Fermentation methods for producing steviol glycoside using high ph and compositions obtained therefrom

Abstract

Disclosed are methods for producing steviol glycosides, such as rebaudioside D and rebaudioside M, using engineered yeast. In some embodiments, the methods include fermenting with a yeast at a high pH such as of about 5.8 or greater. In some embodiments, the methods can be carried out by first growing the yeast at a lower first pH, and then adjusting the pH to a higher pH.

1 . A method for increasing the production of a first, lower molecular weight steviol glycoside, relative to a second, higher molecular weight steviol glycoside, in an engineered yeast, comprising fermenting the engineered yeast capable of producing the first steviol glycoside and the second steviol glycoside at a pH of 5.8 or greater in a fermentation medium. 
     
     
         2 . The method of  claim 1 , wherein the pH is in the range of 5.8 to 8. 
     
     
         3 . The method of  claim 1 , wherein the pH is in the range of 6.0 to 7.5. 
     
     
         4 . The method of  claim 1 , wherein the pH is in the range of 6.5 to 7.5. 
     
     
         5 . The method of  claim 1 , wherein the pH is in the range of 5.8 to 6.5. 
     
     
         6 . The method of  claim 1 , wherein the pH is in the range of 5.8 to 6.2. 
     
     
         7 . The method of  claim 1 , wherein the fermentation medium comprises a nitrogen source selected from the group consisting of ammonium hydroxide, yeast extract, urea, and ammonium sulfate. 
     
     
         8 . The method of  claim 7 , wherein the nitrogen source comprises 90% (wt) or greater ammonium hydroxide or urea. 
     
     
         9 . The method of  claim 1 , further comprising continuous or batch addition of additional fermentation material to the medium comprising the engineered yeast. 
     
     
         10 . The method of  claim 1  performed for a period of time of up to 150 hours. 
     
     
         11 . The method of  claim 10  performed for a period of time in the range of 8 to 88 hours. 
     
     
         12 . The method of  claim 11  performed for a period of time in the range of 22 to 48 hours. 
     
     
         13 . The method of  claim 1 , wherein the fermentation medium has a biomass in the range of 20-120 g dcw/L. 
     
     
         14 . The method of  claim 1 , wherein the engineered yeast produces a ratio of the first steviol glycoside to the second steviol glycoside at the pH of 5.8 or greater that is increased by 10% or greater than a ratio of the first steviol glycoside to the second steviol glycoside when the engineered yeast are fermented at a pH of 5.0. 
     
     
         15 . The method of  claim 1 , wherein the first steviol glycoside is rebaudioside D, and the second steviol glycoside is rebaudioside M. 
     
     
         16 . The method of  claim 15 , wherein the ratio of rebaudioside D to rebaudioside M is in the range of 1:20 to 1:1. 
     
     
         17 . The method of  claim 1 , further comprising growing the engineered yeast in a first medium at a first pH, wherein the first pH is the same as the pH of the fermentation medium. 
     
     
         18 . The method of  claim 1 , further comprising growing the engineered yeast in a first medium at a first pH, wherein the first pH is lower than the pH of the fermentation medium. 
     
     
         19 . The method of  claim 18 , wherein the first pH is a pH less than 5.8. 
     
     
         20 . The method of  claim 18 , wherein the first pH is a pH less than 5.5. 
     
     
         21 . A method for increasing the production of a first, lower molecular weight steviol glycoside relative to a second, higher molecular weight steviol glycoside in an engineered yeast, the method comprising a step of fermenting engineered yeast capable of producing one or more steviol glycoside(s) at a pH of 5.8 or greater in a fermentation medium, wherein the engineered yeast produces a ratio of the first and second steviol glycosides at pH of 5.8 or greater that is greater than a ratio of the first and second steviol glycosides produced at a pH that is less than 5.8. 
     
     
         22 . The method of  claim 21  wherein the ratio of the first and second steviol glycosides at pH of 5.8 or greater is 10% or greater than the ratio of the first and second steviol glycosides produced at a pH that is less than 5.8. 
     
     
         23 . The method of  claim 22  wherein the ratio of the first and second steviol glycosides at pH of 5.8 or greater is 25% or greater than the ratio of the first and second steviol glycosides produced at a pH that is less than 5.8.