Media and fermentation methods for producing polysaccharides in bacterial cell culture

Abstract

The present invention relates to media and fermentation methods for producing polysaccharides in bacterial cell culture. In one aspect, the invention relates to a complex culture medium comprising a vegetable hydrolysate, a yeast extract, and a carbon source. In another aspect, the invention relates to a defined media having a total amino acid concentration greater than about 50 mM. A further aspect of the invention relates to the use of fed batch and perfusion fermentation methods for cultivating polysaccharide-producing bacteria.

1 . A polysaccharide-producing bacterial cell culture medium comprising a vegetable hydrolysate, a yeast extract, and a carbon source. 
     
     
         2 . The medium of  claim 1 , wherein the vegetable hydrolysate is a soy hydrolysate. 
     
     
         3 . The medium of  claim 2 , wherein the soy hydrolysate is selected from the group consisting of HYPEP 1510 (Kerry Group Services Ltd.), HYPEP 4601 (Kerry Group Services Ltd.), HYPEP 5603 (Kerry Group Services Ltd.), HY-SOY (Kerry Group Services Ltd.), AMI-SOY (Kerry Group Services Ltd.), N-Z-SOY (Kerry Group Services Ltd.), N-Z-SOY BL4 (Kerry Group Services Ltd.), N-Z-SOY BL7 (Kerry Group Services Ltd.), SHEFTONE D (Kerry Group Services Ltd.), SE50M, SE50MK, soy peptone, BACTO soytone (Difco Laboratories Inc.), NUTRISOY 2207 (ADM), NUTRISOY (ADM), NUTRISOY flour (ADM), and soybean meal. 
     
     
         4 . The medium of  claim 3 , wherein the soy hydrolysate is HYPEP 1510 (Kerry Group Services Ltd.). 
     
     
         5 . The medium of any one of  claims 1 - 4 , wherein the concentration of the vegetable hydrolysate is between about 5 g/L and about 75 g/L. 
     
     
         6 . The medium of  claim 5 , wherein the concentration of the vegetable hydrolysate is between about 10 g/L and about 50 g/L. 
     
     
         7 . The medium of  claim 6 , wherein the concentration of the vegetable hydrolysate is about 28 g/L. 
     
     
         8 . The medium of any one of  claims 1 - 7 , wherein the yeast extract is a yeast autolysate, an ultrafiltered yeast extract, or a synthetic yeast extract. 
     
     
         9 . The medium of  claim 8 , wherein the yeast extract is an ultrafiltered yeast extract. 
     
     
         10 . The medium of  claim 9 , wherein the ultrafiltered yeast extract is AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE (Kerry Group Services Ltd.), HY-YEST 412 (Kerry Group Services Ltd.), HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry Group Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), or HY-YEST 504 (Kerry Group Services Ltd.). 
     
     
         11 . The medium of any one of  claims 1 - 10 , wherein the concentration of yeast extract is between about 1 g/L to about 50 g/L. 
     
     
         12 . The medium of  claim 11 , wherein the concentration of yeast extract is between about 5 g/L to about 25 g/L. 
     
     
         13 . The medium of  claim 12 , wherein the concentration of yeast extract is about 10 g/L. 
     
     
         14 . The medium of any one of  claims 1 - 13 , wherein the carbon source is selected from the group consisting of glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and mannose. 
     
     
         15 . The medium of  claim 14 , wherein the carbon source is glucose. 
     
     
         16 . The medium of any one of  claims 1 - 15 , wherein the concentration of the carbon source is between about 25 g/L to about 100 g/L. 
     
     
         17 . The medium of  claim 16 , wherein the concentration of the carbon source is between about 50 g/L to about 90 g/L. 
     
     
         18 . The medium of  claim 17 , wherein the concentration of the carbon source is about 80 g/L. 
     
     
         19 . The medium of any one of  claims 1 - 18 , wherein the medium comprises soy hydrolysate, an ultrafiltered yeast extract, and glucose. 
     
     
         20 . The medium of any one of  claims 1 - 19 , wherein the medium further comprises a phosphate-containing ingredient. 
     
     
         21 . The medium of  claim 20 , wherein the phosphate-containing ingredient is Na 2 HPO 4 , K 2 HPO 4 , or KH 2 PO 4 . 
     
     
         22 . The medium of any one of  claims 1 - 21 , wherein the medium further comprises at least one amino acid, vitamin, nucleoside, or inorganic salt. 
     
     
         23 . A polysaccharide-producing bacterial cell culture medium having a total amino acid concentration greater than about 50 mM. 
     
     
         24 . The medium of  claim 23 , wherein the medium comprises a total glycine concentration of between about 1.5 mM and about 60.0 mM. 
     
     
         25 . The medium of  claim 24 , wherein the total glycine concentration is between about 5.0 mM and about 15.0 mM. 
     
     
         26 . The medium of  claim 25 , wherein the total glycine concentration is about 7.5 mM. 
     
     
         27 . The medium of any one of  claims 23 - 26 , wherein the medium comprises a total arginine concentration of between about 1.0 mM and about 30.0 mM. 
     
     
         28 . The medium of  claim 27 , wherein the total arginine concentration is between about 1.0 mM and about 20.0 mM. 
     
     
         29 . The medium of  claim 28 , wherein the total arginine concentration is about 4.0 mM. 
     
     
         30 . The medium of any one of  claims 23 - 29 , wherein the medium comprises a total cysteine concentration of between about 0.1 mM and about 5.0 mM. 
     
     
         31 . The medium of  claim 30 , wherein the total cysteine concentration is between about 0.1 mM and about 3.5 mM. 
     
     
         32 . The medium of  claim 31 , wherein the total cysteine concentration is about 0.4 mM. 
     
     
         33 . The medium of any one of  claims 23 - 32 , wherein the medium comprises a total serine concentration of between about 5.0 mM and about 75.0 mM. 
     
     
         34 . The medium of  claim 33 , wherein the total serine concentration is between about 5.0 mM and about 15.0 mM. 
     
     
         35 . The medium of  claim 34 , wherein the total serine concentration is about 7.5 mM, or about 10 mM. 
     
     
         36 . The medium of any one of  claims 23 - 35 , wherein the medium comprises a total glutamine concentration of between about 1.0 mM and about 30.0 mM. 
     
     
         37 . The medium of  claim 36 , wherein the total glutamine concentration is between about 1.0 mM and about 20.0 mM. 
     
     
         38 . The medium of  claim 37 , wherein the total glutamine concentration is about 4.0 mM. 
     
     
         39 . The medium of any one of  claims 23 - 38 , wherein the medium comprises a total concentration of tyrosine of between about 0.1 mM and about 5.0 mM. 
     
     
         40 . The medium of  claim 39 , wherein the total tyrosine concentration is between about 1.0 mM and about 3.5 mM. 
     
     
         41 . The medium of  claim 40 , wherein the total tyrosine concentration is about 2.9 mM or about 3.0 mM. 
     
     
         42 . The medium of any one of  claims 23 - 41 , wherein the medium comprises a total concentration of asparagine of between about 5.0 mM and about 50.0 mM. 
     
     
         43 . The medium of  claim 42 , wherein the total asparagine concentration is between about 10.0 mM and about 30.0 mM. 
     
     
         44 . The medium of  claim 43 , wherein the total asparagine concentration is about 20.0 mM. 
     
     
         45 . The medium of any one of  claims 23 - 41 , wherein the medium does not contain asparagine. 
     
     
         46 . The medium of any one of  claims 23 - 45 , wherein the medium further comprises a potassium salt. 
     
     
         47 . The medium of  claim 46 , wherein the potassium salt is potassium chloride or potassium sulfate. 
     
     
         48 . The medium of  claim 46  or  claim 47 , wherein the total concentration of potassium salt is between about 0.1 g/L and about 25 g/L. 
     
     
         49 . The medium of  claim 48 , wherein the total potassium salt concentration is between about 0.2 g/L and about 1.25 g/L. 
     
     
         50 . The medium of  claim 49 , wherein the total potassium salt concentration is about 0.9 g/L. 
     
     
         51 . The medium of any one of  claims 23 - 50 , wherein the medium further comprises a carbon source. 
     
     
         52 . The medium of  claim 51 , wherein the carbon sources is selected from the group consisting of glucose, dextrose, mannitol, lactose, sucrose, fructose, galactose, raffinose, xylose, and mannose. 
     
     
         53 . The medium of  claim 52 , wherein the carbon sources is glucose. 
     
     
         54 . The medium of any one of  claims 51 - 53 , wherein medium comprises a total concentration of the carbon source of between about 25 g/L and about 100 g/L. 
     
     
         55 . The medium of  claim 54 , wherein the total concentration of the carbon source is between about 25 g/L and about 80 g/L. 
     
     
         56 . The medium of  claim 55 , wherein the total concentration of the carbon source is about 50 g/L. 
     
     
         57 . The medium of any one of  claims 23 - 56 , wherein the medium further comprises sodium bicarbonate. 
     
     
         58 . The medium of  claim 57 , wherein the medium comprises a concentration of sodium bicarbonate of between about 0.1 g/L and about 20 g/L. 
     
     
         59 . The medium of  claim 58 , wherein the concentration of sodium bicarbonate is between about 0.5 g/L and about 1.0 g/L. 
     
     
         60 . The medium of  claim 59 , wherein the concentration of sodium bicarbonate is about 0.84 g/L. 
     
     
         61 . The medium of any one of  claims 23 - 60 , wherein the medium further comprises a yeast extract. 
     
     
         62 . The medium of  claim 61 , wherein the yeast extract is selected from the group consisting of a yeast autolysate, an ultrafiltered yeast extract, and a synthetic yeast extract. 
     
     
         63 . The medium of  claim 62 , wherein the yeast extract is an ultrafiltered yeast extract. 
     
     
         64 . The medium of  claim 63 , wherein the ultrafiltered yeast extract is AMBERFERM 5902 (Sensient Technologies Corp.), BD DIFCO (BD Biosciences), HYPEP YE (Kerry Group Services Ltd.), ULTRAPEP YE (Kerry Group Services Ltd.), HY-YEST 412 (Kerry Group Services Ltd.), HY-YEST 441 (Kerry Group Services Ltd.), HY-YEST 444 (Kerry Group Services Ltd.), HY-YEST 455 (Kerry Group Services Ltd.), or HY-YEST 504 (Kerry Group Services Ltd.). 
     
     
         65 . The medium of any one of  claims 61 - 64 , wherein the concentration of yeast extract is between about 1 g/L to about 50 g/L. 
     
     
         66 . The medium of  claim 65 , wherein the concentration of yeast extract is between about 5 g/L to about 25 g/L. 
     
     
         67 . The medium of  claim 66 , wherein the concentration of yeast extract is about 10 g/L. 
     
     
         68 . The medium of any one of  claims 23 - 67 , wherein the medium comprises at least about 50 mM of amino acids, a potassium salt, a carbon source, and optionally, a yeast extract. 
     
     
         69 . The medium of  claim 68 , wherein the medium comprises at least about 50 mM of amino acids, between about 5.0 mM and about 15.0 mM of glycine, between about 0.2 g/L and about 1.25 g/L of a potassium salt, between about 25 g/L and about 80 g/L of a carbon source, and between about 5 g/L to about 25 g/L of a yeast extract. 
     
     
         70 . The medium of  claim 69 , wherein the medium comprises at least about 60 mM of amino acids, about 7.5 mM of glycine, about 0.9 g/L of potassium chloride, 50 g/L of glucose, and about 10 g/L of an ultrafiltered yeast extract. 
     
     
         71 . A method of cultivating a polysaccharide-producing bacteria comprising a) adding a medium of any one of  claims 1 - 70  to a bioreactor, b) seeding the medium with a polysaccharide-producing bacteria, and c) cultivating the bacteria by fermentation, wherein said cultivation comprises the addition of a nutrient at a constant rate to the medium. 
     
     
         72 . The cultivation method of  claim 71 , wherein the nutrient is a carbon source. 
     
     
         73 . The cultivation method of  claim 72 , wherein the carbon source is glucose. 
     
     
         74 . The cultivation method of any one of  claims 71 - 73 , wherein the cultivated bacteria have a cell density of at least 9.0. 
     
     
         75 . The cultivation method of any one of  claims 71 - 74 , wherein the cultivated bacteria have a polysaccharide concentration of at least about 250 mg/L. 
     
     
         76 . The cultivation method of any one of  claims 71 - 75 , wherein the polysaccharide-producing bacteria is selected from the group consisting of  Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium , and  Enterococcus faecalis.    
     
     
         77 . A method of cultivating a polysaccharide-producing bacteria comprising a) adding a medium of any one of  claims 1 - 70  to a bioreactor, b) seeding the medium with a polysaccharide-producing bacteria, and c) cultivating the bacteria by perfusion, wherein the cultivation comprises (i) removing spent medium from the culture, (ii) adding fresh medium, and (iii) retaining the bacteria. 
     
     
         78 . The cultivation method of  claim 77 , wherein the rate of perfusion is between about 0.07 VVH to about 2.00 VVH. 
     
     
         79 . The cultivation method of  claim 78 , wherein the rate of perfusion is between about 0.67 VVH to about 1.33 VVH. 
     
     
         80 . The cultivation method of  claim 79 , wherein the rate of perfusion is about 1.20 VVH. 
     
     
         81 . The cultivation method of  claim 77 , wherein the rate of perfusion is varied. 
     
     
         82 . The cultivation method of  claim 81 , wherein the perfusion starts at a first rate and the rate is increased to a second rate. 
     
     
         83 . The cultivation method of  claim 81 , wherein the perfusion starts at a first rate and the rate is decreased to a second rate. 
     
     
         84 . The cultivation method of any one of  claims 77 - 83 , wherein the duration of perfusion is between about 1 hour and about 15 hours. 
     
     
         85 . The cultivation method of  claim 84 , wherein the duration of perfusion is between about 1 hour and about 10 hours. 
     
     
         86 . The cultivation method of  claim 85 , wherein the duration of perfusion is about 7 hours. 
     
     
         87 . The cultivation method of any one of  claims 77 - 86 , wherein the cell growth of the cultivated bacteria is at least 2-fold greater than the cell growth in a batch fermentation system. 
     
     
         88 . The cultivation method of any one of  claims 77 - 87 , wherein the cultivated bacteria have reached a cell density of at least 20.0. 
     
     
         89 . The cultivation method of any one of  claims 77 - 88 , wherein the cultivated bacteria have reached a polysaccharide concentration of at least about 600 mg/L. 
     
     
         90 . The cultivation method of any one of  claims 77 - 89 , wherein wherein the polysaccharide-producing bacteria is selected from the group consisting of  Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium , and  Enterococcus faecalis.