Glucoamylase-modified yeast strains and methods for bioproduct production

Abstract

Genetically engineered yeast with a heterologous glucoamylase and fermentation methods are described. The engineered yeast can have multiple exogenous nucleic acid sequences which each have a different sequence, but that encode the same or a similar glucoamylase protein that is heterologous to the yeast. The engineered yeast exhibit desirable bioproduct production profiles during a fermentation process. A fermentation medium with a starch material can be fermented with the engineered yeast to provide high ethanol titers, low glycerol titers, or both.

1 - 52 . (canceled) 
     
     
         53 . An engineered host cell comprising at least first, second, third, and fourth exogenous nucleic acids each comprising a sequence encoding a common heterologous glucoamylase polypeptide, or encoding two or more different heterologous glucoamylase polypeptides having 90% or greater sequence identity to each other, wherein the first, second, third, and fourth exogenous nucleic acids have nucleic acid sequences that are different from one another. 
     
     
         54 . The host cell of  claim 53 , wherein the heterologous glucoamylase polypeptide(s) is from a yeast or fungal organism selected from the group consisting of  Amorphotheca resinae, Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Aspergillus kawachii, Aspergillus shirousami, Blastobotrys adeninivorans, Candida albicans, Rhizopus oryzae, Schizosaccharomyces pombe , and  Saccharomycopsis fibuligera.    
     
     
         55 . The host cell of  claim 53 , wherein the heterologous glucoamylase polypeptide(s) is an enzymatically active portion of a glucoamylase polypeptide from  Saccharomycopsis fibuligera.    
     
     
         56 . The host cell of  claim 53 , wherein the glucoamylase polypeptide(s) has 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NO: 11. 
     
     
         57 . The host cell of  claim 53 , wherein the first, second, third, and fourth exogenous nucleic acids encode two or more different glucoamylase polypeptides having 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to each other. 
     
     
         58 . The host cell of  claim 53 , wherein the host cell is a yeast is selected from the group consisting of  Saccharomyces cerevisiae, Isaatchenkia orientalis, Candida utilis, Pichia Stipitis, Yarrowia lipolytica, Kluyveromyces marxiannus , and  Kluyeromyces lactis.    
     
     
         59 . The host cell of  claim 53 , wherein the host cell can be used to make a bioproduct selected from the group consisting of alcohols, diols, polyols, fatty acids, monacyl glycerides, diacyl glycerides, triacyl glycerides, sweeteners, sophorolipids, and mixtures thereof. 
     
     
         60 . The host cell of  claim 53 , wherein the host cell can be used to make ethanol. 
     
     
         61 . The host cell of  claim 53 , wherein the host cell can be used to make an organic acid or an amino acid. 
     
     
         62 . The host cell of  claim 53 , wherein the host cell can be used to make lactic acid. 
     
     
         63 . An engineered host cell comprising two or more exogenous nucleic acids each comprising a sequence encoding a common heterologous glucoamylase polypeptide, or encoding two or more different glucoamylase polypeptides having 90% or greater sequence identity to each other, wherein the two or more exogenous nucleic acids have nucleic acid sequences that are different from one another. 
     
     
         64 . The host cell of  claim 63 , wherein the heterologous glucoamylase polypeptide(s) is from a yeast or fungal organism selected from the group consisting of  Amorphotheca resinae, Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Aspergillus kawachii, Aspergillus shirousami, Blastobotrys adeninivorans, Candida albicans, Rhizopus oryzae, Schizosaccharomyces pombe , and  Saccharomycopsis fibuligera.    
     
     
         65 . The host cell of  claim 63 , wherein the heterologous glucoamylase polypeptide(s) is an enzymatically active portion of a glucoamylase polypeptide from  Saccharomycopsis fibuligera.    
     
     
         66 . The host cell of  claim 63 , wherein the glucoamylase polypeptide(s) has 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NO: 11. 
     
     
         67 . The host cell of  claim 63 , wherein the first, second, third, and fourth exogenous nucleic acids encode two or more different glucoamylase polypeptides having 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to each other. 
     
     
         68 . The host cell of  claim 63 , wherein the host cell is a yeast is selected from the group consisting of  Saccharomyces cerevisiae, Isaatchenkia orientalis, Candida utilis, Pichia Stipitis, Yarrowia lipolytica, Kluyveromyces marxiannus , and  Kluyeromyces lactis.    
     
     
         69 . The host cell of  claim 63 , wherein the host cell can be used to make a bioproduct selected from the group consisting of alcohols, diols, polyols, fatty acids, monacyl glycerides, diacyl glycerides, triacyl glycerides, sweeteners, sophorolipids, and mixtures thereof. 
     
     
         70 . The host cell of  claim 63 , wherein the host cell can be used to make ethanol. 
     
     
         71 . The host cell of  claim 63 , wherein the host cell can be used to make an organic acid or an amino acid. 
     
     
         72 . The host cell of  claim 63 , wherein the host cell can be used to make lactic acid.