Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)

Definitions

• 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.
• 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. Lacto - coccus 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 BifidoJacteriuin 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.
• 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 decarboxyla- tion (Rl, Fig. l) of ⁇ -acetolactate which is formed from two molecules of pyruvate by the action of o.-acetolactate synthase (R2, Fig. 1) .
• 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 o.-acetolactate synthase affinity for pyruvate.
• Overproduction of cv-acetolactate synthase in Lactococcus lactis as an approach for increased production of diacetyl has been disclosed by Platteuw et al . 1995.
• 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.
• 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.
• PDC pyruvate dehydrogenase complex
• 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.
• an increased pyruvate pool may in turn lead to an increased flux from pyruvate towards acetoin and diacetyl via the intermediate ⁇ t-acetolac- tate.
• 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 ⁇ -acetolactate.
• 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.
• 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.
• act encoding the Pfl activase flanks the pfl gene in E. coli , H. influenzae and C.
• 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.
• Clostridium acetobutylicum an adhE analogue, aad has been cloned and characterized.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• modifications of starter cultures would increase the pyruvate pool and consequently, the formation of diacetyl or other compounds derived from the conversion of o.-acetolactate. Increasing one or both dehydrogenase activities will most likely direct the conversion of acetyl CoA from acetate to acetaldehyde or ethanol .
• the starting point for the invention is the achievement of the isolation and sequencing of the entire adhE and pfl genes of Lactococcus lactis .
• the present invention provides novel means for metabolically engineering lactic acid bacteria, and lactic acid bacteria being modified by such means.
• the inven- tion 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
• ACDH acetaldehyde dehydrogenase
• the invention pertains to a recombinant replicon comprising the above DNA sequence and to a recombinant lactic acid bacterial cell comprising such a replicon.
• 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.
• 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
• 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.
• the facultative anaerobe Escherichia coli is capable of carry- ing out mixed-acid fermentation during anaerobic growth in the absence of exogenous electron acceptors.
• 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.
• 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.
• ACDH acetaldehyde dehydrogenase
• ADH alcohol dehydrogenase
• capability of converting acetyl CoA into ethanol pyruvate formate-lyase deactivase activity.
• the coding sequence for the multifunctional polypeptide is also referred
• the DNA sequence coding for the multi-functional polypeptide may be derived from any lactic acid bacterium.
• 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, Leucono - stoc species and Pediococcus species.
• 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.
• 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 .
• the lactic acid bacterium-derived DNA sequence codes for a multifunctional polypeptide that is at least 30% identical with the gene pro- ducts of the adhE gene of E. coli (FASTA, GCG Wisconsin accession No. P17547) or the aad gene of Clostridiu acetobutylicum (FASTA, GCG Wisconsin accession No. P33744) or the gene product of the sequence of Table 1.4 herein (SEQ ID N0:3).
• 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.
• 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 charac- teristics.
• 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 x SSC at room temperature for 30 minutes and subsequently once in 3 x SSC; 0.1% SDS at 65°C for 30 minutes .
• the DNA sequence according to the invention comprises the sequence as shown herein in Table 1.4
• acetaldehyde dehydrogenase activity whereby acetyl CoA is converted into acetaldehyde
• ADH alcohol dehydrogenase
• capability of converting acetyl CoA into ethanol and pyruvate formate-lyase deactivase activity.
• 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 activi- ties to be expressed by the thus modified sequence.
• 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 modifica- tion 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.
• 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 syntheti- cally prepared sequence. Additionally, the above term "mutant and variant” includes any mutein of the sequence.
• 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 orga- nism.
• 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.
• the coding sequence is under the control of a regulatable promoter.
• a 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.
• a promoter sequence as defined above may further comprise sequences whereby the activity of the promoter becomes regulated.
• 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.
• the invention relates, as it is mentioned above, to a recombinant replicon comprising the above DNA sequence coding for the multifunctional polypeptide.
• replicon designates a DNA sequence which is capable of autonomous replication in a lactic acid bacterium.
• 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.
• the replicon may comprise further sequences including marker sequences and linker sequences for the insertion of genes coding for desirable gene products.
• 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.
• 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.
• the invention also provides a recombinant lactic acid bacterial cell comprising the above replicon.
• 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 Bifidobacte- rium species and a Leuconostoc species.
• 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.
• 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.
• 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.
• 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 recog- nizable 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.
• the above lactic acid bacterial cell can also be provided in the form of a culture for the production of an aroma or antimi- crobially active compound.
• 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.
• ACDH acetaldehyde dehydrogenase
• ADH alcohol dehydrogenase
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• a metabolically engineered cell may as a result of these modifications produce increased amounts of i.a. acetaldehyde, ethanol and/or acetate.
• 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 modifi- cations, including random or site-directed mutagenesis followed by selection of the desired mutants.
• 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 modifi- cations of sequences regulating the production and/or the activity of the multifunctional polypeptide.
• suitable modifi- cations of sequences regulating the production and/or the activity of the multifunctional polypeptide is by operably linking the coding sequence to a promoter sequence having a stronger promoter activity than the native promoter for the coding sequence.
• 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 promo- ter activity.
• 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.
• 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.
• at least one metabolite selected from the group consisting of acetaldehyde, ethanol, formate, acetate, acetoin, diacetyl and 2,3 butylene glycol.
• 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.
• 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.
• 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.
• the gene product of pfl will also be referred to as a Pfl polypeptide.
• 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.
• 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.
• the coding sequence is under the control of a regulatable promoter as defined hereinbefore and being regulatable as also described above.
• 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.
• 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.
• 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 .
• ACDH acetaldehyde dehydrogenase
• ADH alcohol dehydrogenase
• pyruvate formate-lyase deactivase activity as defined hereinbefore .
• 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 Lacto coccus lactis subspecies lactis, Lactococcus lactis subspecies lactis biovar diacetylactis and Lactococcus lactis subspecies cremoris.
• Lactococcus lactis including Lacto coccus lactis subspecies lactis, Lactococcus lactis subspecies lactis biovar diacetylactis and Lactococcus lactis subspecies cremoris.
• the Pfl polypeptide as encoded by the pfl gene of Lactococcus lactis subspecies lactis biovar di acetylactis 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).
• the corresponding pfl gene in Lactococcus lactis subspecies lactis MG1363 differs from the DB1341 gene in only about 5% of the nucleotides.
• 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 mgl363- 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.
• 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.
• 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).
• 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.
• 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 x SSC at room temperature for 30 minutes and subsequently once in 3 x SSC; 0.1% SDS at 65°C for 30 minutes.
• 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.
• a recombinant replicon comprising the above Pfl -encoding DNA sequence.
• a replicon can be derived from a plasmid, a lactic acid bacterial bacteriophage or a lactic acid bacterial chromosome .
• 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.
• 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.
• 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.
• 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.
• 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.
• the level of expression of the Pfl polypeptide can also be reduced by modifying or regulating regulatory sequences controlling the production of the polypeptide.
• 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.
• 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 .
• a cell being modified in this manner will be a metabolically engineered cell, since under conditions where the pyr ⁇ vate 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 ⁇ -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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the lactic acid bacterial cells according to the invention are useful as food starter cultures.
• 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.
• 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.
• Fig. 1 illustrates selected metabolic pathways in citrate fermenting lactic acid bacteria
• 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 ⁇ ZAP clones adhEl 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 lactococ- cal ribosome binding site.
• Arrows show the sequencing strategy for clone 1 (middle) ;
• 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) ;
• Fig. 4 is an overview of the cloned Lactococcus lactis DB1341 strain ( . lactis subspecies lactis biovar diacetylactis) pfl gene (open arrow box) .
• the nucleotide positions of relevant -.6 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 plfl (open box) shows the fragment of the ⁇ ZAP clone of the DB1341 genomic library containing a pfl gene fragment.
• the cloned PCR fragment of the L is an overview of the cloned Lactococcus lactis DB1341 strain ( . lactis subspecies lactis biovar diacetylactis) pfl gene (open arrow box) .
• lactis subspecies lactis MG1363 pfl fragment is shown as a line (MGpfll) .
• 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 pfll-250 and pfll-390 is shown as a dotted box (pflup- l);
• 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;
• 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;
• 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; Fig.
• 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 posi- tion of Ec ⁇ Rl sites;
• 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) ;
• 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 no - Lactococcus lactic acid bacteria;
• 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
• 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, H ⁇ rsholm, Den- mark.
• 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 (erm, 50 ⁇ g/ml) and plasmid DNA was isolated for genetic complementation.
• E. coli strain NZNlll ⁇ pfl ' ; Idh: :Tn5; kan R ) is unable to grow in the absence of 0 2 due to the accumulation of NADH derived from the lack of fermentative enzyme activities encoded by the pfl and Idh genes (Mat-Jan et al . , 1989).
• 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 NZNlll had occurred.
• Protein extraction was carried out adding 100 ⁇ l 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 ⁇ g/ml) which had been washed in fresh ice cold LB, and frozen at -80°C for 15 min. Pellets were dissolved and trans- ferred to Eppendorf tubes.
• Lysozyme (5 mg) was added and samples were incubated on ice for 30 min. Subsequently, glass beads (100 ⁇ M, Sigma; 100 ⁇ l ) 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 ⁇ l 100 mM MOPS, pH 6.5; 100 ⁇ l 120 mM Na-Pyruvate; 50 ⁇ l 2.56 mM NADH and 50 ⁇ l H 2 0.
• Plasmid DNA was isolated from clone 1 and used to retransform E. coli NZNlll.
• 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 transfor- mants was obtained in both procedures (see Table l.l below) Thus, clone 1 did not result from artifact cloning and can indeed complement the defect in strain NZNlll.
• Table 1.1 Retransformation of clone 1 into E. coli NZNlll
• NZNlll competent cells were electroporated with the corresponding plasmid, and one half of the cell mixture was plated onto LB + kan + erm and incubated without 0 2
• 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 N0:1). Table 1.2. Sequence of the insert in clone 1
• 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).
• 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) .
• 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).
• Table 1.3. Homology search FASTA. GCG Wisconsin package version 8. Genetics Computer Group
• AdhE The region of homology to AdhE corresponds to the central region, where the ADH domain is possibly located. Only homology to the best score is shown.
• 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).
• 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) .
• I I Ih I I :::: M I ::::::: MMMMMMI MhhMIMIhM adhe_e AGAPIOLIG IDQPSVELSNAI- HHPDINLILATGGPGMVKAAYSSGKPAIGVGAGNTPV
• Ml : :
• MMMIMMMMMIM MM : MUM M 11 M:: llh: MM: adhe_c IAEPIGVVAAIIPVTNPTSTTIFKSLISLIO'RNGIFFSPHPRA-KSTILAAKTILDAAVK 100 110 120 130 140 150
• : M :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: adhe_c EKLSPVIAMYEADNFDDAL-aCAv LINLGGLGHTSGIYADEIKARDKIDRFSSAMKTVRT 340 350 360 370 380 390
• PCR was used to characterize the adhE homologue of strain MG1363.
• Primers adhE-mgl 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. kb fragment, named MGadhESTOP (Fig. 3) .
• adhemgl363 SEQ ID NO: 8; adhedbl341 : SEQ ID NO: 9; adhe_ec: SEQ ID NO: 10; aad_ca: SEQ ID NO: 11
• 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) .
• V T P F A V I T D D E T H V K Y 3701 CClACrr GCTGACTATC-AATTGAC-ACCTC-AAGTTGCCATTGTTGACCCTGA 3750
• 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 adhPl (position 1069-1088 in Table 1.4) and primer adhP2 (posi- tion 1775-1756 in Table 1.4) .
• primers contain a Xhol and a BamHI recognition site at the 5' end.
• the PCR fragment was digested with .Xhol 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 trans - formants were selected on SGM17 plates containing 1 ⁇ g/ml erythromycin and 80 ⁇ g/ml X-gal (Madsen et al . , 1996) . Homologous integration leads to an adhE gene which is interrupted after amino acid residue Asp 543 . About 100 blue trans - formants 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.
• a 616 bp adhE fragment was amplified from the DB1341 chromosome using primer orf3Pl (posi- tion 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 lie 861 .
• 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.
• Lactococcus lactis subspecies lactis biovar diace- tylactis strains DBKAS4 and DBKAS5, respectively and of Lactococcus lactis subspecies lactis strains MGKAS4 and MG AS5, respectively were deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Masche- roder Weg lb, D-38 124 Braunschweig, Germany on 18 July 1996 under the accession Nos DSM 11084, DSM 11085, DSM 11081 and DSM 11082, respectively.
• adhE mutant strain was obtained by PCR using MG1363 DNA as template and primers adhPl-JChoI (sequence 5' -GGCCGCTCGA- GGTTGAACGTGCTGGTGAAGG-3 ' spanning position 2657-2676 in the MG1363 adhE sequence) (SEQ ID NO: 32) and adhP2 -BainHI (sequence 5 ' -TAGTAGGATCCGGGTCAGGTTGGACTGAGCC-3 ' ; spanning position 3363 - 3344 in the MG1363 adhE sequence) (SEQ ID NO: 33) .
• a 700 bp fragment was digested with Xhol and BamHI, cloned into likewise digested pSMA500 and transformed into E. co ⁇ 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 Asp S43 .
• MGKAS14 A sample of MGKAS14 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg lb, D-38 124 Braunschweig, Germany on 10 July 1997 under the accession No. DSM 11654. 2. Physiological characterization of MGKAS14
• MGKAS14 Physiological studies of MGKAS14 was carried by cultivating the strain in anaerobiosis in M17 medium supplemented with either glucose (GM17) or galactose (GalM17) .
• GM17 glucose
• GalM17 galactose
• 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.
• ⁇ ZAP 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 ⁇ vector BamHI arms. Average insert size was estimated to be 3 kb.
• the 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 and is shown in bold, underline (stem) or dotted underline (loop) .
• MIIMIIM smpf 1 CAGGTGACCCAAC-ATTTATTACGACTTCTATGGCTGGTATGGGAGCTGATGGACGTCACC 1270 1280 1290 1300 1310 1320
• TGCTTTC-t-AGACGTTTACACGCGCAC ( -AAAGTATTGGAGGTTATGACGTCCTTCTT ⁇ -ATT
• dbpfl complementary strand corresponding to nucleotides 1979-9 of SEQ ID NO: 15; hi3281: SEQ ID NO: 18
• 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 pfl sequence shown in Table 3.2
• the Pfl protein of Streptococcus mutans was not recorded in th searched protein databases.
• : Ml MM I
• : Ml
• dbpfl corresponds to amino acid residue ⁇ 1-772 of SEQ ID NO: 16; pflb_e: correspond ⁇ to amino acid residues of SEQ ID NO: 14
• ANTVDSLSAIKYAKVKTLR DENGYI YDYEVEGDFPRYGEDDDRADDIAKL
• 11 pf lb_h - FGPGANPMHGRDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDAEAQRRNLAG 640 650 660 670 680 690
• the highest homology value obtained when analysing the sequence from clone pfll 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. Sequence comparisons indicated that the fragment included in clone pfll encompasses 367 amino acids of the C- terminal regio of the L. lactis pfl gene. Therefore, about 1.3 kb of the 5'- end of the pfl gene was lacking.
• High stringency hybridization (washing steps at 65°C, 2 x 30 min in 2 x SSC, then 1 x 30 min in 0.1 x SSC; 0.1 % SDS) resulted in the isolation of twelve positive clones.
• pfll4 Sequence analysis of pfll4 confirmed that it included a pfl fragment that lacked the Sau3AI site at position 1 in clone pfll, but showed sequence identity from position 30 onwards in clone pfl (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.
• This PCR fragment was re-amplified from J ⁇ coRI-digested and religated DB 1341 DNA using modified primers pfll-250 (including an Xhol site at the 5' -end) and pfll-390 (including a BamHI site at the 5' -end) and the amplified product was digested with Xhol and BamHI and ligated into vector pGE digested with the same enzymes and transformation of E. coli DH5 ⁇ 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.
• E. coli DH5o. strain pflup-1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg lb, D-38 124 Braunschweig, Germany on 18 July 1996 under the accession No. DSM 11087.
• TATO -AATGA .C-ACACTTTCCATGATTATTTGAGAGATTTCCGAAC-AAG 900
• sequence included an open reading frame, designated orfA encoding a putative 37 kDa protein with no relevant homology to any sequence in available databases.

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