Bifunctional phosphoketolase-phosphotransacetylase fusion polypeptides

A technology of phosphotransacetylase and phosphoketolase, applied in the direction of transferase, acyltransferase, enzyme, etc., can solve the problems affecting the production rate of ethanol, unfavorable and other problems

Pending Publication Date: 2019-10-22
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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  • Bifunctional phosphoketolase-phosphotransacetylase fusion polypeptides
  • Bifunctional phosphoketolase-phosphotransacetylase fusion polypeptides
  • Bifunctional phosphoketolase-phosphotransacetylase fusion polypeptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0165] Materials and methods

[0166] Liquefaction preparation

[0167] The liquefied product (ground corn slurry) was added by adding 600ppm urea, 0.124SAPU / g ds FERMGEN TM 2.5X (acid fungal protease), 0.33GAU / g ds glucoamylase (Trichoderma glucoamylase variant), and 1.46 SSCU / g ds GC626 (Aspergillus alpha-amylase), adjusted to pH 4.8 to prepare.

[0168] Preparation of non-liquefied cornmeal substrate

[0169] Non-liquefied corn flour was prepared by adding desalinated water to corn flour obtained by grinding dry corn kernels and adjusting the pH to 4.8. In addition, add 600ppm urea, 0.124SAPU / g ds FERMGEN TM 2.5X (acid fungal protease), 0.22 mg / g ds TrGA (Trichoderma glucoamylase), and 0.033 mg / g ds AcAA (Aspergillus alpha-amylase).

[0170] Serum bottle assay

[0171] Yeast cells were inoculated into 2 mL of YPD in a 24-well plate and cells were grown overnight to reach an OD between 25-30. Aliquots of 2.5 mL of liquefaction were added to serum vials (Chemglass, C...

example 2

[0185] Plasmid pZK41W-GLAF12 with PKL-PTA fusion gene 1

[0186] The synthetic phosphoketolase and phosphotransacetylase fusion gene 1GvPKL-L1-LpPTA comprises a phosphoketolase (GvPKL; SEQ ID NO: 1) and the codon-optimized coding region of phosphotransacetylase (LpPTA; SEQ ID NO:2) from Lactobacillus plantarum. The amino acid sequence of the resulting fusion polypeptide is figure 2 (SEQ ID NO:5), linker regions are shown in bold italics:

[0187] image 3 Plasmid pZK41W-GLAF12 is shown containing three cassettes to express the GvPKL-L1-LpPTA fusion polypeptide, acetylated acetaldehyde dehydrogenase (DjAADH) from Desulfospira joergensenii and acetyl-CoA synthase (McACS) from Methanosa manecalis . Both DjAADH and McACS were codon optimized. The expression of GvPKL-L1-LpPTA is under the control of HXT3 promoter and FBA1 terminator. The expression of DjAADH is under the control of TDH3 promoter and ENO2 terminator. The expression of McACS is under the control of PDC1 promo...

example 3

[0191] Plasmid pZK41W-GLAF22 with PKL-PTA fusion gene 2

[0192] Synthetic GvPKL-L2-LpPTA comprised the same phosphoketolase and phosphotransacetylase as in Example 2 joined with a synthetic linker 2 (L2; SEQ ID NO 4). The amino acid sequence of the resulting fusion polypeptide is Figure 4 (SEQ ID NO:6), linker regions are shown in bold italics. Plasmid pZK41W-GLAF22 contains a cassette to express GvPKL-L2-LpPTA. Plasmid pZK41W-GLAF22 (not shown) is similar to plasmid pZK41W-GLAF12 but has a different linker.

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Abstract

Described are compositions and methods relating to bifunctional phosphoketolase-phosphotransacetylase fusion polypeptides and the use thereof in starch hydrolysis processes for alcohol production.

Description

[0001] priority [0002] This application claims U.S. Provisional Application Serial No. 62 / 435,212, filed December 16, 2016; U.S. Provisional Application Serial No. 62 / 520,604, filed June 16, 2017; and U.S. Provisional Application Serial No., filed September 11, 2017 62 / 556,788, each of which is hereby incorporated by reference in its entirety. technical field [0003] The compositions and methods of the invention relate to bifunctional phosphoketolase-phosphotransacetylase fusion polypeptides and their use in the production of alcohol during starch hydrolysis. Background technique [0004] 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 impac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N9/10C12N9/88C12P7/06
CPCC12N9/1029C12N9/88C12N15/62C12P7/06C12Y401/02009C12Y203/01008Y02E50/10
Inventor Q·Q·朱P·J·泰尼森B·U·科扎克K·霍兰德斯
Owner DANISCO US INC
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