LIVE BIOTHERAPEUTICS FOR THE TREATMENT OF CARBOHYDRATE DISORDERS
20200261521 ยท 2020-08-20
Inventors
Cpc classification
A23V2002/00
HUMAN NECESSITIES
A23V2200/32
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A23L33/21
HUMAN NECESSITIES
A23V2200/32
HUMAN NECESSITIES
A23L33/30
HUMAN NECESSITIES
International classification
A23L33/00
HUMAN NECESSITIES
A23L33/21
HUMAN NECESSITIES
Abstract
The present invention provides method for treating a metabolic disorder, dietary intolerance, or malabsorption in a subject that causes an excess of a metabolite or a dietary substance, particularly a carbohydrate such as galactose or fructose, that the subject does not tolerate by administering an effective amount of an adaptively evolved yeast strain to the subject or to food that reduces the excess of the metabolite or dietary substance. Specifically, the invention is directed to methods for treating the metabolic disorder, such as galactosemia or fructosemia, in which an excess of the carbohydrate is present. The invention also provides isolated, adaptively evolved yeast strains that degrades a metabolite or dietary ingredient, such as galactose or fructose, and methods for preparing such isolated, adaptively evolved yeast strains.
Claims
1. A method for treating dietary intolerance, malabsorption or a metabolic disorder in a subject, comprising: administering an effective amount of an adaptively evolved microorganism to the subject or to food containing a metabolite or a dietary substance in an amount that the subject does not tolerate, wherein the effective amount of the adaptively evolved microorganism reduces the amount of the dietary substance.
2. The method of claim 1, wherein the metabolite or dietary substance is a carbohydrate.
3. The method of claim 2, wherein the carbohydrate is galactose or fructose.
4. The method of claim 1, wherein the adaptively evolved microorganism is a yeast.
5. The method of claim any of the previous claims, wherein the yeast is selected from the group consisting of a Saccharomyces sp., a Saccharomyces sp., a Kluyveromyces sp., a Pichia sp., and a Metschnikowia sp.
6. The method of claim 4, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Kluyveromyces marxianus, Pichia kudriavzevii, and Metschnikowia reukaufii.
7. The method of claim 6, wherein the carbohydrate is galactose and the isolated, adaptively evolved microorganism is Saccharomyces cerevisiae strain Y_C202_1, Saccharomyces cerevisiae strain Y_C201_1, or Kluyveromyces marxianus strain K_219, or the carbohydrate is fructose and the isolated adaptively evolved microorganism is Pichia kudriavzevii strain G1_1A, Saccharomyces cerevisiae strain G2_1A, Saccharomyces uvarum strain G3_1A, or Metschnikowia reukaufii strain G4_1A.
8. A composition, comprising an adaptively evolved microorganism strain that degrades a dietary substance or a metabolite, wherein the dietary substance is a carbohydrate.
9. The composition of claim 8, wherein the carbohydrate is galactose or fructose
10. The composition of claim 8, wherein the isolated, adaptively evolved microorganism is a yeast.
11. The composition of claim 8, wherein the isolated, adaptively evolved microorganism is selected from the group consisting of a Saccharomyces sp., a Saccharomyces sp., a Kluyveromyces sp., a Pichia sp., and a Metschnikowia sp.
12. The composition of claim 10, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Kluyveromyces marxianus, Pichia kudriavzevii, and Metschnikowia reukaufii.
13. The composition of claim 12, wherein the carbohydrate is galactose and the yeast is a strain selected from Saccharomyces cerevisiae strain Y_C202_1, Saccharomyces cerevisiae strain Y_C201_1, and Kluyveromyces marxianus strain K_219, or the carbohydrate is fructose and the yeast is a strain selected from Pichia kudriavzevii strain G1_1A, Saccharomyces cerevisiae strain G2_1A, Saccharomyces uvarum strain G3_1A, or Metschnikowia reukaufii strain G4_1A.
14. The composition of claim 8, wherein the microorganism is adaptively evolved by a method comprising the steps of: a) growing the microorganism under an adaptive condition for a plurality of microorganism cell generations, wherein the microorganism heritably adapts to the adaptive condition; and b) confirming heritable adaptation of the microorganism, thereby adaptively evolving the microorganism strain.
15. The composition of claim 14, wherein the adaptive condition is the presence of the dietary substance or the metabolite and the heritable adaptation is degradation of the dietary substance or the metabolite.
16. The composition of claim 15, wherein metabolite or dietary substance is a carbohydrate.
17. The composition of claim 16, wherein the carbohydrate is galactose and the adaptive condition comprises galactose or galactose plus glucose or fructose, or the carbohydrate is fructose and the adaptive condition comprises fructose.
18. A composition of claim 8, further comprising a pharmaceutically acceptable excipient, carrier enteric coating, food or a combination thereof.
19. A method for adaptively evolving a microorganism for treatment of a carbohydrate-related metabolic disorder or dietary intolerance, comprising the steps of: a) providing a microorganism; b) growing the microorganism under an adaptive condition comprises the presence of a carbohydrate, wherein the organism adapts to the adaptive condition; and c) confirming adaptation of the microorganism, thereby adaptively evolving the microorganism.
20. The method of claim 19, wherein the metabolic disorder is galactosemia, the carbohydrate is galactose and the adaptive condition is the presence of galactose or galactose plus glucose, or the dietary intolerance is fructose intolerance or the metabolic disorder is fructosemia, the carbohydrate is fructose and the adaptive condition is the presence of fructose.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0030]
[0031]
[0032] Serum samples were collected at 0-hour pre-administration and at 0.5, 1, 1.5, 2, 4, and 8-hour post administration (horizontal axis, in hours).
DETAILED DESCRIPTION
[0033] Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided below, definitions of common terms in molecular biology may also be found in Rieger et al. 1991 Glossary of genetics: classical and molecular, 5th Ed., Berlin: Springer-Verlag; and in Current Protocols in Molecular Biology, F. M. Ausubel et al. Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998 Supplement).
[0034] It is to be understood that as used in the specification and in the claims, a or an can mean one or more, depending upon the context in which it is used. Thus, for example, reference to a cell can mean that at least one cell can be utilized.
[0035] Also, for the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
[0036] Also, for the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Definitions
[0037] About as used herein means that a number referred to as about comprises the recited number plus or minus 1-10% of that recited number. For example, about 100 cells can mean 95-105 cells or as few as 99-101 cells depending on the situation. Whenever it appears herein, a numerical range such as 1 to 20 refers to each integer in the given range; e.g., 1 to 20 cells means 1 cell, 2 cells, 3 cells, etc., up to and including 20 cells. Where about modifies a range expressed in non-integers, it means the recited number plus or minus 1-10% to the same degree of significant figures expressed. For example, about 1.50 to 2.50 mM can mean as little as 1.35 M or as much as 2.75M or any amount in between in increments of 0.01. Where a range described herein includes decimal values, such as 1.2% to 10.5%, the range refers to each decimal value of the smallest increment indicated in the given range; e.g. 1.2% to 10.5% means that the percentage can be 1.2%, 1.3%, 1.4%, 1.5%, etc. up to and including 10.5%; while 1.20% to 10.50% means that the percentage can be 1.20%, 1.21%, 1.22%, 1.23%, etc. up to and including 10.50%.
[0038] As used herein, the term substantially refers to a great extent or degree. For example, substantially all typically refers to at least about 90%, frequently at least about 95%, often at least 99%, and more often at least about 99.9%.
[0039] Dietary intolerance and food intolerance are used interchangeably herein to refer to food sensitivity that occurs when a person has difficulty digesting a particular food and has an unpleasant physical reaction to them. It can lead to symptoms such as intestinal gas, bloating, spasm, cramping, abdominal pain, nausea, vomiting and diarrhea. Food intolerances involve the digestive system rather than the immune system, which is responsible for food allergy. Some food intolerance is caused by the lack or inadequacy of a particular digestive enzyme. Certain foods, particularly certain carbohydrates, can be frequent sources of food intolerance.
[0040] Malabsorption is a disorder that occurs when a person is unable to absorb nutrients from his or her diet, such as carbohydrates, fats, minerals, proteins, or vitamins, which can lead to nutrient deficiencies. Malabsorption may be global, with impaired absorption of almost all nutrients, or partial, with malabsorption of only specific nutrients. Causes of malabsorption include damage to the intestine from infection, inflammation, trauma, and surgery as well as specific diseases such as celiac disease, Crohn's disease, chronic pancreatitis, and cystic fibrosis.
[0041] Effective amount, as used herein, refers to the amount of a compound or other substance that is sufficient in the presence of the remaining components to effect the desired result, such as reduction in a metabolite by at least about 50%, usually at least about 70%, typically at least about 90%, frequently at least about 95% and most often, at least about 99%. In other aspects of the invention, an effective amount of a biotherapeutic can refer to that concentration of the biotherapeutic that is sufficient in the presence of the remaining components to effect the desired result. The effective amount of a biotherapeutic or other substance is readily determined by one of ordinary skill in the art.
[0042] Adaptive evolution as used herein, refers a process of heritably changing a cell or population of cells by exposing the cell(s) to selective pressure and supporting survival of those that adapt to the pressure condition, without using artificial genetic engineering. Adaptive evolution is effected by naturally occurring mechanisms rather than by direct physical manipulation of the genome.
[0043] Adaptively evolved microorganism refers to an organism that has been heritably changed by a process of adaptive evolution.
[0044] Selective pressure and selection pressure are used interchangeably herein to refers to a condition under which organisms with certain phenotypes have either a survival benefit or disadvantage. For example, pressure to utilize a particular nutrient or substrate (a pressure selection agent), such as galactose, can be applied by including a physiologically significant amount of galactose in growth media with the result that those best able to utilize the galactose will preferentially survive.
[0045] A genetically modified organism or GMO is any organism whose genetic material has been altered using genetic engineering techniques, such as recombinant DNA, site-directed mutagenesis, gene transfer, and CRISPR. Genetic engineering excludes methods and processes that occur naturally.
[0046] Microbiota, as used herein, refers to the ecological community of commensal, symbiotic and pathogenic microorganisms found in and on all multicellular organisms studied to date from plants to animals. A microbiota includes bacteria, archaea, protists, fungi and viruses. A microbiota can include all the microorganisms in and on a host, or can refer specifically to those populating a specific niche, such as the gut (i.e., gastrointestinal or digestive tract including the esophagus, stomach, small and large intestines), skin, mouth, reproductive tract or the like.
[0047] The term microbiome describes the collective genomes of a microbiota.
[0048] The present invention is based on the observation that microorganisms populating the gut (gut microbiota) can complement defective host metabolism and/or remove intolerable dietary substances by acting directly in the human digestive tract and thereby contribute various functions to a symbiotic relationship with a human host including metabolism of carbohydrates.
[0049] Organisms such as Lactobacilli are the most common microorganisms in the human GI tract and have been widely used as delivery systems in the form of live biotherapeutics either as colonizers and/or delivery expression systems for therapeutic molecules. Yeasts represent another group of organisms that have been exploited as live biotherapeutic products given their generally recognized as safe (GRAS) status as well as their potential to restore gut function after long term antibiotic therapy. The present invention provides a live biotherapeutic approach to significantly minimize one or more symptoms related to fructose exposure.
[0050] The present invention provides a novel approach to treatment of carbohydrate consumption-related disorders, particularly as it relates to gastrointestinal pathologies and genetic-based systemic pathologies. This novel approach detoxifies carbohydrate (e.g., galactose, fructose) in the gut by the use of non-pathogenic microorganisms that minimize build-up of the carbohydrate and mitigate one or more of the symptoms and complications associated with elevated concentrations of the carbohydrate.
[0051] The present invention provides compositions and methods for the management of certain metabolic diseases, particularly diseases caused by deficient carbohydrate metabolism, such as galactosemia and fructosemia. The invention also provides methods for producing microorganisms (e.g., yeast and bacterial strains) that are effective at complementing deficient carbohydrate metabolism, thereby permitting treatment or reduction in symptoms and/or effects of disorders caused by deficient carbohydrate metabolism.
[0052] Also provided are tools for management of galactosemia, fructosemia and other metabolic diseases that develop a microbiota highly effective at complementing metabolic deficiencies by converting potentially toxic substances present in or derived from food to non-toxic and/or beneficial substances. In the case of galactosemia, the invention provides microorganisms that can replace a host (e.g. human patients) deficient metabolism of galactose with organisms that are highly effective at degrading galactose present in the gut that originates from food, or directly in the food prior to consumption. To achieve these goals the invention provides microorganisms, particularly yeasts, that are viable and metabolically active for degrading galactose in the gut prior to its uptake into the blood stream by transporters located in the small intestine.
[0053] Similarly, in the case of fructose intolerance, malabsorption or metabolic disease, the invention provides microorganisms, which can replace deficient metabolism of fructose with organisms that are highly effective at degrading fructose present in the gut that originates from food, or directly in the food prior to consumption.
[0054] Also, considering the digestive transit kinetic, in certain embodiments, galactose-degrading and other yeast strains of the invention can act upon target molecules (e.g. certain carbohydrates) within a relatively short period of time in the gut before potential washout. Gastric emptying lasts an average of 1 h (Maurer et al., The SNMMI and EANM Practice Guideline for Small-Bowel and Colon Transit 1.0. J Nuclear Med 54:2004-13 (2013)), whereas small intestine and colonic transit times are carried out for an average of 1 to 6 h and 72 h, respectively (Read et al., Simultaneous measurement of gastric emptying, small bowel residence and colonic filling of a solid meal by the use of the gamma camera. Gut 27:300-8 (1986)). Moreover, formulations known in the art, such as enteric coatings, could further improve the recovery of viable yeast cells.
[0055] Advantageously, microorganism such as yeast and certain bacteria, can colonize and become a stable component of the gut microbiota. In certain aspects, inventive yeast strains and other microorganisms of the invention, have the ability to colonize the gastrointestinal tract of a human and to degrade galactose before its uptake into the blood stream. In such aspects of the invention, an alternative route of administration, e.g. via a nasogatric or orogastric tube, enema, or endoscopic administration may be suitable for populating the intestines. In other aspects, the microorganisms are stable through transit of the gastrointestinal tract, and can colonize the gut when administered orally. Thus, the biotherapeutic microorganisms of the invention can be long-acting, and in some cases may be a persistent or, in the absence of interventions that reduce or eliminate such microorganisms (e.g., antibiotic treatment) an ongoing and permanent treatment for dietary intolerance, malabsorption and/or metabolic disease. The persistence of microorganism colonization of the gut contrasts with therapeutic use of isolated dietary enzymes, such as lactase, which are unstable, degraded when passing through the digestive tract must be administered with each consumption of offending or intolerable food. In some embodiments, an effective dose of a biotherapeutic of the invention can be delivered by once, once-daily, once-weekly, once-monthly or as needed dosing.
[0056] Although dietary galactose restrictions are life-saving for infants, a strict dietary treatment does not appear to prevent all adult patients from developing complications of galactosemia. The cause of the complications has been explained by an endogenous production of galactose, amounting to 1 gram per day in adults. Berry et al., Endogenous synthesis of galactose in normal men and patients with hereditary galactosaemia. Lancet 346:1073-74 (1995); Bosch Classic galactosemia: Dietary dilemmas. J Inherit Metab Dis 34:257-60(2010)). Despite this observation, galactose reduction continues to be the mainstay of therapy with diet recommendations varying from very strict excluding milk together with certain fruits and vegetables to more liberal excluding dairy products only. Also, even if new modes of treatment are identified such as inhibitor agents of the defective galactose metabolic enzymes, diet restriction will likely remain the basis of galactosemia therapy because experts consider that it is still judicious to restrict galactose intake to manage its toxicity in adults. In this context, a yeast-based approach would permit broader diet flexibility. In addition, a yeast-based approach may alleviate some of the gastrointestinal symptoms associated with galactosemia (Shaw et al., Gastrointestinal Health in Classic Galactosemia. JIMD Rep 33:27-32 (2016)), as the diet would be less restrictive and thus supportive of a more diverse microbiome with potentially positive outcome.
[0057] The present invention represents the first yeast-based approach to manage galactosemia that can significantly reduce galactose concentrations. Thus, in one embodiment, the invention provides an adaptively evolved yeast strain and methods for using the strain to degrade galactose in foods to safe levels that permit access to a simple and palatable diet.
[0058] The gut microbiota includes a large number of microorganisms, which may include bacteria, archaea, protists, fungi and viruses and/or viruses. Any of these are potentially suitable for use in adaptive evolution strategies of the invention. However, yeast are particularly attractive therapeutic agents for minimizing toxicity associated with high levels of galactose for a number of reasons. Yeast are natural components of the microbiome (Nash et al., The gut mycobiome of the Human Microbiome Project healthy cohort. Microbiome 5:153 (2017)), that can express a galactose metabolic pathway similar to that found in humans. Sellick et al., Chapter 3 Galactose Metabolism in YeastStructure and Regulation of the Leloir Pathway Enzymes and the Genes Encoding Them Int Rev Cell Mol Biol 269:111-50 (2008). From a practical standpoint, certain yeast have the advantage over other microorganisms of a GRAS status (e.g. Generally Recognized As Safe) with cultures that can easily be established from a single, homogenous colony, just like bacteria, but devoid of DNA sequences that could potentially promote gene transfer to other gut microorganisms. Yeast has also been found to be able to establish itself in the gastrointestinal tract in areas where bacteria may not thrive. Czerucka et al., Yeast as probioticsSaccharomyces boulardii. Alimentary Pharmacol Therapeutics, 26:767-78 (2007). Moreover, yeast has already been used in human medicine, for instance for the treatment of antibiotic-associated diarrhea. Kelesidis et al., Therapeutic Adv Gastroenterol 5:111-25 (2011); Tung et al., Prevention of Clostridium difficile Infection with Saccharomyces boulardii: A Systematic Review. Can J Gastroenterol 23:817-21 (2009).
[0059] The present invention provides microorganisms, such as yeast, adaptively evolved and evaluated for reducing toxicity originating from food sources, particularly carbohydrates such as galactose and fructose. The present invention also provides methods for generating and selecting microorganisms (e.g. yeast strains) that preferentially or exclusively utilize galactose even in presence of other carbohydrates. In other aspects, the invention provides methods for generating and selecting microorganisms (e.g. yeast strains) that preferentially or exclusively utilize galactose even in presence of other carbohydrates.
[0060] Microorganisms suitable for use in the methods of the invention are generally non-pathogenic and frequently generally recognized as safe (GRAS) when introduced into the gastrointestinal tract of a human, typically by oral consumption, but can be by any route, such via a nasogastric tube, an orogastric tube, an enema or an endoscope. In certain aspects, orally administered microorganisms can be directed to a particular region of the gastrointestinal tract, e.g. by enterically coating the microorganism.
[0061] Yeast contemplated for use in the methods of the invention include, but are not limited to: Saccharomyces species (spp.) such as Brettanomyces spp. such as B. bruxellensis; S. cerevisiae, S. boulardii, S. fragilis, S. bayanus, S. beticus, S. eubayanus, S. fermentati, S. jurei, S. kudriavzevi, S. paradoxus, S. pastorianus, and S. uvarudm, S. cerevisiae x S. eubayanus x S. uvarum, S. eubayanus x S. uvarum; Saturnispora spp. such as S. zaruensis; Pichia (Scheffersomyces) spp. such as P. stipites, P. cecembensis; Schizosaccharomyces spp. such as S. cryophilus, S. japonicus, S. octosporus, and S. pombe; Saccharomycodes spp. such as S. ludwigii; Debaromyces spp, such as D. hansenii; Kazachstania spp. such as K unispora; Kluyveromyces spp. such as K marxianus, K wickerhamii, K lactis, and K. lodder, K. wickerhamii; K. dobzhanskii; Torulaspora spp. such as T. delbrueckii; Yarrowia spp. such as Y. lipolytica and Zygosaccharomyces spp. such as Z kombuchaensis. Kluyveromyces marxianus; Hanseniaspora spp. such as H. opuntiae, H. thailandica, H uvarum; Limtongozyma cylindracea Saccharomyces sp.; Metschnikowia spp. such as M. bicuspidate, M. chrysomelidarum, M. cibodasensis, M. colchici, M. gelsemii, M. gruessii, M. henanensis, M. kofuensis, M. maroccana, M. noctiluminum, M. peoriensis, M. rancensis, M. reukaufii, M. vanudenii, M. viticola, M. zobellii; Hanseniaspora spp. such as H. opuntiae, H. thailandica, and H. uvarum.
[0062] Additional fungi that may be useful in certain aspects of the invention include: Aspergillus spp. such as A. oryzae, A. sojae, and A. tamarii; Penicillium spp. such as P. camemberti and P. roqueforti; Rhizopus spp. such as R. oligosporus and R. oryzae.
[0063] In certain embodiments of the invention, bacteria may be used instead of fungi. Such bacteria include, but are not limited to: Lactobacillus spp. such as L. acidophillus, L. amylovorus, L. bulgaricus, L. brevis, L. casei, L. crispatus, L. curvatus, cL. Delbrueckii, L. fermentum, L. gallinarum, L. gasseri, L. helveticus, L. jensenii, L. johnsonii, L. kefiranofaciens, L. latis, L. parcasei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius, and L. sakei; Bifidobacteria spp. such as B. adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. lactis, and B. longum; Bacillus spp. such as B. cereus, B. clausii, B. coagulans, B. infantis, B. pumilus, B. subtilis, and B. coagulans; Lactococcus spp. such as L. lactis; Enterococcus spp., such as E. durans and E. faecium; Eubacterium spp. such as E. faecium; Leuconostoc spp. such as L. citreum, L. cremoris, L. gasicomitatum, L. gelidum, L. kimchi and L. mesenteroides; Oenococcus spp. such as O. oeni; Pedicoccus spp. such as P. acidilactici, P. pediococcus, and P. pentosaceus; Propionibacterium spp. such as P. acidipropionici, P. freudenreichii, P. jenseniil, and P. shermanii; Sporolactobacillus spp. such as S. inulinus; Streptococcus spp. such as S. thermophilus; Tetragenococcus spp. such as S. halophilus; and Weissella spp. such as W. confuse, W. kimcii and W. koreensis.
[0064] The present invention provides method for adaptively evolving a microorganism, including the steps of providing a microorganism; growing the microorganism under an adaptive condition, wherein the organism adapts to the adaptive condition; and confirming adaptation of the microorganism, thereby adaptively evolving the microorganism. Typically, there is a clonal population of cells of the microorganism that can be isolated by plating cells of the microorganism on an agar plate under conditions that yields single colonies of the cells; growing the plated cells on the agar plate; and picking a single colony of the cells from the plated agar plate, thereby isolating the clonal population of cells. Other methods of isolating a clonal population of cells, such as limiting dilution, are known in the art.
[0065] Growing the microorganism under an adaptive condition can, for example include inoculating a culture medium comprising the adaptive condition with the isolated microorganism; growing the microorganism in the culture medium to prepare an actively growing adaptive culture; diluting the actively growing adaptive culture with fresh medium comprising the adaptive condition; and repeating the growth and dilution steps from 1 to 100 time. The growth and dilution steps can be repeated at any suitable interval that maintains the culture in a rapidly growing state based on time, such as weekly, twice weekly, every second day, daily, twice daily or every 8 hours or based on OD. In certain embodiments, the serial dilution process is repeated every 12-24 hours, such as every 16 hours. The serial dilution process can be repeated as many times as necessary to obtain adaptation of the microorganism. In certain aspects, the serial dilution process is repeated 7-10 times, in other aspects, it is repeated 10-25 times or more.
[0066] Typically, the adaptation results in a change in growth efficiency of the microorganism and confirming adaptation of the microorganism is based on detecting the change in growth efficiency, such as by measuring generation time, specific growth rate, or doubling time and observing a change in at least one of these parameters. In certain aspects, cell growth is monitored prior to each serial dilution, e.g. by measuring the optical density of the culture, which is a function of cell density.
[0067] The frequency of serial dilution will depend on the generation time of the organism under the adaptive condition, and may require at least 20 cell generations, at least 50 cell generations, at least 75 cell generations to obtain a desired change The adaptation can, for example, result in the increase in synthesis of a substance (e.g., an enzyme, vitamin or co-factor) or it can be an increase in metabolism of a food or nutrient, such as the ability of the microorganism to use a carbon source (e.g., a specific carbohydrate).
[0068] In specific embodiments of the invention, the microorganism adaptively evolves to utilize galactose as a carbon source and the adaptive condition includes the presence of galactose, and can also include presence of glucose.
[0069] In other embodiments of the invention, the microorganism adaptively evolves to utilize fructose as a carbon source and the adaptive condition includes the presence of fructose, and can also include presence of glucose.
[0070] The invention also provides adaptively evolved microorganism prepared according to the methods described herein. In one embodiment, adaptively evolved microorganism metabolizes galactose, typically in the presence of glucose. In one aspect, the invention provides adaptively evolved microorganisms derived from Saccharomyces cerevisiae. Advantageously, adaptively evolved microorganisms of the invention degrade galactose in present in food, such as milk.
[0071] The conditions encountered in the gastrointestinal tract, including the gastric fluid environment, intestinal fluid environment, more particularly the gastric fluid environment followed by an intestinal fluid environment, can be hostile to microbiological growth.
[0072] In certain aspects of the invention, the adaptively evolved microorganisms are capable of colonizing the gastrointestinal tract thereby providing long-term galactose metabolism. In certain embodiments of the invention, at least 10, 20, 30 or 40% of the cells will demonstrate properties indicative of the ability to colonize the gastrointestinal tract. The methods can allow the subject to consume galactose containing or galactose producing (upon digestion) food without low absorption of galactose from the food into the bloodstream.
[0073] In certain aspects the adaptively evolved microorganism is administered prior to consuming food, such as with meals three times per day. The effective amount of the adaptively evolved microorganism reduces the amount of galactose by at least 50% and/or eliminates absorption of at least 90% of the galactose in the consumed food. In embodiments of the invention where the microorganism colonizes the gastrointestinal tract, long-term galactose reduction or elimination from food-based sources may be achieved.
[0074] Also provided by the invention are compositions containing one or more galactose metabolizing enzyme from the adaptively evolved microorganisms described herein. Such compositions can be pharmaceutical composition suitable for administration to a subject and/or they can be suitable for addition to food. Effective amounts of either composition reduce the amount of galactose in food either prior to or after consumption of the food.
EXAMPLES
Deposit of Microorganisms
[0075] Applicant is making a deposit with ______ of the following strains on ______ 2020, under the terms of the Budapest Treaty: Saccharomyces cerevisiae clone Y_C202_1 Accession No. ______; Saccharomyces cerevisiae clone Y_C201_1 Accession No. ______; Kluyveromyces marxianus clone K_219 Accession No. ______; Pichia kudriavzevii clone G1_1A Accession No. ______; Saccharomyces cerevisiae clone G2_1A Accession No. ______; Saccharomyces uvarum clone G3_1A Accession No. ______; and Metschnikowia reukaufii clone G4_1A Accession No. ______.
Example 1. Identification of Yeast Strains Capable of Degrading Galactose
[0076] A primary goal of the present invention was to develop yeast strains highly effective at metabolizing galactose. However, galactose-metabolizing yeasts are uncommon; yeasts typically prefer glucose, a carbohydrate source known to strongly suppress the expression of genes needed to metabolize other carbohydrates such as galactose (See Escalante-Chong et al., Galactose metabolic genes in yeast respond to a ratio of galactose and glucose. Proc Nat'l Acad Sci, USA 112:1636-41 (2015)). Wild type yeast strains may thus be prevented from utilizing any carbohydrates when glucose is present. Even if a yeast does have the capability to use other carbohydrate sources, it may occur late in the growth process, only after glucose has been completely depleted. Therefore, a particular type of galactose-metabolizing yeast was developed that degrades galactose in the presence of glucose. The present invention provides methods for adaptively evolving yeast according to this process.
[0077] To assess the ability of a yeast strain to degrade galactose, its growth was evaluated on media containing galactose in presence or absence of glucose. The following strains were tested: the commercially available strains Saccharomyces cerevisiae (N) (Natureland, Saccharomyces boulardii (SB) (Jarrow, Santa Fe Springs, Calif.), and Saccharomyces boulardii (B) (Biocodex, Redwood City, Calif.). Additional strains included in the screening were isolated from food containing large amounts of galactose such as dairy products and legumes stored at room temperature for over two weeks.
[0078] Cultures of various strains were initiated from a single colony on agar plates or from glycerol stocks, and grown in liquid YP medium (1% yeast extract, 2% peptone; Teknova) by incubation at 30 C. with agitation at 125 rpm (Murakami & Kaeberlein Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells. J Visual Exp (27) (2009)). Overnight yeast cultures initiated in duplicate in liquid YP medium were used as pre-cultures to initiate growth efficiency experiments in liquid CM (Synthetic Complete Minimal Medium, 0.5% Ammonium Sulfate, Teknova) containing 2% galactose alone as the sole carbon source, 2% glucose alone as the sole carbon source, or galactose and glucose. Culture growth of cultures set at 30 C. under static conditions was monitored over time by measuring optical density (OD) at 600 nm (OD.sub.600) using a spectrophotometer.
[0079] Growthwas evaluated for several strains. As illustrated in Table 1, one of the evolved clone exhibited the lowest doubling time, which remained at the same level independently of the carbohydrate source and growth conditions.
TABLE-US-00001 TABLE 1 Doubling Time of Yeast Strains under Static Growth Conditions in Media Containing Galactose alone, Glucose alone, and Galactose and Glucose. Galactose + Galactose Glucose Glucose Avg. Avg. Avg. Strains (h) SD (h) SD (h) SD Y1_Parent 4.57 0.03 4.43 0.05 4.40 0.01 Evolved Clone 4.05 0.02 4.17 0.08 4.21 0.02 Strain N 6.05 0.03 4.46 0.02 4.23 0.01 Strain SB 7.79 0.06 4.68 0.00 4.75 0.03 Strain B 8.85 0.02 4.96 0.02 4.63 0.01 Each data point represents the averages (Avg.) and standard deviation (SD) of quadruplicate values obtained for two independent cultures per strain.
Example 2: Adaptive Evolution Results in Superior Yeast Strains
[0080] Adaptive evolutionwas applied to yeast strains, a method that can increase traits of a given strain owing to random mutations in the genome. Clones derived from parental strains that offer a phenotypic advantage are naturally selected when grown under selective pressure. Yeast can be subjected to adaptive evolution with changes that can be observed in a short time period because yeast grows rapidly as single cells in simple media, with the entire life cycle completed in culture similarly to bacteria.
[0081] Adaptive evolution was carried out by daily serial dilution of independent clonal populations of parental strains (See akar et al., Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties. FEMS Yeast Res 12:171-82 (2011)). Parallel cultures were independently grown at 30 C. under agitation at 125 rpm in 15 mL CM medium in 50 mL vented tubes or 3 mL in 14 mL culture tubes supplemented with 2% galactose as a pressure selection agent for about 16 to 30 hours before transferring the cultures to fresh medium at a 1:30 dilution. The procedure of serial dilution was repeated until a change in growth was detected by OD.sub.600.
[0082] A volume of 100 L of a 1:1,000 dilution of the last daily serial dilution of the culture with the greatest OD was plated on YP-galactose agar medium (Teknova) and incubated at 30 C. Isolated, adaptively evolved clones were randomly selected from the plate to start duplicate cultures grown at 30 C. under agitation at 125 rpm in 3 mL CM medium supplemented with 2% galactose alone or 2% galactose and glucose.
[0083] To assess the yeast strains' and clones' ability to degrade galactose, the concentration of galactose in spent medium was determined using the colorimetric Galactose Assay Kit (Cat. No. EGAL-100; BioAssay Systems, Hayward, Calif.). All reactions were performed in 96-well microplates and the absorbance was read at 570 nm using volumes and concentrations recommended by the kit. The samples and standards were mixed with the enzyme and the substrate, which reacts with galactose to produce hydrogen peroxide. Hydrogen peroxide reacts with the colorless substrate, which, in presence of horseradish peroxidase (HRP), produces a pink-colored product with an intensity proportional to galactose concentration. Sample concentrations were determined by comparison to a standard curve generated with known quantities of galactose.
[0084] Utilization and degradation of galactose was assessed by measuring the consumption of galactose in spent culture medium. Amongst the adaptively evolved strains generated by serial dilution from two parent strains, some clones exhibited a greater level of reduction of galactose concentration compared to the parent strains. Table 2, below, lists the percentage of galactose reduction in spent culture medium for the adaptively evolved strains and the parent strains when cultured for 8 hours at 30 C. in CM medium supplemented with 2% galactose alone or with galactose and glucose.
[0085] This data set illustrates that the adaptively evolved clones were able to significantly degrade galactose. More importantly, some clones were also able to utilize galactose even in presence of glucose, demonstrating that they were not affected by glucose catabolite repression. Strain Y1_2 exhibited a reduction greater than 94.4% of galactose concentration compared to less than 1% reduction for the parental strain Y1 in spent medium at the 8 hour time point. This difference is statistically significant, confirming that adaptive evolution produced a more efficient strain.
TABLE-US-00002 TABLE 2 Utilization of Galactose of Adaptively Evolved Clones (Y1_1 to Y1_7; Y2_1 to Y2_7) Compared to the Parent Clones Y1 and Y2. Clones No: Y1_1 Y1_2 Y1_3 Y1_4 Y1_5 Y1_6 Y1_7 Y1_Parent Galactose 69.4% 61.6% 83.6% 60.2% 73.2% 55.7% 64.1% 32.1% Gal + Glu 83.2% 94.4% 85.0% 70.7% 61.9% 32.3% 72.9% 0.1% Clones No.: Y2_1 Y2_2 Y2_3 Y2_4 Y2_5 Y2_6 Y2_7 Y2_Parent Galactose 32.4% 42.5% 43.2% 51.9% 45.9% 49.3% 50.7% 27.3% Gal + Glu 31.9% 29.9% 32.9% 38.7% 46.0% 58.2% 56.9% 34.4% Values represent averages of percent reduction of galactose concentration from two independent cultures grown for 8 hours at 30 C.
Example 3: Isolation and Identification of Galactose-Degrading Strains
[0086] This study was focused on isolating yeast strains that would have the ability to degrade galactose. Yeasts were isolated from galactose-rich food samples such as dairy products and legumes. Various species were isolated by cultivating the samples in culture medium containing galactose at room temperature for a period of time of at least 2 days. The isolates were then subjected to adaptive evolution eventually followed by UV treatment to generate clones with superior galactose-detoxification performance. Taxonomy was assigned to isolates by a molecular approach based on targeted sequencing of Internal Transcribed Spacers (ITS).
[0087] Strains were isolated mainly from two sources: legumes (chickpeas, beans) and dairy product (kefir). For legumes, the seeds were collected into sterile flasks. For milk product, an aliquot of home-made kefir was utilized as inoculum. After cultivation for approximately 2 weeks at laboratory temperature with an agitation of 125 rpm in liquid minimal medium supplemented with 2% galactose (Teknova) and 50 ug/mL chloramphenicol (Teknova, Cat C0380), cultures were serially diluted and seeded again in liquid medium with selection and incubated at 30 C. with agitation of 125 rpm. This cycle was performed at least 3 times until plating serially diluted cultures on YP-2% galactose agar plates. Single colonies obtained after incubation at 30 C. for 2-5 days were sequentially streaked multiple times to obtain pure isolates.
[0088] For taxonomy assignment, the targeted metagenomic sequencing method was employed. Briefly, the isolates were processed for DNA extraction using the ZymoBIOMICS96 MagBead DNA Kit and the DNA samples were prepared for targeted sequencing using targeted Internal Transcribed Spacers primer sets (ITS2, Zymo Research, Irvine, Calif.). The final library was sequenced on Illumina MiSeg. Taxonomy analyses were performed on publicly available sequences using the BLAST program under default settings (http://www.ncbi.nlm.nih gov/BLAST).
[0089] The ITS sequences obtained from pool cultures and from isolate cultures were analyzed to identify isolate phylogeny. Table 3 reports the outcome of this analysis (Hits) based on the homology of ITS sequences amplified from pool cultures and purified isolate cultures with publicly available sequences. The Table lists the species exhibiting the greatest score and 100% sequence identity with ITS sequences obtained from this study.
TABLE-US-00003 TABLE 3 Blast Results of Sequences Obtained after Amplification of Internal Transcribed Spacers (ITS) from Pool Cultures and from Isolate Cultures. Source Hits List of potential assignment for pool Saccharomyces sp, Saccharomyces uvarum, isolated from legume Saccharomyces cerevisiae, Saccharomyces cerevisiae Saccharomyces eubayanus Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces jurei, Saccharomyces sp. boulardii, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Pichia sp. Taxonomic assignment as per Blast search Saccharomyces cerevisiae for isolate Yi derived from legume pool List of potential assignment for pool Saccharomyces sp., Saccharomyces uvarum, isolated from dairy product Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces eubayanus Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces jurei, Saccharomyces euhayanus, Saccharomyces kudriavzevi, Pichia sp., Kluyveromyces marxianus, Kluyveromyces lactis, Kluyveromyces dohzhanskii, Kluyveromyces wickerhamii, Kluyveromyces sp., Kazachstania unispora Taxonomic assignment as per Blast search Saccharomyces sp. for isolate Ki derived from legume pool
[0090] Adaptive evolution was carried out on the isolates by daily serial dilution conducted for at least 30 cycles, corresponding to approximately 180 generations of the parental strain Yi isolated from legumes. Two clones identified as lead clones, clone Y-C201 and clone Y-C202 were subjected to UV treatment as follows. A volume of 100 L of a 1:1,000 dilution from an overnight 3 mL-culture grown at 30 C. under agitation at 125 rpm in CM medium supplemented with 2% galactose was plated on YP-2% galactose agar medium. The petri dishes were exposed to UV rays (235 nm) in the biosafety cabinet at a distance of 20 cm. The plates were then incubated in the dark for at least 3 days at 30 C. Isolated clones were randomly selected from the UV-treated plates to start duplicate cultures. After multiple rounds of screening tests evaluating growth and galactose consumption in presence or absence of glucose, three lead clones were selected: clone Y-C201-1 deriving from clone Y-C201 and clones Y-C202-1 and Y-C202-2 deriving from Y-C202.
Example 4: Degradation of Galactose in Food and Beverages
[0091] As part of evaluating the feasibility of a yeast-based approach as a treatment to mitigate the effects of elevated concentrations of galactose in foods and beverages, several evolved clones were tested for their capability of degrading galactose when present in food. Milk was tested because it represents the most challenging food for galactosemia patients considering its high level of galactose (2-4 g per 100 mL of milk). Food spiked with galactose was tested in parallel.
[0092] For this study, three evolved yeast strains obtained by adaptive evolution followed by UV treatment, Clone Y-C201-1, Clone Y-C202-1, and Clone Y-C202-2, one evolved yeast strain obtained by adaptive evolution, Clone Y-C202, as well as the initial parent strain Yi were compared for their galactose consumption activity. Cultures were initiated from a single colony on agar plates and grown in 15 mL of liquid YP medium (1% yeast extract, 2% peptone; Teknova, Hollister, Calif.) in a 50-mL mini-bioreactor by incubation at 30 C. with an agitation of 225 rpm supplemented with 2% galactose (Teknova). Strain Saccharomyces boulardii (SB) was prepared similarly to the evolved clones except that it was grown in YP medium supplemented with 2% glucose.
[0093] The testing of galactose consumption was started with yeast cells obtained from a culture volume containing 1.010.sup.9 Colony Forming Units (CFU) pelleted by centrifugation at 1000 rpm (Sorval, RT7) for 10 min at room temperature. Cell pellets were resuspended either in 1.0 mL of milk already pre-treated with lactase (LACTAID milk where lactose is transformed into galactose and glucose) or in 1 mL rodent diet (Teklad, Envigo) spiked with a solution of 5% galactose or a solution of 5% galactose+1% glucose. All the reactions were incubated at 37 C. Aliquots of the reactions were taken at multiple time points and stored at 20 C. until galactose concentration determination.
##STR00001##
Conversion of Lactose to Galactose and Glucose
[0094] As shown in Table 4, Table 5, and Table 6, the evolved clones were able to rapidly decrease galactose concentration present in milk and in spiked-diet whereas strain SB did not decrease the galactose concentration. More importantly, the evolved clones were also able to utilize galactose even in presence of glucose, demonstrating that there were not affected by glucose catabolite repression.
TABLE-US-00004 TABLE 4 Galactose concentration (mM) in milk after exposure to strains for a period of 1 hour Time point Y- Y- Y- Y- (hr) C201-1 C202 C202-1 C202-2 Yi SB Milk 0.1 125.07 131.25 135.96 135.12 162.10 180.24 171.24 1 16.50 11.23 34.68 40.75 156.51 172.22 187.96
TABLE-US-00005 TABLE 5 Galactose concentration (mM) in diet spiked with 5% galactose and 1% glucose after exposure to strains for a period of 0.1 to 3 hours Diet 5% Time Galactose point Y- Y- Y- Y- 1% (hr) C201-1 C202 C202-1 C202-2 Yi SB Glucose 0.1 286.89 269.65 262.67 284.94 314.39 305.63 324.16 1 167.19 126.95 132.29 137.21 327.20 312.06 334.32 2 4.18 2.54 2.47 2.33 318.41 308.60 327.24 3 1.95 2.85 2.85 3.48 287.49 310.62 307.31
TABLE-US-00006 TABLE 6 Galactose concentration (mM) in diet spiked with 5% galactose after exposure to yeast strains for a period of 0.1 to 3 hours Time Diet point Y- Y- Y- Y- 5% (hr) C201-1 C202 C202-1 C202-2 Yi SB Galactose 0 284.17 268.96 224.74 244.88 232.57 304.52 313.17 1 169.98 142.31 129.43 123.04 176.86 301.41 322.39 2 3.83 2.37 2.30 2.12 169.46 306.93 315.51 3 1.81 2.71 2.78 3.20 165.41 306.26 332.58
Example 5: Tolerance to Gastrointestinal Conditions
[0095] The most convenient way to deliver a yeast-based therapeutic is orally. During transit through the gastrointestinal tract, an orally delivered agent will be confronted with a variety of simultaneous or sequential adverse conditions, such as the internal body temperature, gastric fluid with acidic pH, and pancreatic fluid with alkaline fluid. A series of in vitro experiments were conducted to assess the potential for survival of the evolved clones when subjected to gastrointestinal conditions.
[0096] Yeast cells were subjected to simulated gastric fluid (SGF) supplemented with 1 mg/mL of pepsin and simulated intestinal fluid (SIF) supplemented with 1 mg/mL of pancreatin to evaluate their tolerance to gastrointestinal conditions. Zhou et al. Statistical investigation of simulated fed intestinal media composition on the equilibrium solubility of oral drugs. Eur J Pharm Sci 99:95-104 (2017). Overnight yeast cultures grown in liquid YP medium supplemented with 2% galactose were harvested by centrifugation for 10 min at 1000 rpm at room temperature. To test for survival in gastric fluid, pellets were re-suspended in 1 mL SGF (Cat No. 7108.16; RICCA Chemical Company, Arlington, Tex.) supplemented with pepsin from porcine gastric mucosa with an activity of 8.60 European Units/mg (Cat No. 41707-1000; ACROS Organics, Geel, Belgium). The pH was measured (pH2) and the reactions were maintained at 37 C. for 240 min. Cells were also tested for survivability in SGF added to dry diet pellet (5 mL of SGF added to 1 g diet, Teklad Diet). To test for survival in intestinal fluid, cells were centrifuged for 10 min at 1000 rpm at room temperature and re-suspended in 1 mL SIF (RICCA Chemical Company, Cat No. 7109.16, pH 6.7-6.9 supplemented with 1 mg/mL of pancreatin from porcine pancreas (Sigma, Cat No. P3292). All reactions were maintained at 37 C. for 240 min. Yeast cells were counted before and after intestinal challenges with Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF). The survival rate was estimated by evaluating the ratio between cell counts of yeast preparations subjected to the simulated fluid challenge over time vs. the same preparations at time zero. Viable cell counts were determined using a hemocytometer and Trypan blue staining.
[0097] As shown in Table 7, the gastric environment had an impact on strain survival. Simulated gastric triggered a cell count decrease of approximately 5% within 20 min but there were no major changes of cell counts when the cells were exposed SGF resuspended in dry diet. Interestingly, the simulated intestinal fluid did not appear to have a significant effect on cells
TABLE-US-00007 TABLE 7 Survivability Time Course (%) of Evolved Clone Y_C202_1 in Gastrointestinal Conditions after Exposure to Simulated Fluids over a period of 0 to 240 min Time point SGF, (min) SGF SIF PBS Diet 0 100.0 100.0 100.0 96.3 20 95.9 100.0 100.0 95.0 40 20.8 100.0 100.0 98.2 60 0.0 100.0 100.0 92.7 120 ND 100.0 100.0 94.6 240 ND 100.0 100.0 95.2 SGF: Simulated Gastric Fluid; SIF: Simulated Intestinal Fluid, SGF, Diet: Simulated Gastric Fluid with Diet; ND: Not Determined.
Example 6: Mitigation of Galactose Accumulation in Serum after a Single Oral Administration of the Evolved Clone
[0098] The objective of this study was to assess the ability of the test article to detoxify galactose in vivo. To that end, galactose concentration was investigated in serum and urine after a single bolus oral administration to animals of 10% galactose with and without the test article.
[0099] Animal care and procedures were approved by the Institutional Animal Care and Use Committee and were followed in accordance to the standards for animal husbandry and care of the U.S. Department of Agriculture's (USDA) Animal Welfare Act. Veterinary care was available throughout the course of the study and animals were examined by the veterinary staff as warranted.
[0100] Fifteen-week-old male Sprague-Dawley rats weighing between 350 and 400 g were acquired from Envigo with a catheter surgically implanted in the jugular vein. Animals were housed individually in polycarbonate cages containing animal bedding during the acclimation period lasting 7 days. Environmental controls were maintained at 18 to 23 C. with humidity of 30% to 70% with automatic lighting on a 12 h/12 h on/off cycle except as required for specimen collection and study conduct. Animals were fed an irradiated chow diet (Pico Lab, Lab Diet 5053) and were provided municipal tap water ad libitum. Animals were housed individually in metabolic cages following the single bolus dose until end of study. No concurrent medication was given.
[0101] The rats were randomly distributed into three groups, each consisting of three animals. Group I was administered with a solution of 10% (=555 mM) galactose; Group II was administered with the test article together with a solution of 10% galactose; and Group III was administered with the test article only. All animals were fasted for a period of 16 hour prior to dosing and all animals were administered a single bolus dose by oral gavage with a total volume of 10 mL/kg. The cell density of the test article was 2E+09 CFU/mL prepared from an overnight culture incubated at 30 C. in orbital shaker and grown in YP medium supplemented with 2% galactose. Blood samples (0.3 mL) were collected serially via catheterized jugular veins at 0, 0.5, 1, 1.5, 2, 4, 8, and 24-hour after dosing for analysis of galactose concentration in serum. Urine samples were collected at intervals from 0 to 4 hours, 4 hours to 8 hours, and 8 hours to 24 hours after the single bolus administration for analysis of galactose concentration. All samples were stored frozen until analysis. Galactose levels in urine and serum samples were determined using the EnzyChrom Galactose Assay Kit (BioAssay Systems, Hayward, Calif.) and analyzed in duplicate in a microplate reader (Molecular Devices).
[0102] As reported in Table 8, galactose concentration rose sharply to a concentration of 10.5 mM after administration of a single bolus dose of 10% galactose (555 mM) and reached its peak level in serum 60 min after administration by oral gavage. There was also a marked increase of galactose concentration in urine, approximately greater than 50% of the loaded dose (Table 9). Interestingly, for the group treated with the test article concurrently to galactose, the concentration of galactose in serum samples and urine samples were low and within the same range than the samples from the control group treated with the test article alone.
[0103] The data indicate that the strain administered to rats resulted in a potent reduction of galactose in serum and in urine. Thus, the strain appears to be effective at detoxifying galactose when delivered orally.
TABLE-US-00008 TABLE 8 Galactose concentrations (mM) in serum collected from animals at 0, 0.5, 1, 1.5, 2, 4, 8, and 24-hour post administration by oral gavage of 10% galactose, of the test article with a solution of 10% galactose, and of the test article alone. Group 2 Time Point Group 1 Test Article + Group 3 (hr) 10% Galactose 10% Galactose Test Article 0 0.52 0.52 0.56 0.5 9.69 0.02 0.54 1 10.49 0.15 0.00 1.5 4.26 0.31 0.42 2 2.19 0.51 0.49 4 0.52 0.06 0.56 8 0.38 0.50 0.52 24 0.39 0.35 0.41 Data points represent means of duplicate values obtained for 3 animals per group.
TABLE-US-00009 TABLE 9 Galactose concentrations (mM) in urine collected from animals at period intervals of [0-4 hours], [4-8 hours], and [8-24 hours] post administration by oral gavage of 10% galactose and of the test article alone and with 10% galactose. Group 1 Group 2 10% Test Article + Group 3 Time Intervals Galactose 10% Galactose Test Article 0 to 4 hr 112.64 6.89 0.039 4 to 8 hr 28.82 0.06 0.19 8 to 24 hr 1.86 0.02 0 Data points represent means of duplicate values obtained for 3 animals per group.
Example 7: Mitigation of Galactose Accumulation in Serum after Multiple Oral Administrations of the Evolved Clone
[0104] The objective of this study was to test the ability of the test article to detoxify galactose when galactose is provided multiple times. To that end, four groups of three Sprague-Dawley male rats were dosed with PBS (with 12.5% galactose in drinking water), test article (with and without 12.5% galactose in drinking water), and test article concurrently with 12.5% galactose via three daily oral gavage administration for 10 Days. Galactose concentration in serum was determined serially over five separate time points.
[0105] Animal care and procedures were approved by the Institutional Animal Care and Use Committee and were followed in accordance to the standards for animal husbandry and care of the U.S. Department of Agriculture's (USDA) Animal Welfare Act. Veterinary care was available throughout the course of the study.
[0106] A total of 12 healthy adult male Wistar rats weighing between 110 to 130 g procured from Envigo (Livermore, Calif.) and maintained in the controlled conditions of 18 to 23 C. with humidity of 30% to 70% with automatic lighting on a 12 h/12 h on/off cycle except as required for specimen collection and study conduct. The rats were fed with standard pellet diet (Teklad 18% protein, Envigo) and water ad libitum throughout the study. The rats were divided into four groups of three animals each. For Group I, the rats were administered the test article alone. For Group II, the rats had access ad libitum to a solution of 10% galactose prepared with tap water and were administered a solution of PBS. For Group III, the rats were administered the test article and had access ad libitum to a solution of 10% galactose prepared with tap water. For Group IV, the rats were administered the test article together with a solution of 10% galactose.
[0107] The dose volume to administer was calculated based on the most recent recorded body weight. Formulated test article for group 1, group 3 and group 4 animals had a cell count averaging 1E+09 CFU/mL. Each group of animals received their respective treatment via oral gavage administration three times daily (10 mL/kg body weight) at 0 hour, 3 hours post 1.sup.st daily dose, and 6 hours post 1.sup.st daily dose starting on Day 0 over a period of 10 days. Drinking water for animals in groups 2 & 3 was replaced with water supplemented with 12.5% Galactose on Day 1.
[0108] A volume of 0.10 mL whole blood was collected via sublingual vein or jugular vein at each time-point at the following time points: Pre-Dose, Day 2 (1 hour after 3.sup.rd dose), Day 4 (1 hour after 3.sup.rd dose), Day 6 (1 hour after 3.sup.rd dose), Day 8 (1 hour after 3.sup.rd dose), and Day 10 (1 hour after 3.sup.rd dose). Blood samples was allowed to clot for >30 minutes under ambient conditions, then was centrifuged at 22 C., 3000 RPM for 10 min. The collected serum was stored frozen at approximately 80 C. until analysis.
[0109] The data obtained in this study shown in
Example 8: Isolation and Identification of Yeast Strains for Fructose Metabolism
[0110] A primary goal of the present invention was to develop yeast strains highly effective at metabolizing fructose. Yeasts were isolated from fructose-containing food such as grapevine berries. Various species were isolated by cultivating the samples in fructose-containing culture medium at room temperature for a period of time of at least 2 days. Isolates were then identified by a molecular approach based on targeted sequencing of Internal Transcribed Spacers (ITS). The isolates were then subjected to adaptive evolution to generate clones with superior fructose-detoxification performance.
[0111] Strains were isolated mainly from grapes from two sources: Sauvignon and Pinot Noir. The berries were collected into sterile flasks and after cultivation for approximately 2 weeks at laboratory temperature with an agitation of 125 rpm in liquid minimal medium CM (Synthetic Complete Minimal Medium, 0.5% Ammonium Sulfate, Teknova) with 50 ug/mL chloramphenicol (Teknova), cultures were serially diluted and seeded again in liquid medium with selection and incubated at 30 C. with agitation of 125 rpm. For selection, 4% fructose (Acros) was added to the medium with or without 2% glucose (Teknova) and 2% raffinose (Teknova). This cycle was performed at least 3 times until plating serially diluted cultures on YP plates (1% yeast extract, 2% peptone; Teknova) supplemented with 4% fructose. Single colonies obtained after incubation at 30 C. for 2-5 days were sequentially streaked multiple times to obtain pure isolates.
[0112] For taxonomy assignment, the targeted metagenomic sequencing method was employed. Briefly, the isolates were processed for DNA extraction using the ZymoBIOMICS-96 MagBead DNA Kit and the DNA samples were prepared for targeted sequencing using targeted Internal Transcribed Spacers primer sets (ITS2, Zymo Research, Irvine, Calif.). The final library was sequenced on Illumina MiSeg. Taxonomy analyses were performed on publicly available sequences using the BLAST program under default settings (http://www.ncbi.nlm.nih.gov/BLAST).
[0113] The ITS sequences obtained from pool cultures and from isolate cultures were analyzed to identify isolate phylogeny. Table 10 reports the outcome of this analysis (Hits) based on the homology of ITS sequences amplified from pool cultures and purified isolate cultures with publicly available sequences. The table lists the species exhibiting the greatest score and 100% sequence identity with ITS sequences obtained from this study.
TABLE-US-00010 TABLE 10 Blast Results of Sequences Obtained after Amplification of Internal Transcribed Spacers (ITS) from Pool Cultures and from Isolate Cultures. Source Hits List of potential assignment for Pool_G1 Pichia kudriavzevii, Pichia cecembensis, Pichia sp. isolated from grapes subjected to selection Brettanoyces bruxellensis, Brettanomyces sp. medium containing 4% fructose Taxonomic assignment as per Blast search Pichia kudriavzevii for isolate G1_1 derived from Pool_G1 culture List of potential assignment for Pool_G2 Saccharomyces sp., Saccharomyces cerevisiae. isolated from grapes subjected to selection Saccharomyces uvarum, Saccharomyces medium containing 4% fructose eubayanus, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces jurei, Saccharomyces sp. boulardii, Saccharomyces kudriavzevii, Pichia sp. Taxonomic assignment as per Blast search Saccharomyces cerevisiae for isolate G2_1 derived from Pool_G2 culture List of potential assignment for Pool_G3 Hanseniaspora sp., Hanseniaspora uvarum, isolated from grapes subjected to selection Hanseniaspora opuntiae, Hanseniaspora medium containing 4% fructose thailandica, Limtongozyma cylindracea Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces uvarum Taxonomic assignment as per Blast search Saccharomyces uvarum for isolate G3_1 derived from Pool_G3 culture List of potential assignment for Pool_G4 Metschnikowia sp., Metschnikowia peoriensis, isolated from grapes subjected to selection Metschnikowia bicuspidata, Metschnikowia medium containing 4% fructose and 2% cibodasensis, Metschnikowia zobellii, raffinose Metschnikowia henanensis, Metschnikowia colchici, Metschnikowia noctiluminum, Metschnikowia rancensis, Metschnikowia maroccana, Metschnikowia vanudenii, Metschnikowia reukaufii, Metschnikowia koreensis, Metschnikowia cibodasensis, Metschnikowia peoriensis, Metschnikowia gelsemii, Metschnikowia bicuspidata, Metschnikowia henanensis, Metschnikowia zobellii, Metschnikowia gruessii, Metschnikowia chrysomelidarum, Metschnikowia colchici, Metschnikowia viticola, Metschnikowia kofuensis, Saturnispora zaruensis Taxonomic assignment as per Blast search Metschnikowia_reukaufii for isolate G4_1 derived from Pool_G4 culture
Example 9: Adaptive Evolution
[0114] Adaptive evolution was applied to yeast strains, a method that can increase traits of a given strain owing to random mutations in the genome. Clones derived from parental strains that offer a phenotypic advantage are naturally selected when grown under selective pressure. Yeast can be subjected to adaptive evolution with changes that can be observed in a short time period because yeast grows rapidly as single cells in simple media, with the entire life cycle completed in culture similarly to bacteria.
[0115] Adaptive evolution was carried out on the isolates by daily serial dilution conducted for at least 28 cycles of parental strains isolated from grapes (See akar et al., Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties. FEMS Yeast Res 12:171-82 (2011). Cultures of various isolates were initiated from a single colony on agar plates or from glycerol stocks, and grown in liquid YP medium by incubation at 30 C. with agitation at 125 rpm (Murakami & Kaeberlein Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells. J Visual Exp (27) (2009)). Parallel cultures were independently grown at 30 C. under agitation at 125 rpm in 15 mL CM medium in 50 mL vented tubes or 3 mL in 14 mL culture tubes supplemented with 2% fructose as a pressure selection agent for about 16 to 30 hours before transferring the cultures to fresh medium at a 1:30 dilution. The procedure of serial dilution was repeated until a change in growth was detected by OD.sub.600. A volume of 100 L of a 1:1,000 dilution of the last daily serial dilution of the culture with the greatest OD was plated on YP-fructose agar medium and incubated at 30 C. Isolated, adaptively evolved clones were randomly selected from the plate to start duplicate cultures grown at 30 C. under agitation at 125 rpm in 3 mL CM medium supplemented with 4% fructose alone or 4% fructose and 0.5 to 2% glucose. After multiple rounds of screening tests evaluating growth and fructose consumption in presence or absence of glucose, several leads were selected. A typical example of data profiles is shown in the table below.
[0116] To assess the yeast strains' and clones' ability to degrade fructose, the concentration of fructose was determined using the colorimetric Fructose Assay Kit (Cat. No. EFRU-100; BioAssay Systems, Hayward, Calif.). All reactions were performed in 96-well microplates and the absorbance was read at 565 nm using volumes and concentrations recommended by the kit. The samples and standards were mixed with the enzyme fructose dehydrogenase, MTT [3-(4,5-dimethylthiaze-syl)-2,5-diphenyltetrazolium bromide] and phenazine methosulfate (PMS), which reacts with fructose to produce a colored compound MTT formazan measurable by direct spectrophotometry. Sample concentrations were determined by comparison to a standard curve generated with known quantities of fructose.
[0117] Utilization and degradation of fructose was assessed by measuring fructose consumption by the strains. Table 11, below, lists the percentage of fructose reduction in spent culture medium for one adaptively evolved strain and the corresponding parent strain G4_1 when grown in presence of 4% fructose (=222 mM) and 1% glucose. This data set illustrates that the adaptively evolved clone was able to significantly degrade fructose. The evolved strain exhibited a reduction greater than 95% of fructose initial concentration compared to less than 5% reduction for the parental strain at the 3-hour time point for the same cell density. This difference is statistically significant, confirming that adaptive evolution produced a more efficient strain.
TABLE-US-00011 TABLE 11 Remaining Fructose Concentration (mM) after Exposure to a Solution of 4% Fructose and 1% Glucose with Parent Clone G4_1 and Evolved Clone G41A Clone Parent G4_1 G4_1A G4_1A Cell Density (CFU/mL) 1.00E + 09 1.00E + 08 1.00E + 09 Time point: 0.2 hour 193.70 mM (88.1%) 168.57 mM (76.7%) 134.51 mM (61.1%) Time point: 2.0 hour 214.26 mM (97.4%) 112.22 mM (51.0%) 29.97 mM (13.6%) Time point: 3.0 hour 212.88 mM (96.8%) 114.98 mM (52.3%) 5.98 mM (2.7%) Values between brackets indicate the percentage of fructose remaining when compared with the average value of the positive control (219.98 mM). CFU: Colony Forming Unit.
Example 10: Degradation of Fructose in Food
[0118] As part of evaluating the feasibility of a yeast-based approach as a treatment to mitigate the effects of elevated concentrations of fructose in foods and beverages, several evolved clones obtained by adaptive evolution were tested for their ability of degrading fructose when present in food.
[0119] For this study, two evolved yeast strains obtained by adaptive evolution, G1_1A and G2_1A were tested for their ability to degrade dietary fructose. The testing of fructose consumption was started with yeast cells obtained from a culture initiated from a single colony on agar plates and grown in 15 mL of liquid YP medium in a 50-mL mini-bioreactor by incubation at 30 C. with an agitation of 225 rpm supplemented with 4% fructose. Cells were pelleted by centrifugation at 1000 rpm (Sorval, RT7) for 10 min at room temperature. Cell pellets were resuspended in 5 mL rodent diet (Teklad, Envigo) spiked with a solution of 10% fructose (=555 mM). Reactions were incubated at 37 C. to mimic human gastrointestinal temperature conditions. Aliquots of the reactions were taken at multiple time points and stored at 20 C. until fructose concentration determination using the colorimetric Fructose Assay Kit (Cat. No. EFRU-100; BioAssay Systems, Hayward, Calif.).
[0120] As shown in Table 12, the evolved clones were able to rapidly decrease fructose concentration when present in diet.
TABLE-US-00012 TABLE 12 Remaining Fructose (%) after Exposure for 0.5 to 3 Hours to a Solution of 10% Fructose with Evolved Clones G1_1A and G2_1A Clone G1_1A G1_1A G1_1A G2_1A G2_1A G2_1A CFU/mL 3.10E+09 6.21E+09 2.17E+10 3.39E+09 6.78E+09 2.37E+10 Time point: 0.5 hr 111.1% 88.2% 45.9% 91.3% 76.8% 37.8% Time point: 2.0 hr 78.0% 53.2% 6.3% 77.2% 53.3% 5.6% Time point: 3.0 hr 60.8% 35.2% 1.5% 59.7% 34.7% 0.5%
Example 11: Tolerance to Gastrointestinal Conditions
[0121] The most convenient way to deliver a yeast-based therapeutic is orally. During transit through the gastrointestinal tract, an orally delivered agent will be confronted to a variety of simultaneous or sequential adverse conditions, such as the internal body temperature, gastric fluid with acidic pH, and pancreatic fluid with alkaline fluid. A series of in vitro experiments were conducted to assess the potential for survival of the evolved clones when subjected to gastrointestinal conditions.
[0122] Yeast cells were subjected to simulated gastric fluid (SGF) supplemented with 1 mg/mL of pepsin and simulated intestinal fluid (SIF) supplemented with 1 mg/mL of pancreatin to evaluate their tolerance to gastrointestinal conditions. (Zhou et al. Statistical investigation of simulated fed intestinal media composition on the equilibrium solubility of oral drugs. Eur J Pharm Sci 99:95-104 (2017). Overnight yeast cultures grown in liquid YP medium supplemented with 4% fructose were harvested by centrifugation for 10 min at 1000 rpm at room temperature. To test for survival in gastric fluid, pellets were re-suspended in 1 mL SGF (Cat No. 7108.16; RICCA Chemical Company, Arlington, Tex.) supplemented with pepsin from porcine gastric mucosa with an activity of 8.60 European Units/mg (Cat No. 41707-1000; ACROS Organics, Geel, Belgium). The pH was measured (pH1 to 2). Reactions were maintained at 37 C. and aliquots were taken at the indicated time points. To test for survival in intestinal fluid, cells were centrifuged for 10 min at 1000 rpm at room temperature and re-suspended in 1 mL SIF (RICCA Chemical Company, Cat No. 7109.16, pH 6.7-6.9 supplemented with 1 mg/mL of pancreatin from porcine pancreas (Sigma, Cat No. P3292). Reactions were maintained at 37 C. and aliquots were taken at the indicated time points. Yeast cells were counted before and after intestinal challenges with Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF). The survival rate was estimated by evaluating the ratio between cell counts of yeast preparations subjected to the simulated fluid challenge over time vs. the same preparations at time zero. Viable cell counts were determined using a hemocytometer and Trypan blue staining. Strain Saccharomyces boulardii (SB) was prepared similarly to the evolved clones except that it was grown in YP medium supplemented with 2% glucose.
[0123] As shown in Tables 13 and 14, the gastric environment had an impact on strain survival. For evolved clones G1_1A and clone G2_1A, SIF had minimal impact on cell counts and SGF reduced the cell count only after 180 min incubation.
TABLE-US-00013 TABLE 13 Survivability Time Course (%) of Four Evolved Clones in Gastrointestinal Conditions after Exposure to SGF over a Period of 90 min Clone Clone Clone Clone Clone G1_1A G2_1A G3_1A G4_1A SB 0 min 100.0 100.0 100.0 98.5 100.0 90 min 95.9 97.2 46.7 0 15.4
TABLE-US-00014 TABLE 14 Survivability Time Course (%) of Two Evolved Clones in Gastrointestinal Conditions after Exposure to Simulated Fluids over a Period of 180 min Clone G1_1A Clone G2_1B Conditions PBS SGF SIF PBS SGF SIF 0 min 98.9 98.2 99.5 99.5 98.4 99.5 60 min 99.5 98.1 99.5 99.5 95.5 99.5 120 min 100.0 91.0 98.5 100.0 91.0 100.0 180 min 98.9 66.7 99.4 98.9 65.2 99.5 SGF: Simulated Gastric Fluid; SIF: Simulated Intestinal Fluid.
Example 12: Mitigation of Fructose Accumulation in Serum after a Single Oral Administration of the Evolved Clone
[0124] The objective of this study was to assess the ability of the test article to detoxify fructose in vivo. To that end, serum fructose concentration was assessed after a single bolus oral administration to animals of 10% fructose and 20% fructose with or without concurrent administration of the test article.
[0125] Fifteen-week-old male Sprague-Dawley rats weighing between 350 and 400 g were acquired from Envigo with a catheter surgically implanted in the jugular vein. Animals were housed individually in polycarbonate cages containing animal bedding during the acclimation period lasting 7 days. Environmental controls were maintained at 18 to 23 C. with humidity of 30% to 70% with automatic lighting on a 12 h/12 h on/off cycle except as required for specimen collection and study conduct. Animals were fed an irradiated chow diet (Teklad, Envigo, Calif.) and were provided municipal tap water ad libitum. No concurrent medication was given. Animal care and procedures were approved by the Institutional Animal Care and Use Committee and were followed in accordance to the standards for animal husbandry and care of the U.S. Department of Agriculture's (USDA) Animal Welfare Act. Veterinary care was available throughout the course of the study and animals were examined by the veterinary staff as warranted.
[0126] The rats were randomly distributed into five groups, each consisting of three animals. Group I and Group II were administered with a solution of 10% (=555 mM) and 20% fructose, respectively; Group III was administered with the test article alone; Group IV and V were administered with a solution of 10% fructose and 20% fructose, respectively, together with the test article. All animals were fasted for a period of 16 hour prior to dosing and all animals were administered a single bolus dose of 10 mL/kg by oral gavage. The cell density of the test article (Evolved Clone G1_1A) was 4.0E+09 CFU/mL prepared from an overnight culture incubated at 30 C. in orbital shaker and grown in YP medium supplemented with 4% fructose. Blood samples (0.3 mL) were collected serially via catheterized jugular veins at 0 pre-dose and at 0.5, 1, 1.5, 2, 4, and 8-hour after dosing for analysis of fructose concentration in serum. All samples were stored frozen until analysis. Fructose levels in serum samples were determined using the EnzyChrom Fructose Assay Kit (BioAssay Systems, Hayward, Calif.) and analyzed in duplicate in a microplate reader (Molecular Devices).
[0127] As shown in
Example 13: Mitigation of Dietary Fructose Induced Symptoms after a Single Bolus Dose of Fructose
[0128] A group of adult subjects who presents one or more symptoms such as abdominal bloating, flatulence, pain, distension, diarrhea and nausea within 2 to 8 hours after drinking a beverage containing 25 g fructose is administered an oral dose of the preparation of the invention prior to the fructose provocation. During the following 8 hours, multiple tests are performed including breath test and blood fructose concentration evaluation. It is observed that the administration of the preparation of the invention decreases the production of hydrogen gas in the respiratory air and it levels off blood fructose level rapidly. These data confirm that preparation of the invention can efficiently detoxify fructose.
Example 14: Mitigation of Dietary Fructose-Induced Symptoms by Multiple Doses of Preparation of the Invention
[0129] An adult patient with dietary fructose intolerance presents with one or more of symptoms such as abdominal bloating, flatulence, pain, distension, diarrhea and nausea. Treatment with the preparation of the invention is initiated by the clinician at an effective dose, which mitigates fructose-induced symptoms. Assessment of symptoms and testing are periodically performed. The dose of the treatment is adjusted as required by the clinician in attendance to manage symptoms of the dietary fructose-related condition. The subject may be treated with other drugs concurrently and may or may not be under restricted diet. Treatment with the preparation of the present invention is able to mitigate one or more symptoms related to dietary fructose.