FR2798670A1 - TRANSFORMED LACTIC BACTERIA TO PROVIDE THEM WITH BREATHING METABOLISM, AND LEAVES INCLUDING SAID BACTERIA - Google Patents

Abstract

The invention concerns lactic acid bacteria which have been provided with a respiratory metabolism, in particular by genetic modifications, such that the expression of at least one gene, either heterologous or homologous, coding for a protein intervening in said respiratory metabolism, is altered. Said bacteria are useful in particular for producing lactic acid ferments.

Description

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LACTIC BACTERIES TRANSFORMED TO CONFER THEM RESPIRATORY METABOLISM, AND LEAVES INCLUDING THE SAME.

 The present invention relates to the improvement of the preservation and acidification properties of lactic starter cultures.

 By lactic sourdough is meant any preparation intended for the inoculation of a medium to be fermented, and comprising at least one strain of lactic acid bacteria belonging in particular to one of the genera Lactococcus, Streptococcus, Enterococcus, Leuconostoc, Lactobacillus, Propionibacteria, or Bifidobacteria, or a mixture of strains belonging to one or more of these genera mentioned above.

 The lactic acid starter used in particular to produce fermented foods and silage products are usually prepared in batch cultures, and are then concentrated and packaged for later use in order to inoculate different food products for fermentation. One of the concerns of the producers of sourdoughs is to obtain a large bacterial biomass, and to maintain a good viability of the bacteria during storage, so that during inoculation, the fermentation starts quickly and gives food products having characteristics reproducible.

 However, many causes of stress can intervene during the different stages of preparation of sourdoughs and alter the survival of lactic acid bacteria. In particular, bacterial viability can be quickly lost if the cultures are kept stationary. One of the causes is the accumulation in the environment of natural products of bacterial metabolism, in particular organic acids such as lactic acid which cause a decrease in pH detrimental to bacterial growth. Another

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 -cause of loss of viability during preparation and storage is the presence of oxygen, which is naturally toxic to lactic acid bacteria; these bacteria have in fact a metabolism of carbohydrates based on fermentation. BERGEY'S manual, 9th edition, edited by HOLT et al. (1994) WILLIAMS and WILKINS Eds.

 To limit the drop in pH, culture media buffered around pH 6 with cations associated with carbonates, hydroxides, phosphates or oxides are usually used for the production of sourdoughs of lactic acid bacteria. However, these additions to the culture medium can cause problems for subsequent productions, for example by promoting the development of phages, or by increasing the solubility of caseins.

 To avoid the toxic effects of oxygen, the preparation and storage of sourdoughs is usually carried out anaerobically; for example, during the preparation of starter cultures in batches, certain steps are carried out under nitrogen, in order to remove traces of oxygen. However, when using sourdoughs, they are frequently exposed to high levels of oxygen. For example, the milk that is used for the preparation of fermented milk products is highly aerated during the transfer process and therefore rich in oxygen. This could be a cause of slower restarting of the leaven.

 It has been reported (A.K. SIJPESTEIJN, Antonie von Leeuwenhoek 36: 335, 1970) that Lactococcus and Leuconostoc grown in the presence of heme and under aeration produce cytochromes and have a respiratory metabolism.

 More recent work [KANEKO et al. Appl.

About. Microbiol., 56: 9, 2644-2649 (1990)], report an improvement in the proliferation of a strain of

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 Lactococcus lactis diacetylactis, cultivated in the presence of hemin and / or Cu2 +. This effect is not attributed to the appearance of respiratory metabolism, but to the activation of diacetyl synthase by hemin and / or Cu2 +, which would preferentially orient fermentative metabolism towards the production of diacetyl, at the expense of lactate.

 The team of inventors recently discovered that in the context of the preparation of lactic starter cultures, the use of a porphyritic compound associated with an aerobic culture made it possible to obtain greater bacterial growth than that obtained during conventional processes, and that in addition, the percentage of viable bacteria in the bacterial population and the duration of survival were also much greater. What is more, when the leaven obtained in this way is used to inoculate a product to be fermented, a very rapid restart of growth and bacterial fermentation is observed, resulting in an acidification of the product much faster than that observed with classic sourdoughs.

This work is described in International Application PCT / IB 9901430, filed on July 26, 1999 on behalf of the NATIONAL INSTITUTE FOR AGRONOMIC RESEARCH.

 The inventors have now shown that the improvements in bacterial yield and viability during storage were due to the acquisition of a respiratory metabolism by L. lactis during culture under aeration and in the presence of a porphyric compound.

During the inoculation of the product to be fermented, the bacteria are also capable of immediately restoring a fermentative metabolism, which results in an increase in restarting performance.

 Respiratory metabolism requires the presence of the enzyme equipment involved in

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 different metabolic pathways, including the tricarboxylic acid cycle (Krebs cycle), the synthesis and use of heme, and the synthesis of cytochromes.

 Using primers derived from the alignment of gene sequences known to be involved in respiration in other bacteria, the inventors sought the presence of homologous genes in L. lactis. They thus identified three genes respectively coding for aconitase (enzyme involved in the Krebs cycle), ferrochelatase (enzyme involved in the biosynthesis of heme by catalyzing the formation of a complex between iron and a precursor porphyric compound heme, the complex thus formed can be incorporated into bacterial cytochromes), and cytochrome D.

 They further showed that these genes were functional in L. lactis. They have in fact found that bacteria in which the aconitase gene or the cytochrome D gene is inactivated no longer exhibit respiratory metabolism when they are cultivated aerobically and in the presence of a porphyritic compound containing iron. Likewise, they observed that the inactivation of the gene coding for ferrochelatase led to the loss of the respiratory metabolism capacities of L. lactis in the case of cultures carried out in the presence of a porphyric compound containing no iron, such as protoporphyrin. , but not in the case of cultures carried out in the presence of a porphyritic compound containing iron, such as heme.

 These observations confirm that the improvement in the performance of lactic starters obtained by the inventors by preparing these sourdoughs aerobically and in the presence of a porphyritic compound is linked to the appearance of respiratory metabolism under these culture conditions.

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 The object of the present invention is to provide other means of conferring a respiratory metabolism on lactic acid bacteria, or of promoting it, in particular in order to improve the performance of lactic starter cultures in a manner comparable to that observed by the inventors during the addition of heme (or other molecules derived from porphyrins). In accordance with the present invention, this object can be achieved by causing or promoting the expression, in a lactic bacterium, of at least one protein participating in this metabolism, for example at least one protein involved in the Krebs cycle, and / or at least one protein involved in the biosynthetic pathway for heme, and / or at least one protein involved in the biosynthetic pathway in cytochromes.

 Advantageously, this can be done by transferring to one lactic acid bacterium one or more of the genes of these proteins, cloned from an aerobic bacterium.

 The present invention relates to a lactic acid bacteria transformed by at least one heterologous gene coding for a protein involved in respiratory metabolism.

 According to a preferred embodiment of the present invention, said gene is chosen from: - genes coding for proteins of the Krebs cycle; - the genes coding for proteins of the heme biosynthesis pathway; - the genes coding for proteins of the biosynthetic pathway of cytochromes.

 According to another preferred embodiment of the present invention, said lactic acid bacterium is chosen from bacteria of the genera Lactococcus, Streptococcus, Enterococcus, Leuconostoc, Lactobacillus, Propionibacteria, or Bifidobacteria. Bacteria

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 preferred are those of the various species of the genus Lactococcus, as well as streptococci of the species Streptococcus thermophilus.

 For a given bacterial species, the gene (s) suitable for conferring on bacteria all or part of the enzymatic equipment necessary for the acquisition of a respiratory metabolism can be identified by a person skilled in the art from the information on the sequences of bacterial genomes available in databases, which makes it possible to identify the genes already present in a given microorganism and the metabolic pathways in which these genes can participate. In the absence of the genomic sequence of the bacterial species of interest, the sequence (s) of one or more neighboring species is (are) usable to determine which genes are probably present. For example, the complete or almost complete genome sequences of several streptococcal species (Streptococcus pneumoniae, Streptococcus pyogenes, and partially Streptococcus mutans) are currently available, and reveal the presence of several of the genes required for respiration. These species are phylogenetically close to lactic bacteria commonly used in the food industry such as thermophilic streptococci, and are also related to lactococci.

 Thus, the transformation of bacteria of the species Lactococcus or Streptococcus by one or more gene (s) coding for one or more protein (s) of the heme biosynthesis pathway can make it possible to obtain bacteria having a respiratory metabolism without the need to add porphyric derivatives to the culture medium.

 The desired genes can be obtained from a strict aerobic bacterium, or from a bacterium

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 optional aerobic. They can easily be identified from the bacterial genomes available on the databases. For example, one can use genes obtained from Bacillus subtilis, which is an aerobic bacterium, and whose complete genomic sequence has been published.

 It is thus possible to supply a lactic acid bacterium with all of the genes necessary to confer a respiratory metabolism on this bacterium. It is also possible, if desired, to bring only part of these genes, for example in order to be able to control in different ways the conditions under which the bacteria will be able to breathe.

 We can thus construct, as non-limiting examples: - a lactic acid bacterium having all of the genes necessary for respiratory metabolism; the switching between a fermentative metabolism and a respiratory metabolism can be controlled by modifying the oxygen content of the culture medium; - a lactic acid bacterium possessing all of the genes coding for the proteins of the Krebs cycle and all of the genes for cytochromes, but devoid of all or part of the genes of the heme biosynthesis pathway; switching from a fermentative metabolism to a respiratory metabolism will then require, in addition to aeration of the medium, the addition of heme or one of its precursors.

 To obtain a transformed lactic acid bacterium according to the invention, the desired gene (s) can be introduced separately, or at least part of them can be grouped into one or more operons ( s).

 For example, to give L. lactis a total or partial capacity for heme biosynthesis, we

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 can transfer one or both of the heme operons from B. subtilis into L. lactis or only some of the genes present on these operons.

 In order to obtain lactic acid bacteria in accordance with the invention, it is also possible to promote the expression of genes intervening in the respiratory metabolism naturally already present in said bacteria. This can be done for example by acting on the cis or trans regulation of the activity of these genes.

 Lactic acid bacteria according to the invention can be obtained by implementing conventional techniques of genetic engineering, known in themselves to those skilled in the art. The desired gene (s) can be associated with functional transcription and translation control sequences in the lactic acid bacteria which it is desired to transform. One can in particular, if desired, place one or more of the transferred genes under transcriptional control of an inducible promoter, in order to make it possible to control the switching between fermentative metabolism and respiratory metabolism.

 The constructs produced are placed in an appropriate vector to introduce them into the lactic acid bacteria concerned. Vectors which can be used to transform lactic acid bacteria of different species, and which make it possible either to maintain the genetic information introduced in the form of a stable independent replicon, or to integrate it into the bacterial chromosome, are known in themselves. In cases where the amount of genetic information to be transferred requires the introduction of large segments of DNA, protoplast fusion or bacterial conjugation techniques can be used.

 The functioning of the respiratory metabolism in the transformed bacterium can be verified by carrying out the culture of said bacterium in

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 conditions allowing the induction of a respiratory metabolism (ie under aeration, and possibly, under conditions of induction of one or more inducible promoters possibly controlling the expression of one or more of the transferred genes and / or in the presence of heme or one of its precursors in the case where the transformed bacterium does not include all of the genes of the heme biosynthetic pathway, etc.), and by measuring the following parameters: i) the pH of the final culture, ii) the products consumed or formed during growth (for example the oxygen consumed, the production of fumarate or that of lactate, the amount of total carbon at the end of culture, which in particular makes it possible to evaluate C02 production during respiration, etc.), iii) bacterial population at the end of growth, iv) survival during long storage, and v) re-acidification properties when the transformed strain is used as a dema culture error for fermentation.

 The present invention also relates to the use of a strain of lactic acid bacteria according to the invention for obtaining a lactic acid starter.

 The present invention also relates to a process for producing lactic sourdough, characterized in that it comprises the culture of at least one strain of lactic acid bacteria in accordance with the invention under conditions allowing the induction of a respiratory metabolism in said strain.

 Said respiratory metabolism induction conditions include aerating the culture; advantageously, this aeration is carried out so as to maintain, throughout the duration of the culture, an oxygen content equal to at least 5 millimoles per liter of culture medium.

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 The bacteria can then be harvested by any means known in themselves; one can for example distribute the culture in suitable packaging and keep it in this form until use; generally, however, it is preferable to separate the bacteria from the culture medium and to concentrate them by centrifugation or by filtration. The collected bacteria can then be packaged for storage.

 The present invention also includes lactic sourdoughs comprising at least one strain of transformed lactic acid bacteria according to the invention.

 These leaven can also include one or more other bacterial strains, of the same species or of different species. Several species or several different strains may have been cultivated simultaneously (if their optimal growth conditions are compatible), or else cultivated separately and brought together after harvest.

 The lactic starters in accordance with the invention can be harvested and stored under the same conditions as the lactic starters of the prior art, and in particular as the lactic starters which are the subject of PCT Application IB / 99 01430; they have conservation and restart properties at least comparable to those of the latter. The invention also includes the use of these lactic acid starter cultures for the preparation of fermented products, and any process for the preparation of a fermented product product comprising the inoculation of a medium to be fermented using a lactic acid starter in accordance with the invention.

 The invention will be further illustrated with the aid of the additional description which follows, which refers to non-limiting examples of obtaining lactic acid bacteria according to the invention.

EXAMPLE 1: OBTAINING A STRAIN OF L. LACTIS EXPRESSING THE GENES NECESSARY TO PRODUCE PROTOHEM IX
The hemA (NADP (H) genes: glutamyl-tRNA reductase, SWISS-PROT access number: P16618), hemL (GSA 2,1-aminotransferase, SWISS-PROT access number: P30949), hemB (Porphobilinogen synthase, access number

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 SWISS-PROT: P30950), hemC (hydroxymethylbilane synthase, SWISS-PROT access number P16616), hemD (Uroporphyrinogen III synthase, SWISSPROT access number: P21248), and hemE (Uroporphyrinogen decarboxylase, SWISS-PROT access number P32395), hemF (Coproporphyrinogen III oxidase, SWISSPROT access number: P54304), hemY (hemG) (Protoporphyrinogen oxidase) (SWISS-PROT: P32397) and hemH (hemF) (ferrochelatase, SWISS-PROT access number: P32396 ) of Bacillus subtilis allow the synthesis of protoheme IX from glutamyl-tRNA.

 The hemACDBL genes contained in a single operon in B. subtilis are amplified by PCR from the strain 168 [ANAGNOSTOPOULOS and SPITZIZEN, J.

Bacteriol. 81,741-746, (1961)] using primers allowing the coding sequence of the gene to be obtained with the ribosome binding site and its terminator: Primer sense: 5'-CTGCTGTTTTTGGTATTGTC-3 ',.

Antisense primer: 5 '-GTATGAACTGGAGAAAATATG-3'.

 Amplification [5 min 96 C, (15 s. 96 C, 15 s.

50 C, 7 min 72 C) 30 times] is carried out with 5 units of Vent Polymerase (New England Biolabs) in the presence of 4 mM of MgSO 4.

 A fragment of 6700 bp is obtained. This fragment is then cloned on the plasmid pBSKS + (STRATAGENE) at the SmaI site in the XLIBlue strain (STRATAGENE) under the control of the promoter P23 [VAN DER VOSSEN et al., Appl.

About. Microbiol., 53,2452-2457, (1984)] previously cloned into this plasmid. The plasmid obtained, called pBSUROIII, is linearized by NotI and integrated into the NotI site of the plasmid pILNewl3 [RENAULT et al., Gene, 183,175-182 (1996)]. The resulting plasmid called pIL-UROIII is introduced into the strain of L. lactis MG1363 [GASSON, J.

Bacteriol., 154, 1-9, (1983)]. The production

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 of uroporphyrinogen III by this strain is determined as previously described by ANDERSON and IVANOVICS, [J.

Gen. Microbiol., 49,31-40, (1967)].

 The hemEHY genes contained in a single operon in B. subtilis are amplified by PCR from strain 168 using primers allowing the coding sequence of the gene to be obtained with the ribosome binding site and its terminator: sense primer.

5'-TTGCCGTATGAAAGGTGGAAATC-3 ', antisense primer: 5'-TCATAACCTGCTGTTCAATTCAATC-3'.

 Amplification [5 min 96 C, (15 s. 96 C, 15 s.

50 C, 4 min 72 C) 30 times] is carried out with 5 units of Wind Polymerase in the presence of 4 mM of MgSO 4. A fragment of 3600 bp is obtained. This fragment is then cloned on the plasmid pBSKS + at the SmaI site in the XLIBlue strain under the control of the promoter P23 previously cloned in this plasmid. The plasmid obtained, called pBS-PIX, is linearized by NotI, then the ends are made blunt by Polymerase I. This fragment is then integrated into the unique SmaI site of the plasmid pNZ2120 [PLATTEUW et al. Appl. About. Microbiol. 62: 1008-13 (1996)]. The resulting plasmid called pLac-PIX is introduced into the strain of L. lactis MG1363 containing the plasmid pILUROIII. The production of protoheme IX by this strain is determined as described by SHIBATA, [Methods of biochemical analysis, D. Glick (Ed.), Interscience, New York, Vol. VII, 77-109, (1959)].

Claims (8)

1) Lactic acid bacteria transformed by at least one heterologous gene coding for a protein involved in respiratory metabolism.  2) lactic acid bacteria according to claim 1, characterized in that said gene is chosen from: - genes coding for proteins of the Krebs cycle; - the genes coding for proteins of the heme biosynthesis pathway; - the genes coding for proteins of the biosynthetic pathway of cytochromes.  3) Lactic acid bacteria according to any one of claims 1 or 2, characterized in that it is chosen from bacteria of the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Propionibacterium, Bifidobacterium, or Enterococcus.  4) lactic acid bacteria according to any one of claims 1 to 3, characterized in that it is a strain of the species Lactococcus or Streptococcus transformed by at least one gene coding for a protein of the biosynthetic pathway of l 'heme.  5) A method for producing a lactic acid starter, characterized in that it comprises the culture of at least one strain of lactic acid bacteria according to any one of claims 1 to 4, under conditions allowing the induction of a respiratory metabolism in said strain 6) lactic acid starter comprising at least one strain of lactic acid bacteria transformed according to any one of claims 1 to 3.  7) Process for preparing a fermented product, characterized in that it comprises the inoculation of a medium to be fermented using a lactic leaven according to claim 6. <Desc / Clms Page number 14>  8) Use of a lactic acid starter according to claim 6 for the preparation of a fermented product.