EP4182033A1 - Isoliertes gen für das enzym glycosyl transferase 2 aus pyrococcus horikoshin ot3 und seine homologe - Google Patents

Isoliertes gen für das enzym glycosyl transferase 2 aus pyrococcus horikoshin ot3 und seine homologe

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Publication number
EP4182033A1
EP4182033A1 EP21740109.0A EP21740109A EP4182033A1 EP 4182033 A1 EP4182033 A1 EP 4182033A1 EP 21740109 A EP21740109 A EP 21740109A EP 4182033 A1 EP4182033 A1 EP 4182033A1
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EP
European Patent Office
Prior art keywords
gene
enzyme
coli
host cell
homologs
Prior art date
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Pending
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EP21740109.0A
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English (en)
French (fr)
Inventor
Sylvain TRANCHIMAND
Thierry Benvegnu
Hala CHAMIEH
Khadija AMIN
Samir TAHA
Ziad ABDEL-RAZZAK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ecole Nationale Superieure De Chimie
Centre National de la Recherche Scientifique CNRS
Universite de Rennes 1
Institut National des Sciences Appliquees de Rennes
Lebanese University
Original Assignee
Ecole Nationale Superieure De Chimie
Centre National de la Recherche Scientifique CNRS
Universite de Rennes 1
Institut National des Sciences Appliquees de Rennes
Lebanese University
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Application filed by Ecole Nationale Superieure De Chimie, Centre National de la Recherche Scientifique CNRS, Universite de Rennes 1, Institut National des Sciences Appliquees de Rennes, Lebanese University filed Critical Ecole Nationale Superieure De Chimie
Publication of EP4182033A1 publication Critical patent/EP4182033A1/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides

Definitions

  • the present invention refers to an isolated gene coding for a Glycosyl transferase 2 enzyme, to the isolated enzyme Glycosyl transferases 2 encoded by this gene, and to a process for producing sulfated glycosaminoglycans (GAGs).
  • GAGs glycosaminoglycans
  • the present invention has utility in pharmaceuticals, cosmetics, nutraceuticals, and food fields.
  • brackets [ ] refer to the listing of references situated at the end of the text.
  • Glycosaminoglycans also known as mucopolysaccharides, are long-chain, unbranched polysaccharides that are found on the cell surface and constitute a major constituent of the extracellular matrix. They are negatively-charged polysaccharide of diverse molecular weights, composed of repeating disaccharide units that consist of either sulfated or non-sulfated monosaccharides.
  • They contain an amino sugar, either glucose based or galactose based (GlcNAc or GalNAc (N- acetylgalactosamine) or a or b D-glucosamine) and an uronic acid (either glucuronic acid and/or a-L-iduronic acid, a C5 epimer of GlcA) except for keratan which does not contain an uronic acid and is composed of an amino sugar and galactose disaccharides.
  • GlcNAc or GalNAc N- acetylgalactosamine
  • a or b D-glucosamine uronic acid
  • uronic acid either glucuronic acid and/or a-L-iduronic acid, a C5 epimer of GlcA
  • chondroitin sulfate CS
  • KS keratan sulfate
  • HA hyaluronic acid also called hyaluronan
  • DS dermatan sulfate
  • HS heparin and heparan sulfate
  • GAGs In vertebrates, all these GAGs are present, where three major classes predominate including (1) heparin and heparin sulfate (HS) that are both composed of alternating 4-linked uronic acid and 4-linked a-GIcNAC but differ only in the relative proportions of their monosaccharide and disaccharide substructures units, (2) hyaluronan (HA) and (3) chondroitin sulfate (CS) and dermatan sulfate (DS). All these GAGs, except for HA, acquire structural variability by modifications that involve sulfation and uronate epimerization, which are the basis for the wide variety of domain structures with biological activities.
  • HS heparin and heparin sulfate
  • HS heparin and sulfate
  • HS heparin sulfate
  • CS chondroitin sulfate
  • DS dermatan sulfate
  • GAGs and their heterogeneities regarding their sulfation content are related to their role as structural components and regulators of a variety of functions of proteins, cells and tissues in the human body and are all contributing factors to the diversity of their biomedical roles by their ability to bind to multiple extracellular proteins whose actions are spread in various pathophysiological events, as inflammation, cancer, sugar metabolism, bone remodeling and resorption, tissue development, regeneration and repair, and wound repair and coagulation.
  • GAGs notably the hyaluronan (HA) and chondroitin sulfate (CS) that represent the top ranked products in industrial biotechnology for biomedical applications, but also in the fields of cosmetics, nutraceuticals and food.
  • HA hyaluronan
  • CS chondroitin sulfate
  • GAGs especially hyaluronan (HA) and chondroitin have been recently produced by metabolic engineering strategy by which their synthases, hyaluronan synthase and chondroitin polymerase respectively, have been cloned and expressed in E. coli (Yu, H. and G.
  • GTs Glycosyl transferases
  • GTs are classified into 110 families and their number is continuously increasing by discovering new GT genes with the evolution of gene database sequences.
  • the study of these enzymes is indispensable for their exploitation in order to develop their roles in biotechnology, in particular for producing GAGs by metabolic engineering strategy.
  • the present invention fulfills these and other needs.
  • the Applicants discovered novel GTs from hyperthermophilic Archaea, more specifically the GT-2 family by a global phylogenomic approach from Archaeal sequenced genomes. This allowed to categorize GT-2 families into separate clusters and devise a classification for all GT-2 families from Archaea and they detected the presence of a novel GT-2 family.
  • these thermostable enzymes are able to catalyze the polymerization of a novel GAG polysaccharide displaying unique properties which give them remarkable industrial relevance.
  • GTs from hyperthermophilic Archaea are interesting for their resistance to heat and denaturants and their remarkable stability which give them an industrial relevance. While GTs from many organisms have been characterized, few examples of GTs from Archaea are described particularly for the large processive GTs. These enzymes are involved in the synthesis of carbohydrate polymers such as chitin, alginate, poly -N- acetylglucosamine (PNAG) and some GAGs such as hyaluronan and chondroitin that play a major role in different tissues and organs and have numerous applications in the fields of pharmaceuticals, cosmetics, nutraceuticals, and foods.
  • PNAG poly -N- acetylglucosamine
  • This family is distinct from the other detected GT-2 families in Archaeal genomes which included the cellulose like synthase subunit A (CesA) family, the dolichol phosphate synthase like family, rhamnosyl transferase, WcaA cell wall biogenesis proteins and the egg head like proteins, Search for homologs and analysis of domain architecture revealed that the closest homologs to this gene family are the prokaryotic hyaluronan synthase and prokaryotic chondroitin polymerase. Multiple sequence alignment of the protein family members and motif comparaison with the other GT-2 detected families show some motifs characteristic for all the GT-2 in general but the novel GT-2 proteins display distinct specific motifs.
  • CesA cellulose like synthase subunit A
  • multiple sequence alignment of protein family members of the PhCS-like can be represented by Weblogo (WebLogo - Abouthttps://weblogo. berkeley.edu).
  • OXD OXD
  • the QxxRW (SEQ ID NO: 29) pentapeptide was found in both the novel GT-2 family and cellulose synthase clusters.
  • a DXT motif was detected in both the novel GT- 2 family and the rhamnosyl like synthase family but not in the cellulose synthase family.
  • the motif DGS present in the prokaryotic chondroitin polymerase is also detected in the N terminus of the protein family sequences.
  • the GT-2 family displays a specific motif located at the N- terminus which consists of the consensus SXLXXXYP (SEQ ID NO: 30) detected only in these family members.
  • a conserved Aspartic Acid was also detected at the C terminus in the novel GT-2 family.
  • the Applicants have selected a protein composed of 314 amino acids from the hyperthermophilic Archaeon Pyrococcus horikoshii.
  • the present invention provides an isolated gene coding for the enzyme Glycosyl transferase 2 from Pyrococcus horikoshii OT3, the nucleotide sequence of said gene comprising or consisting of sequence SEQ ID NO: 1 , or its homologs from hyperthermophilic Archaea.
  • the present invention provides an isolated enzyme Glycosyl transferases 2, encoded by the isolated gene defined above.
  • the isolated enzyme comprises or consists to the sequence SEQ ID NO : 2, or an homologous sequence from hyperthermophilic Archaea.
  • glycosyl transferase 2 refers to an enzyme having glycosyl transferase activity, which catalyzes the biosynthesis of disaccharides, oligosaccharides, polysaccharides and/or glycoconjugates by the transfer of a sugar residue from activated donor molecules to appropriate acceptor molecules to form new glycosidic linkages.
  • the term “gene” refers to a segment of DNA involved in producing a polypeptide, that may or may not include regions preceding and following the coding region, e.g. 5' untranslated (51 UTR) or “leader” sequences and 3' UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • a gene may refer to one that is naturally occurring, or a synthetic gene, as long as they keep the glycosyl transferase activity.
  • isolated gene refer to a nucleic acid or a polypeptide that is removed from at least one other material or component with which it is naturally associated as found in nature.
  • isolated » is synonym to « separated » or « purified ceremoni
  • An isolated polynucleotide, thereof, includes, but is not limited to, a culture broth containing the polynucleotide inserted in a heterologous host cell, or to a culture broth containing secreted polypeptide expressed in a cell, for example an heterologous host cell.
  • the techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art, including isolation from genomic DNA, preparation from cDNA, or a combination thereof.
  • the cloning of the polynucleotides of the present disclosure from such genomic DNA can be effected, e.g., by using the polymerase chain reaction (PCR) or probe screening of expression libraries to detect cloned DNA fragments with shared structural features, as illustrated for example in Sambrook et al , MOLECULAR CLONING: A LABORATORY MANUAL, 2 nd ed., Cold Spring Harbor, 1989, and 3 rd ed., 2001 ([12]).
  • the gene or enzyme of the invention may be isolated from Pyrococcus horikoshii OT3 (Accession number PH_RS02140). Pyrococcus horikoshii is a hyperthermophilic Archaeon that belongs to the phylum Euryarchaeota within the order Thermococcales and thrive in hydrothermal vents of the deep sea. It is characterized by particular enzymes that display high thermostability allowing them to resist the elevated temperature and extreme pressure conditions giving them high resistance to denaturation and proteolysis.
  • thermophilic proteins have enabled them to be used in numerous biotechnological applications (Kumar, S. and R. Nussinov, How do thermophilic proteins deal with heat? Cellular and Molecular Life Sciences CMLS, 2001. 58(9): p. 1216-1233 ([3])).
  • it may be isolated from any ortholog including hyperthermophilic Archaea and halophiles, for example those listed in Table 1 below.
  • homolog refers to DNA sequences sharing ancestry, because of either a speciation event (orthologs) or a duplication event (paralogs). Homologous sequences are inferred from a phylogenetic tree of the GT-2 protein gene families wherein homologous protein sequences are devised into separate families. Each family originates from a single node representing the ancestral protein and share specific distinct protein motifs.
  • homolog refers also to a mutant gene having substantially the same the glycosyl transferase activity as GT2 from hyperthermophilic Archaea Pyrococcus horikoshii OT3, and having a nucleotide sequence that is at least about 80% identical, or at least about 85% identical, or at least about 90% identical, or at least about 91 % identical, or at least about 92% identical, or at least about 93% identical, or at least about 94% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, at least about 99% identical, to SEQ ID NO:1or any of the homologs of table 1.
  • nucleotide sequence of SEQ ID NO: 1 may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having glycosyl transferase activity from strains of different genera or species according to methods well known in the art.
  • probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • nucleic acid probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at Ieast35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length.
  • the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length.
  • nucleic acid probes which are at least 600 nucleotides, at least preferably at least 700 nucleotides, more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length. Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present disclosure.
  • a genomic DNA or cDNA library prepared from such other organisms may, therefore, be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having alpha- glucosidase activity.
  • Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is used in a southern blot.
  • hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleotide sequence shown in SEQ ID NO: 1, or SEQ ID NO:2, their complementary strands, or subsequences thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using X-ray film.
  • the Glycosyl transferase 2 polypeptides of the instant disclosure are encoded by polynucleotide or nucleic acid sequences that hybridizes, for example under stringent conditions, to an isolated nucleic acid which comprises consecutive nucleotides having a sequence selected from the group consisting of the sequence SEQ ID NO: 1 or sequences from table 1, and a variant of any one having at least about 50% identity to SEQ ID NO: 1 , for example at least 60%, or 70%, or 80%, or 90%, or 95% or more identity to SEQ ID NO: 1 , or any of the homologs from table 1 under conditions of high stringency .
  • hybridization refers to the process by which one strand of nucleic acid forms a duplex with, i.e. , base pairs with, a complementary strand, as occurs during blot hybridization techniques and PCR techniques.
  • Hybridized, duplex nucleic acids are characterized by a melting temperature (T m ), where one half of the hybridized nucleic acids are unpaired with the complementary strand.
  • the present invention includes nucleic acids that encode any recombinant or engineered enzyme having deletions, insertions, or substitutions that do not alter substantially the activity of the enzyme. These deletions, insertions, or substitutions may especially be due to the degeneracy of the genetic code, where a plurality of nucleic acids may encode the same polypeptide having the same amino acid sequence.
  • Another object of the invention relates to a vector, especially a plasmid vector comprising a gene coding for the enzyme Glycosyl transferase 2 from Pyrococcus horikoshii OT3, the nucleotide sequence of said gene comprising or consisting of the sequence SEQ ID NO : 1, or its homologs from hyperthermophilic Archaea.
  • a recombinant vector refers to any suitable vector that can be transformed into and replicated within a host cell for expressing the Glycosyl transferase 2 enzyme.
  • a recombinant vector may be selected from the group comprising or consisting of a plasmid, a cosmid, a phage, an integrated cassette or a virus vector.
  • the recombinant vector may further comprise a purified nucleic acid segment having a coding region encoding enzymatically active chondroitin sulfate synthase.
  • plasmid refers to a circular double- stranded
  • ds DNA construct used as a cloning vector, and which forms an extra chromosomal self-replicating genetic element in many bacteria and some eukaryotes.
  • Vectors can be transferred to a host cell using known transformation techniques, such as those disclosed below.
  • the plasmid may be any plasmid known in the art and adapted to the particular use according to the invention, for example pBAD-TOPO ® (InvitrogenTM) or pET102/D- TOPO ® (InvitrogenTM), pALTER-Ex1/2 (Promega), pCAL-n (Stratagene), peT series (Novagen), pGEX series (Pharmacia).
  • the vector may also comprises elements allowing the expression of said gene in a cell.
  • a vector comprising a nucleic acid encoding an glycosyl transferase polypeptide of the present disclosure can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector.
  • the vector may also be suitably transformed into an expression host, such that the encoding polynucleotide is expressed as a functional alpha- glucosidase enzyme.
  • a vector useful for this purpose typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes.
  • the expression vector usually comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes.
  • the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the alpha-glucosidase to a host cell organelle such as a peroxisome, or to a particular host cell compartment.
  • the nucleic acid sequence of the alpha- glucosidase is operably linked to the control sequences in proper manner with respect to expression.
  • a polynucleotide encoding a glycosyl transferase polypeptide of the present invention can be operably linked to a promoter, which allows transcription in the host cell.
  • the promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • Examples of promoters for directing the transcription of the DNA sequence encoding a glycosyl transferase, especially in a bacterial host include at least one promoter chosen among an arabinose inducible promoter, especially a weak arabinose inducible promoter, a T7 inducible promoter, especially a strong T7 inducible promoter, the promoter of the lac operon of E.
  • the vector is a plasmid vector comprising an arabinose inducible promoter and/or a T7 inducible promoter, operationally linked to said isolated gene.
  • the coding sequence can be operably linked to a signal sequence.
  • the DNA encoding the signal sequence may be a DNA sequence naturally associated with the glycosyl transferase gene of interest to be expressed, or may be from a different genus or species as the alpha-glucosidase.
  • An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding an alpha- glucosidase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell.
  • the vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g. ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • a selectable marker e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g. ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • the plasmid of the invention may comprise at least one other sequence encoding another protein, possibly placed in tandem with the GT-2 gene.
  • the other coding sequence may code for any protein that may be usefully co-expressed with the GT-2 gene of the invention, for example an enzyme usually involved in the synthesis of GAGs. It may be for example at least one enzyme selected among UDP-glucose dehydrogenase KfoF and UTP-glucose-1-P-Uridyltransferase gaLU.
  • the plasmid vector may comprise or consist of nucleotide sequence SEQ ID NO: 3 or SEQ ID NO : 4.
  • Another object of the invention relates to a host cell comprising a recombinant polynucleotide encoding Glycosyl transferase 2 from Pyrococcus horikoshii OT3, or its homologs from hyperthermophilic Archaea.
  • An isolated cell either comprising a recombinant polynucleotide comprising sequence SEQ ID NO : 1, or an expression vector, especially a plasmid vector, as defined above, is advantageously used as a host cell in the recombinant production of a glycosyl transferase 2.
  • the cell may be transformed with the DNA construct encoding the enzyme, for example by integrating the DNA construct, in one or more copies, in the host chromosome. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector in connection with the different types of host cells.
  • Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g. , lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion.
  • General transformation techniques are known in the art. See, e.g. Sambrook et al. ([12]).
  • Suitable host cell may be any cell known to possess the metabolic pathway necessary for the biosynthesis of precursors required for GAG production. It may be for example Archaea, Gram-positive and Gram negative Bacteria, including Bacillus sp., Lactococcos lactis, Agrobacterium sp., and Escherichia coli. or even eukaryotic systems such as yeast or Chinese hamster ovary, African green monkey kidney cells, VERO cells, or the like.
  • the host cell may be E. Coli, for example E. Coli selected among AaraC E.coli cells, in particular LMG194 E. Coli strain, BL21 (DE3), BL21 (DE3) codon plus, BL21 (C41) and BL21 (C43) E. Coli strains.
  • E. Coli selected among AaraC E.coli cells, in particular LMG194 E. Coli strain, BL21 (DE3), BL21 (DE3) codon plus, BL21 (C41) and BL21 (C43) E. Coli strains.
  • cells of Pyrococcus horikoshii OT3 are excluded from the “host cells”.
  • Another object of the invention relates to a process for producing sulfated glycosaminoglycans (GAGs), said method comprising the steps of: a) culturing a host cell as defined above in a culture medium containing glucose or any sugar or sugar acid synthesized from glucose, and under conditions compatible with the production of said GAGs by said cell; and b) recovering sulfated GAGs from the culture medium of step (a).
  • GAGs glycosaminoglycans
  • Any host cell as defined above may be used in the process of the invention.
  • the medium used to cultivate the host cells may be any conventional medium suitable for growing the host cell and obtaining expression of a glycosyl transferase 2 enzyme. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
  • Host cells may be cultured under suitable conditions that allow expression of a glycosyl transferase 2.
  • Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
  • protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or sophorose or using an auto-induction media which enables regulated protein expression in E. coli without monitoring the culture or adding inducer during cell growth (Studier, F.W. (2005) Protein Expr. Purif. 41 , 207-34.2005 ([13])).
  • An expression host also can be cultured in the appropriate medium for the host, under aerobic or anaerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g. from about 25 °C to about 75°C
  • the process of the invention may comprises the following steps:
  • pBAD Thermo Fisher Scientific, Invitrogen
  • pBAD episomal plasmid vector comprising a sequence coding for the enzyme GT2 from hyperthermophilic Archaea Pyrococcus horikoshii OT3 or its homologs from hyperthermophilic Archaea under the control of arabinose promoter or peT episomal plasmid vector comprising a sequence coding for the enzyme GT2 from Pyrococcus horikoshii OT3 or its homologs from hyperthermophilic Archaea under the control of T7 promoter ; b.
  • pBAD episomal plasmid vector comprising a sequence coding for the enzyme GT2 from Pyrococcus horikoshii OT3 or its homologs from hyperthermophilic archaea and a sequence coding for the enzyme UDP- glucose dehydrogenase KfoF in tandem under the control of the arabinose promoter or peT episomal plasmid vector comprising a sequence coding for the enzyme GT2 from Pyrococcus horikoshii OT3 or its homologs from hyperthermophilic archaea and a sequence coding for the enzyme UDP- glucose dehydrogenase KfoF in tandem under the control of the T7 promoter ; c.
  • pBAD episomal plasmid vector comprising a sequence coding for the enzyme GT2, a sequence coding for the enzyme UDP-glucose dehydrogenase KfoF, a sequence coding for the enzyme UTP-glucose-1-P- Uridyltransferase GalU under the control of the arabinose promoter or peT episomal plasmid vector comprising a sequence coding for the enzyme GT2, a sequence coding for the enzyme UDP-glucose dehydrogenase KfoF, a sequence coding for the enzyme UTP-glucose-1-P-Uridyltransferase GaLU under the control of the T7 promoter ;
  • Escherichia coli bacterial host cells are transformed with the plasmid vector able to produce the glycosaminoglycan and are pre-selected on ampicillin petri dishes plate
  • Glycosaminoglycan recovered from the culture medium.
  • a GAG polypeptide secreted from the host cells can be used, with minimal post-production processing, as a whole broth preparation. However, to increase the yield of GAG production, a fermentation process may be performed.
  • a classical batch fermentation is a closed system, where the composition of the medium is set at the beginning of the fermentation, and the composition is not altered during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism(s). In other words, the entire fermentation process takes place without addition of any components to the fermentation system throughout.
  • a batch fermentation qualifies as a "batch" with respect to the addition of the carbon source.
  • attempts are often made to control factors such as pH and oxygen concentration throughout the fermentation process.
  • the metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped.
  • cells progress through a static lag phase to a high growth log phase and finally to a stationary phase, where growth rate is diminished or halted. Left untreated, cells in the stationary phase would eventually die. In general, cells in log phase are responsible for the bulk of production of product.
  • a suitable variation on the standard batch system is the "fed-batch fermentation" system. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when it is known that catabolite repression would inhibit the metabolism of the cells, and/or where it is desirable to have limited amounts of substrates in the fermentation medium.
  • Continuous fermentation is another known method of fermentation. It is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant density, where cells are maintained primarily in log phase growth. Continuous fermentation allows for the modulation of one or more factors that affect cell growth and/or product concentration. For example, a limiting nutrient, such as the carbon source or nitrogen source, can be maintained at a fixed rate and all other parameters are allowed to moderate.
  • a limiting nutrient such as the carbon source or nitrogen source
  • the produced GAG may be subjected to different purification protocols. Separation and concentration techniques are known in the art and conventional methods can be used to prepare a concentrated solution or broth comprising GAG polypeptide of the invention, such as filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction, or chromatography, or the like, are generally used.
  • the purification method may include a SDS treatment, followed by ethanol precipitation and anionic exchange column DEAE. Detection of submicrogram quantities of glycosaminoglycans may be performed with any method known in the art, for example on agarose gels by sequential staining with toluidine blue and Stains-AII.
  • both agarose gel electrophoresis and sequential toluidine blue/Stains-AII staining can be performed to analyze all the complex glycosaminoglycans and the specificities of the glycosaminoglycan-degrading enzymes.
  • analytical chemistry technique may be performed in quality control and research for determining the content and purity of the product, as well as its molecular structure.
  • FACE analysis for determining the presence and relative abundance of individual (oligo)saccharide, nuclear magnetic resonance (NMR), FTIR (Fourier Transform Infrared Spectroscopy) analysis or mass spectrometry may be performed.
  • Another object of the present invention is a sulfated glycosaminoglycan obtained by a process as defined above.
  • This sulphated GAG may be used for producing pharmaceutical, cosmetic, nutraceutical and and food products, for example as illustrated in Kubaski, F., FI. Osago, R.W. Mason, S. Yamaguchi, FI. Kobayashi, M. Tsuchiya, T. Orii, and S. Tomatsu, Glycosaminoglycans detection methods:
  • FIG. 1 represents model of the 3D structure of PhCS-like from Pyrococcus horikoshii.
  • the model was determined by homology with the chondroitin polymerase, (PBD ID: 2Z87).
  • the SWISS-MODEL template library was searched with BLAST (Camacho, C., G. Coulouris, V. Avagyan, N. Ma, J. Papadopoulos, K. Bealer, and T.L. Madden, BLAST+: architecture and applications. BMC Bioinformatics, 2009. 10: p. 421 ([4])) and HHBIits (Remmert, M., A. Biegert, A. Hauser, and J.
  • HHblits lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods, 2011. 9(2): p. 173-5 ([5])) for evolutionary related structures matching the target sequence.
  • the Model was built based on the target- template alignment using ProMod3 (Guex, N., M.C. Peitsch, and T. Schwede, Automated comparative protein structure modeling with SWISS- MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis, 2009. 30 Suppl 1: p. S162-73 ([6])).
  • FIG. 2 represents schematic structure of PhCS-like predicted transmembrane domains.
  • the transmembrane domains were predicted by the PSIPRED program (MEMSAT-membrane topology prediction).
  • the catalytic motifs represented by YNE, VXDXS, DXD and LWRQRKRW were found to be located at the cytoplasmic N-terminal side.
  • Figure 3 represents the pBAD-TOPO R plasmid.
  • FIG. 4 represents the pET102/D-TOPO R plasmid.
  • FIG. 5 represents the diagram showing the TOPO cloning sites of pBAD-TOPO ® vector (SEQ ID NO: 26). Restriction sites are labeled to indicate the actual cleavage site.
  • FIG. 6 represents the diagram showing the TOPO cloning sites of pET102/D-TOPO ® vector (SEQ ID NO: 27). Restriction sites are labeled to indicate the actual cleavage site.
  • FIG. 7 represents 1 % agarose gel electrophoresis showing the amplified genes.
  • the genes Ph-CS-like (1&2) genes of length 946 bp, respectively were successfully amplified.
  • the first well represents the ladder purchased from biotechrabbit.
  • FIG. 8 represents denaturing electrophoresis gels showing results from 0.2% L-arabinose induced expression of membranous protein expressed in recombinant LMG-194 E. coli strains in a shake-flask culture.
  • the LMG-pBAD-Ph-CS-like expresses Ph-CS-like from Pyrococcus of 37 kDa.
  • the wild type LMG-194 represents the negative control (N.C.).
  • the first well represents the protein ladder SDS-PAGE Standards, Broad range (in kDa.).
  • FIG. 9 shows estimated concentration (mg/L) of the produced GAG from the recombinant E. coli LMG-194 strains in shake flask experiments.
  • the GAG concentration produced from LMG-pBAD-Ph-CS- like was estimated in the presence (+) or absence (-) of 0.2% L-arabinose as revealed by the CTAB turbidimetric test.
  • the wild type LMG-194 was used as a negative control. Experiments were reproduced in duplicate.
  • - Figure 10 represents the estimated concentration (mg/L) of produced GAG from the two recombinant E. coli LMG-194 strains in shake flask versus bioreactor fermentation.
  • the GAG concentration produced from LMG-pBAD-Ph-CS-like was estimated in the presence (+) or absence (-) of 0.2% L-arabinose as revealed by the CTAB turbidimetric test. Experiments were reproduced in duplicate.
  • - Figure 11 represents the estimated concentration (mg/L) of produced GAG in the recombinant BL21 codon plus strains in shake flask cultures.
  • the concentration of produced GAG from pET102-Ph-CS-like was estimated in the absence and presence of IPTG as determined by the CTAB turbidimetric method.
  • the BL21 codon plus-pET102 carrying empty vector was used as a negative control. Experiments were reproduced in triplicate.
  • - Figure 12 represents the estimated concentration (mg/L) of produced GAG in the recombinant C43 (DE3) strains in shake flask cultures.
  • the GAG yield produced from C43-peT102-Ph-CS-like was estimated in the absence and presence of IPTG as determined by the CTAB turbidimetric method.
  • the C43-pET102 carrying empty vector was used as negative control. Experiments were reproduced in duplicate.
  • - Figure 13 represents hyaluronidase digestion of the purified polymer on agarose gel after staining with Stains-AII (A) and after CTAB turbidimetric test for mass estimation of the reaction product (mg) formed after FIAase digestion from the DEAE purified fractions from LMG-pBAD-Ph-CS-like (B). Experiments were reproduced in duplicate.
  • - Figure 14 Agarose gel electrophoresis of standard commercial GAGs and the heterologous biopolymers stained with (B)toluidine blue.
  • Commercial GAGs (1: hyaluronic acid; 2: chondroitin 6-sulfate) and the heterologous biopolymers (3: combined fractions 14 and 15 from E. coli LMG-PhCS-like.
  • - Figure 15 represents FTIR characterization of purified biopolymer produced by recombinant E. coli strains represented by LMG-pBAD-PhCS- like.
  • - Figure 16 represents FTIR characterization of chondroitin 6-sulfate purchased from Carbosynth.
  • - Figure 17 represents One-dimensional NMR spectral analysis of (a) chondroitin 6-sulfate (CS) and (b) hyaluronic acid (FIA) standards.
  • - Figure 19 represents mass spectroscopy using ESI ionization (negative ion mode) to identify GAG oligosaccharides. Chondroitin sulphate oligosaccharides and disaccharides were obtained after hyaluronidase digestion. The products were compared with the standard of chondroitin-6- sulphated products (a) CS-C, (b) polymer from LMG-pBAD-PhCS-like and (1) and after (2) digestion with hyaluronidase. - Figure 20 represents the general strategy for metabolic engineering of E.coli for the production of chondroitin sulfate.
  • Example 1 Genetic engineering of E. coli for the production of a novel glycosaminoglycan
  • Ampicillin Sigma-Aldrich, Cat No.10835242001: The stock solution was prepared at 50 mg/mL in water and used at a final concentration of 100 pg/mL.
  • Chloramphenicol (Sigma-Aldrich, Cat No. C0378): The stock solution was prepared at a concentration of 50 mg/mL in ethanol and used at a final concentration of 35 pg/mL.
  • Kanamycin (Sigma-Aldrich, Cat No. 60615): The stock solution was prepared at a concentration of 100 mg/mL in water and used at a final concentration of 100 pg / mL.
  • Doxycycline (Sigma-Aldrich, Cat No. D1822): The stock solution was prepared at a concentration of 20 mg/mL in water and used at a final concentration of 10 pg/mL.
  • DH5-a strain (Invitrogen, Cat No. 18265017): This cloning strain is developed by D. Hanahan with multiple mutations that enable high-efficiency transformations. The genotype is F-endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 cp80dlacZAM15 D (lacZYA-argF) U169, hsdR17 (rK-mK +) / l- [44]
  • TOP10 strain (Invitrogen, Cat No. C404003): This strain is used also for general cloning of blunt-end PCR products into the pBADTOPO® vector.
  • the genotype is: F- mcrA A(mrr-hsdRMS-mcrBC) O80lacZAM15 AlacX74 recA1 araD139 A(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG.
  • LMG-194 strain (Invitrogen, Cat No.
  • E. coli B BL21 codon plus strain (DE3) RIPL (Stratagene, Cat No.230280): This strain, derivative of E. coli B, is a general protein expression strain that lack both the Lon protease and the OmpT protease.
  • the genotype of E. coli B [F- ompT hsdS(rB - mB - ) dcm+ Tetr gal A(DE3) endA Hte [argU proL Camr ] [argU ileY leuW Strep/Specr ]. It harbors a prophage DE3 derived from a bacteriophage A, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter. The cells also harbor extra copies of the argU, ileY, and leuW as well as the proL tRNA genes.
  • C43 (DE3) (Lucigen, Cat No. 60446-1): This strain is derived from the T7 RNA polymerase (P)-based BL21 (DE3) protein production strain. It is a mutant strain of BL21 (DE3) and carries mutations that lower T7 RNAP expression levels that result in strongly reduced T7 RNAP accumulation levels.
  • the pBAD-TOPO ® (Invitrogen, Catalog No. K4300-01 ) is a vector of 4,126 base pairs. It is shown in Figure 3.
  • the pBAD plasmids harbors Ph-CS-like genes from Pyrococcus horikoshii.
  • the corresponding gene sequence of Ph- CS-like, Pyrococcus horikoshii OT3 (Accession number PH_RS02140) is SEQ ID NO: 1 .
  • the PCR product was amplified with a stop codon within the open reading frame which provides the expression of the gene without the 6x-Histidine tag. It was designated LMG-pBAD-PhCS-like.
  • the selection marker is Ampicillin.
  • the promoter is a weak promoter (araBAD). Induction is possible by L-arabinose.
  • Figure 3 shows the TOPO cloning sites of pBAD-TOPO ® vector. Restriction sites are labeled to indicate the actual cleavage site.
  • the pET102/D-TOPO ® vector (Invitrogen, Catalog No. K102-01) is based on the strong T7 promoter, with IPTG (isopropyl-1 -thio-B-D-galactoside) as the inducible agent.
  • Target protein expression may be initiated by transferring the plasmid into an expression host containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control (DE3 strain). The expression is induced by the addition of IPTG to the bacterial culture.
  • the gene of interest was cloned as a fusion to the only /V-terminal His-Patch thioredoxin for increased translation efficiency and solubility of heterologous proteins. Selection marker is Ampicillin. Its structure is shown in Figure 4.
  • the diagram of Figure 5 shows the TOPO cloning sites of pET 102/D-TOPO ® vector. Restriction sites are labeled to indicate the actual cleavage site.
  • the CCMB80 buffer was prepared by adding 0.98 g KOAc (10 mM), 11.8 g CaCI 2. 2H 2 0 (80 mM), 4.0 g MnCI 2. 4H 2 0 (20 mM), 2.0 g MgCI 2. 6H 2 0 (10 mM), 100 g glycerol (10%), and water to a final volume of 1 L.
  • the pH of solution was adjusted to 6.4 then sterile filtered and stored at 4°C.
  • a single colony of DFI5-a or TOP10 (Invitrogen) was inoculated into 5 ml_ of SOB medium and incubated overnight at 37°C with vigorous shaking at 200-220 rpm.
  • the preculture was diluted by 1/100 to reach an optical density of O ⁇ boo of 0.3.
  • the cells were then centrifuged at 3000 rpm and 4°C for 10 min. The supernatant was discarded and the cells were resuspended in 10 mL of ice cold CCMB80 buffer with a gentle swirl. Then the resuspended cells were incubated on ice for 20 min, centrifuged as before then resuspended in 5 mL of ice cold CCMB80 buffer. Each 100 pL was aliquoted into microcentrifuge tube and freezed at -80°C.
  • the DNA fragment coding for chondroitin synthase-like from Pyrococcus horikoshii was amplified by PCR using the forward and reverse primers listed in table 2.
  • the PCR was carried out using a mixture called "RedTaq Ready Mix” (purchased from Sigma) that contains the Taq DNA polymerase enzyme, the dNTPs and a buffer formed of Mg ++ and NaCI.
  • Each reaction was carried out in a total volume of 40 pi with 20 mI of RedTaq Ready Mix, 1 mI primer forward, 1 mI primer reverse, 17.5 mI sterile water.
  • the CS-like gene amplification program was as follows: initial denaturation at 95 ° C for 1 min, followed by 30 cycles of denaturation at 95 ° C for 1 min, hybridization at 58 ° C for 30 seconds and extension at 72 ° C for 1 min, followed by a final extension for 7 min at 72 ° C needed for the addition of a single deoxyadenosine at the 3 'end of the PCR product required for the TOPO cloning and to ensure the complete extension of the amplified fragments.
  • the PCR products were migrated on 1% agarose by agarose gel electrophoresis to confirm the expected size of the desired DNA fragment.
  • the TOPO Cloning reaction was performed by adding 2 pi of fresh PCR product, 1 mI of vector pBAD, 1 mI of salt solution which allow to increase the transformation efficiency and 2 mI of sterile water, then incubated for 15 min at room temperature and placed on ice to proceed in the transformation of One Shot TOP10 chemically competent E. coli.
  • 3mI of the TOPO cloning reaction was added into a vial of One Shot TOP10 chemically competent E. coli and mixed gently, incubated on ice for 30 minutes then the cells were heat shocked for 1min 30 s at 42°C and transferred to ice immediately.
  • PCR amplification of KfoF and galU The genome of E. coli LMG-194 was used as a template for the amplification of KfoF and galU genes by PCR (PCR1) using specific primers as shown in Table 3. These primers were deisgned to recognize KfoF (UDP-glucose dehydrogenase NCBI-GenelD:946571) and galU (UDP-glucose- phosphorylase NCBI-GenelD:945730) and that added in addition a ribosome binding site (RBS) to increase internal translational efficiency (Table 3). All the primers to be used with the HiFi DNA assembly kit (BioLabs, Cat No.
  • E5520S were generated through the program Nebbuilder available online www.nebuilder.neb.com. Serial cloner was also used to check for the obtained reading frames from the final construct of the plasmid containing the operons (Serial cloner, version 2.6; http://serialbasics.free.fr/Serial Cloner.html ). After the first PCR, a second PCR (PCR2) was applied on the PCR1 products to add specific ends that overlap to the vector or to the adjacent gene.
  • PCR2 was applied on the PCR1 products to add specific ends that overlap to the vector or to the adjacent gene.
  • PCR2 added to the KfoF gene one overlapping end with the plasmid pBAD and another overlapping end either with the plasmid (to have a KfoF-galU operon) or with the GalU gene (to have a KfoF-galU operon).
  • PCR2 added one overlapping end with the KfoF gene and another overlapping end with the pBAD plasmid.
  • PCR reactions were performed using a mixture called "RedTaq Ready Mix” (purchased from Sigma) containing the Taq DNA polymerase enzyme, the dNTPs and a buffer of Mg 2+ and NaCI.
  • the PCR conditions are summarized in the Table 3.
  • Table 3 Conditions of PCR1 and PCR2 for KFOF and GALU genes.
  • Table 4 shows the primers design for PCR1 and PCR2 of KFOF and GALU genes.
  • Each reaction was carried out in a total volume of 40 mI_ with 20 mI_ of RedTaq Ready Mix, 1 mI_ forward primer, 1 mI_ reverse primer, 1 mI_ matrix DNA and 17.5 mI_ sterile water.
  • the PCR1 and PCR2 products were migrated on 1 % agarose gel electrophoresis at 100 V for 1 h to confirm the presence of desired DNA fragment with the correct length using the 1 kb DNA ladder (NIPPON Genetics Europe, Cat No. MWD1 P).
  • DNA cloning by PCR DNA cloning method was achieved by PCR.
  • the necessary primers needed for the amplification and linearization of vectors harboring Ph-CS-like gene from Pyrococcus and the primers required to add specific ends to KFOF gene to overlap with PhCS-like genes from one side and with the pBAD vector from the other side were designed. These overlapping sequences are useful for the cloning reactions of the two genes in the plasmid pBAD.
  • the primers used for the amplification of vectors as well as the KFOF gene (3 and 4) are listed in Table 5.
  • the Long Range PCR allows the amplification of DNA lengths up to 15 kb or longer genomic DNA.
  • Each reaction was carried out in a total volume of 50 pL with 5 pL of dNTP mix, 1 pL of Long PCR Enzyme Mix, 5 pL of 10X Long PCR buffer with 15 mM MgC , 2 pL of DMSO, 1 pL forward primer, 1 pL reverse primer, 0.5 pL of template DNA and 34.5 pL of sterile water.
  • the amplification program consists of three steps cycling protocol listed in the Table 6.
  • Table 62 Three-step cycling protocol.
  • the PCR products were migrated on 1% agarose by agarose gel electrophoresis for 1 h at 100 V to confirm the expected size of the desired DNA fragment.
  • the linearized plasmids were digested with Dpnl enzyme (Thermo SCIENTIFIC, Cat No. ER1701) that cleaves the parental template Dam methylase-methylated plasmid DNA from E. coli.
  • Dpnl enzyme Thermo SCIENTIFIC, Cat No. ER1701
  • 5 pL of PCR product was mixed with 1 pL of 10X buffer Tango (Thermo SCIENTIFIC, Cat No. BY5) and 1 pL Dpnl enzyme with 3 mI_ of sterile water. Each mix was incubated for 1 h at 37°C and heat- inactivated by incubating at 80°C for 20 min.
  • the linearized plasmids (obtained by PCR or restriction enzymes) were purified by gel extraction kit (Sigma-Aldrich, Cat No. NA1111 ). After purification, the KFOF and GALU genes were cloned into the linear pBAD plasmid according to the manufacturer's instructions using the NEBuilder HiFi DNA Assembly protocol (BioLabs, Cat No. E5520S) to obtain synthetic operons CS-like- oF and CS-like-KfoF-GalU for each strain. Then, the competent DFI5-a strains were transformed by thermal shock with each reaction of assembly. The pUC vector was included as a positive control to verify the transformation efficiency of competent cells. The transformed DFI5-a bacteria were incubated at 37°C for 40 min, then inoculated on TB agar plates supplemented with ampicillin 100 pg/mL and incubated overnight at 35°C.
  • the first step of cloning reaction consists of amplifying the target genes.
  • the DNA fragment coding for Ph-CS-like genes from Pyrococcus was amplified by PCR, using the forward primer containing on the 5’ end the 4 base pair sequences (CACC), necessary for directional cloning of the forward primer, in a way such our gene of interest will be optimally expressed and fused in frame with the vector (Table 9).
  • PCR was carried out with 50 mI_ of reaction mixture containing 1 mI_ template DNA, 2.5 mI_ each primer (10 mM), 19 mI_ of sterile water, and 25 mI_ Taq DNA polymerase (Sigma-Aldrich). The PCR assay was performed for 40 cycles under the following conditions, See Table 10.
  • Table 10 PCR conditions for the amplification of Ph-CS-like gene in pET 102 vector.
  • the TOPO cloning reaction was performed by adding 4 pl_ of fresh PCR product, 1 mI_ of vector pET102/D-TOPO ® and 1 mI_ of salt solution, then incubated for 15 min at room temperature and placed on ice to proceed in the transformation of One Shot TOP10 chemically competent E. coli.
  • 3 mI_ of the TOPO cloning reaction was added into a vial of One Shot TOP10 chemically competent E. coli and mixed gently, then incubated on ice for 30 min. The cells were then heat shocked for 1 min 30 s at 42°C and transferred to ice immediately.
  • PhCS-like recombinant protein expressed in LMG-194 BL21 codon plus and C43 (DE3) E. coli expression strains on SDS-PAGE, bacteria transformed with the appropriate vector harboring Ph-CS-like genes from Pyrococccus were grown overnight in shake flask experiments at 37°C with shaking at 235 rpm in SOB medium supplemented with the appropriate antibiotics.
  • SDS sodium dodecyl sulfate
  • polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length.
  • SDS denatures secondary and nondisulfide-linked tertiary structures and coats them with a negative charge that correlates with their length, allowing molecular weights to be estimated.
  • the staining of the gel subsequent to electrophoresis reveals the characteristic band positions.
  • the stacking gel is the upper part for loading the sample. Proteins are well lined up when they reach the lower gel (Resolving gel).
  • the stacking gel (6 %) was prepared in total volume of 5 ml_ by mixing 30 % acrylamide solution, 0.126 M Tris-CI pH 6.8, 0.0035 M SDS, 0.00219 M APS, 0.0144 TEMED and H2O.
  • the resolving gel (12.5 %) was prepared in a total volume of 7.5 ml_ by mixing 30 % acrylamide solution, 0.4 M Tris-CI pH 8.8, 0.003 M SDS, 0.002 M APS, 0.0096 M TEMED (N,N,N',N'-Tetramethylethylenediamine) and H2O.
  • the gel was stained with Brilliant Blue R Staining (0.25 g Coomassie Brilliant Blue R (Sigma-Aldrich, Cat No. 1.12553) in 45% methanol and 10% acetic acid) for 20 min with slight agitation at room temperature to make the separated proteins appear as distinct colored bands on the gel. Finally, the gel was destained with 10% acetic acid and 40% ethanol. The size of the protein was estimated in comparison with the molecular weight marker bands (Thermoscientific, Cat No. 26619).
  • CTM assay Cetyltrimethylammonium bromide turbidimetric method
  • CTM assay was performed to estimate the concentration of the produced reaction product. This test is based on the formation of turbidity between the negatively charged polyanionic polysaccharide reaction product resulting from the polymerizing activity of the recombinant enzyme, and cetyltrimethylammonium bromide (CTAB), which is a cation surface active agent.
  • CTM assay Cetyltrimethylammonium bromide turbidimetric method
  • the turbidity resulting from the precipitation is related to the concentration of reaction products and can be titrated by spectrophotometry and the concentration of the formed reaction products (in this case, resulting from the polymerizing activity of the recombinant enzyme) present in each reaction was calculated based on a calibration line of HA.
  • the CTAB reagent (2.5 g) was dissolved in 100 ml_ of 2% (w/v) NaOH. Different concentrations of HA standard solutions ranging from 0 to 1 mg/mL were prepared in order to check the linearity range of the standard curve. 250 pL of each concentration was introduced into a cuvette containing 500 pl_ of CTM reagent and was incubated at 37°C for 10 min. Same conditions were applied to the samples. Absorbance was read at 600 nm against the blank (HA solution replaced by 0.05 M NaCI solution) and plotted against HA standard concentrations to constitute calibration lines. 2.2. Agarose gel electrophoresis
  • Ph-CS-like gene from Pyrococcus were induced with 0.2% of L-arabinose.
  • the LMG-194 araAC with empty plasmid was served as a negative control for the production experiments.
  • Each fresh transformant was grown overnight at 37°C with shaking at 235 rpm in SOB medium supplemented with 100 pg/mL of ampicillin and 10 pg/mL of doxycycline. Cultures were then diluted by 100 and grown to reach an optical density (O ⁇ boo) of 0.6 upon which gene expression was induced using 0.2% L-arabinose. The cultures were left to grow under 30°C for an additional 18 to 24 h.
  • a fed-batch fermentation process was developed using a 2 L bioreactor (MINIFOR laboratory fermentor) in TB medium.
  • the culture conditions were controlled in term of temperature (37°C), pH (7), oxygenation (22) and agitation (4).
  • the transformed LMG-194 E. coli cells LMG-pBAD-PhCS-like were induced with 0.2% L-arabinose when reached an O ⁇ booq ⁇ 0.6.
  • Another strategy was used to increase the production was by adding 10 g.L -1 glucose and 2.5 g.L -1 phosphate in the fermentation culture of LMG-pBAD-PhCS-like after 6 h of induction with 0.2% L-arabinose.
  • the transformed E. coli BL21 codon plus and C43 (DE3) with either empty pET102 vector or with the plasmids harboring Ph-CS-like gene from Pyrococcus were grown overnight at 37°C with shaking at 235 rpm in SOB medium supplemented with ampicillin (100 pg/mL) and chloramphenicol (25 pg/mL).
  • the cultures were then diluted by 100 and grown to reach an optical density (O ⁇ boo) of 0.6 upon which gene expression was induced using 1 mM isopropyl-1 -thio-B-D-galactoside (IPTG) purchased from Himedia.
  • IPTG isopropyl-1 -thio-B-D-galactoside
  • Fermentation brothes from cultures obtained after 24 h of induction were added to an equal volume of 0.1% of SDS and incubated at room temperature for 10 min and then centrifuged for 10 min at 6000 rpm. The obtained supernatant was treated with 2 volumes of cold absolute ethanol and incubated overnight at 4°C. The precipitate was then collected by centrifugation (3000 rpm for 10 min) and resuspended in 1/10 volume of 0.1 M NaCI by agitation for 10 min and the concentration of the obtained biopolymer was estimated by CTM assay based on the formation of turbidity between the negatively charged formed polyanionic polysaccharide and the cationic CTAB. Therefore, the turbidity resulting from the precipitation is titrated by spectrophotometry and is related to the concentration of the produced GAG.
  • the produced biopolymer was then subjected to different purification protocols to remove all the contaminants from the fermentation broth.
  • Trichloroacetic acid and charcoal treatment after proceeding with SDS treatment and ethanol precipitation, the nucleic acids and bacterial derived proteins were removed by lowering the pH of the broth from 6 to 2 by addition of trichloroacetic acid (10%) and subsequent charcoal treatment (2%) for 1 h followed by centrifugation at 7000 rpm for 30 min at 4°C. Then the supernatant was passed through a 0.45 pm filter and desalted in 300 kDa cut-off cassette (vivaspin 20 ml_) then concentrated by adding 2 volume of ethanol to be further lyophilized for NMR analysis.
  • samples were first treated with 1 mg/L of Pierce Universal Nuclease (Thermo SCIENTIFIC, Cat. No. 88700) and incubated for 1 h at 37°C for cell lysis and then with 2.5 mg/mL of proteinase K (Biotechrabbit, Cat. No. BR1100901) and incubated for 2 h at 56°C. Then each sample was passed through HiTrap Capto DEAE 5 ml_ with liquid chromatography AKTATM system.
  • the liquid chromatography system such as AKTATM can separate substances based on their charges using an ion exchange resin containing positive charged group such as HiTrap Capto DEAE 5 ml_.
  • the gels were stained with either stains-all for 24 h at room temperature to be then destained with 10% ethanol for 1 h or with 0.1% toluidine blue prepared in a solution of 1% acetic acid, 50% ethanol and 49% water and incubated for 15 min at room temperature to be then destained with the same solution without toluidine blue. 3.1.1.2. Analysis of the produced glycosaminoglycan by hyaluronidase digestion
  • glycosaminoglycan was performed using hyaluronidase from bovine testes (Sigma-Aldrich cat. No. H 3506). This enzyme catalyzes the hydrolysis of (1-4) linkages between /V-acetylhexosamine and D- glucuronate residues in chondroitin, chondroitin-4-sulfate, chondroitin-6- sulfate, and hyaluronic acid.
  • the hyaluronidase was prepared at 1 mg/mL in HSE buffer (20 mM sodium phosphate buffer, pH 7.0, with 77 mM sodium chloride and 0.1 mg/mL BSA) or in 50 mM of ammonium acetate buffer (1.84 mL glacial acetic acid, 1.48 g sodium acetate in 350 mL H2O, pH 4.5).
  • IR infrared radiation
  • Thermo-Nicolet, Avatar 320- FT-IR infrared radiation
  • Potassium bromide (KBr, spectroscopic grade) is typically used as the window material because it is transparent in the IR. Alternatively, samples can be contained within a KBr matrix and pressed to form a pellet that is then analysed.
  • the signal processing is a Fourier transform. The acquisition conditions were as follow: Scanning from 0 to 4000 cm-1, 32 scans, resolution of 1 cm-1. Collection of the spectrum in Transmittance after acquisition of a spectrum background. For processing: simple FT then subtraction of the background spectrum from the product spectrum. Auto indexing of peaks or manual.
  • Mass spectrometry for the detection of disaccharides after hyaluronidase digestion
  • MS Mass spectrometry
  • MS analysis was performed on the chondroitin sulfate as well as the lyophilized fractions containing the polymer of interest before and after digestion with hyaluronidase (See paragraph 3.1.1.2).
  • the lyophilized fractions containing the polymer of interest were diluted in a total volume of 100 pL in water and applied to MS, Shimadzu LCMS-2020 Prominence UFLC system with a single quadrupole mass spectrometer using ESI ionization (negative ion mode).
  • the pBAD expression system is used for the expression of heterologous genes in E. coli under the control of a weak promoter induced by L- arabinose. This system was chosen for the expression of the PhCS-like protein and production of its corresponding glycosaminoglycan because it is generally used for the production of biomolecules that results from a strong competition between cell growth and biomolecule accumulation.
  • the pBAD system was successfully used for the production of recombinant hyaluronic acid by an E. coli strain expressing a FIAS synthase from a Streptococcus ([1]). Recombinant E.
  • the pET system offers an advantage over the pBAD system that it features a strong T7 promoter that permits high-level, IPTG inducible expression by which a recombinant protein can accumulate to up to 50% of total cellular proteins. This can be used to achieve a maximal yield of GAG production.
  • the choice of vector and expression host can significantly increase the activity and the amount of target protein present in the soluble fraction.
  • the pET102/D-TOPO ® vector generally allows to clone the gene of interest as a fusion to /V-terminal His-Patch thioredoxin for increased translation efficiency and solubility of heterologous proteins.
  • the thioredoxin is a 11 .7 kDa protein that was originally isolated from E.
  • thioredoxin can increase translation efficiency.
  • the thioredoxin protein has been mutated to contain a metal binding domain, and is termed “His-Patch thioredoxin”.
  • this system takes advantage of the high activity and specificity of the bacteriophage T7 RNA polymerase that allows regulated expression of heterologous genes in E. coli lambda DE3 lysogen which carries the gene for T7 RNA polymerase. Accordingly, two strains were used for production experiments, the BL21 codon plus and the C43 (DE3).
  • the BL21 codon plus is generally used for high-level protein expression and easy induction in T7 expression systems. It harbors a prophage DE3 derived from a bacteriophage l, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter.
  • the cells harbor extra copies of tRNA coding genes (AUA, AGG, AGA, CUA, CCC and GGA) compensating for the rare tRNA and is used to overcome the expression problems coming from codon bias and therefore improves the expression of foreign protein.
  • Such strain lacks also both the Lon protease and the OmpT protease that can degrade the expressed foreign protein.
  • the C43 (DE3) is usually effective for the expression of toxic and membrane proteins as it carries mutations that lower T7 RNAP expression levels that results in strongly reduced T7 RNAP accumulation levels. As a consequence, membrane protein production stress is alleviated, thereby increasing membrane protein yields without toxic effects.
  • the cells were then incubated at 37°C to reach an optical density (OD) of 0.6 at 600 nm, upon which a final concentration of 0.2% of L-arabinose or 1 mM IPTG was added to induce the expression of the genes of interest in pBAD or pET expression system, respectively.
  • OD optical density
  • Samples from non-induced and induced strains were taken at different times of induction, denatured at 95°C and loaded on the polyacrylamide gel for further separation on the basis of molecular masses. The Coomassie- brilliant blue was used to stain the proteins.
  • the cells were then incubated at 37°C to reach an optical density (OD) of 0.6 at 600 nm, upon which a final concentration of 0.2% of L-arabinose was added to induce the expression of the genes of interest.
  • OD optical density
  • the concentration of 0.2% L-arabinose was chosen based on previous experiments performed in the laboratory where it has been found that raising the concentration of L-arabinose above 0.2% did not result in further increase in the concentration of the obtained biopolymer as determined by the CTAB turbidimetry assay (CTM assay). This test is based on the formation of turbidity between the negatively charged polyanionic polymers (like HA and chondroitin) and CTAB, a cation surface active agent.
  • the turbidity resulting from the precipitation is related to the polymer concentration and can be titrated by spectrophotometry and the concentration was calculated based on a HA standard curve.
  • the temperature was then down shifted from 37 to 30°C after induction to allow better expression of the recombinant proteins.
  • the fementation broth was treated with SDS followed by ethanol precipitation and resuspension in NaCI to further determine the concentration of the produced GAG by the CTM assay. Induced and non-induced cultures were included to confirm that the produced polysaccharide is specific only to the induced-system.
  • the process transfers from shake flasks to a bioreactor had a visible effect on the concentration showing around 1 to 2-fold increase in GAG production, from 71.01 mg/L to 180.55 mg/L in LMG-pBAD-PhCS-like (see Figure 10).
  • the agarose gel electrophoresis constitutes a sensitive method to visualize the non radiolabelled GAGs in submicrogram quantities in a complex polysaccharide mixture by sequential stains-all/toluidine blue staining.
  • the carbocyanine based dye Stains-all (1-Ethyl-2-[3-(1-ethylnaphto[1 ,2- d]thiazoline-2-ylidene)-2-methyl-propenyl]naphto[1 ,2-d]thiazolium bromide) has been increasingly used to stain and identify the non-sulfated hyaluronic acid and other GAGs in submicrogram quantities with basis on their specific colors, at least on a screening level.
  • the non-sulfated HA was stained in bright-blue and the sulfated GAGs such as dermatan sulfate was stained in purple; heparin in yellow and keratan sulfate in light- red.
  • GAG showed a faster migration pattern than a sample of purified commercial hyaluronic acid migrated along the samples.
  • the color of the produced biopolymer corresponded more to the color code known for the chondroitin and not for hyaluronic acid. Both of the migration pattern and the color of the biopolymer revealed that it is more biochemically similar in composition to chondroitin than to hyaluronic acid.
  • the GAG polysaccharide resulting from the recombinant BL21 codon plus E. coli strain was analyzed after SDS treatement, ethanol precipitation and fractionation by DEAE purification. Results showed a contaminating polymer present in the induced and the negative control strain BL21 codon plus that could not be even resolved after purification by anion exchange chromatography. This could explain the presence of an endogenous polymer synthesized by E.coli BL21 codon plus itself and the production was not specific to the induced recombinant strain.
  • LMG-194 strain using the inducible plasmid backbone of pBAD was the best choice to produce the following biopolymer. Further characterization methods were examined to identify the structure of the following GAG. 3.3.2.1.1. Analysis of the produced glycosaminoglycan by hyaluronidase digestion
  • the agarose gel electrophoresis can analyze all the complex GAGs and the specificities of the glycosaminoglycan-degrading enzymes. To go deeper into the nature of the produced GAG from the recombinant LMG-194 E. coli strain, enzymatic digestion by hyaluronidase (HAase) was performed on the purified fractions containing the polysaccharide of interest and analyzed by agarose gel electrophoresis after staining with Stains-all.
  • HAase hyaluronidase
  • the hyaluronidase enzyme can hydrolyze randomly the (1-4)-linkages between /V-acetyl- -D-glucosamine and D-glucuronate residues in hyaluronate and the (1-4)- -D-glycosidic linkages between /V-acetyl-galactosamine or N- acetylgalactosamine sulfate and glucuronic acid in chondroitin, chondroitin 4- and 6-sulfates, and dermatan.
  • the ability of HAase to attack the produced GAG by the recombinant E. coli strains would confer essential features about the nature of this polysaccharide.
  • HA HA.
  • GlucNAcp-(1-4)-Glucuronate or GalNAc (sulfated or not) p-(1-4)-Glucuronate linkages.
  • GalNAc sulfated or not
  • a glycosaminoglycan containing different uronic acid such as GalNAc (sulfated or not) p-(1-4)-lduronate linkages found in Dermatan sulfate could not be excluded at this stage.
  • the formation of this polymer requires the presence of a downstream epimerase enzyme which is activated after chondroitin biosynthesis.
  • glycosaminoglycan The occurrence of such a glycosaminoglycan is restricted to eukaryotes and is not documented in any of the glycosaminoglycans isolated from prokaryotes previously (Paul. L DeAngelis, Microbial glycosaminoglycan glycosyltransferases, glycobiology, 2002 ([14])).
  • CTM turbidity assay was performed on the lyophilized fractions 14 and 15, eluted between 0.35 and 0.45 M NaCI respectively by DEAE purification.
  • This test was done to screen the hydrolysis activity of hyaluronidase against the produced GAG by which the amount of turbidity developed when CTAB is added is proportional to the amount of the formed reaction product.
  • the results were in agreement with those of agarose gel.
  • the total mass of reaction product was decreased from 6.73 to 3.37 mg and from 5.07 to 2 mg in the fractions 14 and 15 respectively of LMG-pBAD-PhCS-like after 24 h of digestion with hyaluronidase.
  • FTIR spectroscopy was applied on the lyophilized fractions after DEAE purification containing the polysaccharide of interest produced from the recombinant E. coli strains LMG-pBAD-PhCS-like. This technique is based on measuring the infrared absorption of a test sample over a narrow range between 400-4000 cm -1 .
  • NMR analysis was performed first on the lyophilized total cell extracts resulting from the fermentation of recombinant E. coli strains carrying Ph- CS-like from Pyrococcus after SDS treatment and ethanol precipitation.

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EP21740109.0A 2020-07-15 2021-07-15 Isoliertes gen für das enzym glycosyl transferase 2 aus pyrococcus horikoshin ot3 und seine homologe Pending EP4182033A1 (de)

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PCT/EP2021/069863 WO2022013399A1 (en) 2020-07-15 2021-07-15 Isolated gene coding for the enzyme glycosyl transferase 2 from pyrococcus horikoshii ot3 or its homologs from hyperthermophilic archaea, host cell expressing it and its use in a process for producing sulfated glycosaminoglycans

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