Enhanced production and purification of a natural high intensity sweetener

Abstract

Recombinant P. pastoris producing natural sweet proteins and methods for engineering these recombinant yeast are described. Methods for enhancing foreign protein production in yeast fermentation and improved methods for purification of foreign proteins produced in yeast fermentation are presented.

What is claimed: 
     
         1 . An expression vector for the production of a brazzein protein by a yeast cell comprising at least two cassettes, each cassette comprising a promoter sequence, a secretion signal sequence, a brazzein protein reading frame sequence, and a termination sequence, wherein the at least two cassettes are present in an expression vector comprising an integration sequence for integration of the cassettes into the yeast genome at a specific site. 
     
     
         2 . A yeast cell comprising the expression vector of  claim 1 . 
     
     
         3 . The yeast cell of  claim 2 , wherein the yeast cell is of the genus  Pichia.    
     
     
         4 . The expression vector of  claim 1 , wherein the brazzein protein reading frame sequence is a reading frame sequence of brazzein Type II or brazzein Type III. 
     
     
         5 . The expression vector of  claim 1 , wherein the promoter is a constitutive promoter. 
     
     
         6 . The expression vector of  claim 1 , wherein the promoter is the pGAP promoter. 
     
     
         7 . The expression vector of  claim 1 , wherein the promoter is selected from the group of promoters consisting of PPHis4, FLD1, PEX8, YPTI, TEF1, and PGK1. 
     
     
         8 . The expression vector of  claim 1 , wherein the promoter and terminator are from the same gene. 
     
     
         9 . The expression vector of  claim 1 , wherein the terminator is AOX1, pGAP, or FLD1. 
     
     
         10 . The expression vector of  claim 1 , wherein the secretion signal sequence is selected from the group consisting of the  S. cerevisiae  mating factor alpha signal sequence, a truncated form of the  S. cerevisiae  mating factor alpha signal sequence, the PHO-1 signal sequence, the PHA-E signal sequence, a matrix metalloproteinase signal sequence, and the beta-glucanase signal sequence. 
     
     
         11 . A recombinant  P. pastoris  cell comprising at least two copies of a gene construct comprising a pGAP promoter sequence, a secretion signal sequence, a brazzein Type II or Type III protein reading frame sequence, and a termination sequence, wherein the copies of the gene construct are present at a preselected integration site within the  P. pastoris  genome. 
     
     
         12 . A method for producing enhanced levels of a brazzein protein comprising the steps of a) transforming one or more cells of  P. pastoris  with the expression vector of  claim 1 ; and b) growing the transformed cell or cells in a fermentation medium under aerobic conditions, wherein the transformed cells produce enhanced levels of a brazzein protein compared with cells containing only one copy of the brazzein protein gene. 
     
     
         13 . The method of  claim 12 , wherein the transformed cell or cells are grown as a fed-batch fermentation culture. 
     
     
         14 . The method of  claim 12 , wherein the transformed cells produce a protein selected from the group consisting of Type I brazzein, Type II brazzein, and Type III brazzein. 
     
     
         15 . The method of  claim 12 , wherein the transformed cells produce a variant of a Type I, II, or III brazzein protein. 
     
     
         16 . A method for enhancing production of a foreign protein in recombinant  Pichia  comprising the steps of a) initiating a culture of the recombinant  Pichia,  under aerobic conditions, b) reducing oxygen levels in the culture to less than 5%, and c) maintaining oxygen levels in the culture at less than 5%. 
     
     
         17 . The method of  claim 16 , wherein the culture is a fed-batch fermentation culture. 
     
     
         18 . The method of  claim 16 , wherein the culture is a batch culture or a chemostat culture. 
     
     
         19 . The method of  claim 16 , wherein the oxygen levels in the culture are reduced and maintained by regulating the agitation speed of the culture medium. 
     
     
         20 . The method of  claim 16 , wherein the oxygen levels in the culture are reduced and maintained by a method selected from the group consisting of limiting the flow of air, altering the amount of oxygen in the gas composition, altering the backpressure of the fermentation reactor, and altering the feed rate. 
     
     
         21 . A method for purifying a protein from a yeast fermentation culture comprising the ordered steps of a) adjusting the pH of the fermentation medium above or below the isoelectric point of the protein to be purified; b) removing the cells from the pH adjusted fermentation medium; c) subjecting the cell-free fermentation medium to ion-exchange chromatography and eluting the protein as a solution; d) passing the eluted protein solution through a first membrane to remove unwanted high molecular weight solutes; and e) concentrating and diafiltering the protein permeate on a second membrane to remove low molecular weight solutes. 
     
     
         22 . The method of  claim 21 , wherein the pH of the eluted protein solution is optimized prior to ultrafiltration step d) to facilitate permeation of the protein through the first membrane. 
     
     
         23 . The method of  claim 22 , wherein the pH of the filtered protein permeate is optimized prior to concentration step e) to facilitate flux through the second membrane. 
     
     
         24 . The method of  claim 21 , wherein the protein to be purified is Type II brazzein; step a) comprises adjusting the pH of the fermentation medium to about 3.7-3.9; step b) comprises microfiltration; step c) comprises cation-exchange chromatography; the pH of the eluted protein solution is adjusted to about 6.0-6.2 prior to step d); and the Type II brazzein is recovered at greater than 95% purity. 
     
     
         25 . The method of  claim 21 , wherein the protein to be purified is Type III brazzein; step a) comprises adjusting the pH of the fermentation medium to about 3.7-3.9; step b) comprises microfiltration; step c) comprises cation-exchange chromatography; the pH of the eluted protein solution is adjusted to about 6.0-6.2 prior to step d); the pH of the protein permeate is adjusted to about 3.8-4.2 prior to step e); and the Type III brazzein is recovered at greater than 95% purity. 
     
     
         26 . The method of  claim 20 , wherein the pH adjusted fermentation medium is first pumped through a microfiltration membrane and the resulting cell-free fermentation medium is subsequently pumped to an ion-exchange column in a continuous process.