Search

EP-4737579-A2 - GLUCOAMYLASE ENGINEERED YEAST AND FERMENTATION METHODS

EP4737579A2EP 4737579 A2EP4737579 A2EP 4737579A2EP-4737579-A2

Abstract

The invention is directed to an engineered yeast including an exogenous nucleic acid encoding a glucoamylase comprising SEQ ID NO:1 and SEQ ID NO:4, or a variant thereof. The engineered yeast are able to provide glucoamylase into a fermentation media and cause degradation of starch material generating glucose for fermentation to a desired bioproduct, such as ethanol. High titers of bioproduct (e.g., 70 g/kg of ethanol) can be achieved, along with low residual glucose levels. Further the yeast exhibit good growth and bioproduct product at temperatures of 32°C or greater.

Inventors

  • MILLER, CHRIS
  • RUSH, BRIAN
  • STANTON, Brynne
  • DUNN, JOSHUA

Assignees

  • Cargill, Incorporated

Dates

Publication Date
20260506
Application Date
20190227

Claims (15)

  1. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 97% or greater sequence identity to SEQ ID NO:4, wherein the yeast is capable of producing ethanol at a rate of 1 g/L *h or greater during a fermentation process.
  2. The engineered yeast of claim 1, wherein the yeast is capable of producing at least 70g/kg of ethanol in a fermentation medium made from a glucose polymer-containing feedstock having a DE of not greater than 50.
  3. The engineered yeast of claim 1, wherein the yeast is capable of producing at least 70g/kg of ethanol in a fermentation medium made from a glucose polymer-containing feedstock having a DE of 30.
  4. The engineered yeast of any of claims 1-3, wherein the glucoamylase comprises a sequence having 98% or greater sequence identity to SEQ ID NO:4.
  5. The engineered yeast of claim 4, wherein the glucoamylase comprises a sequence having 99% or greater sequence identity to SEQ ID NO: 4.
  6. The engineered yeast of any of the previous claims wherein there are 2 - 8 copies of the exogenous nucleic acid in a cell of the engineered yeast.
  7. The engineered yeast of any of the previous claims wherein there are 2 - 6 copies of the exogenous nucleic acid in a cell of the engineered yeast.
  8. The engineered yeast of any of the previous claims which is a Saccharomyces cerevisiae yeast.
  9. The engineered yeast of any of the previous claims which is tolerant to growth in a fermentation medium having a concentration of ethanol of greater than 90 g/L.
  10. The engineered yeast of any of the previous claims which is tolerant to growth at temperatures in the range of greater than 31 °C - 35 °C.
  11. A fermentation method for producing a fermentation product, comprising: forming a fermentation medium from a glucose polymer-containing feedstock; and fermenting the fermentation medium using an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 97% or greater sequence identity to SEQ ID NO: 4, wherein fermenting produces a bioproduct.
  12. The fermentation method of claim 11, wherein the glucose polymer-containing feedstock or the fermentation medium, at the beginning of fermentation, has a DE of about 50 or less.
  13. The fermentation method of claims 11 or 12 wherein the glucose polymer-containing feedstock comprises glucose polymer having a degree of polymerization of 4 or greater and is present in an amount of 75% weight or greater total fermentable carbohydrates in the feedstock.
  14. The fermentation of any of claims 11-13 wherein ethanol is produced to a concentration of 70 g/L or greater in the medium.
  15. The fermentation method of any of claims 11-14 comprising adding supplemental glucoamylase to the feedstock, or supplemental glucoamylase to the medium during the fermentation period.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62/636,716, filed February 28, 2018, and entitled "GLUCOAMYLASE ENGINEERED YEAST AND FERMENTATION METHODS", which application is hereby incorporated by reference herein in its entirety. SEQUENCE LISTING The entire contents of the ASCII text file entitled "N00570_Sequence_Listing_ST25.txt," created on February 27, 2019, and having a size of 123 kilobytes is incorporated herein by reference. FIELD OF THE INVENTION The current invention relates to yeast engineered with exogenous glucoamylase nucleic acids and fermentations methods for producing a bioproduct such as ethanol. BACKGROUND Many fermentation feedstocks are derived from plant sources (e.g., corn mash) where the carbohydrates are predominantly in the form of starch polymers. The starch polymers in such feedstocks must be treated to low molecular weight sugars that can be consumed by the yeast and used for growth and bioproduct production. Typical treatments include acid and/or enzymatic hydrolysis where the polymer chain is hydrolyzed to generate the sugars that can be used by the yeast. Starch degrading enzymes such as alpha amylases and glucoamylases can be added to convert the polymer to simple sugars. However, such enzyme additions can add significant cost and complexity to the fermentation process. Heterologous expression and functionality of enzymes in yeast to aid in starch hydrolysis can be challenging, as it is difficult to know if the nucleic acid will be expressed properly and a functional enzyme will form, and if an active form of the enzyme will be secreted from the cell. It is also challenging to engineer yeast for growth and bioproduct production at non-optimal conditions, such as high temperatures, and in high bioproduct titers. For example, while ethanol production by fermentation is a well know industrial process, maintaining ethanol rates, titers, and yields while at the same time engineering the yeast to reduce reliance on supplemental enzymes, growth under non-optimal conditions (e.g., temperature), and minimizing by-product formation can be technically difficult. Increased ethanol concentration and accumulation of undesirable byproducts can also be detrimental to cell health. SUMMARY OF THE INVENTION The invention relates to engineered yeast and fermentation methods, wherein the engineered yeast are able to secrete a heterologous glucoamylase (GA) into a fermentation medium and provide glucoamylase activity (E.C. 3.2.1.3) on a fermentation substrate. The invention also relates to methods of for producing bio-derived products, such as ethanol, via fermentation using the engineered yeast. In one aspect, experimental studies associated with the current application identified fungal glucoamylase genes that, when introduced exogenously into yeast, allowed it to grow well on feedstocks containing low glucose and high starch amounts, and produce high levels of bioproduct. The results indicated that the engineered yeast were able to secrete glucoamylase into the fermentation medium and that the glucoamylase was enzymatically active towards the starch to generate sufficient glucose for growth and bioproduct production. Other benefits associated with the disclosure include improved fermentation and bioproduct production at elevated fermentation temperatures. Yet other benefits associated with the disclosure include reduced amounts of glucose at the end of the fermentation period. In one aspect, the invention provides an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 81% or greater sequence identity to SEQ ID NO: 1 (Rhizopus microsporus glucoamylase). In an embodiment, the engineered yeast is capable of producing ethanol at a rate of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 g/L*h or greater during a fermentation process. In another embodiment, the engineered yeast is capable of producing (a) at least 70g/kg of ethanol in a fermentation medium made from a glucose polymer-containing feedstock having (i) a DE of about 50 or less. In another embodiment, the engineered yeast is capable of producing (a) 90 g/kg or greater, 120 g/kg or greater, 130 g/kg or greater, or 140 g/kg or greater of ethanol in a fermentation medium made from a glucose polymer-containing feedstock having (i) a DE of about 30. In embodiments, the amount of 70g/kg of ethanol may be produced within 48 hours of inoculation in a fermentation medium with the feedstock. In embodiments, the glucose concentration may not be greater than 5% (wt) in the fermentation medium at the beginning (inoculation) of the fermentation process. In embodiments, the feedstock may provide an amount of glucose polymer-containing feedstock sufficient to produce 70g/kg ethanol, for example about 20 wt% glucose-polymer feedstock in the medium. In embodiments, the feedstock may have one or more of the following properties: