Processing

Please wait...

Settings

Settings

Goto Application

1. WO2020136237 - WEISSELLA VIRIDESCENS STRAIN AND USES THEREOF FOR THE SYNTHESIS OF EXOPOLYSACCHARIDES

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

Weissella viridescens strain and uses thereof for the synthesis of

exopolvsaccharides

The present invention relates to a strain of the species Weissella viridescens with accession number CECT 9735 that has the ability to produce exopolysaccharides (EPS) from sucrose. Therefore, the present invention could be framed within the field of industrial biotechnology, in particular the production of biopolymers.

STATE OF THE ART

Exopolysaccharides (EPS) are polymers of monosaccharides produced by bacteria able to demonstrate strong or weak adherence to the cell surface or are released into the extracellular medium forming a matrix. Depending on their chemical composition, EPS can be classified as homopolysaccharides, consisting of units of a single type of monosaccharide, or heteropolysaccharides, consisting of units of several monosaccharides. These monosaccharides are mainly glucose, fructose, galactose, ramnose or arabinose and can be linked by different types of bonds (a or b), both in the main chain and in the branches. Depending on the composition of monosaccharides, the type of bond and the carbon position of the main bond, there is a wide variety of polymers with different names and produced by different bacteria. It is interesting to note that new structures of these compounds are continually being described.

Currently, there is interest in investigating and characterizing EPS due to their positive or negative influence on foods containing these bacteria, in addition to the potential health benefits attributed to them.

Numerous EPS-producing lactic-acid bacteria belonging to different genera have been identified (Zafar S. B., et al. Journal of Genetic Engineering and Biotechnology. 2018; (16): 17-22). These include those of the genus Weissella, which are capable of producing some EPS or not, depending on the specific species (Fusco V., et al. Frontiers in Microbiology. 2015; (6): 155).

Knowledge and characterization of bacteria, as well as the EPS they produce, helps the development of industrial processes to produce these compounds. For example, patent WO20151 17624A1 describes a method of producing an EPS of interest, dextran, using the bacterium Weissella cibaria.

Therefore, the state of the art evidences the need to provide useful micro-organisms for the industrial production of EPS of interest with higher yields than those described in the prior art, for their appropriate and efficient industrial use.

DESCRIPTION OF THE INVENTION

The inventors of the present invention have identified a strain of the species Weissella viridescens isolated from meat products which, surprisingly, is capable of producing exopolysaccharides (EPS), with a higher yield than that known to date for bacteria of the same genus, using sucrose as carbon source. This capacity is bestowed on the said strain by the presence of a nucleotide sequence that encodes an enzyme capable of producing the said EPS, more specifically, the nucleotide sequence encoding that enzyme is found in a mobile element of DNA, more specifically, that nucleotide sequence is found in a plasmid, outside the genome of the strain. This feature allows the application of this strain to obtain EPS in processes with a superior yield to other microorganisms producing these compounds, as will be demonstrated below.

Therefore, in a first aspect, the present invention relates to a lactic-acid bacterial strain, referred to hereinafter as, strain of the invention, preferably of the genus Weisella, more preferably of the species Weissella viridescens, and even more preferably of the strain W. viridescens CECT 9735.

The strain of the invention was deposited on October 3, 2018 under the Budapest Treaty in the Spanish Type Culture Collection (CECT), as International Depositary Authority (Building 3 CUE, Parc Cientific Universitat de Valencia, C/ Catedratico Agustin Escardino, 9, 46980, Paterna, Valencia, Spain). The accession number assigned is CECT 9735 (hereinafter“strain of the invention CECT 9735” or“ Weissella viridescens CECT 9735”).

1/1/. viridescens CECT 9735 is a Gram-positive bacterium of spherical or lenticular irregular rod shape. The strain of the invention belongs to the Kingdom: Bacteria; Phyla: Firmicutes; Class: Bacilli; Order: Lactobacillales; Family: Leuconostocaceae; Genus: Weissella. The said strain was isolated from meat products.

As shown throughout this document, the strain of the invention is capable of producing EPS, with a higher yield than any known so far for bacteria of the same genus. This is due to the presence of a nucleotide sequence encoding an enzyme capable of producing said EPS, which is located in a mobile element, specifically in a plasmid, and not in its genome.

Thus, in another preferred embodiment, the strain of the invention comprises the polynucleotide of the invention comprising SEQ ID NO: 1. In another more preferred embodiment, the strain of the invention comprises the polynucleotide comprising SEQ ID NO: 1 , preferably in a mobile element or plasmid, more preferably, where the said mobile element or plasmid is extrachromosomal. In another more preferred embodiment, the mobile element or extracromosomal plasmid comprising the polynucleotide of the invention is defined in SEQ ID NO: 3.

Thus, in another aspect, the invention relates to a sequence of nucleotides, hereinafter, the nucleotide sequence of the invention or polynucleotide of the invention, which encodes for an enzyme capable of producing EPS and comprising SEQ ID NO: 1.

In another particular embodiment, the nucleotide sequence of the polynucleotide of the invention consists of the nucleotide sequence shown in SEQ ID NO: 1.

In another particular embodiment, the polynucleotide of the invention is a substantially homologous and functionally equivalent variant of the polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 1. In this regards, the polynucleotide of the invention corresponds to: a) nucleic acid molecules of the isolated polynucleotide sequence consisting of SEQ ID NO: 1 , or its complementary chains, b) nucleic acid molecules whose complementary chain is capable of hybridizing under astringent

conditions with a polynucleotide sequence of (a), c) nucleic acid molecules whose sequence differs from a) and/or (b) due to the degeneration of the genetic code.

As used in the description, a polynucleotide is "substantially homologous" to the polynucleotide of SEQ ID NO: 1 when its nucleotide sequence has a degree of identity with the nucleotide sequence shown in SEQ ID NO: 1 of at least 90%, more preferably of, at least, 91 %, 92%, 93%, 95%, 96%, 97% or 98%, and still more preferably of at least 99%. The degree of identity between two nucleotide sequences can be determined by conventional methods, for example, by using standard algorithms of sequence alignment known in the prior art, such as, for example, BLAST (Altschul S. F. et al. 1990). By way of example, a polynucleotide that is substantially homologous to the polynucleotide whose nucleotide sequence comprises, or consists of, the nucleotide sequence shown in SEQ ID NO: 1 can be constructed on the basis of the nucleotide sequence shown in said SEQ ID NO: 1 by introducing, for example, nucleotides or codons encoding amino acids constituting substitutions, which are conservative or non-conservative with respect to the amino acids encoded by the nucleotide sequence shown in SEQ ID NO: 1 . Other illustrative examples of possible modifications include the insertion of one or more nucleotides into the sequence, the addition of one or more nucleotides at either end of the sequence, or the deletion of one or more nucleotides at either end or inside the sequence. In the definition used in this description, a polynucleotide is "functionally equivalent" to the polynucleotide whose nucleotide sequence comprises, or consists of, the nucleotide sequence shown in SEQ ID NO: 1 when encoding a protein capable of producing EPS.

In a particular embodiment, the polynucleotide of the invention is a substantially homologous and functionally equivalent variant of the invention, that is, it is a polynucleotide whose nucleotide sequence has a degree of identity of at least 90%, more preferably of at least 91 %, 92%, 93%, 94%, 95%, 96%, 97% or 98%, and even more preferably at least 99%, with respect to the nucleotide sequence shown in said SEQ ID NO: 1 and that encodes a peptide capable of producing and secreting EPS. More preferably, the polynucleotide of the invention comprises SEQ ID NO: 1 , more preferably, the polynucleotide of the invention consists of SEQ ID NO: 1 .

The polynucleotide of the invention can be found either as such or as part of a gene construction, henceforth gene construction of the invention, which comprises said polynucleotide of the invention. The genetic construction of the invention can incorporate, operatively linked, a regulatory sequence of the expression of the polynucleotide of the invention, thus constituting an expression cassette. As used in this description, the expression "operatively linked" means that the peptide of the invention, encoded by the polynucleotide of the invention, is expressed in the correct reading frame controlled by the expression control sequences or regulators. The control sequences are sequences that control and regulate the transcription and, when applicable, the translation of the peptide of the invention, and include promoter sequences, coding sequences for transcriptional regulators, ribosome binding sequences (RBS) and/or transcription terminator sequences. In one particular embodiment, said expression control sequence is functional in prokaryote cells and organisms, for example, bacteria, etc., while, in another particular embodiment, such an expression control sequence is functional in cells and eukaryotic organisms, for example, insect cells, plant cells, mammalian cells, etc. Advantageously, the construction of the invention further comprises a marker or gene encoding a motif or a phenotype which permits the selection of the host cell transformed with such construction. The genetic construction of the invention can be obtained by the use of techniques widely known in the state of the art (Sambrook et al., 2001 ).

The expression "gene construction " or "nucleic acid construction", as used herein, refers to a functional unit necessary for the transfer or the expression of a nucleotide sequence or gene of interest, in the present document, the nucleic acid sequence of the invention as described above, and regulatory sequences, including, for example, a promoter, operationally bound to the sequence that encodes the protein. Therefore, for the purposes of this invention, gene construction relates to a bicatenarian nucleic acid molecule, which is isolated from a natural nucleic acid or which is artificially modified to contain segments of nucleic acids. The expression “nucleic acid construction" is synonymous with the expression "expression cassette" when the nucleic acid construction contains the control sequences required for the expression of the coding sequence.

The genetic construction of the invention can be inserted into an appropriate vector. Therefore, in another aspect, the invention is related to a vector, such as a recombinant vector, hereinafter vector of the invention, which comprises the polynucleotide of the invention or the gene construction of the invention. The choice of vector will depend on the host cell in which it to be inserted later. In a particular embodiment, the vector of invention is an expression vector. The term "expression vector", also known as "expression construction" or "plasmid", refers to a linear or circular DNA molecule comprising the nucleic acid sequence of the invention and which is operatively linked to additional segments allowing the transcription of the encoded peptide. A plasmid is usually used to insert a specific gene into a target cell. Once the expression vector is inserted in the cell, the protein that is encoded by the gene is produced by the cell transcription and translation machinery. The vector is often genetically engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to an efficient transcription of the gene carried in the expression vector. The purpose of an expression vector or plasmid is to produce large quantities of stable messenger RNA and therefore protein. Expression vectors are basic tools of biotechnology and protein production, such as enzymes. The expression vector of the invention is inserted in a host cell so that the vector is retained as an integral part of the chromosome or as an extra-chromosomal self-replicating vector. In a preferred embodiment of the invention, the expression vector or plasmid is retained as an extra-chromosomal self-replicating vector or plasmid. Illustrative, non-limiting examples of vectors in which the polynucleotide of the invention or the gene construction of the invention can be inserted are plasmids, phages, cosmids, phagemids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), human artificial chromosomes (HAC) or viral vectors, such as adenovirus, retrovirus or lentivirus. The vector of the invention can be obtained by conventional methods known by technicians in the art (Sambrook et al., 2001 ). In a particular embodiment, said vector is a useful vector for transforming competent cells of Escherichia coli. The vector of the invention can be used to transform cells that can be transformed by that vector. Said cells may be prokaryotes or eukaryotes. The vector of the invention can be used to transform eukaryotic cells, such as yeast, for example, Aspergillus niger, Pichia pastoris, etc., or microalgae, for example, Chlamydomonas reinhardtii, etc., or prokaryotic cells, such as bacteria, for example,

E. coli, Pseudomonas putida, Lactobacillus sp., Bifidobacterium sp., Bacillus subtilis, etc.

More preferably, the plasmid of the invention comprises the sequence SEQ ID NO: 1 .

Thus, the gene construction of the invention that comprises the SEQ ID NO: 1 can be used to obtain transgenic cells capable of expressing the peptide encoded by said SEQ ID NO: 1 .

In this respect, another aspect of the present invention is related to a transgenic host cell, henceforth the transgenic cell of the invention, which comprises the polynucleotide of the invention, the gene construction of the invention, the expression cassette of the invention, or the plasmid of the invention, and is able to express the protein of the invention (defined below). The term "cell", as used here, refers to the smallest unit that maintains the fundamental properties of life, and, comprises prokaryotic, eukaryotic cells, as well as cells of single-celled (e.g., bacteria) and multicellular (e.g., animals, plants) organisms. Thus, by way of illustration, the transgenic cell of the invention can be a eukaryotic cell, such as a plant tissue cell, a yeast, a microalgae, etc., a prokaryotic cell, such as a bacterium, for example, E. coli, etc. The transgenic cells of the invention can be obtained by conventional methods known by technicians in the art (Sambrook et al., 2001 ). In a particular embodiment, this transgenic cell of the invention is a recombinant host cell, which has been transformed with the vector of invention.

For the purposes of the present invention, the terms“endogenous” or“homologous” refer both to genes and to proteins that are naturally found in a cell, that is to say, without any human intervention. In addition, these terms also refer to those same genes or proteins that, once isolated from the organism, can be reintroduced (transgene) through genetic engineering.

For the purposes of this invention, the term“heterologous” refers to a nucleic acid derived from a different species or, if derived from the same species, a nucleic acid that is substantially modified from its native form. For example, a promoter that is operatively linked to a heterologous structural gene belongs to a different species from which the structural gene was originally obtained, provided it originates from deliberate human intervention. If they belong to the same species, they must be substantially modified in their original form in one to several heterologous genes. A heterologous protein may originate from a different species or from the same species, provided it originates from deliberate human intervention.

As used herein, the term "recombinant" refers to a polynucleotide or polypeptide that is not naturally produced in a host cell. In certain embodiments, the "recombinant cells" or“transgenic cells” express genes that are not found in an identical form as the native or wild (i.e., non-recombinant) form of the cell and/or express native genes whose expression would otherwise be increased, decreased or knocked-out due to the deliberate human intervention. Recombinant cells contain at least one recombinant polynucleotide or polypeptide. A nucleic acid construction comprising the nucleic acid itself and the elements necessary for its expression, nucleic acid (e.g. a polynucleotide), cell or polypeptide are referred to here as "recombinant" when of non natural, artificial or processed origin.

In the transgenic cell of the invention more than one copy of the nucleic acid sequence of the present invention can be inserted to increase the production of the gene product. The number of copies of the polynucleotide can be increased by integrating at least one additional copy of the sequence into the transgenic host cell or by including a selectable marker gene amplifiable with the polynucleotide, where cells containing amplified copies of the selectable marker gene and therefore additional copies of the polynucleotide can be selected by growing the cells in the presence of the appropriate selectable agent. The procedures used to bind the elements described above in order to construct the recombinant expression vectors referred to in this invention are well known by skilled persons in the art.

In another preferred embodiment, the transgenic cells of the invention are characterized by the overexpression of the peptide encoded by the nucleotide sequence SEQ ID NO: 1 , with the ability to secrete EPS. More preferably, the transgenic cells of the invention are characterized by an increase in the production of EPS compared to parental or wild-type cells that do not comprise the nucleotide

sequence of the invention SEQ ID NO: 1 , which encodes for the protein comprising the SEQ ID NO: 2 and which is capable of producing and secreting EPS.

For the purposes of this invention, the terms “increased expression” or “overexpression” may be used interchangeably throughout this document and refer to any form of expression that is additional to or greater than the original expression level in a parental or wild-type cell. For the purposes of the present invention, in increasing order of preference, overexpression is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% compared to expression in the parental or wild-type cells. Methods to increase the expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Nucleic acid isolates can also serve as promoters or enhancers, insertable in an appropriate position in a non-heterologous form of a polynucleotide in order to upregulate the expression of a nucleic acid that encodes the polypeptide of interest. For example, endogenous promoters can be altered in vivo by mutation, deletion and/or substitution; also isolated promoters can be inserted in a polynucleotide sequence that encodes for a gene of interest to control its expression.

For the purposes of this invention, the scope of these terms is intended to cover the increase in the expression of both homologous and heterologous enzymes.

In some embodiments, the increase in expression includes a high transcription rate and/or a high gene level compared to the homologous transcription rate of that gene. In some other embodiments, a heterologous gene is introduced into a host cell to induce increased expression of a gene encoding a homologous enzyme. In certain embodiments, the heterologous gene is a gene that has been modified to increase the expression of the gene product. In some embodiments, the term also covers polypeptide secretion from a cell.

An expert in the art knows the different tools and routine techniques for overexpression or increased expression of a gene and/or protein.

The "host cell” or "transgenic cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction and similar to the genetic construction of the invention. The host cell can be eukaryotic, such as a mammal, insect, plant, bacterial, or fungal cell. In a preferred embodiment, the host cell is a bacterial cell. In a more preferred embodiment, the bacterial host cell is selected from the list consisting of: Bacillus subtilis, Escherichia coli, Pseudomonas pCitida, Lactobacillus sp and Bifidobacterium sp, among others.

In another aspect, the invention is related to a peptide, hereinafter "peptide of the invention", whose amino acid sequence corresponds to the amino acid sequence encoded by the polynucleotide of the invention.

The term "peptide", as used herein, refers to an amino acid chain bound by peptide bonds, regardless of their length, and includes both amino acid chains of 10 or less amino acids (in some publications identified as "oligopeptides") and amino acid chains of more than 10 amino acids, for example, over 100 amino acids.

In a particular embodiment, the peptide of the invention comprises the sequence of amino acids shown in the SEQ ID NO: 2. The amino acid sequence shown in the SEQ ID NO: 2 corresponds to the sequence encoded by the polynucleotide whose nucleotide sequence is shown in the SEQ ID NO: 1 . Said peptide is capable of secreting EPS. In another preferred embodiment, the peptide of invention consists of the SEQ ID NO: 2.

According to the meaning used in this description, a peptide is "substantially homologous" to the peptide comprising the SEQ ID NO: 2, when its amino acid sequence has a degree of identity with respect to the amino acid sequence shown in the SEQ ID NO: 2 of at least 90%, more preferably of at least 91 %, 92%, 93%, 95%, 96%, 97% or 98%, and even more preferably of at least 99%. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by using standard algorithms of sequence alignment, known in the prior art, such as, for example, BLAST (Altschul S. F. et al. 1990). By way of illustration, a peptide that is substantially homologous to the peptide whose nucleotide sequence comprises, or consists of, the amino acid sequence shown in the SEQ ID NO: 2 can

be constructed on the basis of the amino acid sequence shown in the SEQ ID NO: 2 by inserting, for example, amino acids that constitute conservative or non conservative substitutions with respect to the amino acids present in the amino acid sequence shown in the SEQ ID NO: 2. Other illustrative examples of possible modifications include the addition of one or more amino acids at either end (amino or carboxyl), the insertion of one or more amino acids in the sequence, or the deletion of one or more nucleotides at either end or inside the sequence. In the meaning used in this description, a peptide is "functionally equivalent" to the peptide whose amino acid sequence comprises, or consists of, the amino acid sequence shown in the SEQ ID NO: 2 when it is able to synthesize and secrete EPS. In a particular embodiment of the peptide comprising the SEQ ID NO: 2 of the invention, it is characterized by a high sequence identity, preferably more than 95% sequence identity, with peptides known in the prior art and where these state-of-the-art peptides have dextransacarase activity. Therefore, the peptide comprising the SEQ ID NO: 2 of the invention may also present such dextransacarase activity. The dextransacarase activity of the peptide of the invention encoded by the polynucleotide that is functionally equivalent to the polynucleotide whose nucleotide sequence comprises, or consists of, the nucleotide sequence shown in SEQ ID NO: 1 , can be determined by conventional determination methods of said activity, such as, for example, through the analysis of the concentration of reducing sugars using the 3,5 dinitrosalicylic acid (DNS) method (Miller, G. L. Analytical Chemistry. 1959; 31 : 426-428).

The term 'dextransacarase' refers to a peptide or enzyme in the glucosyltransferase Group (E. C. 2.4.1 .5) capable of catalysing the synthesis of an exopolysaccharide using sucrose (a-D-glucopyranosyl-(1 -2)^-D-fructofuranoside) as a substrate.

In another particular embodiment, the peptide of the invention is a substantially homologous and functionally equivalent variant of the peptide whose amino-acid sequence is shown in the SEQ ID NO: 2.

The person skilled in the art will understand that the amino acid sequences referred to in this description can be chemically modified, for example, by means of chemical modifications which are physiologically relevant, such as, phosphorylations, acetylations, etc. Also, the person skilled in the art will understand that there are peptide fragments satisfying such conditions within these variants which are substantially homologous and functionally equivalent to the peptide whose amino acid sequence is displayed in the SEQ ID NO: 2.

The peptide of the invention can be obtained by conventional methods known by the technicians in the art. Although the said peptide of the invention can be isolated from the strain of the invention, particularly from strain Weissella viridescens, preferably W. viridescens strain CECT 9735, in another particular embodiment, said peptide of the invention can also be obtained by methods based on recombinant DNA technology.

In another aspect, the invention is related to a procedure to obtain a peptide of the invention, which includes cultivating the CECT 9735 strain of the invention, or the transgenic cell of the invention, under conditions that allow the production of said peptide and, if desired, recover said peptide from the culture medium. The conditions for optimising the culture of these cells will depend on the cell used. The expert in the art knows such conditions. The procedure for producing the peptide of the invention includes, optionally, the isolation and purification of said peptide of the invention.

In another aspect, the present invention relates to the use of the CECT 9735 strain, or the transgenic host cell of the invention, as described above, for EPS production.

The term“exopolysaccharide” or "EPS" means a polymer composed of more than 10 monosaccharides secreted by an organism. These units may be glucose, mannose, ribose, arabinose, etc. linked by o-glucosidic bonds in both the main chain and the branched chains. EPS are classified mainly according to their composition in heteropolysaccharides (if they consist of more than one type of monosaccharide) and homopolysaccharides (consisting of a single type of monosaccharide). The properties of these EPS are determined by: (i) the type of bond and monosaccharide units forming it; (ii) its degree and type of branching; and (iii) its molecular mass and conformation.

In another aspect, the present invention relates to a bacterial EPS isolated from a Weissella viridescens CECT 9735 culture.

As shown in Example 4, CECT 9735 strain of the invention produces a significant increase in EPS production of almost 50% compared to other strains of the same genus and/or species, such as W. cibaria CECT 7032, W. cibaria CECT 4710 or W. viridescens CECT 283.

In order to obtain said EPS production yields, the CECT 9735 strain of the invention is cultured under appropriate conditions. Thus, in another aspect, the present invention relates to a method for producing EPS characterized by the following steps: a) culture the bacterial strain of the invention or the transgenic host cell of the invention, in a culture medium that allows its growth; and

b) extraction of the EPS synthesised by the cell of step a), preferably by

precipitation.

The term“culture medium” means a solid and/or liquid food containing the nutrients required for bacterial growth.

In order to obtain EPS by growing the strain of the invention, or the transgenic host cell of the invention, the culture medium must contain sources of carbon, nitrogen and inorganic salts, using the procedures known in the art. The culture medium may contain other additional components commonly used in the prior art and considered necessary for the growth of the strain or of the transgenic cell of the invention and the EPS synthesis obtained by the cultured of the cells mentioned above.

In a preferred embodiment of the method for obtaining EPS by growing the strain CECT 9735, this is characterized in that the stage a) is carried out at a pH of between 6.5 to 7.5, preferably at 7.2; and at a temperature in the range from 28°C to 35°C, preferably at 30°C.

In another particular embodiment, the CECT 9735 strain of the invention is cultured under anaerobic conditions.

The strain of the invention or the transgenic host cell of the invention can be cultivated in a suitable, solid or liquid nutrient medium to produce the enzymes of the invention, using well-known procedures in the art. For example, the cell can be grown on a small scale or on a large scale (including continuous, discontinuous or batch fermentation, discontinuous or fed-batch or solid state culture) carried out in a laboratory or industrial bioreactor, in an appropriate medium and under conditions that enable the enzyme of the invention to be expressed and/or isolated. If the enzyme is secreted, together with other enzymes or proteins in the nutrient medium, they can be recovered directly from the medium.

With regard to the techniques for isolating the synthesized EPS, the precipitation of the EPS with cold ethanol is preferably, although any technique known by an expert in the art is applicable whereby the EPS of interest can be separated from the culture medium, correctly maintaining its physical-chemical properties.

In another aspect, the present invention relates to an EPS characterised by glucose, mannose and arabinose, in a molar ratio of approximately 3.1 : 1 : 0.1.

In another preferred embodiment, the EPS has an elemental composition, in percentages by weight, of approximately at least 30-45% C; 1 -10%H; 1 -15% N, and 0.05-2% S. More preferably, the EPS of the invention has an elemental composition, in percentages by weight, of at least 37.7% C, 3.1 % H, 5.25% N and 0.08% S. In another preferred embodiment, the EPS comprises an average molecular weight (Mw) of between 100 and 150 kDa, an average molecular mass number (Mn) of between 50 and 100 kDa and a polydispersity index of between 1 .5 and 2.

In another particular embodiment, the EPS has a Fourier-transformed infrared spectrum (in cm-1) where the maximum absorbance was observed at a peak at 1013.8 cm-1, followed by a wide peak at 3298 cm-1. Intense bands were also observed at approximately 2910, 2069, 1648.7, 1456, 1412, 1351 , 1237 and 912 cm 1.

In another preferred embodiment, the EPS includes the following bonds:



In a particular embodiment, EPS comprises at least 70% glucose, 20% mannose, and at least 3% arabinose.

Another aspect of the invention relates to compositions comprising the EPS of the invention, hereinafter, compositions of the invention.

EPS are widely used in the chemical, food and pharmaceutical industry for their emulsifying and functional properties, biocompatibility and biodegradability. EPS are part of many products such as herbicides, functional foods, nutraceuticals, cosmeceuticals, pharmaceutical active substances (such as immunomodulators and anticoagulants) or insecticides, among others.

In one particular embodiment, the EPS of the invention is used in the food industry, preferably as a thickening agent, stabilizing agent, emulsifying agent, gelling agent, such as hydrocolloids, such as inclusion bodies (for example, for the controlled release of vitamins, aromatic compounds, etc.).

In another particular embodiment, the EPS of the invention is used in the pharmaceutical and biomedical industry and environmental engineering, such as for example bio-adhesive, thickening agent, stabilizing agent, emulsifying agent, agent, gelling agent, binder (for example, in toothpaste, lipstick, creams, implants, etc). In particular, the benefits described in the field of medicine include the use of EPS composed of mannose and glucose in the form of highly soluble fibres. Said fibres have an exceptional capacity to capture water, forming highly viscous solutions. Glucomannans are efficient satiating agents, intestinal transit regulators (improving constipation by increasing faecal volume), hypocholesterolemic agents (interfering with the transport of cholesterol and bile acids) and regulators of insulin metabolism.

In a preferred embodiment, EPS is used in the food industry, preferably as an excipient and/or additive.

In another preferred embodiment, the composition of the invention may also include other EPS and/or active substance, as well as excipients and / or additives.

Thus, the compositions of the invention can be compositions for use in personal care, pharmaceutical compositions, domestic products, industrial products or foodstuffs.

Throughout the description and claims, the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For experts in the art, other objects, advantages and characteristics of the invention shall be made known partially from the description and partially from the practice of the invention. The following examples and figures are provided as way of example, and are no way intended to limit this invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Photographs of serial cultivation of the CECT 9735 strain under anaerobic conditions at 30°C in medium VWA sucrose at pH 7.2 and pH 5. Detail of serial cultivation 4 (A) and serial cultivation 5 (B) of the CECT 9735 strain cultivated at pH 5.

Fig. 2. Photograph of a gel obtained by electrophoresis. 1. molecular weight marker 1 Kb; 2. genomic DNA of the CECT 9735 strain of the invention grown under conditions with loss of EPS production capacity (pH 5, 30°C, anaerobiosis); 3. Plasmid DNA of the CECT 9735 strain of invention grown under conditions with loss of EPS production capacity (pH 5, 30°C, anaerobiosis); 4. Genomic DNA of the CECT 9735 strain of the invention grown under conditions with EPS production capacity (pH 7.2, 30°C, anaerobiosis); 5. Plasma DNA of the CECT 9735 strain of the invention grown under conditions with EPS production capacity (pH 7.2, 30°C, anaerobiosis); 6. Negative control, mixture of PCR components, but no DNA.

Fig. 3. EPS production (g/L) by culturing the strains W. viridescens CECT 9735, W. cibaria CECT 7032, l/l cibaria CECT4710, W. viridescens CECT 283 and W. viridescens P28B.8.

EXAMPLES

Below the invention will be illustrated by tests performed by the inventors, which show the advantages of the products of the invention.

Example 1. Identification and characterisation of EPS-producing bacterial strain CECT 9735.

A meat product isolates were grown on MRS culture medium and genomic DNA was isolated followed by direct amplification by PCR of the 16S rRNA gene. Subsequently, the 16S rRNA gene was partially sequenced, with readings in both directions, and the sequences were analysed.

Sequence analysis was performed using BLAST. The result obtained from collecting the extension of the overlapping fragment, the similarity percentage and the sequence accession number of the species type strain with the highest degree of identity, revealed that the bacterium belongs to the genus Weisella and the species Weissella viridescens with a similarity of 1014/1014 pb (100%) on the sequence AB023236 (NRIC type strain 1536) and, specifically, W. viridescens CECT 9735.

Example 2. Identification and characterisation of the gene encoding the enzyme capable of producing EPS in strain CECT 9735.

Genomic DNA was isolated from the CECT 9735 strain of the invention and the genome of said strain was subjected to complete sequencing. Once the DNA was purified and concentrated, the DNA being clean of inhibitors, metals and pigments, the sequencing libraries were prepared. For this step the DNA was fragmented and selected according to its size. The samples were sequenced using the PacBio platform, using P6-C4 polymerase and a 4-hour data acquisition time. Once the quality sequences were obtained, the Pacific Bioscience recommendations were followed with the aim of performing a de novo assembly. Blast sequence analysis of the results obtained demonstrated that the bacterium possesses a homologous

sequence with genes encoding dextransacarase and that it is located in a plasmid or extracromosomal mobile element, not forming part of the cell genome.

The complete sequence of the plasmid comprising the gene encoding the enzyme responsible for the EPS synthesis corresponds to SEQ ID NO: 3.

In addition to the nucleotide sequence SEQ ID NO: 1 encoding the enzyme responsible for EPS synthesis comprising SEQ ID NO: 2, the inventors have identified another 7 nucleotide sequences further encoding 7 additional genes in this plasmid.

The nucleotide sequence of the invention encoding the peptide responsible for EPS synthesis represents in strain W. viridescens CECT 9735 of the invention 25% of the complete sequence of the plasmid (SEQ ID NO: 3), with 3042 bp of the gene coinciding with the base pairs of the plasmid from 3042 bp to 1 bp.

Comparing the nucleotide sequence (SEQ ID NO: 1 ) that encodes the enzyme responsible for EPS synthesis (SEQ ID NO: 2) of the strain of the invention, to the sequences recorded in the NCBI database by Blast analysis, it is observed that the sequence is preserved in different strains of W. cibaria and in a strain of Lactobacillus fermentum with a homology of between 96% and 99%, but no homology has been found with any strain of W. viridescens (Table 1 ).

Table 1. Homology of the dextransacarase gene in other species.



Max. Score Maximum score; Score. Total: Total Score; GenBank access number at the GenBank database.

After the identification of the strains of the prior art that comprise a nucleotide sequence encoding an enzyme able to produce EPS and further showing a high sequence homology with the nucleotide sequence (SEQ ID NO: 1 ) encoding the enzyme responsible for EPS synthesis (SEQ ID NO: 2) of the invention, Blast analysis was performed to check for the possible existence of bacterial strains that comprise plasmids containing the nucleotide sequence encoding the enzyme responsible for EPS synthesis of the invention. The results show the existence of strains of species W. cibaria that align with the complete plasmid sequence of the strain of the invention and that have a high identity with it (Table 2).

Table 2. Homology of the plasmid of the invention compared to other plasmids present in other bacterial strains.


Max. Score Maximum score; Score. Total: Total Score; GenBank accession number at the GenBank database.

Below it is shown the analysis of the presence of plasmids outside the genome in different strains of Weissella spp., whose genomes are completely or partially annotated in the databases analysed (NCBI) (Table 3). The result reveals that only five of them have plasmid annotations outside their genome.

Table 3. Strains of Weissella spp. that contain plasmids outside their genome.


As mentioned above, none of the strains known to date of the species W. viridescens, nor of the genus Weisella, have a plasmid outside the genome comprising the nucleotide sequence SEQ ID NO: 1 capable of encoding an enzyme (SEQ ID NO: 2) responsible for EPS synthesis, as described in the present invention.

Example 3. Production and identification of EPS synthesized by strain CECT 9735.

The culture conditions for modulating the loss or non-loss of EPS production capacity by CECT 9735 strain of the invention are given below. For this purpose, serial cultivation was carried out in both liquid and solid medium for production and growth at different incubation temperatures: 30eC and 37eC, under anaerobic conditions.

The WVA production medium, both liquid and solid, comprisel O g/L peptone, 8 g/L meat extract, 2 g/L K2HP04, 5 g/L NaC2H302*3H20, 0.05 g/L MnS04 *4H20, 0.2 g/L MgS04 *7H20, 2 g/L (NH4)3C6H707, 1 ml/L Tween 80, 0,04 g/L Red chlorophenol, 20 g/L Sacarose, 12 ml/L HCI 1 N, pH = 6,5; and WVA growth medium, both liquid and solid, comprise 10 g/L peptone, 8 g/L meat extract, 2 g/L K2HP04, 5 g/L NaC2H302*3H20, 0,05 g/L MnS04 *4H20, 0.2 g/L MgS04 *7H20, 2 g/L (NH4)3C6H707,

1 ml/L Tween 80, 0,04 g/L Red chlorophenol, 20 g/L Sacarose, 12 ml/L HCI 1 N, pH = 7.2.

EPS production capacity after each serial cultivation was evaluated in a solid production medium.

The results show that no loss of EPS production capacity is observed after performing more than 20 serial cultivations under the above conditions, but the strain of the invention grows better when grown at a temperature of 30°C, with a higher production of EPS being observed in plates grown at 30°C than in plates grown at 37°C. Therefore, this is established as the optimal temperature for the evaluation of EPS synthesis by growing the strain of the invention.

The ability of pH to influence the loss of EPS production capacity by the strain of the invention was further tested. To do so, serial cultivations were carried out in the production medium, evaluating in parallel the growth in liquid or solid medium and at two different pH levels: 5 and 7.2. Incubation was performed at 30°C under anaerobic conditions.

The results demonstrate that the CECT 9735 strain of the invention loses its production capacity after five serial cultivations on solid WVA sucrose production medium when cultured at 30eC in anaerobic conditions and at pH 5, whereas when grown at 30eC in anaerobic conditions at pH de 7,2, it continues to synthesize EPS (Fig. 1 ). Therefore, the optimal conditions for EPS synthesis by CECT 9735 strain of the invention are growth in anaerobic conditions at a temperature of 30 ° C and at a pH of 7.2.

Then, we evaluated possible correlation between the loss of EPS production capacity and the presence of the nucleotide sequence (SEQ ID NO: 1 ) encoding an enzyme (SEQ ID NO: 2) capable of secreting EPS. To do so, the presence of the gene in the plasmid of the strain of the invention was identified by means of DNA amplification using PCR techniques. To avoid nonspecific amplifications, two pairs of primers were designed (Table 4) and DNA was amplified with the outermost pair of primers (SEQ ID NO: 4 and SEQ ID No: 5) in the coding sequence of the enzyme of the invention and, on the amplicon obtained, a second amplification was performed with the innermost pair of primers (SEQ ID No: 6 and SEQ ID No: 7) in the sequence of that gene.

Table 4. Primers designed to amplify the nucleotide sequence (SEQ ID NO: 1 ) encoding the enzyme (SEQ ID NO: 2) responsible for synthesis of EPS of the invention.


Fig. 2 shows that the nucleotide sequence (SEQ ID NO: 1 ) of the CECT 9735 strain of the invention is detected only in the DNA plasmid (line 5 of Fig. 2), showing the nucleotide sequence (SEQ ID NO: 1 ) and not in the extraction of genomic DNA (line 4 of Fig. 2) extraction. As seen in Fig. 2, in the strain of the invention CECT 9735, cultivated under conditions in which it loses EPS production ability, SEQ ID NO: 1 is not detected in either genomic DNA (Line 2 of the Fig. 2) or in plasmid DNA (Fig Line 3. 2) extraction. This corroborates that the loss of the nucleotide sequence (SEQ ID NO: 1 ) is correlated with the loss of EPS production capacity.

Once the optimal culture conditions for the CECT 9735 strain of the invention were established, the next step was to identify the EPS secreted. This polymer has been characterized in terms of its elemental composition, bond types, molecular size and infrared spectrum.

The polymer produced by the CECT 9735 strain of the invention is mainly composed of a mixture of glucose and mannose. The approximate percentage of each of these saccharides is 70%, most preferably 73% for glucose and 20%, more preferably 24% for mannose.

Table 5. Composition of the EPS secreted by strain CECT 9735 of the invention.


Recovery 101.3

In accordance with this result, the types of bonds presented are glucose-mannose bonds deduced by the method of obtaining partially methylated alditol acetate (PMAA)

(Table 6).

Table 6. Types of bonds presented by the EPS synthesized by CECT 9735 strain of the invention.

Ratio Ara:Man:Glu (0,1 :1 :3,1 )


Fourier-transform infrared spectroscopy (in cm 1) of the EPS of the invention, showed a maximum absorbance peak at 1013.8 cm-1 , followed by a broad peak at 3298 cnr 1. Intense bands were also observed at approximately 2910, 2069, 1648.7, 1456, 1412, 1351 , 1237 and 912 cm-1.

In addition, we compared the EPS of the invention, with an EPS obtained from cultures of the strain l/l I. cibaria CECT 7032. In the case of W. cibaria CECT 7032, the synthesized EPS contains less than 5% mannose and is composed primarily of glucose (Table 7).

Table 7. Composition of EPS secreted by strain 1/1/. cibaria CECT 7032


Recovery 69.3

The bonds presented by the EPS produced by strain W. cibaria CECT 7032 are mainly glucose, deduced using the partially methylated alditol acetate (PMAA) method

(Table 8).

Table 8. The bonds presented by the EPS produced by strain 1/1/. cibaria CECT 7032.

Ratio Man:Glu (1 :15,1 )


Regarding the molecular weight of the polymer, the EPS produced by the strain of the invention has a single peak, mean molecular weights of Mw =126.1 kDa and Mn=66.6 kDa and a polydispersion index of 1 .9 (Table 9).

Table 9. Molecular weight and polydispersion index of EPS obtained by CECT 9735 strain of the invention.


Medium 126.1 ±7.8 66.6±8.2 1 .9±0.12

In the case of W. cibaria CECT 7032, two peaks were obtained with different molecular weight sizes. Peak 1 : Mw = 43753kDa and Mn = 30173 kDa and a polydispersion index of 1 .45. Peak 2: Mw = 1057kDa and Mn = 700 kDa and a polydispersion index of 15.2 (Table 10).

Table 10. Molecular weight and EPS polydispersion index obtained by strain W. cibaria CECT 7032.


Medium 43753±66 30173±55 1 .45±0,05 1057±77 700±45 1 .51 ±0,02

Both peaks have molecular weights greater than those of the EPS produced by CECT 9735 strain of the invention, which confers on the EPS secreted by that strain different physical characteristics to those of the EPS secreted by strain W. cibaria CECT 7032.

Example 4. Production yields of EPS obtained by growing strain CECT 9735 of the invention compared to other known strains.

The production of EPS in different strains of the genus Weissella has been evaluated, specifically in 2 strains of the species W. cibaria and 2 strains of W. viridescens, comparing them to strain W. viridescens CECT 9735 of the invention.

For this purpose, starting with a preinoculum of each of the analysed strains, they are inoculated in VW growth medium and grown at pH 6.5, at 30°C for 16h in anaerobic conditions. From the preinoculum, they are inoculated on VW production medium and are grown at pH 7.2, under anaerobic conditions at 30°C.

After 48 h, the culture is centrifuged and the supernatant is recovered. Two volumes of EtOH are added and cold precipitated for 16h. Once the EPS is precipitated, it is recovered by centrifugation and freeze dried.

As seen in Fig. 3 and Table 11 , strain CECT 9735 of the invention is capable of producing 50% more EPS cultivated in the same conditions as the strains of W. cibaria or even strains of the same species, W. viridescens, which have low EPS production capacity. This result is totally unexpected, as it is the first description of EPS production capacity in the species W. viridescens and, furthermore, it is the first description of the presence of a nucleotide sequence encoding an enzyme capable of producing EPS in that bacterial species.

Table 11. EPS production by different strains of the genus Weissella.

Media (g/L) SD