Yeast strain having improved capability for fermenting xylose in the presence of acetic acid

Abstract

The invention relates to a method for selecting a yeast strain having improved capability for fermenting a pentose, advantageously xylose, in the presence of organic acid, advantageously acetic acid, in non-dissociated form, in which at least one yeast strain that is capable of fermenting said pentose is consecutively cultured in the following two media: a first growth medium comprising said pentose as the only carbon source and said organic acid in non-dissociated form; a second growth medium comprising another carbon source as the only carbon source, advantageously glucose, free of said organic acid in non-dissociated form, the consecutive culture in at least said two growth media being repeated at least twice, in the presence of rising concentrations of organic acid in non-dissociated form.

Claims

1. A yeast strain characterized in that it was deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-4953 on Jan. 29, 2015.

2. A yeast obtained by culturing the strain according to claim 1.

3. The yeast strain according to claim 1, further comprising one or more genetic modifications which do not negatively impact xylose metabolism in the presence of acetic acid.

4. A method for producing fermentation products or ethanol comprising the following steps: Incubation of a material or medium containing xylose with the yeast strain according to claim 1; Fermentation under anaerobic or semi-anaerobic conditions; and Recovery of the one or more fermentation products, or ethanol.

5. The method according to claim 4, characterized in that the material or medium also contains glucose.

6. The yeast strain of claim 1, further comprising at least one copy of an exogenous gene encoding xylose isomerase.

7. The yeast strain according to claim 1, further comprising at least one supernumerary copy of a GAL2 gene.

8. The yeast strain of claim 1, further comprising a modification which suppresses aldose reductase activity.

9. The yeast strain according to claim 1, further comprising at least one supernumerary copy of a xylulokinase (XKS1) gene or a modification of a promoter resulting in overproduction of xylulokinase.

10. The yeast strain according to claim 1, further comprising at least one supernumerary copy of a gene in the pentose phosphate pathway or a modification of a promoter resulting in overproduction of a gene product in the pentose phosphate pathway.

11. The yeast strain according to claim 10, wherein the gene of the pentose phosphate pathway is RPE1, RKL1, TKL1, TAL1, or a combination of any of the foregoing.

12. The yeast strain according to claim 1, comprising a modification resulting in the absence of xylose reductase activity in the yeast.

13. The yeast strain according to claim 12, comprising a mutation in a XR or XYL1 gene.

14. The yeast strain according to claim 1, comprising a modification that enables the yeast to ferment arabinose.

Description

LEGENDS FOR THE FIGURES

(1) FIG. 1 shows the progression of the mass loss (expressed in g/kg) as a function of fermentation time for various strains, in a medium containing 50 g/L xylose and 5 g/L acetic acid at pH=5.

(2) FIG. 2 shows the impact of acetic acid concentration (expressed in g/L at pH 5) on the mass loss rate (production of CO.sub.2 from xylose), during fermentation of various strains, in a medium containing 50 g/L xylose.

(3) FIG. 3 shows the progression of the mass loss (expressed in g/kg) as a function of fermentation time for various strains, in a medium containing 55 g/L glucose, 45 g/L xylose and 5 g/L acetic acid (at pH 5).

EXAMPLE EMBODIMENTS

(4) I) Material and Methods:

(5) 1) Strain:

(6) Yeast: Saccharomyces cerevisiae

(7) Strain: I-4829 or I-4966

(8) 2) Culture Medium and Conditions:

(9) 2-a) Directed Evolution:

(10) Medium 1: YFX50=

(11) Xylose 50 g/L; Yeast extract 5 g/L; Di-ammonium phosphate 4.7 g/L; Citric acid 11.4 g/L; Trisodium citrate 13.5 g/L; ZnSO.sub.4 21.2 mg/L; MgSO.sub.4 7H.sub.2O 1 g/L; Thiamine 18.24 mg/L; Pyridoxine 5.28 mg/L; Biotin 1.76 g/L; Pantothenate 3.8 mg/L; Niacin 20 mg/L; Meso-inositol 50 mg/L; Riboflavin 1 mg/L; Para-aminobenzoate 1.2 mg/L; Tween 80, 1 g/L.

(12) The yeasts are cultivated in this medium with xylose as the only carbon source and with acetic acid whose concentration increases over the cycles (see Results section).

(13) The pH of the medium is kept at 5.

(14) The culture is done at 32° C. with stirring at 100 rpm in flasks stoppered by caps which serve to reduce the supply of oxygen in the medium and allow CO.sub.2 which is produced throughout this culture in excess pressure to escape. Under these conditions, the culture lasts about seven days.

(15) Medium 2 (YPG):

(16) 10 g/L of yeast extract; 10 g/L de peptone; 20 g/L of glucose as the only carbon source.

(17) The culture is done at 30° C. with stirring at 150 rpm in baffled flasks stoppered by porous caps which allow the supply of oxygen into the medium. Under these conditions, the culture lasts 24 hours.

(18) Medium 3:

(19) 3.4 g/L of DIFCO® yeast nitrogen base; 5 g/L of ammonium sulfate; 10 g/L of glycerol as the only carbon source.

(20) The culture is done at 30° C. with stirring at 150 rpm in baffled flasks stoppered by porous caps which allow the supply of oxygen into the medium. Under these conditions, the culture lasts 24 to 48 hours.

(21) 2-b) Selection of the Most Effective Strains:

(22) At the end of the eighth cycle, the cells were spread at a concentration of 2000 cells per dish (150 mm diameter) of agar medium (YPG+agar).

(23) The resulting colonies were then cultivated in Deep Well format (=96−well microplate) in a YPG medium.

(24) After 72 hours of culture at 30° C., the colonies were transferred into a YFX50-Ac-5000 medium whose composition is indicated below:

(25) TABLE-US-00001 YFX50-Ac-5000 g/kg mL/kg Distilled water qs 1000 Xylose 50 Yeast extract type J 5 DAP 4.7 Citric acid 11.4 Trisodium citrate 13.5 Acetic acid 5 pH 5 ZnSO.sub.4 (10.6 g/L) 2 MgSO.sub.4 7H.sub.2O (400 g/L) 2.5 Thiamine Vit B1 (18.24 g/L) 1 Pyrodixine Vit B6 (5.28 g/L) 1 Biotin (1.76 g/L) + KOH 1 Pantothenate (3.8 g/L) 1 Nicotinic acid (8 g/L) 2.5 Meso-inositol (50 g/L) 1 Riboflavin (1 g/L) 1 Para-aminobenzoate (1.2 g/L) 1 Tween 80 1

(26) In practice, it involves the same culture medium as the first growth medium used in the method according to the invention, with an acetic acid concentration (quantity added into the culture medium at pH 5) equal to 5 g/L.

(27) Likewise, the culture conditions are similar:

(28) Conditions:

(29) pH: 5

(30) Temperature: 32° C.

(31) O.sub.2 conditions: without oxygen supply (anaerobic)

(32) Stirring: 100 rpm

(33) Length of the culture: 72 hours

(34) Pre-Culture/Incubation: Take up the colonies for the liquid cultures from Deep Well (96-well micro plates); 0.25 g/kg equivalent DM for liquid cultures.
2-c) Validation of the Isolated Strains

(35) Medium: YFP-5000 10 g/L of yeast extract; 10 g/L of bacto-peptone; 55 g/L of glucose; 45 g/L de xylose; 5 g/L of acetic acid (quantity added to the culture medium at pH 5); KOH.
Conditions:

(36) pH: 5

(37) Temperature: 32° C.

(38) O.sub.2 conditions: without oxygen supply

(39) Stirring: 100 rpm

(40) Length of the culture: up to 72 hours

(41) Pre-Culture/Incubation: 0.25 g/kg eq DM of yeast previously propagated in YPG medium.

(42) 3) Evaluation of the Mass Loss:

(43) The ethanol production is measured indirectly by a measurement of the mass lost from the fermentation flask, since this mass loss is directly correlated with alcohol production. It is expressed in grams per kilogram of medium.

(44) 4) Evaluation of the CO.sub.2Production Speed:

(45) Starting with the determination of the mass loss (corresponding to CO.sub.2 production), it is possible to determine the xylose consumption rate (expressed in g/L) which is twice the mass loss.

(46) The maximum CO.sub.2 production rate is expressed in g.Math.kg.sup.−1.Math.h.sup.−1.Math.g.sup.−1 of DM (dry material).

(47) II) Results:

(48) 1) Selection of Strains of Interest:

(49) 1-a) Directed Evolution:

(50) The directed evolution was done in Single Batch Repeat mode: Cycles 1 to 3: medium 1 with 3 g/L of acetic acid (added into the medium at pH 5) and then medium 2 and then medium 3; Cycles 4 to 6: medium 1 with 4 g/L of acetic acid and then medium 2 and then medium 3; Cycle 7: medium 1 with 5 g/L of acetic acid and then medium 2 and then medium 3; Cycle 8: medium 1 with 6 g/L of acetic acid and then medium 2 and then medium 3;
1-b) Selection of the Most Effective Strains:

(51) After isolation of the clones on solid medium and their pre-culture in liquid medium, they are seeded in the Deep Well type micro plates containing the medium YFX50-Ac-5000 (50 g/kL of xylose and 5 g/L of acetic acid at pH=5).

(52) After 72 hours of fermentation in this medium, the selected strains (16 in total) are those having the best growth, by analysis of the cellular density of the medium. Strains CNCM I-4071 and CNCM I-4829 served respectively as negative and positive control for the capability of the cells to use xylose for their growth.

(53) These 16 strains were evaluated on the kinetics on this same medium. The results are shown in FIG. 1 which shows the mass loss as a function of the fermentation time. As expected, since this medium contained only xylose (no glucose), the observed profile for all strains tested is single phase. On the other hand, it appears that the derivative at the beginning of the curve varies depending on the strains. This can be formulated using the equation ax+b, where a varies according to the strains.

(54) FIG. 1 shows that strain E9 shows the best performance. The strain was deposited with Institut Pasteur (Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number CNCM 1-4953 on Jan. 29, 2015 and was used in the remainder the experiments.

(55) 2) Properties of Strain 1-4953:

(56) 2-1) in Xylose Medium:

(57) The first step consisted of determining the impact of the directed evolution on the capability of this strain to resist acetic acid during xylose fermentation. To do that, the test consisted of measuring the maximum CO.sub.2 production rate during the xylose fermentation on a YFX50-Ac medium. In order to obtain a dose-response curve, various acetic acid concentrations were added to the fermentation medium.

(58) FIG. 2 shows that this strain has a maximum xylose consumption rate equal to twice the mass loss during fermentation which is about 15% greater than the rate for the strain I-4829 without acetic acid. This gap is even greater when the acetic acid dose is 4 g/L. In fact, at this concentration, the xylose consumption rate is reduced by 85% for strain I-4829, compared to only 15% for strain I-4953. This means that in the presence of 4 g/L acetic acid and at pH 5, strain I-4953 has the same xylose consumption rate as strain I-4829 without acetic acid.

(59) 2-2) in Glucose+Xylose Medium:

(60) In order to better understand the impact of the directed evolution on the fermentation in general, the mass loss as a function of fermentation time was tracked for a medium containing glucose, xylose and acetic acid.

(61) The results are shown in FIG. 3 which clearly shows the double effect of acetic acid on yeasts capable of fermenting xylose: During the first phase, called the “glucose phase”, strain 1-4538 shows a fermentation initiation delay of about 24 hours. For the other strains (I-4749, I-4829 and I-4953), the lag is only 5 to 6 hours. This observation means that the isolated strain has not lost resistance to acetic acid during glucose fermentation. When xylose fermentation picks up the relay, the kinetics of strains I-4538, I-4749 and I-4829 are comparable. In contrast, for strain I-4953, it is notable that CO.sub.2 production (and consequently ethanol production) is faster and greater than that observed for the 3 other strains.

(62) These results confirm that a strain more resistant to acetic acid during xylose fermentation was obtained and that it is therefore globally more effective.

(63) III) Conclusions:

(64) With the method described, it was therefore possible to select at least one stable strain, I-4953, which retained its capability for fermenting glucose with a limited initiation delay, while showing improved xylose fermentation kinetics in the presence of acetic acid.