Patent Application
20240026406
The text of the computer readable sequence listing filed herewith, titled “38573-601_SEQUENCE_LISTING_ST25”, created Sep. 15, 2021, having a file size of 388,374 bytes, is hereby incorporated by reference in its entirety.
The present disclosure provides xanthanase polypeptides, compositions, and uses thereof. The present disclosure also provides polynucleotides, expression vectors, host cells, and genetically modified organisms (e.g., bacteria) encoding xanthanase or xanthan-utilizing gene loci.
Xanthan gum (XG) is an exopolysaccharide produced by Xanthamonas campestris that has been increasingly used as a food additive at concentrations of 0.05-0.5% (w/w) to increase stability, viscosity, and other properties of processed foods. Xanthan gum may also be included in foods as a replacement for gluten at up to gram quantities per serving. The polymer backbone is similar to (mean cellulose, having β-1,4-linked glucose residues, however, xanthan gum contains trisaccharide branches on alternating glucose residues consisting of an α-1,3-mannose, β-1,2-glucuronic acid, and terminal β-1,4-mannose. Xanthan gum has also been used extensively in non-food industries. For example, the oil and gas industry uses xanthan gum in drilling fluid or mud for its rheological properties and in the secondary and tertiary recovery of petroleum.
Disclosed herein are polypeptides comprising a truncated xanthanase, wherein the truncated xanthanase comprises a glycoside hydrolase family 5 endoglucanase domain and three carbohydrate binding domains. In some embodiments, the polypeptides comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptides comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 33. Also disclosed herein are polynucleotides comprising a nucleic acid sequence encoding the polypeptides, expression vectors comprising the polynucleotides operably linked with a promoter and host cells comprising the polynucleotides or expression vectors.
Further disclosed herein are compositions comprising the polypeptides disclosed herein. In some embodiments the compositions are cleaning compositions. In some embodiments the compositions are wellbore servicing compositions. The compositions may be liquids, gels, powders, granulates, pastes, sprays, bars, or unit doses. Also disclosed are methods comprising contacting an object or a surface with the polypeptide disclosed herein or a composition thereof.
Additionally, methods of making intermediate sized xanthan gums and/or pentasaccharide repeating units of xanthan gum are disclosed. The methods comprise contacting xanthan gum or a composition comprising xanthan gum with the polypeptides disclosed herein or compositions thereof.
Additionally, genetically modified organisms (e.g., bacteria) and compositions thereof are disclosed. In some embodiments, the genetically modified organisms comprise the polypeptides or polynucleotides disclosed herein. In some embodiments the genetically modified organisms comprise a heterologous xanthan-utilization gene or gene locus, wherein the heterologous xanthan-utilization gene or gene locus comprises one or more nucleic acids encoding a xanthan or xanthan oligonucleotide degrading enzyme. In some embodiments, the xanthan or xanthan oligonucleotide degrading enzyme comprises a glycoside hydrolase family 5 enzyme from Ruminococcaceae UCG13. The bacteria, for example, may be in the genus Bacteroides, Parabacteroides, Alistipes, Prevotella, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacteria, Escherichia, or Lactobacillus.
Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The present disclosure provides a polypeptide comprising a xanthanase (an enzyme capable of degrading xanthan gum) which can hydrolyze xanthan gum in a single step compared to known xanthanase enzymes which typically require two enzymes. The enzyme generates xanthan degradation products, including pentasaccharide repeating units and intermediate sized xanthan gums, poly- and oligo-saccharides of average molecular weight less than native xanthan gum but more than a single pentasaccharide repeating unit. Additionally, two genetic loci from two microbes have been identified as having xanthan-degrading activity which may be introduced alone or with the xanthanase polypeptide to into heterologous bacteria for use as probiotics in subjects who suffer from gastrointestinal or metabolic diseases or inject a larger than average level of xanthan gum.
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
“Polynucleotide” or “oligonucleotide” or “nucleic acid,” as used herein, means at least two nucleotides covalently linked together. The polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. Polynucleotides may be single- or double-stranded or may contain portions of both double stranded and single stranded sequence. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide” and “protein” are used interchangeably herein.
A “polysaccharide” or “oligosaccharide” is a linked sequence of two or more monomeric carbohydrates connected by glycosidic bonds. The polysaccharides can be natural, synthetic, or a modification or combination of natural and synthetic. polysaccharide may be modified by the addition of sugars, lipids or other moieties not included in the main chain of the polysaccharide.
An “expression vector,” as used herein, refers to a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. The term “operably linked” means a configuration in which a control sequence (e.g., a promoter) is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
The term “bacterial artificial chromosome” or “BAC” as used herein refers to a bacterial DNA vector. BACs, such as those derived from E. coli, may be utilized for introducing, deleting, or replacing DNA sequences of non-human mammalian cells or animals via homologous recombination. E. coli can maintain complex genomic DNA as large as 500 kb or greater in the form of BACs (see Shizuya and Kouros-Mehr, Keio J Med. 2001, 50(1):26-30), with greater DNA stability than cosmids or yeast artificial chromosomes. In addition, BAC libraries of human DNA genomic DNA have more complete and accurate representation of the human genome than libraries in cosmids or yeast artificial chromosomes. BACs are described in further detail in U.S. application Ser. Nos. 10/659,034 and 61/012,701, which are hereby incorporated by reference in their entireties.
The term “host cell,” as used herein, refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
As used herein, “genetically modified” refers to an organism (e.g., a bacterium) which has a modification to introduce a nucleic acid that does not naturally occur in the organism or to introduce additional copies or modified forms of nucleic acids that naturally occur in the organism. The nucleic acid can be integrated in one or more copies into a genome or one or more copies of the nucleic acid can remain episomal, e.g., in a plasmid, phagemid or artificial chromosome.
The term “textile,” as used herein, refers to any textile material including yarns, yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and towelling. The textile may be cellulose based such as natural cellulosics, including cotton, flax/linen, jute, ramie, sisal or coir, or manmade cellulosics (e.g., originating from wood pulp) including viscose/rayon, ramie, cellulose acetate fibers (tricell), lyocell or blends thereof. The textile or fabric may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymer such as nylon, aramid, polyester, acrylic, polypropylene, and spandex/elastane, or blends thereof as well as blend of cellulose based and non-cellulose based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g., rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell).
A “wellbore,” as used herein, refers to any hole drilled to aid in the exploration and/or recovery of natural resources, including oil, gas, or water. For example, a wellbore may be the hole that forms a well. A wellbore can be encased, for example by materials such as steel and cement, or it may be uncased.
As used herein, “treat,” “treating” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a composition described herein to an appropriate control subject. The term also means a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the cell proliferation. As such, “treating” means an application or administration of the compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.
As used herein, the terms “providing,” “administering,” “introducing,” are used interchangeably herein and refer to the placement of the compositions of the disclosure into a subject by a method or route which results in at least partial localization of the composition to a desired site. The compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.
Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The present disclosure provides a polypeptide comprising a truncated xanthanase. The xanthanase has activity on xanthan gum, both native and lyase-treated xanthan gum. In contrast to other known xanthanases, the truncated xanthanase cleaves the reducing end of the non-branching backbone glucosyl residue of xanthan gum (
The truncated xanthanase may comprise a glycosyl hydrolase 5 endoglucanase domain and three carbohydrate binding domains. The glycosyl hydrolase 5 endoglucanase domain comprises an amino acid sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, or 95%) sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 33.
The present disclosure also provides nucleic acids encoding the polypeptides described herein. In some embodiments, the polynucleotides disclosed herein can be introduced into an expression vector, such that the expression vector comprises a promoter operably linked to the polynucleotides encoding the peptides or polypeptides described herein. The expression vector may allow expression of the peptides or polypeptides in a suitable expression system using techniques well known in the art, followed by isolation or purification of the expressed peptide or polypeptide of interest. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art and any such expression system can be used. Alternatively, a polynucleotide encoding a peptide of the invention can be translated in a cell-free translation system.
The selection of promoter will depend on the expression system being used. For example, suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus lichemformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene.
The expression vector may contain other control, selectable marker, or tag sequences. Control sequences include additional components necessary for the expression of a polynucleotide, including but not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription or translation terminator. The control sequence(s) may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
The selectable marker and any other parts of the expression construct may be chosen from those available in the art. In some embodiments, the selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like and thereby permits easy selection of transformed, transfected, transduced, or the like cells. The selectable markers are primarily dictated by the host cell being used. For example, bacterial selectable markers commonly include markers that confer resistance to antibiotics, for example ampicillin, kanamycin, chloramphenicol, or tetracycline.
Various types of expression vectors are available in the art and include, but are not limited to, bacterial, viral, and yeast vectors. For example, the vector may include a plasmid, cosmid, bacteriophage, p1-derived artificial chromosome (PAC), bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or mammalian artificial chromosome (MAC). The various vectors may be selected based on the size of polynucleotide inserted in the construct.
Also provided is a host cell comprising the polynucleotides or the expression vectors described herein. The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote. The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
In some embodiments, the host cell is a gastrointestinal microbiota (gut flora) microorganism that is modified to express and/or secrete the polypeptides described herein. Such host cells find use in populating gastrointestinal systems of host organisms (e.g., people, livestock, etc.) to regulate (e.g., increase) that ability of the host organism to digest or otherwise process xanthan gum. These host cells find particular use in subject that have a high dietary intake of xanthan gum (e.g., human subject on a low gluten or gluten-free diet). Host cells that find use in such application include, for example, bacteria belonging to the genera Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, and/or Bifidobacterium. Such host cells may be introduced into a subject by any suitable methodology including, but not limited to, administration of probiotics containing the host cells and fecal microbiota transplantation. In some embodiments, endogenous gastrointestinal microbiota are genetically modified.
The present disclosure further provides compositions comprising the polypeptides described herein and methods of use thereof. The composition may take on any desired form (e.g., liquid, gel, powder, granulate, paste, spray, bar, unit dose, microcapsule, and the like). The compositions and the polypeptides described herein may be used in any application which requires or it is beneficial to degrade or remove xanthan gum.
In some embodiments, the composition is a cleaning composition. The cleaning composition includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and dish washing detergents); hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops, floors, tables, walls, and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-treaters.
The cleaning compositions may comprise one or more additional enzymes, such as proteases, amylases, lipases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, peroxidaes, catalases, mannanases, redox enzymes, or any mixture thereof. The cleaning compositions may also comprise one or more components selected from surfactants, builders, chelating agents, bleaching components (e.g., precursors, activators, catalysts), antibacterial agents, antifungal agents, polymers, degreasers, corrosion inhibitors, stabilizers, antioxidants, colorants, fragrances, foaming agents, emulsifiers, moisturizers, abrasives, binders, viscosity controlling agents, and pH controlling agents. One of skill in the art is capable of selecting the additional components based on the desired functionality of the composition.
In some embodiments, the composition is a well treatment composition or a wellbore servicing composition. Xanthan gum is commonly used for increasing the viscosity of drilling fluids (e.g., drilling mud, drill-in fluids, or completion fluids). Compositions comprising a xanthanase, such as those disclosed herein, may be used to decrease viscosity of the fluids and/or clean well bores and wellbore filter cakes. Filter cakes are coatings on the walls of the wellbore that limit drilling fluid losses, preserve the integrity of the drilling fluid, prevent formation damage, and provide a balanced density. To form a filter cake, the drilling fluid is often intentionally modified with a weighting material including barite, iron oxide, or calcium carbonate and some particles of a size slightly smaller than the pore openings of the formation. It is these particles which may contain xanthan gum and improve viscosity and emulsification properties of the drilling fluid.
The well treatment composition or wellbore servicing composition may also comprise one or more additional components selected from chelating agents; converting agents (carbonate, nitrate, chloride, formate, or hydroxide salts); other polymer removal agents (persulfate salt, a perborate salt, a peroxide salt, and other enzymes, for example, amylases, glucanases, mannanases, cellulases, oxidoreductases, hydrolases, lyases); organic solvents; surfactants; binders; an aqueous liquid, which may be water, brine, seawater, or freshwater; fragrances; colorants; dispersants; pH control agents or acidifying agents; water softeners or scale inhibitors; bleaching agents; crosslinking agents; antifouling agents; antifoaming agents; anti-sludge agents; corrosion inhibitors; viscosity modifying agents; friction reducers; freeze point depressants, iron-reducing agents; and biocides. One of skill in the art is capable of selecting the additional components based on the desired functionality of the components. Exemplary compositions and methods of using well treatment or wellbore servicing compositions can be found in U.S. Pat. Nos. 5,881,813, 6,110,875, and 9,890,321 and U.S. Patent Publications 2020/0131432 and 2020/0115609; each incorporated herein by reference in its entirety.
The present disclosure provides methods of cleaning utilizing the polypeptides or compositions disclosed herein. The methods comprise contacting an object or a surface with the polypeptides or compositions disclosed herein. In some embodiments, the methods further comprise at least one or both of rinsing the object or surface and drying the object or surface. In some embodiments, the object or surface comprises a textile, a plate, tile, dishware, silverware, glass, a wellbore, or wellbore filter cake.
The process of contacting can be done in a variety of different ways, depending on the composition and the subject or object being cleaned. For example, the composition can be diluted into water to for a cleaning solution which is then contacting the surface or object as commonly done in dishwashing, laundry, and floor cleaning applications. The composition may be directly applied to the surface or object as a spray, liquid, foam, or solid, as is common for fabric spot treatments and hard surface cleansers. The contacting may be carried out for any period of time and may include a soaking period in which the object or surface remains in contact with the composition for a period of time, for example, for at least about 1 hour, at least about 4 hours, at least about 8 hours, at least about 16 hours, or at least about 24 hours.
For cleaning of a wellbore or wellbore filter cake, the composition can be injected into the wellbore to dissolve the filter cake within, the composition can be injected directly at the site of a filter cake, the composition can circulate in the wellbore for a period of time, or the composition may be left in the wellbore in a static manner to soak the wellbore or filter cake.
The present disclosure provides methods of modifying xanthan gum in a subject (e.g., in a digestive tract of a subject). In some embodiments, polypeptides are provided to the subject. In some embodiments, the polypeptides are provided orally such that they are made available in the digestive tract (e.g., mouth, stomach, small intestine, large intestine, etc.) at a concentration sufficient to digest xanthan gum present in the subject. In some such embodiments, purified polypeptides are provided in a capsule or other carrier that releases the peptides at a desired location in the digestive tract. In some embodiments, polypeptides are made available by expressing them in a host cell in a subject. In some embodiments, the host cell is a gastrointestinal microbiota microorganism. The polypeptide may be transiently or stably expressed in the microorganism. A nucleic sequence encoding the polypeptide may be under the control of a promoter that provides optimized expression (e.g., overexpression) of the polypeptide. In some embodiments, the promoter is an inducible promoter that permits control over the timing and/or level of expression. In some embodiments, the polypeptide is encoded by a nucleic acid sequence that further encodes a signal sequence such that the translated polypeptide contains the signal sequence. Signal sequences find use, for example to increase extracellular secretion of the polypeptide.
The present disclosure also provides methods of making intermediate sized xanthan gums and/or pentasaccharide repeating units of xanthan gum. The methods comprise contacting xanthan gum or a composition comprising xanthan gum with the disclosed truncated xanthanase or compositions thereof. The contacting may be done for various lengths of time or under various conditions which facilitate activity of the xanthanase. One of skill in the art can monitor the reaction and the products produced by using any carbohydrate analysis method known in the art, including but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), and quantitative size exclusion or molecular sieve chromatography.
The truncated xanthanase cleaves the reducing end of the non-branching backbone glucosyl residue of xanthan gum. The length or molecular weight of the intermediate sized xanthan gums and/or the relative percentage of pentasaccharide repeating units of xanthan gum formed can be regulated by changing the length of time in which the enzyme is in contact with the xanthan gum, the temperature of the reaction, and/or the quantity of the enzyme.
The intermediate sized xanthan gums and/or pentasaccharide repeating units of xanthan gum may be purified and employed in a number of applications or, alternatively, further modified using chemical modifications known in the art for xanthan gum and other starches. The intermediate sized xanthan gums and/or pentasaccharide repeating units of xanthan gum may be utilized in applications in which rheological and viscosity characteristics different from those conferred by native xanthan gum are desired. For example, the intermediate sized xanthan gums and/or pentasaccharide repeating units of xanthan gum may be employed in drilling fluids/muds, cosmetics, water-based paints, construction and building materials, food products, drug delivery compositions, hydrogels, and tissue engineering (See Kumar, A., et al., Carbohydr Polym 180:128-144 (2018) and Ramburrun, et al., Expert Opin. Drug Deliv. 14, 291-306 (2017), both incorporated herein by reference in their entirety).
The present disclosure provides genetically modified bacteria. In some embodiments, the genetically modified bacteria comprise the truncated xanthanase polypeptides or polynucleotides disclosed herein. In some embodiments, the genetically modified bacteria comprise a heterologous xanthan-utilization gene or gene locus.
The heterologous xanthan-utilization gene or gene locus may comprise one or more nucleic acids encoding a xanthan or xanthan oligosaccharide degrading enzyme. The xanthan or xanthan oligosaccharide degrading enzyme may comprise a glycoside hydrolase, a xanthan or polysaccharide lyase, a mannanase, or a carbohydrate esterase.
In some embodiments, the xanthan-utilization gene or gene locus comprises a gene encoding a glycoside hydrolase family 5 enzyme from Ruminococcaceae UCG13. In some embodiments, the glycoside hydrolase family 5 enzyme may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or 3. In some embodiments, the glycoside hydrolase family 5 enzyme may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 33.
The heterologous xanthan-utilization gene or gene locus may further comprise one or more nucleic acids encoding at least one or all of: one or more carbohydrate uptake proteins; one or more carbohydrate esterases; a polysaccharide lyase family protein (PL); a glycoside hydrolase family 88 (GH88); a glycoside hydrolase family 94 (GH94); and a glycoside hydrolase family 38 (GH38). In some embodiments, the heterologous xanthan-utilization gene or gene locus further comprises one or more nucleic acids encoding each of: one or more carbohydrate uptake proteins; one or more carbohydrate esterases; a polysaccharide lyase family protein (PL); a glycoside hydrolase family 88 (GH88); a glycoside hydrolase family 94 (GH94); and a glycoside hydrolase family 38 (GH38).
Carbohydrate uptake proteins include any proteins or enzymes necessary for the import of carbohydrates, including xanthan oligosaccharides, into the bacterial cell. Carbohydrate uptake proteins may include, but are not limited to, carbohydrate binding proteins and carbohydrate transporters. In some embodiments, the carbohydrate uptake proteins include transporters capable of transporting xanthan oligosaccharides produced by the xanthanase described herein.
Polysaccharide lyases (or eliminases) are a class of enzymes that act to cleave certain activated glycosidic linkages present in polysaccharides. These enzymes act through an eliminase mechanism, rather than through hydrolysis, resulting in unsaturated oligosaccharide products. Polysaccharide lyases are endogenous to various microorganisms, bacteriophages, and some eukaryotes. The polysaccharide lyases have been classified into approximately 40 families available through the Carbohydrate Active enZyme (CAZy) database.
In some embodiments, the polysaccharide lyase family protein comprises a polysaccharide lysase family 8 protein. In some embodiments, the polysaccharide lyase family protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 4.
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to formation of sugar hemiacetal or hemiketal products. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. The glycoside hydrolases have been classified into more than 100 families available through the Carbohydrate Active enZyme database. Each family contains proteins that are related by sequence, and by extension, tertiary structure. A number of glycoside hydrolases may be used in the heterologous xanthan-utilization gene or gene locus disclosed herein.
In some embodiments, the glycoside hydrolase is from the glycoside hydrolase family 88 (GH88). In some embodiments, the glycoside hydrolase family 88 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 8.
In some embodiments, the glycoside hydrolase is from the glycoside hydrolase family 94 (GH94). In some embodiments, the glycoside hydrolase family 94 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 5.
In some embodiments, the glycoside hydrolase is from the glycoside hydrolase family 38 (GH38). In some embodiments, the glycoside hydrolase family 38 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 6 or SEQ ID NO: 7.
Carbohydrate esterases are a group of enzymes which release acyl or alkyl groups attached by ester linkage to carbohydrates. The carbohydrate esterases catalyze deacetylation of both O-linked and N-linked acetylated saccharide residues (esters or amides). The carbohydrate active enzyme database has 16 classified families of carbohydrate esterases. In some embodiments, the carbohydrate esterase used herein is capable of deacetylating xanthan oligosaccharides produced by the xanthanase described herein. The heterologous xanthan-utilization gene or gene locus may include one or more carbohydrate esterases. In some embodiments, the one or more carbohydrate esterases independently comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the heterologous xanthan-utilization gene or gene locus includes two carbohydrate esterases, ones having an amino acid sequence having at least 70% identity to SEQ ID NO: 9 and the other having an amino acid sequence having at least 70% identity to SEQ ID NO: 10.
The heterologous xanthan-utilization gene or gene locus may further comprise, in addition or alternatively, one or more nucleic acids encoding at least one or all of: one or more carbohydrate uptake proteins; a polysaccharide lyase family protein (PL); a glycoside hydrolase family 88 (GH88); a glycoside hydrolase family 92 (GH92); and a glycoside hydrolase family 3 (GH3). In some embodiments, the heterologous xanthan-utilization gene or gene locus further comprises two carbohydrate uptake proteins. In some embodiments, the heterologous xanthan-utilization gene or gene locus further comprises each of two carbohydrate uptake proteins and at least one or all of: a polysaccharide lyase family protein (PL); a glycoside hydrolase family 88 (GH88); a glycoside hydrolase family 92 (GH92); and a glycoside hydrolase family 3 (GH3). In some embodiments, the heterologous xanthan-utilization gene or gene locus further comprises each of two carbohydrate uptake proteins, a polysaccharide lyase family protein (PL), a glycoside hydrolase family 88 (GH88), a glycoside hydrolase family 92 (GH92), and a glycoside hydrolase family 3 (GH3).
The carbohydrate uptake proteins may include members of the starch utilization system (Sus) of Bacteroides. The Sus includes the requisite proteins for binding and processing carbohydrates at the surface of the cell and, the subsequent oligosaccharide transport across the membrane for further degradation. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. The carbohydrate uptake protein may include SusC or SusD or homologs or variants thereof from Bacteroides known in the art (See, for example, Xu, et al., PLoS Biol. 2007 July; 5(7): e156 and Foley, et al., Cell Mol Life Sci. 2016 July; 73(14): 2603-2617, both incorporated by reference herein in their entirety. In some embodiments, the one or more carbohydrate uptake proteins independently comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the one or more carbohydrate uptake proteins independently comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 43 or SEQ ID NO: 44.
In some embodiments, the polysaccharide lyase family protein comprises a polysaccharide lysase family 2 protein. In some embodiments, the polysaccharide lyase family protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 14. In some embodiments, the polysaccharide lyase family protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 42.
In some embodiments, the glycoside hydrolase is from the glycoside hydrolase family 88 (GH88). In some embodiments, the glycoside hydrolase family 88 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 16. In some embodiments, the glycoside hydrolase family 88 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 38.
In some embodiments, the glycoside hydrolase is from the glycoside hydrolase family 92 (GH92). In some embodiments, the glycoside hydrolase family 92 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 17. In some embodiments, the glycoside hydrolase family 92 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 39.
In some embodiments, the glycoside hydrolase is from the glycoside hydrolase family 3 (GH3). In some embodiments, the glycoside hydrolase family 3 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 13. In some embodiments, the glycoside hydrolase family 3 protein comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 35 or SEQ ID NO: 36.
The heterologous xanthan-utilization gene or gene locus may further comprise additional genes encoding proteins and enzymes involved in xanthan-utilization including, but not limited to, glucokinases, mannose-6-phophate isomerases, phosphoglucomutases, other glycoside hydrolases (e.g., other glycoside hydrolase family 5 proteins), environmental sensors, and signaling proteins (e.g., response regulators). For example the gene locus may further comprise a glucokinase protein having an amino acid sequence having at least 70% identity to SEQ ID NO: 18 or 20, a transporter protein having an amino acid sequence having at least 70% identity to SEQ ID NO: 26-29, a transcriptional regulator having an amino acid sequence having at least 70% identity to SEQ ID NO: 25, a response regulator having an amino acid sequence having at least 70% identity to SEQ ID NO: 24, an isomerase having an amino acid sequence having at least 70% identity to SEQ ID NO: 22 or 23, a kinase having an amino acid sequence having at least 70% identity to SEQ ID NO: 21, a carbohydrate-binding module protein (e.g. Carbohydrate-binding module family 11 protein) having an amino acid sequence having at least 70% identity to SEQ ID NO: 19, and/or an environmental sensor (e.g. hybrid two-component system (HTCS) protein) having an amino acid sequence having at least 70% identity to SEQ ID NO: 30 or 40.
The heterologous xanthan-utilization gene locus may comprise a nucleic acid sequence having an amino acid sequence having at least 70% identity to SEQ ID NO: 31, 32, or 45.
The bacteria may be from the genus Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacteria, Escherichia, and/or Lactobacillus.
In some embodiments, the genetically modified bacterium is in the genus Bacteroides, including but not limited to, B. acidifaciens, B. amylophilus, B. asaccharolyticus, B. barnesiae, B. bivius, B. buccae, B. buccalis, B. caccae, B. capillosus, B. capillus, B. cellulosilyticus, B. chinchilla, B. clarus, B. coagulans, B. coprocola, B. coprophilus, B. coprosuis, B. corporis, B. denticola, B. disiens, B. distasonis, B. dorei, B. eggerthii, B. endodontalis, B. faecichinchillae, B. faecis, B. finegoldii, B. fluxus, B. forsythus, B. fragilis, B. furcosus, B. galacturonicus, B. gallinarum, B. gingivalis, B. goldsteinii, B. gracilis, B. graminisolvens, B. helcogenes, B. heparinolyticus, B. hypermegas, B. intermedius, B. intestinalis, B. johnsonii, B. levvi, B. loescheii, B. macacae, B. massiliensis, B. melaninogenicus, B. merdae, B. microfusus, B. multiacidus, B. nodosus, B. nordii, B. ochraceus, B. oleiciplenus, B. oralis, B. oris, B. oulorum, B. ovatus, B. paurosaccharolyticus, B. pectinophilus, B. pentosaceus, B. plebeius, B. pneumosintes, B. polypragmatus, B. praeacutus, B. propionicifaciens, B. putredinis, B. pyogenes, B. reticulotermitis, B. rodentium, B. ruminicola, B. salanitronis, B. salivosus, B. salyersiae, B. sartorii, B. splanchnicus, B. stercorirosoris, B. stercoris, B. succinogenes, B. suis, B. tectus, B. termitidis, B. thetaiotaomicron, B. uniformis, B. ureolyticus, B. veroralis, B. vulgatus, B. xylanisolvens, B. xylanolyticus, B. zoogleoformans, and any combination thereof.
In some embodiments, the genetically modified bacterium is a gram-positive gut commensal bacteria. The gram-positive gut commensal bacteria may be from the genus Enterococcus, Staphylococcus, Lactobacillus, Clostridium, Peptostreptococcus, Peptococcus, Streptococcus, Bifidobacterium, and/or Faecalibacterium. In some embodiments, the gram-positive gut commensal bacteria may be Lactobacillus reuteri or Clostridium scindens.
In some embodiments, the genetically modified bacteria may comprise the polynucleotide on a plasmid, a bacterial artificial chromosome or integrated into the genome of the bacterium.
Also provided are compositions comprising the genetically modified bacteria described herein. In some embodiments, the composition is a pharmaceutical composition (e.g., probiotic composition) further comprising excipients and/or pharmaceutically acceptable carriers. The excipients and/or pharmaceutically acceptable carriers may facilitate delivery of the genetically modified bacteria to a subject, for example a subject's gastro-intestinal tract, in a viable and metabolically-active condition, for example in a condition capable of colonizing and/or metabolizing and/or proliferating in the gastrointestinal tract.
The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
Excipients and carriers may include any and all solvents, dispersion media, coatings, and isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and/or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, corn starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The composition can comprise additional components, such as vitamins, minerals, carbohydrates, and a mixture thereof.
The composition may take on many forms. In some embodiments, the composition comprises encapsulating (e.g., in tablets, caplets, microcapsules) the genetically modified bacteria for enhanced delivery and survival in the gastric and/or gastrointestinal tract of a subject. In some embodiments, the composition is a foodstuff including liquids (e.g., drinks), semi-solids (e.g., jellies, yogurts, puddings, smoothies, and the like) and solids.
The disclosure also provides, a method of treating a disease or disorder comprising administering a therapeutically or prophylactically effective dose of the genetically modified bacteria or compositions thereof to a subject in need thereof. The specific dose level may depend upon a variety of factors including the age, body weight, and general health of the subject, time of administration, and route of administration. An “effective amount” is an amount that is delivered to a subject, either in a single dose or as part of a series, which achieves a medically desirable effect. For therapeutic purposes, and effect amount is the quantity which, when administered to a subject in need of treatment, improves the prognosis and/or state of the subject and/or that reduces or inhibits one or more symptoms to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of the disease or disorder. For prophylaxis purposes, an effective amount is that amount which induces a protective result without significant adverse side effects.
The frequency of dosing the effective amount can vary, but typically the effective amount is delivered daily, either as a single dose, multiple doses throughout the day, or depending on the dosage form, dosed continuously for part or all of the treatment period.
The genetically modified bacteria may be administered at about 104 to about 1010 cfu per dose, about 105 to about 109 cfu per dose, about 105 to about 107 cfu per dose, or about 109 cfu per dose.
The disease or disorder may comprise a gastrointestinal disease or disorder including diseases and disorders that cause inflammation in the gastrointestinal system including, but not limited to, Irritable Bowel Syndrome, diarrhea, Crohn's disease, ulcerative colitis, and gluten intolerance or Celiac's disease. The treatment may be combined with gluten-free or low carbohydrate diets that are high in xanthan gum.
In some embodiments, the administration is oral. The genetically modified bacteria may be administered with food (e.g., concomitantly with food, within an hour of before or after consuming food).
Culturing and phylogenetic analysis of xanthan degrading cultures Xanthan degrading cultures were grown in Defined Medium (DM), which was generally prepared as a 2×stock then mixed 1:1 with 10 mg/mL carbon source (e.g., xanthan gum). Cultures were grown in an anaerobic chamber (10% H2, 5% CO2, and 85% N2) maintained at 37° C. Each liter of prepared DM medium (pH=7.2) contained 13.6 g KH2PO4, 0.875 g NaCl, 1.125 g (NH4)2SO4, 2 mg each of adenine, guanine, thymine, cytosine, and uracil, 2 mg of each of the 20 essential amino acids, 1 mg vitamin K3, 0.4 mg FeSO4, 9.5 mg MgCl2, 8 mg CaCl2, 5 μg Vitamin B12, 1 g L-cysteine, 1.2 mg hematin with 31 mg histidine, 1 mL of Balch's vitamins, 1 mL of trace mineral solution, and 2.5 g beef extract.
Each liter of Balch's vitamins was prepared with 5 mg p-Aminobenzoic acid, 2 mg folic acid, 2 mg biotin, 5 mg nicotinic acid, 5 mg calcium pantothenate, 5 mg riboflavin, 5 mg thiamine HCl, 10 mg pyridoxine HCl, 0.1 mg cyanocobalamin, 5 mg thioctic acid. Prepared Balch's vitamins adjusted to pH 7.0, filter sterilized with 0.22 μm PES filters, and stored in the dark at 4° C.
Each L of trace mineral solution was prepared with 0.5 g EDTA (Sigma, ED4SS), 3 g MgSO4·7H2O, 0.5 g MnSO4·H2O, 1 g NaCl (Sigma, S7653), 0.1 g FeSO4·7H2O (Sigma, 215422), 0.1 g CaCl2, 0.1 g ZnSO4·7H2O, 0.01 g CuSO4·5H20, 0.01 g H3BO3 (Sigma, B6768), 0.01 g Na2MoO4·2H2O, 0.02 g NiCl2·6H2O. Prepared trace mineral solution was adjusted to pH 7.0, filter sterilized with 0.22 μm PES filters, and stored at room temperature.
Samples that showed growth on xanthan gum, as evidenced by loss of viscosity and increased culture density, were subcultured 10 times by diluting an active culture 1:100 into fresh DM-XG medium. For the original culture, multiple samples were stored for gDNA extraction and analysis while for the larger sample set, samples were stored after 10 passages; samples were harvested by centrifugation, decanted, and stored at −20° C. until further processing.
Frozen cell pellets were resuspended in 500 μL Buffer A (200 mM NaCl, 200 mM Tris-HCl, 20 mM EDTA) and combined with 210 μL SDS (20% w/v, filter-sterilized), 500 μL phenol:chloroform (alkaline pH), and ˜250 μL acid-washed glass beads (212-300 μm; Sigma). Samples were bead beaten on high for 2-3 minutes with a Mini-BeadBeater-16 (Biospec Products, USA), then centrifuged at 18,000 g for 5 mins. The aqueous phase was recovered and mixed by inversion with 500 μL of phenol:chloroform, centrifuged at 18,000 g for 3 mins, and the aqueous phase was recovered again. The sample was mixed with 500 μL chloroform, centrifuged, and then the aqueous phase was recovered and mixed with 0.1 volumes of 3 M sodium acetate (pH 5.2) and 1 volume isopropanol. The sample was stored at −80° C. for ≥30 mins, then centrifuged at ≥20,000 g for 20 mins at 4° C. The pellet was washes with 1 mL room temperature 70% ethanol, centrifuged for 3 mins, decanted, and allowed to air dry before resuspension in 100 μL sterile water. Resulting samples were additionally purified using the DNeasy Blood & Tissue Kit (QIAGEN, USA). Illumina sequencing, including PCR and library preparation, were performed by the University of Michigan Microbial Systems Molecular Biology lab as described by Kozich et al (Appl. Environ. Microbiol. 79, 5112-5120 (2013), incorporated herein by reference in its entirety). Barcoded dual-index primers specific to the 16S rRNA V4 region were used to amplify the DNA. PCR reactions consisted of 5 μL of 4 μM equimolar primer set, 0.15 μL of AccuPrime Taq DNA High Fidelity Polymerase, 2 μL of 10× AccuPrime PCR Buffer II (Thermo Fisher Scientific, catalog no. 12346094), 11.85 μL of PCR-grade water, and 1 μL of DNA template. The PCR conditions used consisted of 2 min at 95° C., followed by 30 cycles of 95° C. for 20 s, 55° C. for 15 s, and 72° C. for 5 min, followed by 72° C. for 10 min. Each reaction was normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, catalog no. A1051001), then pooled and quantified using the Kapa Biosystems Library qPCR MasterMix (ROX Low) Quantification kit for Illumina platforms (catalog no. KK4873). After confirming the size of the amplicon library using an Agilent Bioanalyzer and a high-sensitive DNA analysis kit (catalog no. 5067-4626), the amplicon library was sequenced on an Ilumina MiSeq platform using the 500 cycle MiSeq V2 Reagent kit (catalog no. MS-102-2003) according to the manufacturer's instructions with modifications of the primer set with custom read 1/read 2 and index primers added to the reagent cartridge. The “Preparing Libraries for Sequencing on the MiSeq” (part 15039740, Rev. D) protocol was used to prepare libraries with a final load concentration of 5.5 μM, spiked with 15% PhiX to create diversity within the run.
MPN/Dilution to extinction experiment An overnight culture was serially diluted in 2× DM. Serial dilutions were split into two 50 mL tubes and mixed 1:1 with either 10 mg/mL xanthan gum or 10 mg/mL monosaccharide mixture (4 mg/mL glucose, 4 mg/mL mannose, 2 mg/mL sodium glucuronate), both of which also had 1 mg/mL L-cysteine. Each dilution and carbon source was aliquoted to fill a full 96-well culture plate (Costar 3370) with 200 p L per well. Plates were sealed with Breathe-Easy gas permeable sealing membrane for microtiter plates (Diversified Biotech, cat #BEM-1). Microbial growth was measured at least 60 hours by monitoring OD600 using a Synergy HT plate reader (Biotek Instruments) and BIOSTACK2WR plate handler (Biotek Instruments).
Maximum OD for each substrate was measured for each culture. Full growth on substrates was conservatively defined as a maximum OD600 of >0.7. For each unique 96 well plate of substrate and dilution factor, the fraction of wells exhibiting full growth was calculated. Fractional growth was plotted against dilution factor for each substrate. Data were fit to the Hill equation by minimizing squared differences between the model and experimental values using Solver (GRG nonlinear) in Excel. For each experiment, a 50% growth dilution factor (GDF 50) was calculated for each substrate at which half of the wells would be predicted to exhibit full growth.
Neutral Monosaccharide analysis. The hot-phenol extraction method originally described by Massie & Zimm (Proc. Natl. Acad. Sci. 54, 1641-1643 (1965), incorporated herein by reference) and modified by Nie (ProQuest Diss. Theses 136 (2016), incorporated herein by reference) was used for collecting and purifying the polysaccharides remaining at different timepoints. Samples were heated to 65° C. for 5 mins, combined with an equal volume of phenol, incubated at 65° C. for 10 mins, then cooled to 4° C. and centrifuged at 4° C. for 15 min at 12,000 g. The upper aqueous layer was collected and re-extracted using the same procedure, dialyzed extensively against deionized water (2000 Da cutoff), and freeze-dried. Neutral monosaccharide composition was obtained using the method described by Tuncil et al. (Sci. Rep. 8, 1-13 (2018), incorporated herein by reference). Briefly, sugar alditol acetates were quantified by gas chromatography using a capillary column SP-2330 (SUPELCO, Bellefonte, PA) with the following conditions: injector volume, 2 μl; injector temperature, 240° C.; detector temperature, 300° C.; carrier gas (helium), velocity 1.9 meter/second; split ratio, 1:2; temperature program was 160° C. for 6 min, then 4° C./min to 220° C. for 4 min, then 3° C./min to 240° C. for 5 min, and then 11° C./min to 255° C. for 5 min.
Thin Layer Chromatography for Localization of Enzyme Activity Overnight cultures were harvested at 13,000 g for 10 minutes. Supernatant fractions were prepared by vacuum filtration through 0.22 μm PES filters. Cell pellet fractions were prepared by decanting supernatant, washing with phosphate buffered saline (PBS), spinning at 13,000 g for 3 mins, decanting, and resuspending in PBS. Intracellular fractions were prepared by taking cell pellet fractions and bead beating for 90 s with acid-washed glass beads (G1277, Sigma) in a Biospec Mini Beadbeater. Lysed culture fractions were prepared by directly bead beating unprocessed culture.
Each culture fraction was mixed 1:1 with 5 mg/mL xanthan gum and incubated at 37° C. for 24 hours. Negative controls were prepared by heating culture fractions to 95° C. for 15 mins, then centrifuging at 13,000 g for 10 mins before the addition of xanthan gum. All reactions were halted by heating to ≥85° C. for 15 mins, then spun at 20,000 g for 15 mins at 4° C. Supernatants were stored at −20° C. until analysis by thin layer chromatography.
Samples (3 μL) were spotted twice onto a 10×20 cm thin layer chromatography plate (Millipore TLC Silica gel 60, 20×20 cm aluminum sheets), with intermediate drying using a Conair 1875 hairdryer. Standards included malto-oligosaccharides of varying lengths (Even: 2, 4, 6, Odd: 1, 3, 5, 7), glucuronic acid, and mannose. Standards were prepared at 10 mM and 3 uL of each was spotted onto the TLC plate. Plates were run in ˜100 mL of 2:1:1 butanol, acetic acid, water, dried, then run an additional time. After drying, plates were incubated in developing solution (100 mL ethyl acetate, 2 g diphenylamine, 2 mL aniline, 10 mL of ˜80% phosphoric acid, 1 mL of ˜38% hydrochloric acid) for ˜30 seconds, then dried, and developed by holding over a flame until colors were observed.
Proteomic analysis Approximately 1 L of xanthan gum culture was grown until it had completely liquified (˜2-3 days). Supernatant was collected by centrifuging at 18,000 g and vacuum filtering through a 0.2 μm PES filter. 4M ammonium sulfate was added to 200-400 mL of filtrate to a final concentration of 2.4M and incubated for 30-60 mins at RT or, for one sample, overnight at 4° C. Precipitated proteins were harvested by centrifugation at 18,000 g for 30-60 mins, then resuspended in 50 mM sodium phosphate (pH 7.5). Three different fractionation protocols were followed, but after every fractionation step, active fractions were identified by mixing ˜500 μL with 10 mg/mL xanthan and incubating at 37° C. overnight; active-fractions were identified by loss of viscosity or production of xanthan oligosaccharides as visualized by TLC.
1. Resuspended protein was filtered and applied to a HiTrapQ column, running a gradient from β-100% B (Buffer A: 50 mM sodium phosphate, pH 7.5; Buffer B: 50 mM sodium phosphate, 1 M NaCl, pH 7.5). Active fractions were pooled and concentrated with a 10 kDa MWCO centricon and injected onto an S-200 16/60 column equilibrated in 50 mM sodium phosphate, 200 mM NaCl, pH 7.5. The earliest fractions to elute with significant A280 absorbance were also the most active fractions; these were pooled and submitted for proteomics.
2. Resuspended protein was filtered and applied to an S-500 column equilibrated in 50 mM sodium phosphate, 200 mM NaCl, pH 7.5. Active fractions eluted in the middle of the separation were pooled and submitted for proteomics.
3. Resuspended protein was filtered and applied to an S-500 column equilibrated in 50 mM sodium phosphate, 200 mM NaCl, pH 7.5. Pooled fractions were applied to a 20 mL strong anion exchange column running a gradient from β-100% B (Buffer A: 50 mM sodium phosphate, pH 7.5; Buffer B: 50 mM sodium phosphate, 1 M NaCl, pH 7.5). Active fractions were pooled and applied to a 1 mL weak anion exchange column (ANX) running a gradient from β-100% B (Buffer A: 50 mM sodium phosphate, 10% glycerol, pH 7.5; Buffer B: 50 mM sodium phosphate, 1 M NaCl, 10% glycerol, pH 7.5). Active fractions were pooled and submitted for proteomics.
Cysteines were reduced by adding 50 ml of 10 mM DTT and incubating at 45° C. for 30 min. Samples were cooled to room temperature and alkylation of cysteines was achieved by incubating with 65 mM 2-Chloroacetamide, under darkness, for 30 min at room temperature. An overnight digestion with 1 μg sequencing grade, modified trypsin was carried out at 37° C. with constant shaking in a Thermomixer. Digestion was stopped by acidification and peptides were desalted using SepPak C18 cartridges using manufacturer's protocol (Waters). Samples were completely dried using vacufuge. Resulting peptides were dissolved in 8 ml of 0.1% formic acid/2% acetonitrile solution and 2 μls of the peptide solution were resolved on a nano-capillary reverse phase column (Acclaim PepMap C18, 2 micron, 50 cm, ThermoScientific) using a 0.1% formic acid/2% acetonitrile (Buffer A) and 0.1% formic acid/95% acetonitrile (Buffer B) gradient at 300 nl/min over a period of 180 min (2-25% buffer B in 110 min, 25-40% in 20 min, 40-90% in 5 min followed by holding at 90% buffer B for 10 min and re-equilibration with Buffer A for 30 min). Eluent was directly introduced into Q exactive HF mass spectrometer (Thermo Scientific, San Jose CA) using an EasySpray source. MS1 scans were acquired at 60K resolution (AGC target=3×106; max IT=50 ms). Data-dependent collision induced dissociation MS/MS spectra were acquired using Top speed method (3 seconds) following each MS1 scan (NCE˜28%; 15K resolution; AGC target 1×105; max IT 45 ms).
Proteins were identified by searching the MS/MS data against a database of all proteins identified in the original culture metagenomes using Proteome Discoverer (v2.1, Thermo Scientific). Search parameters included MS1 mass tolerance of 10 ppm and fragment tolerance of 0.2 Da; two missed cleavages were allowed; carbamidomethylation of cysteine was considered fixed modification and oxidation of methionine, deamidation of asparagine and glutamine were considered as potential modifications. False discovery rate (FDR) was determined using Percolator and proteins/peptides with a FDR of ≤1% were retained for further analysis.
Kinetics of GH5-30 Lyase-treated xanthan gum was generated by mixing 5 mg/mL xanthan gum with 0.5 U/mL of Bacillus sp. Xanthan lyase (E-XANLB, Megazyme) in 30 mM potassium phosphate buffer (pH 6.5). After incubating overnight at 37° C., an addition 0.5 U/mL of xanthan lyase was added. Both lyase-treated and native xanthan gum were dialyzed extensively against deionized water, heated in an 80° C. water bath to inactivate the lyase, and centrifuged at 10,000 g for 20 mins to remove particulate. Supernatants were collected and stored at 4° C. until use. Kinetic measurements were conducted using a slightly modified version of the low-volume bicinchoninic acid (BCA) assay for glycoside hydrolases used by Arnal et al (Protein-Carbohydrate Interactions. Methods and Protocols (eds. Abbott, D. W. & Lammerts van Bueren, A.) 1588, 209-214 (2017), incorporated herein by reference). Briefly, AEX and SEC purified GH5 was diluted to a 10× stock of 5 μM enzyme, 50 mM sodium phosphate, 300 mM sodium chloride, and 0.1 mg/mL bovine serum albumin, pH=7.5. Reactions were 20 μL of enzyme stock mixed with 180 μL of various concentrations 37° C. xanthan gum. Negative controls were conducted with heat-inactivated enzyme stock. Timepoints were taken by quenching reactions with dilute, ice-cold, BCA working reagent. Reactions and controls were run with 4 independent replicates and compared to a glucose standard curve. Enzyme released reducing sugars were calculated by subtracting controls from reaction measurements.
Growth curves of isolates on XG oligos Pure isolates from the xanthan culture were obtained by streaking an active culture onto a variety of agar plates including LB and brain heart infusion with the optional addition of 10% defibrinated horse blood (Colorado Serum Co.) and gentamycin. After passaging isolates twice on agar plates, individual colonies were picked and grown overnight in tryptone-yeast extract-glucose (TYG) broth medium, then stocked by mixing with 0.5 volumes each of TYG and molecular biology grade glycerol and storing at −80° C. DM without beef extract (DM−BE), with the addition of a defined carbon source, was used to test isolates for growth on xanthan oligosaccharides. Some isolates (e.g., Parabacteroides distasonis) required the inclusion of 5 mg/mL beef extract (Sigma, B4888) to achieve robust growth on simple monosaccharides; in these cases, beef extract was included across all carbon conditions. Unless otherwise specified, carbon sources were provided at a final concentration of 5 mg/mL. Isolates were grown overnight in TYG media, subcultured 1:50 into DM−BE-glucose and grown overnight, then subcultured 1:50 into DM−BE with either various carbon sources. Final cultures were monitored for growth by measuring increase in absorbance (600 nm) using 96-well plates.
Extended metagenome analysis/comparison methodology Individual MAGs in each sample were searched by BlastP for the presence of proteins similar to those encoded by the XG-degrading PUL of R. UCG13 and B. intestinalis. This was done using the amino acid sequences of the proteins in the R. UCG13 and B. intestinalis PULs as the search homologs; both BlastP probes were searched against all the individual MAGs in the different samples with the default threshold e-value of le-5.
R. UCG13 and B. intestinalis/cell. XG Loci in Metagenomes Available cohorts of human gut metagenomic sequence data (National Center for Biotechnology Information projects: PRJNA422434, PRJEB10878, PRJEB12123, PRJEB12124, PRJEB15371, PRJEB6997, PRJDB3601, PRJNA48479, PRJEB4336, PRJEB2054, PRJNA392180, and PRJNA527208) were searched for the presence of xanthan locus nucleotide sequences from R. UCG13 (92.7 kb) and B. intestinalis (17.9kb) using the following workflow: Each xanthan locus nucleotide sequence was used separately as a template and then magic-blast v1.5.0 was used to recruit raw Illumina reads from the available metagenomic datasets with an identity cutoff of 97%. Next, the alignment files were used to generate a coverage map using bedtools v2.29.0 to calculate the percentage coverage of each sample against each individual reference. Metagenomic data sample was considered a to be positive for a particular xanthan locus if it had at least 70% of the corresponding xanthan locus nucleotide sequence covered.
The R. UCG13 locus and B. intestinalis XG locus were used as the query in a large-scale search against the assembled scaffolds of isolates, metagenome assembled genomes (bins), and metagenomes included into the Integrated Microbial Genomes & Microbiomes (IMG/M) comparative analysis system. Within the LAST software package, version 1066, the ‘lastal’ tool was used with default thresholds to search the 2 loci against 72,491 public high-quality isolate genomes, and 102,860 bins from 13,415 public metagenomes, and 21,762 public metagenomes in IMG/M. Metagenome bins were generated using the binning analysis method described in Clum, A. et al. The DOE JGI Metagenome Workflow. bioRxiv (2020), incorporated herein by reference.
Ruminococcaceae UCG13 —Glycosyl Hydrolase 5 (aka XGD26-15, aka GH5-30) Following 16s rDNA gene content determination and metagenomic sequencing of a multi-species xanthan-degrading community, sequence-specific oligonucleotide primers were designed and used to amplify the GH5 sequence from genomic DNA isolated from the multi-species culture. The PCR product for the protein was inserted into a C-terminal His-tagged expression construct using the Lucigen Expresso™ T7 Cloning and Expression System. The engineered plasmid containing the GH5-30 His-tagged sequence was transformed into BL21 (DE3) chemically competent cells. Seed cultures were grown overnight, followed by inoculation of 1 L of either LB or TB media, grown at 37° C. to an OD of ˜0.6-0.8, then induced with 250 μM IPTG and cooled to 18° C. for overnight (12-18 hr) expression. Cells were harvested by centrifugation, lysed with sonication, and recombinant protein was purified using standard His-tagged affinity protein purification protocols employing sodium phosphate buffers and either nickel or cobalt resin for immobilized metal affinity chromatography.
In general, pentameric xanthan oligosaccharides were produced by incubating ≥0.1 mg/mL GH5 with 5 mg/mL xanthan gum in PBS in approximately 1L total volume. For xanthan tetrasaccharides, ˜0.5 U/mL of Xanthan lyase (E-XANLB, Megazyme) was included. After incubating 2-3 days at 37° C. to allow complete liquefication, reactions were heat-inactivated, centrifuged at ≥10,000 g for 30 mins, and the supernatant was vacuum filtered through 0.22 μm PES sterile filters. Supernatants were loaded onto a column containing ˜10 g of graphitized carbon (Supelclean™ ENVI-Carb™, 57210-U Supelco), washed extensively with water to remove salt and unbound material, then eluted in a stepwise fashion with increasing concentrations of acetonitrile. Fractions were dried, weighed, and analyzed by LC-MS and fractions that contained the most significant yield of desired products were combined.
Highly pure products were obtained by reconstituting samples in 50% water:acetonitrile and applying to a Luna® 5 μm HILIC 200 Å LC column (250×10 mm) (OOG-4450-NO, Phenomenex). A gradient was run from 90-20% acetonitrile, with peaks determined through a combination of evaporative light scattering, UV, and post-run analytical LC-MS (Agilent qToF 6545) of resulting fractions.
NMR spectra were collected using an Agilent 600 NMR spectrometer (1H: 600 MHz, 13C: 150 MHz) equipped with a 5 mm DB AUTOX PFG broadband probe and a Varian NMR System console. All data analysis was performed using MestReNova NMR software. All chemical shifts were referenced to residual solvent peaks [1H (D2O): 4.79 ppm].
Enzyme Reaction Analysis All enzyme reactions were similar to preparative methods. carried out in 15-25 mM sodium phosphate buffer, 100-150 mM sodium chloride, and sometimes included up to 0.01 mg/mL bovine serum albumin (B9000S, NEB) to limit enzyme adsorption to pipettes and tubes. All R. UCG13 or B. intestinalis enzymes were tested at concentrations from 1-10 μM. Cellobiose reactions were tested using 1 mM cellobiose at pH 7.5, while all other reactions used 2.5 mg/mL pentasaccharide (produced using RuGH5a) and were carried out at pH 6.0. Reactions were heat-inactivated and centrifuged incubated overnight at 37° C., halted by heating at ≥95° C. for 5-10 minutes, and centrifugation at ≥20,000 g for 10 mins. Supernatants were mixed 1:1 with 4 parts acetonitrile and to yield an 80% acetonitrile solution, centrifuged for 10 mins at ≥20,000 g, and transferred into sample vials. 15 μL of each sample was injected onto a Luna® Omega 3 μm HILIC 200 Å, LC column (100×4.6 mm) (00D-4449-E0, Phenomenex). An Agilent 1290 Infinity II HPLC system was used to separate the sample using solvent A gradient was run from 90-20(100% water, 0.1% formic acid) and solvent B (95% acetonitrile, 5% water, with 0.1% formic acid added) at a flow rate of 0.4 mL/min over the course of ˜10-40 mins. Products were detected by collecting mass spectra. Prior to injection and following each sample the column was equilibrated with 80% B. After injection, samples were eluted with a 30 minute isocratic step at 80% B, a 10 minute gradient decreasing B from 80% to 10%, and a final column wash for 2 min at 10% B. Spectra were collected in negative mode on a MS Detector info, using an Agilent 6545 LC/Q-TOF.
Metagenomics analysis Seven samples (15-mL) were collected at four time points (referred to as T1, T2, T3 and T4) during growth of two biological replicates of the original XG-degrading culture. Cells were harvested by centrifugation at 14,000×g for 5 min and stored a −20° C. until further use. A phenol:chloroform:isoamyl alcohol and chloroform extraction method was used to obtain high molecular weight DNA. The gDNA was quantified using a Qubit™ fluorimeter and the Quant-iT™ dsDNA BR Assay Kit (Invitrogen, USA), and the quality was assessed with a NanoDrop One instrument (Thermo Fisher Scientific, USA). Samples were subjected to metagenomic shotgun sequencing using the Illumina HiSeq 3000 platform at the Norwegian Sequencing Center (NSC, Oslo, Norway). Samples were prepared with the TrueSeq DNA PCR-free preparation and sequenced with paired ends (2×150 bp) on one lane. Quality trimming of the raw reads was performed using Cutadapt v1.3, to remove all bases on the 3′-end with a Phred score lower than 20 and exclude all reads shorter than 100 nucleotides, followed by a quality filtering using the FASTX-Toolkit v.0.0.14 (hannonlab.cshl.edu/fastx_toolkit/). Retained reads had a minimum Phred score of 30 over 90% of the read length. Reads were co-assembled using metaSPAdes v3.10.1 with default parameters and k-mer sizes of 21, 33, 55, 77 and 99. The resulting contigs were binned with MetaBAT v0.26.3 in “very sensitive mode”. The quality (completeness, contamination, and strain heterogeneity) of the metagenome assembled genomes (MAGs) was assessed by CheckM v1.0.7 with default parameters. Contigs were submitted to the Integrated Microbial Genomes and Microbiomes system for open reading frames (ORFs) prediction and annotation. Additionally, the resulting ORF were annotated for CAZymes using the CAZy annotation pipeline. This MAG collection was used as a reference database for mapping of the metatranscriptome data, as described below. Taxonomic classifications of MAGs were determined using both MiGA and GTDB-Tk.
Human fecal samples (20) from a second enrichment experiment (unbiased towards the cultivation of Bacteroides) as well as two enrichments with mouse fecal samples were processed for gDNA extraction and library preparation exactly as described above. Metagenomic shotgun sequencing was conducted on two lanes of both Illumina HiSeq 4000 and Illumina HiSeq X Ten platforms (Illumina, Inc.) at the NSC (Oslo, Norway), and reads were quality trimmed, assembled and binned as described above. Open reading frames were annotated using PROKKA v1.14.0 and resulting ORFs were further annotated for CAZymes using the CAZy annotation pipeline and expert human curation. Completeness, contamination, and taxonomic classifications for each MAG were determined as described above. AAI comparison between the human R. UCG13 and the R. UCG13 found in the two mouse samples was determined using CompareM (github.com/dparks1134/CompareM).
Extracted DNA from a second enrichment experiment on XG using the original culture was prepared for long-reads sequencing using Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-LSK109) according to the manufacture protocol. The DNA library was sequenced with the ONT MinION Sequencer using a R9.4 flow cell. The sequencer was controlled by the MinKNOW software v3.6.5 running for 6 hours on a laptop (Lenovo ThinkPad P73 Xeon with data stored to 2Tb SSD), followed by base calling using Guppy v3.2.10 in ‘fast’ mode. This generated in total 3.59 Gb of data. The Nanopore reads were further processed using Filtlong v0.2.0 (github.com/rrwick/Filtlong), discarding the poorest 5% of the read bases, and reads shorter than 1000 bp.
The quality processed Nanopore long-reads were assembled using CANU v1.9 with the parameters corOutCoverage=10000 corMinCoverage=0 corMhapSensitivity=high genomeSize=5m redMemory=32 oeaMemory=32 batMemory=200. An initial polishing of the generated contigs were carried out using error-corrected reads from the assembly with minimap2 v2.17-x map-ont and Racon v1.4.14 with the argument —include-unpolished. The racon-polished contigs were further polished using Medaka v1.1.3 (github.com/nanoporetech/medaka), with the commands medaka_consensus--model r941_minfast_g303_model.hdf5. Finally, Minimap2-ax sr was used to map quality processed Illumina reads to the medaka-polished contigs, followed by a final round of error correction using Racon with the argument —include-unpolished. Circular contigs were identified by linking the contig identifiers in the polished assembly back to suggestCircular=yes in the initial contig header provided by CANU. These contigs were quality checked using CheckM v1.1.3 and BUSCO v4.1.4. Circular contigs likely to represent chromosomes (>1 Mbp) were further gene-called and functionally annotated using PROKKA v1.13 and taxonomically classified using GTDB-tk v1.4.0 with the classify_wf command. Barrnap v0.9 (github.com/tseemann/barmap) was used to predict ribosomal RNA genes. Average nucleotide Identity (ANI) was measured between the short-reads and long-reads MAGs using FastANI v1.1 with default parameters. Short-reads MAGs were used as query while long-reads MAGs were set as reference genomes. Short-reads MAG1 showed an Average Nucleotide Identity (ANI) of 99.98% with the long-reads ONTCirc01, while short-reads MAG2 showed an ANI of 99.99% with the long-reads ONT_Circ02. Phylogenetic analysis revealed that ONT_Circ02 encoded four complete 16S rRNA operons, three of which were identical to the aforementioned R. UCG13 OTU.
Temporal metatranscriptomic analysis of the original XG-degrading community. Cell pellets from 6 mL samples collected at T1-T4 during growth of two biological replicates of the original XG-degrading culture were supplemented with RNAprotect Bacteria Reagent (Qiagen, USA) following the manufacturer's instructions and kept at −80° C. until RNA extraction. mRNA extraction and purification were conducted as described in Kunath et al. (ISME J. 13, 603-617 (2019). Samples were processed with the TruSeq stranded RNA sample preparation, which included the production of a cDNA library, and sequenced on one lane of the Illumina HiSeq 3000 system (NSC, Oslo, Norway) to generate 2×150 paired-end reads. Prior to assembly, RNA reads were quality filtered with Trimmomatic v0.36, whereby the minimum read length was required to be 100 bases and an average Phred threshold of 20 over a 10 nt window, and rRNA and tRNA were removed using SortMeRNA v.2.1b. Reads were pseudo-aligned against the metagenomic dataset using kallisto pseudo-pseudobam. Of the 58089 ORFs (that encode proteins with >60 aa) identified from the metagenome of the original XG-degrading community, 7549 (13%) were not found to be expressed, whereas 50540 (87%) were expressed, resulting in a reliable quantification of the expression due to unique hits (reads mapping unambiguously against one unique ORF).
Plasmid Design and Protein Purification Plasmid constructs to produce recombinant proteins were made with a combination of synthesized DNA fragments (GenScript Biotech, Netherlands) and PCR amplicons using extracted culture gDNA as a template. In general, sequences were designed to remove N-terminal signaling peptides and to add a histidine tag for immobilized metal affinity chromatography (IMAC) (in many cases using the Lucigen MA101-Expresso-T7-Cloning-&-Expression-System). Plasmid assembly and protein sequences are described in source and supplemental data.
Constructs were transformed into HI-Control BL21(DE3) cells and single colonies were inoculated in 5 mL overnight LB cultures at 37° C. 5 mL cultures were used to inoculate 1 L of Terrific Broth (TB) with selective antibiotic, grown to OD ˜0.8-1.1 at 37° C., and induced with 250 μM IPTG. B. intestinalis enzymes were expressed at RT, while R. UCG13 enzymes were expressed at 18° C. overnight. Cells were harvested by centrifugation and pellets were stored at −80° C. until further processing. Proteins were purified using standard IMAC purification procedures employing sonication to lyse cells. R. UCG13 proteins were purified using 50 mM sodium phosphate and 300 mM sodium chloride at pH 7.5; B. intestinalis proteins were purified using 50 mM Tris and 300 mM sodium chloride at pH 8.0. All proteins were eluted from cobalt resin using buffer with the addition of 100 mM imidazole, then buffer exchanged to remove imidazole using Zeba 2 mL 7 kDa MWCO desalting columns. Protein concentrations were determined by measuring A280 and converting to molarity using calculated extinction coefficients.
qPCR/and RNA-Seq on B. intestinalis and Original Community
For qPCR, B. intestinalis was grown as before but cells were harvested by centrifugation at mid-exponential phase, mixed with RNA Protect (QIAGEN), and stored at −80° C. until further processing. At collection, average OD600 values were ˜0.8 and ˜0.6 for glucose- and oligosaccharide-grown cultures, respectively. RNeasy mini kit buffers (QIAGEN) were used to extract total RNA, purified with RNA-binding spin columns (Epoch), treated with DNase I (NEB), and additionally purified using the RNeasy mini kit. SuperScript III reverse transcriptase and random primers (Invitrogen) were used to perform reverse transcription. Target transcript abundance in the resulting cDNA was quantified using a homemade qPCR mix. Each 20 uL reaction contained 1× Thermopol Reaction Buffer (NEB), 125 uM dNTPs, 2.5 mM MgSO4, 1X SYBR Green I (Lonza), 500 nM gene specific or SI 7/8)65 nM 16S rRNA primer and 0.5 units Hot Start Taq Polymerase (NEB), and 10 ng of template cDNA. Results were processed using the ddCT method in which raw values were normalized to 16S rRNA values, then xanthan oligosaccharide values were compared to those from glucose to calculate fold-change in expression.
For RNA-seq, total RNA was used from the B. intestinalis growths used for qPCR. For the community grown on XG or PGA, 5 mL cultures of DM-XG or DM-PGA were inoculated with a 1:100 dilution of a fully liquified DM-XG culture. PGA cultures were harvested at mid-log phase at OD600˜0.85 whereas XG cultures were harvested at late-log phase at OD600˜1.2 to allow liquification of XG, which was necessary to extract RNA from these cultures. As before, cultures were harvested by centrifugation, mixed with RNA Protect (Qiagen) and stored at −80° C. until further processing. RNA was purified as before except that multiple replicates of DM-XG RNA were pooled together and concentrated with Zymo RNA Clean and Concentrator™-25 to reach acceptable concentrations for RNA depletion input. rRNA was depleted twice from the purified total RNA using the MICROBExpress™ Kit, each followed by a concentration step using the Zymo RNA Clean and Concentrator™-25. About 90% rRNA depletion was achieved for all samples. B. intestinalis RNA was sequenced using NovaSeq and community RNA was sequenced using MiSeq. The resulting sequence data was analyzed for differentially expressed genes following a previously published protocol76. Briefly, reads were filtered for quality using Trimmomatic v0.3968. Reads were aligned to each genome using BowTie2 v2.3.5.177. For the Bacteroides intestinalis transcriptome reads were aligned to its genome, while for the community data reads were aligned to either the B. intestinalis genome or the closed Ruminococcaceae UCG-13 metagenome assembled genome (MAG). Reads mapping to gene features were counted using htseq-count (release_0.11.1)78. Differential expression analysis was performed using the edgeR v3.34.0 package in R v.4.0.2 (with the aid of Rstudio v1.3.1093). The TMM method was used for library normalization79. Coverage data was visualized using Integrated Genome Viewer (IGV)80.
Xanthan gum (XG) has the same β-1,4-linked backbone as cellulose, but contains trisaccharide branches on alternating glucose residues consisting of an α-1,3-mannose, β-1,2-glucuronic acid, and terminal β-1,4-mannose. The terminal β-D-mannose and the inner α-D-mannose are variably pyruvylated at the 4,6-position or acetylated at the 6-position, respectively, with amounts determined by specific strain and culture conditions (
A group of 80 healthy 18-20 year-old adults were surveyed using a bacterial culture strategy originally designed to enrich for members of the Gram-negative Bacteroidetes, a phylum that generally harbors numerous polysaccharide-degrading enzymes. Based on increased bacterial culture turbidity and decreased viscosity of medium containing XG as the main carbon source, the initial survey revealed that just 1 out of 80 people sampled were positive for these characteristics. Growth analysis of a culture from the single positive subject revealed that bacterial growth was dependent on the amount of XG provided in the medium, demonstrating specificity for this nutrient (
Despite the two most abundant bacteria, including R. UCG13 and a Bacteroides OTU, being present as >20% relative abundance, pure cultures that could degrade XG were unable to be isolated using different solid media effective for Gram-positive and-negative bacteria. Correspondingly, replicate experiments in which the active multi-species community was diluted to extinction in microtiter plates containing medium with either XG, or an equal amount of its component monosaccharides, loss of growth on XG was observed at higher dilutions than simple sugars (growth dilution factor 50 (GDF50): dilution factor at which 50% of wells would grow (
A second survey was completed with a new group of 60 healthy adults. This time, feces were directly sampled within 24 hr after sample collection in anaerobic preservation buffer and using no pre-enrichment or antibiotics. In contrast to the previous results, this experiment revealed that the ability to degrade XG was substantially more frequent, as a greater percentage of people sampled harbored bacterial populations that grew to appreciable levels on XG and decreased its characteristic viscosity. Twenty of these samples were independently passaged 10 times each (one 1:200 dilution per day) and the resulting community structure was analyzed. While all of the passaged cultures contained multiple OTUs (between 12-22 OTUs with relative abundance ≥0.5%) as well as commonalities at the genus level, the only OTU common across all cultures at this threshold was the OTU corresponding to R. UCG13 (
To identify XG-degrading genes within the bacterial consortium, a temporal multi-omic analysis was applied to samples taken from the original XG-degrading culture. Two replicates of the original culture were grown in liquid medium with XG and timepoints were harvested for metagenomic, metatranscriptomic and monosaccharide analysis of residual polysaccharide (
Xanthan lyases typically remove the terminal pyruvylated mannose prior to depolymerization, leaving a 4,5 unsaturated residue at the glucuronic acid position, although some tolerate non-pyruvylated mannose. This same locus also contained two GH5 endoglucanases with the potential to cleave the xanthan gum backbone, a GH88 to remove the unsaturated glucuronic acid residue produced by the PL8, and two GH38s which could potentially cleave the alpha-D-mannose. Two carbohydrate esterases (CEs) could remove the acetylation from the mannose and possibly the terminal pyruvate, although the latter activity has not been described. SignalP 5.0 predicted SPI motifs for the two GH5s and one of the CEs (1026424, plasmid 13-8D that is an acetylase), while the other enzymes lacked membrane localization and secretion signals. In addition to putative enzymes to cleave the glycosidic bonds contained within xanthan gum, this locus also contained proteins predicted to be involved in sensing, binding, and transporting the released sugars or oligosaccharides.
Colocalization and expression of genes that saccharify a common polysaccharide as discrete polysaccharide utilization loci (PULs) is common in the Gram-negative Bacteroidetes. Although not present in all xanthan gum-degrading cultures, a MAG was obtained for a strain of B. intestinalis, which was the most abundant OTU in the original xanthan culture (up to 51.0% of the original culture, 26.0% and 32.7% in samples 32 and 11 respectively, 8 other samples ranging from 0.3-4.4%). This MAG contained a putative xanthan PUL that was highly expressed during growth on XG (
Although less dramatic, several microbes showed increased expression of CAZymes over the culture time course, suggesting that other microbes may cross-feed on either XG oligosaccharides released by the primary degraders, or on additional substrates produced by XG consumers (
To investigate the cellular location of the enzymes responsible for xanthan degradation, the original culture was grown in XG medium and separated into filtered cell-free supernatant, cells that were washed to remove supernatant and resuspended or lysed, or lysed cells with supernatant. Incubation of these fractions with XG and subsequent analysis by thin layer chromatography (TLC) revealed that the cell-free supernatant was capable of depolymerizing XG into large oligosaccharides, while the intracellular fraction was required to further saccharify these products into smaller components. Liquid chromatography-mass spectrometry (LC-MS) analysis of the cell-free supernatant incubated with XG revealed the presence of pentameric oligosaccharides matching the structure of xanthan gum.
Three independent cultures were grown in liquid medium containing XG and cell-free supernatants were subjected to ammonium sulfate precipitation. Each of the resuspended protein preparations was able to hydrolyze XG as demonstrated by a complete loss of viscosity overnight. Each sample was fractionated with a variety of purification methods, collecting and pooling xanthan-degrading fractions for subsequent purification steps and taking three different purification paths (
The R. UCG13 GH5 consists of an N-terminal signal peptide sequence, its main catalytic domain which does not classify into any of the GH5 subfamilies, and 3 tandem carbohydrate binding modules (CBMs), which are often associated with CAZymes and assist in polysaccharide degradation (
Analysis of the reaction products showed that R. UCG13 GH5 (R. UCG13 GH5a) releases pentasaccharide repeating units of XG, with various acetylation and pyruvylation (including di-acetylation as previously described), and larger decasaccharide structures (
Although R. UCG13 was recalcitrant to culturing efforts, several bacteria were isolated from the original consortium, including the Bacteroides intestinalis strain that was the most abundant (
To further test the role of the identified B. intestinalis PUL in XG degradation, the recombinant forms of the enzymes it contains were tested for XG degradation. The carbohydrate esterase domain C-terminal to the PL2 bimodular protein was able to remove acetyl groups from acetylated xanthan pentasaccharides (
Additional metagenomic sequencing was performed on 20 additional XG-degrading communities and it was found that the R. UCG13 XG utilization locus is extremely well conserved across these cultures with high amino acid identity and only one variation in gene content, insertion of a GH125 coding gene (
Bacteroides intestinalis Xanthan Gum Utilization Locus. Primers are designed and used to amplify the entire B. intestinalis xanthan gum utilization locus, with overlapping ends to facilitate assembly. PCR fragments of the locus are assembled and circularized into the linearized Bacteroides genomic insertion vector, pNBU2, using Gibson assembly and the NEBuilder HiFi DNA Assembly kit. The pNBU2 vector can be used to insert DNA into one of two tRNA-Serine sites in numerous Bacteroides genomes (Martens, E. C., et al., Cell Host Microbe 4, 447-457 (2008), incorporated herein by reference). After assembly and transformation into Lucigen TransforMax EC100D pir+ electrocompetent E. coli, the plasmid is transformed into S17-1 1 pir E. coli for conjugation into Bacteroides thetaiotaomicron and additional Bacteroides spp by conjugation. B. theta strains with the inserted xanthan utilization locus are tested for the ability to grow on xanthan gum oligosaccharides, indicative of gain of function. Strains that successfully grow on xanthan oligosaccharides with the transferred/engineered locus are tested for their abilities to colonize animal digestive tracts and the pre-existing gut microbiome, the dose (cfu/ml by oral gavage or lyophilized bacteria in capsule) of invading, recombinant B. theta and the dosage of xanthan pentasaccharides administered to the animals can be systematically varied.
The Ruminococcaceae UCG13 GH5-30 enzyme can be transferred into Bacteroides spp. This is accomplished by genetically engineering an insertion of this gene into the B. intestinalis PUL that confers xanthan oligosaccharide metabolism thereby making expression of the GH5-30 gene regulated the same as other xanthan-degrading functions. To adapt this enzyme to be expressed on the surface of the Gam-negative Bacteroides cell, its native secretion signals are removed and recombined with an N-terminal domain of the B. theta surface protein SusF, for which the signal sequence required for secretion and trafficking to the cell surface has been determined. This process results in an active extracellular GH5-30 capable of depolymerizing xanthan gum and engineered Bacteroides that are not only capable of utilizing xanthan oligosaccharides but are fully capable of depolymerizing and growing on native xanthan gum.
Ruminococcaceae UCG13 Xanthan Gum Utilization Locus Gram-positive microbes are potentially superior organisms for production of secreted peptides and proteins. The minimal xanthan gum utilization locus from R. UCG13 may be transferred to Gram positive microbes that are genetically tractable, including but not limited to Lactobacillus reuteri and Clostridium scindens to engineer gram-positive probiotics that can successfully colonize the gastrointestinal tract with co-feeding of xanthan gum.
In contrast to characterized PL8 xanthan lyases, the R. UCG13 PL8 showed no activity on the complete XG polymer but removed the terminal mannose from xanthan pentasaccharides produced by R. UCG13 GH5 (
Using each locus as a query, several publicly available fecal metagenome datasets collected from worldwide populations were searched. All modern populations sampled displayed some presence of the R. UCG13 XG locus, with the Chinese and Japanese cohorts being the highest (up to 51% in one cohort) (
Although the size of the hunter-gatherer datasets is relatively small, excluding the possibility of a false negative suggests several equally intriguing hypotheses. Most obviously, inclusion of XG in the modern diet may have driven either the colonization or expansion of R. UCG13 (and to a lesser extent B. intestinalis) into the gut communities of numerous human populations. This is in concordance with previous observations that found that a set of volunteers fed xanthan gum for an extended period produced stool with increased probability and degree of xanthan degradation. Alternatively, the modern microbiome is drastically different than that of hunter-gatherers and these differences simply correlate with the abundance of R. UCG13, rather than any causal effect of XG in the diet. Another possible hypothesis is that the microbiomes of hunter-gatherer populations can degrade XG but use completely different microbes and pathways.
To further probe the presence of the identified XG utilization genes in other environments, an expanded LAST search of both loci was conducted in 72,491 sequenced bacterial isolates and 102,860 genome bins extracted from 13,415 public metagenomes, as well as 21,762 public metagenomes that are part of the Integrated Microbial Genomes & Microbiomes (IMG/M) database using fairly stringent thresholds of 70% alignment over the query and 90% nucleotide identity. This search yielded 35 hits of the R. UCG13 locus in human microbiome datasets, including senior adults, children, and an infant (12-months of age, Ga0169237_00111). 12 hits of the B. intestinalis XGOs locus were also found, all in human microbiome samples except for a single environmental sample from a fracking water sample from deep shales in Oklahoma, USA (81% coverage, 99% identity) (
To investigate the prevalence of XG-degrading populations beyond the human gut microbiome, a mouse experiment using feed with 5% XG showed increased levels of short chain fatty acids propionate and butyrate, suggesting the ability of members of the mouse microbiome to catabolize and ferment XG43. After culturing mouse feces from this experiment on XG media and confirming its ability to depolymerize XG, the community structure in two samples (M1741 and M737) was metagenomically characterized, revealing a microbial species related to R. UCG13 (AAI values between the human R. UCG13 and the mouse R. UCG13 were 75.7% and 75.2% for M1741 and M737, respectively) as well as a XG locus with strikingly similar genetic architecture to the human XG locus (
An additional targeted search of the R. UCG13 locus in several animal- and plant-associated microbiomes was performed and homologous loci were found in both cow (5 positive samples) and goat (one positive sample) microbiomes. Together, these data show that the R. UCG13 XG locus is more broadly present in mammalian gastrointestinal microbiomes.
Another strain that had a candidate PUL for XG degradation was B. salyersiae (
To further test the role of the identified B. salyersiae PUL in XG degradation, the gene expression of the enzymes was tested when grown on XGOs. As shown in
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.
Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.
This application claims the benefit of U.S. Provisional Application Nos. 63/079,318, filed Sep. 16, 2020, and 63/195,983, filed Jun. 2, 2021, the contents of which are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/50494 | 9/15/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63079318 | Sep 2020 | US | |
| 63195983 | Jun 2021 | US |
