BE1021472B1 - METHOD OF MODIFYING SPECIES OF SPOROLACTBACILLUS BY GENETIC ENGINEERING - Google Patents

Abstract

The invention relates to a method of modifying strains of Sporolactobacilli by genetic engineering, comprising introducing a cloned DNA into a replicon, inside cells of a strain of Sporolactobacilli. The invention further relates to genetically modified strains of Sporolactobacilli and methods using the genetically modified strains of Sporolactobacilli.

Description

METHOD OF MODIFYING A SPECIES OF SPOROLACTBACILLUS BY GENETIC ENGINEERING FIELD OF THE INVENTION

The present invention relates to a method of genetic modification of Sporolactobacillus species, genetically modified species of Sporolactobacilli and processes using genetically modified species of Sporolactobacilli.

STATE OF THE ART

Global demand for lactic acid and its derivatives is steadily increasing. This growing demand is justified by the use of lactic acid in a wider and wider range of applications in the fields of nutrition, cosmetics, pharmaceuticals, as well as in the chemical and plastic industries.

Lactic acid bacteria are known to produce lactic acid as the main metabolic product of fermentation of carbohydrates. Lactic acid bacteria consume mainly glucose or sucrose / sucrose as raw material for fermentation. However, the cost of these raw materials is constantly increasing and becomes a major disadvantage in the production of lactic acid by fermentation.

In addition, some lactic acid bacteria are not fully stereospecific in the production of lactic acid, which may also be a disadvantage, particularly in the production of polylactic acid (PLA), since it is known that Physical properties of PLA depend on its isomeric composition.

Accordingly, it would be desirable to have lactic acid bacteria which are capable of producing lactic acid or one of its stereoisomers from abundant and therefore inexpensive raw materials.

SUMMARY OF THE INVENTION

In their quest to solve the aforementioned problems, the present inventors have isolated a strain of Sporolactobacillus, LMG P-26831, which by analysis of its 16 RNA has been identified as being phylogenetically close to Sporolactobacillus vineae species (Chang et al. , 2008, International Journal of Systematics and Evolution Microbiology, 58, 2316-20). The present inventors have found, through extensive experiments and tests, a method for modifying the strain Sporolactobacillus vineae LMG P-26831 by introducing a DNA cloned into a replicon, inside the cells of the strain Sporolactobacillus vineae LMG P -26,831. In addition to these findings, the inventors observed that this species of

Sporolactobacilli can be genetically engineered, thereby satisfying one or more of the above-mentioned problems of the state of the art.

Accordingly, a first aspect of the present invention relates to a method of genetic engineering modification of Sporolactobacillus species, comprising the step of introducing DNA into cells of a Sporolactobacillus species. The methods applying the principles of the present invention advantageously make it possible to obtain genetically modified species of Sporolactobacilli.

For example, these methods have the advantage of providing genetically modified species of Sporolactobacillus capable of consuming easily available and inexpensive substrates such as lignocellulosic substrates or substrates derived from amylase. These substrates can be an economically very attractive alternative to the use of sucrose / sucrose for industrial fermentation processes.

In addition, the inventors have found that the methods applying the principles of the present invention provide genetically modified species of Sporolactobacillus capable of producing industrially interesting compounds such as lactic acid or one of its stereoisomers.

In fact, the present methods make it possible to provide Sporolactobacillus species capable not only of consuming inexpensive substrates such as, for example, xylose and substrates derived from amylase, but also of producing exclusively lactic acid L or lactic acid D. Therefore, the methods of the present invention advantageously provide genetically modified species of Sporolactobacilli having improved industrial application.

No genetic engineering protocol for Sporolactobacillus species has been described so far.

Although there are different methods for the genetic modification of bacterial strains, it is known that in order to perform a successful genetic modification of a strain of bacteria it is necessary to have at least three tools: a replicon, a marker selection and transformation protocol, and also that when these three tools at least are effective for one bacterial species, it can not automatically be assumed that these tools will be effective for another bacterial species. When a transformation method does not yield a transformant, this may be caused for example by an inappropriate replicon, and / or by an inappropriate selection marker and / or by the use of an inappropriate or suboptimal transformation protocol. and / or other factors. No replicon, selection marker, or specific genetic modification method has so far been described for any strain of Sporolactobacilli.

As mentioned above, the present inventors isolated a strain of Sporolactobacilli, which, by the analysis of its 16S RNA, was identified as being phylogenetically related to Sporolactobacillus vineae species. Therefore, a second aspect of this patent relates to a strain Sporolactobacillus vineae characterized by a sequence of 16S RNA as it appears in SEQ No. 1 or a strain of Sporolactobacillus having at least 96% identity with the sequence 16S RNA as set forth in SEQ ID No. 1. A strain of Sporolactobacillus vineae as described above was deposited under the accession number LMG P-26831 to the BCCM-LMG Collection of Bacteria on January 18th. 2012. The strain of Sporolactobacillus vineae LMG P-26831 is characterized by a 16S RNA sequence as shown in SEQ ID No. 1.

The present inventors have found, through intense experimentation, a method for genetically engineering the strain of Sporolactobacillus vineae LMG P-26831. Based on these findings, the inventors have observed that this species of Sporolactobacilli can be genetically engineered by the methods taught in the present invention. In some embodiments, the inventors have thus considered the methods taught herein, in the case where Sporolactobacillus species can be Sporolactobacillus vineae. Therefore, there is also disclosed herein a method of genetically engineered modification of Sporolactobacillus vineae species, including the introduction of DNA cloned into a replicon, into cells of a species of Sporolactobacillus vineae.

Further, in certain embodiments, the methods taught in the present invention in which the Sporolactobacillus species can be a strain of Sporolactobacillus vineae characterized by a 16S RNA sequence as given in SEQ ID No. 1 are provided. is also included in certain embodiments, the methods as taught in the present invention wherein the Sporolactobacillus species may be a strain of Sporolactobacillus having at least 95% identity with the 16S RNA sequence as it SEQ ID No. 1 In addition, in some embodiments, the Sporolactobacillus species may be a strain of Sporolactobacillus vineae deposited under accession number LMG-P26831 in the BCCM-LMG Collection of Bacteria.

Another aspect of the present invention relates to genetically modified species of Sporolactobacilli obtained by the methods taught herein. Therefore, the genetically modified species of Sporolactobacilli obtained by a method of genetic modification of Sporolactobacillus species, including the introduction of DNA cloned into a replicon, into the cells of a species of Sporolactobacillus are also disclosed. Sporolactobacilli, as for example the species of the vineae strain or the strain deposited as mentioned above. These genetically modified Sporolactobacillus species make it possible to produce compounds of commercial value in a low cost process.

Also included are certain embodiments for obtaining genetically modified species of Sporolactobacilli, according to the methods described herein, wherein said species of Sporolactobacilli as defined above are capable of producing at least one compound selected from the group of compounds lactic acid, lactic acid L, lactic acid D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S- malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate. Preferably, at least one compound is lactic acid L or lactic acid D. Therefore, in some preferred embodiments, genetically modified Sporolactobacillus species obtained by the methods taught herein, in which the species are also provided, are provided. Sporolactobacilli species are capable of producing lactic acid L or lactic acid D. These Sporolactobacillus species advantageously allow the production of industrially interesting compounds by fermentation from inexpensive substrates such as lignocellulosic biomass including arabinose and xylose or substrates from potato, corn or wheat. These species of Sporolactobacillus therefore reduce the costs of fermentation.

Specifically disclosed in certain embodiments of the present method are genetically modified species of Sporolactobacilli as hereinafter defined wherein the Sporolactobacillus species are a genetically modified strain of Sporolactobacillus vineae. Are more expressly disclosed in certain embodiments of the genetically modified species of Sporolactobacillus as defined below, wherein the Sporolactobacillus species are a genetically modified strain of Sporolactobacillus vineae characterized by a 16S RNA sequence as given in SEQ ID NO. In certain specific embodiments are also presented genetically modified species of Sporolactobacillus as defined below, in which the Sporolactobacillus species are a genetically modified strain of Sporolactobacillus having at least 95% identity with the RNA sequence. SEQ ID No. 1 is provided in certain specific embodiments. The genetically modified species of Sporolactobacilli as defined below, in which the Sporolactobacillus species are a genetically modified strain of Sporolactobacillus vineae deposited under accession number LMG P-26831 to the BCCM-LMG Bacteria Collection.

The present inventors have found through experimentation that Sporolactobacillus species can be considered as moderately thermophilic bacteria.

Therefore, in some embodiments of the present methods for the genetic engineering of Sporolactobacillus species, the replicon may be a heat-sensitive replicon. Such a heat-sensitive replicon can advantageously improve the efficiency of the transformation of Sporolactobacillus cells. Such a heat-sensitive replicon may be added to the Sporolactobacillus strain at a permissive temperature, and then growth conditions may be modified, for example, at a non-permissive temperature to render the replicon incapable of replicating itself. This, preferably in combination with antibiotic selection, may result in selection pressure in favor of events of chromosomal integration caused by homologous or non-homologous recombination. Therefore, a heat-sensitive replicon may allow integration of the transformed DNA into the chromosome.

In some embodiments, the replicon may be a plasmid. In some other embodiments, the heat-sensitive replicon may be plasmid pNW33N. These plasmids can improve the efficiency of transformation of Sporolactobacillus cells. For example, as illustrated in Example 1 in the example section, pNW33N gave more transformed colonies than other plasmids. In some embodiments, the heat-sensitive replicon is dependent on the RepB protein, a functional fragment, or a variant thereof for performing thermosensitive replication.

In some embodiments, the methods of the present invention may, after introduction of the DNA into cells of a Sporolactobacillus species, comprise steps of: culturing the cells on selective medium at a permissive temperature for replication to select transformed cells capable of growing on said selective medium at said permissive temperature, and then culturing said transformed cells at a non-permissive temperature for replication, to select transformed cells capable of growing at said non-permissive temperature permissive. Therefore, the invention describes a method for genetic modification of Sporolactobacillus species, comprising the steps of: (a) introducing cloned DNA into a heat-sensitive replicon into cells of a species of Sporolactobacilli, (b) culturing the cells on a selective medium at a permissive temperature for replication to select transformed cells capable of growing on said selective medium at said permissive temperature, and (c) culturing said transformed cells at a non-permissive temperature for the replication to select transformed cells capable of developing at said non-permissive temperature. These culture steps can allow the integration of DNA into the bacterial chromosome of the Sporolactobacillus species. Advantageously, such integration into the chromosome guarantees stable modification of the genetic material of the Sporolactobacillus species. For example, such integration into the chromosome may allow modification such as the introduction of a desired functionality that can be transmitted in the offspring. In addition, the methods taught here may at least partially circumvent the need for high transformation efficiency.

In some embodiments of the method of the present invention, non-permissive temperature culture for replication may allow DNA to be introduced into the Sporolactobacillus chromosome. Such chromosomal integration makes it possible to obtain genetically modified Sporolactobacillus species in which the modification can be transmitted in cell progeny. In preferred embodiments, non-permissive temperature culture for replication may allow DNA to be introduced into the Sporolactobacillus chromosome by homologous recombination.

Some embodiments further provide the methods as taught herein, wherein the DNA may encode one or more desired features. In some embodiments, the DNA may comprise one or more sequences encoding one or more polypeptides capable of producing at least one compound selected from the group consisting of: lactic acid, lactic acid L, lactic acid D, acetolactate, diacetyl, acetome , 2, 3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate. In preferred embodiments, the DNA may comprise one or more sequences coding for one or more polypeptides capable of producing lactic acid L or lactic acid D. These methods advantageously make it possible to obtain Sporolactobacillus species. having improved industrial application possibilities. For example, such a method can make it possible to obtain species of Sporolactobacillus capable of producing an industrially interesting compound, for example from inexpensive substrates from potatoes, corn or wheat.

As mentioned above, non-permissive temperature culture for replication may allow DNA to be introduced into the chromosome of Sporolactobacilli. In some embodiments, the introduction of DNA into the Sporolactobacillus chromosome may inactivate a sequence encoding an undesired functionality of the Sporolactobacillus chromosome. The undesirable functionality may be D-lactate dehydrogenase, L-lactate dehydrogenase, D-2-hydroxisocaproate dehydrogenase or L-2-hydroxisocaproate dehydrogenase. These methods have the advantage that Sporolactobacillus species can be obtained with improved industrial properties. For example, these methods can provide a strain of Sporolactobacillus that is an exclusive producer of lactic acid L or lactic acid D.

Another aspect relates to a process for the preparation of at least one compound selected from the group consisting of lactic acid, lactic acid L, lactic acid D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate, wherein a genetically modified Sporolactobacillus species such as defined is used. Preferably, at least one compound is lactic acid L or lactic acid D. The invention also describes a process for the preparation of lactic acid L or lactic acid D, in which a species of Sporolactobacillus genetically modified as defined here is used. These processes allow the production of industrially interesting compounds by fermentation for example from cheap raw materials. Therefore, these industrially valuable compounds can be produced naturally and economically.

The aspects above and below and the preferred embodiments of the invention are described in the following sections and in the appended claims. The object of the appended claims is hereby expressly incorporated in this specification.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a phylogenetic tree that compares 16S RNA sequences of different species of Sporolactobacillus. Sporolactobacillus strain LMG P-26831 is called Fidel.

Figure 2 presents a schematic representation of the metabolic conversion of D-xylose to pyruvate.

Figure 3 presents a schematic representation of the metabolic conversion of amylose or amylopectin to pyruvate.

Figure 4 presents a schematic representation of the metabolic conversion of pyruvate to lactic acid.

Figures 5a and 5b respectively show a schematic representation of the stereo-specific metabolic conversion of pyruvate respectively lactic acid L or lactic acid D.

Figure 6 shows an agarose gel illustrating (1) the EcoRI-restricted plasmid pNW33N prior to purification; SL: Smart Ladder: commercial molecular weight marker for easy determination of DNA fragment size (Eurogentec, MW-1700-10).

Figure 7 shows an agarose gel illustrating (1) the EcoRI-restricted plasmid pNW33N after purification; SL: Smart Ladder: commercial molecular weight marker for easy determination of DNA fragment size (Eurogentec, MW-1700-10).

Figure 8 shows an agarose gel, illustrating internal fragments of (1) L-LDH, (2) L-HicDH, (3) D-LDH62 and D-LDH65 (4) restricted by EcoRI.

Figure 9 provides a schematic overview of the strategy for obtaining genetically modified Sporolactobacillus species with clean and stable deletion of a gene.

Figure 10 shows a colored Petri dish with vapors of I2 DETAILED DESCRIPTION OF THE INVENTION

The terms "comprising", "includes" and "consisting of" in this document are synonymous with "including", "consist" or "containing", "contains" and are included or opened and do not exclude additional members , not mentioned, elements or steps of the method. The terms also include "consisting of" and "consisting essentially of". The enumeration of ranges of numerical values by their extreme points includes all the numbers and fractions subsumed in the respective ranges, as well as the extreme points cited.

The term "about" as used hereinafter, with reference to a measurable value such as a parameter, an amount, a time duration and the like, is intended to encompass variations in the specified value, in particular variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1% or less and even more preferably +/- 0.1% or less, insofar as such variations are in the invention disclosed herein. It must be understood that the value to which the term "about" refers is itself also precisely and preferably disclosed.

Whereas the term "one or more", as one or more members of a group of members, is self-evident, by means of examples, the term includes in particular a reference to one of these members, or to two or more such members, such as, for example, all> 3,> 4,> 5,> 6 or> 7 etc. of these members, and up to all these members.

All documents cited in this specification are incorporated by reference in their entirety.

Unless otherwise indicated, all terms used in the disclosure of the invention, including technical and scientific terms, have the meaning as commonly understood by one skilled in the art to which this invention belongs. For more information, the definitions of certain expressions may be included to better appreciate the teachings of the present invention.

As mentioned above, the present inventors have found, in accordance with the first aspect of the present invention, a method for genetic modification of Sporolactobacillus species including introduction of DNA cloned into a replicon, followed by inside cells of a Sporolactobacillus species. Therefore, the invention relates largely to such a method for the genetic engineering of Sporolactobacillus species, genetically modified Sporolactobacillus species and methods using genetically modified Sporolactobacillus species.

The term "replicon" in this document refers to an oligonucleotide sequence such as a DNA or RNA sequence that replicates from a single origin of replication. For example, the term replicon may include a plasmid, a transposon, a bacteriophage or a prokaryotic chromosome. Preferably, the replicon used in the methods described herein is a plasmid.

The term "DNA" of the syntax "having cloned DNA in a replicon" refers herein to "transforming DNA" and refers to a DNA molecule that is to be introduced into the cells of a strain of Sporolactobacilli. The DNA or transforming DNA may be introduced into the cells of a Sporolactobacillus strain as a single-stranded nucleic acid sequence or as a double-stranded nucleic acid sequence. The DNA or transforming DNA can be introduced into the cells of a Sporolactobacillus strain in a circular form or in a linear form. The DNA or transforming DNA can be introduced into the cells of a strain of Sporolactobacillus cloned in a replicon or not.

The replicon may include transforming DNA that may be homologous to the Sporolactobacillus chromosome and / or that may encode one or more desired functionalities. It is understood by those skilled in the art that the replicon may include one or more, such as two, three, four or more DNA sequences that may be homologous to the chromosome of Sporolactobacilli, and / or that the replicon may encode one or more desired functionalities, or that more than one replicon such as two, three, four or more replicons can be introduced into the cells of a Sporolactobacillus species for example sequentially, i.e. a replicon to the times, or more than one replicon, such as two, three, four, or more replicons at the same time.

In some embodiments, the step of introducing the cloned DNA into a replicon into the cells of a Sporolactobacillus species may be carried out by transformation or conjugation. The transformation can be carried out by electroporation or transformation of naturally competent cells. In certain preferred embodiments, the introduction of cloned DNA into a replicon into cells of a Sporolactobacillus species is accomplished by electroporation. Electroporation advantageously improves the efficiency of the transformation of Sporolactobacillus cells. Transformation of Sporolactobacilli by electroporation can be accomplished by passing a high voltage electrical discharge through a cell suspension comprising a replicon.

The term "transformation" generally refers to the process of genetic alteration of a cell resulting in the entry of exogenous DNA from its environment, within it, through the cell membrane and its incorporation. In the context of the present invention, the exogenous DNA includes nucleic acids that are introduced into Sporolactobacilli either as a single strand, or double stranded, in a linear form or in a circular form, including the Transforming DNA cloned into a replicon.

In methods of applying the principles of the present invention, the replicon may be a heat sensitive replicon. The term "heat sensitive" in this document refers to the ability of the replicon to replicate at a permissive temperature and the inability of the replicon to replicate at a non-permissive temperature. Generally, the origin of replication of the heat-sensitive replicon is functional at the permissive temperature, but not functional at the non-permissive temperature.

In some embodiments of the present methods, the replicon may be a plasmid. Examples of suitable but non-limiting plasmids may be PMSR10 (Rhee et al., 2007, Plasmid, 58 (1): 13-22); PMG36Cm (van de Guchte et al., 1989, Appl., Environ., Microbiol., 55 (1): 224-8); PGK13 (Broadbent and Kondo, 1991, Appl., Microbiol., 57 (2): 517-24); pHP13 (Haima et al., 1987, Mol Gen Genet., 209 (2): 335-42); PAM401 (Ainley and Key, 1990, Plant Molecular Biology, 14 (6): 949-967); pAMB22 (Zukowski and Miller, 1986, Gene, 46 (2-3): 247-55); pGKV2 (Kok et al., 1985, Appl., Environ., Microbiol., 50 (1): 94-101); PNZ8048 (Sanchez et al., 2008, Appl., Environ., Microbiol., 74 (8): 2471-9); pBS42 (Wells et al., 1983, Nucleic Acids Res., 11 (22): 7911-25); pHPS9 (Haima et al., 1990, Mol Gen Genet., 223 (2): 185-91); pNW33N (Kurt et al., 2005, Biotechnol Lett., 27 (15): 1117-21) and pG + host9 (Maguin et al., 1996, J. Bacteriol., 178 (3): 931-935; et al., 2003, Microbiology, 149: 1503-1511).

The heat-sensitive replicon may be any plasmid capable of replicating in a Sporolactobacillus strain at a permissive temperature and unable to replicate in a Sporolactobacillus strain at a non-permissive temperature. The heat-sensitive replicon may for example be pNW33N or pG + host9. Preferably, the heat-sensitive replicon is the plasmid pNW33N.

In certain embodiments, the methods of the present invention may, after the introduction of DNA into cells of a Sporolactobacillus species, comprise the steps of: culturing the cells on a selective medium at a permissive temperature for replication to select transformed cells capable of growing on said selective medium at said permissive temperature, and culturing said transformed cells at a non-permissive temperature for replication to select transformed cells capable of growing at said non-permissive temperature. A method for genetic modification of Sporolactobacillus species, comprising the following steps: (a) introduction of a cloned DNA into a thermosensitive replicon into cells of a Sporolactobacillus species, is thus disclosed here; culturing the cells on a selective medium at a permissive temperature for replication to select transformed cells capable of growing on said selective medium at said permissive temperature, and (c) culturing said transformed cells at a non-permissive temperature for replication so as to to select transformed cells capable of developing at said non-permissive temperature.

In some embodiments, culturing the cells on a selective medium and at a permissive temperature allows the selection of transformants, i.e., cells that have taken up the replicon comprising the transforming DNA. Preferably, the transformed cell colonies are isolated before culturing transformed cells at the non-permissive temperature. This isolation can advantageously make it possible to check the introduction of the transforming DNA.

In some embodiments, the transformed cells may be cultured at the non-permissive temperature to allow the selection of integrants, i.e., cells that have integrated the replicon or transforming DNA into their genetic material. . The step of culturing cells transformed at a non-permissive temperature for replication can be performed on a selective medium. Thus, some embodiments provide a method of genetically engineered modification of Sporolactobacilli species, comprising the steps of: (a) introducing a cloned DNA into a heat-sensitive replicon into cells of a Sporolactobacillus species, (b) ) culturing the cells on a selective medium at a permissive temperature for replication to select transformed cells capable of growing on said selective medium at said permissive temperature, and (c) culturing said transformed cells on a selective medium at a non-permissive temperature for replication to select transformed cells capable of growing at said non-permissive temperature. The Sporolactobacillus species may be a moderately thermophilic Sporolactobacillus species. Thus, certain embodiments provide a method for modifying moderately thermophilic Sporolactobacilli species by genetic engineering, comprising the steps of: (a) introducing cloned DNA into a heat-sensitive replicon into cells of a Sporolactobacillus species moderately thermophilic, (b) culturing the cells on a selective medium at a permissive temperature for replication to select transformed cells capable of growing on a selective medium at said permissive temperature, and (c) culturing said transformed cells at a specific temperature. non-permissive temperature for replication to select transformed cells capable of growing at said non-permissive temperature.

The permissive temperature for culturing Sporolactobacillus cells in step (b) can vary from 20 ° C to 40 ° C, preferably between 34 ° C and 37 ° C. The permissive temperature for culturing Sporolactobacillus cells in step (b) is preferably 37 ° C if the heat-sensitive replicon is pNW33N. The permissive temperature for culturing Sporolactobacillus cells in step (b) is preferably 34 ° C if the heat-sensitive replicon is pG + host9.

Sporolactobacilli cells can be cultured at a permissive temperature in step (b) for a period of about 4 days to about 14 days. For example, cells can be cultured in step (b) at a permissive temperature of 37 ° C for a period of about 4 days to about 8 days, typically about 5 days. For example, cells can be cultured in step (b) at a permissive temperature of 34 ° C for a period of about 7 days to about 14 days, usually about 10 days.

In one embodiment, after step (b) the Sporolactobacillus cells may be cultured one or more times as one, two, three, four, five, six, seven, eight, nine, ten or more times in a medium. liquid or solid culture. In a preferred embodiment, following step (b), the method thus comprises several cultures and subcultures of the cells as defined in point (b).

In one embodiment, after step (b) the method may comprise the collection of transformed cells. In another application, after step (b) the method may include harvesting transformed cells and verifying the introduction of the transformant DNA or replicon into Sporolactobacillus cells. The verification of the incorporation of the transforming DNA can be carried out by polymerase chain reaction (PCR).

The non-permissive temperature for culturing Sporolactobacillus cells in step (c) may vary from 30 ° C. to 50 ° C. The non-permissive temperature for culturing Sporolactobacillus cells in step (b) is preferably 42 ° C. if the heat-sensitive replicon is pNW33N. The non-permissive temperature for culturing Sporolactobacillus cells in step (b) is preferably 37 ° C if the heat-sensitive replicon is pG + host9.

In one embodiment, the cells may be cultured in step (c) until the replicon is integrated into the Sporolactobacillus chromosome preferentially by homologous recombination. The cells may be cultured in step (c) for a period of about 1 day to about 10 days (?). For example, the cells can be cultured in step (c) for a period of about 2 days to about 6 days, usually for 4 days. If not, the cells can be cultured in step (c) for about 2 to 8 generations, for example, for about 3 to 6 generations, usually for about 4 generations. The term "generation" refers to the time required for doubling the cell population and is measured as a doubling of the optical density to 600nm.

In one embodiment, after step (c) the cells may be cultured one or more times as one, two, three, four, five, six, seven, eight, nine, ten or more times in a culture medium. liquid or solid. In a preferred embodiment, after step (c), the method thus comprises cell subcultures as defined in (c).

In one embodiment, after step (c) the method may comprise the collection of the transformed cells. In another embodiment, after step (c) the method may include harvesting transformed cells and verifying the incorporation of the transforming DNA into the chromosomes of Sporolactobacillus cells. The verification of the incorporation of the transforming DNA can be carried out by polymerase chain reaction (PCR).

The methods applying the principles of the present invention may relate to culturing (eg, maintaining and / or multiplying) cells in the presence of culture media, such as the use of liquid or solid culture media. Such culture media can advantageously support maintenance (eg, survival, genotypic, phenotypic and functional stability) and cell multiplication.

A suitable culture medium may be M17 (Merck, Biokar), or GYP agar with SOSOZ (Chang et al., 2008, International Journal of Systematic and Evolutionary Microbiology, 58, 2316-20). The culture media may include one or more antibiotics to facilitate the selection of Sporolactobacillus cells that have the replicon among the majority of the untransformed cells. The term "selective medium" in this document refers to the culture medium as defined herein comprising one or more antibiotics.

The replicon may include an antibiotic resistance gene. The antibiotic resistance gene may be present on the transforming DNA or another fragment of the replicon. Alternatively, the antibiotic resistance gene may be present on another replicon. Such an antibiotic resistance gene makes it possible to select Sporolactobacillus cells which have taken the replicon from the majority of the non-transformed cells. In some embodiments of the present method, the replicon comprises a resistance gene for one or more of the following antibiotics: chloramphenicol, kanamycin, erythromycin and lincomycin. In preferred embodiments, the replicon comprises a chloramphenicol resistance gene. In addition, in the preferred embodiments, the replicon comprises an erythromycin resistance gene and lincomycin. The combination of erythromycin and lincomycin advantageously decreases the appearance of false positives, that is to say cells that develop on the selective medium, but have not returned to the replicon.

In some embodiments of the present method, the selective medium comprises the culture medium as defined herein and one or more antibiotics among chloramphenicol, kanamycin, erythromycin and lincomycin. In preferred embodiments, the selective medium is chloramphenicol. In addition, in preferred embodiments, the selective medium comprises erythromycin and lincomycin. The selective medium may include one or more antibiotics such as chloramphenicol, kanamycin, erythromycin and lincomycin at concentrations sufficient to kill or impair the growth of Sporolactobacillus cells that have not resumed replicon. In general, chloramphenicol may be included in the culture medium at a concentration ranging from 0.1 to 100 μg / ml, preferably from 5 to 25 μg / ml, for example, at about 20, 15, 12.5, 10, 7 , 5 or 5 μg / ml. As a general rule, kanamycin may be included in the culture medium at a concentration ranging from 0.1 to 100 μg / ml, for example between 1 and 50 μg / ml, for example, at approximately 45, 40, 35, 30 or 25 μg / ml, or about 20 μg / ml or less, for example, at about 15, 10 or 7.5 μg / ml. In addition, the erythromycin may be included in the culture medium at a concentration ranging from 0.1 to 100 μg / ml, for example ranging from 1 to 50 μg / ml, for example, at approximately 45, 40, 35, 30 or 25 μg / ml, or about 20 μg / ml or less, for example, at about 15, 12.5, 10, 7.5, 5 or 2.5 μg / ml. Lincomycin may be included in the culture medium at a concentration ranging from 0.1 to 100 μg / ml, for example from 5 to 50 μg / ml, for example, at approximately 45, 40, 35, 30 or 25 μg. / ml, or about 20 μg / ml or less, for example, at about 12.5, 15, 10, 7.5 or 5 μg / ml. as a general rule, erythromycin can be included in the culture medium at a concentration ranging from 0.1 to 100 μg / ml, for example between 1 and 50 μg / ml, for example at approximately 30, 20, 10 or 5 pg / ml and lincomycin can be included in the culture medium at a concentration ranging from 0.1 to 100 μg / ml, for example ranging from 5 to 50 μg / ml, for example, at approximately 20, 12.5 or 10 μg / ml. / ml. These values refer to the respective antibiotic concentrations after their introduction into the culture media.

Some embodiments provide the methods described herein wherein the Sporolactobacillus species may be a strain of Sporolactobacillus having at least 95% identity with the 16S RNA sequence as set forth in SEQ ID No. 1. For example, in methods applying the principles of the present invention, the Sporolactobacillus species may be a strain of Sporolactobacillus having at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% identity with the 16S RNA sequence as shown in SEQ ID No. 1.

The terms "16S RNA", "16S ribosomal RNA" or "16S rRNA" can be used interchangeably and refer to a component of the small subunit of prokaryotic ribosomes. Generally, 16S RNA sequences can be used in the construction of phylogenies. A phylogenetic tree comparing 16S RNA sequences of Sporolactobacillus species is shown in Figure 1.

In some embodiments, the heat-sensitive replicon may release from the RepB protein, a functional fragment, or a variant thereof to effect thermosensitive replication. In certain other embodiments, the heat-sensitive replicon may contain a DNA sequence encoding a polypeptide having a heat-sensitive replication functionality such as RepB. The origin of replication exemplified by RepB or the initiator protein RepB is a rolling circle replication initiation protein (or tounant). The exemplary RepB has been described as originating from the plasmid ρΑΜαΙΔΙ which is not capable of replicating independently in Enterococcus faecalis, but is capable of replicating in Bacillus subtilis (Francia and Clewell, 2002, J. Bacteriol., 184 ( 18), 5187-93, Perkins and Youngman, 1983, J. Bacteriol 155 (2): 607-15).

The exemplary RepB includes, but is not limited to, a RepB having the primary amino acid sequence as annotated under accession numbers Q7B6N7 or C7WLN5 in Uniprot / Swissprot (http://www.uniprot.org/uniprot/ ). The exemplary RepB protein sequence may also be as annotated according to NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NP 863351.1. The exemplary RepB protein sequence can also be as annotated under EMBL-Bank (http://www.ebi.ac.uk/ena/data/view/) EMBL-CDS AAN03827.1.

The reference hereinafter to any protein, polypeptide or peptide as RepB may also include fragments thereof. The term "fragment" of protein, polypeptide or peptide generally refers to an N-terminal and / or C-terminal truncated form of said protein, polypeptide or peptide. Without limitation, a nucleic acid, protein, polypeptide or peptide fragment may have at least 5%, or at least about 10%, for example,> 20%,> 30% or> 40%, as preferentially> 50%, for example,> 60%,> 70% or> 80%, or even more preferentially> 90% or> 95% of the nucleotide sequence of said nucleic acid or of said amino acid sequence of this protein, polypeptide or peptide.

The reference hereinafter to any protein, polypeptide or peptide as RepB may also encompass its variants. The term "variant" of nucleic acids, proteins, polypeptide or peptide refers to the nucleic acid sequence, polypeptides, proteins or peptides (i.e., the nucleotide sequence or amino acid sequence, respectively) which is substantially identical (i.e., largely but not totally identical) to the sequence of said defined nucleic acids, proteins or polypeptide, for example, having at least about 80% identical or at least about 85% identical, for example, preferably at least 90% identical, preferably at 91%, 92% identical, more preferably at least about 93% identical, for example at least 94% identical, even more preferably at least about 95% identical, for example, at least 96% identical, even more preferably at least about 97% identical, for example, at least identical to 98% and more preferably at least 99% identical. Preferably, a variant can display these degrees of identity to the defined nucleic acids, proteins, polypeptides or peptides, when the entire sequence of the defined nucleic acids, proteins, polypeptides or peptides is analyzed by sequence alignment (e.g., identity overall sequence). Also included among the fragments and variants of said nucleic acids, proteins, polypeptides or peptides, the fusion products of said nucleic acids, proteins, polypeptides or peptides with another nucleic acid, protein, polypeptide or peptide, generally unrelated to the so-called nucleic acid, protein, polypeptide or peptide. Sequence identity can be determined using appropriate algorithms to perform sequence alignments. For example, but not limited to, the algorithms that can be used include those that are based on the search tool. Basic Local Alignment Search Tool (BLAST) initially described by Altschul et al., 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 Sequence" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example using the published default parameters or other suitable parameters (such as, for example, for the BLASTN algorithm: cost to open a gap = 5, cost to extend a gap = 2 , the penalty for an incompatibility = -2, reward for a match = 1, deviation x_dropoff = 50, expected value = 10.0, word size = 28, or for the BLASTP algorithm: matrix = Blosum62, cost to open a breach = 11, cost of 'extend gap = 1, expected value = 10.0, word size = 3).

The degree of identity between two amino acid sequences corresponds to the percentage of identical amino acids between these two sequences. The degree of identity is determined using the BLAST algorithm which is described in Altschul, et al., 1990 (J. Mol Biol 215: 403-10). The BLAST analysis software is available for free access on the National Biotechnology Information Center Web site (National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/) . The parameters W, T and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The default BLAST program has the following parameters: "Word Length" (W) equal to 11, matrix BLOSUM62 (see Henikoff & Henikoff, Proc Natl Acad Sci USA 89: 10915 (1989)) alignment ( B) of 50, expectation (E) of 10, M = 5, N = 4 and a comparison on both strands.

An alternative polypeptide, protein or peptide may be a homolog (e.g., orthologue or paralogue) of said polypeptide, protein or peptide. In the present document, the term "homology" generally refers to a structural similarity between two macromolecules, particularly between two proteins or polypeptides, of identical or different taxa, wherein said similarity is due to shared ancestry. Where the present specification refers to or encompasses fragments or variants of polypeptides, proteins or peptides, this preference denotes variants and / or fragments that are "functional", i.e., that retain at least partially the biological activity or the intended functionality of the respective proteins, peptides or polypeptides. For example and without limitation, a functional fragment and / or a functional variant of RepB must retain at least part of the biological activity of RepB. Preferably, a functional fragment and / or a functional variant may retain at least about 20%, for example at least 30%, or at least about 40% or at least about 50%, for example, at least 60%, more preferably at least about 70%, for example, at least 80%, even more preferably at least about 85%, still more preferably at least about 90% and even more preferably at least about 95% or even about 100% or more of the contemplated biological activity or functionalities relative to the corresponding protein, polypeptide or peptide.

In some embodiments, non-permissive temperature culture for replication may allow DNA to be introduced into the Sporolactobacillus chromosome. DNA can be introduced into the Sporolactobacillus chromosome by non-homologous recombination or homologous recombination. The term "homologous recombination" generally refers to a type of genetic recombination in which nucleotide sequences are exchanged between two identical, similar or homologous DNA molecules. The term "non-homologous recombination" generally refers to a type of genetic recombination between DNA molecules that do not contain any sequence homology. The term non-homologous recombination may include ectopic recombination. Homologous recombination may be preferable because in a homologous recombination situation it is possible to know where the recombination will take place. Homologous recombination may also be preferred because recombination allows the removal of functionality, or the addition and deletion of functionality at the same time.

In some embodiments, where homologous recombination is desired, the transforming DNA also includes a DNA sequence that is homologous to a target sequence present in the genome of the Sporolactobacillus strain that is to be genetically modified. Those skilled in the art will understand that it is not necessary to have 100% identity in order to obtain homologous recombination. A percentage identity between the nucleic acids of sequences such as DNA or RNA sequences of about 90% will be sufficient. For example, the nucleic acid sequences may be at least 91% identical, for example at least 92% identical, preferably at least about 93% identical, for example at least 94% identical, more preferably at least at least about 95%, for example at least 96% identical, more preferably at least about 97% identical, for example at least 98% identical and even more preferably at least 99% identical. In some embodiments, the transforming DNA is surrounded by two homologous sequences which are both of sufficient length to allow homologous recombination. This length must be at least approximately 200bp, for example, it may vary between 200bp and 1500Pb, preferably between 200bp and 1000bp.

In some embodiments, the DNA may include one or more genes or sequences encoding one or more polypeptides. The coding genes can be used with regulatory sequences that are functional in cells, for example their promoter sequences. The regulatory sequences may be sequences that are natively associated with the coding sequences, or may be homologous sequences thereof. The coding gene may be fused to any functional promoter or regulatory sequence in Sporolactobacillus species. Suitable promoter sequences may include the sequences present in the species of Sporolactobacilli, hybrid promoters from different native promoters of Sporolactobacillus species or artificial promoters.

In methods of applying the principles of the present invention, the DNA may encode one or more desired functionalities. The transforming DNA may comprise one or more sequences encoding one or more polypeptides capable of metabolically converting or degrading a substrate to glucose, fructose or pyruvate. For example, the transforming DNA may comprise one or more sequences encoding one or more polypeptides selected from the group consisting of glucoamylase, cellulase, fructosan degradases, hemicellulase, exoglucanases, D-cellodextranases, cellobiohydrolases, α-amylase, α-glucosidase, pullulanase, pullulanase helper, glucan 1,4-α-glucosidase, glucose kinase, sucrose phosphorylase, glucokinase, endoglucanase, β-glucosidase, cellobiose phosphorolase, endochitinase, chitodextrinase, endochitinase, exochitinase, N-acetylglucosaminidase, fructose-6P-transaminase, N -acetylglucoasmine deacetylase, β-1,3 (4) -endoglucanase, P1, 3-endoglucanase, pI, 6-glucosidase, P1, 3-exoglucanase, mannanase, mannosidase, pectate lyase, rhamnogalacturon lyase, rhamnogalacturon hydrolase, exopectate lyase, endoxylanase , xylosidase, α-galactosidase, α-glucoronidase, β-glucoronidase, arabinofuranosidase, arabitan endo 1,5-a arabinosidase, arabinogalactan endo 1,4-β-galactosidase, acetoxylan esterase, β-galactosidase, carboxyl esterase, endocellulase, exocellulases, processive endocellulases, cellobiohydorases, β-xylosidases, β-D-mannanases, β-D-mannosidases, endopolygalacturonases, exopolygalacturonases, rhamnogalacturonan hydrolases, pectin lyases, pectate lyases, rhamnogalacturonan lyases, polygalacturonases, exoplygalacturonases, rhamnogalacturonases, endoxylogalacturonan hydrolases, αD-xylosidases, arabinofuranosidases, arabinoxylan, arabinofuranohydrolases, endoarabinases, exoarabinases, inulinase, exoinulinase, endoinulinase, dextransucrase, sucrose: 1,6-α-D-glucan-6-α-glucosyltransferase, altemansucrase, sucrose: 1 , 6 (1,3) -α-D-glucan-6 (3) -α-D-glucosyltransferase, mutan- (sucrose: 1,3-α-D-glucan-3-α-D-glucosyltransferase), reuteransucrase, sucrose: 1.4 (6 ) -α-D-glucan-4 (6) -α-D-glucosyltransferase, destransucrase, inulosucrase, sucrose: 2,1-beta-D-fructan-1-β-D-fructosyltransferase, glucosyltransferase, fructosyltransferase, glycoside hydrolase, levansucrases. For example, the transforming DNA may comprise one or more sequences encoding one or more polypeptides selected from the group consisting of invertases, ilulinases, levansucrases and fructosane degradases. The transforming DNA may comprise one or more sequences encoding one or more polypeptides having the xylose degradation capability. For example, the transforming DNA may comprise one or more sequences coding for xylose isomerase and / or xylulokinase, preferably xylose isomerases and / or xylulokinase, which are resistant to heat. A schematic representation of the metabolic conversion of D-xylose to pyruvate is illustrated in FIG. 2. The transforming DNA may comprise one or more sequences coding for one or more polypeptides having the ability to degrade starch or its derivatives partially. hydrolyzed. Starch consists of amylose, i.e., a D-glucose polymer with alpha-1,4 bonds and amylopectin, i.e., a glucose polymer with alpha-1, 4 and alpha-1, 6 side chains. Complete starch degradation requires the concerted action of several enzymes: alpha-amylase (disruption of alpha-1 1, 4), pullulanase (breakdown of alpha-1, 6 bonds) and glucoamylase (low molecular weight maltodextrin exoglucosidase with maltase activity). Therefore, the transforming DNA may comprise one or more sequences encoding one or more polypeptides selected from the group consisting of glucoamylase, cellulase and pululanase. For example, the transforming DNA may comprise one or more sequences encoding one or more glucoamylase-type hydrolytic enzymes, preferably a heat-resistant glucoamylase-type hydrolytic enzymes. For example, the transforming DNA may comprise a sequence coding for the glucoamylase of Thermoanaerobacterium thermosaccharolyticum DSM571 / LMG2811 (Ganghofner et al., 1998, Biosci Biotechnol Biochem., 62 (2): 302-308, Ducki et al. 1998, J. Gen. Appl Microbiol., 44 (5): 327-335). This exoglucosidase has a maximum activity on maltoheptaose (100%), but also degrades the following substrates: total starch (79%), amylose (88%), pullulan (5%), maltotetraose (85%), maltotriose (20% ), isomaltose (2%) and maltose (8%). A schematic representation of the metabolic conversion of amylose or amylopectin to pyruvate is illustrated in FIG. 3. The transforming DNA may comprise one or more sequences coding for one or more polypeptides capable of producing at least one compound from glucose, fructose or pyruvate.

The "polypeptide capable of producing a compound" syntax, as used hereinafter, may include polypeptides that can produce said compound or polypeptide involved in the production of said compound, i.e., producing a compound intermediate in the metabolic pathway for the production of this compound. The transforming DNA may comprise one or more sequences encoding one or more polypeptides capable of producing primary, secondary or tertiary metabolic compounds derived from glycolysis or the Krebs cycle (citric acid cycle). The transforming DNA may comprise one or more sequences encoding one or more polypeptides capable of producing at least one compound selected from the group consisting of lactic acid, lactic acid-L, lactic acid-D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, succinic acid , 3-hydroxypropanoic acid, glucaric acid, glycerol, 1,3-propanediol, xylitol, manitol, fumaric acid, aspartic acid, glutamic acid, itaconinc acid, 2,5-furan dicarboxylic acid, levulinic acid, 3-hydroxybutyrolactone, sorbitol, glucuronic acid, xylulose, α-ketoglutarate, glutamate, succinyl-CoA, malate, oxalic acid, acrylic acid, 3-hydroxypropanoic acid, butyric acid, malic acid, adipic acid, ascorbic acid, glutaric acid, gluconic acid, 1,4-diaminobutane , succindiamide, 1,4-butanediol, n-butanol, isobutanol, succinonitrile, dimethylsuccinate, N-methylpyrrolidone, γ-butyrolactone, 2-pyrrolidone, tertrahydorfuran, glucose-6-phosphate, phosphoenolpyruvate, pyruvate, menaquinol, ubiquinol, 1 , 4-dicarboxylic acid, acetyl phosphate, acetyl-CoA, cis-aconitate, acetaldehyde, 2-oxaloglutarate, poly lactic acid (Poly Lactic Acid = PLA), lactyl-CoA, inulin, inulotriose, inulotetraose, inulopentaose, bioethanol, inulooligosaccharides , Histidine, Isoleucine, Leucine, Lysine, Methionine,

Phenylalanine, Threonine, Tryptophan, Valine, Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamine, Glycine, Omithine, Proline, Selenocysteine, Serine, Taurine, Tyrosine, Glucose, Fructose and Pyruvate.

For example, the transforming DNA may comprise one or more sequences encoding one or more polypeptides capable of producing at least one compound selected from the group consisting of lactic acid, lactic acid-L, lactic acid-D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate. Preferably, the transforming DNA may comprise one or more sequences coding for one or more polypeptides capable of producing lactic acid-L or lactic acid-D. A schematic representation of the metabolic conversion of pyruvate to lactic acid is shown in Figure 4.

In some embodiments of the present method, the transforming DNA may comprise one or more D-lactate dehydrogenase 62 (D-LDH62) sequences of the strain Sporolactobacillus LMG P-26831 given in SEQ No. 2 and D-lactate dehydrogenase. 65 (D-LDH65) of Sporolactobacillus LMG P-26831 strain as shown in SEQ ID NO: 4 (?). The numbers in the names D-LDH62 or D-LDH65 denote the contig on which these genes are found. In some embodiments, the transforming DNA may comprise one or more L-lactate dehydrogenase (LDH-L) sequences of the strain Sporolactobacillus LMG P-26831 as shown in SEQ ID No. 6 and L-2- hydroxisocaproate dehydrogenase (L-HicDH) of the strain Sporolactobacillus LMG P-26831 as it appears in SEQ No. 8.

In some embodiments of the present methods, the introduction of DNA into the Sporolactobacillus chromosome may inactivate a sequence encoding an undesirable functionality of the Sporolactobacillus chromosome. Inactivation of the coding sequence for undesirable functionality may result from disruption of said sequence by the transforming DNA. On the other hand, the inactivation of the coding sequence for undesirable functionality may result from the replacement of said sequence by the transforming DNA.

In some embodiments, the undesirable functionality may be D-lactate dehydrogenase (LDH-D). In other embodiments, the undesirable functionality may be L-lactate dehydrogenase (LDH-L), D-2-hydroxisocaproate dehydrogenase (D-HicDH) or L-2-hydroxisocaproate dehydrogenase (L-HicDH). Advantageously, these methods make it possible to obtain species of Sporolactobacillus capable of producing exclusively lactic acid-L or exclusively lactic acid-D.

A schematic representation of the stereo-specific metabolic conversion of pyruvate to lactic acid-L or lactic acid-D by Sporolactobacillus LMG strain P-26831 is shown in Figures 5A and 5B respectively.

In some embodiments, the undesirable functionality may be D-lactate dehydrogenase 62 (D-LDH62) of Sporolactobacillus LMG strain P-26831 as set forth in SEQ ID No. 2. In certain other embodiments, the functionality of undesirable may be D-lactate dehydrogenase 65 (D-LDH65) of the strain Sporolactobacillus LMG P-26831 as shown in SEQ ID No. 4. In some embodiments, the undesirable functionality may be L-lactate dehydrogenase ( LDH-L) of the strain Sporolactobacillus LMG P-26831 as shown in SEQ ID No. 6. In certain other embodiments, the undesirable functionality may be L-2-hydroxisocaproate dehydrogenase (L-HicDH) of the Sporolactobacillus strain. LMG P-26831 as it appears in SEQ ID No. 8.

In some embodiments of the present methods, the introduction of DNA into the Sporolactobacillus chromosome may inactivate the D-LDH62 coding sequence of Sporolactobacillus LMG P-26831 strain as shown in SEQ ID No. 2 and US Pat. introduction of the same or another DNA into the chromosome of Sporolactobacillus can inactivate the coding sequence for D-LDH65 of Sporolactobacillus LMG strain P-26831 as it appears in SEQ # 4. In some other modes of In carrying out the present methods, the introduction of DNA into the Sporolactobacillus chromosome can inactivate the L-LDH coding sequence of the strain Sporolactobacillus LMG P-26831 as shown in SEQ ID No. 6 and the introduction of same or another DNA sequence in the chromosome of Sporolactobacillus can inactivate the coding sequence for L-HicDH of Sporolactobacillus strain LMG P-26831 as it appears in SEQ ID No. 8. These methods allow to obtain a strain of Sporolactobacillus LMG P-26831 capable of producing exclusively lactic acid-L or exclusively lactic acid-D.

In some embodiments of the present methods, the introduction of DNA into the Sporolactobacillus chromosome may inactivate a coding sequence for an undesirable chromosome functionality of these Sporolactobacilli and simultaneously the DNA may encode one or more desired functionalities. Advantageously, these methods make it possible to obtain species of Sporolactobacillus producing abundantly and exclusively lactic acid-L or lactic acid-D. Other types of genetic modification could also be considered to meet other industrial requirements. For example, phage attack of industrial bacteria such as Sporolactobacillus species is a major problem for the industry. Therefore, there is a need for methods that make industrial strains less susceptible to phage attack. Therefore, in some embodiments of the present methods, the transforming DNA may include one or more sequences capable of increasing the resistance of Sporolactobacillus species to bacteriophages. For example, the transforming DNA may comprise short, regularly spaced, clustered palindromic repeat (CRISPR) sequences, CRISPR-associated sequences (Cas genes), and genes encoding one or more enzymes having a ability to restrict or modify DNA.

Another aspect relates to a strain of Sporolactobacillus vineae characterized by a 16S RNA sequence as shown in SEQ ID No. 1. Also included in some embodiments is a strain of Sporolactobacillus having at least 96% identity with the 16s RNA sequence as shown in SEQ ID No. 1. For example, a Sporolactobacillus strain having at least 97%, at least 98%, at least 99%, at least 99.5, at least 99%, 9% identity with the 16S RNA sequence as it appears in SEQ ID No. 1.

Another aspect relates to a genetically modified species of Sporolactobacillus obtained by the methods described herein. Some embodiments provide genetically modified species of Sporolactobacilli obtained by the methods of the invention, wherein Sporolactobacillus species are capable of producing at least one compound selected from the group consisting of: lactic acid, lactic acid-L, acid lactic acid-D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, succinic acid, 3-hydroxypropanoic acid, glucaric acid, glycerol, 1,3-propanediol, xylitol, manitol, fumaric acid, aspartic acid, glutamic acid, itaconinc acid, 2,5-dicarboxylic acid iuran, levulinic acid, 3-hydroxybutyrolactone, sorbitol, glucuronic acid, xylulose, α-ketoglutarate, glutamate, succinyl-CoA, malate, oxalic acid, acrylic acid, 3-hydroxypropanoic acid, butyric acid, malic acid, adipic acid, ascorbic acid, glutaric acid, gluconic acid, 1,4-diaminobutane, succindiamide, 1,4-butanediol, n-butanol, isobutanol, succinonitrile, dimethylsuccinate, N-methylpyrrolidone, γ-butyrolactone, 2 pyrrolidone, tertrahydorfuran, glucose-6-phosphate, phosphoenolpyruvate, pyruvate, menaquinol, ubiquinol, 1,4-dicarboxylic acid, acetyl phosphate, acetyl-CoA, cis-aconitate, acetaldehyde, 2-oxaloglutarate, poly lactic acid (PLA) , lactyl-CoA, inulin, inulotriose, inulotetraose, inulopentaose, bioethanol, inulooligosaccharides, Histidine, Isoleucine, Leucine, Lysine, methionine, phenylalanine, threonine, tryptophan, Valine, Alanine, Arginine, Asparagine, aspartic acid, cysteine, glutamic acid, Glutamine , Glycine, Omithine, Praline, Selenocysteine, Serine, Taurine, Tyrosine, Glucose, Fructose and Pyruvate. In addition, some embodiments provide genetically modified species of Sporolactobacilli obtained by the methods taught herein, wherein Sporolactobacillus species are capable of producing at least one compound selected from the group consisting of: lactic acid, lactic acid, Lactic acid-D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2 -oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate. Some preferred embodiments provide genetically engineered species of

Sporolactobacilli obtained by the methods taught herein, wherein Sporolactobacillus species are capable of producing L-lactic acid or D-lactic acid.

Some embodiments provide genetically engineered species of

Sporolactobacilli obtained by the methods described in the invention, in which the Sporolactobacillus species are capable of producing one or more polypeptides selected from the following D-LDH62 of the strain Sporolactobacillus LMG P-26831 as it appears in SEQ No. 3 , D-LDH65 of the strain Sporolactobacillus LMG P-26831 as shown in SEQ ID No. 5, L-LDH of the strain Sporolactobacillus LMG P-26831 as it appears in SEQ ID No. 7 and L-HicDH of strain Sporolactobacillus LMG P-26831 as shown in SEQ ID No. 9.

Some embodiments provide genetically engineered species of

Sporolactobacilli obtained by the methods described herein, in which one or more sequences selected from the D-LDH62 coding sequence of the strain Sporolactobacillus LMG P-26831 as shown in SEQ ID No. 2 and the coding sequence for the D- LDH65 of strain strain Sporolactobacillus LMG P-26831 as shown in SEQ ID No. 4 were inactivated from Sporolactobacillus LMG strain P-26831 chromosome. Some more embodiments provide genetically modified species of Sporolactobacilli obtained according to the methods of the invention, in which one or more sequences selected from the sequence coding for L-LDH of the strain Sporolactobacillus LMG P-26831 as it appears. in SEQ # 6 and the coding sequence for L-HicDH of Sporolactobacillus LMG strain P-26831 as shown in SEQ ID No. 8 were inactivated from the chromosome of Sporolactobacillus LMG P-26831 strain. This genetically modified strain of Sporolactobacillus LMG P-26831 may be able to produce exclusively lactic acid-L or exclusively lactic acid-D.

Some embodiments provide genetically modified species of Sporolactobacilli obtained by the methods described herein, wherein the Sporolactobacillus species are capable of producing xylose isomerase and / or xylulokinase, preferably heat resistant xylose isomerase and / or xylulokinase. . Advantageously, these heat-resistant xylose isomerase and / or xylulokinase are functional at a pH compatible with Sporolactobacillus species.

Some embodiments provide genetically engineered species of

Sporolactobacilli obtained by the methods of the invention, in which the Sporolactobacillus species are able to produce hydrolytic enzymes of the glucoamylase type, preferentially hydrolytic enzymes of the glucoamylase type that are resistant to heat. Some embodiments provide genetically engineered species of

Sporolactobacilli obtained by the methods described herein, in which the species of

Sporolactobacilli are able to produce the glucoamylase of Thermoanaerobacterium thermosaccharolyticum DSM571 / LMG2811. Advantageously, these hydrolytic enzymes of the glucoamylase type, such as the glucoamylase of Thermoanaerobacterium thermosaccharolyticum DSM571 / LMG2811, are functional at a pH compatible with the Sporolactobacillus species.

Genetically modified species of Sporolactobacilli, as defined herein, are further disclosed in certain embodiments, wherein the Sporolactobacillus species are a genetically modified strain of Sporolactobacillus having at least 95% identity with the 16S RNA sequence as that it appears in SEQ ID No. 1. For example, also described are genetically modified species of Sporolactobacillus as defined herein, in which the Sporolactobacillus species are a genetically modified strain of Sporolactobacillus having at least 96%, at least 97%, at least 98%, at least 99% at least 99.5, at least 99.9% identity with the 16S RNA sequence as shown in SEQ ID NO: 1.

Another aspect relates to a process for preparing at least one compound selected from the group consisting of: lactic acid, lactic acid-L, lactic acid-D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2 oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate, wherein a genetically modified Sporolactobacillus species as defined herein is used. The preparation of a compound as defined in the invention, using a genetically modified Sporolactobacillus species as described, may be carried out by the fermentation of raw material.

The process can be carried out under conditions that allow the production of the desired compound. For example, the process can be performed in batch or in continuous mode. In some embodiments, the process may be conducted at temperatures above 40 ° C. In certain other embodiments, the process may be carried out at a pH below 6.0. In certain preferred embodiments, the process may be carried out at temperatures above 40 ° C and at pH below 6.0.

These methods advantageously make it possible to cultivate Sporolactobacillus species as defined in the invention at relatively high temperatures, which reduce the risk of contamination and therefore the cost of sterilization. In addition, these methods make it possible to cultivate Sporolactobacillus species as defined herein at a relatively low pH which allows the production of acidic compounds without the use of large amounts of buffering agents. These methods using Sporolactobacillus species as defined in the invention can ensure high yields of commercially valuable compounds and low carbon losses.

Genetically modified Sporolactobacillus species as defined herein can allow vigorous growth in relatively simple culture media helping to keep raw materials at their lowest level and therefore cost-effective. Also, genetically modified Sporolactobacillus species as defined in the invention can further help to avoid the fermentation process under strictly anaerobic or aerobic conditions, which are known for their high costs due to the need for a dispersion of gas.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that, in light of the foregoing description, several alternatives, modifications, and variations will occur to those skilled in the art. Accordingly, it is intended to include all such alternatives, modifications and variations thereof in the spirit and scope of the appended claims.

The above aspects and embodiments are supported by the following non-limiting examples.

EXAMPLES

The above aspects and embodiments are supported by the following non-limiting examples.

Example 1: Plasmids for the modification of Sporolactobacillus strains

Several plasmids were tested for electroporation transformation of the strain of Sporolactobacillus vineae LMG P-26831 (Table 1).

The following plasmids gave no colony of transformant cells: pMSRIO (Rhee et al., 2007, Plasmid, 58 (1): 13-22), pGIZ906-pldh (Lorquet et al., 2004, J. Bacteriol. 186 (12): 3749-3759).

The following plasmids gave some colonies: Kana-pMSRIO (= pMSRIO see reference above, to which was added a kanamycin resistance gene), pMG36Cm, pGK13, pHP13, pAM401, pAMB22, pGKV2, pNZ8048, pBS42 and pHPS9 .

Plasmid pNW33N and plasmid pNW33N comprising the erythromycin resistance gene gave the most transformant colonies of Sporolactobacillus vineae LMG strain P-26831 compared with the plasmids mentioned above.

Table 1: Plasmids used for the transformation of the strain Sporolactobacillus vineae LMG P-26831

Example 2: Preparation of a plasmid comprising a heat-sensitive replicon and an internal fragment of L-lactate dehydrogenase (LDH-L), L-2-hydroxyisocaproate dehydrogenase (L-HicDH), D-lactate dehydrogenase 62 (D-LDH62) D-lactate dehydrogenase 65 (D-LDI65).

Four plasmids were constructed for genetic modification of Sporolactobacillus strains: pNW33N comprising an internal fragment of L-LDH; pNW33N comprising an internal fragment of L-HicDH; pNW33N comprising an internal fragment of D-LDH62 and pNW33N comprising an internal fragment of D-LDH65.

Construction of Plasmids The amplification of an internal fragment of L-lactate dehydrogenase (LDH-L) of the strain Sporolactobacillus vineae LMG P-26831 was carried out according to a protocol described by Platteeuw et al. (1995, Appl.Environ.Microbiol., 61: 3967-3971). The L. lactis L-LDH sequence was compared to the L-LDH sequence of Sporolactobacillus vineae LMG strain P-26831 using Clone Manager and Clustal software. The primers below were chosen as shown in Table 2.

Table 2: Primer Sequences for L-LDH of Sporolactobacillus vineae strain LMG P-26831 (Floating tail with BamHI restriction site (underlined))

The amplification of an internal fragment of L-2-hydroxyisocaproate dehydrogenase (L-HicDH) of the strain Sporolactobacillus vineae LMG P-26831 is based on the similarity of the structures between L-HicDH and L-LDH, and the sequences have been aligned as described above. The primers below were chosen as shown in Table 3.

Table 3: Primer sequences for L-HicDH of Sporolactobacillus vineae strain LMG P-26831 (floating tail with BamHI restriction site (underlined)).

The amplification of an internal fragment of D-lactate dehydrogenase D-LDH62 and D-LDH65 (with reference to the contigs on which they are found) of the strain Sporolactobacillus vineae LMG P-26831 was carried out according to a protocol described by Bernard and al. (1994, J. Biochem., 224, 439-446). The primers below were chosen as shown in Table 4.

Table 4: Primer sequences for D-LDH62 and D-LDH65 of Sporolactobacillus vineae strain LMG P-26831 (floating tail with BamHI restriction site (underlined)).

PCR amplification

The PCR was carried out with the Phusion enzyme (high processivity) in the following reaction mixture: 1 μl of chromosomal DNA of the strain Sporolactobacillus vineae LMG P-26831 (concentration: 10 ng / μl), 1 μl dNTP (20 mM) ; 0.5 μΐ primer 1 (100 μΜ); 0.5 μΐ primer 2 (100 μΜ); 10 μΐ HF Phusion buffer (5x); 0.5 μΐ Phusion enzyme (Finnzymes, F - 530 L) and water up to 50 μΐ.

For PCRs carried out from colonies, that is to say, without extraction of the chromosomal DNA, 1 colony was recovered with a toothpick and deposited in 50 μΐ of water. The conditions of the PCR were as follows: 5 μΐ of a colony dilluée in water, 1 μΐ dNTP (20 mM); 5 μΐ primer 1 (100 μΜ); 0.5 μΐ primer 2 (100 μΜ); 10 μΐ buffer (x 5); 0.5 μΐ Phusion enzyme and water up to 50 μΐ. The device was the PERKIN-ELMER Amp ® PCR System. The program had a denaturation of 5 minutes at 98 ° C, 25 cycles of 30 seconds at 96 ° C, 30 seconds at x ° C (hybridization temperature chosen according to Tm) and 30 seconds at 72 ° C and an ultimate elongation from 5 minutes to 72 ° C.

Amplification of inserts Since there must be a large amount of insert in order to cleave into pNW33N, the PCR fragments obtained were cloned for amplification in pGEMTeasy (Promega # A1360), according to the supplier's recommendations.

Vector Restrictions pNW33N pNW33N is described as a shuttle vector between Escherichia coli, Bacillus subtilis and Geobacillus stearothermophilus and comprises a multiple cloning site derived from plasmid pUC19; an origin of functional replication in E. coli from plasmid pUC19; a "rolling circle" origin of replication named RepB and originating from the plasmid pTHT15 of Geobacillus stearothermophilus; and a chloramphenicol resistance gene which is derived from a plasmid of Staphylococcus aureus and which is referred to as chloramphenicol acetyltransferase (Cat).

A restriction with EcoRI of the vector pNW33N was carried out for 2 hours at 37 ° C. and under the following conditions: 1.5 μΐ of pNW33N, 2 μΐ 10 x BSA, 1.5 μΐ EcoRI, 2 μl of buffer and 13 μl of 'water. Restricted pNW33N DNA was gel deposited for recovery in a final volume of 50 μΐ (Figure 6, lane 1) (QIAquick Qiagen Kit). After purification (QIAquick PPK Qiagen kit), an aliquot was deposited on a gel for quantification (Figure 7, lane 1).

Restriction of PCR fragments of the internal fragments of the L-LDH, L-HicDH, D-LDH62 and D-LDH65 genes of LMG strain P-26831

The primers used for PCR amplification have a BamHI site at their 5 'end which could be used for cloning. However, it is the EcoRI restriction sites of pGEMTeasy that have been used for cloning.

The pGEMTeasy comprising the insert was restricted by EcoRI for 2 hours at 37 ° C. and under the following conditions: 1.5 μΐ of pGEMTeasy, 2 μΐ 10 x BSA, 1.5 μΐ EcoRI, 2 μl of buffer and 13 μl of water, then the inserts were recovered on gel (Qiagen Qiaquick gel extraction kit Ref 28706). An aliquot was deposited on a gel for quantification. Lanes 1, 2, 3 and 4 of Figure 8 correspond respectively to the restricted internal fragments of LDH-L, L-HicDH, D-LDH62 and D-LDH65.

Ligation of the vector pNW33N with the internal fragments of the L-LDH, D-LDH62 and D LDH65 genes of the LMG strain P-26831

The ligation of the restricted pNW33N vector with the restricted internal fragments of the LDH-L, L-Hic-DH, D-LDH62 and D-LDH65 genes of the LMG P-26831 strain was carried out at 16 ° C for 16 h and in a final volume of 12.2 μl: 5 μl of vector, 5 μl insert, 1.2 μl of ligase buffer and 1 μl ligase (New England Biolabs, M0202).

Transformation

An aliquot of 2 μl of each of the 4 ligations described above was transformed by a standard protocol into a 50 μl aliquot of electrocompetent Escherichia coli cells (E. coli strain GM2173, (dam-dcm-)). Selection was on LB agar plates containing 20 μg / ml chloramphenicol (Cm), corresponding to the resistance marker of pNW33N.

The E. coli obtained were cultured, and their plasmid was extracted by the MiniPrep method (PLN350-1KT, GenElute mini prep Kit, Sigma-Aldrich). The plasmids were validated by diagnostic restrictions and sequencing. Noon DNA preparations were made (740573.100, DNA purification plasmid Nucleobond AX 100, Macherey-Nagel).

Dialysis of plasmids constructed before electroporation

In order to remove the salts and reduce the risk of having an electric arc during electroporation, 50 μΐ of noon preparation was dialyzed for 30 minutes on a membrane (VSWP02500, MF-filtering membrane 0.025 μm) against 30 ml of sterile deionized water.

The 4 plasmids constructed were: pNW33N comprising an internal fragment of L-LDH; pNW33N comprising an internal fragment of L-HicDH; pNW33N comprising an internal fragment of D-LDH62 and pNW33N comprising an internal fragment of D-LDH65.

Example 3 Genetic Modification of the Strain Sporolactobacillus vineae LMG P-26831

Preparation of equipment

Ml7 culture medium: 42.5 g of M17 (Merck Ref 1.15029.0500) was added to 800 ml of water. The pH was adjusted to 5.5 by the addition of HCl. The volume was then increased to 1 L and subdivided into as many bottles as needed, then filtered on 0.2pm filter. Just before use, sterile glucose was added at a final concentration of 2% (= 10 ml of the 40% glucose stock in 200 ml M17). СаСЬ (1 M): 7.351 g was added to 50 ml of water, the mixture was then filtered through a 0.2 μm filter.

MgCl (1 M): 10.165 g was added to 50 ml of water; the mixture was then filtered through a 0.2 μm filter. PEG1000 (30%): 60 g of PEG1000 was brought to 200 ml with distilled water. The mixture is filtered and then stored at 4 ° C.

Regeneration medium: 2.1 g of M17 (Merck Ref 1.15029.0500) was added to 8.56 g of sucrose; 0.5 g of glucose; 1 ml of MgCl 2 (1 M) and 50 μl of CaCl 2 (1 M) in 40 ml of water. The pH of the mixture was adjusted to 5.5 and the solution was then brought to 50 ml with distilled water. This solution was then aliquoted with 1.5 ml into the required number of 2 ml Eppendorfs and preincubated at 37 ° C.

Competent cells of the strain Sporolactobacillus vineae LMG P-26831

A preculture of the strain Sporolactobacillus vineae LMG P-26831 was carried out by inoculation in culture medium containing CaCO 3 and cultivated overnight at 42 ° C.

The centrifuge was cooled to 4 ° C. The optical density of the preculture was measured at a wavelength of 600 nm (OD600) by solubilizing the excess of CaCO 3 (10 × dilution: 100 μl of culture in the culture medium + 800 μl of the physiological fluid ( 9 g / 1 of NaCl in water) + 100 μl of HCl).

Depending on the optical density of the preculture, the required inoculum volume can be calculated for the culture to have an OD between 0.15 and 0.17. This amount of pre-culture was inoculated into a 500 ml pre-heated flask of M17 at pH 5.5 containing 2% glucose. The culture was carried out at 37 ° C.

When the culture reached an OD600 of approximately 0.25-0.35 (doubling), the cells were centrifuged for 5 min at 4000 rpm and 40 C. The cell pellet was washed three times with water with 1 volume of the initial culture, three times with water with 1/2 x volume of the initial culture (collect the tubes) and three times with PEG1000 (30%) with 1/4 x volume of the culture initial.

The cell pellet was then gently resuspended at 4 ° C. The cells were resuspended in 1-1.5 ml (for 400 ml of initial culture medium) of cold PEG1000 (30%). About 5.109-1010 CFU / ml were obtained. These competent cells can be used immediately or stored at -80 ° C for at least one year.

Electro-transformation 75μ1 of electrocompetent cells were placed in a 1 mm electroporation cuvette kept on ice. 7.5 μl of the plasmid was added. Each of the 4 plasmids constructed (pNW33N comprising an internal fragment of L-LDH, pNW33N comprising an internal fragment of L-HicDH, pNW33N comprising an internal fragment of D-LDH62 and pNW33N comprising a fragment of D-LDH65) was used separately. The controls were: no DNA + pulse; DNA + no impulse; empty plasmid + pulse. Electroporation was performed under the following conditions: 1.5KV (fixed value), 25pF and 400Ω (time constant = 7 or 8). Immediately after electroporation, the cells were transferred to 1.5 ml of preheated regeneration medium at 37 ° C and incubated at 37 ° C for 3 hours.

The cells were centrifuged at room temperature (2000 rpm / 10 min), the regeneration medium was removed, the pellet was resuspended in the medium remained at the bottom of the tube (about 100 μΐ) and spread on a box of Petri M17 containing chloramphenicol at 10 μg / ml.

The dishes were incubated in anaerobic jars at 37 ° C for 5 days or more. Selection and validation of transformants

When the colonies appeared on the plates, they were subcultured several times on a box containing chloramphenicol 10 μg / ml (CmlO). Colony PCR was then performed with primers that hybridize within the plasmid (eg on the chloramphenicol resistance conferring gene) to verify that the plasmid was indeed inside the cells. This was a first level of validation.

The second level of validation consisted of a liquid culture of the transformants in M17 pH5.5 medium with 2% glucose and CmlO, followed by the extraction of plasmid DNA (mini prep) and a PCR (identical to that of first level of validation).

The third level of validation was the use of the plasmid DNA extracted at the second level of validation to perform a return transformation in E. coli, followed by a DNA extraction (mini prep). This DNA was subjected to a diagnostic restriction to verify that it was in fact the expected plasmid.

Obtaining integrants

Liquid cultures of the transformants were moved from 37 ° C to 42 ° C (again in the presence of the antibiotic CmlO). At 42 ° C., the RepB origin of replication of the plasmid pNW33N is no longer functional, and the only way that the plasmids can be maintained in the cell (and thus the only way for the cell to preserve the resistance marker at the same time). antibiotic) is that the plasmid integrates into the bacterial genome: preferably by homologous recombination at the site of the "internal" fragment of the target gene, or more rarely ectopically. The integration of the plasmid into the target genes has the consequence of disabling it or in other words, making a Knock Out (KO) thereof. After several culture cycles at 42 ° C, the liquid culture was plated on a petri dish to obtain isolated colonies.

Validation of integrants

The isolated colonies were subjected to colony PCR to verify, by amplifying the edge fragments that the plasmid was indeed integrated within the bacterial genome and at the expected site. The resulting mutants are termed non-clean and unstable because they contain exogenous sequences and if antibiotic selection is lifted, they can return to the wild-type genotype by homologous recombination and thereby lose the plasmid.

In the exemplified method, 8.times.10.sup.6 cells (divided into about 25 aliquots, i.e., about 3.2.109 cells per aliquot) were engaged. The viability after all competent cell preparation and electroporation steps was 3.107 (i.e., 1.2 x 106 cells per aliquot). The number of transformants obtained is of the order of 1 per aliquot, that is to say a conversion rate of 1.10'6.

This transformation rate is low; however, this method made it possible to obtain transformants in a reproducible manner. In addition, these transformants were validated by the return transformations in E. coli.

Therefore, the exemplary method made it possible to transform by electroporation different plasmids comprising an internal fragment of the different target genes of the strain Sporolactobacillus vineae LMG P-26831, thus obtaining knock outs by homologous recombination of each of these target genes. Therefore, this method was able to genetically modify the strain of Sporolactobacillus vineae LMG P-26831.

EXAMPLE 4 Genetic Modification of the Sporolactobacillus vineae LMG P-26831 Strain by Clean and Stable Knockout of an Undesirable Functionality of the Chromosome of

Sporolactobacillus

FIG. 9 is a schematic overview of the strategy implemented to obtain genetically modified Sporolactobacillus strains with a clean and stable deletion of a target gene.

In FIG. 9a, a plasmid containing the "Amont-Cat-Aval" sequence, with "Upstream" being the nucleotide sequence corresponding to the 1500 bp upstream of the start codon of the target gene; with "Cat" being the chloramphenicol resistance gene under the control of the P32 promoter; and with "Aval" being the nucleotide sequence corresponding to 1500 bp downstream of the stop codon of the target gene, is introduced into a Sporolactobacillus cell by electroporation. Homologous recombination, for example in the upstream region, allows the Amont-Cat-Aval fragment to be integrated into the bacterial genome (Figure 9 (b)). If a second homologous recombination takes place in the same fragment, it allows to return to the starting situation (Figure 9 (a)), or if it takes place in the other fragment (here in the downstream region), this can give rise to a situation in which the target gene is on the plasmid and the cat gene is in the bacterial genome (Figure 9 (c)). The selection of erythromycin-sensitive and chloramphenicol-resistant bacteria isolates mutants in which the target gene has been replaced by the cat gene and the target gene has been lost with the plasmid (Figure 9 (d)).

In Figure 9 (e), a plasmid containing the "Amont-Aval" sequence is introduced into a Sporolactobacillus cell by electroporation and homologous recombination, for example in the upstream region, allows it to be integrated into the bacterial genome (Figure 9 (f)). If the second recombination takes place in the same region, it returns to the situation in Figure 9 (e), or if it occurs in the other regions (here in the downstream region), this will result in a situation in which the cat gene is on the plasmid and there is no more sequence in the Sporolactobacillus genome where the target gene was (Figure (g)).

In Figure 9 (h), the selection of erythromycin and chloramphenicol sensitive bacteria isolates mutants in which the target gene has been completely deleted and the cat gene has been lost with the plasmid. This mutant is called "clean and stable".

A knockout of the L-HicDH gene from the Sporolactobacillus genome has been achieved. For this target gene, two plasmids were constructed according to the procedure described in Example 2: (i) pNW33N comprising as an "Amont-Cat-Aval" insert and (ii) pNW33N comprising an "Amont-Aval" insert.

The primers of Example 4 with a name that contains the term "lox" have an (underlined) sequence that is homologous to the lox primers used to amplify the P32-Cat. This sequence made it possible to carry out a recovery PCR to "join" the 3 PCR fragments, namely, an upstream and P32-Cat fragment and a downstream fragment together in a single large PCR fragment. The tail of the primers of Example 4 containing a restriction site for cloning is indicated in bold.

Table 5: Sequences of sense (Uplox66) and antisense (Dnlox71) primers used to amplify P32-Cat

Table 6: Sequences of sense (UP1) and antisense (UP2_lox) primers used to amplify the upstream region of L-HicDH

Table 7: Sequences of forward (DNllox) and antisense (DN2) primers used to amplify the downstream region of L-HicDH

Sporolactobacillus strain LMG P-26831 was transformed by electroporation with plasmid pNW33N "Amont-Cat-Aval" according to the procedure described in Example 3 (Figure 9 (a)). Integrants were obtained by increasing the temperature from 37 ° C to 42 ° C for 4 generations and about 4 joins (Figure 9 (b)). Excisors were obtained by decreasing the temperature from 42 ° C to 37 ° C for 4 to 10 generations and about 4 to 10 days. The site of the excision was verified by PCR amplification of the edge fragments: the recombination can either give rise to the situation with the plasmid pNW33N "Amont-Aval" (Figure 9 (e)) or give rise to a situation where the target gene is on the plasmid and the cat gene is in the bacterial genome (Figure 9 (c)). These events were selected by selecting bacteria with resistance to chloramphenicol but being sensitive to erythromycin. This selection was carried out by plating chloramphenicol-containing liquid cultures on Petri dishes containing chloramphenicol in order to obtain isolated colonies. These colonies were reinoculated onto Petri dishes containing chloramphenicol, with or without erythromycin to isolate mutants in which the target gene was replaced by the cat gene and the target gene was lost with the plasmid (Figure 9 (d) ). Mutants lacking the target gene of L-HicDH were obtained which are stable but not clean because they contain an exogenous sequence, ie the Cat gene (chloramphenicol resistance).

Stable and clean mutants were then obtained by transformation with plasmid pNW33N "Upstream" according to the procedure described in Example 3 (Figure 9 (e)). Integrants are obtained by increasing the temperature from 37 ° C to 42 ° C (Figure 9 (f)). Excisors are obtained by decreasing the temperature from 42 ° C. to 37 ° C. The site of the excision was verified by PCR amplification of the edge fragments: the homologous recombination can either give rise to the situation with the plasmid pNW33N "Upstream-downstream" (Figure 9 (e)) or give rise to a situation where the cat gene is on the plasmid and the sequence "Downstream-Amont" is in the bacterial genome (Figure 9 (g)). These events were selected by selecting bacteria sensitive to erythromycin and chloramphenicol. This selection was performed by plating liquid cultures on petri dishes with or without chloramphenicol and erythromycin for the purpose of obtaining isolated colonies. These colonies were reinoculated on petri dishes with or without chloramphenicol and erythromycin to isolate mutants in which the cat gene was lost together with the plasmid (Figure 9 (h)). Mutants without the target L-fficDH gene were obtained that are stable and clean because they contain no exogenous sequences; the sequence between the start and stop codons of the target gene has been deleted and the stop codon is just behind the start codon.

This strategy is repeated in order to obtain Sporolactobacillus strains with a clean and stable knockout of L-LDH, D-LDH62 or D-LDH65. For L-LDH, two plasmids were constructed with the primers given in Tables 5, 8 and 9. For D-LDH62, two plasmids were constructed with the primers given in Tables 5, 10 and 11. LDH65, two plasmids were constructed with the primers given in Tables 5,12 and 13.

Table 8: Sequences of sense (UP1) and antisense (UP2_lox) primers used to amplify the upstream region of L-LDH

Table 9: Sequences of sense (DN1 lox) and antisense (DN2) primers used to amplify the region downstream of L-LDH

Table 10: Sequences of the forward (UP1) and antisense (UP2_lox) primers used to amplify the upstream region of the LDH62

Table 11: Sequences of forward (DNllox) and antisense (DN2) primers used to amplify the region downstream of D-LDH62

Table 12: Sequences of the forward (UP1) and antisense (UP2_lox) primers used to amplify the upstream region of the D-LDH65

Table 13: Sequences of sense (DN1 lox) and antisense (DN2) primers used to amplify the region downstream of D-LDH65

Example 5 Production of lactic acid-L or enantiomerically pure lactic acid-D

As schematically illustrated in Figure 5A, in order to obtain Sporolactobacillus species capable of producing L-lactate enantiopur, the D-lactate dehydrogenase genes such as D-LDH62 and D-LDH65 are deleted from the Sporolactobacillus genome.

The strain Sporolactobacillus LMG P-26831 is genetically modified by deleting the genes of D-LDH62 and D-LDH65 from the genome of Sporolactobacillus with the protocol described in Example 4. This strain of Sporolactobacillus LMG P-26831 genetically modified is used in a process for the preparation of lactic acid-L.

As schematically illustrated in FIG. 5B, in order to obtain Sporolactobacillus species capable of producing enantiomerically pure D-lactate, the L-lactate dehydrogenase and / or L-2-hydroxyisocaproate dehydrogenase genes are deleted from the Sporolactobacillus genome.

The strain Sporolactobacillus LMG P-26831 is genetically modified by removing the L-LDH and L-HicDH genes from the Sporolactobacillus genome with the protocol described in Example 4.

This genetically modified strain of Sporolactobacillus LMG P-26831 is used in a process for the preparation of lactic acid-D.

Table 14 shows the decrease in l-lactic acid production by the 2 delta mutants L-HicDH, Delta-L-LDH: P32Cat 2A and 3B relative to the wild-type strain (WT = Wild type).

Table 14: Decrease in lactic acid production-L

EXAMPLE 6 Expression in Sporolactobacillus vineae of a heterologous glucoamylase

In order to obtain a Sporolactobacillus strain capable of hydrolyzing short glucose polymers, expression of a heterologous glucoamylase was induced in LMG strain P-26831.

A plasmid was constructed which comprises pNW33N as a vector and has as insert the Bacillus coagulons glucosamylase gene promoter 36D1 + the Thermoanaerobacterium thermosaccharolyticum glucoamylase gene.

This plasmid was introduced into the strain of Sporolactobacillus LMG P-26831 by the electro-transformation protocol described in Example 3.

The presence of the plasmid in the emerging transformants was verified by PCR on the plasmid. Expression of active glucoamylase from the existing multicopy plasmid in the cytoplasm (no homologous recombination was used here) was verified by growth of the cells on Petri dishes containing starch. After growing for several days, the starch present in the petri dishes was stained by the I2 vapors. As can be seen in Figure 10, there is a ring of degradation around the transformants (7.9, 8.6, 8.7, 8.8, 8.9 and positive control), but not around others (the first five and 7.8, 8.2) indicating that the starch has been partially hydrolysed.

EXAMPLE 7 Expression in Sporolactobacillus vineae of a heterologous alpha-amylase

In order to obtain a strain of Sporolactobacillus capable of hydrolyzing starch, expression of a heterologous ALPHA-amylase was induced in LMG strain P-26831.

Two different plasmids were used: -pGIT008 which contains a L. plantarum promoter + the B. licheniformis alpha-amylase gene -pGIP312.4 which contains an E. coli promoter. faecalis + the B. licheniformis alpha-amylase gene

These plasmids were introduced into the LMG P-26831 strain by the electro-transformation protocol described in Example 3.

The presence of one of the plasmids in the emerging transformants was verified by PCR on the plasmid. Expression of active alpha-amylase from the existing multicopy plasmid in the cytoplasm (no homologous recombination was used here) was verified by growth of the cells on Petri dishes containing starch. After growth for several days, the starch present in the Petri dishes was stained with I 2 vapors.

conclusions

Together, the above examples show that the method of the present invention makes it possible to genetically modify Sporolactobacillus strains. This method advantageously makes it possible to construct strains of Sporolactobacillus genetically modified species such as, for example, Sporolactobacillus strains which are capable of producing lactic acid or one of its stereoisomers from abundant and therefore inexpensive raw materials.

Claims (21)

A method of modifying genetically engineered strains of Sporolactobacillus, comprising introducing a DNA cloned into a replicon into cells of a strain of Sporolactobacilli. The method of claim 1, wherein the replicon is a heat-sensitive replicon. 3. Method according to claim 2, wherein the method comprises, after introduction of the DNA into the cells of a Sporolactobacillus species, the steps of: - culturing the cells on a selective medium at a permissive temperature for replication to select transformed cells capable of growing on said selective medium at said permissive temperature, and - culturing said transformed cells at a non-permissive temperature for replication to select transformed cells capable of growing at said non-permissive temperature. 4. A method according to any one of claims 1 to 3, wherein the replicon is a plasmid. 5. Method according to any one of claims 1 to 4, wherein the Sporolactobacillus strain is Sporolactobacillus vineae. 6. Method according to any one of claims 1 to 5, wherein the strain of Sporolactobacillus is a strain of Sporolactobacillus vineae, characterized by a 16S RNA sequence as indicated in SEQ No. 1 or a strain of Sporolactobacillus having at least minus 95% identity with the 16S RNA sequence as it appears in SEQ ID No. 1. 7. Method according to any one of claims 1 to 6, wherein the Sporolactobacillus strain is a strain of Sporolactobacillus vineae deposited under accession number LMG P-26831 to the BCCM-LMG Bacteria Collection. The method of any one of claims 2 to 7, wherein the heat-sensitive replicon is plasmid pNW33N. The method of any one of claims 2 to 8, wherein the thermosensitive replication is dependent on the RepB protein, a functional fragment or a variant thereof for thermosensitive replication. The method according to any one of claims 3 to 9, wherein the non-permissive temperature culture for replication allows the introduction of DNA into the Sporolactobacillus chromosome, preferably by homologous recombination. The method of any one of claims 1 to 10, wherein the DNA encodes one or more desired functions. The method according to any one of claims 1 to 11, wherein the DNA comprises one or more sequences coding for one or more polypeptides capable of producing at least one compound selected from the group consisting of lactic acid, lactic acid-L lactic acid-D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate, preferentially, wherein the DNA comprises one or more sequences coding for one or more polypeptides capable of producing lactic acid-L or lactic acid-D. 13. A method according to any one of claims 10 to 12, wherein the introduction of DNA into the chromosome of a Sporolactobacillus inactivates a coding sequence for an undesirable function of the chromosome of this Sporolactobacillus. The method according to claim 13, wherein the undesirable function is D-lactate dehydrogenase, L-lactate dehydrogenase, D-2-hydroxisocaproate dehydrogenase or L-2-hydroxisocaproate dehydrogenase. 15. Genetically modified Sporolactobacilli strains obtained by a method according to any one of claims 1 to 14. 16. Genetically modified Sporolactobacilli strains obtained by a method according to any one of claims 12 to 14, wherein the Sporolactobacillus species are capable of producing at least one compound selected from the group consisting of lactic acid, lactic acid-L lactic acid-D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2- oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate, preferentially, wherein the Sporolactobacillus strains are capable of producing lactic acid-L or lactic acid-D. 17. Genetically modified Sporolactobacilli strains according to claim 15 or 16, wherein the Sporolactobacillus strains are a genetically modified strain of Sporolactobacillus vineae and preferably a genetically modified strain of Sporolactobacillus vineae characterized by a 16S RNA sequence, as indicated in SEQ ID No. 1, a genetically modified Sporolactobacillus strain having at least 95% identity with the 16S RNA sequence as it appears in SEQ ID No. 1, or a genetically modified strain of Sporolactobacillus vineae deposited under the number for LMG P-26831 Accession at the BCCM-LMG Bacteria Collection. 18. Process for preparing at least one compound selected from the group consisting of lactic acid, lactic acid-L, lactic acid-D, acetolactate, diacetyl, acetoin, 2,3-butenediol, 1,2-propanediol, acetate , formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate, glucose, fructose and pyruvate, wherein the genetically modified strains of Sporolactobacillus of claims 16 or 17 are used. 19. The method of claim 18, wherein at least one compound is lactic acid-L or lactic acid-D. 20. A strain of Sporolactobacillus vineae characterized by a sequence of 16s RNA as indicated in SEQ No. 1 or a strain of Sporolactobacillus having at least 96% identity with the 16S RNA sequence as it appears in SEQ Ш No 1. 21. The Sporolactobacillus vineae strain according to claim 20, deposited under the accession number LMG P-26831 to the BCCM-LMG Bacteria Collection.