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Fermentative glycerol-free ethanol production

a technology of glycerol-free ethanol and fermentation yeast, which is applied in the field of recombinant yeast cells, can solve the problems of not being suitable for wild-type ethanol producing microorganisms, not all being suitable substrates for wild-type microorganisms, and acetic acid has been reported to negatively affect the kinetics and/or stoichiometry, so as to reduce the enzymatic activity

Active Publication Date: 2016-06-07
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018]Advantageously, in accordance with the invention ethanol is produced in a molar ratio of glycerol:ethanol of less than 0.04:1, in particular of less than 0.02:1, preferably of less than 0.01:1. Glycerol production may be absent (undetectable), although at least in some embodiments (wherein NADH-dependent glycerol synthesis is reduced yet not completely prohibited) some glycerol may be produced as a side-product, e.g. in a ratio glycerol to ethanol of 0.001:1 or more.

Problems solved by technology

Lignocellulosic biomass is abundant, however is in general not readily fermented by wild-type ethanol producing micro-organisms, such as S. cerevisiae.
The resultant hydrolysate is often a mixture of various monosaccharides and oligosaccharides, which may not all be suitable substrates for the wild-type micro-organism.
In particular, acetic acid has been reported to negatively affect the kinetics and / or stoichiometry of sugar fermentation by wild-type and genetically modified S. cerevisiae strains and its toxicity is strongly augmented at low culture pH (Helle et al.
A major challenge relating to the stoichiometry of yeast-based ethanol production is that substantial amounts of glycerol are invariably formed as a by-product.
The fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD+to NADH occurs elsewhere in metabolism.
Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, which is undesired as it reduces overall conversion of sugar to ethanol.
Further, the presence of glycerol in effluents of ethanol production plants may impose costs for waste-water treatment.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Materials and Methods

Strain Construction and Maintenance

[0104]The Saccharomyces cerevisiae strains used (Table 1) originate from the CEN.PK family, which was previously identified as a suitable background for combined genetic and physiological studies (van Dijken et al. (2000) Enzyme Microb. Technol. 26:706-714).

[0105]

TABLE 1Saccharomyces cerevisiae strains usedStrainRelevant genotypeSource / referenceCEN.PK113-5DMATa ura3 GPD1 GPD2EUROSCARF straincollection, Frankfurt,GermanyIME076 (reference)MATa ura3 GPD1 GPD2p426_GPD(URA3)CEN.PK102-3AMATa ura3 leu2 GPD1 GPD2EUROSCARF straincollection, Frankfurt,GermanyRWB0094MATa ura3 leu2 gpd1(−1,1133)::loxP-BIRD Engineering,KanMX-loxP gpd2(−2,1281)::hphMX4RotterdamIMZ008MATa ura3 leu2 gpd1(−1,1133)::loxP gpd2(−2,1281)::hphMX4 YEplac181(LEU2)IMZ132MATa ura3 leu2 gpd1(−1,1133)::loxP gpd2(−2,1281)Deposited at(CBS125049)::hphMX4 YEplac181(LEU2)Centraalbureau voorpUDE43(URA3 pTHD3::mhpFSchimmelcultures on(E. coli)::CYC1t)Jul. 16, 2009IMZ127MATa ura3 ...

example 2

[0125]This example relates to the fermentative glycerol-free ethanol production by a gpd1Δ gpd2ΔSaccharomyces. cerevisiae strain expressing a codon optimized version (SEQ ID NO: 28) of the wild type dmpF gene from Pseudomonas sp. CF600 (GenBank No: CAA43226) (Shingler et al. (1992) J. Bacteriol. 174:71 1-24).

[0126]For this purpose, the same procedures and techniques used for the construction and evaluation of the performance of the strain IMZ132 (gpd1Δ gpd2Δ mhpF) were used. These procedures and techniques are described in Materials and Methods section of Example 1. In this work, transformation of strain IMZ008 (gpd1Δ gpd2Δura3Δ) with the URA3-bearing dmpF expression plasmid pUDE47 yielded the prototrophic, dmpF-expressing strain IMZ130. For the construction of plasmid pUDE47, a codon optimized copy of the dmpF gene (EMBL accession number X60835.1) from Pseudomonas sp. CF600 was ligated into p426_GPD plasmid. Codon optimization for the expression in S. cerevisiae and ligation into t...

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PUM

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Abstract

The present invention relates to a yeast cell, in particular a recombinant yeast cell, the cell lacking enzymatic activity needed for the NADH-dependent glycerol synthesis or the cell having a reduced enzymatic activity with respect to the NADH-dependent glycerol synthesis compared to its corresponding wild-type yeast cell, the cell comprising one or more heterologous nucleic acid sequences encoding an NAD+-dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) activity. The invention further relates to the use of a cell according to the invention in the preparation of ethanol.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of copending U.S. Ser. No. 13 / 061,695 having an international filing date of 23 Jul. 2010, which is a national phase of PCT application PCT / NL2010 / 050475 having an international filing date of 23 Jul. 2010, which claims benefit of European patent application No. 09166360.9 filed 24 Jul. 2009. The contents of the above patent applications are incorporated by reference herein in their entirety.SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE[0002]The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 313632011701SeqList.txt, date recorded: Feb. 25, 2014, size: 38,064 bytes).BACKGROUND OF THE DISCLOSURE[0003]The present invention relates to a recombinant yeast cell having the ability to produce a desired fermentation product, to the construction of said yeast cell by genetic modificat...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N15/81C12N1/18C12N9/02C12P7/06C12P7/10
CPCC12N15/815C12N1/18C12N9/0008C12P7/06C12P7/10Y02E50/16Y02E50/17Y02P20/52
Inventor PRONK, JACOBUS THOMASVAN MARIS, ANTONIUS JEROEN ADRIAANGUADALUPE MEDINA, VICTOR GABRIEL
Owner DSM IP ASSETS BV
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