Metabolically engineered lactic acid bacteria and means for providing same

Abstract
The complete DNA sequences for adhE and pfl genes of Lactococcus lactis, recombinant replicons comprising one or both of these genes or comprising mutants or variants hereof including mutants in which the genes are inactivated, and recombinant lactic acid bacteria comprising such a replicon are provided. The gene sequences and/or sequences regulating the expression of the genes can be modified to provide metabolically engineered lactic acid bacteria which have an enhanced or reduced production of one or more metabolites resulting from citrate and/or sugar fermentation. Such metabolically modified cell are useful as starter cultures in the manufacturing of food products and animal feed having improved flavour and/or shelf life, including dairy products, or they can be used directly in the manufacturing of a lactic acid bacterial metabolite.
Description


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---------+---------+---------+---------+---------+200ThrLysLysAlaAlaProAlaAlaLysLysValLeuSerAlaGluGluLysAGCCGCAAAATTCCAAGAAGCTGTTGCTTATACTGACAAATTAGTCAAAA201---------+---------+---------+---------+---------+250 AlaAlaLysPheGlnGluAlaValAlaTyrThrAspLysLeuValLysLysAAGCACAAGCTGCTGTTCTTAAATTTGAAGGATATACACAAACTCAAGTC251---------+---------+---------+---------+---------+300  AlaGlnAlaAlaValLeuLysPheGluGlyTyrThrGlnThrGlnValGATACTATTGTCGCTGCAATGGCTCTTGCAGCAAGCAAACATTCTCTAGA301---------+---------+---------+---------+---------+350AspThrIleValAlaAlaMetAlaLeuAlaAlaSerLysHisSerLeuGluACTCGCTCATGAAGCCGTTAACGAAACTGGTCGTGGTGTTGTCGAAGACA351---------+---------+---------+---------+---------+400 LeuAlaHisGluAlaValAsnGluThrGlyArgGlyValValGluAspLysAATGACAAAACTGTTGGTGTCATTTCTGAAAACAAGGTTGCTGGATCTGT401---------+---------+---------+---------+---------+450  AspThrLysAsnHisPheAlaSerGluSerValTyrAsnAlaIleLysAATGACAAAACTGTTGGTGTCATTTCTGAAAACAAGGTTGCTGGATCTGT451---------+---------+---------+---------+---------+500AsnAspLysThrValGlyValIleSerGluAsnLysValAlaGlySerValTGAAATCGCAAGCCCTCTCGGTGTACTTGCTGGTATCGTTCCAACGACTA501---------+---------+---------+---------+---------+550 GluIleAlaSerProLeuGlyValLeuAlaGlyIleValProThrThrAsnATCCAACATCAACAGCAATCTTTAAATCTTTATTGACTGCAAAAACACGT551---------+---------+---------+---------+---------+600  ProThrSerThrAlaIlePheLysSerLeuLeuThrAlaLysThrArgAATGCTATTGTTTTCGCTTTCCACCCTCAAGCTCAAAAATGTTCAAGCCA601---------+---------+---------+---------+---------+650AsnAlaIleValPheAlaPheHisProGlnAlaGlnLysCysSerSerHisTGCAGCAAAAATTGTTTACGATGCTGCAATTGAAGCTGGTGCACCGGAAG651---------+---------+---------+---------+---------+700 AlaAlaLysIleValTyrAspAlaAlaIleGluAlaGlyAlaProGluAspACTTTATTCAATGGATTGAAGTACCAAGCCTTGACATGACTACCGCCTTG701---------+---------+---------+---------+---------+750  PheIleGlnTrpIleGluValProSerLeuAspMetThrThrAlaLeuATTCAAAACCGTGGACTTGCAACAATCCTTGCAACTGGTGGCCCAGGAAT751---------+---------+---------+---------+---------+800IleGlnAsnArgGlyLeuAlaThrIleLeuAlaThrGlyGlyProGlyMet —GGTAAACGCCGCACTCAAATCTGGTAACCCTTCACTCGGTGTTGGAGCTG801---------+---------+---------+---------+---------+850 ValAsnAlaAlaLeuLysSerGlyAsnProSerLeuGlyValGlyAlaGlyGTAATGGTGCTGTTTATGTTGATGCAACTGCAAATATTGAACGTGCCGTT851---------+---------+---------+---------+---------+900  AsnGlyAlaValTyrValAspAlaThrAlaAsnIleGluArgAlaValGAAGACCTTTTGCTTTCAAAACGTTTTGATAATGGGATGATTTGTGCCAC901---------+---------+---------+---------+---------+950GluAspLeuLeuLeuserLysArgPheAspAsnGlyMetIleCysAlaThrTGAAAATTCAGCTGTTATTGATGCTTCAGTTTATGATGAATTTATTGCTA951---------+---------+---------+---------+---------+1000 GluAsnSerAlaValIleAspAlaSerValTyrAspGluPheIleAlaLysAAATGCAAGAACAAGGCGCTTATATGGTTCCTAAAAAAGACTACAAAGCT1001---------+---------+---------+---------+---------+1050  MetGlnGluGlnGlyAlaTyrMetValProLysLysAspTyrLysAlaATTGAAAGTTTCGTTTTTGTTGAACGTGCTGGTGAAGGTTTTGGAGTAAC1051---------+---------+---------+---------+---------+1100IleGluSerPheValPheValGluArgAlaGlyGluGlyPheGlyValThrTGGTCCTGTTGCCGGTCGTTCTGGTCAATGGATTGCTGAACAAGCTGGTG1101---------+---------+---------+---------+---------+1150 GlyProValAlaGlyArgSerGlyGlnTrpIleAlaGluGluAlaGlyValTCAAAGTTCCTAAAGATAAAGATGTCCTTCTTTTTGAACTTGATAAGAAA1151---------+---------+---------+---------+---------+1200  LysValProLysAspLysAspValLeuLeuPheGluLeuAspLysLysAATATTGGTGAAGCACTTTCTTCTGAAAAACTTTCTCCTTTGCTTTCAAT1201---------+---------+---------+---------+---------+1250AsnIleGlyGluAlaLeuSerSerGluLysLeuSerProLeuLeuSerIleCTACAAAGCTGAAACACGTGAAGAAGGAATTGAGATTGTACGTAGCTTAC1251---------+---------+---------+---------+---------+1300 TyrLysAlaGluThrArgGluGluGlyIleGluIleValArgSerLeuLeuTTGCTTATCAAGGTGCTGGACATAATGCTGCAATTCAAATCGGTGCAATG1301---------+---------+---------+---------+---------+1350  AlaTyrGlnGlyAlaGlyHisAsnAlaAlaIleGlnIleGlyAlaMetGATGATCCATTCGTTAAAGAATATGGCGAAAAAGTTGAAGCTTCTCGTAT1351---------+---------+---------+---------+---------+1400AspAspProPheValLysGluTyrGlyGluLysValGluAlaSerArgIleCCTCGTTAACCAACCAGATTCTATTGGTGGGGTCGGAGATATCTATACTG1401---------+---------+---------+---------+---------+1450 LeuValAsnGlnProAspSerIleGlyGlyValGlyAspIleTyrThrAspATGCAATGCGTCCATCACTTACACTTGGAACTGGTTCATGGGGGAAAAAT1451---------+---------+---------+---------+---------+1500  AlaMetArgProSerLeuThrLeuGlyThrGlySerTrpGlyLysAsnTCACTTTCACACAATTTGAGTACATACGATCTATTGAATGTTAAAACAGT1501---------+---------+---------+---------+---------+1550SerLeuSerHisAsnLeuSerThrTyrAspLeuLeuAsnValLysThrValGGCTAAACGTCGTAATCGCCCACAATGGGTTCGTTTGCCAAAAGAAATTT1551---------+---------+---------+---------+---------+1600 AlaLysArgArgAsnArgProGlnTrpValArgLeuProLysGluIleTyrACTACGAAAAAAATGCAATTTCTTACTTACAAGAATTGCCACACGTCCAC1601---------+---------+---------+---------+---------+1650  TyrGluLysAsnAlaIleSerTyrLeuGlnGluLeuProHisValHisAAAGCTTTCATCGTTGCTGACCCTGGTATGGTTAAATTTGGTTTCGTTGA1651---------+---------+---------+---------+---------+1700LysAlaPheIleValAlaAspProGlyMetValLysPheGlyPheValAspTAAAGTTTTGGAACAACTTGCTATCCGCCCAACTCAAGTTGAAACAAGCA1701---------+---------+---------+---------+---------+1750 LysValLeuGluGlnLeuAlaIleArgProThrGlnValGluThrSerIleTTTATGGCTCTGTTCAACCTGACCCAACTTTGAGCGAAGCAATTGCAATC1751---------+---------+---------+---------+---------+1800  TyrGlySerValGlnProAspProThrLeuSerGluAlaIleAlaIleGCTCGTCAAATGAAACAATTTGAACCTGACACTGTCATCTGTCTTGGTGG1801---------+---------+---------+---------+---------+1850AlaArgGlnMetLysGlnPheGluProAspThrValIleCysLeuGlyGlyTGGTTCTGCTCTCGATGCCGGTAAGATTGGTCGTTTGATTTATGAATATG1851---------+---------+---------+---------+---------+1900 GlySerAlaLeuAspAlaGlyLysIleGlyArgLeuIleTyrGluTyrAspATGCTCGTGGTGAAGCTGACCTTTCTGATGATGCAGGTTTGAAAGAACTT1901---------+---------+---------+---------+---------+1950  AlaArgGlyGluAlaAspLeuSerAspAspAlaSerLeuLysGluLeuTTCCAAGAATTAGCTCAAAAATTTGTCGATATTCGTAAACGTATTATTAA1951---------+---------+---------+---------+---------+2000PheGlnGluLeuAlaGlnLysPheValAspIleArgLysArgIleIleLysATTCTACCATCCACATAAAGCACAAATGGTTGCAATTCCTACTACTTCTG2001---------+---------+---------+---------+---------+2050 PheTyrHisProHisLysAlaGlnMetValAlaIleProThrThrSerGlyGTACTGGTTCTGAAGTGACTCCATTTGCAGTTATCACTGATGATGAAACT2051---------+---------+---------+---------+---------+2100  ThrGlySerGluValThrProPheAlaValIleThrAspAspGluThrCATGTTAAGTACCCACTTGCTGACTACCAATTAACACCACAAGTTGCCAT2101---------+---------+---------+---------+---------+2150HisValLysTyrProLeuAlaAspTyrGlnLeuThrProGlnValAlaIleTGTTGACCCTGAGTTTGTTATGACTGTACCAAAACGTACTGTTTCTTGGT2151---------+---------+---------+---------+---------+2200 ValAspProGluPheValMetThrValProLysArgThrValSerTrpSerCTGGTATTGATGCGATGTCACACGCGCTTGAATCTTACGTTTCTGTTATG2201---------+---------+---------+---------+---------+2250  GlyIleAspAlaMetSerHisAlaLeuGluSerTyrValSerValMetTCTTCTGACTATACAAAACCAATTTCACTTCAAGCGATCAAACTTATCTT2251---------+---------+---------+---------+---------+2300SerSerAspTyrThrLysProIleSerLeuGlnAlaIleLysLeuIlePheTGAAAACTTGACTGAGTCTTATCATTATGACCCAGCGCATCCAACTAAAG2301---------+---------+---------+---------+---------+2350 GluAsnLeuThrGluSerTyrHisTyrAspProAlaHisProThrLysGluAAGGACAAAAAGCCCGCGAAAACATGCACAATGCTGCAACACTCGCTGGT2351---------+---------+---------+---------+---------+2400  GlyGlnLysAlaArgGluAsnMetHisAsnAlaAlaThrLeuAlaGlyATGGCCTTCGCTAATGCTTTCCTTGGAATTAACCACTCACTTGCTCATAA2401---------+---------+---------+---------+---------+2450MetAlaPheAlaAsnAlaPheLeuGlyIleAsnHisSerLeuAlaHisLysAATTGGTGGTGAATTTGGACTTCCTCATGGTCTTGCCATTGCCATCGCTA2451---------+---------+---------+---------+---------+2500 IleGlyGlyGluPheGlyLeuProHisGlyLeuAlaIleAlaIleAlaMetTGCCACATGTCATTAAATTTAACGCTGTAACAGGAAACGTTAAACGTACC2501---------+---------+---------+---------+---------+2550  ProHisValIleLysPheAsnAlaValThrGlyAsnValLysArgThrCCTTACCCACGTTATGAAACATATCGTGCTCAAGAGGACTACGCTGAAAT2551---------+---------+---------+---------+---------+2600ProTyrProArgTyrGluThrTyrArgAlaGlnGluAspTyrAlaGluIleTTCACGCTTCATGGGATTTGCTGGTAAAGATGATTCAGATGAAAAAGCTG2601---------+---------+---------+---------+---------+2650 SerArgPheMetGlyPheAlaGlyLysAspAspSerAspGluLysAlaValTGCAAGCTCTGGTTGCTGAACTTAAGAAACTGACTGATAGCATTGATATT2651---------+---------+---------+---------+---------+2700  GlnAlaLeuValAlaGluLeuLysLysLeuThrAspSerIleAspIleAATATCACCCTTTCAGGAAATGGTATCGATAAAGCTCACCTTGAACGTGA2701---------+---------+---------+---------+---------+2750AsnIleThrLeuSerGlyAsnGlyIleAspLysAlaHisLeuGluArgGluACTTGATAAATTGGCTGACCTTGTTTATGATGATCAATGTACTCCTGCTA2751---------+---------+---------+---------+---------+2800 LeuAspLysLeuAlaAspLeuValTyrAspAspGlnCysThrProAlaAsnATCCTCGTCAACCAAGAATTGATGAGATTAAACAGTTGTTGTTAGATCAA2801---------+---------+---------+---------+---------+2850  ProArgGlnProArgIleAspGluIleLysGlnLeuLeuLeuAspGlnTACTAATAATCTGTTGATAAAATTATTAAAACGCTCTGATGAATTCGTCA2851---------+---------+---------+---------+---------+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 500orfB  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)MG1363GM1705.3GalM17042MGKAS13GM170 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.



REFERENCES

[0192] 1. Arnau, J., F. Jørgensen, S. Madsen, A. Vrang and H. Israelsen. 1997. Cloning, expression and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase. Submitted for publication.


[0193] 2. Chen, Y.-M. and C. C. Lin. 1991. Regulation of the adhE gene, which encodes ethanol dehydrogenase in Escherichia coli. J. Bacteriol. 173:8009-8013.


[0194] 3. Chippaux, M., F. Casse and M.-C. Pascal. 1972. Isolation and phenotypes of mutants from Salmonella typhimurium defective in formate hydrogenlyase activity. J. Bacteriol. 110:766-768.


[0195] 4. Christiansen, L. and S. Pedersen. 1981. Cloning, restriction endonuclease mapping and post-transcriptional regulation of rspA, the structural gene for ribosomal protein S1. Mol. Gen. Genet. 181:548-551.


[0196] 5. Crow, V. L. and G. G. Pritchard. 1977. Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation. J. Bacteriol. 131:82-91.


[0197] 6. Donkersloot, J. A. and J. Thompson. 1995. Cloning, expression, sequence analysis, and site-directed mutagenesis of the Tn5306-encoded N5-(Carboxyethyl)ornithine synthase from Lactococcus lactis K1. J. Biol Chem 270:12226-12234.


[0198] 7. Fleischmann, R. D., M. D. Adams, O. White, R. A. Clayton, E. F. Kirkness, A. R. Kerlavage, C. J. Bult, J. F. Tomp, B. A. Dougherty and J. M. Merrick et al. 1995. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496-512.


[0199] 8. Frey, M., Rothe, M., Volker Wagner, A. F. and Knappe, J. 1994. Adenosylmethionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen atom. J. Biol. Chem. 269: 12432-12437.


[0200] 9. Goodlove, P. E., P. R. Cunningham, J. Parker, and D. P. Clark. 1989. Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli. Gene 85:209-214.


[0201] 10. Holo, H. and I. F. Ness. 1989. High-frequency transformation by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbial. 55:3119-3123.


[0202] 11. Kessler, D., I. Leibrecht and J. Knappe. 1991. Pyruvate-formate-lyase-deactivase and acetyl CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE. FEBS Lett. 281:59-63.


[0203] 12. Kessler, D., W. Herth and J. Knappe. 1992. Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli. J. Biol. Chem. 267:18073-18079.


[0204] 13. Madsen, S. M., B. Albrechtsen, E. B. Hansen and H. Israelsen. 1996. Cloning and transcriptional analysis of two threonine biosynthetic genes from Lactococcus lactis MG1614. J. Bacteriol 178:3689-3694.


[0205] 14. Mat-Jan, F., K. Y. Alam and D. P. Clark. 1989. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase. J. Bacteriol. 171:342-348.


[0206] 15. Nair, R. V., G. N. Bennett and E. T. Papoutsakis. 1994. Molecular characterization of an aldehyde/alcohol dehydrogenase from Clostridium acetobutylicum ATCC 824. J. Bacteriol. 176:871-881.


[0207] 16. Pecher, A., H. P. Blaschkowski, K. Knappe and A. Böck. 1982. Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene. Arch. Microbiol. 132:365-371.


[0208] 17. Platteeuw, C., J. Hugenholtz, M. Starrenburg, I. van Alen-Boerrigter and W. M. de Vos. 1995. Metabolic engineering of Lactococcus lactis: influence of the overproduction of α-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions. Appl. Environ. Microbiol. 61:3967-3971.


[0209] 18. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.


[0210] 19. Sauter, M. and Sawers, R. G. 1990. Transcriptional analysis of the gene encoding pyruvate formate-lyase-activating enzyme of Escherichia coli. Mol. Microbiol. 4: 355-363.


[0211] 20. Sawers, G., A. F. Wagner and A. B{umlaut over (ock)}. 1989. Transcription initiation at multiple promoters of the pfl gene by Eσ70-dependent transcription in vitro and heterologous expression in Pseudomonas putida in vivo. J. Bacteriol. 171: 4930-4937.


[0212] 21. Sawers, G. and A. Böck. 1988. Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12. J. Bacteriol. 170:5330-5336.


[0213] 22. Sawers, G. and A. Böck. 1989. Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression. J. Bacteriol. 171:2485-2498.


[0214] 23. Snoep, J. L., M. J. T. de Mattos, M. J. C. Starrenburg and J. Hugenholtz. 1992. Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and α-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis. J. Bacteriol. 174: 4838-4841.


[0215] 24. Suppmann, B. and G. Sawers. 1994. Isolation and characterization of hypophosphite-resistant mutants of E. coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter. Mol. Microbiol 11:965-982.


[0216] 25. Takahashi, S., K. Abbe and T. Yamada. 1982. Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties. J. Bacteriol. 149:1034-1040.


[0217] 26. Varenne, S., F. Casse, M. Chippaux and M. C. Pascal. 1975. A mutant of Escherichia coli deficient in pyruvate formate-lyase. Mol. Gen. Genet. 141:181-184.


[0218] 27. deVos, W. M. and G. Simons. 1994. Gene cloning and expression systems in lactococci. In: Gasson, M., Vos W. de (eds) Genetics and biotechnology of lactic acid bacteria, Chapman and Hall, London, pp. 52-105.


[0219] 28. Weidner, G. and G. Sawers. 1996. Molecular characterization of the genes encoding formate-lyase and its activating enzyme of Clostridium pasteurianum. J. Bacteriol. 178:2440-2444.


[0220] 29. Westerfeld, W. W. 1945. A calorimetric determination of blood acetoin. J. Biol. Chem. 161: 495-502.


[0221] 30. Wong, K. K., K. L. Suen and H. S. Kwan. 1989. Transcription of pfl is regulated by anaerobiosis, catabolite repression, pyruvate, and oxrA: pfl::Mu dA operon fusions of Salmonella typhimurium. J. Bacteriol. 171:4900-4905.


[0222] 31. Yamamoto, Y., Y. Sato, S. Takahashi-Abbe, K. Abbe, T. Yamada and H. Kizaki. 1996. Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans. Infect. Immun. 64:385-391.


Claims
  • 1. 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.
  • 2. A DNA sequence according to claim 1 further comprising sequences regulating the expression of the coding sequence and/or the activity of its gene product.
  • 3. A DNA sequence according to claim 1 which is derived from a lactic acid bacterium 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.
  • 4. A DNA sequence according to claim 3 which is derived from Lactococcus lactis.
  • 5. A DNA sequence according to claim 1 coding for a polypeptide which is at least 30% identical with a polypeptide which is selected from the group consisting of the gene product of the adhE gene of E. coli as recorded in FASTA, GCG Wisconsin under the accession No. P17547, the gene product of the aad gene of Clostridium acetobutylicum as recorded in FASTA, GCG Wisconsin under the accession No. P33744 and of the DNA sequence of SEQ ID NO:3.
  • 6. A DNA sequence according to claim 1 which comprises the coding sequence of SEQ ID NO:3 or SEQ ID NO:30, or a mutant or variant hereof which codes 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.
  • 7. A recombinant replicon comprising the DNA sequence of claim 1.
  • 8. A replicon according to claim 7 which is selected from a plasmid capable of replicating in a lactic acid bacterium and a lactic acid bacterial chromosome.
  • 9. A recombinant lactic acid bacterial cell comprising the replicon of claim 7.
  • 10. A lactic acid bacterial cell according to claim 9 which is 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.
  • 11. A lactic acid bacterial cell according to claim 9 which is in the form of a starter culture composition for the production of a food product or an animal feed, or in the form of a culture for the production of an aroma or antimicrobially active compound.
  • 12. A lactic acid bacterial cell according to claim 9 wherein the DNA sequence comprising the sequence coding for the multi-functional 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.
  • 13. A lactic acid bacterial cell according to claim 12 wherein said modification of the DNA sequence results in the cell producing increased amounts of a metabolite selected from the group consisting of acetaldehyde, acetate and ethanol.
  • 14. A lactic acid bacterial cell according to claim 9 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 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.
  • 15. A lactic acid bacterial cell according to claim 14 wherein said modification of the DNA sequence results in the cell producing an increased amount of a metabolite selected from the group consisting of acetaldehyde, ethanol, formate, acetate, α-acetolactate, acetoin, diacetyl and 2,3 butylene glycol.
  • 16. 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 an oral Streptococcus species.
  • 17. A DNA sequence according to claim 16 comprising at least one regulatory sequence regulating the expression of the pyruvate formate-lyase polypeptide or coding for a gene product regulating the pyruvate formate-lyase activity of the polypeptide.
  • 18. A DNA sequence according to claim 17 wherein the regulating gene product is selected from a pyruvate formate-lyase activase and a pyruvate formate-lyase deactivase.
  • 19. A DNA sequence according to claim 18 wherein the 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 in claim 1.
  • 20. A DNA sequence according to claim 16 which is derived from a lactic acid bacterium 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.
  • 21. A DNA sequence according to claim 20 which is derived from Lactococcus lactis.
  • 22. A DNA sequence according to claim 16 which comprises the coding sequence of SEQ ID NO:15 or SEQ ID NO:30, or a mutant or variant hereof which codes for a polypeptide having pyruvate formate-lyase activity.
  • 23. A recombinant replicon comprising the DNA sequence of claim 16.
  • 24. A replicon according to claim 23 which is selected from a plasmid capable of replicating in a lactic acid bacterium and a lactic acid bacterial chromosome.
  • 25. A recombinant lactic acid bacterial cell comprising the replicon of claim 23.
  • 26. A lactic acid bacterial cell according to claim 25 which is 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.
  • 27. A lactic acid bacterial cell according to claim 25 which is in the form of a starter culture composition for the production of a food product or an animal feed.
  • 28. A lactic acid bacterial cell according to claim 25 wherein the DNA sequence is modified whereby its production of pyruvate formate-lyase is reduced or inhibited or whereby the enzyme is produced in a modified form having a reduced pyruvate formate-lyase activity.
  • 29. A lactic acid bacterial cell according to claim 28 wherein said modification of the DNA sequence results in that the cell produces increased amounts of a metabolite selected from the group consisting of α-acetolactate, acetoin, diacetyl and 2,3 butylene glycol.
  • 30. A lactic acid bacterial cell according to claim 25 wherein the DNA sequence is modified whereby its production of pyruvate formate-lyase is enhanced or whereby the enzyme is produced in a modified form having an increased pyruvate formate-lyase activity.
  • 31. A lactic acid bacterial cell according to claim 30 wherein said modification of the DNA sequence results in the cell producing increased amounts of formate.
  • 32. A recombinant lactic acid bacterial cell comprising the DNA sequence of claim 1 and the DNA sequence of claim 16.
  • 33. A recombinant lactic acid bacterial cell according to claim 32 wherein at least one of said DNA sequences is modified so as to modify the expression of pyruvate formate-lyase or the activity hereof.
  • 34. A method of producing a lactic acid bacterial metabolite, the method comprising cultivating a lactic acid bacterium according to any of claims 12, 15, 30 or 32 under conditions where the metabolite is produced and isolating the metabolite from the culture.
  • 36. A method of producing an animal feed, the method comprising the step of admixing to the feed starting materials a starter culture of a lactic acid bacterium according to claim 9 or 26 and keeping the mixture under conditions allowing the starter culture to be metabolically active.
  • 37. An isolated DNA sequence derived from a lactic acid bacterium, said sequence coding for a product having a formate transporter activity.
  • 38. A DNA sequence according to claim 37 which is the open reading frame orfA isolated from Lactococcus lactis strain DB1341 where it is located upstream of the pfl gene (SEQ ID NO:34).
Continuations (1)
Number Date Country
Parent 08981097 Dec 1997 US
Child 10267989 Oct 2002 US