Engineered probiotics for expression of fiber-synthesizing enzymes in gut
The present disclosure provides solutions to various challenges in health, including health challenges related to high consumption of carbohydrate and/or low consumption of soluble fiber. The present disclosure provides, among other things, bacteria engineered for expression of a fiber-synthesizing enzyme, e.g., for expression of the enzyme in the gut (e.g., in the intestine). In particular embodiments, the present disclosure provides bacteria engineered for expression of a fiber-synthesizing enzyme that consumes carbohydrate in the process of synthesizing fiber, e.g., in the gut. The present disclosure further includes formulations of isolated fiber-synthesizing enzymes for administration to subjects.
1 . A method of decreasing the amount or concentration of a carbohydrate in the gut of a subject comprising delivering to the gut of to the subject a bacterium engineered to express an integrated heterologous nucleic acid sequence encoding a fiber-synthesizing enzyme, wherein the fiber-synthesizing enzyme synthesizes fiber from a carbohydrate substrate, where in the enzyme is not a trehalose-6-phosphate synthase and the fiber is not trehalose.
2 . A method of increasing the amount or concentration of a fiber in the gut of a subject comprising delivering to the gut of to the subject a bacterium engineered to express an integrated heterologous nucleic acid sequence encoding a fiber-synthesizing enzyme, wherein the fiber-synthesizing enzyme synthesizes fiber from a carbohydrate substrate, where in the enzyme is not a trehalose-6-phosphate synthase and the fiber is not trehalose.
3 . A method of treating a subject in need of decreased amount or concentration of a carbohydrate in the gut or increased amount or concentration of a fiber in the gut comprising delivering to the gut of to the subject a bacterium engineered to express an integrated heterologous nucleic acid sequence encoding a fiber-synthesizing enzyme, wherein the fiber-synthesizing enzyme synthesizes fiber from a carbohydrate substrate, where in the enzyme is not a trehalose-6-phosphate synthase and the fiber is not trehalose.
4 . The method of claim 3 , wherein the subject is suffering from a condition positively correlated or associated with consumption of carbohydrate.
5 . The method of claim 3 , wherein the subject is suffering from a condition negatively correlated or associated with consumption of fiber.
6 . The method of any one of claims 3-5 , wherein the subject is suffering from a condition selected from cardiovascular disease, heart disease, high blood pressure, high blood cholesterol, high blood glucose, diabetes, obesity, dysbiosis of the gut, inflammatory bowel disease, irritable bowel syndrome (IBS), diverticulitis, colorectal cancer, intestinal cancer, bloating, cramping, gas, hemorrhoids, and diarrhea.
7 . The method of claim 1 , wherein the fiber is a soluble fiber.
8 . The method of claim 1 , wherein the synthesized fiber comprises glucose, fructose, galacturonic acid, N-acetyl-D-glucosamine, and/or galactose monomers.
9 . The method of claim 1 , wherein the carbohydrate substrate is selected from one or more of UDP-glucose, UDP-galactose, UDP-fucose, ADP-α-D-glucose, UDP-galacturonic acid, UDP-N-acetyl-alpha-D-glucosamine, galactinol, lactose, glucose, and sucrose and/or wherein the carbohydrate decreased in amount or concentration in the gut is selected from one or more of glucose, galactose, sucrose, fructose, and/or lactose.
10 . The method of claim 1 , wherein the synthesized fiber is a fructooligosaccharide and/or is selected from laminaribiose, callose, curdlan, oat beta-glucan, laminarin, pleuran, lentinan, yeast beta glucan, trehalulose, inulin, kestose, nystose, levan, raffinose, stachyose, verbascose, globotriose, human milk oligosaccharides (HMOs), cellobiose, cellulose, microcellulose, cotton, maltose, amylose, starch, glycogen, amylopectin, pectin, chitin.
11 . The method of claim 1 , wherein the fiber-synthesizing enzyme is selected from a sucrase, an inulosucrase, a levansucrase, a 1,3-beta-glucan synthase, a 1,3;1,4-beta-D-glucan synthase, a 1,6-beta-glucan synthase, a sucrose isomerase, a 1,6-alpha-galactosyltransferase, a trehalulose synthase, an α-1,4-galactosyltransferase, an alpha-1,2-fucosyltransferase, a beta-galactosidase, a b-D-Galactosidase, a cellulose synthase, a maltose synthase, a starch synthase, a starch-branching enzyme, a glycogen synthase, a galacturonosyltransferase, and a chitinoligosaccharide synthase.
12 . The method of claim 1 , wherein the fiber-synthesizing enzyme is operatively linked with a secretion polypeptide.
13 . The method of claim 1 , wherein the bacterium is a spore-forming bacterium and/or is in a spore form.
14 . The method of claim 1 , wherein the bacterium is a probiotic bacterium.
15 . The method of claim 1 , wherein the bacterium is of a genus selected from Bacillus, Bifidobacterium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus , and Streptococcus , optionally wherein the bacterium is of the genus Bacillus , optionally wherein the bacterium is of the species B. subtilis.
16 . The method of claim 1 , wherein the bacterium is of a strain characterized in that it does not colonize the gut.
17 . The method of claim 1 , wherein the method comprises administering the engineered bacterium to a subject that has consumed carbohydrate and/or the carbohydrate substrate within a preceding period, wherein the preceding period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
18 . The method of claim 1 , wherein the method comprises administering the engineered bacterium to a subject that has not consumed carbohydrate and/or the carbohydrate substrate.
19 . The method of claim 1 , wherein the method comprises administering the engineered bacterium to a subject that has not consumed carbohydrate and/or the carbohydrate substrate within a preceding period, wherein the preceding period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
20 . The method of claim 1 , wherein the subject consumes carbohydrate and/or the carbohydrate substrate during a period subsequent to administration of the engineered bacterium, wherein the subsequent period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
21 . The method of claim 1 , wherein the method prevents accumulation of sugar in the colon of the subject.
22 . The method of claim 1 , wherein fiber-synthesizing enzymes and/or enzymes encoded by heterologous nucleic acid sequences comprised by the engineered bacterium consist of the fiber-synthesizing enzyme.
23 . The method of claim 1 , wherein the administration comprises oral administration of a composition comprising the engineered bacterium.
24 . The method of claim 1 , wherein the administration comprises administration of about 104 to about 1012 colony forming units of the engineered bacterium.
25 . The method of claim 1 , wherein the nucleic acid sequence encoding the expression product is operatively linked with a constitutive promoter.
26 . The method of claim 1 , wherein the nucleic acid sequence encoding the expression product is operatively linked with a flagellin gene promoter.
27 . The method of claim 26 , wherein the flagellin gene promoter comprises a mutation in a CsrA binding site, wherein the mutation in the CsrA binding site inhibits binding of CsrA to mRNA transcripts encoding the fiber-synthesizing enzyme but does not preclude expression of the fiber-synthesizing enzyme.
28 . The method of claim 26 , wherein the engineered bacterium comprises a mutation of an endogenous flgM gene that reduces inhibition of a sigma factor by FlgM.
29 . The method of claim 27 , wherein the engineered bacterium comprises a mutation of an endogenous flgM gene that reduces inhibition of a sigma factor by FlgM.
30 . The method of claim 2 , wherein the fiber is a soluble fiber.
31 . The method of claim 2 , wherein the synthesized fiber comprises glucose, fructose, galacturonic acid, N-acetyl-D-glucosamine, and/or galactose monomers.
32 . The method of claim 2 , wherein the carbohydrate substrate is selected from one or more of UDP-glucose, UDP-galactose, UDP-fucose, ADP-α-D-glucose, UDP-galacturonic acid, UDP-N-acetyl-alpha-D-glucosamine, galactinol, lactose, glucose, and sucrose and/or wherein the carbohydrate decreased in amount or concentration in the gut is selected from one or more of glucose, galactose, sucrose, fructose, and/or lactose.
33 . The method of claim 2 , wherein the synthesized fiber is a fructooligosaccharide and/or is selected from laminaribiose, callose, curdlan, oat beta-glucan, laminarin, pleuran, lentinan, yeast beta glucan, trehalulose, inulin, kestose, nystose, levan, raffinose, stachyose, verbascose, globotriose, human milk oligosaccharides (HMOs), cellobiose, cellulose, microcellulose, cotton, maltose, amylose, starch, glycogen, amylopectin, pectin, chitin.
34 . The method of claim 2 , wherein the fiber-synthesizing enzyme is selected from a sucrase, an inulosucrase, a levansucrase, a 1,3-beta-glucan synthase, a 1,3;1,4-beta-D-glucan synthase, a 1,6-beta-glucan synthase, a sucrose isomerase, a 1,6-alpha-galactosyltransferase, a trehalulose synthase, an α-1,4-galactosyltransferase, an alpha-1,2-fucosyltransferase, a beta-galactosidase, a b-D-Galactosidase, a cellulose synthase, a maltose synthase, a starch synthase, a starch-branching enzyme, a glycogen synthase, a galacturonosyltransferase, and a chitinoligosaccharide synthase.
35 . The method of claim 2 , wherein the fiber-synthesizing enzyme is operatively linked with a secretion polypeptide.
36 . The method of claim 2 , wherein the bacterium is a spore-forming bacterium and/or is in a spore form.
37 . The method of claim 2 , wherein the bacterium is a probiotic bacterium.
38 . The method of claim 2 , wherein the bacterium is of a genus selected from Bacillus, Bifidobacterium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus , and Streptococcus , optionally wherein the bacterium is of the genus Bacillus , optionally wherein the bacterium is of the species B. subtilis.
39 . The method of claim 2 , wherein the bacterium is of a strain characterized in that it does not colonize the gut.
40 . The method of claim 2 , wherein the method comprises administering the engineered bacterium to a subject that has consumed carbohydrate and/or the carbohydrate substrate within a preceding period, wherein the preceding period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
41 . The method of claim 2 , wherein the method comprises administering the engineered bacterium to a subject that has not consumed carbohydrate and/or the carbohydrate substrate.
42 . The method of claim 2 , wherein the method comprises administering the engineered bacterium to a subject that has not consumed carbohydrate and/or the carbohydrate substrate within a preceding period, wherein the preceding period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
43 . The method of claim 2 , wherein the subject consumes carbohydrate and/or the carbohydrate substrate during a period subsequent to administration of the engineered bacterium, wherein the subsequent period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
44 . The method of claim 2 , wherein the method prevents accumulation of sugar in the colon of the subject.
45 . The method of claim 2 , wherein fiber-synthesizing enzymes and/or enzymes encoded by heterologous nucleic acid sequences comprised by the engineered bacterium consist of the fiber-synthesizing enzyme.
46 . The method of claim 2 , wherein the administration comprises oral administration of a composition comprising the engineered bacterium.
47 . The method of claim 2 , wherein the administration comprises administration of about 104 to about 1012 colony forming units of the engineered bacterium.
48 . The method of claim 2 , wherein the nucleic acid sequence encoding the expression product is operatively linked with a constitutive promoter.
49 . The method of claim 2 , wherein the nucleic acid sequence encoding the expression product is operatively linked with a flagellin gene promoter.
50 . The method of claim 49 , wherein the flagellin gene promoter comprises a mutation in a CsrA binding site, wherein the mutation in the CsrA binding site inhibits binding of CsrA to mRNA transcripts encoding the fiber-synthesizing enzyme but does not preclude expression of the fiber-synthesizing enzyme.
51 . The method of claim 49 , wherein the engineered bacterium comprises a mutation of an endogenous flgM gene that reduces inhibition of a sigma factor by FlgM.
52 . The method of claim 50 , wherein the engineered bacterium comprises a mutation of an endogenous flgM gene that reduces inhibition of a sigma factor by FlgM.
53 . The method of claim 3 , wherein the fiber is a soluble fiber.
54 . The method of claim 3 , wherein the synthesized fiber comprises glucose, fructose, galacturonic acid, N-acetyl-D-glucosamine, and/or galactose monomers.
55 . The method of claim 3 , wherein the carbohydrate substrate is selected from one or more of UDP-glucose, UDP-galactose, UDP-fucose, ADP-α-D-glucose, UDP-galacturonic acid, UDP-N-acetyl-alpha-D-glucosamine, galactinol, lactose, glucose, and sucrose and/or wherein the carbohydrate decreased in amount or concentration in the gut is selected from one or more of glucose, galactose, sucrose, fructose, and/or lactose.
56 . The method of claim 3 , wherein the synthesized fiber is a fructooligosaccharide and/or is selected from laminaribiose, callose, curdlan, oat beta-glucan, laminarin, pleuran, lentinan, yeast beta glucan, trehalulose, inulin, kestose, nystose, levan, raffinose, stachyose, verbascose, globotriose, human milk oligosaccharides (HMOs), cellobiose, cellulose, microcellulose, cotton, maltose, amylose, starch, glycogen, amylopectin, pectin, chitin.
57 . The method of claim 3 , wherein the fiber-synthesizing enzyme is selected from a sucrase, an inulosucrase, a levansucrase, a 1,3-beta-glucan synthase, a 1,3;1,4-beta-D-glucan synthase, a 1,6-beta-glucan synthase, a sucrose isomerase, a 1,6-alpha-galactosyltransferase, a trehalulose synthase, an α-1,4-galactosyltransferase, an alpha-1,2-fucosyltransferase, a beta-galactosidase, a b-D-Galactosidase, a cellulose synthase, a maltose synthase, a starch synthase, a starch-branching enzyme, a glycogen synthase, a galacturonosyltransferase, and a chitinoligosaccharide synthase.
58 . The method of claim 3 , wherein the fiber-synthesizing enzyme is operatively linked with a secretion polypeptide.
59 . The method of claim 3 , wherein the bacterium is a spore-forming bacterium and/or is in a spore form.
60 . The method of claim 3 , wherein the bacterium is a probiotic bacterium.
61 . The method of claim 3 , wherein the bacterium is of a genus selected from Bacillus, Bifidobacterium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus , and Streptococcus , optionally wherein the bacterium is of the genus Bacillus , optionally wherein the bacterium is of the species B. subtilis.
62 . The method of claim 3 , wherein the bacterium is of a strain characterized in that it does not colonize the gut.
63 . The method of claim 3 , wherein the method comprises administering the engineered bacterium to a subject that has consumed carbohydrate and/or the carbohydrate substrate within a preceding period, wherein the preceding period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
64 . The method of claim 3 , wherein the method comprises administering the engineered bacterium to a subject that has not consumed carbohydrate and/or the carbohydrate substrate.
65 . The method of claim 3 , wherein the method comprises administering the engineered bacterium to a subject that has not consumed carbohydrate and/or the carbohydrate substrate within a preceding period, wherein the preceding period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
66 . The method of claim 3 , wherein the subject consumes carbohydrate and/or the carbohydrate substrate during a period subsequent to administration of the engineered bacterium, wherein the subsequent period is a period of 24 hours 12 hours, 6 hours, 3 hours, or 1 hour.
67 . The method of claim 3 , wherein the method prevents accumulation of sugar in the colon of the subject.
68 . The method of claim 3 , wherein fiber-synthesizing enzymes and/or enzymes encoded by heterologous nucleic acid sequences comprised by the engineered bacterium consist of the fiber-synthesizing enzyme.
69 . The method of claim 3 , wherein the administration comprises oral administration of a composition comprising the engineered bacterium.
70 . The method of claim 3 , wherein the administration comprises administration of about 104 to about 1012 colony forming units of the engineered bacterium.
71 . The method of claim 3 , wherein the nucleic acid sequence encoding the expression product is operatively linked with a constitutive promoter.
72 . The method of claim 3 , wherein the nucleic acid sequence encoding the expression product is operatively linked with a flagellin gene promoter.
73 . The method of claim 72 , wherein the flagellin gene promoter comprises a mutation in a CsrA binding site, wherein the mutation in the CsrA binding site inhibits binding of CsrA to mRNA transcripts encoding the fiber-synthesizing enzyme but does not preclude expression of the fiber-synthesizing enzyme.
74 . The method of claim 72 , wherein the engineered bacterium comprises a mutation of an endogenous flgM gene that reduces inhibition of a sigma factor by FlgM.
75 . The method of claim 73 , wherein the engineered bacterium comprises a mutation of an endogenous flgM gene that reduces inhibition of a sigma factor by FlgM.