Compositions and methods for increasing ethanol production by yeast using gcy1 and dak1

A technology of yeast and Saccharomyces, which is applied in the field of glycerol dehydrogenase-dihydroxyacetone kinase bifunctional fusion polypeptide, which can solve the problems affecting the production rate of ethanol and disadvantages

Pending Publication Date: 2020-12-18
DANISCO US INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, decreased glycerol synthesis may lead to an increase in other metabolic by-products such as acetate
Acetic acid is an undesirable by-product as it adversely affects ethanol production rate, titer and yield of yeast fermentation

Method used

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  • Compositions and methods for increasing ethanol production by yeast using gcy1 and dak1
  • Compositions and methods for increasing ethanol production by yeast using gcy1 and dak1
  • Compositions and methods for increasing ethanol production by yeast using gcy1 and dak1

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0181] Materials and methods

[0182] Liquefaction preparation

[0183] The liquefied product (ground corn slurry) was added by adding 600ppm urea, 0.124SAPU / g ds FERMGEN TM Prepared with 2.5X (acid fungal protease), 0.33 GAU / g ds TrGA variant glucoamylase, and 1.46 SSCU / g ds GC626 (Aspergillus alpha-amylase), adjusted to pH 4.8.

[0184] AnKom assay

[0185] 300 μL of the concentrated overnight yeast culture was added to each of the multiple ANKOM bottles filled with 30 g of the prepared liquefaction to achieve a final OD of 0.3. The flasks were then incubated for 65 hours at 32°C with shaking (150 RPM).

[0186] HPLC analysis

[0187] Samples from serum bottles and AnKom experiments were collected in Eppendorf tubes by centrifugation at 14,000 RPM for 12 minutes. The supernatant was filtered with a 0.2 μΜ PTFE filter and then used for HPLC (Agilent Technologies 1200 series) analysis under the following conditions: Bio-Rad Aminex HPX-87H column operating at 55°C. 0.6ml...

example 2

[0191] Construction of a plasmid carrying a fusion gene encoding N-terminal glycerol dehydrogenase and C-terminal dihydroxyacetone kinase

[0192] Synthetic GCY1-L1-DAK1 and GCY1-L2-DAK1 are fusion genes comprising codon-optimized glycerol fused via linker 1 (SEQ ID NO:3) and linker 2 (SEQ ID NO:4), respectively Dehydrogenase (GCY1, SEQ ID NO:2) and dihydroxyacetone kinase (DAK1, SEQ ID NO:1). The amino acid sequences of fusion polypeptides GCY-L1-DAK1 and GCY1-L2-DAK1 are represented by SEQ ID NO: 5 and 6, respectively.

[0193] Plasmid pZKIIC-YL1K contains an expression cassette to express the GCY1-L1-DAK1 fusion polypeptide under the control of the ACT1 promoter (YFL039C locus) and the FBA1 terminator (YKL060C locus). Plasmid pZKIIC-YL1K was designed to integrate the expression cassette into Saccharomyces chromosome II at positions 345856 and 350891. The functional and structural composition of plasmid pZKIIC-YL1K is described in Table 2.

[0194] Table 2. Functional and...

example 3

[0198] Construction of a plasmid with a fusion gene encoding N-terminal dihydroxyacetone kinase and C-terminal glycerol dehydrogenase

[0199] Synthetic DAK1-L1-GCY1 and DAK1-L2-GCY1 are fusion genes comprising codon-optimized two genes fused by linker 1 (SEQ ID NO:3) and linker 2 (SEQ ID NO:4), respectively. Hydroxyacetone kinase (DAK1, SEQ ID NO:1) and glycerol dehydrogenase (GCY1, SEQ ID NO:2). The amino acid sequences of the fusion polypeptides DAK-L1-GCY1 and DAK1-L2-GCY1 are represented by SEQ ID NO: 7 and 8, respectively.

[0200] Plasmids pZKIIC-KL1Y and pZKIIC-KL2Y contain expression cassettes to express DAK-L1-GCY1 and DAK1-L2-GCY1 fusion polypeptides under the control of the ACT1 promoter (YFL039C locus) and the FBA1 terminator (YKL060C locus). Both pZKIIC-KL1Y and pZKIIC-KL2Y were designed to integrate the expression cassette into Saccharomyces chromosome II at positions 345856 and 350891. The functional and structural composition of plasmids pZKIIC-KL1Y and pZKI...

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Abstract

Described are compositions and methods relating to yeast expressing glycerol dehydrogenase and dihydroxyacetone kinase polypeptides in combination with an exogenous phosphoketolase pathway, as well asto bifunctional glycerol dehydrogenase-dihydroxyacetone kinase fusion polypeptides, and their various and combined uses in starch hydrolysis processes for alcohol production.

Description

technical field [0001] The compositions and methods of the invention relate to yeast expressing glycerol dehydrogenase and dihydroxyacetone kinase polypeptides in combination with an exogenous phosphoketolase pathway, and to glycerol dehydrogenase-dihydroxyacetone kinase bifunctional fusion polypeptides; and Use of the described compositions and methods in a starch hydrolysis process for alcohol production. Background technique [0002] Yeast-based ethanol production is based on the conversion of sugars to ethanol. The current annual fuel ethanol production achieved by this method is about 90 billion liters worldwide. It is estimated that approximately 70% of the cost of ethanol production is feedstock. Because ethanol is produced in such large quantities, even a small increase in yield can have a huge economic impact on the industry. The conversion of one mole of glucose to two moles of ethanol and two moles of carbon dioxide is redox neutral with a maximum theoretical y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N9/02C12N15/62
CPCC12N9/0004C12N9/12C12N15/62Y02E50/10C12N9/0006C12P7/06C12Y101/01006
Inventor Q·Q·朱P·J·M·特尼森
Owner DANISCO US INC
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