Pentose-fermenting strain with optimized propagation

Patent metadata

Field	Value
Publication number	US-2016348064-A1
Application number	US-201515116756-A
Country	US
Kind code	A1
Filing date	Feb 16, 2015
Priority date	Feb 17, 2014
Publication date	Dec 1, 2016
Grant date	—

Abstract

The present invention relates to a method for obtaining a strain capable of efficiently propagating in a low nutritive potential medium, capable of metabolising pentoses and of resisting fermentation inhibitors, comprising the following steps; a) growth of a strain of recombinant yeast with a strain of wild yeast lacking any impairments, the recombinant yeast strain comprising at least one copy of an exogenous gene of xylose isomerase and at least one additional copy of a gene of D-xylulokinase included in the genome and linked to a single sexual characteristic of the strain, b) at least two cycles of genome shuffling by sporulation and or random hybridization, c) selection of the population obtained in step b) according to a suitability criterion of the strains to metabolize xylose, d) selection of the population obtained in step c) according to a suitability criterion of the strains to grow in a Pref type medium, a medium with low nutritive value. The invention also relates to a cell, a yeast or a strain of recombinant yeast obtained according to the method, and to the use of such a cell for the production of bioethanol.

First claim

1 . A process for obtaining a yeast strain suited to propagating effectively on a medium with low nutritional potential, suited to metabolize pentoses and with fermentation inhibitor resistance, comprising the steps of:
a) crossing a recombinant yeast strain with a wild yeast strain devoid of deficiencies, wherein the recombinant yeast strain includes at least one copy of an exogenous xylose isomerase gene and at least one additional copy of a D-xylulokinase gene incorporated into the genome and linked to one of the mating traits of the strain, b) performing at least two cycles of genome shuffling by random sporulation and/or hybridization c) selecting a population obtained in step b) according to a criterion of ability of strains to metabolize xylose, and according to a criterion of fitness of the strains to grow in a medium low in nitrogenous bases, a low nutrient-value medium.

2 . The process according to claim 1 wherein the recombinant yeast strain of step a) is the yeast strain deposited at the CNCM (National Collection of Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number I-4538.

3 . The process according to claim 1 wherein the selecting according to the criterion of ability of strains to metabolize xylose includes a step of selection on a YFX medium.

4 . The process according to claim 1 , wherein the selecting according to the criterion of ability of strains to metabolize xylose comprises the steps of: measuring the percentage of xylose converted into ethanol by at least one hybrid, in anaerobic conditions over 60 hours in a fermentation medium comprising 55 g of glucose and 45 g of xylose per kg of said medium and selecting at least one hybrid that converts at least 70% of the xylose into ethanol in 60 hours.

5 . The process according to claim 1 , wherein the selecting according to the criterion of fitness of the strains to grow in a medium low in nitrogenous bases, a low nutrient-value medium comprises measuring the propagation of the strain on a medium of low nutritional value including a protein hydrolysate and devoid of nitrogenous bases.

6 . The process according to claim 5 wherein the middle to low nutritive value is the Pref. medium as defined in table I.

7 . The process according to claim 1 , further comprising a step of genetic transformation by the addition of at least one copy of the GAL2 gene.

8 . Yeast comprising at least one copy of an exogenous xylose isomerase gene and at least one additional D-xylulokinase gene incorporated into the genome and linked to one of the mating traits of the strain, able to grow on a low nutritional value medium comprising a protein hydrolysate and devoid of nitrogenous bases, which could be obtained by the process according to claim 1 .

9 . Yeast according to claim 8 , said yeast being of the genus Saccharomyces.

10 . The yeast according to claim 9 , said yeast being of the species Saccharomyces cerevisiae.

11 . The yeast according to claim 9 , further comprising at least one additional copy of the GAL2 gene.

12 . The yeast according to claim 11 , said yeast being of the species Saccharomyces cerevisiae.

13 . The yeast strain derived from a yeast according to claim 8 , characterized in that it is suited to metabolize pentoses while resisting to fermentation inhibitors.

14 . Yeast resulting from culture of a yeast strain according to claim 13 .

15 . A method for producing at least one fermentation product comprising the steps of:
a) fermenting, in anaerobic or semi-anaerobic conditions, a medium comprising one source of xylose, by a yeast according to claim 8 , and b) obtaining ethanol.

16 . The process according to claim 15 in which the fermenting is performed by a yeast of the genus Saccharomyces, the yeast comprising at least one copy of an exogenous xylose isomerase gene and at least one additional D-xylulokinase gene incorporated into the genome and linked to one of the mating traits of the strain, able to grow on a low nutritional value medium comprising a protein hydrolysate and devoid of nitrogenous bases.

17 - 18 . (canceled)

19 . The process according to claim 1 , wherein the selecting c) comprises:
c1) selecting a population obtained in step b) according to a criterion of ability of strains to metabolize xylose, and c2) selecting a population obtained in step c1) according to a criterion of fitness of the strains to grow in a medium low in nitrogenous bases, a low nutrient-value medium.

20 . The process according to claim 1 , wherein the selecting c) comprises
c1) selecting a population obtained in step b) according to a criterion of fitness of the strains to grow in a medium low in nitrogenous bases, a low nutrient-value medium, and c2) selecting a population obtained in step c1) according to a criterion of ability of strains to metabolize xylose.

21 . The yeast according to claim 10 comprising a strain deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number I-4749.

22 . The yeast according to claim 12 comprising a strain deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number I-4829.

Assignees

• Lesaffre & Cie
• LESAFFRE et COMPAGINE

Inventors

• Desfougeres Thomas
• Pignede Georges
• Techel Jennifer

Classifications

• C12Y207/01017

  Xylulokinase (2.7.1.17) · CPC title

• C12N9/1205

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title

• C12N9/92

  Glucose isomerase {(5.3.1.5; 5.3.1.9; 5.3.1.18)} · CPC title

• C12N1/36 Primary

  Adaptation or attenuation of cells · CPC title

• C12Y207/01006

  Galactokinase (2.7.1.6) · CPC title