+---------+---------+---------+150AsnIlePheGluGlnAlaTrpAspGlyPheLysGlyThrAsnTrpArgAsp-TAAAGCAAGCGTTACTCGCTTTGTACAAGAAAACTACAAACCATATGATG151---------+---------+---------+---------+---------+200 LysAlaSerValThrArgPheValGlnGluAsnTyrLysProTyrAspGly-GTGATGAAAGCTTTCTTGCTGGGCCAACAGAACGTACACTTAAAGTAAAG201---------+---------+---------+---------+---------+250 AspGluSerPheLeuAlaGlyProThrGluArgThrLeuLysValLys-AAAATTATTGAAGATACAAAAAATCACTACGAAGAAGTAGGATTTCCCTT251---------+---------+---------+---------+---------+300LysIleIleGluAspThrLysAsnHisTyrGluGluValGlyPheProPhe-CGATACTGACCGCGTAACCTCTATTGATAAAATCCCTGCTGGATATATCG301---------+---------+---------+---------+---------+350 AspThrAspArgValThrSerIleAspLysIleProAlaGlyTyrIleAsp-ATGCTAATGATAAAGAACTTGAACTCATCTATGGGATGCAAAATAGCGAA351---------+---------+---------+---------+---------+400 AlaAsnAspLysGluLeuGluLeuIleTyrGlyMetGlnAsnSerGlu-CTTTTCCGCTTGAATTTCATGCCAAGAGGTGGACTTCGTGTTGCTGAAAA401---------+---------+---------+---------+---------+450LeuPheArgLeuAsnPheMetProArgGlyGlyLeuArgValAlaGluLys-GATTTTGACAGAACACGGTCTCTCAGTTGACCCAGGCTTGCATGATGTTT451---------+---------+---------+----------+--------+500 IleLeuThrGluHisGlyLeuSerValAspProGlyLeuHisAspValLeu-TGTCACAAACAATGACTTCTGTAAATGATGGAATCTTTCGTGCTTATACT501---------+---------+---------+---------+---------+550 SerGlnThrMetThrSerValAsnAspGlyIlePheArgAlaTyrThr-TCAGCAATTCGTAAAGCACGTCATGCTCATACTGTAACAGGTTTGCCAGA551---------+---------+---------+---------+---------+600SerAlaIleArgLysAlaArgHisAlaHisThrValThrGlyLeuProAsp-TGCTTACTCTCGTGGACGTATCATTGGTGTCTATGCACGTCTTGCCCTTT601---------+---------+---------+---------+---------+650 AlaTyrSerArgGlyArgIleIleGlyValTyrAlaArgLeuAlaLeuTyr-ACGGTGCTGATTACCTTATGAAGGAAAAAGCAAAAGAATGGGATGCAATC651---------+---------+---------+---------+---------+700 GlyAlaAspTyrLeuMetLysGluLysAlaLysGluTrpAspAlaIle-ACTGAAATTAACGAAGAAAACATTCGTCTTAAAGAAGAAATTAATATGCA701---------+---------+---------+---------+---------+750ThrGluIleAsnGluGluAsnIleArgLeuLysGluGluIleAsnMetGln-ATACCAAGCTTTGCAAGAAGTTGTAAACTTTGGTGCCTTATATGGTCTTG751---------+---------+---------+---------+---------+800TyrGlnAlaLeuGlnGluValValAsnPheGlyAlaLeuTyrGlyLeuAsp-ATGTTTCACGTCCAGCTATGAACGTAAAAGAAGCAATCCAATGGGTTAAC801---------+---------+---------+---------+---------+850 ValSerArgProAlaMetAsnValLysGluAlaIleGlnTrpValAsn-ATCGCTTATATGGCAGTATGTCGTGTCATTAATGGAGCTGCAACTTCACT851---------+---------+---------+---------+---------+900IleAlaTyrMetAlaValCysArgValIleAsnGlyAlaAlaThrserLeu -TGGACGTGTTCCAATCGTTCTTGATATCTTTGCAGAACGTGACCTTGCTC902---------+---------+---------+---------+---------+950 GlyArgValProIleValLeuAspIlePheAlaGluArgAspLeuAlaArg-GTGGAACATTTACTGAACAAGAAATTCAAGAATTTGTTGATGATTTCGTT951---------+---------+---------+---------+---------+1000 GlyThrPheThrGluGlnGluIleGlnGluPheValAspAspPheVal-TTGAAGCTTCGTACAATGAAATTTGCTCGTGCAGCTGCTTATGATGAACT1001---------+---------+---------+---------+---------+1050LeuLysLeuArgThrMetLysPheAlaArgAlaAlaAlaTyrAspGluLeu-TTATTCTGGTGACCCAACATTCATCACAACATCTATGGCTGGTATGGGTA1051---------+---------+---------+---------+---------+1100 TyrSerGlyAspProThrPheIleThrThrSerMetAlaGlyMetGlyAsn-ATGACGGACGTCACCGTGTCACTAAAATGGACTACCGTTTCTTGAACACA1101---------+---------+---------+---------+---------+1150 AspGlyArgHisArgValThrLysMetAspTyrArgPheLeuAsnThr-CTTGATACAATCGGAAATGCTCCAGAACCAAACTTGACAGTCCTTTGGGA1151---------+---------+---------+---------+---------+1200LeuAspThrIleGlyAsnAlaProGluProAsnLeuThrValLeuTrpAsp-TTCTAAACTTCCTTACTCATTCAAACGTTATTCAATGTCTATGAGCCACA1201---------+---------+---------+---------+---------+1250 SerLysLeuProTyrSerPheLysArgTyrSerMetSerMetSerHisLys-AGCATTCTTCTATTCAATATGAAGGTGTTGAAACAATGGCTAAAGATGGA1251---------+---------+---------+---------+---------+1300 HisSerSerIleGlnTyrGluGlyValGluThrMetAlaLysAspGly- S a u 3 A ITATGGCGAAATGTCATGTATCTCTTGTTGTGTCTCACCACTTGATCCAGA1301---------+---------+---------+---------+---------+1350TyrGlyGluMetSerCysIleSerCysCysValSerProLeuAspProGlu-AAATGAAGAAGGACGTCATAACCTCCAATACTTTGGTGCGCGTGTAAACG1351---------+---------+---------+---------+---------+1400 AsnGluGluGlyArgHisAsnLeuGlnTyrPheGlyAlaArgValAsnVal-TCTTGAAAGCAATGTTGACTGGTTTGAACGGTGGTTATGATGACGTTCAT1401---------+---------+---------+---------+---------+1450 LeuLysAlaMetLeuThrGlyLeuAsnGlyGlyTyrAspAspValHis-AAAGATTATAAAGTATTCGACATCGAACCTGTTCGTGACGAAATTCTTGA1451---------+---------+---------+---------+---------+1500LysAspTyrLysValPheAspIleGluProValArgAspGluIleLeuAsp-CTATGATACAGTTATGGAAAACTTTGACAAATCTCTCGACTGGTTGACTG1501---------+---------+---------+---------+---------+1550 TyrAspThrValMetGluAsnPheAspLysSerLeuAspTrpLeuThrAsp-ATACTTATGTTGATGCAATGAATATCATTCATTACATGACTGATAAATAT1551---------+---------+---------+---------+---------+1600 ThrTyrValAspAlaMetAsnIleIleHisTyrMetThrAspLysTyr-AACTATGAAGCAGTTCAAATGGCCTTCTTGCCTACTAAAGTTCGTGCTAA1601---------+---------+---------+---------+---------+1650AsnTyrGluAlaValGlnMetAlaPheLeuProThrLysValArgAlaAsn-CATGGGATTTGGTATCTGTGGATTCGCAAATACAGTTGATTCACTTTCAG1651---------+---------+---------+---------+---------+1700MetGlyPheGlyIleCysGlyPheAlaAsnThrValAspSerLeuSerAla-CAATTAAATATGCTAAAGTTAAAACATTGCGTGATGAAAATGGCTATATC1701---------+---------+---------+---------+---------+1750 IleLysTyrAlaLysValLysThrLeuArgAspGluAsnGlyTyrIle- S a u 3 A ITACGATTACGAAGTAGAAGGTGATTTCCCTCGTTATGGTGAAGATGATGA1751---------+---------+---------+---------+---------+1800TyrAspTyrGluValGluGlyAspPheProArgTyrGlyGluAspAspAsp-TCGTGCTGATGATATTGCTAAACTTGTCATGAAAATGTACCATGAAAAAT1801---------+---------+---------+---------+---------+1850 ArgAlaAspAspIleAlaLysLeuValMetLysMetTyrHisGluLysLeu-TAGCTTCACACAAACTTTACAAAAATGCTGAAGCTACTGTTTCACTTTTG1851---------+---------+---------+---------+---------+1900 AlaSerHisLysLeuTyrLysAsnAlaGluAlaThrValSerLeuLeu-ACAATTACATCTAACGTTGCTTACTCTAAACAAACTGGTAATTCTCCAGT1901---------+---------+---------+---------+---------+1950ThrIleThrSerAsnValAlaTyrSerLysGlnThrGlyAsnSerProVal-ACATAAAGGAGTATTCCTCAATGAAGATGGTACAGTAAATAAATCTAAAC1951---------+---------+---------+---------+---------+2000 HisLysGlyValPheLeuAsnGluAspGlyThrValAsnLysSerLysLeu- E c o R ITTGAATTCTTCTCACCAGGTGCTAACCCATCTAATAAAGCTAAGGGTGGT2001---------+---------+---------+---------+---------+2050 GluPhePheSerProGlyAlaAsnProSerAsnLysAlaLysGlyGly- E c o R ITGGTTGCAAAACCTTCGCTCATTGGCTAAGTTGGAATTCAAAGATGCAAA2051---------+---------+---------+---------+---------+2100TrpLeuGlnAsnLeuArgSerLeuAlaLysLeuGluPheLysAspAlaAsn-TGATGGTATTTCATTGACTACTCAAGTTTCACCTCGTGCACTTGGTAAAA2101---------+---------+---------+---------+---------+2150 AspGlyIleSerLeuThrThrGlnValSerProArgAlaLeuGlyLysThr-CTCGTGATGAACAAGTGGATAACTTGGTTCAAATTCTTGATGGATACTTC2151---------+---------+---------+---------+---------+2200 ArgAspGluGlnValAspAsnLeuValGlnIleLeuAspGlyTyrPhe-ACACCAGGTGCTTTGATTAATGGTACTGAATTTGCAGGTCAACACGTTAA2201---------+---------+---------+---------+---------+2250ThrProGlyAlaLeuIleAsnGlyThrGluPheAlaGlyGlnHisValAsn-CTTGAACGTAATGGACCTTAAAGATGTTTACGATAAAATCATGCGTGGTG2252---------+---------+---------+---------+---------+2300 LeuAsnValMetAspLeuLysAspValTyrAspLysIleMetArgGlyGlu-AAGATGTTATCGTTCGTATCTCTGGTTACTGTGTCAATACTAAATACCTC2301---------+---------+---------+---------+---------+2350 AspValIleValArgIleSerGlyTyrCysValAsnThrLysTyrLeu-ACACCAGAACAAAAACAAGAATTAACTGAACGTGTCTTCCATGAAGTTCT2351---------+---------+---------+---------+---------+2400ThrProGluGlnLysGlnGluLeuThrGluArgValPheHisGluValLeu-TTCAAACGATGATGAAGAAGTAATGCATACTTCAAACATCTAATTCTTAA2401---------+---------+---------+---------+---------+2450 SerAsnAspAspGluGluValMetHisThrSerAsnIleEndAATTTAATGAATATTCGGTCTGTCAGTTTTACTGACAGACTTTTTTTTAC2451---------+---------+------{overscore (---)}+---------+---------+2500GAAAAAATTAATCATAATAGTTAAAAACTATTGTTTTTAGTTTAAGAAAG2501---------+---------+---------+---------+---------+2550TTAAATTTTATGCTAAAATAGATGAATGAAAATGGTAATTGGATTGACAG2551---------+---------+---------+---------+---------+2600GCGGAATTGCGATGGGAAATCAACGGTGGTTGATTTTTTGATTCTGAGGG2601---------+---------+---------+---------+---------+2650TTATCAAGTGATTGATGCTGACAAAGTTGTCCGTCAATTTACAAGAACCT2651---------+---------+---------+---------+---------+2700GGCGGAAAACTTTACAAGGCAATATTAGAAACTTACGGTTTAGATTTTAT2701---------+---------+---------+---------+---------+2750TGCTGACAATTGGACAGTTAAATCGTGAAAAATTAGGAGCTTTAGTTTTT2751---------+---------+---------+---------+---------+2800TCTGATTCAAAAGAGCGCGAGAAATTATCAAACTTACAAGATGAAATTAT2801---------+---------+---------+---------+---------+2850TCGTACAGAATTATATGATAGACGTGATGACTTATTAAAAAAAATGACTG2851---------+---------+---------+---------+---------+2900ACAAGTCTGTCAGTAAAAATTTTGATTCAAAGAGTCAAGGAAAAAATCTG2901---------+---------+---------+---------+---------+2950TCAGTAAATAAGCCAATATTTATGGATATTCCGTTATTAATTGAATACAA2951---------+---------+---------+---------+---------+3000TTATACCGGATTTGATGAAATATGGTTGGTCAGCTTACCTGAAAAAATAC3001---------+---------+---------+---------+---------+3050AATTAGAAAGACTGATGGCAAGAAATAAGTTTACGGAAGAAGAAGCTAAA3051---------+---------+---------+---------+---------+3100AAACGAATTTCTTCACAAATGCCATTGTCAGAAAAACAAAAAGTCGCTGA3101---------+---------+---------+---------+---------+3150TGTCATTCTGGATAATTCTGGAAAGATTGAAGCACTAAAAAAACAAATCC3151---------+---------+---------+---------+---------+3200AGCGAGAACTAGCTAGGATAGAAGAACAGAAATAGAGGTGAATCGCACGA3201---------+---------+---------+---------+---------+3250AAACAGTTAATTGGAAAGGAATTTATTTATAACATGGATTGGCTGCTTTT3251---------+---------+---------+---------+---------+3300TTGTAGGTTCATCATTTTCACTCGTCATGCCTTTCTCCCCTTGTATATTC3301---------+---------+---------+---------+---------+3350AAGGACTGGGTGAAGCGGTGGGAATTTGAACTTTACTCAGGGTTACTTTT3351---------+---------+---------+---------+---------+3400TCTTTGCCAGCCTTA3401---------+----- 3415

[0136]

12

TABLE 3.3

DNA homology (FASTA, GCG Wisconsin Package Version 8, Genetics Computer Group)

using the complete L. lactis DB1341 pf1 sequence shown in TABLE 3.2

Only the two highest scores (S. mutans and H. influenzae pf1 genes, designated

smpf1 and hi3281, respectively) are shown.

(Nucleotide) FASTA of: dbpf1.seq from: 1 to: 3415 Jul. 19, 1996 10:11

The best scores are: init1 initn opt . . .

empro: smpf1 D50491 Streptococcus mutans pf1 . . . 4335 5345 4996

empro: hi32812/rev U32812 Hae. influenzae focA 652 1077 1299

empro: ecpf1 X08035 E. coli pf1 (pyruvate form. 429 735 1214

empro: cppf1act X93463 C. pasteurianum pf1 and act 309 487 744

emnew: cef13b12/rev Z70683 Caenorhabditis eleg. 94 168 128

dbpf1.seq

empro: smpf1

ID SMPFL standard; DNA; PRO; 3067 BP.

AC D50491;

NI g1129081

DT 23 DEC. 1995 (Rel. 46, Created)

DE Streptococcus mutans pf1 gene for pyruvate formate-lyase . . . .

SCORES Init1: 4335 Initn: 5345 Opt: 4996

71.8% identity in 2608 bp overlap

10 20 30 40 50

dbpf1. GAATTCTGTTTGCTATTCTCAAACTGTATGATATAATGAAGTTGTAATTTGA

||||||||||| ||| | | |||| |||

smpf1 AAGCAAGTTCTTTCGCTTGTGTAACCGGTTACTGTATGATAGAATATAATCGTAAATTGT

200 210 220 230 240 250

60 70 80 90

dbpf1. AACAGA-----------AAGAACAAAGGAGATTTCAA-AATGAAAAC---CGAAGTTACG

|||||| |||| | ||| || |||| ||| ||| | || ||

smpf1 AACAGATTAACTGTTACTAGAATAGAGGGGAACTCAATTATGGCAACTGTCAAAACTAAC

260 270 280 290 300 310

100 110 120 130 140 150

dbpf1. GAAAATATCTTTGAACAAGCTTGGGATGGTTTTAAAGGAACCAACTGGCGCGATAAAGCA

| | |||||| |||| ||||| || ||||||||||| ||||| || | ||||

smpf1 ACTGACGTTTTTGAAAAAGCCTGGGAAGGCTTTAAAGGAACTGACTGGAAAGACAGAGCA

320 330 340 350 360 370

160 170 180 190 200 210

dbpf1. AGCGTTACTCGCTTTGTACAAGAAAACTACAAACCATATGATGGTGATGAAAGCTTTCTT

||| || |||||||||| ||||| ||||||| |||||||| || | ||||| ||||||

smpf1 AGCATTTCTCGCTTTGTTCAAGACAACTACACTCCATATGACGGAGGCGAAAGTTTTCTT

380 390 400 410 420 430

220 230 240 250 260 270

dbpf1. GCTGGGCCAACAGAACGTACACTTAAAGTAAAGAAAATTATTGAAGATACAAAAAATCAC

|| || || || |||||| ||||| | | || ||| | | ||||| || ||| ||

smpf1 GCCGGCCCTACTGAACGTTCACTTCACATCAAAAAAGTCGTAGAAGAAACTAAAGCGCAT

440 450 460 470 480 490

280 290 300 310 320 330

dbpf1. TACGAAGAAGTAGGATTTCCCTTCGATACTGACCGCGTAACCTCTATTGATAAAATCCCT

||||||||| | | ||||| | ||||| || | || ||||||| | | |||||

smpf1 TACGAAGAAACACGTTTTCCAATGGATAC---ACGTATTACATCTATTGCTGATATCCCA

500 510 520 530 540 550

340 350 360 370 380 390

dbpf1. GCTGGATATATCGATGCTAATGATAAAGAACTTGAACTCATCTATGGGATGCAAAATAGC

|| || ||||| ||| || ||| |||| | || | ||| || |||||

smpf1 GCAGGTTATAT---------TGACAAGGAAAATGAATTGATTTTTGGTATCCAAAACGAT

560 570 580 590 600

400 410 420 430 440 450

dbpf1. GAACTTTTCCGCTTGAATTTCATGCCAAGAGGTGGACTTCGTGTTGCTGAAAAGATTTTG

|||||||| |||| |||||||||| ||| || |||| | ||||||| ||||

smpf1 GAACTTTTTAAGCTGAACTTCATGCCAAAAGGCGGTATTCGCATGGCTGAAACAGCTTTG

610 620 630 640 650 660

460 470 480 490 500 510

dbpf1. ACAGAACACGGTCTCTCAGTTGACCCAGGCTTGCATGATGTTTTGTCACAAACAATG--A

| |||||| ||| | ||||| | | | ||||| | | | || || ||| |

smpf1 AAAGAACATGGTTATGAACCAGACCCTGCCGTTCATGAAATCT--TTACCAAATATGCAA

670 680 690 700 710 720

520 530 540 550 560 570

dbpf1. CTTCTGTAAATGATGGAATCTTTCGTGCTTATACTTCAGCAATTCGTAAAGCACGTCATG

| | || |||||||| |||||||||||||| |||||| ||||| ||||||||||

smpf1 CAACCGTTAATGATGGTATCTTTCGTGCTTACACTTCAAACATTCGCCGTGCACGTCATG

730 740 750 760 770 780

580 590 600 610 620 630

dbpf1. CTCATACTGTAACAGGTTTGCCAGATGCTTACTCTCGTGGACGTATCATTGGTGTCTATG

| || |||||||| ||| | |||||||| |||||||| |||||||| ||||| || ||||

smpf1 CCCACACTGTAACTGGTCTCCCAGATGCATACTCTCGCGGACGTATTATTGGAGTTTATG

790 800 810 820 830 840

640 650 660 670 680 690

dbpf1. CACGTCTTGCCCTTTACGGTGCTGATTACCTTATGAAGGAAAAAGCAAAAGAATGGGATG

| |||||||| || || |||||||| ||| | ||| | ||||||| || || ||| |

smpf1 CCCGTCTTGCTCTCTATGGTGCTGACTACTTGATGCAAGAAAAAGTGAACGACTGGAACT

850 860 870 880 890 900

700 710 720 730 740 750

dbpf1. CAATCACTGAAATTAACGAAGAAAACATTCGTCTTAAAGAAGAAATTAATATGCAATACC

|||| |||||||| | |||||| ||||||||| |||||||| ||| | ||||| |

smpf1 CAATTGCTGAAATTGATGAAGAATCAATTCGTCTTCGTGAAGAAATCAATCTTCAATATC

910 920 930 940 950 960

760 770 780 790 800 810

dbpf1. AAGCTTTGCAAGAAGTTGTAAACTTTGGTGCCTTATATGGTCTTGATGTTTCACGTCCAG

| || | ||||| || || |||| | ||||||||||||||| || |

smpf1 AGGCACTTGGCGAAGTAGTGCGGTTGGGTGATCTGTATGGTCTTGATGTTCGCAAACCTG

970 980 990 1000 1010 1020

820 830 840 850 860 870

dbpf1. CTATGAACGTAAAAGAAGCAATCCAATGGGTTAACATCGCTTATATGGCAGTATGTCGTG

||||||| || || ||||| ||||||||| |||| ||||| | |||||| || || || |

smpf1 CTATGAATGTTAAGGAAGCTATCCAATGGATTAATATCGCCTTTATGGCTGTCTGCCGCG

1030 1040 1050 1060 1070 1080

880 890 900 910 920 930

dbpf1. TCATTAATGGAGCTGCAACTTCACTTGGACGTGTTCCAATCGTTCTTGATATCTTTGCAG

| || ||||| ||||||||||| ||||||||||| |||||||||||||||||||||||||

smpf1 TTATCAATGGTGCTGCAACTTCTCTTGGACGTGTCCCAATCGTTCTTGATATCTTTGCAG

1090 1100 1110 1120 1130 1140

940 950 960 970 980 990

dbpf1. AACGTGACCTTGCTCGTGGAACATTTACTGAACAAGAAATTCAAGAATTTGTTGATGATT

||||||||||||||||||| || || |||||| |||||| |||||||| |||||||| |

smpf1 AACGTGACCTTGCTCGTGGCACTTTCACTGAATCAGAAATCCAAGAATTCGTTGATGACT

1150 1160 1170 1180 1190 1200

1000 1010 1020 1030 1040 1050

dbpf1. TCGTTTTGAAGCTTCGTACAATGAAATTTGCTCGTGCAGCTGCTTATGATGAACTTTATT

||||| |||| |||||||| | |||||||| ||| | |||||||| ||||||||||

smpf1 TCGTTATGAAACTTCGTACGGTTAAATTTGCACGTACTAAGGCTTATGACGAACTTTATT

1210 1220 1230 1240 1250 1260

1060 1070 1080 1090 1100 1110

dbpf1. CTGGTGACCCAACATTCATCACAACATCTATGGCTGGTATGGGTAATGACGGACGTCACC

| |||||||||||||| || || || ||||||||||||||||| ||| ||||||||||

smpf1 CAGGTGACCCAACATTTATTACGACTTCTATGGCTGGTATGGGAGCTGATGGACGTCACC

1270 1280 1290 1300 1310 1320

1120 1130 1140 1150 1160 1170

dbpf1. GTGTCACTAAAATGGACTACCGTTTCTTGAACACACTTGATACAATCGGAAATGCTCCAG

|||| ||||| ||||||||||||||||| || || ||||||| || || ||||||||||

smpf1 GTGTTACTAAGATGGACTACCGTTTCTTAAATACGCTTGATAATATTGGCAATGCTCCAG

1330 1340 1350 1360 1370 1380

1180 1190 1200 1210 1220 1230

dbpf1. AACCAAACTTGACAGTCCTTTGGGATTCTAAACTTCCTTACTCATTCAAACGTTATTCAA

|||| ||||| || || |||||| |||| | |||||||| ||| | ||||| |

smpf1 AACCTAACTTAACCGTTCTTTGGTCAAGTAAATTGCCTTACTCTTTCCGTCATTATTGTA

1390 1400 1410 1420 1430 1440

1240 1250 1260 1270 1280 1290

dbpf1. TGTCTATGAGCCACAAGCATTCTTCTATTCAATATGAAGGTGTTGAAACAATGGCTAAAG

||||||||||||||||||||||||| ||||||||||||||||| ||| ||||||||||

smpf1 TGTCTATGAGCCACAAGCATTCTTCAATTCAATATGAAGGTGTCACAACTATGGCTAAAG

1450 1460 1470 1480 1490 1500

1300 1310 1320 1330 1340 1350

dbpf1. ATGGATATGGCGAAATGTCATGTATCTCTTGTTGTGTCTCACCACTTGATCCAGAAAATG

| || ||||| ||||||||||||||||| || ||||| || || |||||||| ||||| |

smpf1 AAGGTTATGGTGAAATGTCATGTATCTCATGCTGTGTATCTCCGCTTGATCCTGAAAACG

1510 1520 1530 1540 1550 1560

1360 1370 1380 1390 1400 1410

dbpf1. AAGAAGGACGTCATAACCTCCAATACTTTGGTGCGCGTGTAAACGTCTTGAAAGCAATGT

|||| | || || || || |||||||||||||| ||||| ||||| | |||||| |

smpf1 AAGATCGTCGCCACAATCTACAATACTTTGGTGCTCGTGTTAACGTTCTTAAAGCACTTC

1570 1580 1590 1600 1610 1620

1420 1430 1440 1450 1460 1470

dbpf1. TGACTGGTTTGAACGGTGGTTATGATGACGTTCATAAAGATTATAAAGTATTCGACATCG

| || ||| | || || ||||| || || ||||| ||||| || |||||||| || |||

smpf1 TTACAGGTCTTAATGGCGGTTACGACGATGTTCACAAAGACTACAAAGTATTTGATGTCG

1630 1640 1650 1660 1670 1680

1480 1490 1500 1510 1520 1530

dbpf1. AACCTGTTCGTGACGAAATTCTTGACTATGATACAGTTATGGAAAACTTTGACAAATCTC

||||| | ||||| ||| | ||||| | ||| || |||| | || ||||| ||| | |

smpf1 AACCTATCCGTGATGAAGTCCTTGATTTTGAAACGGTTAAAGCTAATTTTGAAAAAGCAC

1690 1700 1710 1720 1730 1740

1540 1550 1560 1570 1580 1590

dbpf1. TCGACTGGTTGACTGATACTTATGTTGATGCAATGAATATCATTCATTACATGACTGATA

| || ||||||||||||||||| || || ||||||||||||||||| || ||||||||||

smpf1 TTGATTGGTTGACTGATACTTACGTGGACGCAATGAATATCATTCACTATATGACTGATA

1750 1760 1770 1780 1790 1800

1600 1610 1620 1630 1640 1650

dbpf1. AATATAACTATGAAGCAGTTCAAATGGCCTTCTTGCCTACTAAAGTTCGTGCTAACATGG

|||||||||||||||| ||||||||||||||||| || || ||| || || ||||

smpf1 AATATAACTATGAAGCCGTTCAAATGGCCTTCTTACCAACACGTGTTAAAGCCAATATGG

1810 1820 1830 1840 1850 1860

1660 1670 1680 1690 1700 1710

dbpf1. GATTTGGTATCTGTGGATTCGCAAATACAGTTGATTCACTTTCAGCAATTAAATATGCTA

|||||||||| || |||||| | ||||||||||||||| | ||||| |||||||||||||

smpf1 GATTTGGTATTTGCGGATTCTCTAATACAGTTGATTCATTATCAGCTATTAAATATGCTA

1870 1880 1890 1900 1910 1920

1720 1730 1740 1750 1760 1770

dbpf1. AAGTTAAAACATTGCGTGATGAAAATGGCTATATCTACGATTACGAAGTAGAAGGTGATT

|| ||| | | ||||||||| |||| || || ||||| || ||| | ||| | |

smpf1 CTGTAAAACCTATTCGTGATGAAGATGGTTACATTTACGACTATGAAACTGTTGGTAACT

1930 1940 1950 1960 1970 1980

1780 1790 1800 1810 1820 1830

dbpf1. TCCCTCGTTATGGTGAAGATGATGATCGTGCTGATGATATTGCTAAA--CTTGTCATGAA

|||||||||| || ||||||||||| |||| || || ||| || ||| | ||||

smpf1 TCCCTCGTTACGGAGAAGATGATGACCGTGTAGACTCAATCGCTGAATGGTTG-CTTGAA

1990 2000 2010 2020 2030 2040

1840 1850 1860 1870 1880 1890

dbpf1. AATGTACCATGAAAAATTAGCTTCACACAAACTTTACAAAAATGCTGAAGCTACTGTTTC

| | |||| | || || ||||| |||||| || | ||||||||||| ||

smpf1 GCT-TTCCATACTCGTCTTGCACGTCATAAACTGTACAAAGATTCCGAAGCTACTGTATC

2050 2060 2070 2080 2090 2100

1900 1910 1920 1930 1940 1950

dbpf1. ACTTTTGACAATTACATCTAACGTTGCTTACTCTAAACAAACTGGTAATTCTCCAGTACA

| | | ||||| || ||||| |||||||| |||||||||||||||||||||||||| ||

smpf1 ATTGCTTACAATCACTTCTAATGTTGCTTATTCTAAACAAACTGGTAATTCTCCAGTTCA

2110 2120 2130 2140 2150 2160

1960 1970 1980 1990 2000 2010

dbpf1. TAAAGGAGTATTCCTCAATGAAGATGGTACAGTAAATAAATCTAAACTTGAATTCTTCTC

|| || || | |||||||||||||||| | || || |||||| | |||||||||||

smpf1 CAAGGGTGTTTACCTCAATGAAGATGGTTCTGTGAACTTGTCTAAAGTAGAATTCTTCTC

2170 2180 2190 2200 2210 2220

2020 2030 2040 2050 2060 2070

dbpf1. ACCAGGTGCTAACCCATCTAATAAAGCTAAGGGTGGTTGGTTGCAAAACCTTCGCTCATT

|||||||||||||||||| ||||||||| || || |||||||||||| | ||||||

smpf1 ACCAGGTGCTAACCCATCAAATAAAGCTTCCGGCGGCTGGTTGCAAAACTTGAACTCATT

2230 2240 2250 2260 2270 2280

2080 2090 2100 2110 2120 2130

dbpf1. GGCTAAGTTGGAATTCAAAGATGCAAATGATGGTATTTCATTGACTACTCAAGTTTCACC

| || | || || | |||||||||||||| |||||||| ||||||||||||||

smpf1 GAAGAAACTTGACTTTGCTCACGCAAATGATGGTATCTCATTGACAACTCAAGTTTCACC

2290 2300 2310 2320 2330 2340

2140 2150 2160 2170 2180 2190

dbpf1. TCGTGCACTTGGTAAAACTCGTGATGAACAAGTGGATAACTTGGTTCAAATTCTTGATGG

|| |||||||| || ||||||||||| | |||||| || ||||||||||||

smpf1 AAAAGCTCTTGGTAAGACATTCGATGAACAAGTTGCTAACTTAGTAACAATTCTTGATGG

2350 2360 2370 2380 2390 2400

2200 2210 2220 2230 2240 2249

dbpf1. ATACTTCACACCAGGTGCT----TTGATTAATGGTACTGAATTTGCAGGTCAACACGTTA

||||| | || | | | | | | | || | | | | || | |||

smpf1 TTACTTTGAAGGCGGCGGTCAACACGTTAACTTGAAC-GTTATGGATCTTAAAGATGTTT

2410 2420 2430 2440 2450 2460

2250 2260 2270 2280 2290 2300 2309

dbpf1. ACTTGAACGTAATGGACCTTAAAGATGTTTACGATAAAATCATGCGTGGTGAAGATGTTA

| || | ||| | | |||||||| || | ||| ||| | |||||

smpf1 ATGACAAGATCATGAATGGTGAAGATGTTATCGTTCGTATC---TCAGGTTACTGTGTTA

2470 2480 2490 2500 2510

2310 2320 2330 2340 2350 2360 2369

dbpf1. TCGTTCGTATCTCTGGTTACTGTGTCAATACTAAATACCTCACACCAGAACAAAAACAAG

| | | | | | || ||| ||| | |||| | || |

smpf1 ACACTAAATACCTTACTAAAGAACAAAAGACTGAAT---TGACACAACGTGTTTTCCATG

2520 2530 2540 2550 2560 2570

2370 2380 2390 2400 2410 2420

dbpf1. AA-TTAACTGAACGTGTCTTCCA--TGAAGTTCTTTCAAACGATGATGAAGAAGTAA--T

|| || || || | | | || | || | ||| | | |||| |||

smpf1 AAGTTCTCTCAATGGATGATGCAGCTACAGACTTGGTTAACAACAAGTAAGAGTTAAACA

2580 2590 2600 2610 2620 2630

2430 2440 2450 2460 2470

dbpf1. GCATA-CTTCAAACATCTAATTCTTAAAA--------TTTAATGAATATTCGG--TCTGT

| || || ||| | || | || ||||| |||| | ||||| | |

smpf1 GTTTAGTTTAAAAGACCTCACTCATAAAAGTGAGGTCTTTACTTTGCTTTCGGGTACGAT

2640 2650 2660 2670 2680 2690

2480 2490 2500 2510 2520 2530

dbpf1. CAGTTTTACTGACAGACTTTTTTTTACGAAAAAATTAATCATAAT-AGTTAAAAACTATT

|| | ||| || |||| | | | ||||| | | || | | || |||||| |

smpf1 CA-AAGCAGTGAGAGCTTTTTATATTCTAAAAACTCA--CAAATTCAGAAAAAAACAGCT

2700 2710 2720 2730 2740 2750

2540 2550 2560 2570 2580 2590

dbpf1. GTTTTTAGTTTAAGAAAGTTAAATTTTATGCTAAAATAGATGAATGAAAATGGTAATTGG

|| | | || |||| | ||||| |||| |||| |||||||| |||||

smpf1 CTTGTGATTT---GAAA---AGCTTTTA-GCTACAATAATATTATGAAAAT--TAATTAT

2760 2770 2780 2790 2800

2600 2610 2620 2630 2640 2650

dbpf1. ATTGACAGGCGGAATTGCGATGGGAAATCAACGGTGGTTGATTTTTTGATTCTGAGGGTT

smpf1 ACTCGCGACACACTGTCATCCACCTATCTTGATGCAGTAAAAATTAGACACCTTGTCTTC

2810 2820 2830 2840 2850 2860

dbpf1.: corresponding to nucleotides 1-2653 of SEQ ID NO:15;

smpf1: SEQ ID NO:17

dbpf1.seq/rev

empr6: hi32812

ID HI32812 standard; DNA; PRO; 10817 BP.

AC U32812; L42023;

NI g1222092

DT Aug. 9, 1995 (Rel. 44, Created)

DE Haemophilus influenzae focA, pf1A, pf1B, rspB, yaaJ, yajF, yeiG . . .

SCORES Init1: 652 Initn: 1077 Opt: 1299

55.4% identity in 1961 bp overlap

1979 1969 1959 1949 1939 1929 1920

dbpf1. CATCTTCATTGAGGAATACTCCTTTATGTACTGGAGAATTACCAGTTTGTTTAGAGTAAG

| || | ||||| |||| ||| |||

hi3281 GTCCGAATGGTGCACCAGCACGACGACCATCAGGGGTGTTACCCGTTTTCTTACCATAAA

2730 2740 2750 2760 2770 2780

1919 1909 1899 1889 1879 1869 1860

dbpf1. CAACGTTAGATGTAATTGTCAAAAGTGAAACAGTAGCTTCAGCATTTTTGTAAAGTTTGT

| |||||||| ||||| || || | || |||| | ||||| |||| |||

hi3281 CTACGTTAGAAGTAATGGTTAATACAGATTGTGTAGGCACTGCATTGCGGTAAGTTTTAA

2790 2800 2810 2820 2830 2840

1859 1849 1839 1829 1819 1809

dbpf1. GTGAAGCTAATTT-TTCATGGTACATTTTCATGACAAGTTTAGCAATATCATCAGCACGA

|| | ||| ||||| || || | | ||| || |||||| ||||

hi3281 GTTTTTGAATTTTCTTCATAAAAC-GTTCAACTAAGTCACAAGCGATGTCATCAACACGG

2850 2860 2870 2880 2890 2900

1799 1789 1779 1769 1759

dbpf1. TCATCATCTTCACCATAACGAGGGAAATCACCTTCTACTTCGTAATCG----------TA

| ||||| | |||||| | || | |||||||| | ||| | ||| ||

hi3281 TTATCATTGTTACCATATTGTGGATATTCACCTTCGATTTCAAAGTCGATTGCTACGTTA

2910 2920 2930 2940 2950 2960

1750 1749 1739 1729 1719

dbpf1. G--------ATATAGCCATTTTCAT------------CACGCAATGTTTTAACTTTAGCA

| | || ||||| || || |||| | || ||||||||| |||

hi3281 GTTGCAACAACATTGCCATCTTTATCTTTGATGTCGCCACGAACTGGTTTAACTTTCGCA

2970 2980 2990 3000 3010 3020

1709 1699 1689 1679 1669 1659

dbpf1. TATTTAATTGCTGAAAGTGAATCAACTGTATTTGCGAATCCACAGATACCAAATCCCATG

||||| |||||||||||||| ||| | | | | | || ||||||| | ||||

hi3281 TATTTGATTGCTGAAAGTGAGTCAGCCGCAACAGAAAGACCTGCGATACCACAAGCCATA

3030 3040 3050 3060 3070 3080

1649 1639 1629 1619 1609 1599

dbpf1. TTAGCACGAACTTTAGTAGGCAAGAAGGCCATTTGAACTGCTTCATAGTTATATTTATCA

|| || | | | || |||||| | ||||| || |||||||||

hi3281 GTACGGTATACATCACGATCATGTAATGCCATTAATGCGGCTTCGTATGAATATTTATCG

3090 3100 3110 3120 3130 3140

1589 1579 1569 1559 1549 1539

dbpf1. GTCATGTAATGAATGATATTCATTGCATCAACATAAGTATCAGTCAACCAGTCGAGAGAT

||| || || || | || | ||| ||||| | | |||||| || | | |

hi3281 TGCATATAGTGGATTACGTTTAAGGCAGTCACATATTGTTTTGCCAACCAATCCATAAAG

3150 3160 3170 3180 3190 3200

1529 1519 1509 1499 1489 1479

dbpf1. TTGTCAAAGTTTTCCATAACTGTATCATAGTCAAGAATTTCGTCACGAACAGGTTC-GAT

| || | ||| |||||||| | || | | ||| ||| || ||| | | |

hi3281 CTATCCATACGAGTCATTACTGTATCGAAATCTAATACTTCATCAGTAATTGGTGCAGTT

3210 3220 3230 3240 3250 3260

1469 1459 1449 1439 1429 1419

dbpf1. GTCGAATACTTTATAATCTTTATGAACGTCATCATAACCACCGTTCAAACCAGTCAACAT

||| | | || || ||||| ||| ||||| | | ||||

hi3281 TTCGGACCTACTTGCATACCTA-ATTTTTCATCGATACCGCCGTTGATTGCGTATAACAA

3270 3280 3290 3300 3310

1409 1399 1389 1379 1369 1359

dbpf1. TGCTTTCAAGACGTTTACACGCGCACCAAAGTATTGGAGGTTATGACGTCCTTCTTCATT

|| |||| | |||| |||| ||||| ||| |||| | | | || || ||||

hi3281 TGTTTTCGCTAAGTTTGCACGTGCACCGAAGAATTGCATTTGTTTAC--CCACAATCAT-

3320 3330 3340 3350 3360 3370

1349 1339 1329 1319 1309 1299

dbpf1. TTCTGGATCAAGTGGTGAGACACAACAAGAGATACATGACATTTCGCCATATCCATCTTT

|||||| |||||||| | ||| | ||| | |||

hi3281 ------------TGGTGATACACAACATGCGATTGCGTAGTCATCGTTGTTGAAGTCTGG

3380 3390 3400 3410 3420

1289 1279 1269 1259 1249 1239

dbpf1. AGCCATTGTTTCAACACCTTCATATTGAATAGAAGA-ATGCTTGTGGCT-CATAGACATT

| |||| ||| | ||| ||||||| || || | | | | | | ||

hi3281 ACGCATTAAATCATCGTTTTCGTATTGAACTGATGAGGTATCAATCGATACTTTTGCACA

3430 3440 3450 3460 3470 3480

1229 1219 1209 1199 1189 1179

dbpf1. GAATAACGTTTGAATGAGTAAGGAAGTTTAGAATCCCAAAGGACTGTCAAGTTTGGTTCT

|| |||||||||| | ||| | || | |||||| | || |||||||| |||

hi3281 GA--AACGTTTGAAGTTTTCAGGTAATTGTTCAGACCAAAGAATGGTTAAGTTTGGCTCT

3490 3500 3510 3520 3530 3540

1169 1159 1149 1139 1129 1119

dbpf1. GGAGCATTTCCGATTGTATCAAGTGTGTTCAAGAAACGGTAGTCCATTTTAGTGACACGG

|||| | | || || | | ||| |||| || | |||| | |||| || ||

hi3281 GGAGAAGTACCCATGTTGTAAAGGGTGTGTAAAATACGGAATGTATTTTTGGTTACTAAT

3550 3560 3570 3580 3590 3600

1109 1099 1089 1079 1069 1059

dbpf1. TGACGTCCGTCATTACCCATACCAGCCATAGATGTTGTGATGAATGTTGGGTCACCAGAA

||| || || ||||||||| || || | | || | |||||||||||||

hi3281 GTACGACCATCTAAACCCATACCTGCGATGGTTTCAGTTGCCCACATTGGGTCACCAGAG

3610 3620 3630 3640 3650 3660

1049 1039 1029 1019 1009 999

dbpf1. TAAAGTTCATCATAAGCAGCTGCACGAGCAAATTTCATTGTACGAAGCTTCAAAACGAAA

| | || ||| || ||| || ||| || | ||||||| |||| ||| ||

hi3281 AATAATTGATCGTATTCAGGTGTACGTAAGAAACGAACCATACGAAGTTTCATAACTAAG

3670 3680 3690 3700 3710 3720

989 979 969 959 949 939

dbpf1. TCATCAACAAATTCTTGAATTTCTTGTTCAGTAAATGTTCCACGAGCAAGGTCACGTTCT

| ||||| |||||||| ||| |||||||| | |||| | ||||||||

hi3281 TGGTCAACTAATTCTTGCGCTTCAGTTTCAGTAATTTTTCCTGCTTTTAAATCACGTTCG

3730 3740 3750 3760 3770 3780

929 919 909 899 889 879

dbpf1. GCAAAGATATCAAGAACGATTGGAACACGTCCAAGTGAAGTTGCAGCTCCA-TTAATGAC

| | |||| || | ||| ||| || | ||| ||||||| ||| || |||

hi3281 ATGTACACGTCAATAAAGGTTGCGGTACGACCGAATGACATTGCAGCACCATTTTGTGAT

3790 3800 3810 3820 3830 3840

869 859 849 839 829 819

dbpf1. ACGACATACTGCCATATAAGCGATGTTAACCCATTGGATTGCTTCTTTTACGTTCATAGC

| | | || | |||||| | || | |||||| || ||||||| | || | ||

hi3281 TTTA-TTGCAGCAAGATAAGCAAAGTACATCCATTGAATGGCTTCTTGAGCATTAGTTGC

3850 3860 3870 3880 3890 3900

809 799 789 779 769 759

dbpf1. TGGACGTGAAACATCAAGACCATATAAGGCACCAAAGTTTACAACTTCTTGCAAAGCTTG

||| |||| |||| |||||| || | | | | | || || || |

hi3281 TGGGTTAGAAATATCATAACCATAGCTTGCTGCCATTTGTTTTAATTGACCTAATGCACG

3910 3920 3930 3940 3950 3960

749 739 729 719 709 699

dbpf1. GTATTGCATATTAATTTCTTCTTTAAGACGAATGTTTTCTTCGTTAATTTCAGTGATTGC

|| ||| |||||||| | |||||| || |||| | || | |

hi3281 GTGTTGTTCTGCGATTTCTTCACGTAAACGAATTGTTGCTTCAAGATTT------ACGCC

3970 3980 3990 4000 4010

689 679 669 659 649 639

dbpf1. ATCCCATTCTTTTGCTTTTTCCTTCATAAG-GTAATCAGCACCGTAAAGGGCAAGACGTG

||| |||| |||||| | | ||| | | | || | | |

hi3281 ATC---TTCTAAATCTTTTT-GTAAAGAAGAGAATTGTGCGTATTTATCTTTCATTAAGA

4020 4030 4040 4050 4060 4070

629 619 609 599 589 579

dbpf1. CATAGACACCAATGATACGTCCACGAGAGTAAGCATCTGGCAAACCTGTTACAGTATGAG

|| |||||| | | ||||| ||| || | | || | || ||

hi3281 AATCTACACCATAAAGTGCTACACGACGGTAGTCACCGATGATACGACCACGACCATAAG

4080 4090 4100 4110 4120 4130

569 559 549 539 529 520

dbpf1. CAT---GACGTGCTTTACGAATTGCTGAAGTATAAGCACGAAAGAT-TCCATCATTTACA

||| || | | | | | || || ||| || || || | | || |

hi3281 CATCTGGAAGACCAGTTAATACCCCAGATTTACGGCAACGTAAAATATCTGGCGTGTAAA

4140 4150 4160 4170 4180 4190

519 509 499 489 479

dbpf1. ----GAAGTCA--TTGTTTGTG------ACAAAACATCATGCAAGCCTGGGTCAACTGAG

||| || ||| || || || | ||| ||| | | |||

hi3281 CATCGAATACACCTTGGTTATGTGTTTTACGGTA-TTCAGTGAAGATTTTTTTCACTTTT

4200 4210 4220 4230 4240 4250

469 459 449 439 429 419

dbpf1. AGACCGTGTT--CTGTCAAAATCTTTTCAGCAACACGAAGTCCACCTCTTGGCATGAAAT

|| | ||| | || || |||| || ||| |||||| || | || |

hi3281 GGATCAAGTTCACGACCATAAACTTTACAAGAAC----CTTCCACCATTTTG-ATACCAC

4260 4270 4280 4290 4300

409 399 389 379 369 359

dbpf1. TCAAGCGGAAAAGTTCGCTATTT-----TGCATCCCATAGATGAGTTCAAGTTCTTTATC

|| | | || | | ||| | |||| | || || ||| ||| ||

hi3281 CGAATGGCATAATGGCACGTTTTAAAGGTTCATCAGTTTGA--AGACCAACGATTTTTTC

4310 4320 4330 4340 4350 4360

349 339 329 319 309

dbpf1. ATTAGCATCGATATATCCAGCAGGGATTTTATCAATAGAGGT-TACGCGGTCAGT--ATC

| | | | || | |||| | | ||| ||| | | || || |||

hi3281 TAAATCTTTGTTAATGTAACCAGGTGCGTGAGAGATAATGGTAGATGGTGTATGTTCATC

4370 4380 4390 4400 4410 4420

299 289 279 269 259 249

dbpf1. GAAGGGAAATCCTACTT--CTTC-GTAGTGATTTTTTGTATCTTCAAT---AATTTTCTT

|| ||| | | | | || | | |||| || |||| || | || |

hi3281 AAAATCTAATGGCGCGTGAGTACGGTTTTCAATTTTAATACCTTCCATCACAGATTCCCA

4430 4440 4450 4460 4470 4480

239 229 219 209 199 189

dbpf1. TACTTTAAGTGTACGTTCTGTTGGCCCAGC-AAGAAAGCTTTCATCACCATCATATGGTT

| || ||| ||| ||||| || || ||||||| ||||| || ||||| ||

hi3281 AAGCTTGGTTGTTGCTTCGGTTGGACCTGCTAAGAAAG-AGTCATCGCCTTCATAAGGGG

4490 4500 4510 4520 4530 4540

179 169 159 149 139 129

dbpf1. TGTAGTTTTCTTGTACAAAGCGAGTAACGCTTGCTTTATCGCGCCAGTTGGTTCCTTTAA

| ||||||| ||| | |||| | || | | || || |||| | | || ||

hi3281 TATAGTTTTTTTGGATAAAGTCACGTACATTGACATTTTCTTGCCAATCGCCACCAGCAA

4550 4560 4570 4580 4590 4600

119 109 99 89 79 69

dbpf1. AACCATCCCAAGCTTGTTCAAAGATATTTTCCGTAACTTCGGTTTTCATTTTGAAATCTC

||||| |||| || || || ||| | | || ||| | | | | || ||

hi3281 AACCAGCCCACGC-----CA---ATTTTTGCATTTCATTAAGTTCTGACATAGTCATTTC

4610 4620 4630 4640 4650

59 49 39 29 19 9

dbpf1. CTTTGTTCTTTCTGTTTCAAATTACAACTTCATTATATCATACAGTTTGAGAATAGCAAA

||||||| ||

hi3281 CTTTGTTAATTAATAAATAAATCTTTAATGTGTTTTGGTTAAATAACGTTGGAATACACC

4660 4670 4680 4690 4700 4710

dbpf1: complementary strand corresponding to nucleotides 1979-9 of SEQ ID NO:15;

hi3281: SEQ ID NO:18

[0137]

13

TABLE 3.4

Protein homology (FASTA, GCG Wisconsin Package Version 8. Genetics Computer Group)

using the complete protein sequence derived from the L. lactis DB1341 pf1 sequence

shown in TABLE 3.2

Only alignment of the L. lactis Pf1 protein (dbpf1.pep) with the best four scores

is shown.

The Pf1 protein of Streptococcus mutans was not recorded in the searched protein

databases.

(Peptide) FASTA of: dbpf1.pep from: 1 to: 788 Jul. 19, 1996 09:11

The best scores are:
init1
initn
Opt . . .

sw:pf1b_ecoli
P09373 escherichia coli. formate ac.
560
1498
1502

sw:pf13_ecoli
P42632 escherichia coli. probable f.
558
1358
1487

sw:pf1b_haein
P43753 haemophilus influenzae. form.
545
1228
1521

sw:pf1_chlre
P37836 chiamydomonas reinhardtii. f.
163
259
306

sw:fasd_ecoli
P46000 escherichia coli. outer memb.
53
113
75

sw:gtf2_strdo
P27470 streptococcus downei (strept.
46
110
75

sw:frap_rat
P42346 rattus norvegicus (rat). fkb
42
101
53

sw:frap_human
P42345 homo sapiens (human). fkbp-r.
42
101
53

dbpf1.pep

sw:pf1b_ecoli

ID PFLB_ECOLI STANDARD; PRT; 759 AA.

AC P09373;

DE FORMATE ACETYLTRANSFERASE 1 (EC 2.3.1.54) (PYRUVATE FORMATE-LYASE 1) . . .

SCORES Init1: 560 Initn: 1498 Opt: 1502

42.2% identity in 732 aa overlap

10 20 30 40 50 59

dbpf1. MKTEVTENIFEQAWDGFKGTNWRDKASVTRFVQENYKPYDGDESFLAGPTERTLKV-KKI

:: ||:||: ::|:::::| |:|:||:||:||||||||:|| | :: :|:

pf1b_e SELNEKLATAWEGFTKGDWQNEVNVRDFIQKNYTPYEGDESFLAGATEATTTLWDKV

10 20 30 40 50

60 70 80 90 100 110

dbpf1. IEDTK-NHYEEVGFPFDTDRVTSIDKIPAGYIDANDKELELIYGMQNSELFRLNFMPRGG

:|::| :: ::: |||: :::|:: |||| :|:|| | |:|::: :: :::| ||

pf1b_e MEGVKLENRTHAPVDFDTAVASTITSHDAGYI---NKQLEKIVGLQTEAPLKRALIPFGG

60 70 80 90 100 110

120 130 140 150 160 170

dbpf1. LRVAEKILTEHGLSVDPGLHDVLSQTMTSVNDGIFRAYTSAIRKARHAHTVTGLPDAYSR

::: | :::: ::|| ::::::: ::: |:|:| :||::| : |:: ::|||||||:|

pf1b_e IKMIEGSCKAYNRELDPMIKKIFTEYRKTHNQGVFDVYTPDILRCRKSGVLTGLPDAYGR

120 130 140 150 160 170

180 190 200 210 220 230

dbpf1. GRIIGVYARLALYGADYLMKEKAKEWDAI-TEIN-----EENIRLKEEINMQYQALQEVV

||||| | |:|||| |||||:| ::::: :::: |::|||:|||: |::|| ::

pf1b_e GRIIGDYRRVALYGIDYLMKDKLAQFTSLQADLENGVNLEQTIRLREEIAEQHRALGQMK

180 190 200 210 220 230

240 250 260 270 280 290

dbpf1. NFGALYGLDVSRPAMNVKEAIQWVNIAYMAVCRVINGAATSLGRVPIVLDIFAERDLARG

:::| || |:| || |::|||||: ::|:|: : |||| |:||::: ||:: |||| |

pf1b_e EMAAKYGYDISGPATNAQEAIQWTYFGYLAAVKSQNGAAMSFGRTSTFLDVYIERDLKAG

240 250 260 270 280 290

300 310 320 330 340 350

dbpf1. TFTEQEIQEFVDDFVLKLRTMKFARAAAYDELYSGDPTFITTSMAGMGNDGRHRVTKMDY

::|||| ||:||::|:||| ::| |:::||||:||||:: |:|::||| ||| ||| ::

pf1b_e KITEQEAQEMVDHLVMKLRMVRFLRTPEYDELFSGDPIWATESIGGMGLDGRTLVTKNSF

300 310 320 330 340 350

360 370 380 390 400 410

dbpf1. RFLNTLDTIGNAPEPNLTVLWDSKLPYSFKRYSMSMSHKHSSIQYEGVETMAKDGYGEMS

|||||| |:| :||||:|:||::||| :||::: ::| : ||:|||: : | | ::

pf1b_e RFLNTLYTMGPSPEPNMTILWSEKLPLNFKKFAAKVSIDTSSLQYENDDLMRPDFNNDDY

360 370 380 390 400 410

420 430 440 450 460 470

dbpf1. CISCCVSPLDPENEEGRHNLQYFGARVNVLKAMLTGLNGGYDDVHKDYKVFDIEPVRDEI

|:|||||: ::: :|:||||:|: |:|| ::||| |: | : ||:::::

pf1b_e AIACCVSPMIVGKQ-----MQFFGARANLAKTMLYAINGGVDEKLKMQVGPKSEPIKGDV

420 430 440 450 460

480 490 500 510 520 530

dbpf1. LDYDTVMENFDKSLDWLTDTYVDAMNIIHYMTDKYNYEAVQMAFLPTKVRANMGFGICGF

|:||:|||::|: :|||:: |::|:|||||| |||:||| ||: :| :|: || |:

pf1b_e LNYDEVMERMDHFMDWLAKQYITALNIIHYMHDKYSYEASLMALHDRDVIRTMACGIAGL

470 480 490 500 510 520

540 550 560 570 580 590

dbpf1. ANTVDSLSAIKYAKVKTLRDENGYIYDYEVEGDFPRYGEDDDRADDIAKLVMKMYHEKLA

: ::||||||||||||::|||:| |:|:||::|::|::| |:||:| ::::: :|::

pf1b_e SVAADSLSAIKYAKVKPIRDEDGLAIDFEIEGEYPQFGNNDPRVDDLAVDLVERFMKKIQ

530 540 550 560 570 580

600 610 620 630 640 650

dbpf1. SHKLYKNAEATVSLLTITSNVAYSKQTGNSPVHKGVFLNEDGTVNKSKLEFFSPGANPSN

: : |::| :| |:|||||||:|:|:|||:| || ::: | : :: :

pf1b_e KLHTYRDAIPTQSVLTITSNVVYGKKTGNTP---------DG--RRAGAPFGPGANPMHG

590 600 610 620 630

660 670 680 690 700 710

dbpf1. KAKGGWLQNLRSLAKLEFKDANDGISLTTQVSPRALGKTRDEQVDNLVQILDGYFTPGAL

::: | :::| |:||| | |:|||| | : |:||||: : : :||: ::|||| ::|

pfLb_e RDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDDEVRKTNLAGLMDGYFHHEAS

640 650 660 670 680 690

720 730 740 750 760 770

dbpf1. INGTEFAGQHVNLNVMDLKDVYDKIMRGEDVIVRISGYCVNTKYLTPEQKQELTERVFHE

|:|:: : :| : | |:::

pf1b_e IEGGQHLNVNVMNREMLLDAMENPEKYPQLTIRVSGYAVRFNSLTKEQQQDVITRTFTQS

700 710 720 730 740 750

dbpf1: corresponds to amino acid residues 1-772 of SEQ ID NO:16;

pf1b_e: corresponds to amino acid residues of SEQ ID NO:14

dbpf1.pep

sw:pf13_ecoli

ID PFL3_ECOLI STANDARD; PRT; 746 AA.

AC P42632;

DE PROBABLE FORMATE ACETYLTRANSFERASE 3 (EC 2.3.1.54) (PYRUVATE FORMATE- . . .

SCORES Init1: 558 Initn: 1358 Opt: 1487

39.8% identity in 741 aa overlap

10 20 30 40 50

dbpf1. MKTEVTENIFEQAWDGFKGTNWRDKASVTRFVQENYKPYDGDESFLAGPTERTLKV-K

::::::::::::|| |||||:|::: :| |:|:||:||:|||||||::| | :: :

pf13_e MKVDIDTSDKLYADAWLGFKGTDWKNEINVRDFIQHNYTPYEGDESFLAEATPATTELWE

10 20 30 40 50 60

60 70 80 90 100 110

dbpf1. KIIEDTK-NHYEEVGFPFDTDRVTSIDKIPAGYIDANDKELELIYGMQNSELFRLNFMPR

|::|::: :: ::: |||: :|:|: |||| :: || | |:|::: :: :: |

pfL3_e KVMEGIRIENATHAPVDFDTNIATTITAHDAGYI---NQPLEKIVGLQTDAPLKRALHPF

70 80 90 100 110

120 130 140 150 160 170

dbpf1. GGLRVAEKILTEHGLSVDPGLHDVLSQTMTSVNDGIFRAYTSAIRKARHAHTVTGLPDAY

||::: :: : ::| ::|:::: :::: ::: |:|:| :|:::: : |:: ::|||||:|

pf13_e GGINMIKSSFHAYGREMDSEFEYLFTDLRKTHNQGVFDVYSPDMLRCRKSGVLTGLPDGY

120 130 140 150 160 170

180 190 200 210 220 230

dbpf1. SRGRIIGVYARLALYGADYLMKEKAKEWDAI------TEINEENIRLKEEINMQYQALQE

:|||||| | |:|||| :||::|:: ::::: :| |::|||:||:: : :|| :

pf13_e GRGRIIGDYRRVALYGISYLVRERELQFADLQSRLEKGEDLEATIRLREELAEHRHALLQ

180 190 200 210 220 230

240 250 260 270 280 290

dbpf1. VVNFGALYGLDVSRPAMNVKEAIQWVNIAYMAVCRVINGAATSLGRVPIVLDIFAERDLA

: :::| ||:|:|||| |::||:||: :||:|: : ||:| ||||:: |||: |||:

pf13_e IQEMAAKYGFDISRPAQNAQEAVQWLYFAYLAAVKSQNGGAMSLGRTASFLDIYIERDFK

240 250 260 270 280 290

300 310 320 330 340 350

dbpf1. RGTFTEQEIQEFVDDFVLKLRTMKFARAAAYDELYSGDPTFITTSMAGMGNDGRHRVTKM

|:::||: ||::|:|::|:| ::| |::::|:|:||||:: |: ::||| ||| |||

pf13_e AGVLNEQQAQELIDHFIMKIRMVRFLRTPEFDSLFSGDPIWATEVIGGMGLDGRTLVTKN

300 310 320 330 340 350

360 370 380 390 400 410

dbpf1. DYRFLNTLDTIGNAPEPNLTVLWDSKLPYSFKRYSMSMSHKHSSIQYEGVETMAKDGYGE

::|:|:||:|:| |||||||:||:::|| :||:|: :| ||:|||: : | :| ::

pf13_e SFRYLHTLHTMGPAPEPNLTILWSEELPIAFKKYAAQVSIVTSSLQYENDDLMRTDFNSD

360 370 380 390 400 410

420 430 440 450 460 470

dbpf1. MSCISCCVSPLDPENEEGRHNLQYFGARVNVLKAMLTGLNGGYDDVHKDYKVFDIEPVRD

|:|||||: ::: :|:||||:|: |::| ::||| |: | :::|::|

pf13_e DYAIACCVSPMVIGKQ-----MQFFGARANLAKTLLYAINGGVDEKLKIQVGPKTAPLMD

420 430 440 450 460 470

480 490 500 510 520 530

dbpf1. EILDYDTVMENFDKSLDWLTDTYVDAMNIIHYMTDKYNYEAVQMAFLPTKVRANMGFGIC

::||||:||:::|: :|||: |::|:|||||| |||:||| ||: :| :|: ||

pf13_e DVLDYDKVMDSLDHFMDWLAVQYISALNIIHYMHDKYSYEASLMALHDRDVYRTMACGIA

480 490 500 510 520 530

540 550 560 570 580 590

dbpf1. GFANTVDSLSAIKYAKVKTLRDENGYIYDYEVEGDFPRYGEDDDRADDIAKLVMKMYHEK

|:: ::|||||||||:||::||||| |:|::|::|:||::|:|:|:|| ::::: :|

pf13_e GLSVATDSLSAIKYARVKPIRDENGLAVDFEIDGEYPQYGNNDERVDSIACDLVERFMKK

540 550 560 570 580 590

600 610 620 630 640 650

dbpf1. LASHKLYKNAEATVSLLTITSNVAYSKQTGNSPVHKGVFLNEDGTVNKSKLEFFSPGANP

: : |:|| :| |:|||||||:|:::|||:| || ::: | : ::

pf13_e IKALPTYRNAVPTQSILTITSNVVYGQKTGNTP---------DG--RRAGTPFAPGANPM

600 610 620 630 640

660 670 680 690 700 710

dbpf1. SNKAKGGWLQNLRSLAKLEFKDANDGISLTTQVSPRALGKTRDEQVDNLVQILDGYFTPG

:::: | :::| |:||| |: |:|||| | : | ||||: : :||| :||||| ::

pf13_e HGRDRKGAVASLTSVAKLPFTYAKDGISYTFSIVPAALGKEDPVRKTNLVGLLDGYFHHE

650 660 670 680 690 700

720 730 740 750 760 770

dbpf1. ALINGTEFAGQHVNLNVMDLKDVYDKIMRGEDVIVRISGYCVNTKYLTPEQKQELTERVF

| ::|:: : :| : | |:::

pf13_e ADVEGGQHLNVNVMNREMLLDAIEHPEKYPNLTIRVSGYACASTH

710 720 730 740

dbplf: corresponds to amino acid residues 1-770 of SEQ ID NO:16;

pf113_e: SEQ ID NO:19

dbpl.pep

sw:pf1b_haein

ID PFLB_HAEIN STANDARD; PRT; 769 AA.

AC P43753;

DE FORMATE ACETYLTRANSFERASE (EC 2.3.1.54) (PYRUVATE FORMATE-LYASE) . . .

SCORES Init1: 545 Initn: 1228 Opt: 1521

42.1% identity in 781 aa overlap

10 20 30 40 50 59

dbpf1. MKTEVTENIFEQAWDGFKGTNWRDKASVTRFVQENYKPYDGDESFLAGPTERTLKV-KKI
(SEQ ID NO:16)

||:|| |::|:::::| |:|:||:||:||:|||||||| | |: :::

pf1b_h SELNEMQKLAWAGFAGGDWQENVNVRDFIQKNYTPYEGDDSFLAGPTEATTKLWESV
(SEQ ID NO:20)

10 20 30 40 50

60 70 80 90 100 110

dbpf1. IEDTK-NHYEEVGFPFDTDRVTSIDKIPAGYIDANDKELELIYGMQNSELFRLNFMPRGG

:|::| :: ::: : ||:: ::| : ::||| :|:|| | |:|::| :: ::|| ||

pf1b_h MEGIKIENRTHAPLDFDEHTPSTIISHAPGYI---NKDLEKIVGLQTDEPLKRAIMPFGG

60 70 80 90 100 110

120 130 140 150 160 170

dbpf1. LRVAEKILTEHGLSVDPGLHDVLSQTMTSVNDGIFRAYTSAIRKARHAHTVTGLPDAYSR

::::| : :| ::|| ::::::: ::: |:|:| :||::| : |:: ::|||||||:|

pf1b_h IKMVEGSCKVYGRELDPKVKKIFTEYRKTHNQGVFDVYTPDILRCRKSGVLTGLPDAYGR

120 130 140 150 160 170

180 190 200 210 220 230

dbpf1. GRIIGVYARLALYGADYLMKEKAKEWDAI-----TEIN-EENIRLKEEINMQYQALQEVV

||||| | |:||||:|:|||:| ::::: :::| |::|||:|||: |::|| ::

pf1b_h GRIIGDYRRVALYGVDFLMKDKYAQFSSLQKDLEDGVNLEATIRLREEIAEQHRALGQLK

180 190 200 210 220 230

240 250 260 270 280 290

dbpf1. NFGALYGLDVSRPAMNVKEAIQWVNIAYMAVCRVINGAATSLGRVPIVLDIFAERDLARG

:::| || |:|:|| |::|||||: :||:|: : |||| |:||::: :|:: |||| |

pf1b_h QMAASYGYDISNPATNAQEAIQWMYFAYLAAIKSQNGAAMSFGRTATFIDVYIERDLKAG

240 250 260 270 280 290

300 310 320 330 340 350

dbpf1. TFTEQEIQEFVDDFVLKLRTMKFARAAAYDELYSGDPTFITTSMAGMGNDGRHRVTKMDY

::|| | ||:||::|:||| ::| |:::||:|:|||| : |:::|||| ||| ||| ::

pf1b_h KITETEAQELVDHLVMKLRMVRFLRTPEYDQLFSGDPMWATETIAGMGLDGRTLVTKNTF

300 310 320 330 340 350

360 370 380 390 400 410

dbpf1. RFLNTLDTIGNAPEPNLTVLWDSKLPYSFKRYSMSMSHKHSSIQYEGVETMAKDGYGEMS

|:|:|| ::|::||||||:||:::|| :|||:: ::| : ||:|||: : | | ::

pf1b_h RILHTLYNMGTSPEPNLTILWSEQLPENFKRFCAKVSIDTSSVQYENDDLMRPDFNNDDY

360 370 380 390 400 410

420 430 440 450 460 470

dbpf1. CISCCVSPLDPENEEGRHNLQYFGARVNVLKAMLTGLNGGYDDVHKDYKVFDIEPVRDEI

|:|||||: ::: :|:||||:|: |::| ::||| |: :::|: ||:

pf1b_h AIACCVSPMIVGKQ-----MQFFGARANLAKTLLYAINGGIDEKLGMQVGPKTAPITDEV

420 430 440 450 460

480 490 500 510 520 530

dbpf1. LDYDTVMENFDKSLDWLTDTYVDAMNIIHYMTDKYNYEAVQMAFLPTKVRANMGFGICGF

||:||||:::|: :|||:: ||:|:|:|||| |||:|||: ||: :| :|: || |:

pf1b_h LDFDTVMTRMDSFMDWLAKQYVTALNVIHYMHDKYSYEAALMALHDRDVYRTMACGIAGL

470 480 490 500 510 520

540 550 560 570 580

dbpf1. ANTVDSLSAIKYAKVKTLR----DENGYI------YDYEVEGDFPRYGEDDDRADDIAKL

: ::||||||||||||::| |::| : |:|:||::|:||::|:|:||||

pf1b_h SVAADSLSAIKYAKVKPVRGDIKDKDGNVVATNVAIDFEIEGEYPQYGNNDNRVDDIACD

530 540 550 560 570 580

590 600 610 620 630 640

dbpf1. VMKMYHEKLASHKLYKNAEATVSLLTITSNVAYSKQTGNSPVHKGVFLNEDGTVNKSKLE

::::: :|::: | |:|| :| |:|||||||:|:|:|||:| || : :

pf1b_h LVERFMKKIQKLKTYRNAVPTQSVLTITSNVVYGKKTGNTP---------DGRRAGAP--

590 600 610 620 630

650 660 670 680 690 700

dbpf1. FFSPGANPSN-KAKGGWLQNLRSLAKLEFKDANDGISLTTQVSPRALGKTRDEQVDNLVQ

|:||||| : ::: | :::| |:||| | |:|||| | : |:||||: ::| ||:

pf1b_h -FGPGANPMHGRDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDAEAQRRNLAG

640 650 660 670 680 690

710 720 730 740 750 760

dbpf1. ILDGYFTPGALINGTEFAGQHVNLNVMDLKDVYDKIMRGEDVIVRISGYCVNTKYLTPEQ

::|||| ::| ::|:: : :| || | |: :: : ::::|:||| |: : || ||

pf1b_h LMDGYFHHEATVEGGQHLNVNV-LNREMLLDAMENPDKYPQLTIRVSGYAVRFNSLTKEQ

700 710 720 730 740 750

770 780

dbpf1. KQELTERVFHEVLSNDDEEVMHTSNIX

:|::::|:| | :

pf1b_h QQDVITRTFTESM

760

dbpf1.pep

sw:pf1_chlre

ID PFL_CHLRE STANDARD; PRT; 195 AA.

AC P37836;

DE FORMATE ACETYLTRANSFERASE (EC 2.3.1.54) (PYRUVATE FORMATE-LYASE) . . .

SCORES Init1: 163 Initn: 259 Opt: 306

38.0% identity in 213 aa overlap

540 550 560 570 580 590

dbpf1. NTVDSLSAIKYAKVKTLRDENGYIYDYEVEGDFPRYGEDDDRADDIAKLVMKMYHEKLAS

|:||:||:||||:|:||: ::: : :|||:

pf1_ch GSFPKYGNDDDRVDEIAEWVVSTFSSKLAK

10 20 30

600 610 620 630 640 650

dbpf1. HKLYKNAEATVSLLTITSNVAYSKQTGNSPVHKGVFLNEDGTVNKSKLEFFSPGANP-SN

:: |:|: :|:|:|||||||:|:|:||::| || ::| | |:||||| :

pf1_ch QHTYRNSVPTLSVLTITSNVVYGKKTGSTP---------DG---RKKGEPFAPGANPLHG

40 50 60 70

660 670 680 690 700 710

dbpf1. KAKGGWLQNLRSLAKLEFKDANDGISLTTQVSPRALGK-TRDEQVDNLVQILDGYFTPGA

:: | |::|:|:||| :: |||| | : |::||: : :|:::||: ||||||: |:

pf1_ch RDAHGALASLNSVAKLPYTMCLDGISNTFSLIPQVLGRGGEHERATNLASILDGYFANGG

80 90 100 110 120 130

720 730 740 750 760 770

dbpf1. LINGTEFAGQHVNLNVMDLKDVYDKIMRGEDVIVRISGYCVNTKYLTPEQKQELTERVFH

::: :: : ::::: : | ::::|:||| |: ||:||: |:::|:||

pf1_ch HHINVNVLNRSMLMDAVEHPEKY------PNLTIRVSGYAVHFARLTREQQLEVIARTFH

140 150 160 170 180 190

780

dbpf1. EVLSNDDEEVMHTSNIX (corresponding to amino acid residues 535-788 of SEQ ID NO:16)

:::

pf1_ch DTM (SEQ ID NO:21)

[0138] The highest homology value obtained when analysing the sequence from clone pfl1 corresponds to the S. mutans pfl gene (Table 3.1), i.e. about 80% at the DNA level, in the region covered by the probe used for library screening and 68.5% for the 1.1 kb pfl fragment analyzed.

[0139] Sequence comparisons indicated that the fragment included in clone pfl1 encompasses 367 amino acids of the C-terminal region of the L. lactis pfl gene. Therefore, about 1.3 kb of the 5′-end of the pfl gene was lacking.

[0140] A 0.6 kb PstI-EcoRI fragment of clone pfl1, spanning from the polylinker (PstI site) and including a fragment spanning from positions 1342-2003 in the sequence shown in Table 3.2, was randomly labelled and used for screening a λZAP genomic library of strain DB1341 (Sambrook et al., 1989) to get the upstream region of the pfl gene. High stringency hybridization (washing steps at 65° C., 2×30 min in 2×SSC, then 1×30 min in 0.1×SSC; 0.1% SDS) resulted in the isolation of twelve positive clones.

[0141] Sequence analysis of clones pfl9, pfl10, pfl19 and pfl20 showed that they included the same pfl fragment as did clone pfl1. Restriction analysis of the above clones showed that they all contained a 460 bp Sau3AI fragment identical to pfl1 (positions 1342-1798 in Table 1.2). Only clone pfl14 showed a different Sau3AI restriction pattern. This clone lacked the above Sau3AI fragment and had a 600 bp fragment that hybridized to the PstI-EcoRI pfl probe, suggesting that rearrangement of the insert occurred during in vivo excision of the plasmid. Sequence analysis of pfl14 confirmed that it included a pfl fragment that lacked the Sau3AI site at position 1 in clone pfl1, but showed sequence identity from position 30 onwards in clone pfl1 (position 1372 in Table 3.2). It is therefore likely that the presence of an intact L. lactis pfl gene is toxic in E. coli and leads to plasmid rearrangement.

[0142] 4. Inverse PCR to Obtain the Complete pfl Sequence of L. lactis DB1341

[0143] To facilitate the characterization of the 5′ region of the L. lactis pfl gene from strain DB1341 inverse PCR was used. EcoRI-digested genomic DNA of strain DB1341 was religated at low concentration (Sambrook et al., 1989) and PCR was carried out using primers pfl1-250 and pfl-390 (see FIG. 4). A 1.6 kb fragment that contained the lacking 421 codons and the upstream region of the L. lactis pfl sequence (positions 1 to 1342 in Table 3.2) was amplified. This PCR fragment was re-amplified from EcoRI-digested and religated DB 1341 DNA using modified primers pfl1-250 (including an XhoI site at the 5′-end) and pfl1-390 (including a BamHI site at the 5′-end) and the amplified product was digested with XhoI and BamHI and ligated into vector pGEM digested with the same enzymes and transformation of E. coli DH5α resulted in strain pflup-1. The L. lactis DB1341 pfl gene encodes a 787 amino acid protein (Tables 3.2, 3.4 and 3.6) with a deduced molecular weight of 89.1 kDa.

[0144] A sample of E. coli DH5α strain pflup-1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession No. DSM 11087.

[0145] 5. Cloning of the pfl Upstream Sequence from L. lactis DB1341

[0146] Inverse PCR was carried out on HhaI-digested and religated chromosomal DNA of strain DB1341, using primers derived from the above sequence (Table 3.2). The HhaI fragment spans about 1.7 kb from position 1 to 1707 in the below sequence which overlaps the sequence shown in Table 3.2 from position 1563 to 1750.

14TABLE 3.5pf1 upstream sequence from L. lactis DB1341HhaI1GCGCCTAGATAAGAAACAGCAACAGCTAAAAGATAGGTATCAAAAGCACT5051TGATTTAAAAATAATGACTTTATCCGATTTTTTGATTCCCAACTCAGATA100101AGAGACTTGCCTTATCAACAATTGCTTGATGAGTCTTTTGGTAAGTCGTT150151TCAAGAGCTAGTTCGGGGAAAGCTCCAACAGCCTCATCAAAGATAATTGG200201GCTATCAGGAAACTGTTCAGCTGATTTTTTAAAGTTTAGATACAAATTTA250251GGGGTTCGTGTTTGAATTTCAAAAAAAATCTCCTCAAGTTAATAAGTTTA300302TTATATCACAAAGTATTCTTTAGACCAATAGTTAATGTAAATGTTTTCTT350351AAGTCGTAGAGAATAAAATTCTCGGAAAAAAAGTCTAAAATCTGCTACAA400401TTAAAGGGACACTAAGAGGATTCCAATCCTCTTTTATCAGGAAAAGAAGG450451GATAGATAGGAAAATGATTAAAAATTATGAACTATCCAACGAAAAAAAAT500 orfA M I K N Y E L S N E K K L(SEQ ID NO:35)501TAATTTCAACCTCTGAAATGAAGAATTTCACCTATGTTCTCAATCCAACA550 I S T S E M K N F T Y V L N P T551CGTGAAGAAATTGGGAATATTTCTGAATACTATGACTTCCCTTTTGACTA600R E E I G N I S E Y Y D F P F D Y601TTTATCAGGAATTTTGGATGACTATGAAAATGCCCGTTTTGAAACAGATG650 L S G I L D D Y E N A R F E T D D651ATAATGATAATAATCTGATTCTCTTACAATATCCTCCACTCTCTAATTAT700 N D N N L I L L Q Y P P L S N Y701GGAGAAGTGGCGACTTTTCCATATTCTTTGGTTTGGACTAAAAATGAATC750G E V A T F P Y S L V W T K N E S751GGTTATTTTAGCACTTAATCATGAGATTGATAATGGCTTAATTTTCGAGC800 V I L A L N H E I D N G L I F E R801GTGAATATGATTATAAACGCTACAAACATCAAGTTATTTTTCAAGTGATG850 E Y D Y K R Y K H Q V I F Q V M851TATCAAATGACACACACTTTCCATGATTATTTGAGAGATTTCCGAACAAG900Y Q M T H T F H D Y L R D F R T R901GCGTCGCAGACTTGAACAGGGAATCAAAAATTCAACAAAGAACGACCAAA950 R R R L E Q G I K N S T K N D Q I951TTGTTGATTTGATTGCCATTCAAGCAAGTTTAATTTATTTTGAAGATGCC1000 V D L I A I Q A S L I Y F E D A1001TTGCACAATAATATGCAAGTACTTCAGGATTTTATTGATTACTTGAGAGA1050L H N N M Q V L Q D F I D Y L R E1051AGATGATGAAGACGGTTTTGCTGAAAAGATTTATGATATTTTTGTCGAAA1100 D D E D G F A E K I Y D I F V E T1101CAGACCAAGCTTATACAGAAACCAAGATTCAGCTCAAGTTACTAGAAAAT1150 D Q A Y T E T K I Q L K L L E N1151CTCCGAGATTTGTTCTCAAACAATGTCTCTAATAACTTGAACATTGTCAT1200L R D L F S N N V S N N L N I V M1201GAAAATCATGACATCAGCTACTTTCGTTCTAGGGATTCCTGCAGTAATTG1250 K I M T S A T F V L G I P A V I V1251TTGGTTTTTACGGAATGAATGTTCCAATTCCTGGTCAAAATTTTAATTGG1300 G F Y G M N V P I P G Q N F N W1301ATGGTTTGGCTTATTTTAGTTCTAGGAATTTTATTATGTGTTTGGGTCAC1350M V W L I L V L G I L L C V W V T1351TTGGTGGTTACATAAAAAAGATATGTTATAAAATGGAGAAAAATCTCCAT1400 W W L H K K D M L Stop1401TTTTTTGCTCTTTGTGAAAAAATTAATTAGTGATTGCAGATTATGAAGTT14501451AGCAATGTTTGTTAAAACTATTTTGTGAATTATTTATGAAAACGTTTTAA15001501AAAAGTATAACAGATATTAAAATAATTGGAACTGTATTAGTAAAGAATCT1550 EcoRI1551GTAATTTCTCTTGAATTCTGTTTGCTATTCTCAAACTGTATGATATAATG16001601AAGTTGTAATTTGAAACAGAAAGAACAAAGGAGATTTCAAAATGAAAACC1650 pf1 M K T1651GAAGTTACGGAAAATATCTTTGAACAAGCTTGGGATGGTTTTAAAGGAAC1700E V T E N I F E Q A W D G F K G T HhaI1701CAACTGGCGCGATAAAGCAAGCGTTACTCGCTTTGTACAAGAAAACTACA1750 N W R D K A S V T R F V Q E N Y KNucleotides 1-1750: SEQ ID NO:34

[0147] The sequence included an open reading frame, designated orfA encoding a putative 37 kDa protein with no relevant homology to any sequence in available databases.

EXAMPLE 4

Characterization of L. lactis orfA Encoding a Putative Transporter Protein

[0148] In gram-negative bacteria, the pfl gene is located downstream of an open reading frame transcribed with focA that codes for a putative membrane-bound formate transporter (Suppmann and Sawers 1994). This genetic organization is conserved in E. coli and H. influenzae but has shown great variation in streptococci (Arnau et al. 1997). In L. lactis, the orfA gene is located immediately upstream of pfl. An open reading frame is also found upstream of the pfl gene in Streptococcus mutans that showed no homology to the L. lactis orfA.

[0149] In E. coli, growth under anaerobiosis results in the synthesis of large amounts of PFL protein, about 3% of the total protein content (Suppmann and Sawers 1994). Consequently, high amounts of formate are formed intracellularly. At physiological intracellular pH in E. coli formate (low pKa, 3.75) is not dissociated and therefore is not membrane-permeable. Thus, there is a requirement for a specific transporter to remove the excess formate in the cells.

[0150] In the following the novel orfA gene of L. lactis and its gene product is characterized.

[0151] 1. The orfA Gene Structure, Protein Homology and Structure

[0152] Sequence analysis of orfA (see Table 3.5. above) showed a “weak” RBS (AGG) and a consensus −10 promoter region upstream of the ATG start codon. No −35 consensus region was identified, suggesting a low expression level for this gene. The deduced protein encoded by orfA, consisting of 306 amino acids and a size of 37 kDa, showed homology (38% identity at the C-terminus) to a 37 kDa putative lactococcal protein (Donkersloot and Thompson 1995) and to a less extent to numerous membrane-bound transporter proteins. A prediction of the structure of OrfA suggested the presence of a large intracellularly located N-terminal region followed by two transmembrane domains, Leu242 to Phe265 and Asn276 to Val294 (FIG. 6). These features are consistent with a possible role of the protein in transport across the cell membrane, although neither sequence homology nor structural similarities with the E. coli FocA protein could be identified. A molecular prediction of the FocA protein showed the presence of six transmembrane domains, but among the related proteins a certain variation in the number of these domains is found. In fact, one of these proteins, the E. coli NirC has four and not six of these domains in its primary sequence (Suppmann and Sawers 1994).

[0153] 2. Expression of orfA

[0154] RNA was isolated from aerobic and anaerobic cultures of L. lactis MG1363 grown in fermenters at 30° C. Using an orfA specific probe (FIG. 7A), Northern blot hybridization was carried out. As shown (FIG. 7B), a low level of expression was observed under the conditions used, which is in agreement with the sequence analysis (lack of −35 region, short RBS) of the upstream region of orfA and with the level of expression expected for a gene coding a membrane associated protein.

[0155] No anaerobic induction was observed in GM17 or GalM17 during exponential growth. In GM17 a lower expression of orfA was detected as compared to GalM17 and virtually no expression of the gene was observed during stationary phase.

[0156] 3. Construction and Analysis of orfA Mutant Strains in L. lactis MG1363

[0157] In order to determine whether orfA is the focA analogue in L. lactis, two mutant strains of MG1363 were constructed. A null mutation was carried out by gene disruption using an internal fragment of the orfA gene (including codons 30-168, FIG. 7A), cloned into the integrative vector pSMA500 and transformed into MG1363. One transformant (MG1363ΔorfA) that formed light blue colonies on X-gal was selected. An orfA multicopy strain was constructed by cloning of the entire coding sequence and promoter region of this gene in pAK80 and transforming into MG1363. As above, a transformant giving blue colonies in X-gal was selected (MG1363 pAK80::orfA).

[0158] In E. coli, a focA null mutant strain was capable of growing at higher sodium hypophosphite concentrations than was the wild type strain. This compound is a formate analogue that is toxic. Thus, transport of hypophosphite into the cytosol via the FocA channel protein is deleterious for the cells (Suppmann and Sawers 1994). If the OrfA protein has a similar function in L. lactis as does FocA in E. coli, then a null mutant should show an increased resistance to hypophosphite and a strain containing multiple copies of the gene should be more sensitive to this compound than the wild type. As shown in FIG. 8, strain MG1363 showed reduced growth when the medium was supplemented with 500 mM of hypophosphite and it did not grow at 600 mM.

[0159] MG1363ΔorfA grew at 600 mM and was unable to grow at higher concentrations. The orfA multicopy strain, MG1363 pAK80::orfA was completely unable to grow at 500 mM hypophosphite. Thus, these results confirmed that OrfA may represent a formate transporter protein in L. lactis.

[0160] The mutant strains constructed included a translational fusion of the orfA gene to the lacLM reporter gene (Madsen et al. 1996). The effect of the addition of formate to the medium on the expression of orfA was studied. To exclude a possible toxic effect of the addition of formate to the medium, a dosis curve was studied. Growth inhibition of the wild type strain was observed at formate concentrations exceeding 10 mM. Exponentially growing cultures (OD600 about 1) were used to measure β-galactosidase after the addition of 10 mM of formate to the growth medium. As shown in the below Table 3.6 similar levels of β-galactosidase were observed in MG1363ΔorfA independently of the addition of formate or the growth conditions.

15TABLE 4.1Analysis of orfA expression in mutant strains of L. lactis strains.a)AerobicAnaerobicSTRAIN+Formate−Formate+Formate−FormateMG1363ΔorfA 9.1 ± 0.3 8.2 ± 0.7 7.5 ± 0.7 6.2 ± 0.1MG1363pAK80::orfA14.6 ± 0.216.7 ± 0.713.2 ± 0.113.2 ± 1.3a)β-galactosidase activity in exponentially growing cultures. At OD600 about 1, formate was added (+formate) and the cultures were incubated further for 15 min before cells were separated by centrifugation and frozen.

[0161] Higher levels were observed in all cases with the multicopy strain MG1363 pAK80::orfA. These levels, about 2-fold higher, did not correlate with the number of copies (5-10 per cell) expected in this strain. A degree of regulation of expression may exist for orfA in L. lactis to ensure an appropriate level of the OrfA protein.

EXAMPLE 5

Isolating and Characterizing the pfl Gene from L. lactis Subspecies lactis MG1363

[0162] 1. Cloning of a Fragment of the pfl Gene

[0163] A pfl fragment was amplified with the above modified primers pfl1-20 and pfl1-1066 from chromosomal DNA of strain MG1363 (see FIG. 4). This fragment was digested and cloned into the vector pGEM digested with XhoI and BamHI, respectively and transformed into E. coli strain DH5α (Stratagene), resulting in strain MGpfl-1. The fragment was sequenced using the relevant primers derived from the sequence of the DB1341 pfl fragment (see FIG. 4).

[0164] The sequence of the MG1363 pfl fragment showed 48 differences (42 base changes and a 6 bp deletion) in the 1 kb region characterized when compared to the corresponding sequence of the DB1341 pfl (below Table 5.1). The deduced Pfl protein fragment encoded by the characterized pfl sequences of strains MG1363 and DB1341 showed high homology. Only four sequence differences are found in a 336 amino acid stretch (below Table 5.1): two amino acid substitutions (Pro447 to Thr473 and Asn486 to Asp486 in Table 5.1) and two adjacent deletions (Asp454-Asp455) encoded by the DB1341 pfl gene. The latter two residues are also present in the protein encoded by the S. mutans pfl gene.

[0165] A sample of E. coli DH5α strain MGpfl-1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession Nos DSM 11088.

16TABLE 5.1Homology between the DNA sequences of a fragment of the pf1 genefragment isolated from L. lactis strains DB1341 (db1341pf1)and a fragment of the pf1 gene of MG1363 (mg1363-pf1)The comparison starts at the position of the Sau3AI sitein the L. lactis DB 1341 pf1 gene (position 1342 in TABLE 3.2).1 50mg1363pf1.......... .......... .......... .......... ..........(SEQ ID NO:22)db1341pf1GATCCAGAAA ATGAAGAAGG ACGTCATAAC CTCCAATACT TTGGTGCGCGconsensus.......... .......... .......... .......... ..........51 100mg1363pf1.......... .......... TGTTACCTGG TTTGAACGGT GGTTAC....db1341pf1TGTAAACGTC TTGAAAGCAA TGTTGACTGG TTTGAACGGT GGTTATGATGconsensus.......... .......... TGTT..CTGG TTTGAACGGT GGTTA.....101 150mg1363pf1..GTTCATAA AGATTATAAA GTATTCGATA TTGAACCTGT TCGTGATGAAdb1341pf1ACGTTCATAA AGATTATAAA GTATTCGACA TCGAACCTGT TCGTGACGAAconsensus..GTTCATAA AGATTATAAA GTATTCGA.A T.GAACCTGT TCGTGA.GAA151 200mg1363pf1ATTCTTGACT ATGATACAGT TATGGAAAAC TTCGACAAAT CACTCAACTGdb1341pf1ATTCTTGACT ATGATACAGT TATGGAAAAC TTTGACAAAT CTCTCGACTGconsensusATTCTTGACT ATGATACAGT TATGGAAAAC TT.GACAAAT C.CTC.ACTG201 250mg1363pf1GTTGACAGAT ACTTATGTTG ATGCAATGAA TATCATTCAC TACATGACTGdb1341pf1GTTGACTGAT ACTTATGTTG ATGCAATGAA TATCATTCAT TACATGACTGconsensusGTTGAC.GAT ACTTATGTTG ATGCAATGAA TATCATTCA. TACATGACTG251 300mg1363pf1ACAAATATAA CTATGAAGCA GTTCAAATGG CCTTCTTGCC TACTAAAGTTdb1341pf1ATAAATATAA CTATGAAGCA GTTCAAATGG CCTTCTTGCC TACTAAAGTTconsensusA.AAATATAA CTATGAAGCA GTTCAAATGG CCTTCTTGCC TACTAAAGTT301 350mg1363pfLCGTGCTAACA TGGGATTTGG TATCTGTGGT TTCGCAAATA CAGTTGATTCdb1341pf1CGTGCTAACA TGGGATTTGG TATCTGTGGA TTCGCAAATA CAGTTGATTCconsensusCGTGCTAACA TGGGATTTGG TATCTGTGG. TTCGCAAATA CAGTTGATTC351 400mg1363pf1ACTTTCAGCG ATTAAATATG CTAAAGTTAA AACTTTGCGT GATGAAAATGdb1341pf1ACTTTCAGCA ATTAAATATG CTAAAGTTAA AACATTGCGT GATGAAAATGconsensusACTTTCAGC. ATTAAATATG CTAAAGTTAA AAC.TTGCGT GATGAAAATG401 450mg1363pf1GCTACATCTA CGATTATGAA GTAGAAGGTG ACTTCCCACG TTATGGTGAAdb1341pf1GCTATATCTA CGATTACGAA GTAGAAGGTG ATTTCCCTCG TTATGGTGAAconsensusGCTA.ATCTA CGATTA.GAA GTAGAAGGTG A.TTCCC.CG TTATGGTGAA451 500mg1363pf1GATGATGACC GTGCTGATGA TATCGCTAAA CTTGTCATGA AAATGTACCAdb1341pf1GATGATGATC GTGCTGATGA TATTGCTAAA CTTGTCATGA AAATGTACCAconsensusGATGATGA.C GTGCTGATGA TAT.GCTAAA CTTGTCATGA AAATGTACCA501 550mg1363pf1TGAAAAATTA GCTTCACACA AACTTTACAA AAATGCTGAA GCTACTGTTTdb1341pf1TGAAAAATTA GCTTCACACA AACTTTACAA AAATGCTGAA GCTACTGTTTconsensusTGAAAAATTA GCTTCACACA AACTTTACAA AAATGCTGAA GCTACTGTTT551 600mg1363pf1CACTTTTGAC AATCACATCT AACGTTGCTT ACTCTAAACA AACTGGTAACdb1341pf1CACTTTTGAC AATTACATCT AACGTTGCTT ACTCTAAACA AACTGGTAATconsensusCACTTTTGAC AAT.ACATCT AACGTTGCTT ACTCTAAACA AACTGGTAA.601 650mg1363pf1TCTCCAGTTC ATAAAGGAGT ATTCCTCAAT GAAGATGGTA CAGTCAACAAdb1341pf1TCTCCAGTAC ATAAAGGAGT ATTCCTCAAT GAAGATGGTA CAGTAAATAAconsensusTCTCCAGT.C ATAAAGGAGT ATTCCTCAAT GAAGATGGTA CAGT.AA.AA651 700mg1363pf1ATCTAAACTT GAATTCTTCT CACCAGGTGC TAACCCATCT AACAAAGCTAdb1341pf1ATCTAAACTT GAATTCTTCT CACCAGGTGC TAACCCATCT AATAAAGCTAconsensusATCTAAACTT GAATTCTTCT CACCAGGTGC TAACCCATCT AA.AAAGCTA701 750mg1363pf1AAGGTGGATG GTTGCAAAAT CTTCGTTCAT TAGCTAAATT GGAATTCAAAdb1341pf1AGGGTGGTTG GTTGCAAAAC CTTCGCTCAT TGGCTAAGTT GGAATTCAAAconsensusA.GGTGG.TG GTTGCAAAA. CTTCG.TCAT T.GCTAA.TT GGAATTCAAA751 800mg1363pf1GATGCAAATG ACGGTATTTC ATTAACTACT CAAGTTTCTC CTCGTGCACTdb1341pf1GATGCAAATG ATGGTATTTC ATTGACTACT CAAGTTTCAC CTCGTGCACTconsensusGATGCAAATG A.GGTATTTC ATT.ACTACT CAAGTTTC.C CTCGTGCACT801 850mg1363pf1TGGTAAAACT CGTGATGAAC AAGTAGATAA CTTGGTTCAA ATTCTTGATGdb1341pf1TGGTAAAACT CGTGATGAAC AAGTGGATAA CTTGGTTCAA ATTCTTGATGconsensusTGGTAAAACT CGTGATGAAC AAGT.GATAA CTTGGTTCAA ATTCTTGATG851 900mg1363pf1GATACTTCAC ACCAGGAGCT TTGATTAATG GTACTGAATT TGCAGGTCAAdb1341pf1GATACTTCAC ACCAGGTGCT TTGATTAATG GTACTGAATT TGCAGGTCAAconsensusGATACTTCAC ACCAGG.GCT TTGATTAATG GTACTGAATT TGCAGGTCAA901 950mg1363pf1CACGTTAACT TGAACGTTAT GGACCTTAAA GATGTTTACG ATAAAATCATdb1341pf1CACGTTAACT TGAACGTAAT GGACCTTAAA GATGTTTACG ATAAAATCATconsensusCACGTTAACT TGAACGT.AT GGACCTTAAA GATGTTTACG ATAAAATCAT951 1000mg1363pf1GCGTGGTGAA GATGTTATCG TTCGTATCTC TGGATACTGT GTTAACACTAdb1341pf1GCGTGGTGAA GATGTTATCG TTCGTATCTC TGGTTACTGT GTCAATACTAconsensusGCGTGGTGAA GATGTTATCG TTCGTATCTC TGG.TACTGT GT.AA.ACTA1001 1050mg1363pf1AATACCTCAC ACCTGAACAA AAACAAGAAT TGACTGAACG TGTCTTCCATdb1341pf1AATACcTCAC ACCAGAACAA AAACAAGAAT TAACTGAACG TGTCTTCCATconsensusAATACCTCAC ACC.GAACAA AAACAAGAAT T.ACTGAACG TGTCTTCCAT1051 1100mg1363pf1GAAGTACTTT CAAACGATGA TGAAGAAGTA ATdb1341pf1GAAGTTCTTT CAAACGATGA TGAAGAAGTA ATGCATACTT CAAACATCTAconsensusGAAGT.CTTT CAAACGATGA TGAAGAAGTA AT........ ..........1101 1150db1341pf1ATTCTTAAAA TTTAATGAAT ATTCGGTCTG TCAGTTTTAC TGACAGACTTconsensus.......... .......... .......... .......... ..........1151 1200db1341pf1TTTTTTACGA AAAAATTAAT CATAATAGTT AAAAACTATT GTTTTTAGTTconsensus.......... .......... .......... .......... ..........1201 1250db1341pf1TAAGAAAGTT AAATTTTATG CTAAAATAGA TGAATGAAAA TGGTAATTGGconsensus.......... .......... .......... .......... ..........1251 1300db1341pf1ATTGACAGGC GGAATTGCGA KTGGGAAATC AACGGTGGTT GATTTTTTGAconsensus.......... .......... .......... .......... ..........db1341pf1:corresponding to nucleotides 1342-2641 of SEQ ID NO:15

[0166]

17

TABLE 5.2

Multialignment of the putative Pf1 protein from L. lactis strains MG1363

(partial sequence; 1) and DB134l (2) with the deduced amino acid sequences

of known cloned bacterial pf1 genes

The L. lactis Pf1 proteins were aligned with the following known Pf1 proteins:

deduced proteins of S. mutans pf1 (3); E. coli pf13 and pf1b genes

(Accession Nos. P42632 and P09373; 4 and 5); H. influenzae Pf1 (6);

C
. pasteurianum Pf1 (7).

Consensus (con) shows conserved positions (bold) among all of the protein

sequences. The four amino acid differences between the MG1363 and DB1341 Pf1

are shown in underlined, bold at the top (1)

60

2
MKTEVTENI FEQAWDGFKG TNWRDKASVT RFVQENYKPY DGDESFLAGP TERTLKVKKI
(SEQ ID NO:16)

3
MATVKTNTDV FEKAWEGFKG TDWKDRASIS RFVQDNYTPY DGGESFLAGP TERSLHIKKV
(SEQ ID NO:24)

4
MKVDIDTSDKL YADAWLGFKG TDWKNEINVR DFIQHNYTPY EGDESFLAEA TPATTELWEK
(SEQ ID NO:19)

5
SELNEK LATAWEGFTK GDWQNEVNVR DFIQKNYTPY EGDESFLAGA TEATTTLWDK
(SEQ ID NO:14)

6
SELNEM QKLAWAGFAG GDWQENVNVR DFIQKNYTPY EGDDSFLAGP TEATTKLWES
(SEQ ID NO:20)

7
LFKQWEGFQD GEWTNDVNVR DFIQKNYKEY TGDKSFLKGP TEKTKKVWDK
(SEQ ID NO:25)

con

W GF W F Q NY Y G SFL T

120

2
IEDTKNHYEE VGFPFDTD-- RVTSIDKIPA GYIDANDKEL ELIYGMQNSE LFRLNFMPRG

3
VEETKAHYEE TRFPMDT--- RITSIADIPA GYID---KEN ELIFGIQNDE LFKLNFMPKG

4
VMEGIRIENA THAPVDFDTN IATTITAHDA GYIN---QPL EKIVGLQTDA PLKRALHPFG

5
VMEGVKLENR THAPVDFDTA VASTITSHDA GYIN---KQL EKIVGLQTEA PLKRALIPFG

6
VMEGIKIENR THAPLDFDEH TPSTIISHAP GYIN---KDL EKIVGLQTDE PLKRAIMPFG

7
AVS-LILEEL KKGILDVDTE TISGINSFKP GYLD---KDN EVIVGFQTDA PLKRITNPFG

con

D I GY E I G Q P G

180

2
GLRVAEKILT EHGLSVDPGL HDVLSQTMTS VNDGIFRAYT SAIRKARHAH TVTGLPDAYS

3
GIRMAETALK EHGYEPDPAV HEIFTKYATT VNDGIFRAYT SNIRRARHAH TVTGLPDAYS

4
GINMIKSSFH AYGREMDSEF EYLFTDLRKT HNQGVFDVYS PDMLRCRKSG VLTGLPDGYG

5
GIKMIEGSCK AYNRELDPMI KKIFTEYRKT HNQGVFDVYT PDILRCRKSG VLTGLPDAYG

6
GIKMVEGSCK VYGRELDPKV KKIFTEYRKT HNQGVFDVYT PDILRCRKSG VLTGLPDAYG

7
GIRMAEQSLK EYGFKISDEM HNIFTNYRKT HNQGVFDAYS EETRIARSAG VLTGLPDAYG

con

G G F Y R TGLPD Y

240

2
RGRIIGVYAR LALYGADYLM KEKAKEWDAI ------TEIN EENIRLKEEI NMQYQALQEV

3
RGRIIGVYAR LALYGADYLM QEKVNDWNSI ------AEID EESIRLREEI NLQYQALGEV

4
RGRIIGDYRR VALYGISYLV RERELQFADL QSRLEKGEDL EATIRLREEL AEHRHALLQI

5
RGRIIGDYRR VALYGIDYLM KDKLAQFTSL QADLENGVNL EQTIRLREEI AEQHRALGQM

6
RGRIIGDYRR VALYGVDFLM KDKYAQFSSL QKDLEDGVNL EATIRLREEI AEQHRALGQL

7
RGRIIGDYRR VALYGIDFLI QEKKKDLSNL -----KGDML DELIRLREEV SEQIRALDEI

con

RGRIIG Y R ALYG L IRLREE AL

300

2
VNFGALYGLD VSRPAMNVKE AIQWVNIAYM AVCRVINGAA TSLGRVPIVL DIFAERDLAR

3
VRLGDLYGLD VRKPAMNVKE AIQWINIAFM AVCRVINGAA TSLGRVPIVL DIFAERDLAR

4
QEMAAKYGFD ISRPAQNAQE AVQWLYFAYL AAVKSQNGGA MSLGRTASFL DIYIERDFKA

5
KEMAAKYGYD ISGPATNAQE AIQWTYFGYL AAVKSQNGAA MSFGRTSTFL DVYIERDLKA

6
KQMAASYGYD ISNPATNAQE AIQWMYFAYL AAIKSQNGAA MSFGRTATFI DVYIERDLKA

7
KKMALSYGVD ISRPAVNAKE AAQFLYFGYL AGVKENNGAA MSLGRTSTFL DIYIERDLEQ

con

YG D PA N E A Q A NG A S GR D ERD

360

2
GTFTEQEIQE FVDDFVLKLR TMKFARAAAY DELYSGDPTF ITTSMAGMGN DGRHRVTKMD

3
GTFTESEIQE FVDDFVMKLR TVKFARTKAY DELYSGDPTF ITTSMAGMGA DGRHRVTKMD

4
GVLNEQQAQE LIDHFIMKIR MVRFLRTPEF DSLFSGDPIW ATEVIGGMGL DGRTLVTKNS

5
GKITEQEAQE MVDHLVMKLR MVRFLRTPEY DELFSGDPIW ATESIGGMGL DGRTLVTKNS

6
GKITETEAQE LVDHLVMKLR MVRFLRTPEY DQLFSGDPMW ATETIAGMGL DGRTLVTKNT

7
GLITEDEAQE VIDQFIIKLR LVRHLRTPEY NELFAGDPTW VTESIAGVGI DGRSLVTKNS

con

G QE D K R R L GDP T G G DGR VTK

420

2
YRFLNTLDTI GNAPEPNLTV LWDSKLPYSF KRYSMSMSHK HSSIQYEGVE TMAKDGYGEM

3
YRFLNTLDNI GNAPEPNLTV LWSSKLPYSF RHYCMSMSHK HSSIQYEGVT TMAKEGYGEM

4
FRYLHTLHTM GPAPEPNLTI LWSEELPIAF KKYAAQVSIV TSSLQYENDD LMRTDFNSDD

5
FRFLNTLYTM GPSPEPNMTI LWSEKLPLNF KKFAAKVSID TSSLQYENDD LMRPDFNNDD

6
FRILHTLYNM GTSPEPNLTI LWSEQLPENF KRFCAKVSID TSSVQYENDD LMRPDFNNDD

7
FRYLHTLINL GSAPEPNMTV LWSENLPESF KKFCAEMSIL TDSIQYENDD IMRPI-YGDD

con

L TL G PEPN T LW LP F S S QYE M

480

1
LPGLNG GY--VHKDYK VFDIEPVRDE
(SEQ ID NO:23)

2
SCISCCVSPL DPENEEGRHN LQYFGARVNV LKAMLTGLNG GYDDVHKDYK VFDIEPVRDE

3
SCISCCVSPL DPENEDRRHN LQYFGARVNV LKALLTGLNG GYDDVHKDYK VFDVEPIRDE

4
YAIACCVSPM VIG-----KQ MQFFGARANL AKTLLYAING GVDEKLKIQV GPKTAPLMDD

5
YAIACCVSPM IVG-----KQ MQFFGARANL AKTMLYAING GVDEKLKMQV GPKSEPIKGD

6
YAIACCVSPM IVG-----KQ MQFFGARANL AKTLLYAING GIDEKLGMQV GPKTAPITDE

7
YAIACCVSAM RVG-----KD MQFFGARCNL AKCLLLAING GVDEKKGIKV VPDIEPITDE

con

I CCVS Q FGAR N K L NG G P

540

1
ILDYDTVMEN FDKSLNWLTD TYVDAMNIIH YMTDKYNYEA VQMAFLPTKV RANMGFGICG

2
ILDYDTVMEN FDKSLDWLTD TYVDAMNIIH YMTDKYNYEA VQMAFLPTKV RANMGFGICG

3
VLDFETVKAN FEKALDWLTD TYVDAMNIIH YMTDKYNYEA VQMAFLPTRV KANMGFGICG

4
VLDYDKVMDS LDHFMDWLAV QYISALNIIH YMHDKYSYEA SLMALHDRDV YRTMACGIAG

5
VLNYDEVMER MDHFMDWLAK QYITALNIIH YMHDKYSYEA SLMALHDRDV IRTMACGIAG

6
VLDFDTVMTR MDSFMDWLAK QYVTALNVIH YMHDKYSYEA ALMALHDRDV YRTMACGIAG

7
VLDYEKVKEN YFKVLEYMAG LYVNTNNIIH FMHDKYAYEA SQMALHDTKV GRLMAFGIAG

con

L V Y N IH YM DKY YEA MA V M GI G

600

1
FANTVDSLSA IKYAKVKTLR DEN------- ---GYIYDYE VEGDFPRYGE DDDRADDIAK

2
FANTVDSLSA IKYAKVKTLR DEN------- ---GYIYDYE VEGDFPRYGE DDDRADDIAK

3
FSNTVDSLSA IKYATVKPIR DED------- ---GYIYDYE TVGNFPRYGE DDDRVDSIAE

4
LSVATDSLSA IKYARVKPIR DEN------- ---GLAVDFE IDGEYPQYGN NDERVDSIAC

5
LSVAADSLSA IKYAKVKPIR DED------- ---GLAIDFE IEGEYPQFGN NDPRVDDLAV

6
LSVAADSLSA IKYAKVKPVR GDIKDKDGNV VATNVAIDFE IEGEYPQYGN NDNRVDDIAC

7
FSVAADSLSA IRYAKVKPIR -EN------- ---GITVDFV KEGDFPKYGN DDDRVDSIAV

con

DSLSA IKYA VK R D G P G D R D A

660

1
LVMKMYHEKL ASHKLYKNAE ATVSLLTITS NVAYSKQTGN SPVHKGVFLN EDGTVNKSKL

2
LVMKMYHEKL ASHKLYKNAE ATVSLLTITS NVAYSKQTGN SPVHKGVFLN EDGTVNKSKL

3
WLLEAFHTRL ARHKLYKDSE ATVSLLTITS NVAYSKQTGN SPVHKGVYLN EDGSVNLSKV

4
DLVERFMKKI KALPTYRNAV PTQSILTITS NVVYGQKTGN TPD------- -----GRRAG

5
DLVERFMKKI QKLHTYRDAI PTQSVLTITS NVVYGKKTGN TPD------- -----GRRAG

6
DLVERFMKKI QKLKTYRNAV PTQSVLTITS NVVYGKKTGN TPD------- -----GRRAG

7
EIVEKFSDEL KKHPTYRNAK HTLSVLTITS NVMYGKKTGT TPD------- -----GRKVG

con

Y T S LTITS NV Y TGN P

720

1
EFFSPGANPS NKA-KGGWLQ NLRSLAKLEF KDANDGISLT TQVSPRALGK TRDEQVDNLV

2
EFFSPGANPS NKA-KGGWLQ NLRSLAKLEF KDANDGISLT TQVSPRALGK TRDEQVDNLV

3
EFFSPGANPS NKA-SGGWLQ NLNSLKKLDF AHANDGISLT TQVSPKALGK TFDEQVANLV

4
TPFAPGANPM HGRDRKGAVA SLTSVAKLPF TYAKDGISYT FSIVPAALGK EDPVRKTNLV

5
APFGPGANPM HGRDQKGAVA SLTSVAKLPF AYAKDGISYT FSIVPNALGK DDEVRKTNLA

6
APFGPGANPM HGRDQKGAVA SLTSVAKLPF AYAKDGISYT FSIVPNALGK DAEAQRRNLA

7
EPLAPGANPM HGRDMEGALA SLNSVAKVPY VCCEDGVSNT FSIVPDALGN DHDVRINNLV

con

PGANP G L S K DG S T P ALG NL

780

1
QILDGYFTPG ALINGTEFAG QHVNLNVMDL KDVYDKIMRG EDV---IVRI SGYCVNTKYL

2
QILDGYFTPG ALINGTEFAG QHVNLNVMDL KDVYDKIMRG EDV---IVRI SGYCVNTKYL

3
TILDGYF--- ------EGGG QHVNLNVMDL KDVYDKIMNG EDV---IVRI SGYCVNTKYL

4
GLLDGYFHHE ADV----EGG QHLNVNVMNR EMLLDAIEHP EKYPNLTIRV SGYACASTH

5
GLMDGYFHHE ASI----EGG QHLNVNVMNR EMLLDAMENP EKYPQLTIRV SGYAVRFNSL

6
GLMDGYFHEE ATV----EGG QHLNVNVLNR EMLLDAMENP DKYPQLTIRV SGYAVRFNSL

7
SIMGGYF--- ------GQGA HHLNVNVLNR ETLIDAMNNP DKYPTLTIRV SGYAVNFNRL

con

GYF H N NV D R SGY

1
TPEQKQELTE RVFHEVLSND DEEV

2
TPEQKQELTE RVFHEVLSND DEEVMHTSNI Z

3
TKEQKTELTQ RVFHEVLSMD DAATDLVNNK Z

4

5
TKEQQQDVIT RTFTQSM

6
TKEQQQDVIT RTFTESM

7
SKDHQKEVIS RTFHEKL

con

[0167] 2. Cloning and Sequencing of the Entire pfl Gene of L. lactis Strain MG1363

[0168] The entire pfl gene sequence was obtained from L. lactis subsp. cremoris strain MG1363 using PCR. Like the pfl coding sequence of L. lactis strain DB1341 the coding sequence of MG1363 comprises 2363 bp and encodes a 787 amino acid PFL protein having a predicted molecular weight of 89.1 kDa.

18TABLE 5.3The complete sequence of the pf1 locus of L. lactis strain MG13631TTGGGCTATAAGGAAATTGTTCTGCTGATTTTTTAAAGTTTAGATATAGG50Nucleotides 1-4191:51TTTAGGGGTTCATGTTTGAATTTCAAAAAAAGTCTCCTCAAGTTAATAAG100SEQ ID NOS:36/38)101TTTATTATATCACAAAGTATTATTTAGACCAACTTCCTTCAAAAAACTTT150151TCGTTAAGGCTTTGAAATAAAATAATGAGAAAAAAATAGGAAAATCTGCT200201ACAATTAGAAGGAGAAGAAGAGGATTTAAATCCTTTTTTATTAGGAAAAG250251AAGGGATAGATAGGCTGATATGATAAAAAATTATGAACTATCCAATGAAA300 orfA M I K N Y E L S N E K(SEQ ID NO:37) Sau3AI301AAAAATTGATCTCAACTTCTGAGATGAAGAATTTCACTTATGTCCTCAAT350 K L I S T S E M K N F T Y V L N351CCAACACGTGAAGAAATTGGGAATATCTCAGAACACTATGATTTTCCTTT400P T R E E I G N I S E H Y D F P F401TGACTATCTATCTGGAATTTTAGATGACTATGAAAATGCCCGTTTTGAAA450 D Y L S G I L D D Y E N A R F E T451CAGATGATAATGACAATAATCTGATTCTTTTGCAATATCCCGCCTTGTCC500 D D N D N N L I L L Q Y P A L S501AACTATGGAGAAGTGGCCACTTTTCCATATTCTTTGGTTTGGACTAAGAA550N Y G E V A T F P Y S L V W T K N551TGAATCGGTTATTTTGGCCCTTAACCATGAAATTGATAATGGTCTCATTT600 E S V I L A L N H E I D N G L I F601TTGAACGAGAATATGATTATAAACGCTATAAACACCAATTGATTTTTCAA650 E R E Y D Y K R Y K H Q L I F Q651GTGATGTCACCAAATGACTCATACTTTTCATGATTATTTGAGAGACTTTAG700V M Y Q M T H T F H D Y L R D F R701AACAAGGCGCCGCCGGCTTGAAGTTGGTATCAAAAATTCAACAAAAAATG750T R R R L E V G I K N S T K N D751ACCAAATTGTTGACTTAATTGCCATTCAAGCGAGTTTGATTTATTTTGAA800 Q I V D L I A I Q A S L I Y F E801GATGCGCTGCACAATAATATGCAAGTTCTCCAGAATTTTATTGATTACTT850D A L H N N M Q V L Q N F I D Y L851ACGAGAAGATGATGAAGATGGTTTTGCCGAAAAAATCTATGATATTTTTG900 R E D D E D G F A E K I Y D I F V901TCGAAACAGACCAAGCTTATACAGAAACCAAGATTCAGCTCAAGTTACTA950 E T D Q A Y T E T K I Q L K L L951GAAAATCTCCGAGATTTGTTCTCAAACATTGTCTCTAATAATTTGAATAT1000E N L R D L F S N I V S N N L N I1001CGTCATGAAAATTATGACCTCAGCAACATTTGTTCTAGGTATTCCGGCGG1050 V M K I M T S A T F V L G I P A V1051TTATTGTCGGCTTTTATGGAATGAATGTTCCGATTCCTGGTCAAAATTTT1100 I V G F Y G M N V P I P G Q N F1101AATTGGATGGTCTGGCTCATTTTGGTGTTTGGAATTTTATTATGTGTTTG1150N W M V W L I L V F G I L L C V W1151GGTTACTTGGTGGCTACACAAAAAAGATATGTTATGAATGGAGAAAATTT1200 V T W W L H K K D M L Stop1201CTCCGTTTTTTTATCTTTGTGAAAAAATTAATTAGTGATAATAAATCATG12501251AAGTTAGCAATGTTTGTCAAAGCTATTTAGTGAATTAATTATGAAAACGT13001301TTTAAAAAAGTATAACAGATATTAAAATAATTGAAACTGTATTAGTAAAG1350 EcoRI1351AATCTGTAATTTCTCTTGAATTCTGTTTGCTATTATCAAACTGTATGATA14001401TAATGAAGTTGTAATTTGAAACAGAAAGAACAAAGGAGATTTCAAAATGA1450 pf1 M K(SEQ ID NO:39)1451AAACCGAAGTTACGGAAAATATCTTTGAACAAGCTTGGGATGGTTTTAAA1500 T E V T E N I F E Q A W D G F K1501GGAACTAACTGGCGCGATAAAGCAAGCGTTACTCGCTTTGTACAAGAAAA1550G T N W R D K A S V T R F V Q E N1551CTACAAACCATATGATGGTGATGAAAGCTTTCTTGCTGGGCCAACAGAAC1600 Y K P Y D G D E S F L A G P T E R1601GTACACTTAAAGTAAAGAAAATTATTGAAGATACAAAAAATCACTACGAA1650 T L K V K K I I E D T K N H Y E1651GAAGTAGGATTTCCCTTTGATACTGACCGCGTAACCTCTATCGATAAAAT1700E V G F P F D T D R V T S I D K I1701TCCTGCTGGATATATTGATGCTAATGATAAAGAACTTGAACTCATCTATG1750 P A G Y I D A N D K E L E L I Y G1751GGATGCAAAATAGCGAACTTTTCCGCTTAAACTTCATGCCAAGAGGTGGT1800 M Q N S E L F R L N F M P R G G1801CTTCGTGTTGCTGAAAAGATTTTGACAGAACACGGTCTTTCAGTTGACCC1850L R V A E K I L T E H G L S V D P1851AGGTTTGCATGATGTTTTGTCACAAACAATGACTTCTGTAAATGATGGAA1900 G L H D V L S Q T M T S V N D G I1901TCTTCCGTGCTTATACTTCAGCAATTCGTAAAGCACGTCACGCTCACACT1950 F R A Y T S A I R K A R H A H T1951GTAACAGGTTTGCCTGATGCATACTCTCGTGGACGTATCATCGGGGTATA2000V T G L P D A Y S R G R I I G V Y2001TGCACGTCTTGCTCTTTATGGAGCTGACTACCTTATGAAGGAAAAAGCAA2050 A R L A L Y G A D Y L M K E K A K2051AAGAATGGGATGCAATCACTGAAATTAATGATGATAACATTCGTCTTAAA2100 E W D A I T E I N D D N I R L K2101GAAGAAATTAACATGCAATACCAAGCTTTGCAAGAAGTTGTAAACTTTGG2150E E I N M Q Y Q A L Q E V V N F G2151TGCTTTGTATGGTCTTGACGTTTCTCGTCCAGCGATGAACGTAAAAGAAG2200 A L Y G L D V S R P A M N V K E A2201CAATCCAATGGGTTAATATTGCATACATGGCAGTTTGTCGTGTTATCAAT2250 I Q W V N I A Y M A V C R V I N2251GGTGCTGCAACTTCACTTGGACGTGTGCCAATCGTTCTTGACATCTTTGC2300G A A T S L G R V P I V L D I F A2301AGAACGTGACCTTGCTCGTGGAACATTTACTGAGCAAGAAATCCAAGAAT2350 E R D L A R G T F T E Q E I Q E F2351TTGTTGATGATTTCATTTTAAAACTTCGTACAATGAAATTTGCTCGTGCT2400 V D D F I L K L R T M K F A R A2401GCTGCTTATGATGAACTTTATTCTGGTGACCCCACGTTCATCACAACATC2450A A Y D E L Y S G D P T F I T T S2451TATGGCTGGTATGGGTAATGACGGACGCCACCGTGTCACTAAAATGGACT2500 M A G M G N D G R H R V T K M D Y2501ATCGTTTCTTGAACACACTTGATACAATCGGAAATGCTCCAGAACCAAAC2550 R F L N T L D T I G N A P E P N2551TTGACAGTTCTTTGGGACTCTAAACTCCCATATTCATTCAAACGTTATTC2600L T V L W D S K L P Y S F K R Y S2601AATGTCTATGAGTCACAAACACTCATCTATCCAATATGAAGGTGTTGAAA2650 M S M S H K H S S I Q Y E G V E T2651CAATGGCTAAAGATGGATATGGCGAAATGTCATGTATCTCTTGTTGTGTC2700 M A K D G Y G E M S C I S C C V2701TCACCACTTGACCCAGAAAATGAAGAAGGACGTCATAATCTCCAATTACTT2750S P L D P E N E E G R H N L Q Y F2751TGGTGCGCGTGTAAACGTCTTGAAAGCAATGTTGACTGGTTTGAACGGTG2800 G A R V N V L K A M L T G L N G G2801GTTACGATGACGTTCATAAAGATTATAAAGTATTCGATATTGAACCTGTT2850 Y D D V H K D Y K V F D I E P V2851CGTGATGAAATTCTTGACTATGATACAGTTATGGAAAACTTCGACAAATC2900R D E I L D Y D T V M E N F D K S2901ACTCAACTGGTTGACAGATACTTATGTTGATGCAATGAATATCATTCACT2950 L N W L T D T Y V D A M N I I H Y2951ACATGACTGACAAATATAACTATGAAGCAGTTCAAAGTGGCCTTCTTGCCT3000 M T D K Y N Y E A V Q M A F L P3001ACTAAAGTTCGTGCTAACATGGGATTTGGTATCTGTGGTTTCGCAAATAC3050T K V R A N M G F G I C G F A N T3051AGTTGATTCACTTTCAGCGATTAAATATGCTAAAGTTAAAACTTTGCGTG3100 V D S L S A I K Y A K V K T L R D3101ATGAAAATGGCTACATCTACGATTATGAAGTAGAAGGTGACTTCCCACGT3150 E N G Y I Y D Y E V E G D F P R3151TATGGTGAAGATGATGACCGTGCTGATGATATCGCTAAACTTGTCATGAA3200Y G E D D D R A D D I A K L V M K3201AATGTACCATGAAAAATTAGCTTCACACAAACTTTACAAAAATGCTGAAG3250 M Y H E K L A S H K L Y K N A E A3251CTACTGTTTCACTTTTGACAATCACATCTAACGTTGCTTACTCTAAACAA3300 T V S L L T I T S N V A Y S K Q3301ACTGGTAACTCTCCAGTTCATAAAGGAGTATTCCTCAATGAAGATGGTAC3350T G N S P V H K G V F L N E D G T EcoRI3351AGTCAACAAATCTAAACTTGAATTCTTCTCACCAGGTGCTAACCCATCTA3400 V N K S K L E F F S P G A N P S N3401ACAAAGCTAAAGGTGGATGGTTGCAAAATCTTCGTTCATTAGCTAAATTG3450 K A K G G W L Q N L R S L A K LEcoRI3451GAATTCAAAGATGCAAATGACGGTATTTCATTAACTACTCAAGTTTCTCC3500E F K D A N D G I S L T T Q V S P3501TCGTGCACTTGGTAAAACTCGTGATGAACAAGTAGATAACTTGGTTCAAA3550 R A L G K T R D E Q V D N L V Q I3551TTCTTGATGGATACTTCACACCAGGAGCTTTGATTAATGGTACTGAATTT3600 L D G Y F T P G A L I N G T E F3601GCAGGTCAACACGTTAACTTGAACGTTATGGACCTTAAAGATGTTTACGA3650A G Q H V N L N V M D L K D V Y D3651TAAAATCATGCGTGGTGAAGATGTTATCGTTCGTATCTCTGGATACTGTG3700 K I M R G E D V I V R I S G Y C V3701TTAACACTAAATACCTCACACCTGAACAAAAACAAGAATTGACTGAACGT3750 N T K Y L T P E Q K Q E L T E R3751GTCTTCCATGAAGTACTTTCAAATGATGATGAAGAAGTAATGCACACTTC3800V F H E V L S N D D E E V M H T S3801AAATATCTAATTCTTAGTATTAAAAAATATAAGGTCTGTCAGTTCTACTG3850 N I Stop3851ACAGACTTTTTTTCTATAAATTAATTATAATAGTTAAAAACTATTATTTT39003901TAGTTTAAGAAAAATAAAATTTGTGCTAAAATAGATGAATGATAAAGGTA39503951ATTGGATTAACAGGCGGAATTGCGAGTGGGAAATCAACGGTGGTTGATTT40004001TTTGATTTCTGAAGGTTATCAAGTAATTGATGCTGACAAAGTTGTTCGTC40504051AGTTGCAAGAACCTGATGGGAAACTTTTTAATGCAATAATGGAAACTTTC41004101GGTTCAGATTTTACTGACGAAAATGGGAAATTAAACCGATGCAAAATTGA41504151GTGCTTAAGTTTTGCTGACCCAAATCAACGTCAAAAATTAT 4191

[0169] Homology searches using the above deduced PFL protein revealed a 790 overall protein sequence identity with the S. mutans PFL and higher than 40% with the E. coli, C. pasteurianum and H. influenzae PFL.

[0170] In the promoter region of the MG1363 pfl gene canonical lactococcal ribosome binding site (AAAGGAG, position +21 to +27). −35 and −10 promoter regions (TTGCTA and TATAAT, respectively were found. A putative rho-dependent transcription terminator was located 24 bp downstream of the pfl stop codon (position 2432 to 2445). Additionally, two sequences (FNR-1 and FNR-2 with significant homology to E. coli FNR-boxes having consensus sequence TTGAT-N4-ATCAA (SEQ ID NO:40) and being involved in regulation of the expression of pfl in E. coli were identified. The MG1363 FNR-1 (GGAGT-N4ATCAA) (SEQ ID NO:41) was also present in strain DB1341. FNR-2 (TTTGC-N4-ATCAA) (SEQ ID NO:42); position −36 to −23 overlaps with the −35 hexamer of the promoter region of the pfl gene.

[0171] The coding sequence of the MG1363 pfl gene showed 102 basepair changes when compared to the corresponding sequence of strain DB1341, but these changes resulted only in four amino acid changes in the PFL primary structure. The lactococcal PFL includes the conserved Gly residue at position 749, flanked by Ser and Tyr residues, which is involved in activation and deactivation of the enzyme in E. coli via free radical formation. This region is present in all PFL proteins characterized to date. The L. lactis sequence ISCCVSP is highly conserved and includes two adjacent Cys residues.

EXAMPLE 6

Construction of pfl Mutant Strains of L. lactis Strains DB1341 and MG1363 by Gene Inactivation and Physiological Characterization of pfl− Strains

[0172] A 460 bp Sau3AI internal fragment (positions 1343 to 1799 in Table 3.2) of the L. lactis DB1341 pfl gene was cloned into BamHI-digested pSMA500 (Madsen et al., 1996), resulting in plasmid pSMAKAS7, and transformed into E. coli MC1000 by electroporation (Sambrook et al., 1989). A transformant (SMAKAS7) containing the recombinant plasmid was isolated. The orientation of the pfl fragment in pSMAKAS7 was confirmed by sequencing. Homologous recombination of pSMAKAS7 into the L. lactis pfl gene allows translational fusion of the reporter lacLM gene (Madsen et al., 1996).

[0173] Plasmid pSMAKAS7 was used to transform L. lactis strains DB1341 and MG1363 by electroporation (Holo and Nes 1989). Two single transformants were isolated (DBKAS7 and MGKAS7, respectively). DBKAS7 became blue on X-gal plates, as expected if homologous integration at the chromosomal pfl locus had occurred, and was further characterized. Integration of pSMAKAS7 by homologous recombination into the DB1341 chromosome would result in a truncated pfl gene, where the N-terminal region of the protein (residues Met1-Asp574) would be separated from the C-terminal domain (residues Asp422-Ile778). PCR analysis was used to confirm that DBKAS7 carries a disrupted pfl gene. The activation site of the E. coli Pfl, a glycine residue at position Gly734 flanked by serine and tyrosine is conserved in all bacterial Pfl proteins characterized (Weidner and Sawers, 1996; Yamamoto et al., 1996), including the L. lactis Pfl (position 2321-2329 of the nucleotide sequence in Table 3.2; Table 3.6). The truncated Pfl protein in strain DBKAS7 would lack an activation site.

[0174] A sample of Lactococcus lactis subspecies lactis biovar diacetylactis strain DBKAS7 and of Lactococcus lactis subspecies lactis strains MGKAS7, respectively were deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession Nos DSM 11086 and DSM 11083, respectively.

[0175] A 495 bp PCR fragment was amplified from MG1363 using primers pfl1-P1MG1363 (5′-GGCCGCTCGA GTTGTGTCTC ACCACTTGAC CC-3′ (SEQ ID NO:43); XhoI site underlined) and pflP2MG1363 (5′-TAGTAGGATCCCATCATCTT CACCATAACG TGG-3′ (SEQ ID NO:44); BamHI site underlined) and cloned into XhoI+BamHI digested pSMA500 and transformed into strain MG1363, resulting in strain MGKAS13.

[0176] MGKAS13 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 10 Jul. 1997 under the accession No. DSM 11653.

[0177] DBKAS7 and MGKAS13 formed blue colonies on X-gal-containing plates. Plasmid integration through homologous recombination was confirmed via PCR in both strains.

[0178] Physiological Analysis of the L. lactis pfl− Strain

[0179] A colorimetric assay (Voges-Proskauer, VP; Westerfeld 1945) was used to study acetoin and diacetyl production in strain DBKAS7. The presence of acetoin and diacetyl in the samples results in the formation of red colour which is monitored by measuring OD520. Overnight cultures of strain DBKAS7 (pfl−) and wild type strain DB1341, grown at 30° C. without aeration in GM17 were used. The VP assay was performed by mixing 200 μl bacterial culture, 100 μl 0.3% (w/v) creatine, 100 μl 5 M NaOH, and 50 μl 5% α-naphthol (dissolved in 2.5 M NaOH immediately before use). The mixture was incubated for 10 min at room temperature, with constant stirring to provide aeration. The reaction was stopped by adding 1 ml 4 mM DTT. After centrifugation to remove cellular debris, OD520 was measured. As shown in Table 6.1. DBKAS7 had approximately a 2-fold increase in the production of acetoin/diacetyl as compared to strain DB1341.

19TABLE 6.1 Voges-Proskauer assay for aroma compounds produced byDB1341 and DBKAS7, respectivelyStrainOD600OD520DB13412.400.082DBKAS72.220.155Overnight cultures were grown at 30° C., without shaking in GM17. The OD600 values represent a measure for growth. The OD520 values are the results of the production of acetoin and diacetyl (Westerfeld 1945).

[0180] Thus, gene inactivation of the pfl gene in the L. lactis strain DB1341 results in an enhanced production of aroma compounds, without affecting the ability to grow.

[0181] Similar levels of formate were obtained in strain DB1341 as in MG1363, and no formate was detected in DBKAS7 under anaerobic conditions, confirming the pfl mutant phenotype in this strain.

[0182]

L. lactis
biovar diacetylactis strains are used as starter cultures due to their ability to produce diacetyl during milk fermentation. A mutation in the pfl gene of DB1341 should result in increased pyruvate levels under anaerobic growth. Thus, if excess pyruvate is directed towards the production of diacetyl and acetoin, a higher level of these metabolites would be expected in strain DBKAS7 grown under anaerobiosis. As shown in Table 6.2. a 7-fold increase in the production of aroma compounds was observed in strain DBKAS7 grown in GM17 and a more than 4-fold increase was detected in GalM17 as compared to the wild type strain, DB1341. This demonstrated the effect of a pfl mutation in the production of diacetyl and acetoin in a L. lactis biovar diacetylactis strain.

20TABLE 6.2 Production of aroma compounds in the L. lactisbiovar diacetylactis pfl− strain,DBKAS7 as compared to the wild type strainVoges-Proskauer assay (diacetyl + acetoin in mMa)StrainGlucoseGalactoseDB13410.2≦0.05DBKAS71.50.2acell extracts from stationary culture (OD600 about 3) were assayed according to Casabadan et al. 1980. Values shown are the mean of two independent experiments.

[0183] Inactivation of the pfl gene leads to a transcriptional fusion of the lacLM reporter gene (Madsen et al. 1996) β-galactosidase levels were measured in overnight cultures of strain MGKAS13 grown in M17 with either glucose (GM17) or galactose (GalM17) (Table 6.2). Using GM17, anaerobic growth was observed, about a 10-fold increase of β-galactosidase units, which is consistent with the induction observed at RNA level. High levels of β-galactosidase were observed under anaerobic growth when growing in the presence of galactose, and a 4-fold induction was observed under anaerobiosis in this medium which is in agreement with the RNA studies.

21TABLE 6.3Characterization of the L. lactis Mg1363 pfl− strain. MGKAS13AerobicAnaerobicGrowthFormateβ-galFormateβ-galStrainmedium(mM)(units)(mM)(units)MG1363GM170—5.3—GalM170—42—MGKAS13GM170 9.50150.0GalM17094.60600.0MGKAS13 should not produce formate under anaerbic conditions as a result of the inactivation of the pfl gene in this strain. In strain MG1363, no formate was detected under aerobic growth in GM17, as it would be expected if the lactococcal PFL is inactivated in the presence of oxygen. Relatively low levels of formate were detected under anaerobic conditions. In GalM17 a 8-fold higher amount of formate was detected in anaerobiosis. No formate was detected in strain MGKAS13 in either of the test media, confirming that this strain carries a pfl null mutation.

EXAMPLE 7

Identification of pfl and adhE Homologues in Non-Lactococcus lactic Acid Bacteria Using Lactococcus lactis pfl and adhE Gene Fragments as Probes

[0184] 1. Southern Hybridization of Genomic DNA from Non-Lactococcus Lactic Acid Bacteria Using a L. lactis pfl Gene Fragment as a Probe

[0185] A PCR fragment including most of the L. lactis pfl coding sequence was obtained by amplification of MG1363 genomic DNA with primers pfl89 and pfl1066 (see FIG. 4). A 2 kb DNA fragment (FIG. 9) was obtained and used as a probe in Southern hybridization experiments using EcoRI-digested total DNA from Streptococcus thermophilus ATCC 19258, Leuconostoc mesenteroides subsp. mesenteroides ATCC 10878 and Lactobacillus acidophilus ATCC 4796 (FIG. 10).

[0186] Hybridization was carried out overnight at 65° C. Filters were washed twice in 5×SSC at room temperature for 30 minutes and subsequently once in 3×SSC; 0.1% SDS at 65° C. for 30 minutes. As shown in FIG. 10C the expected EcoRI genomic fragment deduced from L. lactis pfl sequence was detected after overnight exposure. After short exposure of the filters (FIG. 10B) only hybridization was detected in S. thermophilus and only weak signals were detected in L. mesenteroides and L. acidophilus after longer exposure (FIG. 10C), indicating lower pfl sequence homology in these bacteria, as would be expected due to their taxonomic distance to L. lactis.

[0187] 2. Southern Hybridisation of Genomic DNA from Non-Lactococcus Lactic Acid Bacteria Using L. lactis adhE Gene Fragment as a Probe

[0188] Two Sau3AI fragments including most of the L. lactis subsp. lactis biovar diacetylactis DB 1341 adhE coding sequence (FIG. 11) were used as a probe in Southern hybridization experiments using EcoRI-digested total DNA from Streptococcus thermophilus ATCC 19258, Leuconostoc mesenteroides subsp. mesenteroides ATCC 10878 and Lactobacillus acidophilus ATCC 4796.

[0189] Hybridization was carried out overnight at 65° C. Filters were washed twice in 5×SSC at room temperature for 30 minutes and subsequently once in 3×SSC; 0.1% SDS at 65° C. for 30 minutes. As shown in FIG. 12, the expected EcoRI genomic fragment (about 5 kb) deduced from the L. lactis MG1363 adhE sequence was detected. Strongly hybridizing bands were also detected in S. thermophilus (5 kb) and L. mesenteroides (5 and 0.4 kb). Weaker hybridizing bands were also detected in L. acidophilus (4.2 and 2 kb, and two minor bands, 2.3 and 5 kb).

[0190] 3. Conclusions

[0191] Using the above L. lactis DNA probes, preliminary restriction maps of the pfl and adhE genes, respectively in the three non-Lactococcus lactic acid bacterial species could be carried out using different restriction digests of the genomic DNA. Two strategies for the cloning of these non-Lactococcus genes can be followed: (i) cloning of DNA fragments isolated from agarose gels corresponding in size to the hybridizing bands detected in Southern analysis; (ii) PCR of conserved regions using primers derived from the corresponding L. lactis sequence.

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	Number	Date	Country
Parent	08981097	Dec 1997	US
Child	10267989	Oct 2002	US

Metabolically engineered lactic acid bacteria and means for providing same

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