METHODS AND COMPOSITIONS FOR ANAEROBIC BACTERIAL FERMENTATION

BACKGROUND

Anaerobic bacteria are bacteria that that grow poorly (or do not grow) in the presence of oxygen. In humans, many types of anaerobic bacteria are found in the gastrointestinal tract. As microbial culturing methods typically occur in atmospheric air (an aerobic environment), the culturing of anaerobic bacteria can be challenging and often requires specialized equipment and techniques. For example, anaerobic bacteria can be cultured in an anaerobic glovebox or other specially sealed container filled with nitrogen. However, currently available techniques are not amenable to the large-scale cultures necessary for the commercial production of therapeutic microbes. Therefore, alternative methods for anaerobic bacterial fermentation would be useful for growing anaerobic bacteria, particularly in large scales.

SUMMARY

Anaerobic bacteria benefit from the presence of carbon dioxide (CO₂) at the start of culturing in the lag phase, but some strains of anaerobic bacteria do not need CO₂to maintain robust growth through log phase. Certain anaerobic strains, e.g., strains described herein, grow better when CO₂is provided throughout growth (e.g., as compared to the rate of growth when CO₂is not provided in log phase). For example, certain such bacteria consume CO₂throughout the fermentation process.

In certain aspects, the culture methods described herein allow for better growth of an anaerobic bacteria strain, e.g., a strain described herein, as compared to conventional methods. For example, in some embodiments, the methods described herein allow growth of the bacteria to an OD of over 4, e.g., over 10, or over 20. For example, in some embodiments, sparging CO₂at about 25% (e.g., and about 75% N₂) rather than at about 5% (e.g., and about 95% N₂) into a bioreactor allows about a 5-fold increase in biomass yield. The CO₂can be introduced in a gas mixture; the gas mixture can also include N₂.

A key feature of certain embodiments of the methods described herein is that they are particularly applicable to large scale production, e.g., in a bioreactor, e.g., in vessels over 1 L in volume. As culture volumes increase, simply providing CO₂into the headspace of a vessel may not suffice to provide sufficient CO₂throughout the culture to achieve optimal growth. In some embodiments, by providing CO₂throughout the culture (e.g., beyond providing CO₂in the headspace), bacterial growth is improved. In some embodiments, CO₂can be provided throughout the culture, e.g., by sparging/bubbling CO₂into the culture; by injecting boluses of CO₂into the culture at intervals (e.g., at 30-minute or one-hour intervals); and/or by adding a carbonate or bicarbonate salt into the culture. Carbonate salts that can be used in embodiments provided herein include, for example, sodium carbonate, potassium carbonate, barium carbonate, carbonic acid, magnesite (magnesium carbonate), sodium percarbonate (adduct with hydrogen peroxide) and calcium carbonate. Bicarbonate salts that can be used in embodiments provided herein include, for example, sodium bicarbonate, potassium bicarbonate, cesium bicarbonate, magnesium bicarbonate, and ammonium bicarbonate. The carbonate or bicarbonate salt can be used at a concentration of, e.g., 0.5 g/L to 10 g/L, e.g., 0.5 to 1 g/L, 1 to 5 g/L, 2 to 8 g/L, about 0.5 g/L, about 1 g/L, about 5 g/L, about 10 g/L. The carbonate or bicarbonate salt can be used in certain embodiments as an alternative or additional source of CO₂(e.g., by adding the salt, a lower percentage of CO₂can be used yet still achieve the same growth benefits as when a higher percentage of CO₂is used). For example, in some embodiments, bacteria can be grown in a bioreactor into which about 25% CO₂(e.g., and about 75% N₂) is sparged into the culture; similar yields can be obtained, e.g., growing the bacteria in a bioreactor into which about 5% CO₂(e.g., and about 95% N₂) is sparged with the addition of sodium bicarbonate (e.g., 0.5-1 g/L).

In certain aspects, provided herein are methods of growing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor comprising carbonate salt. In some embodiments, is sodium carbonate, potassium carbonate, barium carbonate, carbonic acid, magnesite (magnesium carbonate), sodium percarbonate (adduct with hydrogen peroxide), or calcium carbonate. In some embodiments, the carbonate is at a concentration of 0.5 g/L to 10 g/L. In some embodiments, the carbonate salt is at a concentration of 0.5 to 1 g/L, 1 to 5 g/L, or 2 to 8 g/L. In some embodiments, the carbonate salt is at a concentration of about 0.5 g/L, about 1 g/L, about 5 g/L, or about 10 g/L.

In certain aspects, provided herein are methods of growing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor comprising bicarbonate salt. In some embodiments, the bicarbonate salt is sodium bicarbonate, potassium bicarbonate, cesium bicarbonate, magnesium bicarbonate, or ammonium bicarbonate. In some embodiments, the bicarbonate salt is at a concentration of 0.5 g/L to 10 g/L. In some embodiments, the bicarbonate salt is at a concentration of 0.5 to 1 g/L, 1 to 5 g/L, or 2 to 8 g/L. In some embodiments, the bicarbonate salt is at a concentration of about 0.5 g/L, about 1 g/L, about 5 g/L, or about 10 g/L.

In certain aspects, provided herein are improved compositions and methods for culturing anaerobic bacteria. For example, in some embodiments provided herein are methods of culturing anaerobic bacteria under anaerobic conditions comprising a greater level of CO₂compared to conventional anaerobic culture conditions (e.g., at a level of greater than 1% CO₂, e.g., at a level of greater than 5% CO₂, such as at a level of about 25% CO₂). In certain embodiments, provided herein are bioreactors comprising anaerobic bacteria being cultured under conditions comprising a greater level of CO₂compared to conventional anaerobic culture conditions (e.g., at a level of greater than 1% CO₂, such as at a level of about 25% CO₂). In some embodiments, the methods and compositions provided herein result in increased bacterial yield compared to conventional culture conditions.

In certain aspects, provided herein are methods of growing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor under an anaerobic atmosphere comprising CO₂. In some embodiments, the anaerobic atmosphere comprises greater than 1% CO₂. In some embodiments, the anaerobic atmosphere comprises greater than 5% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 8% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 20% CO₂. In some embodiments, the anaerobic atmosphere comprises from 8% to 40% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 10% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 20% CO₂. In some embodiments, the anaerobic atmosphere comprises from 10% to 40% CO₂. In some embodiments, the anaerobic atmosphere comprises from 20% to 30% CO₂. In some embodiments, the anaerobic atmosphere comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% CO₂. In some embodiments, the anaerobic atmosphere comprises about 25% CO₂.

In certain aspects, provided herein are methods of growing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor into which CO₂in a gas mixture is introduced into the culture. In some embodiments, the gas mixture comprises greater than 1% CO₂. In some embodiments, the gas mixture comprises greater than 5% CO₂. In some embodiments, the gas mixture comprises at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% CO₂. In some embodiments, the gas mixture comprises at least 8% CO₂. In some embodiments, the gas mixture comprises at least 20% CO₂. In some embodiments, the gas mixture comprises from 8% to 40% CO₂. In some embodiments, the gas mixture comprises at least 10% CO₂. In some embodiments, the gas mixture comprises at least 20% CO₂. In some embodiments, the gas mixture comprises from 10% to 40% CO₂. In some embodiments, the gas mixture comprises from 20% to 30% CO₂. In some embodiments, the gas mixture comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, CO₂. In some embodiments, the gas mixture comprises about 25% CO₂.

In certain aspects, provided herein are methods of culturing anaerobic bacteria under anaerobic conditions comprising a lower level of N₂compared to conventional anaerobic culture conditions (e.g., at a level of less than 95% N₂, e.g., at a level of less than 90% N₂, such as at a level of about 75% N₂). In certain embodiments, provided herein are bioreactors comprising anaerobic bacteria being cultured under conditions comprising a lower level of N₂compared to conventional anaerobic culture conditions (e.g., at a level of less than 95% N₂such as at a level of about 75% N₂). In some embodiments, the methods and compositions provided herein result in increased bacterial yield compared to conventional culture conditions.

In certain aspects, provided herein are methods of culturing anaerobic bacteria under anaerobic conditions comprising introducing a gas mixture comprising a lower level of N₂compared to conventional anaerobic culture conditions (e.g., a gas mixture of less than 95% N₂, e.g., of less than 90% N₂, of about 75% N₂). In certain embodiments, provided herein are bioreactors comprising anaerobic bacteria being cultured under conditions comprising introducing a gas mixture comprising a lower level of N₂compared to conventional anaerobic culture conditions (e.g., a gas mixture of less than 95% N₂such as of about 75% N₂). In some embodiments, the methods and compositions provided herein result in increased bacterial yield compared to conventional culture conditions. In some embodiments, the gas mixture comprises no more than 75%, no more than 76%, no more than 77%, no more than 78%, no more than 79%, no more than 80%, no more than 81%, no more than 82%, no more than 83%, no more than 84%, no more than 85%, no more than 86%, no more than 87%, no more than 88%, no more than 89%, no more than 90%, no more than 91%, no more than 92%, no more than 93%, or no more than 94% N₂. In some embodiments, the gas mixture comprises from 75% to 94% N₂. In some embodiments, the gas mixture comprises about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, or about 94% N₂.

In certain aspects, provided herein are methods of growing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor into which an anaerobic gas mixture comprising N₂is introduced. In some embodiments, the gas mixture comprises less than 95% N₂. In some embodiments, the gas mixture comprises less than 90% N₂. In some embodiments, the gas mixture comprises less than 95%, less than 92%, less than 90%, less than 87%, less than 85%, less than 82%, less than 80%, less than 77% N₂. In some embodiments, the gas mixture comprises less than 85% N₂. In some embodiments, the gas mixture comprises less than 80% N₂. In some embodiments, the gas mixture comprises from 65% to 85% N₂. In some embodiments, the gas mixture comprises from 70% to 80% N₂. In some embodiments, the gas mixture comprises about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72% about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% N₂. In some embodiments, the gas mixture comprises about 75% N₂.

In some embodiments, the anaerobic atmosphere and/or gas mixture consists essentially of CO₂and N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises about 25% CO₂and about 75% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises about 20% CO₂and about 80% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises about 30% CO₂and about 70% N₂.

In certain aspects, provided herein are methods of culturing anaerobic bacteria, the method comprising the steps of a) purging a bioreactor with an anaerobic gas mixture comprising greater than 1% CO₂; and b) culturing the anaerobic bacteria in the bioreactor purged in step a). In certain embodiments, the anaerobic gas mixture is added to the bioreactor during step b). In some embodiments, the anaerobic gas mixture comprises at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% CO₂. In some embodiments, the anaerobic gas mixture comprises greater than 5% CO₂. In some embodiments, the anaerobic gas mixture comprises at least 8% CO₂. In some embodiments, the anaerobic gas mixture comprises at least 20% CO₂. In some embodiments, the anaerobic gas mixture comprises from 8% to 40% CO₂. In some embodiments, the anaerobic gas mixture comprises from 20% to 30% CO₂. In some embodiments, the anaerobic gas mixture comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, CO₂. In some embodiments, the anaerobic gas mixture comprises about 25% CO₂. In some embodiments, the anaerobic gas mixture comprises about 100% CO₂.

In some embodiments, the anaerobic gas mixture consists essentially of CO₂and N₂. In some embodiments, the anaerobic gas mixture comprises about 25% CO₂and about 75% N₂. In some embodiments, the anaerobic atmosphere comprises about 20% CO₂and about 80% N₂. In some embodiments, the anaerobic atmosphere comprises about 30% CO₂and about 70% N₂.

In some embodiments, the methods provided herein further comprises the step of inoculating a growth media with the anaerobic bacteria, wherein the bacteria are cultured in the growth media according to the methods provided herein. In some embodiments, the volume of the inoculated anaerobic bacteria is between 0.01% and 10% v/v of the growth media (e.g., about 0.1% v/v of the growth media, about 0.5% v/v of the growth media, about 1% v/v of the growth media, about 5% v/v of the growth media).

In some embodiments, the growth media is at least about 1 L in volume, at least about 5 L in volume, at least about 10 L in volume, at least about 15 L in volume, at least about 20 L in volume, at least about 30 L in volume, at least about 40 L in volume, at least about 50 L in volume, at least about 100 L in volume, at least about 200 L in volume, at least about 250 L in volume, at least about 500 L in volume, at least about 750 L in volume, at least about 1000 L in volume, at least about 1500 L in volume, at least about 2000 L in volume, at least about 2500 L in volume, at least about 3000 L in volume, at least about 3500 L in volume, at least about 4000 L in volume, at least about 5000 L in volume, at least about 7500 L in volume, at least about 10,000 L in volume, at least about 15,000 L in volume, at least about 20,000 L in volume, at least about 50,000 L in volume, at least about 100,000 L in volume, at least about 150,000 L in volume, at least about 200,000 L in volume, at least about 250,000 L in volume, at least about 300,000 L in volume, at least about 350,000 L in volume, at least about 400,000 L in volume, or at least about 500,000 L in volume.

In some embodiments, the anaerobic bacteria is cultured for at least 5 hours (e.g., at least 10 hours). In some embodiments, the anaerobic bacteria is cultured for 10-24 hours. In some embodiments, the anaerobic bacteria is cultured for 14 to 16 hours. In some embodiments, the method further comprises the step of inoculating about 5% v/v of the cultured bacteria in a growth media. In some embodiments, the growth media is about 20 L in volume. In some embodiments, the anaerobic bacteria is cultured for 10-24 hours. In some embodiments, the anaerobic bacteria is cultured for 12-14 hours. In some embodiments, the anaerobic bacteria is cultured at least until a stationary phase is reached.

In some embodiments, the anaerobic bacteria is cultured at a temperature of 35° C. to 42° C. In some embodiments, the anaerobic bacteria is cultured at a temperature of 35° C. to 39° C. In some embodiments, the anaerobic bacteria is cultured at a temperature of about 37° C. In some embodiments, the anaerobic bacteria is cultured at a pH of 5.5 to 7.5. In some embodiments, the anaerobic bacteria is cultured at a pH of about 6.5.

In some embodiments, the anaerobic bacteria is cultured in a bioreactor. In some embodiments, culturing the anaerobic bacteria comprises agitating the culture at a RPM of 50 to 1000. In some embodiments, culturing the anaerobic bacteria comprises agitating the culture at a RPM of 100 to 700. In some embodiments, culturing the anaerobic bacteria comprises agitating the culture at a RPM of 50 to 300. In some embodiments, the anaerobic bacteria is agitated at a RPM of about 150.

In some embodiments, an anaerobic gas mixture is continuously added to the bioreactor during culturing. In some embodiments, the continuously added anaerobic gas mixture is added at a rate of 0.01 to 1 vvm. In some embodiments, the continuously added anaerobic gas mixture is added at a rate of 0.01 to 0.1 vvm. In some embodiments, the continuously added anaerobic gas mixture is added at a rate of 0.02 vvm. In some embodiments, CO₂is continuously added during culturing. In some embodiments, CO₂is added at a rate of 0.002 vvm to 0.1 vvm. In some embodiments, CO₂is added at a rate of about 0.002 vvm. In some embodiments, CO₂is added at a rate of about 0.02 vvm. In some embodiments, the continuously added anaerobic gas mixture comprises at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% CO₂. In some embodiments, the anaerobic gas mixture comprises greater than 5% CO₂. In some embodiments, the continuously added anaerobic gas mixture comprises at least 8% CO₂. In some embodiments, the continuously added anaerobic gas mixture comprises at least 20% CO₂. In some embodiments, the anaerobic atmosphere comprises from 8% to 40% CO₂. In some embodiments, the continuously added anaerobic gas mixture comprises at least 10% CO₂. In some embodiments, the continuously added anaerobic gas mixture comprises at least 20% CO₂. In some embodiments, the anaerobic atmosphere comprises from 10% to 40% CO₂. In some embodiments, the continuously added anaerobic gas mixture comprises from 20% to 30% CO₂. In some embodiments, the continuously added anaerobic gas mixture comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% CO₂. In some embodiments, the continuously added anaerobic gas mixture comprises about 25% CO₂.

In some embodiments, the anaerobic atmosphere consists essentially of CO₂and N₂. In some embodiments, the continuously added anaerobic gas mixture comprises about 25% CO₂and about 75% N₂. In some embodiments, the anaerobic atmosphere comprises about 20% CO₂and about 80% N₂. In some embodiments, the anaerobic atmosphere comprises about 30% CO₂and about 70% N₂.

In certain embodiments of the methods provided herein, herein the anaerobic bacteria are cultured in a pressurized bioreactor. In some embodiments, the bioreactor is pressurized at least at 100,000 Pascal. In some embodiments, the bioreactor is pressurized at least at 100,000 Pascal, 125,000 Pascal, 150,000 Pascal, 175,000 Pascal, 200,000 Pascal, or 225,000 Pascal. In some embodiments, the bioreactor is pressurized at most at 2,225,000 Pascal. In some embodiments, the bioreactor is pressurized at most at 2,000,000 Pascal, 2,025,000 Pascal, 2,050,000 Pascal, 2,075,000 Pascal, 2,100,000 Pascal, 2,150,000 Pascal, 2,200,000 Pascal, or 2,225,000 Pascal. In some embodiments, the bioreactor is pressurized from about 100,000 Pascal to about 2,100,000 Pascal. In some embodiments, the bioreactor is pressurized from about 101,325 Pascal to about 2,026,500 Pascal. Generally, but in no way wishing to be bound by theory, operating at increased pressures allows a significant increase in the rate of CO₂transfer from the gas phase to the liquid phase.

In certain embodiments, the methods provided herein comprise introducing a gas to the bioreactor with a diffusion sparger. In some embodiments, the gas is introduced with sintered or porous spargers. In other embodiments, the gas is introduced with perforated plates or other apparatus to introduce microbubbles. Generally, but in no way wishing to be bound by theory, the introduction of smaller and more diffuse bubbles allows a significant increase in the rate of CO₂transfer from the gas phase to the liquid phase.

In some embodiments the anaerobic bacteria is cultured in growth media. In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, maltodextrin (e.g., glucidex, e.g., glucidex 21 D), glucose, and/or hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises about 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises about 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises about 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises about 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises about 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises about 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises about 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 20 g/L to 30 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21 D). In some embodiments, the growth media comprises about 25 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21 D). In some embodiments, the growth media comprises 5 g/L to 15 g/L glucose. In some embodiments, the growth media comprises about 10 g/L glucose. In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises about 0.02 g/L hemoglobin.

In some embodiments the anaerobic bacteria is cultured in growth media. In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucose, and/or hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises about 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises about 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises about 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises about 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises about 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises about 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises about 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 5 g/L to 15 g/L glucose. In some embodiments, the growth media comprises about 10 g/L glucose. In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises about 0.02 g/L hemoglobin.

In some embodiments the anaerobic bacteria is cultured in growth media. In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, maltodextrin (e.g., glucidex, e.g., glucidex 21 D), and/or hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises about 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises about 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises about 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises about 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises about 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises about 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises about 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 20 g/L to 30 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21 D). In some embodiments, the growth media comprises about 25 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21 D). In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises about 0.02 g/L hemoglobin.

In some embodiments, the method further comprises the step of harvesting the cultured bacteria (e.g., when a stationary phase is reached). In some embodiments, the method further comprises the step of centrifuging the cultured bacteria after harvesting (e.g., to produce a cell paste). In some embodiments, the method further comprises diluting the cell paste with a stabilizer solution to produce a cell slurry. In some embodiments, the method further comprises the step of lyophilizing the cell slurry to produce a powder. In some embodiments, the method further comprises irradiating the powder with gamma radiation.

In certain aspects, provided herein are bioreactors comprising anaerobic bacteria under an anaerobic atmosphere comprising at least about 1% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% CO₂. In some embodiments, the anaerobic gas mixture comprises greater than 5% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 8% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 10% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 20% CO₂. In some embodiments, the anaerobic atmosphere comprises from 8% to 40% CO₂. In some embodiments, the anaerobic atmosphere comprises from 10% to 40% CO₂. In some embodiments, the anaerobic atmosphere comprises from 20% to 30% CO₂. In some embodiments, the anaerobic atmosphere comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, CO₂. In some embodiments, the anaerobic atmosphere comprises about 25% CO₂.

In certain aspects, provided herein are bioreactors comprising anaerobic bacteria under an anaerobic atmosphere comprising less than 95% N₂. In some embodiments, the anaerobic atmosphere comprises less than 90% N₂. In some embodiments, the anaerobic atmosphere comprises less than 95%, less than 92%, less than 90%, less than 87%, less than 85%, less than 82%, less than 80%, less than 77% N₂. In some embodiments, the anaerobic gas mixture comprises less than 85% N₂. In some embodiments, the anaerobic atmosphere comprises less than 80% N₂. In some embodiments, the anaerobic atmosphere comprises from 65% to 85% N₂. In some embodiments, the anaerobic atmosphere comprises from 70% to 80% N₂. In some embodiments, the anaerobic atmosphere comprises about 65%, about 66%, about 67%, about 28%, about 69%, about 70%, about 71%, about 72% about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% N₂. In some embodiments, the anaerobic atmosphere comprises about 75% N₂.

In some embodiments, the anaerobic atmosphere consists essentially of CO₂and N₂. In some embodiments, the anaerobic atmosphere comprises about 25% CO₂and about 75% N₂. In some embodiments, the anaerobic atmosphere comprises about 20% CO₂and about 80% N₂. In some embodiments, the anaerobic atmosphere comprises about 30% CO₂and about 70% N₂.

In some embodiments, the bioreactor is at least about 1 L in volume, at least about 5 L in volume, at least about 10 L in volume, at least about 15 L in volume, at least about 20 L in volume, at least about 30 L in volume, at least about 40 L in volume, at least about 50 L in volume, at least about 100 L in volume, at least about 200 L in volume, at least about 250 L in volume, at least about 500 L in volume, at least about 750 L in volume, at least about 1000 L in volume, at least about 1500 L in volume, at least about 2000 L in volume, at least about 2500 L in volume, at least about 3000 L in volume, at least about 3500 L in volume, at least about 4000 L in volume, at least about 5000 L in volume, at least about 7500 L in volume, at least about 10,000 L in volume, at least about 15,000 L in volume, at least about 20,000 L in volume, at least about 30,000 L in volume, at least about 50,000 L in volume, at least about 100,000 L in volume, at least about 150,000 L in volume, at least about 200,000 L in volume, at least about 250,000 L in volume, at least about 300,000 L in volume, at least about 350,000 L in volume, at least about 400,000 L in volume, at least about 450,000 L in volume or at least about 500,000 L in volume. In some embodiments, the bioreactor is an about 20 L bioreactor, an about 3500 L bioreactor, an about 20,000 L bioreactor, an about 50,000 L bioreactor, an about 100,000 L bioreactor, an about 200,000 L bioreactor, an about 300,000 L bioreactor, an about 400,000 L bioreactor or an about 500,000 L bioreactor. In some embodiments, the bioreactor is an about 20 L bioreactor, an about 3500 L bioreactor, an about 20,000 L bioreactor, or an about 400,000 L bioreactor.

At all scales, mass transfer of CO₂can be important and is determined by a variety of factors. For example, mass transfer of CO₂can be modulated by other factors including, but not limited to, increasing gas flow, increasing the concentration of CO₂in the gas, increasing media agitation, agitator geometry, reactor geometry, and using a scintillator or other device to create smaller CO₂gas bubbles. Alternatively, addition of bicarbonate or other sources of CO₂can be implemented prior to or during culture growth. In certain embodiments, a combination specific to the vessel hardware/configuration can be used to optimize growth.

In some embodiments, the bioreactor further comprises a growth media. In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, maltodextrin (e.g., glucidex, e.g., glucidex 21 D), glucose, and hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises about 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises about 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises about 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises about 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises about 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises about 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises about 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 20 g/L to 30 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21 D). In some embodiments, the growth media comprises about 25 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21 D). In some embodiments, the growth media comprises 5 g/L to 15 g/L glucose. In some embodiments, the growth media comprises about 10 g/L glucose. In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises about 0.02 g/L hemoglobin.

In some embodiments the anaerobic bacteria is cultured in growth media. In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucose, and/or hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises about 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises about 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises about 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises about 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises about 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises about 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises about 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 5 g/L to 15 g/L glucose. In some embodiments, the growth media comprises about 10 g/L glucose. In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises about 0.02 g/L hemoglobin.

In some embodiments the anaerobic bacteria is cultured in growth media. In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, maltodextrin (e.g., glucidex, e.g., glucidex 21 D), and/or hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises about 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises about 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises about 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises about 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises about 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises about 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises about 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 20 g/L to 30 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21 D). In some embodiments, the growth media comprises about 25 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21 D). In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises about 0.02 g/L hemoglobin.

In some embodiments, the anaerobic bacteria are selected from bacteria of the genus Actinomyces, Bacteroides, Clostridium, Fusobacterium, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, or Veillonella. In some embodiments, the anaerobic bacteria are from the genus Prevotella. In some embodiments, the anaerobic bacteria are from a strain of Prevotella bacteria comprising one or more proteins listed in Table 1. In some embodiments, the anaerobic bacteria are from a strain of Prevotella substantially free of a protein listed in Table 2. In some embodiments, the anaerobic bacteria are from a strain of Prevotella bacteria comprising one or more of the proteins listed in Table 1 and that is free or substantially free of a protein listed in Table 2.

In some embodiments, the Prevotella bacteria are of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, or Prevotella veroralis.

In some embodiments, the Prevotella is Prevotella Strain B 50329 (NRRL accession number B 50329). In some embodiments, the Prevotella strain is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329.

In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria comprising a protein listed in Table 1 and/or a gene encoding a protein listed in Table 1. In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria free or substantially free of a protein listed in Table 2 and/or a gene encoding a protein listed in Table 2.

In some aspects, provided herein is a stabilizer that stabilizes bacterial compositions and methods of making and using such a stabilizer. In some embodiments, the stabilizer comprises at least one of sucrose, dextran 40k, cysteine HCl, and water. In some embodiments, the stabilizer comprises sucrose (e.g., about 200 g/kg sucrose), dextran 40k (e.g., about 200 g/kg dextran 40k), cysteine HCl (about 4 g/kg cysteine HCl), and water (e.g., about 596 g/kg water). In some aspects, provided herein are bacterial compositions comprising a stabilizer provided herein and bacteria (e.g., a Prevotella strain disclosed herein), and methods of preparing the same. In some embodiments, the bacterial composition comprises sucrose, dextran 40k, and cysteine HCl. In some embodiments, the bacterial composition comprises 1.5% sucrose, 1.5% dextran 40k, and 0.03% cysteine HCl. In certain embodiments, the bacterial composition is prepared by combining and mixing bacteria with a certain percentage of the stabilizer in liquid suspension. In some embodiments, the percentage of the stabilizer solution used to mix with bacteria is about 10%. In some embodiments, the bacteria in the bacterial composition are anaerobic bacteria. In some embodiments, the anaerobic bacteria are Prevotella histicola. In some such embodiments, the anaerobic bacteria are Prevotella histicola Strain B 50329. In some embodiments, the bacterial composition is lyophilized to form a powder.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of an exemplary manufacturing process for anaerobic bacteria, including, e.g., Prevotella histicola.

FIG. 2 is a schematic of an exemplary manufacturing process described herein.

FIG. 3 is a plot showing that reduced rates of sparging (bubbling) of 95% N₂, 5% CO₂gas (0.1 vvm vs. 0.02 vvm) results in decreased growth potential of Prevotella histicola Strain B 50329 anaerobic bacteria. (vvm stands for Volume of gas per Volume of vessel per Minute).

FIG. 4 is a plot showing that the presence of CO₂is necessary for initiating Prevotella histicola Strain B 50329 growth, as well as the effect of various amounts of CO₂(0%, 5%, 25%, 100%) on Prevotella histicola growth potential. (vvm stands for volume of gas per volume of vessel per minute).

FIG. 5 is a plot showing that Prevotella histicola Strain B 50329 consumes CO₂.

FIG. 6 is a plot showing that maltodextrin in combination with glucose can support growth of Prevotella histicola Strain B 50329 better than glucose alone.

DETAILED DESCRIPTION
Definitions

As used herein, “anaerobic conditions” are conditions with reduced levels of oxygen compared to normal atmospheric conditions. For example, in some embodiments anaerobic conditions are conditions wherein the oxygen levels are partial pressure of oxygen (pO₂) no more than 8%. In some instances, anaerobic conditions are conditions wherein the pO₂is no more than 2%. In some instances, anaerobic conditions are conditions wherein the pO₂is no more than 0.5%. In certain embodiments, anaerobic conditions may be achieved by purging a bioreactor and/or a culture flask with a gas other than oxygen such as, for example, nitrogen and/or carbon dioxide (CO₂).

The term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pre-treatment state.

As used herein, “engineered bacteria” are any bacteria that have been genetically altered from their natural state by human intervention and the progeny of any such bacteria. Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution.

The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. The term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.

“Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).

The term “increase” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10{circumflex over ( )}9 fold, 10{circumflex over ( )}4 fold, 10{circumflex over ( )}5 fold, 10{circumflex over ( )}6 fold, and/or 10{circumflex over ( )}7 fold greater after treatment when compared to a pre-treatment state. Properties that may be increased include immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites, and cytokines.

“Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species. In some embodiments the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. For 16S, OTUs that share ≥97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See e.g. Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ross R P, and O'Toole P W. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. For complete genomes, MLSTs, specific genes, other than 16S, or sets of genes OTUs that share ≥95% average nucleotide identity are considered the same OTU. See e.g., Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof. Operational Taxonomic Units (OTUs) with taxonomic assignments made to, e.g., genus, species, and phylogenetic Glade are provided herein.

“Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.

Manufacturing Process

In certain aspects, provided herein are methods of culturing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor under an anaerobic atmosphere comprising CO₂, e.g., greater than 1% CO₂(e.g., greater than 5% CO₂). In certain aspects, provided herein are methods of growing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor into which a gas mixture comprising CO₂is introduced e.g., greater than 1% CO₂(e.g., greater than 5% CO₂). In some embodiments, provided herein are methods culturing anaerobic bacteria, the method comprising the steps of a) purging a bioreactor with an anaerobic gas mixture comprising greater than 1% CO₂; and b) culturing the anaerobic bacteria in the bioreactor purged in step a) (e.g., while a gas mixture comprising greater than 1% CO₂is introduced into the bioreactor).

In certain aspects, provided herein are methods of culturing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor under an anaerobic atmosphere comprising N₂, e.g., less than 95% N₂(e.g., less than 90% N₂). In certain aspects, provided herein are methods of culturing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor into which a gas mixture comprising N₂is introduced, e.g., less than 95% N₂(e.g., less than 90% N₂). In some embodiments, provided herein are methods culturing anaerobic bacteria, the method comprising the steps of a) purging a bioreactor with an anaerobic gas mixture comprising less than 95% N₂; and b) culturing the anaerobic bacteria in the bioreactor purged in step a) (e.g., while a gas mixture comprising less than 95% N₂is introduced into the bioreactor).

Schematic representations providing exemplary manufacturing methods according to certain embodiments provided herein are depicted in FIGS. 1 and 2.

In certain embodiments, culturing anaerobic bacteria according to a method provided herein results in an improved yield of anaerobic bacteria. In certain embodiments, the yield is improved by a factor of at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold or 3.0-fold. In some embodiments, the yield is improved by a factor of between 1.5-fold and 4.0-fold. In some embodiments, the yield is improved by a factor of between 2-fold and 3-fold.

In some embodiments, the methods provided herein reduce contamination of the anaerobic bacteria culture. For example, the methods provided herein can prevent the outgrowth or overgrowth of a contaminant in the anaerobic bacteria culture. Contaminants can include, e.g., bacterial strains present in air flow or gas flow and/or environmental strains, e.g., present at a manufacturing facility.

In certain aspects, provided herein are methods of growing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor under an anaerobic atmosphere comprising CO₂. In some embodiments, the anaerobic atmosphere comprises greater than 1% CO₂. In some embodiments, the anaerobic atmosphere comprises at least about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, CO₂. In some embodiments, the anaerobic atmosphere comprises greater than 5% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 8% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 10% CO₂. In some embodiments, the anaerobic atmosphere comprises at least 20% CO₂. In some embodiments, the anaerobic atmosphere comprises from 8% to 40% CO₂. In some embodiments, the anaerobic atmosphere comprises from 10% to 40% CO₂. In some embodiments, the anaerobic atmosphere comprises from 20% to 30% CO₂. In some embodiments, the anaerobic atmosphere comprises about 25% CO₂.

In certain aspects, provided herein are methods of growing anaerobic bacteria comprising culturing the anaerobic bacteria in a bioreactor into which an anaerobic gas mixture comprising CO₂is introduced. In some embodiments, the anaerobic gas mixture comprises greater than 1% CO₂. In some embodiments, the anaerobic gas mixture comprises at least about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, CO₂. In some embodiments, the anaerobic gas mixture comprises greater than 5% CO₂. In some embodiments, the anaerobic gas mixture comprises at least 8% CO₂. In some embodiments, the anaerobic gas mixture comprises at least 10% CO₂. In some embodiments, the anaerobic gas mixture comprises at least 20% CO₂. In some embodiments, the anaerobic gas mixture comprises from 8% to 40% CO₂. In some embodiments, the anaerobic gas mixture comprises from 10% to 40% CO₂. In some embodiments, the anaerobic gas mixture comprises from 20% to 30% CO₂. In some embodiments, the anaerobic gas mixture comprises about 25% CO₂.

In some embodiments, the anaerobic atmosphere and/or gas mixture comprises less than 95% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises less than 95%, less than 92%, less than 90%, less than 87%, less than 85%, less than 82%, less than 80%, less than 77% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises less than 85% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises less than 80% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises from 65% to 85% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises from 70% to 80% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises about 75% N₂.

In certain aspects, provided herein are methods of culturing anaerobic bacteria, the method comprises the steps of a) purging a bioreactor with an anaerobic gas mixture comprising greater than 1% CO₂; and b) culturing the anaerobic bacteria in the bioreactor purged in step a). In some embodiments the method comprises introducing the anaerobic gas mixture into the bioreactor during step b). In some embodiments, the anaerobic gas mixture comprises greater than 1% CO₂. In some embodiments, the anaerobic gas mixture comprises at least about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, CO₂. In some embodiments, the anaerobic gas mixture comprises at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, CO₂. In some embodiments, the anaerobic gas mixture comprises from 5% to 35% CO₂, 10% to 40% CO₂, 10% to 30% CO₂, 15% to 30% CO₂, 20% to 30% CO₂, 22% to 28% CO₂, or 24%, to 26% CO₂. In some embodiments, the anaerobic gas mixture comprises greater than 5% CO₂. In some embodiments, the anaerobic gas mixture comprises at least 8% CO₂. In some embodiments, the anaerobic gas mixture comprises at least 10% CO₂. In some embodiments, the anaerobic gas mixture comprises at least 20% CO₂. In some embodiments, the anaerobic gas mixture comprises from 8% to 40% CO₂. In some embodiments, the anaerobic gas mixture comprises from 10% to 40% CO₂. In some embodiments, the anaerobic gas mixture comprises from 20% to 30% CO₂. In some embodiments, the anaerobic gas mixture comprises about 25% CO₂. In some embodiments, CO₂gas is continuously added during culturing.

In some embodiments, the anaerobic gas mixture comprises CO₂and N₂in a ratio of about 1:99, about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, about 10:90, 11:89, about 12:88, about 13:87, about 14:86, about 15:85, about 16:84, about 17:83, about 18:82, about 19:81, about 20:80, 21:79, about 22:78, about 23:77, about 24:76, about 25:75, about 26:74, about 27:73, about 28:72, about 29:71, about 30:70, 31:69, about 32:68, about 33:67, about 34:66, about 35:65, about 36:64, about 37:63, about 38:62, about 39:61, or about 40:50 CO₂to N₂.

In certain aspects, provided herein are bioreactors comprising anaerobic bacteria under an anaerobic atmosphere comprising at least about 1% CO₂and/or into which an anaerobic gas mixture comprising at least about 1% CO₂is added. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises greater than 5% CO₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises at least 8% CO₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises at least 20% CO₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises from 8% to 40% CO₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises from 10% to 40% CO₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises from 20% to 30% CO₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises about 25% CO₂.

In certain aspects, provided herein are bioreactors comprising anaerobic bacteria under an anaerobic atmosphere comprising less than 95% N₂and/or into which an anaerobic gas mixture comprising less than 95% N₂is added. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises less than 90% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises less than 85% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises from 65% to 85% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises from 70% to 80% N₂. In some embodiments, the anaerobic atmosphere and/or gas mixture comprises about 75% N₂.

In some embodiments, the methods and compositions provided herein include the culturing of anaerobic bacteria in growth media. In some embodiments the growth media may contain sugar, yeast extracts, plant based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and/or vitamins.

The sugar source present in the growth media can affect growth. For example, use of maltodextrin (e.g., glucidex, e.g., glucidex 21D) can provide better growth than use of glucose, e.g., at the same concentration of each sugar source, e.g., about 10 g/L. Alternatively, maltodextrin and glucose can both be used in the growth media, e.g., glucose at 10 g/L and maltodextrin at 25 g/L. For example, use of maltodextrin (e.g., glucidex, e.g., glucidex 21D) and glucose can provide better growth than use of glucose alone, e.g., at the same total concentration, e.g., about 35 g/L total sugar. In some embodiments, the growth media comprises glucose. In some embodiments, the growth media comprises maltodextrin. In some embodiments, the growth media comprises glucose and maltodextrin.

In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, maltodextrin (e.g., glucidex, e.g., glucidex 21D), glucose, and/or hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 20 g/L to 30 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21D). In some embodiments, the growth media comprises 25 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21D). In some embodiments, the growth media comprises 5 g/L to 15 g/L glucose. In some embodiments, the growth media comprises 10 g/L glucose. In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises 0.02 g/L hemoglobin.

In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucose, and/or hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 5 g/L to 15 g/L glucose. In some embodiments, the growth media comprises 10 g/L glucose. In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises 0.02 g/L hemoglobin.

In some embodiments, the growth media comprises yeast extract, soy peptone A2SC 19649, Soy peptone E110 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, maltodextrin (e.g., glucidex, e.g., glucidex 21D), and/or hemoglobin. In some embodiments, the growth media comprises 5 g/L to 15 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 12.5 g/L Soy peptone E110 19885. In some embodiments, the growth media comprises 1 g/L to 2 g/L dipotassium phosphate. In some embodiments, the growth media comprises 1.59 g/L Dipotassium phosphate. In some embodiments, the growth media comprises 0.5 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.91 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.5 g/L L-cysteine-HCl. In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises 0.5 g/L ammonium chloride. In some embodiments, the growth media comprises 20 g/L to 30 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21D). In some embodiments, the growth media comprises 25 g/L maltodextrin (e.g., glucidex, e.g., glucidex 21D). In some embodiments, the growth media comprises 0.01 g/L to 0.05 g/L hemoglobin. In some embodiments, the growth media comprises 0.02 g/L hemoglobin.

In some embodiments, the media is sterilized. Sterilization may be by Ultra High Temperature (UHT) processing, autoclaving or filtering. The UHT processing is performed at very high temperature for short periods of time. The UHT range may be from 135-180° C. For example, the medium may be sterilized from between 10 to 30 seconds at 135° C.

In some embodiments, inoculum can be prepared in flasks or in smaller bioreactors where growth is monitored. For example, the inoculum size may be between approximately 0.1% v/v and 5% v/v of the total bioreactor volume. In some embodiments, the inoculum is 0.1-3% v/v, 0.1-1% v/v, 0.1-0.5% v/v, or 0.5-1% v/v of the total bioreactor volume. In some embodiments, the inoculum is 0.1% v/v, 0.2% v/v, 0.3% v/v, 0.4%, v/v, 0.5% v/v, 0.6% v/v, 0.7% v/v, 0.8% v/v, 0.9% v/v, 1% v/v, 1.5% v/v, 2% v/v, 2.5% v/v, 3% v/v 4%, v/v, or 5% v/v of the total bioreactor volume.

Depending on the application and need for material, bioreactor volume can be at least 1 L, 2 L, 10 L, 80 L, 100 L, 250 L, 1000 L, 2500 L, 3500 L, 5000 L, 10,000 L, 20,000 L, 50,000 L, 100,000 L, 200,000 L, 300,000 L, 400,000 L or 500,000 L.

In some embodiments, before the inoculation, the bioreactor is prepared with growth medium at desired pH and temperature. The initial pH of the culture medium may be different that the process set-point. pH stress may be detrimental at low cell centration; the initial pH could be between pH 7.5 and the process set-point. For example, pH may be set between 4.5 and 8.0, preferably 6.5. During the fermentation, the pH can be controlled through the use of sodium hydroxide, potassium hydroxide, or ammonium hydroxide. The temperature may be controlled from 25° C. to 45° C., for example at 37° C.

In certain embodiments, anaerobic conditions are created by reducing the level of oxygen in the bioreactor by introducing or purging the bioreactor with nitrogen, carbon dioxide or gas mixtures (N₂and CO₂) in order to establish an anaerobic atmosphere in the bioreactor.

In some embodiments, the atmosphere comprises at least about 2% to about 40% CO₂, about 5% to 35% CO₂, about 10% to 30% CO₂, about 15% to 30% CO₂, about 20% to 30% CO₂, about 22% to 28% CO₂, or about 24% to 26% CO₂. In some embodiments, the anaerobic gas mixture comprises greater than 5% CO₂. In some preferred embodiments, the atmosphere comprises about 25% CO₂.

In some embodiments, the atmosphere comprises at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% CO₂. In some embodiments, the anaerobic gas mixture comprises greater than 5% CO₂. In some preferred embodiments, the atmosphere comprises at least about 25% CO₂.

In some embodiments, the atmosphere comprises 65% to 85% N₂or 70% to 80% N₂. In some embodiments, the anaerobic gas mixture comprises less than 95% N₂. In some preferred embodiments, the atmosphere comprises about 75% N₂.

In some embodiments, the atmosphere comprises about 65%, about 66%, about 67%, about 28%, about 69%, about 70%, about 71%, about 72% about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% N₂. In some embodiments, the anaerobic gas mixture comprises less than 95% N₂. In some preferred embodiments, the atmosphere comprises about 75% N₂.

In some embodiments, the gas mixture (CO₂and N₂) provides an atmosphere in the bioreactor comprising CO₂and N₂in a ratio of about 1:99, about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, about 10:90, 11:89, about 12:88, about 13:87, about 14:86, about 15:85, about 16:84, about 17:83, about 18:82, about 19:81, about 20:80, 21:79, about 22:78, about 23:77, about 24:76, about 25:75, about 26:74, about 27:73, about 28:72, about 29:71, about 30:70, 31:69, about 32:68, about 33:67, about 34:66, about 35:65, about 36:64, about 37:63, about 38:62, about 39:61, or about 40:50 CO₂to N₂. In some embodiments, the mixed gas composition provides an atmosphere in the bioreactor comprising CO₂and N₂in a ratio of about 25:75.

In some embodiments, depending on strain and inoculum size, the bioreactor fermentation time can vary. For example, fermentation time can vary from approximately 5 hours to 48 hours. In some embodiments, fermentation time may be from about 5 hours to about 24 hours, about 8 hours to about 24 hours, about 8 hours to about 18 hours, about 8 hours to about 16 hours, about 8 hours to about 14 hours, about 10 hours to about 24 hours, about 10 hours to about 18 hours, about 10 hours to about 16 hours, about 10 hours to about 14 hours, about 10 hours to about 12 hours, about 12 hours to about 24 hours, about 12 hours to about 18 hours, about 12 hours to about 16 hours, or about 12 hours to about 14 hours. In some embodiments, fermentation time may be from about 12 hours to about 96 hours, from about 12 hours to about 72 hours, from about 12 hours to about 60 hours, from about 24 hours to about 96 hours, from about 24 hours to about 72 hours, from about 24 hours to about 60 hours, from about 24 hours to about 48 hours, from about 36 hours to about 96 hours, from about 36 hours to about 72 hours, from about 36 hours to about 60 hours, or from about 36 hours to about 48 hours.

In some embodiments, fermentation culture is continuously mixed with addition of a mixed gas composition of CO₂and N₂. In some embodiments, the mixed gas composition provides an atmosphere in the bioreactor comprising CO₂and N₂in a ratio of about 1:99, about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, about 10:90, 11:89, about 12:88, about 13:87, about 14:86, about 15:85, about 16:84, about 17:83, about 18:82, about 19:81, about 20:80, 21:79, about 22:78, about 23:77, about 24:76, about 25:75, about 26:74, about 27:73, about 28:72, about 29:71, about 30:70, 31:69, about 32:68, about 33:67, about 34:66, about 35:65, about 36:64, about 37:63, about 38:62, about 39:61, or about 40:50 CO₂to N₂. In some embodiments, the mixed gas composition provides an atmosphere in the bioreactor comprising CO₂and N₂in a ratio of about 25:75.

In certain embodiments, harvest time may be based on either glucose level is below 2 g/L or when stationary phase is reached.

In some embodiments, once fermentation complete, the culture is cooled (e.g., to 10° C.) and centrifuged collecting the cell paste. A stabilizer may be added to the cell paste and mixed thoroughly. Harvesting may be performed by continuous centrifugation. Product may be resuspended with various excipients to a desired final concentration. Excipients can be added for cryo protection or for protection during lyophilization. Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants. Prior to lyophilization, droplets of cell pellets may be mixed with excipients and submerged in liquid nitrogen.

In certain embodiments, the cell slurry may be lyophilized. Lyophilization of material, including live bacteria, may begin with primary drying. During the primary drying phase, the ice is removed. Here, a vacuum is generated and an appropriate amount of heat is supplied to the material for the ice to sublime. During the secondary drying phase, product bound water molecules may be removed. Here, the temperature is raised higher than in the primary drying phase to break any physico-chemical interactions that have formed between the water molecules and the product material. The pressure may also be lowered further to enhance desorption during this stage. After the freeze-drying process is complete, the chamber may be filled with an inert gas, such as nitrogen. The product may be sealed within the freeze dryer under dry conditions, preventing exposure to atmospheric water and contaminants. The lyophilized material may be gamma irradiated (e.g., 17.5 kGy).

Anaerobic Bacteria

In some aspects, provided herein are methods and compositions for culturing anaerobic bacteria. In certain aspects, the anaerobic bacteria used in the methods and compositions provided herein are selected from bacteria of the genus Actinomyces, Bacteroides, Clostridium, Fusobacterium, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, or Veillonella.

In some embodiments, the anaerobic bacteria are Prevotella bacteria of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella melanogenica, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, or Prevotella veroralis.

In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) proteins listed in Table 1 and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) genes encoding proteins listed in Table 1. In some embodiments, the Prevotella bacteria comprises all of the proteins listed in Table 1 and/or all of the genes encoding the proteins listed in Table 1.

TABLE 1

Exemplary Prevotella proteins

Seq. ID. No.
Name
Uniprot ID
Amino Acid Sequence

1
Cluster:
G6ADE1
MNLKTFTKTVLCFALFAVSAITAKAADHLAIVGE

Uncharacterized

AVWGGWDLVKATAMVKSPNNPDVFMATVHLNAGK

protein

GFKFLTEREWGKLEYRSGASDVVLKSGIRYKLYA

SIGASEDGKFKVSESANYEIICDLARKTVEVKKV

AYQAKEIRYAALWMIGDATAGDWDYNNGVLLSQD

SGNPTCYTATVELKEGEFKFTTNKQWGYDHSVYI

FRDVNDQNKIVFGGEDNKWRITEDGMYNVTVDVP

TKTISIKQIDDPAGHKPQFGNDVILVGDATIAGW

NLDNAIYLEHTGQAGRVFKTTTYLEAGKGFKFLS

MLSYDDIDYRPANNTVLNPGVPGTFVPSLPSSTD

TKFSVERSGNYDIVCNMNNRTVVVTLSENQVLVN

YPALWLIGSATSAGWNPGKAVELKRSEADPAVYT

ARVQLKKGEFKILTSKNVGFDQPTYYRDSTNEHR

IVFGVDGDEVAKKDCKWTLSENAEGTYDVTVDIE

AMTIFCDKVNMDEPSVESTDKELILIGDATYSAW

DLPKSIVMTPVGPTTFKAVTHLEAGKEFKFLTEL

AWKRYEYRAESLRKELQEGSMSMLVPYRYTNDKD

DKDHDFKFVVKESGNYEIVCDLYIPALIIRKVRY

QDTPVTYSSLWIVGSATPGGWTIERGIKMTQDEN

YPTKFTAKANLVPGELKFATNKFADFTQDFFFRG

KDDYTAVLGGNDNKWNITEAGTYSVTIDVASKRV

TITKPARNAPTGISTVDSSDEAPAEYFTLNGIKV

TTPSSGIYIKRQGGRTTKVVMK

2
Nicotinamide_
P24520
MDTYQILDIIGCIVGLIYIYQEYKASIWLWMTGI

riboside_

IMPVIYMFVYYEAGLYADFGMQIYYTLAAIYGYL

transporter_PnuC

YWKLGKKKGTEDKEIPITHFPRRYIIPAIIVFFV

LWIALYYILICFTNSTVPVLDSFGNALSFIGLWA

LAKKYLEQWWIWIVVDAELSALYIYKGIPFTAML

YALYTVIAVAGYFKWRRYIKQQK

3
Pectate_trisaccharide-
Q8GCB2
MRVRLYKNILLFLFLWVNTLACVSADTSRTVESQ

lyase

PIENGLIITESKGWLETIYAKWKPVAEADGYYVY

VKGGQYADYSKVDSELIRVYNGYVRVDIPGLKAG

TYSLKIVAVKGGKETQSSEVTGLKVLNYVREGFA

HKNYSGVGAYNDDGTLKSGAVVIYVNKDNAKTVS

AHLGKTTFIGLQAILNAYQKGNITTPLSVRILGL

LRNGDTDTFGSSTEGIQIKGKQADSEMNITIEGI

GEDASIYGFGFLVRNAKSVEFRNLGIMRAMDDGV

SLDTNNSNIWIHHMDLFYGKASGGDHIKGDGSID

VKTDSKYVTIDNCHFWDTGKTSMCGMKKETGPNY

ITYHHNWFDHSDSRHARVRTMSVHLWNNYYDGCA

KYGIGATMGCSVFSENNYFRATKNPILISKQGSD

AKGTGKFSGEPGGMVKEYGSLFTEKGAESTYTPI

SYADNNSSFDFYHAISRNEKVPASVKTLNGGNIY

NNFDTDAALMYSYTPDATALVPSQVTGFYGAGRL

NHGSLQFKFNNAVEDTNSTPIPALEALIDAYSGK

4
Glycosyltransferase_
Q9AET5
MKYNIAYCIEGFYNHGGMERILSVCANLLSDIYS

Gtf1

ITIIVANQRGREHAYNLAQNVNVVDLGVSCKNYK

EEYKKSLTRYLQDHQFSVVISLAGLELFFLPQIK

DGSKKVMWFHFAFDVSKMFLSERFHGWKLNLLYY

IHTIRRIYFAKKFDTIVVLSKSDCDSWSRFCNNV

KYIYNPITIDRKVISNLSEESVIAVGRLGWQKGF

DFLIDSWVLVDDKHPDWHLDIFGEGPDRLELQHQ

IDRKGLHDKVRLCGVTKQIEEEYGKHSIYVMSSR

AEGFPLALLEASSCGLPMISFNCHQGPNEIIQEG

ENGFLVDKVGDIYTLSDRICKLIEDNNLRNMMGK

KALDSSFRFEGEVIKKDWISLLKQLI

5
Cluster: Protein
A0A096B759
MKRLFFMFLFLGTITMNSLAQEEKPIKYETKNFS

TonB

LPDKMPLYPGGDGALRAFLSLNLHYPEKAQAFGV

EGRSLMKFCVSSDGSIKDISAVDCKITNYNRTEF

NKLPLSKQESLKKECAKAFAKEAARVIRLMPKWE

PAELNGKKMNVYYSLPFTFKLR

6
Cluster:
G6AEN6
MNYPLFIARKIYNGGDRTRKVSKPAIRIATIGVA

Uncharacterized

IGLAVMIISVGVVLGFKHTIRNKVVGFGSDITVA

protein

NFLTLQSSEQYPIQITDSLVKSLQITPGIKHVQR

YDYTQGILKTDNDFLGVLLKGVGPDFDSTFIHEN

MVEGSLPHFHDNESQQKIVISKTIADKLNLKVGQ

RIFAYFINKQGVRTRKFTITGIYATNMKQFDSQI

CFTDIYTTNKLNGWEPDQYSGAELQVDNFSQLTP

ISMRVLNKVKNTVDHYGGTYSSENIIEQNPQIFS

WLDLMDMNVWIILALMISVAGVTMISGLLIIILE

RTQMIGILKALGSRNRQIRHIFLWFATFIIGKGL

LWGNIIGLGCILFQSWTGLVKLDPQTYYVNTVPV

EINIPLIIALNMVTMLVCLVILIAPSYLISHIHP

AKSMHYE

7
Bifunctional_
P9WHG9
MEDKFIYTDKERKLSYQILDELKDTLDKSFLEND

(p)ppGpp_synthase/

LPMLQVQLKDSVAKNTIHRNVFGLNPILCSLQTA

hydrolase_RelA

AIAVKDIGLKRDSVIAILLHQSVQDGYITLEDID

NRFGKSVAKIIHGLIRIQTLYQKNPIIESENFRN

LLLSFAEDMRVILIMIADRVNLMRQIRDAEDKEA

QHKVAEEASYLYAPLAHKLGLYQLKRELEDLSLK

YLEHDAYYLIKDKLNATKASRDAYINQFIAPVRE

RLTAGGLRFHIKGRTKSIHSIWQKMKKQKCGFEG

IYDLFAIRIILDAPLEKEKIQCWQAYSIVTDMYQ

PNPKRLRDWLSVPKSNGYECLHITVLGPEKKWVE

VQIRTERMDEIAEHGLAAHWRYKGIKEEGGLDDW

LASIRAALEAGDNLEVMDQFKSDLYEKEIYVFTP

KGDLLKFPKGATILDFAYHIHSKVGNQCVGGKIN

AKNVSLRTELHSGDTVEILTSATQKPKAEWLKIV

KSSRAKAKIRLALKETQIKDGLYAKELLERRFKN

KKIEIEESTMGHLLRKLGFKEVSEFYKQVADEKL

DPNYIIEEYQKVYNHDHNLNQPKETESAENFEFE

NPTNEFLKKNDDVLVIDKNLKGLDFSLAKCCHPI

YGDPVFGFVTVNGGIKIHRTDCPNAPEMRKRFGY

RIVKARWSGKGSSQYAITLRVIGNDDIGIVSNIT

NVISKDEKIVMRSINIDSHDGLFSGNLVVLLDDN

SKLNMLIKKLRTVKGVKQVTRI

8
Vitamin_B12_
P06609
MKRRIFLFVALSVSIVILFGLNLIIGSVHIPLSD

import_system_

ILTILSGSFTGKESWRFIIWDSRLPQALTAMLCG

permease_protein_BtuC

SSLAVCGLMLQTAFRNPLAGPDVFGISSGASLGV

ALVMLLLGGTVETSMFTASGFLAILIVAFAGAIL

VTAFILFLSSVVRNSVLLLIVGIMVGYVASSAVT

LLNFFSSEDGVKGYIVWGMGNFGGVSMSHIPLFA

FLCLAGIIASFLLVKPLNILLLGPQYAESLGISI

RRIRNILLVVVGILTAVTTAFCGPISFIGLAAPH

VARLLFRTENHQKLLPGTLLVGTVVALLCNLICF

LPRESGMIPLNAVTPLIGAPIIIYVIMKRH

9
NADH-
P33599
MKLENKEFGFDSFATEMARLKNEKHFDYLVTVVG

quinone_

EDFGTEEGLGCIYILENTSTHERCSVKQLAKKVG

oxidoreductase_

EEFVIPSVIKLWADADLLEREVYDFYGIKFLGHP

subunit_C/D

DMRRLFLRNDFKGYPLRKDYDMDPAKNMYTTEDD

VELDTTTEWNLDKNGELVGTQHALFTDDNFVVNI

GPQHPSTHGVLRLQTVLDGETVTNIYPHLGYIHR

GIEKLCEQFTYPQTLALTDRMNYLSAMMNRHALV

GVIEEGMGIELSERILYIRTIMDELQRIDNHLLY

TACCAQDLGALTAFLYGMRDREHVLNVMEETTGG

RLIQNYYRIGGLQADIDPNFVSNVKELCKYLRPM

IQEYVDVFGDNVITHQRFEGVGVMDEKDCISYGV

TGPAGRASGWKNDVRKYHPYAMYDKVNFEEITLT

NGDSMDRYFCHIKEIYQSLNIIEQLIDNIPEGEF

YIKQKPIIKVPEGQWYFSVEGASGEFGAYLDSRG

DKTAYRLKFRPMGLTLVGAMDKMLRGQKIADLVT

TGAALDFVIPDIDR

10
FKBP-
P45523
MRTSTQSKDMGKKQEYKLRNEEFLHNISKKDSIK

type_peptidyl-

TLPHGIFYEIIKEGSGEGTVQPRSIVICNYRGSL

prolyl_cis-

ISGQVFDDSWQKPTPEAFRLNELITGLQIALCAM

trans_isomerase

HKGDSWRIYIPYQEGYGSKRNADIPAFSTLIFDI

ELINIA

11
Putative_acetolactate_
P9WKJ3
MADNKIAKESVKREVIAGERLYTLLVYSENVAGV

synthase_small_

LNQIAAVFTRRQVNIESLNVSASSIEGIHKYTIT

subunit

AWSDAATIEKITKQVEKKIDVIKADYYEDSDLFI

HEVGLYKIATPILLENAEVSRAIRKRNARMMEVN

PTYSTVLLAGMTDEVTALYHDLKNFDCLLQYSRS

GRVAVTRGFSEPVSDFLKSEEESSVL

12
Serine/threonine_
P0AGE4
MKKKVKIGLLPRVIIAILLGIFFGYFMPTPLARV

transporter_SstT

FLTFNGIFSQFLGFMIPLIIIGLVTPAIADIGKG

AGKLLLVTVIIAYVDTVVAGGLAYGTGLCLFPSM

IASTGGAMPHIDKATELAPYFSINIPAMADVMSG

LVFSFMLGLGIAYGGLTATKNIFNEFKYVIEKVI

AKAIIPLLPLYIFGVFLNMAHNGQAQQILLVFSQ

IIIVILVLHVFILVYQFCIAGAIIRRNPFRLLWN

MMPAYLTALGTSSSAATIPVTLEQTMKNGVGKEI

AGFVVPLCATIHLSGSAMKITACALTICLLVGLP

HDPALFIYFILMLSIIMVAAPGVPGGAIMAALAP

LASILGFNSEAQALMIALYIAMDSFGTACNVTGD

GAIALVVNKMFGKKER

13
Cluster:
G6AJ07
MKKLLLLVCAAVMSLSASAQAGDKALGAQLVFGS

Uncharacterized

ETNSLGFGVKGQYYFTDHIRGEGSFDYFLKNKGI

protein

SMWDINANVHYLFDVADKFKVYPLAGLGYTNWSY

KYEYAGAPVVEGSDGRLAVNLGGGVEYELTKNLN

VNAEAKYQIISNYNQLVLGVGVAYKF

14
Heterocyst_
P22638
MHFYCTKSSLDTMSERYVKRMIAKLASQGKTVIS

differentiation_ATP-

IAHRFSTIMDAKHIILLAKGKVVAEGTHQELLKT

binding_protein

SEDYRKLWSDQNDEID

15
UDP-2,3-
Q9I2V0
MKNVYFLSDAHLGSLAIAHRRTQERRLVRFLDSI

diacylglucosamine_

KHKASAVYLLGDMFDFWDEYKYVVPKGFTRFLGK

hydrolase

VSELTDMGVEVHFFTGNHDLWTYGYLEEECGVIL

HRKPVTMEIYGKVFYLAHGDGLGDPDPMFQFLRK

VFHNRVCQRLLNFFHPWWGMQLGLNWAKKSRLKR

ADGKEMPYLGEDKEYLVRYTKDYMRSHKDIDYYI

YGHRHIELDLTLSGKVRMLILGDWIWQFTYAVFD

GEHMFLEEYIEGESKP

16
Anaerobic_glycerol-
P0A9C0
MNSKQNDNYDVIIIGGGITGAGTARDCALRGLKV

3-phosphate_

LLVEKFDFTNGATGRNHGLLHSGARYAVTDPESA

dehydrogenase

TECIKENMVLRRIAKHCIEETDGLFITLPEDDIN

YQKTFVEACARAGISANIISPEEALRLDPSVNPD

LLGAVRVPDASVDPFHLTTANVLDARQHGADVLT

YHEVVAILTSNGRVEGVRLRNNHTGEEIEKHAVL

VINAAGIWGHDIAKMADIKINMFPAKGTLLVFGH

RVNKMVINRCRKPANADILVPDDAVCVIGTTSDR

VPYDTVDNLKITSEEVDTLIREGEKLAPSLATTR

ILRAYAGVRPLVAADNDPTGRSISRGIVCLDHEK

RDGLTGMITITGGKMMTYRLMAEQATDLACKKLG

INKTCETATTPLPGTAGKDSDNPHHTYSTAHKAA

KGRQGNRVKEIDERTEDDRALICECEEVSVGEAK

YAIEELHVHDLLNLRRRTRVGMGTCQGELCACRA

AGVMCENGVKVDKAMTDLTKFINERWKGMRPVAW

GSTLDEAQLTTIIYQGLCGLGI

17
Anaerobic_glycerol-
P13033
MRYDTIIIGGGLSGLTAGITLAKAGQKVCIVSAG

3-phosphate_

QSSLHFHSGSFDLLGYDADGEVVTHPLQAIADLK

dehydrogenase

AEHPYSKIGISNIEHLASQAKTLLCEAGISVMGN

YEQNHYRVTPLGTLKPAWLTTEGYAMIDDPEILP

WKKVELLNIQGFMDFPTQFIAENLRMMGVECQIK

TFTTDELSTARQSPTEMRATNIAKVLANKDALSK

VSERINAISGDPDALLLPAVLGFSNAESLDEMKQ

WIKKPVQYIATLPPSVSGVRTTILLKRLFAQAGG

TLLIGDSATTGQFSGNHLVSITTDHLPDEKLYAD

HFILASGSFMSHGIRSNYAGVYEPVFKLDVDAAE

KRDDWSVTNAFEAQPYMEFGVHTDKDFHATKDGK

NIENLYAIGSVLSGHNSIKHADGTGVSLLTALYV

AKKITGKG

18
Anaerobic_glycerol-
P0A996
MAEGIQLKNISGNNLEQCLKCSICTAYCPVSAVE

3-phosphate_

PKYPGPKQSGPDQERYRLKDSKFFDEALKMCLNC

dehydrogenase

KRCEVACPSGVRIADIIQASRITYSTHRPIPRDI

MLANTDFVGTMANMVAPIVNATLGLKPVKAVLHG

VMGIDKHRTFPAYSSQKFETWYKRMAAKKQDSYS

KHVSYFHGCYVNYNFPQLGKDLVKIMNAVGYGVH

LLEKEKCCGVALIANGLSGQARRQGKVNIRSIRK

AAEQNRIVLTTSSTCTFTMRDEYEHLLDIKTDDV

RENITLATRFLYRLIEKGDIKLAFRKDFKMRTAY

HSACHMEKMGWIIYSTELLKMIPGLELIMLDSQC

CGIAGTYGFKKENYQRSQEIGEGLFKQIKELNPD

CVSTDCETCKWQIEMSTGYEVKNPISILADALDV

EETIKLNQ

19
Glycerol_uptake_
P18156
MMIKNIVLSIPISLIIYLNHLIMEYSMTTQFLME

facilitator_protein

LIGTLILVLFGDGVCACVTLNKSKGQKAGWVVIT

IAWGLAVCMGVLVAGPYTGAHLNPAVSIGLAVAG

MFPWSSVPYYIVAQMIGGFLGGLLVWFFYKDHYD

ATDDEAAKLGTFCTSPAIRNYKMNFLSEVIATLV

LVFIIISFSVDGNTGDAEHFKFGLAALGPIPVTL

LIIALGMSLGGTTGYAMNPARDLSPRLAHAVCMK

GDNDWSYSWIPVLGPIIGAIIAGFCGAALLLV

20
Serine/threonine-
Q97PA9
MSEKIIPSNEPAQAASEPIKASYTEYTVIPSQGY

protein_kinase_StkP

CQFVKCKKGDQPVVLKGLKEAYRERVLLRNALKR

EFKQCQRLNHPGIVRYQGLVDVEGYGLCIEEEYV

DGRTLQAYLKESHTDDEKITIVNQIADALRYAHQ

QGVAHRNLKPSNILITKQGDHVKLIDFNVLSLDD

VKPTADTTRFMAPELKDETMTADGTADIYSLGTI

MKVMGLTLAYSEVIKRCCAFKRSDRYSDIDEFLA

DFNHDGSSFSMPKIGKGTVVIGFIAVVVIALAAL

AYNYGGALVDQVGKIDVTSIFKSDAETAPEDSAM

VKSVEQNNNDSVADEAPATGKLAFMNTMKPALYK

DLDRLFAKHSDDRAKLNRAIKVYYRGLIQANDTL

DNEQRAELDRVFGNYVKQKKAALK

21
Cluster: D-alanyl-
G6AHI1
MLVAQLFVGVLQAQKPVQNRRQAVGQSMERQGLV

D-alanine dipeptidase

NVKAVVPSIKVALMYARTDNFCHRMALS

22
Anaerobic_C4-
P0ABN5
MITGLVIIQLLIVLALIFIGARVGGIGLGIYGMI

dicarboxylate_

GVFILVYGFGLAPGSAPIDVMMIIVAVITAASAL

transporter_DcuA

QASGGLEYLVGVAAKFLQKHPDHITYFGPITCWL

FCVVAGTAHTSYSLMPIIAEIAQTNKIRPERPLS

LSVIAASLGITCSPVSAATAALISQDLLGAKGIE

LGTVLMICIPTAFISILVAAFVENHIGKELEDDP

EYKRRVAAGLINPEAACEEVQKAENEHDPSAKHA

VWAFLFGVALVILFGFLPQLRPEGVSMSQTIEMI

MMSDAALILLVGKGKVGDAVNGNIFKAGMNAVVA

IFGIAWMGNTFYVGNEKILDAALSSMISSTPILF

AVALFLLSIMLFSQAATVTTLYPVGIALGINPLL

LIAMFPACNGYFFLPNYPTEVAAIDFDRTGTTRV

GKYVINHSFQIPGFITTIVSILLGVLMVQFFR

23
L-asparaginase_2
P00805
MRILKITFVTVLALVMSTVVFAQKPKIRIIATGG

TIAGVSASATSSAYGAGQVGVQTLIDAVPQIKDI

ADVSGEQLVNIGSQDMNDEVWLKLAKRINDLLNK

EGYDGVLITHGTDTMEETAYFLSLTVHTDKPVVM

VGSMRPSTAISADGPANLYNGICTLVDPSSKGHG

VMVCMNNELFEAKSVIKTHTTDVSTFKGGLYGEM

GYVYNGKPYFLHKPVAKQGLTSEFNVDNLTSLPK

VGIVYGYANCSPLPIQAFVNAKFDGIVLAGVGDG

NFYKDVFDVALKAQNSGIQIVRSSRVPFGPTNLN

GEVDDAKYHFVASLNLNPQKARVLLMLALTKTKD

WQKIQQYFNEY

24
Trehalose_synthase/
P9WQ19
MALACAMTMSASAQMGTNPKWLGDAIFYQIYPSS

amylase_TreS

YMDTDGNGIGDLPGITQKLDYIKSLGVNAIWLNP

VFESGWFDGGYDVIDFYKIDPRFGTNTDMVNLVK

EAHKRGIKVCLDLVAGHTSTKCPWFKESANGDRN

SRYSDYFIWTDSISEADKKEIAERHKEANPASST

HGRYVEMNAKRGKYYEKNFFECQPALNYGFAKPD

PNQPWEQPVTAPGPQAVRREMRNIMAFWFDKGVD

GFRVDMASSLVKNDWGKKEVSKLWNEMREWKDKN

YPECVLISEWSDPAVAIPAGFNIDFMIHFGIKGY

PSLFFDRNTPWGKPWPGQDISKDYKFCYFDKAGK

GEVKEFVDNFSEAYNATKNLGYIAIPSANHDYQR

PNIGTRNTPEQLKVAMTFFLTMPGVPFIYYGDEI

GMKYQMDLPSKEGSNERAGTRTPMQWTSGPTAGF

STCNPSQLYFPVDTEKGKLTVEAQQNDPRSLLNY

TRELTRLRHSQPALRGNGEWILVSKESQPYPMVY

KRTSGGETVVVAINPSDKKVSANIAHLGKAKSLI

MTGKASYKTGKTEDAVELNGVSAAVFKIAE

25
Ribitol-5-
Q720Y7
MNIAVIFAGGSGLRMHTKSRPKQFLDLNGKPIII

phosphate_cytidyl-

YTLELFDNHPGIDAIVVACIESWIPFLEKQLRKF

yltransferase

EINKVVKIVPGGESGQASIYNGLCAAEAYIKSKN

VASEDTTVLIHDGVRPLITEETITDNINKVAEVG

SCITCIPATETLVVKQHDGSLEIPSRADSLIARA

PQSFLLSDILTAHRRAIDEKKNDFIDSCTMMSHY

GYRLGTIIGPMENIKITTPTDFFVLRAMVKVHED

QQIFGL

26
UDP-Glc: alpha-D-
B5L3F2
MTEKKSVSIVLCTYNGTKYLQEQLDSILAQTYPL

GlcNAc-

HEIIIQDDGSTDNTWQILEKYEEKYPLIHIYHNE

diphosphoundecaprenol

GTHGVNANFLSAMHRTTGDFIAIADQDDIWETDK

IANQMTTIGNKLLCSGLTRPFSSDGSFAYFDNRP

RNVSIFRMMFLGLPGHTMLFRRELLRMMPPVTHS

FFNVSLYDAALSILAASHDSIAFCNKVLVNFRRH

ADATTYNDYSRSLPSWQNGLYELLWGLRHYHQAR

SIALPIYRGKLALMEGITTNYHDFIEAKAIMRLE

TQKGLWAFLRLQYLLTKNHQRLFQTSGGSFIKMI

RAWLYPVMQLYMYHHALRRCK

27
UDP-N-
P33038
MESFIIEGGHRLSGTIAPQGAKNEALEVICATLL

acetylglucosamine

TTEEVIIRNIPNILDVNNLIKLLQDIGVKVKKLG

ANDFSFQADEVKLDYLESIDFVKKCSSLRGSVLM

IGPLLGRFGKATIAKPGGDKIGRRRLDTHFLGFK

NLGARFVRIEDRDVYEIQADKLVGDYMLLDEASV

TGTANIIMSAVMAEGTTTIYNAACEPYIQQLCHL

LNAMGAKITGIASNLITIEGVTSLHGAEHRILPD

MIEVGSFIGMAAMVGDGVRIKDVSIPNLGLILDT

FRRLGVQIIEDEDDLIIPRQDHYVIDSFIDGTIM

TISDAPWPGLTPDLISVLLVVATQAQGSVLFHQK

MFESRLFFVDKLIDMGAQIILCDPHRAVVVGHDH

AKKLRAGRMSSPDIRAGIALLIAALTAEGTSRID

NIAQIDRGYENIEGRLNALGAKVQRVEIC

28
Sensor_protein_EvgS
P30855
MERSGNFYKAIRLGYILISILIGCMAYNSLYEWQ

EIEALELGNKKIDELRKEINNINIQMIKFSLLGE

TILEWNDKDIEHYHARRMAMDSMLCRFKATYPAE

RIDSVRHLLEDKERQMCQIVQILEQQQAINDKIT

SQVPVIVQKSVQEQPKKSKRKGFLGIFGKKEEAK

PTVTTTMHRSFNRNMRTEQQAQSRRLSVHADSLA

ARNAELNRQLQGLVVQIDGKVQTDLQKREAEITA

MRERSFIQIGGLTGFVILLLVISYIIIHRNANRI

KRYKQETADLIERLQQMAKRNEALITSRKKAVHT

ITHELRTPLTAITGYAGLIQKNFNADKTGMYIRN

IQQSSDRMREMLNTLLSFFRLDDGKEQPNFSTCR

ISSIAHTLESEFMPIAINKGLALTVTNHTDAVVL

TDKERILQIGNNLLSNAIKFTENGAVSLTMGYDN

GMLKLIVKDTGSGMTEEEQQRVFGAFERLSNAAA

KDGFGLGLSIVQRIVTMLGGTIQLKSEKGKGSRF

TVEIPMQSAEELPERINKTQIHHNRTLHDIVAID

NDKVLLLMLKEMYAQEGIHCDTCTNAAELMEMIR

RKEYSLLLTDLNMPDINGFELLELLRTSNVGNSR

IIPIIVTTASGSCNREELLERGFSDCLLKPFSIS

ELMEVSDKCAMKGKQNEKPDFSSLLSYGNESVML

DKLIAETEKEMQSVRDGEQRKDFQELDALTHHLR

SSWEILRADQPLRELYKQLHGSAVPDYEALNNAV

TAVLDKGSEIIRLAKEERRKYENG

29
Phosphate-
Q7A5Q2
MKRSRFYITVGLILSLTLLMSACGQKKAKDGRTD

binding_protein_PstS

TPTSGTIKFASDESFSPIVEELLQNYQFRYPQAH

LLPIYTDDNTGMKLLLDQKVNLFITSHAMTKGED

AILRGKGPIPEVFPIGYDGIAFIVNRSNPDSCIT

VDDVKKILQGKIAKWNQLNPKNNRGSIEVVFDNK

ASATLHYVVDSILGGKNIKSENIVAAKNSKSVID

YVNKTPNAIGVIGSNWLNDHRDTTNTTFKKDVTV

ASISKATVASPSNSWQPYQAYLLDGRYPFVRTIY

ALLADPHKALPYAFANYIANPIGQMIIFKAGLLP

YRGNINIREVEVKNQ

30
Bifunctional_purine_
P9WHM7
MAGTKRIKTALISVFHKDGLDDLLKKLDEEGVQF

biosynthesis_protein_

LSTGGTQQFIESLGYECQKVEDVTSYPSILGGRV

PurH

KTLHPKIFGGILARRDNEEDQKQMVEYTIPAIDL

VIVDLYPFEQTVASGASAQDIIEKIDIGGISLIR

AGAKNFKDVVIVPSKAEYPVLLQLLNTKGAETEI

EDRKMFAERAFGVSSHYDTAIHSWFAAE

31
Multidrug_efflux_
P0AE06
MEEEKGGRIGQRPYILKIITERNYIIIIDMKKAK

pump_subunit_AcrA

ILLFVTALVAVLTSCGGGQKGLPTSDEYPVITIG

ASNAQLKTTYPATIKGVQDVEVRPKVSGFITKLN

IHEGEYVHAGQVLFVIDNSTYQAAVRQAQAQVNS

AQSAVAQAKANVVQANASLNSANAQAATSRLTYN

NSQNLYNNKVIGDYELQSAKNTYETAQASVRQAQ

SGIASAQAAVKQAEAGVRQAQAMLSTAKDNLGFC

YVKSPASGYVGSLPFKEDALVSASSAQPVTTISN

TSTIEVYFSMTEADVLKLSRTDDGLSNAIKKFPA

VSLLLADGSTYNHEGAIVKTSGMIDATTGTINVI

ARFPNPEHLLKSGGSGKIVIAKNNNRALLIPQEA

VTQVQNKMFVYKVDAKDKVHYSEITVDPQNDGIN

YIVTSGLKMGERIVSKGVSSLEDGAKIKALTPAE

YEEAIKKAEKLGENQSSASGFLKTMKGDSK

32
Cell_division_protein_
Q81X30
MAKRRNKARSHHSLQVVTLCISTAMVLILIGMVV

FtsX

LTVFTSRNLSSYVKENLTVTMILQPDMSTEESAA

LCQRIRSLHYINSLNFISKEQALKEGTRELGANP

AEFAGQNPFTGEIELQLKANYANNDSIKNIEREL

RTYRGVSDITYPQNLVESVNHTLGKISLVLLVIA

ILLTIVSFSLMNNTIRLSIYARRFSIHTMKLVGA

SWGFIRAPFLRRAVMEGLVSALLAIAVLGVGLCL

LYDYEPDITKVLSWDVLVITAGVMLAFGVLIATF

CSWLSVNKFLRMKAGDLYKI

33
Fe(2+)_transporter_
Q9PMQ9
MKLSDLKTGETGVIVKVLGHGGFRKRIIEMGFIQ

FeoB

GKQVEVLLNAPLRDPVKYKIMGYEVSLRHSEADQ

IEVISAEEARQLEQAKADNEPQQGALSNNIPDES

DHALTPFELTDAANRKSKVINVALVGNPNCGKTS

LFNFASGAHERVGNYSGVTVDAKVGRANYEGYEF

HLVDLPGTYSLSAYSPEELYVRKQLVEKTPDVVI

NVIDASNLERNLYLTTQLIDMHVRMVCALNMFDE

TEQRGDNIDYQKISELFGIPMVPTVFTNGRGVKE

LFHQVIAVYEGKEDETSQFRHIHINHGHELEGGI

KNIQEHLRAYPDICQRYSTRYLAIKLLEHDKDVE

ELIKPLKDSDEIFKHRDIAAQRVKEETGNESETA

IMDAKYGFIHGALEEADYSTGQKKDTYQTTHFID

QILTNKYFGFPIFFLILFIMFTATFVIGQYPMDW

IDGGVSWLGDFISSNMPDGPVKDMLVDGIIGGVG

AVIVFLPQILILYFFISYMEDSGYMARAAFIMDK

LMHKMGLHGKSFIPLIMGFGCNVPAVMATRTIES

RRSRLVTMLILPLMSCSARLPIYVMITGSFFALK

YRSLAMLSLYVIGILMSVIMSRVFSRFLVKGEDT

PFVMELPPYRFPTWKAIGRHTWEKGKQYLKKMGG

IILVASIIVWALGYFPLPDKPDMGQQERQEHSFI

GQIGHAVEPVFRPQGFNWKLDVGLLAGVGAKEIV

ASTMGVLYSNDDSFKDDNSFSSEGGKYVKLHKQI

TQDVANLHGVSYNEAEPIATLTAFCFLLFVLLYF

PCIATIAAIKGETGSWGWALFAAGYTTLLAWVVS

AIVFQVGMLFIG

34
Pneumolysin
Q04IN8
MKKNLLKAVLPASLALFAVTFGSCSQDGQLTGTK

EDTGERVLDNTREIQNYLRTLPLAPMMSRASDPV

PSDDGTTVPVDEGTSKTEEKGVLNGIPGSWVKTT

RRYKMTQAFDESFLFDPTSDIVYPGCVLKGGTIA

NGTYAIITSHETGDVTFSINLSPANPQEARETSA

TVHNIRKSEYQEVWNKWANMQWKESPITTIESVE

KINSQEELATKLGVAVNSPVANGSLNFGFNFNKK

KNHILARLIQKYFSVSTDAPKKGNIFESIDKEAL

DGYQPVYISNINYGRIIYLSVESDEDEKVVDEAI

NFAMNQIKGVDVSVSADQSLHYRKVLANCDIRIT

VLGGGQTIQKEVLKGDIDSFQRFLNADIPMEQMS

PISFSLRYAVDNSQARVVTSNEFTVTQRDFVPEF

KKVRMQLQVLGFSGTNTGPFPNLDREAGLWGSIS

LSLNGQDNELVKISQSNPFFFNYREKKETMHPIG

FGGIVTVEFDKDPNESLEDFVDHQKMTFVSDLHS

TRSIYNYNFGRTTFTHTLGTLYTKYKGDDPIFVL

ESNNKNVKIHTYVKVLDMKFFN

35
Cluster:
G6AG77
MTKFIYAMSLFLLAAISIKAQPIQKTSGCLLHGS

Uncharacterized

VVSSTDATAIAGATVRLYQLKKLVGGTVSDASGN

protein

FDVKCPSSGSLQLRITAVGFKEVDTTLNVPTVTP

LSIYMRAGKHAMDEVTVTASEKRGMTSTTVIGQT

AMEHLQPSSFADLLALLPGGMTKIPALGSANVIT

LREAGPPSSQYATSSLGTKFVIDGQAIGTDANMQ

YIAGSFQGDADNSRNHVSYGVDMREIPTDNIEKV

EVVRGIPSVKYGELTSGLINITRKRSQSPLLLRL

KADEYGKLVSVGKGFLLSGKWNLNVDGGLLDARK

EPRNRFETYRRLTFSARLRRKWNLGERYVLEWSG

ATDYSLNIDNVKTDPEIQIHREDSYRSSYLKMGM

NHRLLLRRKALVGLQSVSLAYSASLASDRIHQTE

AVALQRDYVVPLAYEGGEYDGLFLPMQYLCDYRV

EGKPFYSTLRGETEWLARTSFISHHITAGGEFLL

NKNYGRGQIFDITKPLHASTARRPRSYKDIPATD

ILSFYAEDKATMPIGKHQLTVMAGLRTTQMLNIP

ASYAVHGKLFTDTRVNVQWDFPSFLGFKSFVSGG

LGMMTKMPTVLDLYPDYVYKDITEMNYWDIRPAY

KRIHIRTYKLNQVNPDLRPARNKKWEIRLGMDKG

AHHFSVTYFHEDMKDGFRSTTTMRPFIYKRYDTS

VINPSALTGPPSLASLPVVTDTLLDGYGRTENGS

RITKQGIEFQYSSPRIPVIQTRITVNGAWFRTLY

ENSIPLFRSAPNVVVGTVAIADRYAGYYMSTDKY

DKQIFTSNFIFDSYVDKLGLILSATAECFWMSNT

KRPATSSTPMGYMDITGTVHPYVEADQSDPYLRW

LVLTGTAGQDMDYRERSYMLVNFKATKRFGRHLS

LSFFADRVFYVAPDYEVNGFIVRRTFSPYFGMEI

GLKI

36
Cell_division_ATP-
P0A9R7
MLIDFKKVNIYQDERLILKDIDFQATEGEFIYLI

binding_protein_FtsE

GRVGSGKSSLLKTFYGELDIDQEDAEKAEVLGES

VLDIKQKRIPALRRQMGIIFQDFQLLHDRSVAKN

LKFVLQATGWKDKEKIKQRIKEVLEQVGMIDKAA

KMPSELSGGEQQRIAIARAFLNNPKIILADEPTG

NLDPETASNIVSILKDTCKNGTTVIMSTHNINLL

SQFPGKVYRCMEQALVPVTNEAQTKDLEEDSTSV

EPLIEPVLEEEAQAEDSKE

37
Di-/tripeptide_
P0C2U3
MFENQPKALYALALANTGERFGYYTMIAVFALFL

transporter

RANFGLEPGTAGLIYSIFLGLVYFLPLIGGIMAD

KFGYGKMVTIGIIVMFAGYLFLSVPLGGGTVAFG

AMLAALLLISFGTGLFKGNLQVMVGNLYDTPELA

SKRDSAFSIFYMAINIGALFAPTAAVKIKEWAET

SLGYAGNDAYHFSFAVACVSLIVSMGIYYAFRST

FKHVEGGTKKTEKAAAAAVEELTPQQTKERIVAL

CLVFAVVIFFWMAFHQNGLTLTYFADEFVSPTST

GVQSMAFDVVNLVMIVFIVYSIMALFQSKTTKAK

GIACAVILAAIAVLAYKYMNVNGQVEVSAPIFQQ

FNPFYVVALTPISMAIFGSLAAKGKEPSAPRKIA

YGMIVAGCAYLLMVLASQGLLTPHEQKLAKAAGE

TVPFASANWLIGTYLVLTFGELLLSPMGISFVSK

VAPPKYKGAMMGGWFVATAIGNILVSVGGYLWGD

LSLTVVWTVFIVLCLVSASFMFLMMKRLEKVA

38
Calcium-
Q47910
MKKILIFVAGLCMSLAASAQIQRPKLVVGLVVDQ

transporting_ATPase

MRWDYLYYYYNEYGTDGLRRLVDNGFSFENTHIN

YAPTVTAIGHSSVYTGSVPAITGIAGNYFFQDDK

NVYCCEDPNVKSVGSDSKEGQMSPHRLLASTIGD

ELQISNDFRSKVIGVALKDRASILPAGHAADAAY

WWDTSAGHFVTSTFYTDHLPQWVIDFNEKNHTAP

NFNIKTSTQGVTMTFKMAEAALKNENLGKGKETD

MLAVSISSTDAIGHVYSTRGKENHDVYMQLDKDL

AHFLKTLDEQVGKGNYLLFLTADHGAAHNYNYMK

EHRIPAGGWDYRQSVKDLNGYLQGKFGIAPVMAE

DDYQFFLNDSLIAASGLKKQQIIDESVEYLKKDP

RYLYVFDEERISEVTMPQWIKERMINGYFRGRSG

EIGVVTRPQVFGAKDSPTYKGTQHGQPFPYDTHI

PFLLYGWNVKHGATTQQTYIVDIAPTVCAMLHIQ

MPNGCIGTARNMALGN

39
Poly-beta-1,6-N-
Q5HKQ0
MDRQVFQTDSRQRWNRFKWTLRVLITIAILLGVV

acetyl-D-

FVAMFALEGSPQMPFRHDYRSVVSASEPLLKDNK

glucosamine_synthase

RAEVYKSFRDFFKEQKMHSNYAKVAARQHRFVGH

TDNVTQKYIKEWTDPRMGIRSAWYVNWDKHAYIS

LKNNLKNLNMVLPEWYFINPKTDRIEARIDQRAL

KLMRRAHIPVLPMLTNNYNSAFRPEAIGRIMRDS

TKRMGMINELVAACKHNGFAGINLDLEELNINDN

ALLVTLVKDFARVFHANGLYVTQAVAPFNEDYDM

QELAKYDDYLFLMAYDEYNAGSQAGPVSSQRWVE

KATDWAAKNVPNDKIVLGMATYGYNWAQGQGGTT

MSFDQTMATALNAGAKVNFNDDTYNLNFSYQDED

DGTLHQVFFPDAVTTFNIMRFGATYHLAGFGLWR

LGTEDSRIWKYYGKDLSWESAARMPIAKIMQLSG

TDDVNFVGSGEVLNVTSEPHAGRIGIVLDKDNQL

IIEERYLSLPATYTVQRLGKCKEKQLVLTFDDGP

DSRWTPKVLSILKHYKVPAAFFMVGLQIEKNIPI

VKDVFNQGCTIGNHTFTHHNMIENSDRRSFAELK

LTRMLIESITGQSTILFRAPYNADADPTDHEEIW

PMIIASRRNYLFVGESIDPNDWQQGVTADQIYKR

VLDGVHQEYGHIILLHDAGGDTREPTVTALPRII

ETLQREGYQFISLEKYLGMSRQTLMPPIKKGKEY

YAMQANLSLAELIYHISDFLTALFLVFLVLGFMR

LVFMYVLMIREKRAENRRNYAPIDPLTAPAVSII

VPAYNEEVNIVRTISNLKEQDYPSLKIYLVDDGS

KDNTLQRVREVFENDDKVVIISKKNGGKASALNY

GIAACSTDYIVCVDADTQLYKDAVSKLMKHFIAD

KTGKLGAVAGNVKVGNQRNMLTYWQAIEYTTSQN

FDRMAYSNINAITVIPGAIGAFRKDVLEAVGGFT

TDTLAEDCDLTMSINEHGYLIENENYAVAMTEAP

ESLRQFIKQRIRWCFGVMQTFWKHRASLFAPSKG

GFGMWAMPNMLIFQYIIPTFSPIADVLMLFGLFS

GNASQIFIYYLIFLLVDASVSIMAYIFEHESLWV

LLWIIPQRFFYRWIMYYVLFKSYLKAIKGELQTW

GVLKRTGHVKGAQTIS

40
ATP_synthase_subunit_
P29707
MSQINGRISQIIGPVIDVYFDTKGENPEKVLPNI

beta,_sodium_ion_

YDALRVKKADGQDLIIEVQQQIGEDTVRCVAMDN

specific

TDGLQRGLEVVPTGSPIVMPAGEQIKGRMMNVIG

QPIDGMSALQMEGAYPIHREAPKFEDLSTHKEML

QTGIKVIDLLEPYMKGGKIGLFGGAGVGKTVLIM

ELINNIAKGHNGYSVFAGVGERTREGNDLIRDML

ESGVIRYGEKFRKAMDEGKWDLSLVDSEELQKSQ

ATLVYGQMNEPPGARASVALSGLTVAEEFRDHGG

KNGEAADIMFFIDNIFRFTQAGSEVSALLGRMPS

AVGYQPTLASEMGAMQERITSTKHGSITSVQAVY

VPADDLTDPAPATTFTHLDATTELSRKITELGIY

PAVDPLGSTSRILDPLIVGKEHYDCAQRVKQLLQ

KYNELQDIIAILGMDELSDDDKLVVNRARRVQRF

LSQPFTVAEQFTGVKGVMVPIEETIKGFNAILNG

EVDDLPEQAFLNVGTIEDVKEKAKQLLEATKA

41
Cluster:
G6AGX5
MNPIYKIITSILFCVLSINTMAQDLTGHVTSKAD

Uncharacterized

DKPIAYATVTLKENRLYAFTDEKGNYTIKNVPKG

protein

KYTVVFSCMGYASQTVVVMVNAGGATQNVRLAED

NLQLDEVQVVAHRKKDEITTSYTIDRKTLDNQQI

MTLSDIAQLLPGGKSVNPSLMNDSKLTLRSGTLE

RGNASFGTAVEVDGIRLSNNAAMGETAGVSTRSV

SASNIESVEVVPGIASVEYGDLTNGVVKVKTRRG

SSPFIVEGSINQHTRQIALHKGVDLGGNVGLLNF

SIEHARSFLDAASPYTAYQRNVLSLRYMNVFMKK

SLPLTLEVGLNGSIGGYNSKADPDRSLDDYNKVK

DNNVGGNIHLGWLLNKRWITNVDLTAAFTYADRL

SESYTNESSNATQPYIHTLTEGYNIAEDYDRNPS

ANIILGPTGYWYLRGFNDSKPLNYSLKMKANWSK

AFGKFRNRLLVGGEWTSSMNRGRGTYYADMRYAP

SWREYRYDALPSLNNIAIYAEDKLSMDVNERQNA

ELTAGIREDITSIPGSEYGSVGSFSPRMNARYVF

RFGQNSWLNSMTLHAGWGRSVKIPSFQVLYPSPS

YRDMLAFASTSDADNRSYYAYYTYPSMARYNANL

KWQRADQWDLGVEWRTKIADVSLSFFRSKVSNPY

MATDVYTPFTYKYTSPAMLQRSGIAVADRRFSID

PQTGIVTVSDASGVKSPVTLGYEERNTYVTNTRY

VNADALQRYGLEWIVDFKQIKTLRTQVRLDGKYY

HYKAQDETLFADVPVGLNTRQSDGRLYQYVGYYR

GGAATTTNYTANASASNGSVSGQVDLNATITTHI

PKIRLIVALRLESSLYAFSRATSSRGYVVSSGNE

YFGVPYDDKTENQTVIVYPEYYSTWDAPDVLIPF

AEKLRWAETNDRGLFNDLAQLVVRTNYPYTLNPN

RLSAYWSANLSVTKEIGRHVSVSFYANNFFNTLS

QVHSTQTGLETSLFGSGYVPSFYYGLSLRLKI

In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria free or substantially free of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) proteins listed in Table 2 and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) genes encoding proteins listed in Table 2. In some embodiments, Prevotella bacteria is free of all of the proteins listed in Table 2 and/or all of the genes encoding the proteins listed in Table 2.

TABLE 2

Other Prevotella proteins

Seq. ID. No.
Name
Uniprot ID
Amino Acid Sequence

42
UDP-Gal: alpha-D-
Q03084
MERIDISVLMAVYKKDNPAFLRESLESIFSQTVEAAEV

GlcNAc-

VLLEDGPLTDALYDVIKSYEAIYSTLKVVSYPENRGLG

diphosphounde-

KTLNDGLLLCKYNLVARMDADDICKPNRLEMEYNWLKS

caprenol

HEDYDVIGSWVDEFTDNKTRVKSIRKVPEAYDEIKNYA

QYRCPINHPTAMYRKAAVLAVGGYLTEYFPEDYFLWLR

MLNNGSKFYNIQESLLWFRYSEETVAKRGGWAYACDEV

RILVRMLKMGYIPFHVFCQSVVIRFTTRVMPLPIRQRL

YNLIRKT

43
ATP_synthase_
A1B8P0
MSQINGRISQIIGPVIDVYFDTKGENPEKVLPKIHDAL

subunit_beta

RVKRANGQDLIIEVQQHIGEDTVRCVAMDNTDGLQRNL

EVVPTGSPIVMPAGDQIKGRMMNVIGQPIDGMEALSME

GAYPIHREAPKFEDLSTHKEMLQTGIKVIDLLEPYMKG

GKIGLFGGAGVGKTVLIMELINNIAKGHNGYSVFAGVG

ERTREGNDLIRDMLESGVIRYGEKFRKAMDEGKWDLSL

VDQEELQKSQATLVYGQMNEPPGARASVALSGLTVAEE

FRDHGGKNGEAADIMFFIDNIFRFTQAGSEVSALLGRM

PSAVGYQPTLASEMGTMQERITSTKHGSITSVQAVYVP

ADDLTDPAPATTFTHLDATTELSRKITELGIYPAVDPL

GSTSRILDPLIVGKDHYECAQRVKQLLQHYNELQDIIA

ILGMDELSDEDKLVVNRARRVQRFLSQPFTVAEQFTGV

KGVMVPIEETIKGFNAILNGEVDDLPEQAFLNVGTIED

VKEKAKRLLEATK

44
Cell_division_
O05779
MPIGNGQKYQLTIINHTEIIMLIDYKKVNIYQDERLIL

ATP-binding_

KDVDFQAETGEFIYLIGRVGSGKSSLLKTIYGELDIDS

protein_FtsE

EDAEKAVVLDESMPNIKRSRIPALRKQMGIIFQDFQLL

HDRSVAKNLKFVLQATGWTSKQKIERRIEEVLAQVGMT

DKKNKMPSELSGGEQQRIAIARALLNTPKIIIADEPTG

NLDPETAANIVSILKDSCQAGTTVIMSTHNINLIDQFP

GKVYRCHEGELHQLTDKKEVSELAEETAPVETIDEPEQ

ND

45
Hemin_transport_
Q56992
MKRNILLFICLATSILLLFGLNLTTGSVQIPFADILDI

system_permease_

LCGRFIGKESWEYIILENRLPQTLTAILCGASLSVCGL

protein_HmuU

MLQTAFRNPLAGPDVFGISSGAGLGVALVMLLLGGTVS

TSIFTVSGFLAILTAAFVGAIAVTALILFLSTLVRNSV

LLLIVGIMVGYVSSSAVSLLNFFASEEGVKSYMVWGMG

NFGAVSMNHIPLFSILCLIGIIASFLLVKPLNILLLGP

QYAESLGISTRQIRNILLVVVGLLTAITTAFCGPISFI

GLAIPHIARLLFRTENHQILLPGIVLSGAAIALLCNFI

CYLPGESGIIPLNAVTPLIGAPIIIYVIIQRR

46
Hexuronate_
O34456
MKKYYPWVLVALLWFVALLNYMDRQMLSTMQEAMKVDI

transporter

AELNHAEAFGALMAVFLWIYGIVSPFAGIIADRVNRKW

LVVGSIFVWSAVTYLMGYAESFDQLYWLRAFMGISEAL

YIPAALSLIADWHEGKSRSLAIGIHMTGLYVGQAVGGF

GATLAAMFSWHAAFHWFGIIGIVYSLVLLLFLKENPKH

GQKSVLQGETKPSKNPFRGLSIVFSTWAFWVILFYFAV

PSLPGWATKNWLPTLFANSLDIPMSSAGPMSTITIAVS

SFIGVIMGGVISDRWVQRNLRGRVYTSAIGLGLTVPAL

MLLGFGHSLVSVVGAGLCFGIGYGMFDANNMPILCQFI

SSKYRSTAYGIMNMTGVFAGAAVTQVLGKWTDGGNLGN

GFAILGGIVVLALVLQLSCLKPTTDNME

47
1,4-alpha-
P9WN45
MVTKKTTTKKAPVKKTSAKTTKVKEPSHIGLVKNDAYL

glucan_branching_

APYEDAIRGRHEHALWKMNQLTQNGKLTLSDFANGHNY

enzyme_GlgB

YGLHQTADGWVFREWAPNATEIYLVGDFNGWNEQEAYQ

CHRIEGTGNWELTLPHDAMQHGQYYKMRVHWEGGEGER

IPAWTQRVVQDEASKIFSAQVWAPAEPYVWEKKTFKPQ

TSPLLIYECHIGMAQDEEKVGTYNEFREKVLPRIIKDG

YNAIQIMAIQEHPYYGSFGYHVSSFFAASSRFGTPEEL

KALIDEAHKNGIAVIMDIVHSHAVKNEVEGLGNLAGDP

NQYFYPGERHEHPAWDSLCFDYGKDEVLHFLLSNCKYW

LEEYHFDGFRFDGVTSMLYYSHGLGEAFCNYADYFNGH

QDDNAICYLTLANCLIHEVNKNAVTIAEEVSGMPGLAA

KFKDGGYGFDYRMAMNIPDYWIKTIKELPDEAWKPSSI

FWEIKNRRSDEKTISYCESHDQALVGDKTIIFRLVDAD

MYWHFRKGDETEMTHRGIALHKMIRLATIAAINGGYLN

FMGNEFGHPEWIDFPREGNGWSHKYARRQWNLVDNEEL

CYHLLGDFDRKMLEVITSEKKFNETPIQEIWHNDGDQI

LAFSRGELVFVFNFSPSHSYSDYGFLVPEGSYNVVLNT

DAREFGGFGFADDTVEHFTNSDPLYEKDHKGWLKLYIP

ARSAVVLRKK

48
Cluster: YihY
D9RW24
MKIDIERIKYFLTVGMFMKTEHSSKRRNMLIRQFQKFY

family protein

LTVKFFFVRDHAASTAQLSFSTIMAIVPIASMIFAIAN

GFGFGQFLEKQFREMLSAQPEAATWLLKLTQSYLVHAK

TGLFIGIGLMIMLYSVFSLIRTVETTFDNIWQVKDSRP

ISRIVIDYTALMFLVPISIIILSGLSIYFYSFVENLNG

LRFLGTIASFSLRYLVPWAILTLMFIVLYVFMPNAKVK

ITKTVAPAMIASIAMLCLQAVYIHGQIFLTSYNAIYGS

FAALPLFMLWILASWYICLFCAELCYFNQNLEYYECLI

DTEDICHNDLLILCATVLSHICQRFANDQKPQTALQIK

TETHIPIRVMTDILYRLKEVNLISENFSPTSDEVTYTP

THDTNNITVGEMIARLESTPASDFALLGFSPKKAWNHD

IYDRVGSIREIYLNELKSINIKELISYSEN

49
Capsule_
P19579
MMKRPSIARWKVIICLLTPILLSFSGIGDNDIDKKKST

biosynthesis_

SKEVDDTLRIVITGDLLLDRGVRQKIDMAGVDALFSPT

protein_CapA

IDSLFHSSNYVIANLECPVTKIRERVFKRFIFRGEPEW

LPTLRRHGITHLNLANNHSIDQGRNGLLDTQEQIKKAG

MIPIGAGKNMEEAAEPVLISTSPRHVWVISSLRLPLEN

FLYLPQKPCVSQESIDSLIMRVKRLRATDKNCYILLIL

HWGWEHHFRATPQQREDAHKLIDAGADAIVGHHSHTLQ

TIETYRGKPIYYGIGNFIFDQRKPMNSRACLVELSITA

EKCKAKALPIEIKNCTPYLSK

50
Peptidoglycan_
B5ZA76
MILLSFDTEEFDVPREHGVDFSLEEGMKVSIEGTNRIL

deacetylase

DILKANNVCATFFCTGNFAELAPEVMERIKNEGHEVAC

HGVDHWQPKPEDVFRSKEIIERVTGVKVAGYRQPRMFP

VSDEDIEKAGYLYNSSLNPAFIPGRYMHLTTSRTWFMQ

GKVMQIPASVSPHLRIPLFWLSMHNFPEWFYLRLVRQV

LRHDGYFVTYFHPWEFYDLKSHPEFKMPFIIKNHSGHE

LEQRLDRFIKAMKADKQEFITYVDFVNRQKK

51
Fumarate_
P0AC47
MAKNISFTIKYWKQNGPQDQGHFDTHEMKNIPDDTSFL

reductase_iron-

EMLDILNEELIAAGDEPFVFDHDCREGICGMCSLYING

sulfur_subunit

TPHGKTERGATTCQLYMRRFNDGDVITVEPWRSAGFPV

IKDCMVDRTAFDKIIQAGGYTTIRTGQAQDANAILISK

DNADEAMDCATCIGCGACVAACKNGSAMLFVSSKVSQL

ALLPQGKPEAAKRAKAMVAKMDEVGFGNCTNTRACEAV

CPKNEKIANIARLNREFIKAKFAD

52
Serine/threonine-
P9WI71
MSENKLSTNEQAQTADAPVKASYTEYKVIPSQGYCMIV

protein_kinase_

KCRKGDQTVVLKTLKEEYRERVLLRNALKREFKQCQRL

PknH

NHSGIVRYQGLVEVDGYGLCIEEEYVEGRTLQAYLKEN

HTDDEKIAIINQIADALRYAHQQGVIHRNLKPSNVLVT

TQGDYVKLIDFSVLSPEDVKPTAETTRFMAPEMKDETL

TADATADIYSLGTIMKVMGLTLAYSEVIKRCCAFKRSD

RYSNVDELLADLNNEGSSFSMPKIGKGTVVLGLIIAVV

IGIGALLYNYGGALIDQVGKIDVSSVFSSDAETAPEDT

VKVNTAEQSDSLSTEAEAPAIGKLAFMNRMKPALYKDL

DNIFEKNSADKAKLTKAIKTYYRGLIQANDTLDNEQRA

EVDRVFGDYVKQKKAALN

53
Carboxy-
O34666
MRKYICLLLFYLFTFLPLSAQQGNDSPLRKLQLAEMAI

terminal_

KNFYVDSVNEQKLVEDGIRGMLEKLDPHSTYTDAKETK

processing_

AMNEPLQGDFEGIGVQFNMIEDTLVVIQPVVNGPSQKV

protease_CtpA

GILAGDRIVSVNDSTIAGVKMARIDIMKMLRGKKGTKV

KLGVVRRGVKGVLTFVVTRAKIPVHTINASYMIRPNVG

YIRIESFGMKTHDEFMSAVDSLKKKGMKTLLLDLQDNG

GGYLQSAVQISNEFLKNNDMIVYTEGRRARRQNFKAIG

NGRLQDVKVYVLVNELSASAAEIVTGAIQDNDRGTVVG

RRTFGKGLVQRPFDLPDGSMIRLTIAHYYTPSGRCIQK

PYTKGDLKDYEMDIEKRFKHGELTNPDSIQFSDSLKYY

TIRKHRVVYGGGGIMPDNFVPLDTTKFTRYHRMLAAKS

IIINAYLKYADANRQALKAQYSSFDAFNKGYVVPQSLL

DEIVAEGKKEKIEPKDAAELKATLPNIALQIKALTARD

IWDMNEYFRVWNTQSDIVNKAVALATGK

54
Cluster:
D9RRG3
MKLTEQRSSMLHGVLLITLFACAAFYIGDMGWVKALSL

Uncharacterized

SPMVVGIILGMLYANSLRNNLPDTWVPGIAFCGKRVLR

protein

FGIILYGFRLTFQDVVAVGFPAIIVDAIIVSGTILLGV

LVGRLLKMDRSIALLTACGSGICGAAAVLGVDGAIRPK

PYKTAVAVATVVIFGTLSMFLYPILYRAGIFDLSPDAM

GIFAGSTIHEVAHVVGAGNAMGAAVSNSAIIVKMIRVM

MLVPVLLVIAFFVAKNVAERDDEAGGSRKINIPWFAIL

FLVVIGFNSLNLLPKELVDFINTLDTFLLTMAMSALGA

ETSIDKFKKAGFKPFLLAAILWCWLIGGGYCLAKYLVP

VLGVAC

55
Cluster: Cna
X6Q2J4
MNKQFLLAALWLSPLGLYAHKANGIGAVTWKNEAPKER

protein B-type

MIRGIDEDKTHQRFTLSGYVKDRNGEPLINATIYDLTT

domain protein

RQGTMTNAYGHFSLTLGEGQHEIRCSYVGYKTLIETID

LSANQNHDIILQNEAQLDEVVVTTDLNSPLLKTQTGKL

SLSQKDIKTEYALLSSPDVIKTLQRTSGVADGMELASG

LYVHGGNGDENLFLLDGTPLYHTNHSLGLFSSFNADVV

KNVDFYKSGFPARYGGRLSSVIDVRTADGDLYKTHGSY

RIGLLDGAFHIGGPIRKGKTSYNFGLRRSWMDLLTRPA

FAIMNHKSDNEDKLSMSYFFHDLNFKLTNIFNERSRMS

LSVYSGEDRLDAKDEWHSNNSSGYNDVDIYVNRFHWGN

FNAALDWNYQFSPKLFANFTAVYTHNRSTVSSSDEWRF

TRPGEKEQLTLTSHGYRSSIDDIGYRAAFDFRPSPRHH

IRFGQDYTYHRFQPQTYNRFDNYQTNSEAKADTIATHS

YNKNVAHQLTFYAEDEMTLNEKWSLNGGVNADVFHISG

KTFATLSPRLSMKFQPTERLSLKASYTLMSQFVHKIAN

SFLDLPTDYWVPTTARLHPMRSWQVAAGAYMKPNKHWL

LSLEAYYKRSSHILQYSSWAGLEPPAANWDYMVMEGDG

RSYGVELDADYNVSNLTLHGSYTLSWTQKKFDDFYDGW

YYDKFDNRHKLTLTGRWNITKKIAAFAAWTFRTGNRMT

IPTQYIGLPDVPAQEQGGLTFNSSDDNTLNFAYEKPNN

VILPAYHRLDIGFDFHHTTKKGHERIWNLSFYNAYCHL

NSLWVRVKIDSNNQMKIRNIAFIPVIPSFSYTFKF

56
Poly-beta-1,6-N-
P75905
MSKQVFQTDSRQRWSYFKWTLRVILTILSLLGIVFLAM

acetyl-D-

FALEGSPQMPFRHDYRNAVTAASPYTKDNKTAKLYKSF

glucosamine_

RDFFKEKKMHNNYAKATIKKQRFIGKADSVTQKYFREW

synthase

DDPRIGVRSAWYVNWDKHAYISLKNNIKHLNMVLPEWF

FINPKTDKVEYRIDKQALRLMRRTGIPVLPMLTNNYNS

DFHPEAIGRIMRDEKKRMALINEMVRTCRHYGFAGINL

DLEELNIQDNDLLVELLKDFSRVFHANGLYVTQAVAPF

NEDYNMQELAKYNDYLFLMAYDEHNIESQPGAVSSQRW

VEKATDWAAKNVPNDKIVLGMATYGYDWANGEGGTTVS

FDQTMAIAQDADAKVKFDDDTYNVNFSYQNTDDGKIHH

VFFTDAATTFNIMRFGAEYHLAGYGLWRLGTEDKRIWR

FYGKDMSWENVARMSVAKLMQLNGTDDVNFVGSGEVLE

VTTEPHPGDISIRIDKDNRLISEEYYRALPSTYTIQRL

GKCKDKQLVITFDDGPDSRWTPTVLSTLKKYNVPAAFF

MVGLQMEKNLPLVKQVYEDGHTIGNHTFTHHNMIENSD

RRSYAELKLTRMLIESVTGHSTILFRAPYNADADPTEH

EEIWPMIVASRRNYLFVGESIDPNDWEPNVTSDQIYQR

VIDGVHHEDGHIILLHDAGGSSRKPTLDALPRIIETLQ

HEGYQFISLEQYLGMGKQTLMPEINKGKAYYAMQTNLW

LAEMIYHVSDFLTALFLVFLALGMMRLIFMYVLMIREK

RAENRRNYAPIDAATAPAVSIIVPGYNEEVNIVRTITT

LKQQDYPNLHIYFVDDGSKDHTLERVHEAFDNDDTVTI

LAKKNGGKASALNYGIAACRSEYVVCIDADTQLKNDAV

SRLMKHFIADTEKRVGAVAGNVKVGNQRNMLTYWQAIE

YTSSQNFDRMAYSNINAITVVPGAIGAFRKEVIEAVGG

FTTDTLAEDCDLTMSINEHGYIIENENYAVALTEAPET

LRQFVKQRIRWCFGVMQAFWKHRSSLFAPSKKGFGLWA

MPNMLIFQYIIPTFSPLADVLMLIGLFTGNALQIFFYY

LIFLVIDASVSIMAYIFEGERLWVLLWVIPQRFFYRWI

MYYVLFKSYLKAIKGELQTWGVLKRTGHVKG

57
Cell_division_
O34876
MAKKRNKARSRHSLQVVTLCISTAMVLMLIGIVVLTGF

protein_FtsX

TSRNLSSYVKENLTITMILQPDMNTEESAALCERIRTL

HYINSLNFISKEQALKDGTKELGANPAEFAGENPFTGE

IEVQLKANYANNDSIRNIVQQLRTYRGVSDITYPQSLV

ESVNQTLGKISLVLLVIAVLLTIISFSLINNTIRLSIY

AHRFSIHTMKLVGGSWSFIRAPFLRRAVLEGLVSALLA

IAVLGIGICLLYEKEPEITKLLSWDALIITAIVMLAFG

VIIATFCAWLSVNKFLRMKAGDLYKI

58
UDP-2,3-
P44046
MKNIYFLSDAHLGSLAIDHRRTHERRLVRFLDSIKHKA

diacylglucosamine_

AAVYLLGDMFDFWNEYKYVVPKGFTRFLGKISELTDMG

hydrolase

VEVHFFTGNHDLWTYGYLEKECGVILHRKPITTEIYDK

VFYLAHGDGLGDPDPMFRFLRKVFHNRFCQRLLNFFHP

WWGMQLGLNWAKRSRLKRKDGKEVPYLGEDKEYLVQYT

KEYMSTHKDIDYYIYGHRHIELDLTLSRKARLLILGDW

IWQFTYAVFDGEHMFLEEYVEGESKP

59
Poly-beta-1,6-N-
P75905
MVGLDVLCYFIHAKGREKECYFERIIYQITCHSRTKCY

acetyl-D-

LCNIMKYSIIVPVFNRPDEVEELLESLLSQEEKDFEVV

glucosamine_

IVEDGSQIPCKEVCDKYADKLDLHYYSKENSGPGQSRN

synthase

YGAERAKGEYLLILDSDVVLPKGYICAVSEELKREPAD

AFGGPDCAHESFTDTQKAISYSMTSFFTTGGIRGGKKK

LDKFYPRSFNMGIRRDVYQELGGFSKMRFGEDIDFSIR

IFKAGKRCRLFPEAWVWHKRRTDFRKFWKQVYNSGIAR

INLYKKYPESLKLVHLLPMVFTVGTALLVLMILFGLFL

QLFPIINVFGSVFIMMGLMPLVLYSVIICVDSTMQNNS

LNIGLLSIEAAFIQLTGYGCGFISAWWKRCVCGMDEFA

AYEKNFYK

60
Enolase
Q8DTS9
MKIEKVHAREIMDSRGNPTVEVEVTLENGVMGRASVPS

GASTGENEALELRDGDKNRFLGKGVLKAVENVNNLIAP

ALKGDCVLNQRAIDYKMLELDGTPTKSKLGANAILGVS

LAVAQAAAKALNIPLYRYIGGANTYVLPVPMMNIINGG

AHSDAPIAFQEFMIRPVGAPSEKEGIRMGAEVFHALAK

LLKKRGLSTAVGDEGGFAPKFDGIEDALDSIIQAIKDA

GYEPGKDVKIAMDCAASEFAVCEDGKWFYDYRQLKNGM

PKDPNGKKLSADEQIAYLEHLITKYPIDSIEDGLDEND

WENWVKLTSAIGDRCQLVGDDLFVTNVKFLEKGIKMGA

ANSILIKVNQIGSLTETLEAIEMAHRHGYTTVTSHRSG

ETEDTTIADIAVATNSGQIKTGSMSRTDRMAKYNQLIR

IEEELGACAKYGYAKLK

61
Outer_membrane_
Q8G0Y6
MKKLFTIAMLLGVTLGIHAQEVYSLQKCRELALQNNRQ

efflux_protein_

LKVSRMTVDVAENTRKAAKTKYLPRVDALAGYQHFSRE

Bepc

ISLLSDDQKNAFSNLGTNTFGQLGGQIGQNLTSLAQQG

ILSPQMAQQLGQLFSNVATPLTQVGNNIGQSINDAFRS

NTKNVYAGGIVVNQPIYMGGAIKAANDMAAIGEQVAQN

NISLKRQLVLYGVDNAYWLAISLKKKEALAIRYRDLAQ

KLNEDVKKMIREGVATRADGLKVEVAVNTADMQIARIQ

SGVSLAKMALCELCGLELNGDIPLSDEGDADLPPTPST

QFDNYTVSSSDTTGLNEARPELRLLQNAVDLSIQNTKL

IRSLYMPHVLLTAGYSVSNPNLFNGFQKRFTDLWNIGI

TVQVPVWNWGENKYKVRASKTATTIAQLEMDDVRKKID

LEIEQNRLRLKDANKQLATSQKNMAAAEENLRCANVGF

KEGVMTVTEVMAAQTAWQTSRMAIIDAEISVKLAQTGL

QKALGGL

62
Phosphoethanol-
Q7CPC0
MKRTFVTKMVKPIEENSLFFMFMLLVGAFTNVSHRNVF

amine_transferase_

GYIELIADVYIICFLLSLCQRTIRQGLVIMLSSVIYVV

CptA

AIIDTCCKTLFDTPITPTMLLLAQETTGREATEFFLQY

LNLKLFFSAADIILFLAFCHIVMAVKKMKFSTSYLKQP

FVAFVLMFTIFVGMALSIYDKVQLYTVKNLSGLEVAVT

NGFAHLYHPVERIVYGLYSNHLIAKQVDGVIMANQQIK

VDSCSFTSPTIVLVIGESANRHHSQLYGYPLPTTPYQL

AMKNGKDSLAVFTNVVSPWNLTSKVFKQIFSLQSVDEK

GDWSKYVLFPAVFKKAGYHVSFLSNQFPYGINYTPDWT

NNLVGGFFLNHPQLNKQMFDYRNVTIHNYDEDLLNDYK

EIISYKKPQLIIFHLLGQHFQYSLRCKSNMKKFGIKDY

KRMDLTDKEKQTIADYDNATLYNDFVLNKIVEQFRNKD

AIIVYLSDHGEDCYGKDVNMAGRLTEVEQINLKKYHEE

FEIPFWIWCSPIYKQRHRKIFTETLMARNNKFMTDDLP

HLLLYLAGIKTKDYCEERNVISPSFNNNRRRLVLKTID

YDKALYQ

63
Dipeptide_and_
P36837
MFKNHPKGLLQAAFSNMGERFGYYIMNAVLALFLCSKF

tripeptide_

GLSDETSGLIASLFLAAIYVMSLVGGVIADRTQNYQRT

permease_B

IESGLVVMALGYVALSIPVLATPENNSYLLAFTIFALV

LIAVGNGLFKGNLQAIVGQMYDDFETEAAKVSPERLKW

AQGQRDAGFQIFYVFINLGALAAPFIAPVLRSWWLGRN

GLTYDAALPQLCHKYINGTIGDNLGNLQELATKVGGNS

ADLASFCPHYLDVFNTGVHYSFIASVVTMLISLIIFMS

SKKLFPMPGKKEQIVNVEYTDEEKASMAKEIKQRMYAL

FAVLGISVFFWFSFHQNGQSLSFFARDFVNTDSVAPEI

WQAVNPFFVISLTPLIMWVFAYFTKKGKPISTPRKIAY

GMGIAGFAYLFLMGFSLVHNYPSAEQFTSLEPAVRATM

KAGPMILILTYFFLTVAELFISPLGLSFVSKVAPKNLQ

GLCQGLWLGATAVGNGFLWIGPLMYNKWSIWTCWLVFA

IVCFISMVVMFGMVKWLERVTKS

64
C4-
Q9I4F5
MQKKIKIGLLPRVIIAILLGLFLGYYLPDPAVRVFLTF

dicarboxylate_

NSIFSQFLGFMIPLIIIGLVTPAIAGIGKGAGKLLLAT

transport_

VAIAYVDTIVAGGLSYGTGTWLFPSMIASTGGAIPHID

protein_2

KATELTPYFTINIPAMVDVMSSLVFSFIAGLGIAYGGL

RTMENLFNEFKTVIEKVIEKAIIPLLPLYIFGVFLSMT

HNGQARQVLLVFSQIIIVILVLHVLILIYEFCIAGAIV

KHNPFRLLWNMLPAYLTALGTSSSAATIPVTLKQTVKN

GVSEEVAGFVVPLCATIHLSGSAMKITACALTICMLTD

LPHDPGLFIYFILMLAIIMVAAPGVPGGAIMAALAPLS

SILGFNEEAQALMIALYIAMDSFGTACNVTGDGAIALA

VNKFFGKKKETSILS

65
Inner_membrane_
P76090
MISVYSIKPQFQRVLTPILELLHRAKVTANQITLWACV

protein_YnbA

LSLVIGILFWFAGDVGTWLYLCLPVGLLIRMALNALDG

MMARRYNQITRKGELLNEVGDVVSDTIIYFPLLKYHPE

SLYFIVAFIALSIINEYAGVMGKVLSAERRYDGPMGKS

DRAFVLGLYGVVCLFGINLSGYSVYIFGVIDLLLVLST

WIRIKKTLKVTRNSQTPE

66
2′,3′-cyclic-
P08331
MKLSTILLSIMLGLSSSTMAQQKDVTIKLIETTDVHGS

nucleotide

FFPYDFITRKPKSGSMARVYTLVEELRKKDGKDNVYLL

DNGDILQGQPISYYYNYVAPEKTNIAASVLNYMGYDVA

TVGNHDIETGHKVYDKWFKELKFPILGANIIDTKTNKP

YILPYYTIKKKNGIKVCVIGMLTPAIPNWLKESIWSGL

RFEEMVSCAKRTMAEVKTQEKPDVIVGLFHSGWDGGIK

TPEYDEDASKKVAKEVPGFDIVFFGHDHTPHSSIEKNI

VGKDVICLDPANNAQRVAIATLTLRPKTVKGKRQYTVT

KATGELVDVKELKADDAFIQHFQPEIDAVKAWSDQVIG

RFENTIYSKDSYFGNSAFNDLILNLELEITKADIAFNA

PLLFNASIKAGPITVADMFNLYKYENNLCTMRLTGKEI

RKHLEMSYDLWCNTMKSPEDHLLLLSSTQNDAQRLGFK

NFSFNFDSAAGIDYEVDVTKPDGQKVRILRMSNGEPFD

ENKWYTVAVNSYRANGGGELLTKGAGIPRDSLKSRIIW

ESPKDQRHYLMEEIKKAGVMNPQPNHNWKFIPETWTVP

AAARDRKLLFGE

67
Fe(2+)_
P33650
MKLSELKTGETGVIVKVSGHGGFRKRIIEMGFIKGKTV

transporter_FeoB

EVLLNAPLQDPVKYKIMGYEVSLRHSEADQIEVLSDVK

THSVGNEEEQEDNQLEMDSTTYDSTDKELTPEKQSDAV

RRKNHTINVALVGNPNCGKTSLFNFASGAHERVGNYSG

VTVDAKVGRAEFDGYVFNLVDLPGTYSLSAYSPEELYV

RKQLVDKTPDVVINVIDSSNLERNLYLTTQLIDMHIRM

VCALNMFDETEQRGDHIDAQKLSELFGVPMIPTVFTNG

RGVKELFRQIIAVYEGKEDESLQFRHIHINHGHEIENG

IKEMQEHLKKYPELCHRYSTRYLAIKLLEHDKDVEQLV

SPLGDSIEIFNHRDTAAARVKEETGNDSETAIMDAKYG

FINGALKEANFSTGDKKDTYQTTHVIDHVLTNKYFGFP

IFFLVLLVMFTATFVIGQYPMDWIEAGVGWLGEFISKN

MPAGPVKDMIVDGIIGGVGAVIVFLPQILILYFFISYM

EDCGYMSRAAFIMDRLMHKMGLHGKSFIPLIMGFGCNV

PAVMATRTIESRRSRLITMLILPLMSCSARLPIYVMIT

GSFFALKYRSLAMLSLYIIGVLMAVAMSRLFSAFVVKG

EDTPFVMELPPYRFPTWKAIGRHTWEKGKQYLKKMGGI

ILVASIIVWALGYFPLPDDPNMDNQARQEQSYIGRIGK

AVEPVFRPQGFNWKLDVGLLSGMGAKEIVASTMGVLYS

NDGSFSDDNGYSSETGKYSKLHNLITKDVATMHHISYE

EAEPIATLTAFSFLLFVLLYFPCVATIAAIKGETGSWG

WALFAAGYTTALAWIVSAVVFQVGMLFM

68
UDP-N-
P9WJM1
MESFIIEGGHQLSGTIAPQGAKNEALEVICATLLTSEE

acetylglucosamine

VIIRNVPDILDVNNLIKLLQDIGVKVKKLAPNEFSFQA

DEVNLDYLESSDFVKKCSSLRGSVLMIGPLLGRFGKAT

IAKPGGDKIGRRRLDTHFLGFKNLGAHFGRVEDRDVYE

IQADKLVGTYMLLDEASITGTANIIMAAVLAEGTTTIY

NAACEPYIQQLCKMLNAMGAKISGIASNLITIEGVKEL

HSADHRILPDMIEVGSFIGIAAMIGDGVRIKDVSVPNL

GLILDTFHRLGVQIIVDNDDLIIPRQDHYVIDSFIDGT

IMTISDAPWPGLTPDLISVLLVVATQAQGSVLFHQKMF

ESRLFFVDKLIDMGAQIILCDPHRAVVVGHDNAKKLRA

GRMSSPDIRAGIALLIAALTAQGTSRIDNIVQIDRGYE

NIEGRLNALGAKIQRAEVC

69
Ribitol-5-
Q8RKI9
MNIAVIFAGGSGLRMHTKSRPKQFLDLNGKPIIIYTLE

phosphate_cytidyl-

LFDNHPNIDAIVVACIESWIPFLEKQLRKFEINKVVKI

yltransferase

IPGGKSGQESIYKGLCAAEEYAQSKGVSNEETTVLIHD

GVRPLITEETITDNIKKVEEVGSCITCIPATETLIVKQ

ADDALEIPSRADSFIARAPQSFRLIDIITAHRRSLAEG

KADFIDSCTMMSHYGYKLGTIIGPMENIKITTPTDFFV

LRAMVKVHEDQQIFGL

In some embodiments, the Prevotella bacteria are from a strain of Prevotella bacteria comprising one or more of the proteins listed in Table 1 and that is free or substantially free of one or more proteins listed in Table 2. In some embodiments, the Prevotella bacteria are from a strain of Prevotella bacteria that comprises all of the proteins listed in Table 1 and/or all of the genes encoding the proteins listed in Table 1 and that is free of all of the proteins listed in Table 2 and/or all of the genes encoding the proteins listed in Table 2.

Stabilizer and Bacterial Compositions

In some aspects, provided herein is a stabilizer that stabilizes bacterial compositions comprises sucrose. In some embodiments, the stabilizer comprises about 100 g/kg, about 110 g/kg, about 120 g/kg, about 130 g/kg, about 140 g/kg, about 150 g/kg, about 160 g/kg, about 170 g/kg, about 180 g/kg, about 190 g/kg, about 200 g/kg, about 210 g/kg, about 220 g/kg, about 230 g/kg, about 240 g/kg, about 250 g/kg, about 260 g/kg, about 270 g/kg, about 280 g/kg, about 290 g/kg, or about 300 g/kg sucrose. In some embodiments, the stabilizer comprises at least 100 g/kg, at least 110 g/kg, at least 120 g/kg, at least 130 g/kg, at least 140 g/kg, at least 150 g/kg, at least 160 g/kg, at least 170 g/kg, at least 180 g/kg, at least 190 g/kg, at least 200 g/kg, at least 210 g/kg, at least 220 g/kg, at least 230 g/kg, at least 240 g/kg, at least 250 g/kg, at least 260 g/kg, at least 270 g/kg, at least 280 g/kg, at least 290 g/kg, or at least 300 g/kg sucrose.

In some embodiments, the stabilizer comprises dextran 40k. In some embodiments, the stabilizer comprises about 100 g/kg, about 110 g/kg, about 120 g/kg, about 130 g/kg, about 140 g/kg, about 150 g/kg, about 160 g/kg, about 170 g/kg, about 180 g/kg, about 190 g/kg, about 200 g/kg, about 210 g/kg, about 220 g/kg, about 230 g/kg, about 240 g/kg, about 250 g/kg, about 260 g/kg, about 270 g/kg, about 280 g/kg, about 290 g/kg, or about 300 g/kg dextran 40k. In some embodiments, the stabilizer comprises at least 100 g/kg, at least 110 g/kg, at least 120 g/kg, at least 130 g/kg, at least 140 g/kg, at least 150 g/kg, at least 160 g/kg, at least 170 g/kg, at least 180 g/kg, at least 190 g/kg, at least 200 g/kg, at least 210 g/kg, at least 220 g/kg, at least 230 g/kg, at least 240 g/kg, at least 250 g/kg, at least 260 g/kg, at least 270 g/kg, at least 280 g/kg, at least 290 g/kg, or at least 300 g/kg dextran 40k.

In some embodiments, the stabilizer comprises cysteine HCl. In some embodiments, the stabilizer comprises about 1.0 g/kg, about 1.1 g/kg, about 1.2 g/kg, about 1.3 g/kg, about 1.4 g/kg, about 1.5 g/kg, about 1.6 g/kg, about 1.7 g/kg, about 1.8 g/kg, about 1.9 g/kg, about 2.0 g/kg, about 2.1 g/kg, about 2.2 g/kg, about 2.3 g/kg, about 2.4 g/kg, about 2.5 g/kg, about 2.6 g/kg, about 2.7 g/kg, about 2.8 g/kg, about 2.9 g/kg, about 3.0 g/kg, about 3.1 g/kg, about 3.2 g/kg, about 3.3 g/kg, about 3.4 g/kg, about 3.5 g/kg, about 3.6 g/kg, about 3.7 g/kg, about 3.8 g/kg, about 3.9 g/kg, about 4.0 g/kg, about 4.1 g/kg, about 4.2 g/kg, about 4.3 g/kg, about 4.4 g/kg, about 4.5 g/kg, about 4.6 g/kg, about 4.7 g/kg, about 4.8 g/kg, about 4.9 g/kg, or about 5.0 g/kg cysteine HCl. In some embodiments, the stabilizer comprises at least 1.0 g/kg, at least 1.1 g/kg, at least 1.2 g/kg, at least 1.3 g/kg, at least 1.4 g/kg, at least 1.5 g/kg, at least 1.6 g/kg, at least 1.7 g/kg, at least 1.8 g/kg, at least 1.9 g/kg, at least 2.0 g/kg, at least 2.1 g/kg, at least 2.2 g/kg, at least 2.3 g/kg, at least 2.4 g/kg, at least 2.5 g/kg, at least 2.6 g/kg, at least 2.7 g/kg, at least 2.8 g/kg, at least 2.9 g/kg, at least 3.0 g/kg, at least 3.1 g/kg, at least 3.2 g/kg, at least 3.3 g/kg, at least 3.4 g/kg, at least 3.5 g/kg, at least 3.6 g/kg, at least 3.7 g/kg, at least 3.8 g/kg, at least 3.9 g/kg, at least 4.0 g/kg, at least 4.1 g/kg, at least 4.2 g/kg, at least 4.3 g/kg, at least 4.4 g/kg, at least 4.5 g/kg, at least 4.6 g/kg, at least 4.7 g/kg, at least 4.8 g/kg, at least 4.9 g/kg, or at least 5.0 g/kg cysteine HCl.

In certain embodiments, the stabilizer is in liquid suspension. In some embodiments, the components of the stabilizer are dissolved in water to prepare the liquid suspension. In some such embodiments, the stabilizer comprises about 500 g/kg, about 510 g/kg, about 520 g/kg, about 530 g/kg, about 540 g/kg, about 550 g/kg, about 560 g/kg, about 570 g/kg, about 580 g/kg, about 590 g/kg, about 600 g/kg, about 610 g/kg, about 620 g/kg, about 630 g/kg, about 640 g/kg, about 650 g/kg, about 660 g/kg, about 670 g/kg, about 680 g/kg, about 690 g/kg, or about 700 g/kg water. In some such embodiments, the stabilizer comprises at least 500 g/kg, at least 510 g/kg, at least 520 g/kg, at least 530 g/kg, at least 540 g/kg, at least 550 g/kg, at least 560 g/kg, at least 570 g/kg, at least 580 g/kg, at least 590 g/kg, at least 600 g/kg, at least 610 g/kg, at least 620 g/kg, at least 630 g/kg, at least 640 g/kg, at least 650 g/kg, at least 660 g/kg, at least 670 g/kg, at least 680 g/kg, at least 690 g/kg, or at least 700 g/kg water.

In some embodiments, the stabilizer comprises sucrose, dextran 40k, cysteine HCl, and water. In some such embodiments, the stabilizer comprises 150 g/kg to 250 g/kg sucrose. In some embodiments, the stabilizer comprises 200 g/kg sucrose. In some embodiments, the stabilizer comprises 150 g/kg to 250 g/kg dextran 40k. In some embodiments, the stabilizer comprises 200 g/kg dextran 40 k. In some embodiments, the stabilizer comprises 2 g/kg to 6 g/kg cysteine HCl. In some embodiments, the stabilizer comprises 4 g/kg cysteine HCl. In some embodiments, the stabilizer comprises the stabilizer comprises 500 g/kg to 700 g/kg water. In some embodiments, the stabilizer comprises 596 g/kg water. In some embodiments, the stabilizer comprises 200 g/kg sucrose, 200 g/kg dextran 40k, 4 g/kg cysteine HCl, and 596 g/kg water.

In some aspects, provided herein are bacterial compositions comprising a stabilizer and bacteria, and methods of preparing same. In certain embodiments, the bacterial composition is prepared by combining bacteria with a certain percentage of the stabilizer in liquid suspension. In some embodiments, the percentage of the stabilizer solution combined with bacteria is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%. In some embodiments, the percentage of the stabilizer solution combined with bacteria is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50%.

In certain aspects, the bacterial compositions provided herein comprise a stabilizer. In some embodiments, the bacterial composition comprises sucrose. In some embodiments, the concentration of sucrose in the bacterial composition is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0%. In some embodiments, the concentration of sucrose in the bacterial composition is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2.0%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, or at least 3.0%.

In some embodiments, the bacterial composition comprises dextran 40k. In some embodiments, the concentration of dextran 40k in the bacterial composition is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0%. In some embodiments, the concentration of dextran 40k in the bacterial composition is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, at least 1.9%, at least 2.0%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, or at least 3.0%.

In some embodiments, the bacterial composition comprises cysteine HCl. In some embodiments, the concentration of cysteine HCl in the bacterial composition is about 0.001%, about 0.005%, about 0.01%, about 0.011%, about 0.012%, about 0.013%, about 0.014%, about 0.015%, about 0.016%, about 0.017%, about 0.018%, about 0.019%, about 0.02%, about 0.021%, about 0.022%, about 0.023%, about 0.024%, about 0.025%, about 0.026%, about 0.027%, about 0.028%, about 0.029%, about 0.03%, about 0.031%, about 0.032%, about 0.033%, about 0.034%, about 0.035%, about 0.036%, about 0.037%, about 0.038%, about 0.039%, about 0.04%, about 0.041%, about 0.042%, about 0.042%, about 0.043%, about 0.044%, about 0.045%, about 0.046%, about 0.047%, about 0.048%, about 0.049%, or about 0.05%. In some embodiments, the concentration of cysteine HCl in the bacterial composition is at least 0.001%, at least 0.005%, at least 0.01%, at least 0.011%, at least 0.012%, at least 0.013%, at least 0.014%, at least 0.015%, at least 0.016%, at least 0.017%, at least 0.018%, at least 0.019%, at least 0.02%, at least 0.021%, at least 0.022%, at least 0.023%, at least 0.024%, at least 0.025%, at least 0.026%, at least 0.027%, at least 0.028%, at least 0.029%, at least 0.03%, at least 0.031%, at least 0.032%, at least 0.033%, at least 0.034%, at least 0.035%, at least 0.036%, at least 0.037%, at least 0.038%, at least 0.039%, at least 0.04%, at least 0.041%, at least 0.042%, at least 0.042%, at least 0.043%, at least 0.044%, at least 0.045%, at least 0.046%, at least 0.047%, at least 0.048%, at least 0.049%, or at least 0.05%.

In some embodiments, the bacterial composition comprises sucrose, dextran 40k, and cysteine HCl. In some such embodiments, the bacterial composition comprises 1% to 2% sucrose. In some embodiments, the bacterial composition comprises 1.5% sucrose. In some embodiments, the bacterial composition comprises 1% to 2% dextran 40k. In some embodiments, the bacterial composition comprises 1.5% dextran 40k. In some embodiments, the bacterial composition comprises 0.01% to 0.05% cysteine HCl. In some embodiments, the bacterial composition comprises 0.03% cysteine HCl. In some embodiments, the bacterial composition comprises 1.5% sucrose, 1.5% dextran 40k, and 0.03% cysteine HCl.

In certain aspects, the bacterial composition comprises bacteria. In some embodiments, the bacteria are anaerobic bacteria. In some embodiments, the anaerobic bacteria are Prevotella histicola. In some such embodiments, the anaerobic bacteria are Prevotella histocola Strain B 50329.

In some embodiments, the bacterial composition is lyophilized to form a powder.

EXAMPLES
Example 1: Exemplary Manufacturing Process of Prevotella histicola and Lyophilized Powder of Prevotella histicola and Stabilizer

Exemplary manufacturing processes of Prevotella histicola are shown in FIG. 1 and FIG. 2. In this exemplary method, the anaerobic bacteria are grown in growth media comprising the components listed in Table 3. The media is filter sterilized prior to use.

TABLE 3

Growth Media

Component
g/L

Yeast Extract 19512
10

Soy Peptone A2SC 19649
12.5

Soy Peptone E110 19885
12.5

Dipotassium Phosphate K2HPO4
1.59

Monopotassium phosphate
0.91

L-Cysteine-HCl
0.5

Ammonium chloride
0.5

Glucidex 21 D (Maltodextrin)
25

Glucose
10

Hemoglobin
0.02

Briefly, a 1 L bottle is inoculated with a 1 mL of a cell bank sample that had been stored at −80° C. This inoculated culture is incubated in an anaerobic chamber at 37° C., pH=6.5 due to sensitivity of this strain to aerobic conditions. When the bottle reaches log growth phase (after approximately 14 to 16 hours of growth), the culture is used to inoculate a 20 L bioreactor at 5% v/v. During log growth phase (after approximately 10 to 12 hours of growth), the culture is used to inoculate a 3500 L bioreactor at 0.5% v/v.

Fermentation culture is continuously mixed with addition of a mixed gas at 0.02 VVM with a composition of 25% CO₂and 75% N₂. pH is maintained at 6.5 with ammonium hydroxide and temperature controlled at 37° C. Harvest time is based on when stationary phase is reached (after approximately 12 to 14 hours of growth).

Alternatively, the fermentation culture is continuously mixed with the addition of 100% CO₂gas at 0.002 VVM. pH is maintained at 6.5 with ammonium hydroxide and temperature controlled at 37° C. Harvest time is based on when stationary phase is reached (after approximately 12 to 14 hours of growth).

Once fermentation is complete, the culture is cooled to 10° C., centrifuged, and the resulting cell paste is collected.

A stabilizer is prepared by combining and mixing the components described in Table 4. In order to prepare a lyophilized powder of Prevotella histicola and the stabilizer, 10% stabilizer is added to the cell paste and mixed thoroughly (Stabilizer Concentration (in slurry): 1.5% Sucrose, 1.5% Dextran, 0.03% Cysteine). The cell slurry is lyophilized.

TABLE 4

Stabilizer Formulation

Component
g/kg

Sucrose
200

Dextran 40k
200

Cysteine HCl
4

Water
596

Example 2: Effect of CO₂Availability on Prevotella histicola Growth

The effect of CO₂availability on the growth of Prevotella histicola Strain B 50329 was tested. Prevotella histicola was cultured under anaerobic conditions with sparging of 95% N₂and 5% CO₂at a rate of either 0.1 volume of gas per volume of vessel per minute (vvm) or 0.02 vvm. As seen in FIG. 3, sparging an increased amount of the gas increased the growth potential of the Prevotella histicola.

The Prevotella histicola strain was then cultured with sparging of pure N₂(0% CO₂), 95% N₂and 5% CO₂, or 75% N₂and 25% CO₂at a rate of 0.02 vvm. As can be seen in FIG. 4, the presence of CO₂is necessary for initiation of Prevotella histicola growth. Sparging increasing concentrations of CO₂increased the growth potential of the Prevotella histicola. Sparging 100% CO₂at a lower rate (0.005 vvm) resulted in an intermediate growth potential for the Prevotella histicola.

At all scales, mass transfer of CO₂is important and determined by a variety of factors. Here we show the impact of scale, agitation, gas concentration, and gas flow rate (Table 5).

TABLE 5

Prevotella histicola growth under various conditions

CO₂
Gas
Final
Final

Scale
Agitation
Conc.
flow rate
OD
TCC

15L
100 RPM
5% CO₂
0.1vvm
14.22
1.1 × 10¹⁰

cells/mL

15L
100 RPM
5% CO₂
0.02vvm
3
NA

15L
100 RPM
25% CO₂
0.02vvm
11.7
7.16 × 10⁹

cells/mL

36L
50 RPM
25% CO₂
0.02vvm
10.99
8.47 × 10⁹

cells/mL

50L
100 RPM
25% CO₂
0.02vvm
30.1
3.77 × 10¹⁰

cells/mL

50L
60 RPM
75% CO₂
0.007vvm
32
NA

36L
70 RPM
25% CO₂
0.02vvm
29
2.33 × 10¹⁰

cells/mL

36L
70 RPM
25% CO₂
0.02vvm
34.4
2.82 × 10¹⁰

cells/mL

36L
70 RPM
25% CO₂
0.02vvm
33.8
2.47 × 10¹⁰

cells/mL

The Prevotella histicola was consuming CO₂during growth. As seen in FIG. 5, when CO₂was added to a freshly inoculated culture of Prevotella histicola, the CO₂concentration increased and the concentration approached equilibrium. As the Prevotella histicola culture grew, the increase of CO₂concentration slowed and then, as the culture entered logarithmic growth, the level of CO₂declined. When the culture stopped logarithmic growth, this decline stopped as mass transfer offset consumption. When the sparging of the CO₂was turned off, the concentration of CO₂in the culture began to immediately to rapidly decline, indicating that that Prevotella histicola consumed CO₂. If no consumption were to occur, such as in sterile media, little to no change of CO₂concentration would be observed in that time frame.

Example 3: Maltodextrin in Combination with Glucose can Support Growth of Prevotella histicola Strain B 50329 Better than Glucose Alone as Sugar Source

The results in FIG. 6 show that, at the same amount of total sugar, maltodextrin (25 g/L) in combination with glucose (10 g/L), compared to glucose (35 g/L) alone, led to increased process yield. Other than the sugars used, the culture conditions were identical. The result show that for equivalent masses of glucose and maltodextrin, Prevotella histicola Strain B 50329 grows better on maltodextrin plus glucose than on glucose alone. Because maltodextrin is just chains of glucose monomers, the results suggest that the cells may be benefiting in growth from some aspect of the chain structure.

INCORPORATION BY REFERENCE

All publications patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

	Number	Date	Country
	62952798	Dec 2019	US
	62850726	May 2019	US

METHODS AND COMPOSITIONS FOR ANAEROBIC BACTERIAL FERMENTATION

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