GB2419600A - Streptococcus with increased rate of glucose metabolism/ lactate synthesis - Google Patents

Abstract

A Streptococcus bacterium with an increased glucose metabolism and/ or an increased rate of lactate synthesis is disclosed. The increase is preferably due to an increased level of expression of the lactate dehydrogenase gene, which may enhance an energy-wasting futile cycle in the bacterium. The bacterium is preferably Streptococcis thermophilus_and may be used in a starter culture in the preparation of foods such as dairy products. A method of preparing a starter culture comprising admixing the bacterium with a dairy product such as milk is also disclosed.

Description

A BACTERIUM

FIELD

The present invention relates to the field of bacteria which are suitable for use in the food industry, in particular the dairy industry.

BACKGROUND

Starter cultures are used extensively in the food industry in the manufacture of fermented products including milk products (such as yoghurt, butter and cheese), meat products, bakery products, wine and vegetable products.

Starter cultures used in the manufacture of many fermented milk products include cultures of bacteria.

Commercial non-concentrated cultures of lactic acid bacteria may be referred to in industry as mother cultures'. These cultures may be propagated at the production site, for example at a dairy, before being added to an edible starting material, such as milk, for fermentation. The starter culture propagated at the production site for inoculation into an edible starting material may be referred to as the bulk starter'.

Typically the bacteria of starter cultures are classified as lactic acid bacteria. Such bacterial starter cultures impart specific features to various dairy products by performing a number of functions.

When a lactic acid bacteria starter culture is added to milk, or any other edible starting material, in appropriate conditions for growth and metabolic activity, the bacteria will start to propagate after a period of time known as the lag phase. During this lag phase the bacteria adapt to the new conditions. Once propagation of the bacteria is initiated it is rapid with concomitant conversion of citrate, lactose or other sugars into lactic acid / lactate (the major acidic metabolite), and possibly other acids including acetate.

These metabolites result in a lowering of the pH of the milk. (In addition, several other metabolites such as e.g. acetaldehyde, a-acetolactate, acetoin, diacetyl and 2,3- butylene glycol (butanediol) may be produced during the growth of the lactic acid bacteria.) When the pH reaches a desired pH value, the fermented product may be cooled in order to reduce or stop further acid development. The desired pH for fermented products may be between pH 3.6 and 5.0 and for cheese the desired pH may be between pH 4.0 and 5.8.

It is known that the rapid production of lactic acid is advantageous for the rapid acidification of inoculated milk.

Generally, the growth rate and the metabolic activity of lactic acid bacterial starter cultures can be controlled by selecting appropriate growth conditions for the strains of the specific starter culture used, such as appropriate growth temperature, oxygen tension and content of nutrients. It is known in the dairy industry that a reduction of the oxygen content of the milk raw material will result in a more rapid growth of the added lactic acid bacteria which in turn results in a more rapid acidification of the inoculated milk. Currently, such a reduction of the oxygen content is carried out by heating the milk in open systems, by deaerating the milk in vacuum or by sparging treatment. Alternative means of reducing the oxygen content include the addition of oxygen scavenging compounds.

Kyla-Nikkila et a! (2000, Applied And Environmental Microbiology, 66:38353841) teach a Lactobacillus helveticus strain wherein the structural gene ldhD was replaced with an additional copy of the structural gene ldhL. Minor, insignificant differences between the growth profiles of the recombinant strains and those of the wild-type were found. Furthermore the amount of lactic acid produced was similar in all cases.

Dartois et a! (1995, Research In Microbiology, 1995, 146:291-302) teach the cloning and characterisation of the D-lactate dehydrogenase gene from Leuconostoc mesenteroides subsp cremoris in E. coli. In addition Dartois et a! suggest that D-LDH deficient strains should be engineered.

Kochhar et al (1992, Biochem and Biophysical Research Communications, 185, 705- 712) disclose the cloning and characterisation of the Lactobacillus bulgaricus D- lactate dehydrogenase gene and overexpression of said gene in E. coli and the purification and characterisation of the recombinant lactate dehydrogenase. In addition, Kochhar et a! (1992, European Journal of Biochemistry, 208 (3) 799-805) teach the cloning and characterisation of the Lactobacillus helveticus D-lactate dehydrogenase gene and overexpression of said gene in E. coli.

The cloning of the Lactobacillus plantarum L-lactate dehydrogenase gene in E. coli and overexpression or no expression of said gene in L. plantarum is disclosed in Ferain et al (1994, Journal of Bacteriology, 176 (3) 596-601). Ferain et a! show that neither an increase nor the absence of L-LDL activity in the modified strains results in a modification of global lactate production.

Yang et a! (1999, Metab. Eng. 2:141-152) teach an E. coli mutant strain overexpressing ldhA. Yang et al teach that the overexpression of LDH results in a slight increase in lactate synthesis. However the majority of the carbon flux is channelled through the acteyl-CoA branch.

Savijoki et a! (1997, Applied and Environmental Microbiology, 63 (7) 28502856) disclose the cloning and characterisation of an ldhL gene from L. helveticus and the overexpression of said gene in E. coli.

Studies have suggested that lactate dehydrogenase has virtually no control over glycolytic flux and the flux to lactate in Lactococcus lactis (Andersen et a!, 2001, Eur J Biochem 268:6379-6389).

In order to study the extent which ATP demand contributes to the control of glycolytic flux Koebmann et a! (2002, Antonie van Leeuwenhoek 82:237-248) depleted the ATP- pooi by introduction of an ATPase. Koebmann et a! demonstrated that in Lactococcus lactis the control on glycolytic flux by ATP demand was close to zero but when non- growing cells were used the ATP demand had a high flux control and the flux could be stimulated more than two fold.

Several attempts have been made to increase the amount of lactic acid produced when lactic acid bacteria are cultured.

However, there is a continuing need to improve lactic acid bacteria for the production of food products.

SUMMARY ASPECTS OF THE PRESENT INVENTION

The present invention is based on the surprising finding that a Streptococcus bacterium which has an increased glucose metabolism and/or increased rate of lactate synthesis is capable of producing an increased amount of lactic acid when said bacterium is cultured under conditions in which said bacterium is metabolically active.

The present invention provides in a one aspect a Streptococcus bacterium wherein said bacterium has an increased glucose metabolism and for an increased rate of lactate synthesis.

In another aspect the present invention provides a starter culture comprising at least a bacterium according to the present invention wherein said bacterium has an increased glucose metabolism and br an increased rate of lactate synthesis.

In another aspect the present invention provides a method of preparing a food comprising the steps of: (i) admixing a bacterium or a starter culture with a dairy medium, and (ii) culturing said admixture under conditions in which said bacterium is metabolically active, wherein said bacterium is a bacterium according to the present invention or a starter culture according to the present invention.

In another aspect the present invention provides a method of preparing a bacterium according to the present invention wherein said bacterium has an increased level of the polynucleotide sequence encoding lactate dehydrogenase and/or an increased level of the lactate dehydrogenase polypeptide sequence.

The present invention also provides a method of preparing a starter culture of a bacterium comprising the steps of: (i) admixing said bacterium with a dairy medium, and (ii) culturing the resulting admixture under conditions in which said bacterium is metabolically active, wherein said bacterium has an increased glucose metabolism and br an increased rate of lactate synthesis is The present invention provides in a further aspect thereof a food wherein said food comprises at least a bacterium according to the present invention or a starter culture according to the present invention In a further aspect the present invention provides the use of a bacterium according to the present invention in the preparation of a starter culture.

In another aspect, the present invention provides the use of a bacterium according to the present invention or a starter culture according to the present invention in the preparation of a food.

The present invention provides in another aspect a food produced by the method according to the present invention wherein said a food is a dairy product.

ADVANTAGES

One advantage is that the bacterium according to the present invention has an increased rate of acidification of milk.

DETAILED ASPECTS OF THE PRESENT iNVENTION

WILD-TYPE

The term "wild-type" is a term of art understood by the skilled person and means a phenotype that is characteristic of most members of the species occurring naturally and contrasting with the phenotype of a mutant or a variant.

GROWTH

Preferably the bacterium is capable of growing when said bacterium is cultured in a dairy medium and wherein said bacterium is cultured under conditions in which said bacterium is metabolically active.

The term "capable of growing" as used herein refers to a bacterium being capable of growth (i.e. propagation) when cultured under appropriate conditions (for example, temperature and nutrients present in the culture medium) which support (i.e. maintain) the bacterium and permit its metabolic activity.

A bacterium "capable of growing normally" refers to a bacterium having a comparable growth rate (i.e. propagation) or a faster rate of growth when compared to the wild- type bacterium, when cultured under appropriate conditions (for example, temperature and nutrients present in the culture medium) which support (i. e. maintain) the bacterium and permit its metabolic activity.

A bacterium "not capable of growing normally" refers to a bacterium having no growth (i.e. no propagation) or a slower rate of growth when compared to the wild- type bacterium, when cultured under appropriate conditions (for example, temperature and nutrients present in the culture medium) which support (i. e. maintain) the bacterium and permit its metabolic activity.

Preferably the bacterium according to the present invention is capable of growing quickly.

The term "growing quickly", as used herein, refers to a bacterium having an increased rate of growth i.e. propagation, when cultured, under appropriate conditions (for example, temperature and nutrients present in the culture medium) which support (i.e. maintain) the bacterium and permit its metabolic activity when compared to the wild- type bacterium.

CULTURE CONDITIONS

As used herein, the term "cultured under conditions in which said bacterium is metabolically active" refers to appropriate conditions (for example, temperature and nutrients present in the culture medium) which support (i.e. maintain) the bacterium and permit its metabolic activity. Such culture conditions may or may not support the growth (i.e. propagation) of the bacterium.

DAIRY MEDIUM

The term "dairy medium" as used here refers to a culture medium which can be inoculated with bacteria (such as the bacterium according to the present invention) and cultured. Said dairy medium permits the metabolic activity of the bacteria, such as the bacterium of the present invention, and optionally the growth (i.e. propagation) of the bacteria - here the culturing and optionally inoculation is carried out under non-sterile conditions.

In one aspect, preferably the culture medium is a diary medium.

Preferably said dairy medium is milk.

Milk can be obtained from a ruminant animal, preferably selected from a group consisting of buffalo, cow, sheep, lama, goat or camel. The milk can also be skimmed or semi-skimmed. Alternatively or in addition the milk may be derived from plants such as soy or rice or it can be synthetically generated milk.

In a preferred embodiment, the milk is one or more of the following: cows' milk, goats' milk, ewes' milk, soy milk, lamas' milk, buffalo cows' milk and yaks' milk. In a highly preferred embodiment, the milk is cows' milk.

STARTER CULTURE

Starter cultures play several important roles in the fermentation of food products.

Typically the principle function of starter cultures is to provide for acidification by converting sugar(s) in a food ingredient (such as a starting material for a food) into acid. The sugars can be a natural part of the food ingredient or can be added to the food ingredient. Typically starter cultures, through the fermentation process(es), contribute to the flavour (such as the tanginess) of the food product, provide safety against food-borne pathogens and contribute to the final texture The term "starter culture" as used here refers to a culture of bacterium which may be used to produce a culture which in turn is used in the preparation of a food. In the present invention a starter culture is typically prepared by inoculating a culture medium (for example a dairy medium) with a bacterium of the present invention and culturing the inoculated medium under conditions (which optionally are sterile) which permit the metabolic activity of the bacteria and optionally the growth (i.e. propagation) of said bacteria.

FOOD

Preferably a starting material of a food described herein is milk.

Preferably a food as described herein is a dairy product. More preferably a dairy product as described herein is one or more of the following: a yoghurt, a cheese (such as an acid curd cheese, a hard cheese, a semihard cheese, a cottage cheese), a buttermilk, quark, a sour cream, kefir, a fermented whey-based beverage, a koumiss, a milk drink and a yoghurt drink. In a highly preferred embodiment the dairy product is one or more of the following: a yoghurt, a yoghurt drink and a cheese.

The bacterium described herein may be used as - or in the preparation of a food.

Here, the term "food" is used in a broad sense and includes feeds, foodstuffs, food ingredients, food supplements, and functional foods.

As used herein the term "food ingredient" includes a formulation, which is or can be added to foods and includes formulations which can be used at low levels in a wide variety of products that require, for example, acidifying or emulsifying.

As used herein, the term "functional food" means a food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a further beneficial effect to consumer. Although there is no legal definition of a functional food, most of the parties with an interest in this area agree that there are foods marketed as having specific health effects.

The term "food" covers food for humans as well as food for animals (i.e. a feed). In a preferred aspect, the food is for human consumption.

The bacterium described herein may be - or may be added to - a food ingredient, a food supplement, or a functional food.

The food may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.

The bacterium described here can be used in the preparation of food products such as one or more of: confectionery products, dairy products, meat products, poultry products, fish products and bakery products.

By way of example, the bacterium can be used as ingredients to soft drinks, a fruit juice or a beverage comprising whey protein, health teas, cocoa drinks, milk drinks and lactic acid bacteria drinks, yoghurt, drinking yoghurt and wine.

The present invention also provides a method of preparing a food, the method comprising admixing the bacterium according to the present invention with a food ingredient (such as a starting material for a food). The method for preparing a food is also another aspect of the present invention.

STREPTOCOCCUS BACTERIUM

Preferably the bacterium is Streptococcus thermophilus. More preferably said bacterium is a Streptococcus thermophilus variant.

As used herein the term "a Streptococcus thermophilus variant" refers to a modified Streptococcus thermophilus strain.

GLUCOSE METABOLISM

The term "increased glucose metabolism" as used herein refers to the bacterium according the present invention having an increased metabolism (i.e. consumption) of glucose when compared to the wild-type bacterium when cultured in a given period of time (when the substrates for such metabolism are not limited). Thus for an equivalent set of culture conditions the bacterium of the present invention metabolises (i.e. uses) an amount of glucose which is preferably 10% greater than that of a wildtype bacterium, preferably greater than 20%, preferably greater than 40%, more preferably greater than 60%. Preferably the amount of glucose in a culture is measured after about 14 hours incubation of the culture, more preferably after about 12 hours, more preferably after about 7 hours, more preferably after about 6 hours, more preferably after about 5 hours, more preferably after about 4 hours and in a highly preferred embodiment after about 3 hours.

RATE OF LACTATE SYNTHESIS

The term "increased rate of lactate synthesis" as used herein refers to the bacterium according to the present invention producing an increased amount of lactate when compared to the wild-type bacterium when cultured in a given period of time (when the substrates for the reaction are not limited). For an equivalent set of culture conditions the bacterium of the present invention produces an amount of lactate which is preferably 10% greater than that of a wild-type bacterium, preferably greater than 20%, preferably greater than 40%, more preferably greater than 60%. Preferably the amount of lactate in a culture is measured after about 14 hours incubation of the culture, more preferably after about 12 hours, more preferably after about 7 hours, more preferably after about 6 hours, more preferably after about 5 hours, more preferably after about 4 hours and in a highly preferred embodiment after about 3 hours.

In one preferred embodiment, the bacterium according to the present invention has an increased glucose metabolism and an increased rate of lactate synthesis.

LACTATE DEHYDROGENASE

Preferably, the bacterium according to the present invention has an increased expression of a lactate dehydrogenase gene and/or an increased level of lactate dehydrogenase polypeptide sequence. More preferably said increased expression of said lactate dehydrogenase gene and/or an increased level of lactate dehydrogenase polypeptide sequence increases glucose metabolism and for increases the rate of lactate synthesis in said bacterium.

As used herein the term "increased expression of lactate dehydrogenase gene and/or an increased level of lactate dehydrogenase polypeptide sequence" refers to the bacterium according to the present invention expressing an increased level (amount) of the polynucleotide encoding lactate dehydrogenase and/or synthesising an increased level (amount) of lactate dehydrogenase polypeptide sequence when compared to the wild- type bacterium when cultured in a given period of time. The increased amount of lactate dehydrogenase polypeptide results in a higher lactate dehydrogenase activity in a cell which in turn results in an increased amount of lactate or pyruvate being produced in a cell (when the substrates for the reaction are not limited). For the production of lactate the following applies. For an equivalent set of culture conditions the bacterium of the present invention produces an amount of lactate which is preferably 10% greater than that of a wild-type bacterium, preferably greater than 20%, preferably greater than 40%, more preferably greater than 60%. Preferably the amount of lactate in a culture is measured after about 14 hours incubation of the culture, more preferably after about 12 hours, more preferably after about 7 hours, more preferably after about 6 hours, more preferably after about 5 hours, more preferably after about 4 hours and in a highly preferred embodiment after about 3 hours.

Preferably the lactate dehydrogenase gene encodes L-lactate dehydrogenase (BC 1.1.1.27).

Preferably the lactate dehydrogenase polypeptide sequence is L-lactate dehydrogenase (BC 1.1.1.27).

The tenn "increased expression of a lactate dehydrogenase gene" as used herein refers to a bacterium according to the present invention producing an increased amount of the polynucleotide sequence encoding the lactate dehydrogenase polypeptide when compared to the wild-type bacterium when cultured over a given period of time (when transcription of genes in a cell is not limited).

The amount of polynucleotide sequence encoding lactate dehydrogenase in a sample may be determined by monitoring the amount of polypeptide sequence produced by way of quantitative PCR. For an equivalent set of culture conditions the amount of the polynucleotide sequence encoding lactate dehydrogenase produced by the bacterium of the present invention is preferably at least 2% more than the wild-type bacterium, preferably greater than 5% more, preferably greater than 10% more, more preferably greater than 20% more. Preferably the amount of polynucleotide sequence encoding lactate dehydrogenase is measured after about 14 hours incubation of the culture, more preferably after about 12 hours, more preferably after about 7 hours, more preferably after about 6 hours, more preferably after about 5 hours, more preferably after about 4 hours and in a highly preferred embodiment after about 3 hours.

Alternatively, the amount of polynucleotide sequence encoding lactate dehydrogenase in a sample may be determined by measuring the specific activity of the lactate dehydrogenase. Lactate dehydrogenase activity can be determined by measuring the amount of lactate produced in a sample (when the substrates for the reaction are not limited). For the production of lactate the following applies. For an equivalent set of culture conditions the bacterium of the present invention produces an amount of lactate which is preferably 30% greater than that of a wild- type bacterium, preferably greater than 40%, preferably greater than 50%, more preferably greater than 60%. Preferably the amount of lactate in a culture is measured after about 14 hours incubation of the culture, more preferably after about 12 hours, more preferably after about 7 hours, more preferably after about 6 hours, more preferably after about 5 hours, more preferably after about 4 hours and in a highly preferred embodiment after about 3 hours.

The term "increased level of lactate dehydrogenase polypeptide sequence" as used herein refers to a bacterium according to the present invention producing an increased amount of the polypeptide encoding lactate dehydrogenase when compared to the wild-type bacterium when cultured in a given time. The increased amount of lactate dehydrogenase results in a higher lactate dehydrogenase activity in a cell which in turn results in an increased amount of lactate or pyruvate being produced in a cell (when the substrates for the reaction are not limited). For the production of lactate the following applies. For an equivalent set of culture conditions the bacterium of the present invention produces an amount of lactate which is preferably 30% greater than that of a wild-type bacterium, preferably greater than 40%, preferably greater than 50%, more preferably greater than 60%. Preferably the amount of lactate in a culture is measured after about 14 hours incubation of the culture, more preferably alter about 12 hours, more preferably after about 7 hours, more preferably after about 6 hours, more preferably after about 5 hours, more preferably after about 4 hours and in a highly preferred embodiment after about 3 hours.

FUTILE CYCLE

The terms "energy-wasting futile cycle", "futile cycle" and "energy wasting cycle" are synonymous. As used herein the term refers to the waste of energy by a metabolic cycle, normally the energy is wasted by hydrolysis of ATP. Futile cycles are naturally not active in bacteria Russell and Cook (1995, Microbiological reviews, 59:48-62) and Portais and Delort (2002, FEMS Microbiology Reviews 26: 375-402).

Examples of futile cycles are as follows: The activity of pyruvate kinase and PEP-synthetase leads to pyruvate kinase producing one energy-richbond (ATP is produced from ADP) and PEP-synthetase using two energy-rich bonds (ATP is split in AMP + Pi) thus a net of one ATP is used. If both enzymes are similarly active then there is no change in the amount of phosphoenolpyruvate and pyruvate but more ATP is used than produced. Such reactions make no sense for the cell, since ATP is wasted, and hence such reactions are called "futile cycles".

Phosphoenolpyruvate P)vate / PEP-S pyruvate The depletion of the ATP pool is the signal for glycolysis, which produces ATP in order to refill the ATP pool. Glycolysis is not part of the futile cycle but, without wishing to be bound by theory, the futile cycle has the effect of increasing the rate of glycolysis.

Another example of a futile cycle is the simultaneous activity of phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase. The high activities of these enzymes leads to an effective loss of one energy-rich bond per cycle (GTP is split into GDP and inorganic phosphate). If both enzymes are similarly active then there is no change in the amount of phosphoenolpyruvate and oxaloacetate but a lot of GTP is used. Such reactions make no sense for the cell, since energy-rich molecules are wasted, and hence such reactions are called "futile cycles".

Phosphoenolpyruvate PEP PEP carboxykinase carboxylase oxaloacetate The depletion of the GTP pool is the signal for glycolysis, which produces energy-rich molecules (ATP), to speed up in order to refill the pool of energy-rich molecules. The speeding up of glycolysis is a consequence of the futile cycle, but is not part of the futile cycle Chao and Liao (1994, Metabolic responses to substrate futile cycling in Escherichia coli. J BioL Chem. 269:5122-5126) showed that the simultaneous overexpression of phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in Escherichia coli stimulated oxygen and glucose consumption, reduced growth yields and resulted in high level excretion of fermentative end products.

Preferably the bacterium according to the present invention has at least one energy- wasting futile cycle. Preferably said energy-wasting cycle is enhanced. More preferably said enhanced energy-wasting futile cycle increases glucose metabolism and/or increases the rate of lactate synthesis in said bacterium.

Preferably an increased expression of lactate dehydrogenase gene and/or increase level of lactate dehydrogenase polypeptide sequence in said bacterium enhances said energy-wasting futile cycle.

Preferably said energy-wasting futile cycle results in an increased glucose metabolism and/or an increased rate of lactate synthesis in said bacterium.

In one embodiment of the present invention, the energy-wasting cycle is introduced into a bacterium according to the present invention. Said introduction is carried out by, for example, recombinant DNA technology.

As used here, the term "energy-wasting cycle is enhanced" refers to a bacterium according to the present invention which uses more ATP or GTP when compared to the wild-type bacterium when cultured for a given period of time. For an equivalent set of culture conditions the bacterium of the present invention preferably uses at least 2% more ATP or GTP than the wild-type bacterium, preferably at least 5% more, preferably at least 10% more, more preferably at least 20% more. Preferably the amount of ATP or GTP used is measured after about 14 hours incubation of the culture, more preferably after about 12 hours, more preferably after about 7 hours, morepreferably after about 6 hours, more preferably after about 5 hours, more preferably after about 4 hours and in a highly preferred embodiment after about 3 hours.

LACTIC ACID

Preferably the bacterium according to the present invention is capable of producing an increased amount of lactic acid when said bacterium is cultured under conditions in which said bacterium is metabolically active.

The term "increased amount of lactic acid" as used herein refers to the bacterium according to the present invention producing an increased amount of lactic acid when compared to the wild-type bacterium when cultured in a given period (when the substrates for the reaction are not limited). For an equivalent set of culture condition the amount of lactic acid produced by the bacterium of the present invention is preferably at least 2% more than the wild-type bacterium, preferably greater than 5% more, preferably greater than 10% more, more preferably greater than 20% more.

Preferably the amount of lactic acid produced is measured after about 14 hours incubation of the culture, more preferably after about 12 hours, more preferably after about 7 hours, more preferably after about 6 hours, more preferably after about 5 hours, more preferably after about 4 hours and in a highly preferred embodiment after about 3 hours.

FAST ACIDIFICATION

Preferably the bacterium according to the present invention is a fast acidifier of milk.

The term "fast acidifier of milk" as used herein refers to a faster acidification rate of milk by the bacterium according to the present invention when compared to the wild- type bacterium when cultured in milk (when the substrates for the reaction are not limited). For an equivalent set of culture conditions, preferably the bacterium of the present invention acidifies 9% reconstituted skim milk powder (said reconstituted skim milk powder is pretreated for 30 mm at 95-100 C before inoculation with the bacteria) after 4 hours incubation at a temperature of 37 C to a pH, which is 3% lower than that of the wild-type bacterium, more preferably 5% lower, more preferably 10% lower, more preferably 20% lower, more preferably 30% lower.

ASSAY TO DETERMiNE GLUCOSE CONCENTRATION Glucose concentration is a sample may be measured using HPLC as described below.

Sample preparation: a) Yoghurt: 10 g +1- 0.02 g of a sample is filled up to 100 ml in a volumetric flask with milliQ water (<0.054 j.tS/cm). The sample is mixed 20 times and diluted with milliQ water to a range of 1050 mg/I. Finally the sample is filtrated through a 0.45 Lm nylon membrane.

b) Fermentation broth: 1 ml of the mixed sample is diluted in a volumetric flask with milliQ water to the range 10-50 mg/i. The sample is mixed 20 times and filtrated through a 0.45 j.tm nylon membrane.

Chromatographic conditions: A DX600 from Dionex, which is controlled by the software PeakNet 6.X, is used.

CarboPac PA100 column, a pre- and ATC-Trap columns are used.

A flow rate of 1 mi/mm and an injection volume of 25j.tl is used.

Detection occurs using an IPAD, gold working electrode.

The elutants are (a) water (milliQ) and (b) 250mM NaOH.

An isocratic flow of 100 mM NaOH is used for 15 mm.

Calibration: External three point calibration (5, 25, 50 mg/I). For the identification of glucose the following spiked samples are used (galactose from Merck, Darmstadt, Germany (art. no. 1.04058.0025), glucose (art. no. 49138) and lactose (art. no. 61340) from Fluka, Taufkirchen, Germany).

ASSAY TO DETERMiNE LACTATE L-lactate dehydrogenase (L-LDH) - E.C. 1.1.1. 27 - catalyses the following reaction: (L)-lactate + NAD(+) = pyruvate + NADH L-lactate dehydrogenase can be referred to as L-Iactic acid dehydrogenase or L-Iactic dehydrogenase.

D-lactate dehydrogenase (D-LDH) - E.C. 1.1.1.28 - catalyses the following reaction: (D)-lactate + NAD(+) = pyruvate + NADH.

D-lactate dehydrogenase can be referred to as D-lactic acid dehydrogenase or D-lactic dehydrogenase.

Concentrations of D- and L-lactate in cultures can be determined as follows using a method described by Lapiere et a!. (1999 - App!. Environm. Microbiol. 65:4002- 4007).

Bacterial cultures are diluted in 100 mM Tris-HC1 (pH 8.0)-150 mM NaC1, kept on ice for 10 to 30mm, and centrifuged at maximal speed for 10 miii. (When necessary, supematants are kept frozen at 20 C until use.) The supematants are further diluted in the same buffer (100 mM Tris-HCI (pH 8. 0)-iSO mM NaC!), mixed with 1 ml of mixture reaction (100 mM Tris-HC1, pH 9.0; 2 mM EDTA; 3% hydrazine; 1 mM NAD) in the presence of D-LDH or L-LDH (20 U/rn!) (Leuconostoc mesenteroides D-LDH can be obtained from Sigma L2395, and rabbit muscle L-LDH can be obtained from Sigma L-2500, St. Louis, Mo.) and incubated for 90 miii at room temperature.

NADH accumulation is monitored by measuring the optical density at 340 nm.

ASSAY TO DETERMINE THE EXPRESSION OF THE LACTATE

DEHYROGENASE

The amount of lactate dehydrogenase in a sample can be determined using a protocol described by Lapiere eta!. (1999 - App!. Environm. Microbiol. 65:4002-4007.).

For the quantification of intracellular D-LDH and L-LDH, cells are washed with a solution containing 50 mM Tris-HC1 (pH 8.0), 100 mM NaC!, 2 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulphonyl fluoride, centrifuged, concentrated five times in the same buffer containing 0.5 mg of lysozyme and 50 j.tg of mutanolysin per ml, and incubated for 20 mm at 37 C.

The solution of lysed cells is cleared by centrifugation, and the D-LDH and L-LDH activities are determined in 100 mM Tris-HCI (pH 8.0), 15 mM NAD, and 500 mM D-lactate (Fluka 71716) or L-lactate (Fluka 71718), respectively, by monitoring the optical density at 342 nm.

The total LDH activity is measured under the same conditions but in the presence of 20 mM pyruvate and 0.15 mM NADH in 100mM potassium buffer at pH 7.5.

ASSAY TO DETER1vI1N THE EXPRESSION OF POLYNUCLEOT1DES

SEQUENCES ENCODING LACTATE DEHYDROGENASE

Real-time or quantitative PCR can be used to determine the amount of polynucleotide sequence encoding lactate dehydrogenase in a sample.

RNA isolation and eDNA synthesis (RT step) RNA extraction and cDNA synthesis are carried out essentially as described by Neretin Ct a!. (2003). Two millilitres of each culture are centrifuged for 5 mm at 14000 r.p.m. at 4 C (Centrifuge 5417R, Eppendorf, Hamburg, Germany), the supematant is discarded, and the cell pellet is immediately frozen at 80 C. For RNA isolation, the pellet is suspended in a 50 ml solution containing lysozyme (10 mg 1.1) and lysostaphin (50 mg) (Gram- CrackerlM Kit, Ambion, Austin, TX, USA). 900 ml RNAwiz extraction reagent (Ambion, Austin, TX, USA) are added to the suspension and are then transferred into a silica-bead containing FastRNA tube-blue (BiolOl, Carlsbad, CA, USA). The tube is shaken for 30 seconds at the speed level 6 in a FastPrep instrument (FP 120; Bio 101, Savant, Holbrook, NY, USA). After shaking, RNA is extracted according to the RNAwiz extraction protocol (Ambion, Austin, TX, USA) with chloroform and isopropanol. Contaminating DNA is removed using a DNA-free Kit (Ambion, Austin, TX, USA). Samples are incubated with DNase for 2 hours at 37 C. The DNase Inactivation Reagent is pelleted by centrifugation to avoid its introduction into PCR. The RNA is diluted to a final volume of 100 ml in RNase- free H20 and stored at -80 C before the reverse-transcription step and the PCR are perfonned.

For reverse transcription 250 U (1.25 ml) of MMLV RTase are used with the supplied 5 x buffer (5 ml) (Promega, Madison, WI, USA), 50 U (1.25 ml) RNasin ribonuclease inhibitor (Promega, Madison, WI, USA), 1.25 ml of 50 mJvI random hexamers (PE Applied Biosystems, Forster City, CA, USA), 5 ml of 2. 5 mM deoxynucleoside triphosphates each (Promega, Madison, WI, USA), 1.25 ml of BSA (3 g 1'), and 10 ml of a RNA sample per 25 ml total reaction volume. To estimate the genomic DNA contamination in isolated RNA, cDNA synthesis is performed in duplicate omitting RTase from the reaction mixture. The reaction conditions are as follows: preheating of the RNA for 10 mm at 75 C, addition of the reaction mixture on ice, and incubation for 10 mm at 25 C, followed by 1 hour at 37 C, enzyme inactivation for 5 mm at 95 C, and rapid cooling to 4 C. cDNA samples are stored at -20 C before PCR analysis. Two cDNA syntheses per RNA sample are performed.

Measurement of ldh expression For measurement of ldh mRNA relative to 16S rRNA, a duplex TaqMan assay was developed. The cDNA quantification is performed using a model Rotor-Gene 3000 quantitative PCR system (Corbett Research, Sydney, Australia). Primers and the TaqMan probe for the ldh gene were designed with computer aid using the software Beacon Designer (Premier Biosoft International, Palo Alto, CA, USA). A 108 bp sized PCR product of the ldh cDNA is amplified using the two primers ldh3O7F (5'GACCTTAGCCACGCACTTC-3') and ldh4l4R (5'-TTGAGGAGCACCAGCAGT- 3') and detected using the TaqMan probe TM1dh391R (5'-FAMCTACAAGGTCAGCATCCGCACAGTCT.BHQ1..3') The primers and TaqMan probe of Neretin et al. (2003) are used for the quantification of 16S rRNA-derived cDNA. The TaqMan probe for 16S rcDNA is labelled with ROX at the 5' end and with BHQ2 (Black Hole Quencher) at the 3' end (Metabion, Munich, Germany). Duplex qPCR reactions are performed using the QuantiTect Probe PCR Kit (Qiagen, Hilden, Germany) in a final volume of 20 j.tl. Thermal cycling conditions consist of a first denaturation step at 95 C for 15 mm followed by 50 cycles at 94 C for 15 sec and 60 C for 45 sec. Fluorescence data are acquired at the end of each annealing step.

The data are analysed using the Rotor-Gene software, version 6.0 and Microsoft Excel.

CT values (C1 = threshold cycle) are determined by manually setting the threshold value at 0.03 in all fluorescence channels applying dynamic tube normalisation and slope correction. Efficiency values are determined using the comparative quantitation feature implemented in Rotor-Gene based on the 2' derivative of the quantification reactions. Reactions are performed at least in triplicates. For the measurement of ldh expression relative to 16S rcDNA the formulae of Pfaffl (2001) is applied (1): E 1XCrigt (con trol-sample) ratio = target) E (control-sample) ref I

ATP ASSAY

One suitable assay to quantify the amount of ATP in a sample is the ATP determination Kit A-22066 (Molecular Probes) which uses bioluminescence.

COMBINATION WITH OTHER MICROORGANTSMS

Accordingly, the bacterium of the present invention may be used in combination with other microorganisms such as bacteria, fimgi and yeasts. Preferably the additional microorganism is a lactic acid bacterium.

The selection of organisms for the starter culture as described herein or another other suitable culture will depend on the particular type of products to be prepared and treated. Thus, for example, for cheese and butter manufacturing, mesophilic cultures of Lactococcus species, Leuconostoc species and Lactobacillus species are widely used, whereas for yoghurt and other fermented milk products, thermophilic strains of Streptococcus species and of Lactobacillus species are typically used.

In one preferred embodiment of the present invention, a dairy medium, starter culture or other culture comprising a bacterium according to the present invention may also comprise one or more further micro-organism. Examples of such further micro- organisms include, but are not limited to, Penicillium spp (such as P. roquefortii, P. glaucum, P camenberti and P. canididum). Geotrichum spp (such as Geotrichum candidum), Torula spp (such as Torula kefir), Saccharomyces spp (such as Saccharomyces kefir and Saccharomyces cerevisiae) and lactic acid bacteria.

In another embodiment of the present invention, a dairy medium, starter culture or other culture comprising a bacterium according to the present invention may also comprise one or more further lactic acid bacterium. Examples of such further lactic acid bacteria include, but are not limited to, Streptococcus species (spp), Lactococcus spp (such as Lactococcus cremoris, Lactococcus lactis, Lactococcus lactis biovar diacetylactis, Lactococcus lactic subsp. lactis and Lactococcus lactis subsp. cremoris), Lactobacillus spp (such as Lactobacillus bulgaricus), Leuconostoc spp, Oenococcus spp, Pediococcus spp, Streptococcus spp (such as Streptococcus therinophilus), Propionibacterium spp Brevibacterjum spp (such as Brevibacterium linens), Enterococcus spp and Bfldobacterjum spp.

LACTIC ACID BACTERIA

As used herein the term "lactic acid bacteria" refers to gram positive, microaerophilic or anaerobic bacteria which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propiomc acid.

The industrially most useful lactic acid bacteria are found among Lactococcus species, such as Lactococcus lactis, Lactobacillus species, Bfidobacterium species, Streptococcus species, Leuconostoc species, Pediococcus species and Propionibacterium species.

Examples of lactic acid bacterial species include, but are not limited to, Lactococcus spp (such as Lactococcus lactis, Lactococcus cremoris, Lactococcus lactis biovar diacetylactis, Lactococcus lactic subsp. lactis and Lactococcus lactis subsp. cremoris), Lactobacillus spp (such as Lactobacillus reuteri Lactobacillus acidophilus, Lactobacillus bulgaricus) , Enterococcus species, Leuconostoc spp, Pediococcus spp, Oenococcus spp, Propionibacterium species Streptococcus spp and Bfidobacterium species (such as Bfldobacterium longum, B/Idobacterjum bfldum, Bfidobacterium lactis, Bfldobacterjum breve, Bfldobacterjum adolescentis, Bfidobacterium dentium).

Mesophilic cultures of lactic acid bacteria commonly used in the manufacture of fermented milk products such as buttermilk, yoghurt or sour cream, and in the manufacture of butter and cheese, for example Brie or Harvati, include Lactococcus cremoris, Lactococcus lactis, Leuconostoc sp., Lactococcus lactis biovar diacetylactis, Streptococcus thermophilus, Lactobacillus bularicus and Lactobacillus helveticus. In addition, probiotic strains such as Byidobacterium lactis, Lactobacillus acidophilus, Lactobacillus casei may be added during said manufacturing to enhance flavour or to promote health.

Mesophilic cultures of lactic acid bacteria commonly used in the manufacture of cheddar and Monterey Jack cheeses include Streptococcus thermophilus, Lactococcus lactis and Lactococcus cremoris or combinations thereof.

Thermophilic cultures of lactic acid bacteria commonly used in the manufacture of Italian cheeses such as Pasta filata or parmesan, include Streptococcus thermophilus and Lactobacillus bularicus. Other Lactobacillus strains such as Lactobacillus helveticus may be added during manufacturing to obtain a desired flavour.

Lactic acid bacteria cultures are commonly used in the food industry as mixed strain cultures comprising one or more species. For a number of mixed strain cultures, such as yoghurt starter cultures comprising strains of Lactobacillus bulgaricus and Streptococcus thermophilus, a symbiotic relationship exists between the species wherein the production of lactic acid is greater compared to cultures of single strain lactic acid bacteria (Rajagopal et a!. J.Dairy Sci., 73, p.894-899, 1990).

Several documents report the stimulation of lactic acid production and bacterial growth, the teachings of which are incorporated herein by reference. Pette and Lolkema (Netherland Milk Dairy Journal, 4, p.197, 1950) report increased lactic acid production in mixed cultures and suggest that L. bulgaricus provides essential growth requirements for stimulation of the growth of S. therm ophilus.

Other compounds found to be stimulatory to S. thermophilus are peptides containing lysine resulting from hydrolysis of casein by Micrococcus caseolyticus (Desmazeaud, M.J. & Hermier, J.H. (1972) European J. Biochem. , p.190) or hepta- and pentapeptides containing histidine obtained by hydrolysis of glucagon by papain (Desmazeaud, M.J. & Hermier, J.H. (1973) Biochemie, p.679).

OTHER MICROORGANISMS

Fungal cultures including yeast cultures and cultures of filamentous fungi, are particularly used in the manufacture of certain types of cheese and beverage. Examples of currently used cultures of fungi include Penicillium roqueforti, Penicillium candidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir and Saccharomyces cerevisiae.

PREPARING CULTURES

An effective amount of a bacterium is added to a starter culture or culture. As used herein, the term "effective amount" describes an amount of a bacterium which is sufficient to ferment detectable amount of sugar compounds present in the milk.

Starter cultures and other cultures may be prepared by techniques well known in the art such as those disclosed in US 4,621,058. By way of example, cultures maybe prepared by the introduction of an inoculum, for example a bacterium, to a growth medium to produce an inoculated medium (such as a starter culture) and ripening the inoculated medium to produce a culture.

Preparing Dried Cultures Dried cultures may be prepared by techniques well known in the art, such as those discussed in US 4, 423, 079 and US 4, 140,800.

Dried cultures may be in the form of solid preparations. Examples of solid preparations include, but are not limited to tablets, pellets, capsules, dusts, granules and powders which may be wettable, spray-dried, freeze-dried or lyophilised.

The cultures may be in the form of concentrates which comprise a substantially high concentration of one or more bacteria. Preferably the concentrates may be diluted with water or resuspended in water or other suitable diluents, for example, an appropriate growth medium or mineral or vegetable oils, for use in the present invention. The dried cultures in the form of concentrates may be prepared according to the methods known in the art, for example by centrifugation, filtration or a combination of such techniques.

Product Any product which is prepared from, or comprises, a culture or starter culture, may be prepared using a bacterium according to the present invention. Such products include, but are not limited to, fruits, legumes, fodder crops and vegetables including derived products, grain and grain-derived products, dairy foods and dairy food-derived products, meat, poultry, seafood, cosmetic and pharmaceutical products. In particular, bacterium according to the present invention may be used in connection with dairy products such as cheeses, for example continental cheese, Italian cheese types or American cheese types and fermented milk products such as buttermilk, sour cream, yoghurt, drinkable yoghurt, quark, kefir, milk drinks and fermented whey-based beverages.

PREPARATION OF MICROORGANISM

A microorganism according to or for use in the present invention may be prepared by mutating or transforming a parent microorganism (which may be a wild-type microorganism) - such that for example one or more nucleic acid or amino acid sequences are altered and/or added and/or deleted and br silenced.

NUCLEOTIDE SEQUENCE

The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand.

The term "nucleotide sequence" or "polynucleotide sequence" as used herein includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably eDNA sequence.

In a preferred embodiment, the nucleotide sequence as described herein does not include the native nucleotide sequence when in its natural environment and when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, we shall call this preferred embodiment the "non- native nucleotide sequence". In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. However, the amino acid sequence as described herein can be isolated and/or purified post expression of a nucleotide sequence in its native organism. Preferably, however, the amino acid sequence as described herein may be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.

As used with reference to the present invention, the terms "expressing", "expression", "expresses", "expressed" and "expressable" are synonymous with the respective terms "transcribing", "transcription", "transcribes", "transcribed" and "transcribable".

In a preferred embodiment the term "polynucleotide sequence" as described herein refers to a polynucleotide sequence encoding a functional polypeptide.

AMINO ACID SEQUENCES

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

In a preferred embodiment the term "polypeptide" as described herein refers to a functional polypeptide. The term "functional polypeptide" refers to the polypeptide as being capable of having the function normally assigned to the polypeptide when the polypeptide is in the appropriate conditions for the polypeptide to function.

The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

The enzyme as described herein may be used in conjunction with other enzymes. Thus there is provided a combination of enzymes wherein the combination comprises the enzyme for use in the present invention and another enzyme.

Preferably the amino acid sequence as described herein is not a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.

VARIANTS/HOMOLOGUES,IDEPJVATWES There is also provided the use of variants, homologues and derivatives of any amino acid sequence of an enzyme described herein or of any nucleotide sequence encoding such an enzyme.

Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".

In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), here it is preferred to express homology in terms of sequence identity.

In the present context, an homologous sequence is taken to include a nucleotide sequence which may be at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding an enzyme (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), here it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compaied with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments S are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestflt package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et a! 1984 Nuc. Acids Research 12 p3 87). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et a!., 1999 Short Protocols in Molecular Biology, 4th Ed Chapter 18), FASTA (Altschul et a!., 1990 J. Mol. Biol. 403-4 10) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et a!., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).

However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMSMicrobiolLett 1999 174(2): 247-50; FEMSMicrobiolLett 1999 177(1): 187-8 and [email protected]).

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using the multiplealignment feature in DNASIS (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.

Alternatively, the sequences may also have deletions, insertions or substitutions of amino acid residues which do not produce a silent change and result in a non- functionally equivalent substance.

Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids can be grouped together based on the properties of their side chain alone. However it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone C.D.

and Barton G.J. (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput.Appl Biosci. 9: 745756)(Taylor W.R.

(1986) "The classification of amino acid conservation" iTheor.Biol. 119; 205-218).

Conservative substitutions may be made, for example according to the table below which describes a generally accepted Venn diagram grouping of amino acids.

SET SUB-SET

Hydrophobic F W Y H K M I L V A G C Aromatic F W Y H _____________ Aliphatic I L V Polar WYHKREDCSTNQ Charged HKRED Positively H K R charged Negatively E D _____________ charged Small VCAGSPTND Tiny AGS Homologous substitution is also provided (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine omithjne (hereinafter referred to as 0), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or - alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.

For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue's nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ eta!, PNAS (1992) 89(20), 9367-937 1 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methyiphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. Here, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences for use in the present invention.

There is also provided the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.

S

Polynucleotides which are not 100% homologous to the sequences described here but fall within the scope can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences described herein.

Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences for use herein.

Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer sofiware known in the art. For example the GCG Wisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

Polynucleot ides (nucleoticle sequences) described here may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides described herein.

Polynucleotides such as DNA polynucleotides and probes as described herein may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

DELETION

As used herein, a "deletion" is defined as a change in the nucleotide sequence in which one or more nucleotides are absent.

INSERTION/ADDITION As used herein, an "insertion" or "addition" is a change in the nucleotide sequence which has resulted in the addition of one or more nucleotides as compared to the naturally occurring promoter.

As used herein, "substitution" results from the replacement of one or more nucleotides or by one or more different nucleotides.

BIOLOGICALLY ACTIVE

Preferably, the variant sequences etc. are at least as biologically active as the sequences described herein.

As used herein "biologically active" refers to a sequence having a similar structural function (but not necessarily to the same degree), and/or similar regulatory function (but not necessarily to the same degree), and/or similar biochemical function (but not necessarily to the same degree) of the naturally occurring sequence.

Alternatively, the variant sequences etc. are not as biologically active as the naturally occurring sequence. For example, a sequence does not have a similar structural function, and/or similar regulatory function, and/or similar biochemical function when compared to the naturally occurring sequence.

HYBRIDISATION

There is also provided sequences that are complementary to the nucleic acid sequences for use here or sequences that are capable of hybridising either to the sequences for use here or to sequences that are complementary thereto.

The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as canied out in polymerase chain reaction (PCR) technologies.

There is also provided the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.

The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein.

Preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under stringent conditions (e.g. 50 C and 0.2xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na3citrate pH 7.0)) to the nucleotide sequences presented herein.

More preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under high stringent conditions (e.g. 65 C and 0.1xSSC {1xSSC = 0.15 M NaC1, 0.015 M Na3citrate pH 7.0)) to the nucleotide sequences presented herein.

There is provided nucleotide sequences that can hybridise to the nucleotide sequences for use here (including complementary sequences of those presented herein).

There is also provided nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences for use here (including complementary sequences of those presented herein).

Also there is provided polynucleotide sequences that are capable of hybridising to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.

In a preferred aspect, there is provided nucleotide sequences that can hybridise to the nucleotide sequence mentioned herein, or the complement thereof, under stringent conditions (e.g. 50 C and O.2xSSC).

There is also provided nucleotide sequences that can hybridise to the nucleotide sequence described herein, or the complement thereof, under high stringent conditions (e.g. 65 C and O.1xSSC).

SITE-DIRECTED MUTAGENESIS

Once an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme-encoding nucleotide sequence has been identified, it may be desirable to mutate the sequence.

Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.

A suitable method is disclosed in Morinaga et a!., (Biotechnology (1984) 2, p646-649).

Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochem istry (1989) , 180, p 147-151). A further method is described in Sarkar and Sommer (Biotechniques (1990), 8, p404-407 - "The megaprimer method of site directed mutagenesis").

RECOMBINANT

In one aspect the sequence for use in the present invention is a recombinant sequence - i.e. a sequence that has been prepared using recombinant DNA techniques.

These recombinant DNA techniques are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press.

SYNTHETIC

In one aspect the sequence for use in the present invention is a synthetic sequence - i.e. a sequence that has been prepared by in Vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms - such as the methylotrophic yeasts Pichia and Hansenula.

EXPRESSION OF ENZYMES

The nucleotide sequence for use in the present invention may be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in enzyme form, in and/or from a compatible host cell.

Expression may be controlled using control sequences e.g. regulatory sequences.

The enzyme produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences may be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.

EXPRESSION VECTOR

The term "expression vector" means a construct capable of in vivo or in vitro expression.

Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably covers stable incorporation into the genome.

The nucleotide sequence for use in the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism.

The vectors for use in the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide for use in the present invention.

Alternatively, the vectors for use in the present invention may be transformed into a suitable host cell as described below to provide prevent the expression of a polypeptide.

The choice of vector e.g. a plasmid, cosmid, or phage vector will often depend on the host cell into which it is to be introduced.

The vectors for use in the present invention may contain one or more selectable marker genes- such as a gene, which confers antibiotic resistance e.g. ampicillin, kanamycin, chloramphemcol or tetracyclin resistance. Alternatively, the selection may be accomplished by cotransformation (as described in W091/1 7243).

Vectors may be used in vitro, for example for the production of RNA or used to transfect, transform, transduce or infect a host cell.

The vector may further comprise a nucleotide sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB! 10, pE194, pAMB1 and p11702.

REGULATORY SEQUENCES

In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell. By way of example, this covers a vector comprising the nucleotide sequence for use in the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.

The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site.

Enhanced expression of the nucleotide sequence encoding the enzyme for use in the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions.

Preferably, the nucleotide sequence as described herein is operably linked to at least a promoter.

Examples of suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.

CONSTRUCTS

The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter.

An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shi-intron or the ADH intron, intermediate the promoter and the nucleotide sequence described herein. The same is true for the term "fused" as used here which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.

The construct may even contain or express a marker, which allows for the selection of the genetic construct.

For some applications, preferably the construct for use in the present invention comprises at least the nucleotide sequence as described herein operably linked to a promoter.

HOST CELLS

The term "host cell" used here includes any cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of an enzyme having the specific properties as defined herein.

Thus, there is provided host cells transformed or transfected with a nucleotide sequence that expresses the enzyme for use in the present invention. The vector will be chosen to be compatible with the host cell.

Examples of suitable bacterial host organisms are lactic acid bacteria, preferably Streptococcus spp, more preferably Streptococcus thermophilus or a variant thereof.

The genotype of the host cell may be modified to improve expression.

Examples of host cell modifications include protease deficiency, supplementation of rare tRNA's, and modification of the reductive potential in the cytoplasm to enhance disuiphide bond formation.

For example, the host cell E. co/i may overexpress rare tRNA's to improve expression of heterologous proteins as exemplified/described in Kane (Curr Opin Biotechnol (1995), 6, 494-5 00 "Effects of rare codon clusters on high-level expression of heterologous proteins in E.coli"). The host cell may be deficient in a number of reducing enzymes thus favouring formation of stable disuiphide bonds as exemplified/described in Bessette (Proc NatlAcadSci USA (1999), 96, 13703-13708" Efficient folding of proteins with multiple disulphide bonds in the Escherichia co/i cytoplasm").

ORGANISM

The term "organism" used here includes any organism that could comprise the nucleotide sequence coding for the enzyme for use in the present invention and/or wherein a promoter can allow expression of the nucleotide sequence for use in the present invention when present in the organism.

Suitable organisms include Streptococcug spp, preferably Streptococcus thermophilus or a variant thereof.

The term "transgenic organism" as used here includes any organism that comprises the nucleotide sequence coding for the enzyme for use in the present invention and/or wherein a promoter can allow expression of the nucleotide sequence for use in the present invention within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.

The term "transgenic organism" does not cover native nucleotide coding sequences in their natural enviromnent when they are under the control of their native promoter which is also in its natural environment.

Therefore, the transgemc organism as described herein includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the enzyme described herein, constructs for use as described herein, vectors for use as described herein, plasmids for use as described herein, cells for use as described herein, or the products thereof.

For example the transgenic organism may also comprise the nucleotide sequence coding for the enzyme for use in the present invention under the control of a heterologous promoter.

TRANSFORMATION OF HOST CELLS/ORGANISM As indicated earlier, the host organism can be a Streptococcus spp, preferably Streptococcus thermophilus or a variant thereof.

Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press).

CULTURiNG AND PRODUCTION Host cells transformed with the nucleotide sequence described herein may be cultured under conditions conducive to the production of the encoded enzyme and which facilitate recovery of the enzyme from the cells and/or culture medium.

The medium used to cultivate the host cells may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the enzyme. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).

The protein produced by a recombinant cell may be displayed on the surface of the cell. If desired, and as will be understood by those of skill in the art, expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).

FUSION PROTEINS

The amino acid sequence for use as described herein may be produced as a fusion protein.

Preferably, the fusion protein will not hinder the activity of the protein sequence.

ADDITIONAL POIs The sequences for use as described herein may also be used in conjunction with one or more additional proteins of interest (POIs) or nucleotide sequences of interest (NOIs).

Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, a-galactosidases, f-galactosidases, a-glucanases, glucan lysases, endo-glucanases, glucoamylases, glucose oxidases, cc-glucosidases, glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhaninogalacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: 02oxidoreductase, EC 1.1.3.5) or combinations thereof. The NOT may even be an antisense sequence for any of those sequences.

The POT may even be a fusion protein, for example to aid in extraction and purification.

The NOT coding for POT may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations, which modify the processing and/or expression of the expression product thereof. By way of further example, the NOT may also be modified to optimise expression in a particular host cell. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.

The NOT coding for the POT may include within it synthetic or modified nucleotides- such as methyiphosphonate and phosphorothioate backbones.

The NOT coding for the POT may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule.

DETECTION

A variety of protocols for detecting and measuring the expression of the amino acid sequences as described herein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.

A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for these procedures.

Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3,8 17,837; US-A-3,850,752; US-A-3,939,350; US-A-3,996,345; US-A- 4,277,437; US-A-4,275,149 and US-A-4,366,241.

Also, recombinant immunoglobulins may be produced as shown in US-A-4,8 16, 567.

GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES

The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratoiy Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. Ct al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press.

Each of these general texts is herein incorporated by reference.

EXAMPLES

The invention is now further illustrated in the following non-limiting examples.

EXAMPLE 1 - Isolation and overexpression of the lactate dehydrogenase (ldh) gene To identify the ldh gene of S. thermophilus, ldh genes of related strains deposited in public databanks were blasted against the partly sequenced genome of S. therm op hilus wild-type strain (www.biol. ucl.ac.be./gene/genome/index.html). A putative LDH sequence in S. thermophilus wild-type was found (see SEQ 1D No 1) which shows high homology to the sequence of the LDH enzymes from S. agalactiae, S.pneumoniae and S. pyogenes.

This putative ldh gene of S. thermophilus is chosen as target for overexpression experiments. For this purpose, chromosomal DNA from S. thermophilus 5945 is isolated and primers according to the putative ldh sequence from S. therm ophilus wild- type are designed (SEQ U) No 2 and 3).

ldh 1 fw: 5'-ccggggatccatgactgcactaaactacac-3' (SEQ ID No 2) ldh 1 rev: 5'-ccgcggatcccgctgcagttagttttttgaagcttcttgg-3' (SEQ ID No 3) PCR on the basis of chromosomal DNA from S. therm ophilus by usage of the primers ldh 1 fw and ldh 1 rev, leads to amplification of a 1 kb PCR fragment, carrying the putative ldh gene without promoter. This fragment is purified and analysed by gel electrophoresis.

For overexpression of the id/i gene, the PCR product is restricted with the enzymes BamHI and PstI and ligated into the vector pOri23 carrying the P23 promoter (Que et al., 2000). E. coli DH5a is transformed with the ligation mixture by electroporation (Harlander & McKay, 1984) and the resulting transformants are selected on TY agar plates containing kanamycin (50 j.gIml). Clones with plasmids which allow the growth on this medium are chosen for plasmid isolation. Isolated plasmids are analysed by gel electrophoresis and controlled by restriction with the enzymes BamHI and PstI.

Plasmids carrying the putative ldh gene as an insert are purified and introduced into S. thermophilus by electroporation (Holo & Nes, 1995). The resulting transformants are selected on M17-lactose agar plates with erythromycin (2.5.tg/ml) and kanamycin (50 tg/ml). These strains and the original strain are grown in M17-lactose medium, the specific LDH activity is determined and lactate formation is measured by the decrease in the pH value.

Compared to the original host strain, the newly constructed strain shows a higher specific activity of LDH and an increased production of lactate in the course of the growth.

EXAMPLE 2- Construction of a futile cycle system In order to accelerate the formation of lactate by S. therm ophilus, an energy-wasting futile cycle within its metabolism is constructed. In a futile cycle, enzymes catalysing a cycling reaction are overexpressed. Within this cycling reaction there is less energy produced than wasted. In order to equalise the loss of energy, the organism accelerates its energy-producing carbon flux, i.e. glycolysis and lactate production.

As a futile cycle in the S. thermophilus strain, the reversible reaction of phosphoenolpyruvate (PEP) to oxaloacetate (OA) is chosen. The enzymes involved in this futile cycle are: (i) PEP carboxylase, encoded by the ppc gene, which catalyses the reaction from PEP to OA, and (ii) PEP carboxykinase, encoded by the pck gene, catalysing the reverse reaction.

Every time this cycle runs there is a waste of energy of one GTP. This forces the energy production via glycolysis resulting in a higher production of lactate.

Genes for PEP carboxylase and PEP carboxykinase are identified and characterised from Corynebacterium glutamicum (see SEQ ID Nos 4 and 5 respectively).

Chromosomal DNA from C. glutamicum ATCC 13032 is isolated. On the basis of the previously determined nucleotide sequences (Eikmanns et a!., 1989; Riedel et a!., 2001) primers pck fly and pc/c rev as well as primers ppc fw and ppc rev are designed and constructed (see SEQ ID Nos 6-9) and the primer pairs are used to amplify thepck gene and theppc gene, respectively. The amplification of the pck gene leads to a 1950 bp PCR product, amplification of the ppc gene to a 3274 bp fragment.

pc/c fly: 5'-ccgcggatccagggggcgagaactctgt-3' (SEQ ID No 6) pck rev: 5'ccgcggatcccgctgcagacaagaaaggctcccacm-3' (SEQ ID No 7) ppc fw: 5'ccgcggatccgtcgacggcggacttgt-3' (SEQ ID No 8) ppc rev: 5'ccgcggatccgctgcagcaacagtggagcccgaaa-3' (SEQ ID No 9) Both PCR products are purified and cloned subsequently into the streptococcal overexpression vector pOri23 (Que et a!., 2000) via the restriction sites BamHI and PstI. The vector constructs are transformed into E. coli DH5a (Harlander & McKay, 1984). Selection is performed on TY agar plates containing kanamycin (50 tg/ml).

Plasmid DNA from the clones is isolated. The plasmids are purified and controlled by restriction with the enzymes BamHI and PstI. Vector constructs carrying thepck gene and the ppc gene are introduced into the S. therinophilus strain via electroporation (lob & Nes, 1995). Selection is performed on M17-lactose agar plates with erythromycin (2.5 jig/mi) and kanamycin (50 jg/ml). Resistant strains and the original strain are grown in M17-lactose medium, the specific PEP carboxylase and PEP carboxykinase activities are determined and lactate formation is measured by decrease of the pH value.

Compared to the original host strain the newly constructed strain will show a higher lactate production in the course of the growth.

EXAMPLE 3-Production of acidified milk - Yoghurt 3.1 Stirred voghurt. produced with bulk starter: 3.1.1 Preparation of the bulk starter: Skimmed milk with 1.5% BIOS 2000 (Danisco) or 67kg of VIS-START (Danisco) in water (933 L) is heated at 95 C for 30 minutes before being cooled to an incubation temperature of 42 C. The mixture is then inoculated with a suitable starter culture (for example, 1 BULK FL 1000-tin of YO-MIX 406 per 500 -1000 litre milk, Danisco) before being incubated for 6-6','2 hours at 42 C until a final pH of 4.4 0.05 is reached.

The mixture is then cooled as quickly as possible to 4-8 C.

3.1.2. Preparation of stirred yoghurt: Processed milk with the desired fat content is homogenised at a pressure of 100-200 bars at 65-70 C. Then the milk is heated at 90-95 C for 5-10 minutes. For a short fermentation the milk is cooled to 42 C whereas for a long fermentation the milk is cooled to 36 C.

For a short fermentation the milk is inoculated with 3-5% bulk starter (described under 3.1.1.) The mixture is then incubated for 3'/z-4'/2 hours at 42 C and the final pH is 4.5 0.05.

For a long fermentation the milk is inoculated with 1-2% bulk starter (described under 3.1.1.). The mixture is then incubated for 12-14 hours at 36 C and the final pH is 4.5 0.05.

The fermentation mixtures are then cooled as quickly as possible to 2025 C. Then, optionally, the yoghurt is mixed with a fruit preparation. The product is filled into cups and cooled to a final temperature of 48 C.

3.2 Stirred voghurt, produced with direct inoculation: Processed milk with the desired fat content, together with skim milk powder if required, is homogenised at a pressure of 150-200 bars at 65-70 C. Then the medium is heated at 90-95 C for 5-10 minutes and afterwards cooled to fennentation temperature.

The processed milk is then inoculated with a suitable starter culture (for example, with Units DIRECT PP YO-MIX 401 per 5,000 litres of processed milk, Danisco) before being incubated for 6-7 hours at 42 C or for 9-11 hours at 38 C or for 14-16 hours at 34 C. The final pH is in all three cases 4.50 0.05. After stirring, the mixture is cooled to approximately 20-25 C. Then, optionally, the yoghurt is mixed with a fruit preparation. The product is filled into cups and cooled to a final temperature of 4-8 C.

EXAMPLE 4- Production of acidified milk - Cheese 4.1 Camembert The raw milk undergoes standardisation and heating at 72- 75 C for 15-30 seconds.

The milk is then cooled to a renneting temperature of 36-39 C (ideally 3 7 C). The milk is then inoculated with a suitable starter culture (for example, milk is inoculated with 100 g PROBAT M4 DIRECT FP + 500 g YOMD(101 DIRECT PP + 10-25 dose rates rehydrated Penicillium candidum NR or SC DIRECT PD per 5000 litre).

After pre-ripening for about 30-35 minutes, rennet is added to the mixture at a dilution of 1:15000.

The mixture is then allowed to coagulate for 5-10 minutes and then allowed to set for 25-30 minutes. The resulting mixture is cut into pieces of 11.5 cm and then left for about 5 minutes. In order to separate the combined curd mass, the mixture is stirred, allowed to rest and then stirred again.

The mixture is washed and the whey is removed.

The mixture is turned over about 5 times within 6-7 hours at a temperature of 24-26 C.

Once a pH of 5.05 0.15 at 14-16 C is reached the cheese is cooled and transferred into brine (alternatively the cheese is cooled in the brine). The minimum pH of the cheese is 4.95 0.1.

The cheese is removed from the brine and allowed to drip off for 1-2 hours. The cheese is then dried at 14-16 C at 70-75% relative air moisture for one day. The cheese is then allowed to ripen at 12-14 C at a relative air moisture of 92-98% for 8-12 days. Afterwards the cheese is packaged.

4.2 Mozzarella The fat content of the milk is adjusted according to the protein content of milk and the intended fat in the dry mass of the cheese.

The milk is pasteurised at 72 C for 15-40 seconds and then tempered at about 41 C ( 1 C).

The milk is then inoculated with a suitable starter culture (for example, the milk is inoculated with 50 Units CHOOZITTM 902 DIRECT FP per 5,000 7500 litre milk, Danisco).

The mixture is pre-ripened until a pH of 6.5 is reached. Then rennet is added according to the supplier's recommendation (temperature = 41 C). Coagulation occurs at about 15 minutes. The mixture is then cut to cobnut size (max. 1 - 1.5 cm), the precheese is stirred for 15 mm, the final cheese for 20-3 0 mm. The mixture is pre- pressed and fermented until a pH of 5.2 0.1 is achieved under whey level. After reaching pH 5.2 the total amount of cheese has to be blanched in hot water as quickly as possible. Stretching and kneading (i.e. filatization) in hot water at 75-85 C of the mass which has been cut into cubes then follows.

The cheese pieces are placed in a salt bath at 15-18 BE at a temperature of less than 18 C. The time the cheese is left in the bath depends on the cheese size. The cheese is then packaged under vacuum in plastic bags.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

REFERENCES

Eikmanns, B. J., Follettie, M. T., Griot, M. U., Sinskey, A. J. (1989). The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence and expression. Mol Gen Genet 218: 330-339.

Harlander, S. K., McKay, L. L. (1986). Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA. App! Environ Microbiol 48: 342-346.

Holo, H., Nes, I. F. (1995). Transformation of Lactococcus by electroporation.

Methods Mo! Biol 47: 195-199.

Neretin,L.N.; Schippers,A.; Pemthaler,A.; Hamann,K.; Amann,R.; Jorgensen, B.B.

(2003): Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR. Environ Microbiol 5: 660- 671.

Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45.

Riedel, C., Rittmann, D., Dangel, P., Möckel, B., Petersen, S., Sahm, H., Eikmanns, B. J. (2001). Characterization of the Phosphoenolpyruvate Carboxykinase Gene from Corynebacterium glutamicum and Significance of the Enzyme for Growth and Amino Acid Production. JMo!Microbiol 3: 573-583.

Que, Y-A., Haeflinger, J-A., Francioli, P., Moreillon, P. (2000). Expression of Staphylococcus aureus Clumping Factor A in Lactococcus lactis subsp. cremoris Using a New Shuttle Vector. Infect Immun 68: 35 163522.

Sambrook, J., Fritsch, E. F., Maniatis, J. (1989). Molecular Cloning: a laboratory manual NY: Cold Spring Harbor.

SEQUENCE LISTING

SEQIDNo 1 Putative ldh gene from S. thermop/zilus 1 ATGACTGCAA CTAAACTACA CAAAAAAGTC ATCCTTGTTG GTGACGGTGC CGTAGGTTCA 61 TCTTACGCTT TCGCACTTGT AAACCAAGGT ATCGCTCAAG AACTAGGTAT CATCGAAATT 121 CCACAATTAT TTGAAAAAGC CGTTGGTGAT GCGCTTGACC TTAGCCACGC ACTTCCTTTC 181 ACTTCACCTA AAAAAATCTA TGCAGCTAAA TATGAAGACT GTGCGGATGC TGACCTTGTA 241 GTTATCACTG CTGGTGCTCC TCAAAAACCA GGTGAGACTC GTCTTGATCT TGTTGGTAAA 301 AACCTTGCAA TCAACWTC AATCGTTACT CAAGTTGTTG AATCAGGATT CAACGGTATT 361 TTCCTTGTAG CTGCTAACCC AGTAGACGTA TTGACTTACT CTACATGGAA GTTCTCAGGA 421 TTCCCTWG AACGCGTTAT CGGTTCAGGT ACTTCACTTG ACTCAGCTCC TTTCCGTCAA 481 GCACTTGCTG AAAAACTTAA TGTCGATGCT CGTTCAGTTC ACGCTTACAT CATGGGTGAA 541 CACGGCGACT CAGAGTTTGC GGTTTGGTCA CACGCTAACA TCGCCGGTGT AAACCTTGAA 601 GAGTTCCTTA AAGACGAAGA AAACGTTCAA GAAGCTGAAT TAGTTGAATT GTTCGAAGGT 661 GTTCGTGATC CAGCTTACAC AATTATCAAC AAAAAAGGTG CTACATACTA CGGTATCGCA 721 GTAGCCCTTG CTCGTATCAC TAAAGCTATC CTTGACGATG AAAATGCAGT ACTTCCATTG 781 TCTGTATTCC AAGAAGGTCA ATATGGTGTA AACAACATCT TTATCCCTCA ACCTGCTATT 841 GTAGGTGCAC ACGGTATCGT ACGTCCAGTA AACATCCCAT TGAACGATGC TGAACAACAA 901 AAGATGAACG CTTCTGCCGA TGAATTGCAA GCTATCATTG ATGAAGCATG GAAAAACCCT 961 GAATTCCAAG AAGCTTCAAA AAACTAA SEQIDNo2 id/i 1 fw: 5 - ccggggatccatgactgcaactaaactacac-3' SEQIDNo3 id/i 1 rev: 5 - ccgcggatcccgctgcagttagttttttgaagcttcttgg-3' SEQIDNo4 ppc gene from C. giutamicum 1 GTCGACGGCG GACTTCTCGG TGGCGCTTCC CTCGACGGTC AAGCATTGCC CAAGCTGGCT 61 GCCAACGCTG CGAGCGTTGC TTAAAGTACA GAGCTTTAAA GCACAGCCTT AAAGCACAGC 121 CTTAAAGCAC AAGCACTGTA GAAGTGCGGT TTTGATGAGC CCATGAAAGC CATCGAAATC 181 AATCGCCCAG CTAAACACCT GTTTTGCTGG GTGATTTTTT ATCTCATGCA CGCCAACACC 241 CTCAATGTGA AAGAGTGTTT AAACTAGTTA TGACTGATTT TTTACGCGAT GACATCAGGT 301 TCCTCGGTCA AATCCTCGGT GAGGTAATTG CGGAACAAGA AGGCCAGGAG GTTTATGAAC 361 TGGTCGAACA AGCGCGCCTG ACTTCTTTTG ATATCQCCAA GGGCAACGCC GAAATGGATA 421 GCCTGGTTCA GGTTTTCGAC GGCATTACTC CAGCCAAGGC AACACCGATT GCTCGCGCAT 481 TTTCCCACTT CGCTCTGCTG GCTAACCTGG CGGAAGACCT CTACGATGAA GAGCTTCGTG 541 AACAGGCTCT CGATGCACCC GACACCCCTC CGGACAGCAC TCTTGATGCC ACCTGGCTGA 601 AACTCAATGA GGGCAATGTT GGCGCAGAAG CTGTGGCCGA TGTGCTGCCC AATGCTCAGG 661 TGGCGCCGGT TCTGACTGCC CACCCAACTG AGACTCGCCG CCGCACTGTT TTTGATGCGC 721 AAAAGTGGAT CACCACCCAC ATGCGTGAAC GCCACGCTTT GCAGTCTGCG GAOCCTACCG 781 CTCGTACGCA AAGCAAGTTG GATGAGATCG AGAAOAACAT CCCCCGTCGC ATCACCATTT 841 TGTGGCACAC CGCGTTGATT CGTGTGGCCC GCCCACGTAT CGAGGACGAG ATCGAAGTAG 901 GGCTGCGCTA CTACAAGCTG AGCCTTTTGG AAGAGATTCC ACGTATCAAC CGTGATGTGG 961 CTGTTGAGCT TCGTGAGCGT TTCGGCGAGG GTGTTCCTTT GAAGCCCGTG GTCAAGCCAG 1021 GTTCCTGGAT TOCTOGAGAC CACGACGGTA ACCCTTATGT CACCGCGGAA ACAGTTGAGT 1081 ATTCCACTCA CCGCGCTGCG GAAACCGTGC TCAAGTACTA TGCACGCCAG CTGCATTCCC 1141 TCGAGCATGA GCTCAGCCTG TCGGACCGCA TGAATAAGGT CACCCCGCAG CTGCTTGCGC 1201 TGGCAGATGC AGGGCACAAC GACGTGCCAA GCCGCGTGGA TGAGCCTTAT CGACGCGCCG 1261 TCCATGGCGT TCGCGGACGT ATCCTCGCGA CGACGGCCGA GCTGATCGGC GAGGACGCCG 1321 TTGAGGGCGT GTGGTTCAAG GTCTTTACTC CATACGCATC TCCGGAAGAA TTCTTAAACG 1381 ATGCGTTGAC CATTGATCAT TCTCTGCGTG AATCCAAGGA CGTTCTCATT GCCGATGATC 1441 GTTTGTCTGT GCTGATTTCT GCCATCGAGA GCTTTGGATT CAACCTTTAC GCACTGGATC 1501 TGCGCCAAAA CTCCGAAAGC TACGAGGACG TCCTCACCGA GCTTTTCGAA CGCGCCCAAG 1561 TCACCGCAAA CTACCGCGAG CTGTCTGAAG CJGAGAAGCT TGAGGTGCTG CTGAAGGAAC 1621 TGCGCAGCCC TCGTCCGCTG ATCCCGCACG GTTCAGATGA ATACAGCGAG GTCACCGACC 1681 GCGAGCTCGG CATCTTCCGC ACCGCGTCGG AGGCTGTTA GAAATTCGGG CCACGGATGG 1741 TGCCTCACTG CATCATCTCC ATGGCATCAT CGGTCACCGA TGTGCTCGAG CCGATGGTGT 1801 TGCTCAAGGA ATTCGGACTC ATCGCAGCCP. ACGGCGACA CCCACGCGGC ACCGTCGATG 1861 TCATCCCACT GTTCGAAACC ATCGAGATC TCCAGGCCGG CGCCGGAATC CTCGACGAAC 1921 TGTGGAAAAT TGATCTCTAC CGCAACTACC TCCTGCAGCG CGACAACGTC CAGGAATCA 1981 TGCTCGGTTA CTCCGATTCC AACAAGGATG GCGGATATTT CTCCGCAAAC TGGGCGCTTT 2041 ACGACGCGGA ACTGCAGCTC GTCGAACTAT GCCGATCAGC CGGGGTCAAC GTTCGCCTGT 2101 TCCACGGCCG TGGTGGCACC GTCCGCCGCG GTGGCGGACC TTCCTACGAC GCGATTCTTG 2161 CCCAGCCCAG GGGGGCTGTC CAAGGTTCCG TGCGCATCAC CGAGCAGGGC GAATCATCT 2221 CCGCTAAGTA CGGCAACCCC GAAACCGCGC GCCGAAACCT CGAAGCCCTG GTCTCAGCCA 2281 CGCTTGAGGC ATCGCTTCTC GACGTCTCCG AACTCACCGA TCACCAACGC GCGTACGACA 2341 TCATGAGTGA GATCTCTGAG CTCAGCTTGA AGAAGTACGC CTCCTTGGTG CACGAGGATC 2401 AAGGCTTCAT CGATTACTTC ACCCATCCA CGCCGCTGCA GGAGATTGGA TCCCTCAACA 2461 TCGGATCCAG GCCTTCCTCA CGCAACCAGA CCTCCTCGGT GGAAGATTTG CGAGCCATCC 2521 CPTGGGTGCT CAGCTGGTCA CAGTCTCGTG TCATGCTGCC AGGCTGGTTT GGTGTCGGAA 2581 CCGCATTAGA GCAGTGGATT GGCGAAGGGG AGCAGGCCAC CCAACGCATT GCCGAGCTGC 2641 AAACACTCAA TGAGTCCTGG CCATTTTTAC CCTCAGTGTT GGATAACATG GCTCAGGTGA 2701 TGTCCAAGGC AGAGCTGCGT TTGGCAAAGC TCTACGCAGA CCTGATCCCA GATACGGAAG 2761 TAGCCGAGCG AGTCTATTCC GTCATCCGCG AGGAGTACTT CCTGACCAAG AAGATGTTCT 2821 GCGTAATCAC CGGCTCTGAT GATCTGCTTG ATGACAACCC ACTTCTCGCA CGCTCTGTCC 2881 AGCGCCGATA CCCCTACCTG CTTCCACTCA ACGTGATCCA GGTAGAGATG ATGCGACGCT 2941 ACCGAAAAGG CGACCAAAGC GAGCAAGTGT CCCGCAACAT TCAGCTGACC ATGAACGGTC 3001 TTTCCACTGC GGTCCGCAAC TCCGGCTAGT CAGCCGGCTG GGTAGTACTC GTGTATACTG 3061 TCTAAATTA TTCGAAATCA GQTGGGCATA AGGTTCACCT GGGTTCTCAA ACGGCAAAGG 3121 AACATTTTCC ACATGCCATT GACGCTTCAA ATCATCCTCG TCGTCGCCAG CCTGCTCATG 3181 ACGGTTTTCG TCTTGCTGCA CAAGGGCAAA GGCGGCGGAC TCTCCAGCCT CTTCGGTGGC 3241 GGTGTGCGT CCAATCTTTC GGGCTCCACT GTTGTTGAAA AGAACCTGGA TC SEQNo.5 pck gene from C. Glutamicum 1 ACGCTAGGGG GCGAGAACTC TGTCGAATGA CACAAAATCT GGAGAAGTAA TGACTACTGC 61 TGCAATCAGG GGCCTTCAGG GCGAGCGCC GACCAAGAAT AAGGAACTGC TGAACTGGAT 121 CGCAGACGCC GTCGAGCTCT TCCAGCCTGA GGCTGTTGTG TTCGTTGATG GATCCCAGGC 181 TGAGTGGGAT CGCATGGCGG AGGATCTTGT TGAAGCCGGT ACCCTCATCA AGCTCAACGA 241 GGAAAAGCGT CCGAACAGCT ACCTAGCTCG TTCCAACCCA TCTGACGTTG CGCGCGTTGA 301 GTCCCGCACC TTCATCTGCT CCGAGAAGGA AGAAGATGCT GGCCCAACCA ACAACTGGGC 361 TCCCCACAG GCAATGAAGG ACGAAATGTC CAAGCATTAC GCTGGTTCCA TGAAGGGGCG 421 CACCATGTAC GTCGTGCCTT TCTGCATGGG TCCAATCAGC GATCCGGACC CTAAGCTTGG 481 TGTGCAGCTC ACTGACTCCG AGTACGTTGT CATGTCCATG CGCATCATGA CCCGCATGGG 541 TATTGAAGCG CTGGACAAGA TCGGCGCGAA CCGCPGCTTC GTCAGGTGCC TCCACTCCGT 601 TGGTCCTCCT TTGGACCAG GCCAGGAAGA CGTTGCATGG CCTTGCAACG ACACCAAGTA 661 CATCACCCAG TTCCCAGAGA CCAAGGAAAT TTGGTCCTAC GGTTCCGGCT ACGGCGGAAA 721 CGCAATCCTG GCAAPCAAGT GCTACGCACT GCGTATCGCA TCTCTCATGG CTCGCGAAGA 781 AGCATGGATG GCTGAGCACA TGCTCATCCT GAAGCTGATC AACCCAGAGG GCAAGGCGTA 841 CCACATCGCA GCAGCATTCC CATCTGCTTG TGGCAAGACC AACCTCGCCA TGATCACTCC 901 AACCATCCCA GGCTGGACCG CTCAGGTTGT TGGCGACGAC ATCGCTTGGC TGAAGCTGCG 961 CGAGGACGGC CTCTACGCAG TTAACCCAGA AAATGGTTTC TTCGGTGTTG CTCCAGGCAC 1021 CAACTACGCA TCCAACCCAA TCGCGATQAA GACCATGGAA CCAGGCAACA CCCTGTTCAC 1081 CACGTGGCA CTCACCGACG ACGGCGACAT CTGGTGGGAA GGCTGGACG GCGACGCCCC 1141 AGCTCACCTC ATTGACTGGA TGGGCAACGA CTGGACCCCA GAGTCCGACG AAAACGCTGC 1201 TCACCCTAAC TCCCGTTACT GCGTAGCAAT CGACCAGTCC CCACAGCPG CACCTGAGTT 1261 CAACGACTGG GAAGGCGTCA AGATCGACGC AATCCTCTTC GGTGGACGTC GCGCAGACAC 1321 CGTCCCACTG GTPACCCAGA CCTACGACTG GGAGCACGGC ACCATGGTTG GTGCACTGCT 1381 CGCTCCGGT CAACCCCAG CTTCCGCAGA AGCAAAGGTC GGCACACTCC GCCACGACCC 1441 AATGGCAATG CTCCCATTCA TTGGCTACAA CGCTGGTGAA TACCTGCAGA ACTGGATTGA 1501 CATGGGTAAC AACGTGGCG ACAAGATGCC ATCCATCTTC CTGGTCAACT GGTTCCGCCG 1561 TCCCGAAGAT GGACGCTTCC TGTGGCCTGG CTTCGGCGAC AACTCTCGCG TTCTGAAGTG 1621 GGTCATCGAC CGCATCGAAG GCCACGTTGG CGCAGACGAG ACCGTTGTTG GACACACCGC 1681 TAAGCCGAA GACCTCGACC TCCACCGCCT CGACACCCCA ATTGAGGATG TCAAGGAAGC 1741 ACTGACCGCT CCTGCAGAGC AGTGGGCAAA CGACGTTGAA GACAACGCCG AGTACCTCAC 1801 TTTCCTCGGA CCACGTGTTC CTGCAGAGGT TCACACCCAG TTCCATGCTC TGAAGGCCCG 1861 CATTTCAGCA GCTCACGCTT AAAGTTCACG CTTAAGAACT GCTAAATAAC AAGAAAGGCT 1921 CCCACCGAAA GTGGGAGCCT TTCTTGTCGT TAAGCGATGA ATT SEQIDNo6 pck fw: 5' ccgcggatccagggggcgagaactctgt-3' SEQIDNo7 pck rev: 5 ccgcggatcccgctgcagacaagaaaggctcccacttt-3' SEQIDNo8 ppc f'v: 5 ccgcggatccgtcgacggcggacttgt-3' SEQIDNo9 ppc rev: 5 ccgcggatccgctgcagcaacagtggagcccgaaa-3'

Claims (24)

1. A Streptococcus bacterium wherein said bacterium has an increased glucose metabolism and br an increased rate of lactate synthesis.

2. The bacterium according to claim 1 wherein said bacterium has an increased expression of a lactate dehydrogenase gene and/or an increased level of lactate dehydrogenase polypeptide sequence.

3. The bacterium according to claim 2 wherein said increased expression of said lactate dehydrogenase gene and/or an increased level of lactate dehydrogenase polypeptide sequence increases glucose metabolism and br increases the rate of lactate synthesis in said bacterium.

4. The bacterium according to any one of claims 1 to 3 wherein said bacterium has at least one energy-wasting futile cycle.

5. The bacterium according to claim 4 wherein said energy-wasting futile cycle is enhanced.

6. The bacterium according to claim 1 or 4 wherein said energy-wasting futile cycle results in an increased glucose metabolism and/or an increased rate of lactate synthesis in said bacterium.

7. The bacterium according to claim 5 wherein said increased expression of said lactate dehydrogenase gene and/or said increased level of lactate dehydrogenase polypeptide sequence in said bacterium enhances said energywasting futile cycle.

8. The bacterium according to any one of claims 1 to 7 wherein said lactate dehydrogenase gene encodes L-lactate dehydrogenase (EC 1.1.1.27) and/or said lactate dehydrogenase polypeptide sequence is L-lactate dehydrogenase (BC 1.1.1.27).

9. The bacterium according to any one of claims 1 to 8 wherein said bacterium is capable of producmg an increased amount of lactic acid when said bacterium is cultured under conditions in which said bacterium is metabolically active.

10. The bacterium according to any one of claims 1 to 9 wherein said bacterium is a fast acidifier of milk.

11. The bacterium according to any one of claims 1 to 10 wherein said bacterium is Streptococcus therm op hilus.

12. A starter culture comprising at least a bacterium according to any one of claims 1 to 11.

13. A food wherein said food comprises at least a bacterium according to any one of claims 1 to 11, or a starter culture according to claim 12 preferably wherein said food is a dairy product.

14. Use of a bacterium according to any one of claims 1 to 11 in the preparation of a starter culture.

15. Use of a bacterium according to any one of claims 1 to 11, or a starter culture according to claim 12 in the preparation of a food.

16. Use according to claim 15 wherein said food is a dairy product.

17. A method of preparing a food comprising the steps of: (i) admixing a bacterium or a starter culture with a dairy medium, and (ii) culturing said admixture under conditions in which said bacterium is metabolically active, wherein said bacterium is a bacterium according to any one of claims 1 to 11 or a starter culture according to claim 12.

18. The method according to claim 17 wherein said dairy medium is milk.

19. The method according to claim 18 wherein said milk is one or more of the following: cows' milk, goats' milk, ewes' milk, soy milk, lamas' milk, buffalo cows' milk and yaks' milk.

20. A food produced by the method according to any one of claims 17 to 19 wherein said food is a dairy product.

21. A method of preparing a bacterium according to any one of claims ito 11 wherein said bacterium has an increased level of the polynucleotide sequence encoding lactate dehydrogenase and/or an increased level of the lactate dehydrogenase polypeptide sequence.

22. A method of preparing a starter culture of a bacterium comprising the steps of: (i) admixing said bacterium with a dairy medium, and (ii) culturing the resulting admixture under conditions in which said bacterium is metabolically active, wherein said bacterium is a bacterium according to any one of claims 1 to 11.

23. The method according to claim 22 wherein said dairy medium is milk.

24. The method according to claim 23 wherein said milk is one or more of the following: cows' milk, goats' milk, ewes' milk, soy milk, lamas' milk, buffalo cows' milk and yaks' milk.