TW202328430A - Yeast cells with improved tolerance to acrylic acid - Google Patents

Abstract

The current disclosure relates to yeast cells that are genetically modified to improve their tolerance to certain platform chemicals, such as acrylic acid, and to methods of preparing and using such yeast cells for the production of acrylic acid and other compounds.

Description

Yeast cells with improved tolerance to acrylic acid

The present invention relates to yeast cells genetically engineered to improve their tolerance to certain platform chemicals, such as acrylic acid, and methods of preparing such yeast cells and using them to produce acrylic acid and other compounds.

Acrylic acid (AA) is an important chemical substance on the market. By 2025, AA's production capacity is expected to reach 9 million tons, and its market size is $20 billion. Its current production depends on petrochemical processes, which are unsustainable and environmentally unfriendly. There has been considerable interest in the production of AA from renewable non-food biomass.

Combinations of biocatalytic and chemical catalytic approaches have been attempted. In one method, lactic acid (LA) is first produced microbially. However, subsequent AA production using dehydration of LA proves difficult due to its resistance to hydrolysis. In recent years, it has been achieved to produce AA by dehydrating 3-hydroxypropionic acid (3-HP) produced by microbial fermentation. Due to its high energy consumption and additional costs caused by catalysts, purification and separation steps, this combined approach does not provide substantial benefits over petroleum-based products.

In order to develop economically attractive methods for producing large quantities of chemicals from renewable resources, three characteristics are considered: high product yield, high productivity and high product potency. This latter characteristic is critical to minimizing capital equipment and downstream separation costs for product purification. The potency of a large number of chemicals in economical fermentation methods usually exceeds 100 g/L.

Some embodiments include a method of preparing yeast with improved tolerance to acrylic acid, comprising allowing the yeast to propagate in the presence of acrylic acid.

Some embodiments include a composition comprising yeast, acrylic acid, and yeast growth medium.

Some embodiments include yeast prepared by a method comprising allowing yeast to propagate in the presence of acrylic acid.

Some embodiments include a yeast tolerant to acrylic acid, comprising a yeast having a non-naturally occurring genetic mutation or modification, wherein the yeast has an optical density at 600 nm in a reference acrylic acid composition of 0.2 The characteristics of the yeast propagating such that the optical density of the yeast increases from 0.2 to 0.4 within 30 hours, wherein the reference acrylic acid composition consists of the following: 1.8 g/L acrylic acid, 20 g/L glucose, 5 g/L (NH ₄ ) ₂ SO ₄ , 3 g/L KH ₂ PO ₄ , 0.5 g/L MgSO ₄ •7H ₂ O, 50 μg/L d-biotin, 1 mg/L D-pantothenate hemicalcium salt, 1 mg /L Thiamine-HCl, 1 mg/L Pyridoxin-HCl (Pyridoxin-HCl), 1 mg/L niacin, 0.2 mg/L 4-aminobenzoic acid, 25 mg/L m-inositol , 3 mg/L FeSO ₄ ∙7H ₂ O, 4.5 mg/L ZnSO ₄ ∙7H ₂ O, 4.5 mg/L CaCl ₂ ∙2H ₂ O, 1 mg/L MnCl ₂ ∙4H ₂ O, 0.3 mg/L CoCl ₂ ∙6H ₂ O, 0.3 mg/L CuSO ₄ ∙5H ₂ O, 0.4 mg/L Na ₂ MoO ₄ ∙2H ₂ O, 1 mg/LH ₃ BO ₃ , 0.1 mg/L KI and 19 mg/L Na ₂ EDTA∙ _2H2O .

Some embodiments include a genetically modified S. cerevisiae ( Saccharomyces cerevisiae ; S. cerevisiae ) having modifications to the wild-type genome such that the Saccharomyces cerevisiae synthesizes: modified ACH1, WAR1, NHA1, TRK1, ALD3 , PMA1, GLG2, MIX23, ATG26, SYT1, SNF3, ARR3, TRS33, GPR1, OCT1, TPO1, MET2, RPC82, or combinations thereof.

Some embodiments include a method of preparing acrylic acid comprising dehydrating hydroxypropionic acid in the presence of engineered yeast described herein.

ALE of yeast is initiated using a composition comprising Adaptive laboratory evolution (ALE) of yeast and acrylic acid and, optionally, a yeast growth medium.

Yeast includes any type of yeast that requires improved tolerance in the presence of acrylic acid, such as Saccharomyces cerevisiae.

Yeast growth medium may contain yeast extract, carbohydrates, peptides and antibiotics, phosphates, sulfates, or combinations thereof. In some embodiments, the yeast growth medium includes yeast extract, carbohydrates, peptides and antibiotics, phosphates and sulfates.

The yeast growth medium may include any suitable yeast extract, such as the cell contents of yeast without cell walls.

Yeast growth medium may include any suitable carbohydrate, including monosaccharides (such as glucose), disaccharides (such as sucrose), polysaccharides (such as starch), and the like. In some embodiments, the carbohydrate includes agar, glucose, or combinations thereof. In some embodiments, the carbohydrate includes agar. In some embodiments, the carbohydrate includes glucose.

The yeast growth medium may include any suitable peptide, such as a peptide, eg, a water-soluble product of partial hydrolysis of a protein.

The yeast growth medium may include any suitable antibiotic, such as G418 disulfate, ampicillin sodium, or combinations thereof.

Yeast growth _medium may include any suitable phosphate, such as _KH2PO4 .

Yeast growth medium may include any suitable sulfate, such as _{( NH4} ) _2SO4 , _MgSO4.7H2O , or combinations _thereof .

The yeast growth medium may include a vitamin solution, for example, in the form of about 0.1 to 10 mL/L, about 0.1 to 2 mL/L, about 2 to 4 mL/L, about 4 to 6 mL/L, about 6 to 8 mL/L, A vitamin solution with a concentration of about 8 to 10 mL/L or about 1 mL/L.

A suitable vitamin solution may contain d-biotin (e.g., in an amount such that the yeast growth medium contains about 1 to 100 μg/L, about 40 to 60 μg/L, or about 50 μg/L of d-biotin), D - Pantothenic acid (e.g., in an amount such that the yeast growth medium contains about 0.1 to 10 mg/L, about 0.5 to 1.5 mg/L, or about 1 mg/L of hemicalcium salt or another form in molar equivalents), thiamine (e.g., in an amount such that the yeast growth medium contains about 0.1 to 10 mg/L, about 0.5 to 1.5 mg/L, or about 1 mg/L of an HCl salt or another molar equivalent), pyridoxine ( For example, in an amount such that the yeast growth medium contains about 0.1 to 10 mg/L, about 0.5 to 1.5 mg/L, or about 1 mg/L of HCl salt or molar equivalents), 1 mg/L of nicotine Acid (e.g., in an amount such that the yeast growth medium contains about 0.1 to 10 mg/L, about 0.5 to 1.5 mg/L, or about 1 mg/L in acid form or molar equivalents in salt form), 4-aminobenzene Formic acid (e.g., in an amount such that the yeast growth medium contains about 0.05 to 0.5 mg/L, about 0.1 to 0.3 mg/L, or about 0.2 mg/L in acid form or molar equivalents in salt form), m-inositol ( For example, in an amount such that the yeast growth medium contains about 5 to 200 mg/L, about 20 to 30 mg/L, or about 25 mg/L), or a combination thereof.

Some examples include d-biotin (e.g., in an amount such that yeast growth medium contains 50 μg/L), D-pantothenate hemicalcium salt (e.g., in an amount such that yeast growth medium contains 1 mg/L), thiamine HCl (e.g., in an amount such that the yeast growth medium contains 1 mg/L), pyridoxine-HCl (e.g., in an amount such that the yeast growth medium contains 1 mg/L), nicotinic acid (e.g., in an amount such that the yeast growth medium contains 1 mg/L) The yeast growth medium contains 1 mg/L), 4-aminobenzoic acid (e.g., in an amount such that the yeast growth medium contains 0.2 mg/L), and m-inositol (e.g., in an amount such that the yeast growth medium contains 25 mg/L).

The yeast growth medium may include a trace metal solution, for example, in the range of about 0.1 to 10 mL/L, about 0.1 to 2 mL/L, about 2 to 4 mL/L, about 4 to 6 mL/L, about 6 to 8 mL/L. L, a trace metal solution with a concentration of about 8 to 10 mL/L or about 1 mL/L.

Suitable trace metal solutions may contain FeSO ₄ (e.g., in the form of ∙7H ₂ O or molar equivalents such that the yeast growth medium contains 0.5 to 10 mg/L, about 2 to 4 mg/L, or about 3 mg/L. in another form), ZnSO ₄ (e.g., in a form such that the yeast growth medium contains about 1 to 10 mg/L, about 4 to 5 mg/L, or about 4.5 mg/L of ∙7H ₂ O or molar equivalents in another form), CaCl ₂ (e.g., in a form such that the yeast growth medium contains about 1 to 10 mg/L, about 4 to 5 mg/L, or about 4.5 mg/L of ∙2H ₂ O or molar equivalents in another form), MnCl ₂ (e.g., in a form such that the yeast growth medium contains about 0.1 to 10 mg/L, about 0.5 to 1.5 mg/L, or about 1 mg/L ∙4H ₂ O or molar equivalents in another form), CoCl ₂ (e.g., in a form such that the yeast growth medium contains about 0.05 to 6 mg/L, about 0.2 to 0.4 mg/L, or about 0.3 mg/L ∙6H ₂ O or molar equivalents in another form), CuSO ₄ (e.g., in a form such that the yeast growth medium contains about 0.05 to 6 mg/L, about 0.2 to 0.4 mg/L, or about 0.3 mg/L ∙5H ₂ O or molar equivalents an amount of another form), Na ₂ MoO ₄ (e.g., in the form of ∙2H ₂ O such that the yeast growth medium contains about 0.05 to 8 mg/L, about 0.3 to 0.5 mg/L, or about 0.4 mg/L, or moles An amount of another form of equivalent), H ₃ BO ₃ (e.g., in a form such that the yeast growth medium contains about 0.1 to 10 mg/L, about 0.5 to 1.5 mg/L, or about 1 mg/L H ₃ BO ₃ or molybdenum). molar equivalents of its salt), KI (e.g., in a form such that the yeast growth medium contains about 0.01 to 1 mg/L, about 0.05 to 0.15 mg/L, or about 0.1 mg/L of KI or molar equivalents of another iodine an amount of salt) and Na ₂ EDTA∙2H ₂ O (e.g., in a form such that the yeast growth medium contains about 1 to 40 mg/L, about 10 to 30 mg/L, or about 19 mg/L ∙2H ₂ O or molar equivalent), or a combination thereof.

In some embodiments, the trace metal solution contains FeSO ₄ (e.g., in the form of ∙7H ₂ O or molybdenum such that the yeast growth medium contains 0.5 to 10 mg/L, about 2 to 4 mg/L, or about 3 mg/L an amount of another form equivalent to 100 mg/L), ZnSO ₄ (e.g., in the form of ∙7H ₂ O such that the yeast growth medium contains about 1 to 10 mg/L, about 4 to 5 mg/L, or about 4.5 mg/L, or molybdenum). an equivalent amount of another form), CaCl ₂ (e.g., in the form of ∙2H ₂ O such that the yeast growth medium contains about 1 to 10 mg/L, about 4 to 5 mg/L, or about 4.5 mg/L, or Mo an amount of another form equivalent to 1 mg/L), MnCl ₂ (e.g., in the form of ∙4H 2 O such that the yeast growth medium contains about 0.1 to 10 mg/L, about 0.5 to 1.5 mg/L, or about 1 mg/L), or MnCl ₂ an amount of another form equivalent to 100 mg/L), CoCl ₂ (e.g., in the form of ∙6H ₂ O such that the yeast growth medium contains about 0.05 to 6 mg/L, about 0.2 to 0.4 mg/L, or about 0.3 mg/L), or Mo An amount of another form equivalent to one ear), CuSO ₄ (e.g., in the form of ∙5H ₂ O such that the yeast growth medium contains about 0.05 to 6 mg/L, about 0.2 to 0.4 mg/L, or about 0.3 mg/L, or molasses An amount of ∙2H 2 O in another form that is equivalent to an ear equivalent), Na ₂ MoO ₄ (e.g., in the form of ∙2H ₂ O such that the yeast growth medium contains about 0.05 to 8 mg/L, about 0.3 to 0.5 mg/L, or about 0.4 mg/L or another form of molar equivalents), H ₃ BO ₃ (e.g., in an amount such that the yeast growth medium contains about 0.1 to 10 mg/L, about 0.5 to 1.5 mg/L, or about 1 mg/L), KI (e.g., in an amount such that the yeast growth medium contains about 0.01 to 1 mg/L, about 0.05 to 0.15 mg/L, or about 0.1 mg/L) and Na ₂ EDTA∙2H ₂ O (e.g., in an amount such that the yeast growth medium contains An amount containing about 1 to 40 mg/L, about 10 to 30 mg/L, or about 19 mg/L of ∙2H ₂ O form or another form in molar equivalents).

Some examples include FeSO ₄ ∙7H ₂ O (e.g., in an amount such that the yeast growth medium contains 3 mg/L), ZnSO ₄ ∙7H ₂ O (e.g., in an amount such that the yeast growth medium contains 4.5 mg/L), CaCl ₂ ∙2H ₂ O (for example, in an amount such that the yeast growth medium contains 4.5 mg/L), MnCl ₂ ∙4H ₂ O (for example, in an amount such that the yeast growth medium contains 1 mg/L), CoCl ₂ ∙6H ₂ O (for example, in an amount such that the yeast growth medium contains 0.3 mg/L), CuSO ₄ ∙5H ₂ O (for example, in an amount such that the yeast growth medium contains 0.3 mg/L), Na ₂ MoO ₄ ∙2H ₂ O (e.g., in an amount such that the yeast growth medium contains 0.4 mg/L), H ₃ BO ₃ (e.g., in an amount such that the yeast growth medium contains 1 mg/L), KI (e.g., in an amount such that the yeast growth medium contains 0.1 mg /L), Na ₂ EDTA∙2H ₂ O (e.g., in an amount such that the yeast growth medium contains 19 mg/L), or a combination thereof.

Some examples include FeSO ₄ ∙7H ₂ O (e.g., in an amount such that the yeast growth medium contains 3 mg/L), ZnSO ₄ ∙7H ₂ O (e.g., in an amount such that the yeast growth medium contains 4.5 mg/L), CaCl ₂ ∙2H ₂ O (for example, in an amount such that the yeast growth medium contains 4.5 mg/L), MnCl ₂ ∙4H ₂ O (for example, in an amount such that the yeast growth medium contains 1 mg/L), CoCl ₂ ∙6H ₂ O (for example, in an amount such that the yeast growth medium contains 0.3 mg/L), CuSO ₄ ∙5H ₂ O (for example, in an amount such that the yeast growth medium contains 0.3 mg/L), Na ₂ MoO ₄ ∙2H ₂ O (e.g., in an amount such that the yeast growth medium contains 0.4 mg/L), H ₃ BO ₃ (e.g., in an amount such that the yeast growth medium contains 1 mg/L), KI (e.g., in an amount such that the yeast growth medium contains 0.1 mg /L) and Na ₂ EDTA∙2H ₂ O (for example, in an amount such that the yeast growth medium contains 19 mg/L).

The pH of the composition of ALE can be adjusted to about 4-6, such as about 5, with KOH or another base, and, for example, with citric acid and/or phosphate buffer, such as 97 mL/L 0.5 M citric acid and 103 mL/L of 1 M Na ₂ HPO ₄ buffer.

In order to prepare yeast with improved tolerance to acrylic acid, the yeast in the composition of ALE is propagated in the presence of acrylic acid. Generally, the ALE process starts with lower concentrations of acrylic acid and gradually increases the concentration of acrylic acid over time to promote the evolution of new yeast organisms with increased tolerance to acrylic acid.

The amount of yeast in the initial ALE composition can be any suitable amount. Yeast concentration may suitably be quantified by OD600, which is the optical density of the ALE composition at a wavelength of 600 nm. In some embodiments, the initial ALE composition has an OD600 of about 0.01 to 0.1, about 0.01 to 0.03, about 0.03 to 0.06, about 0.06 to 0.1, about 0.04 to 0.06, or about 0.05.

The ALE process typically begins with an acrylic acid concentration that is slightly tolerant to yeast, such as at least about 0.05 g/L, about 0.05 to 1 g/L, about 0.1 to 0.3 g/L, about 0.3 to 0.6 g/L, about 0.6 to 1 g/L, about 0.05 to 0.1 g/L, about 0.1 to 0.2 g/L, about 0.2 to 0.3 g/L, about 0.3 to 0.4 g/L, about 0.4 to 0.5, about 0.5 to 0.6 g/L, about 0.6 to 0.7 g/L, about 0.7 to 0.8 g/L, about 0.8 to 0.9 g/L, or about 0.9 to 1 g/L.

During the ALE process, the yeast in the ALE composition is propagated. This can occur at any suitable temperature, such as at about 1°C to 200°C, about 5°C to 100°C, about 100°C to 150°C, about 150°C to 200°C, about 1°C to 10°C, about 10°C Occurs at a temperature of 20°C, about 20°C to 30°C, about 25°C to 35°C, about 30°C to 40°C, about 40°C to 60°C, about 60°C to 80°C, or about 80°C to 100°C.

The addition of acrylic acid can be done by a stepwise continuous method (e.g., adding small amounts of acrylic acid continuously over time) or by a stepwise method (e.g., adding increasing amounts of acrylic acid at precise intervals), but in a stepwise method, the intervals and amounts of acrylic acid can be varied freely. One addition changes to another.

The rate of increase in acrylic acid concentration can be any suitable rate over a 24-hour period, such as about 0.005 to 0.5 g/L, about 0.005 to 0.01 g/L, about 0.01 to 0.02 g/L, about 0.02 to 0.03 g/L, about 0.03 to 0.04 g/L, about 0.04 to 0.05 g/L, about 0.05 to 0.06 g/L, about 0.06 to 0.07 g/L, about 0.07 to 0.08 g/L, about 0.08 to 0.09 g/L, about 0.09 to 0.1 g/L, about 0.1 to 0.2 g/L, about 0.2 to 0.3 g/L, about 0.3 to 0.4 g/L, or about 0.4 to 0.5 g/L, or equivalent rates over a longer or shorter period of time. For example, 0.005 to 0.5 g/L over a 24-hour period would be equivalent to 0.0025 to 0.25 g/L over a 12-hour period or 0.01 to 1 g/L over a 48-hour period.

For the continuous addition of acrylic acid, the increase rate of acrylic acid concentration may be, for example, 58 to 5787 ng∙L ^-1 ∙s ^-1 , about 58 to 116 ng∙L ^-1 ∙s ^-1 , or about 116 to 231 ng∙L ^-1 ∙s ^-1 , about 231 to 347 ng∙L ^-1 ∙s ^-1 , about 347 to 463 ng∙L ^-1 ∙s ^-1 , about 463 to 579 ng∙L ^-1 ∙s ^-1 , about 579 to 694 ng∙L ^-1 ∙s ^-1 , approximately 694 to 810 ng∙L ^-1 ∙s ^-1 , approximately 810 to 926 ng∙L ^-1 ∙s ^-1 , approximately 926 to 1042 ng∙L ^-1 ∙s ^-1 , about 1042 to 1157 ng∙L ^-1 ∙s ^-1 , about 1157 to 2315 ng∙L ^-1 ∙s ^-1 , about 2315 to 3472 ng∙L ^-1 ∙s ^-1 , about 3472 to 4630 ng ∙L ^-1 ∙s ^-1 or approximately 4630 to 5787 ng∙L ^-1 ∙s ^-1 .

For stepwise additions of acrylic acid, the concentration of acrylic acid can be increased at any suitable interval, such as at least 1 hour, about 1 hour to about 4 weeks, about 1 to 200 hours, about 1 to 8 hours, about 8 to 16 hours, about 16 to 24 hours, about 24 to 30 hours, about 30 to 36 hours, about 36 to 48 hours, about 48 to 72 hours, about 72 to 96 hours, about 1 to 7 days, about 7 to 14 days, about 2 to 4 weeks , about 20 to 30 hours or about 24 hours.

For adding acrylic acid at 24 hour intervals, the increase in acrylic acid can be any suitable amount, such as about 0.005 to 0.5 g/L, about 0.005 to 0.01 g/L, about 0.01 to 0.02 g/L, about 0.02 to 0.03 g/L, About 0.03 to 0.04 g/L, about 0.04 to 0.05 g/L, about 0.05 to 0.06 g/L, about 0.06 to 0.07 g/L, about 0.07 to 0.08 g/L, about 0.08 to 0.09 g/L, about 0.09 to 0.1 g/L, about 0.1 to 0.2 g/L, about 0.2 to 0.3 g/L, about 0.3 to 0.4 g/L, or about 0.4 to 0.5 g/L. For other intervals, the increase in acrylic acid can be within any of the above ranges, appropriately increased or decreased to an equivalent increase rate. For example, 0.005 to 0.5 g/L over a 24-hour period would be equivalent to 0.0025 to 0.25 g/L over a 12-hour period or 0.01 to 1 g/L over a 48-hour period.

Gradual addition of acrylic can be done in a series of breeding cycles. The propagation cycle involves increasing the concentration of acrylic acid, adding additional yeast growth medium, and allowing the yeast to propagate again. Any suitable number of breeding cycles may be performed, such as additional 1 to 100, 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 Or 90 to 100 reproductive cycles. The additional propagation cycle is the first increase in acrylic acid concentration, the addition of additional yeast growth medium, and allowing the yeast to propagate after an interval in which the yeast has been allowed to propagate under initial conditions. The breeding cycle can be performed over any suitable period of time, such as at least 1 hour, about 1 hour to about 4 weeks, about 1 to 200 hours, about 1 to 8 hours, about 8 to 16 hours, about 16 to 24 hours, about 24 to 30 hours, about 30 to 36 hours, about 36 to 48 hours, about 48 to 72 hours, about 72 to 96 hours, about 1 to 7 days, about 7 to 14 days, about 2 to 4 weeks, about 20 to 30 hours Or about 24 hours.

Although the propagation cycle can be performed in a variety of ways, in some embodiments, a volume of the ALE composition is diluted, such as by transfer, into fresh medium with a higher acrylic acid concentration, such as an increase in acrylic acid corresponding to that described in the above paragraph. State the rate of increase in acrylic acid concentration. Dilutions can be made to achieve a yeast level, such as a yeast level associated with an OD600 of about 0.01 to 0.2, about 0.01 to 0.05, about 0.05 to 0.1, about 0.1 to 0.15, about 0.15 to 2, about 0.08 to 0.12, or about 0.1 .

ALE can be continued until the desired level of acrylic tolerance is achieved. In some embodiments, ALE is discontinued after about 5 to 20, about 5 to 10, about 10 to 15, or about 15 to 20 propagation cycles without an observable increase in yeast growth rate. In some embodiments, ALE is stopped after there is no observable increase in growth rate for about 3 to 28 days, about 7 to 21 days, about 12 to 16 days, or about 14 days.

Some embodiments include a method of preparing yeast with improved tolerance to acrylic acid, comprising allowing the yeast to propagate in the presence of acrylic acid. In some embodiments, yeast is propagated in the presence of at least 0.05 g/L acrylic acid. In some embodiments, the concentration of acrylic acid is increased after the yeast has been allowed to propagate in the presence of acrylic acid for at least about 1 hour. Regarding any of the above embodiments, in some embodiments, the method further includes performing at least one additional propagation cycle, wherein the propagation cycle includes increasing the concentration of acrylic acid, adding additional yeast growth medium, and allowing the yeast to propagate again. In some embodiments, an additional 1 to 100 breeding cycles are performed. In some embodiments, an additional 30 to 40 breeding cycles are performed. Regarding any of the above embodiments in this paragraph, in some embodiments, the yeast is propagated at a temperature of about 5°C to about 100°C. With respect to any of the above embodiments in this paragraph, in some embodiments, the yeast is propagated for about 1 hour to about 200 hours in each propagation cycle. With respect to any of the above embodiments in this paragraph, in some embodiments, the yeast is propagated for about 20 hours to about 30 hours in each propagation cycle. With regard to any of the above embodiments in this paragraph, in some embodiments, the reproduction cycle is stopped after there is no observable increase in growth rate during a two-week period.

The methods described herein can be used to prepare altered yeast, such as yeast that is tolerant to acrylic acid (eg, Saccharomyces cerevisiae). In some embodiments, such yeast comprises yeast having a genetic mutation or modification that is not naturally occurring, wherein the yeast has an optical density of 0.2 in a reference acrylic composition when the yeast has an optical density of 0.2 at 600 nm. Characteristics that cause the optical density of the yeast to increase from 0.2 to 0.4 within 30 hours, wherein the reference acrylic acid composition consists of the following: 1.8 g/L acrylic acid, 20 g/L glucose, 5 g/L (NH ₄ ) ₂ SO ₄ , 3 g/L KH ₂ PO ₄ , 0.5 g/L MgSO ₄ •7H ₂ O, 50 μg/L d-biotin, 1 mg/L D-pantothenic acid hemicalcium salt, 1 mg/L thiamine- HCl, 1 mg/L pyridoxine-HCl, 1 mg/L nicotinic acid, 0.2 mg/L 4-aminobenzoic acid, 25 mg/L m-inositol, 3 mg/L FeSO ₄ ∙7H ₂ O , 4.5 mg/L ZnSO ₄ ∙7H ₂ O, 4.5 mg/L CaCl ₂ ∙2H ₂ O, 1 mg/L MnCl ₂ ∙4H ₂ O, 0.3 mg/L CoCl ₂ ∙6H ₂ O, 0.3 mg/L CuSO ₄ ∙5H ₂ O, 0.4 mg/L Na ₂ MoO ₄ ∙2H ₂ O, 1 mg/LH ₃ BO ₃ , 0.1 mg/L KI and 19 mg/L Na ₂ EDTA∙2H ₂ O.

The yeast described herein may be suitable for use in the production of acrylic acid, such as by fermenting hydroxypropionic acid in the presence of yeast.

In some embodiments, genetically modified or mutated yeast does not produce acyl-CoA compounds of the formula R-(C=O)-COA, where R is a carbon chain of 5 atoms or less.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has a modification in its genome that produces an amino acid sequence that is resistant to one of the proteins or peptides in Table 1 below. Transformation. Table 1. protein or peptide Abbreviation Acetate-CoA-transferase and acetyl-CoA hydrolase ACH1 PDR12 transcription factor WAR1 Na+/H+ antiporter NHA1 Potassium ion transmembrane transporter TRK1 aldehyde dehydrogenase ALD3 plasma membrane ATPase PMA1 glycogen core protein glucosyltransferase GLG2 Mitochondrial intermembrane space CX(n)C motif protein MIX23 Sterol 3-beta-glucosyltransferase ATG26 Arf protein guanine nucleotide exchange factor (GEF) SYT1 Plasma membrane low glucose sensor SNF3 Antimonite and arsenite transmembrane transporter ARR3 Core components of transport protein particle (TRAPP) complexes I, II and IV TRS33 G protein coupled receptor GPR1 mitochondrial intermediate peptidase OCT1 Major facilitator superfamily of polyamine transporters TPO1 L-homoserine-O-acetyltransferase MET2 RNA polymerase III subunit C82 RPC82

In some embodiments, the yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae having modifications to its genome that result in modifications to the amino acid sequence of ACH1, such as: R334G, A367T, T383K, V353F , G393E, fsN137, H439Y, Q275K or combinations thereof; or modification of the amino acid sequence of WAR1, such as W936L.

In some embodiments, the yeast with increased tolerance to acrylic acid can be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequence of NHA1, such as: H164D, E290K, or a combination thereof; Or modification of the amino acid sequence of TRK1, such as: S144F, R8975, N750K, P11635, L764F or combinations thereof.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequence of ALD3, such as F295L; to the amino acid sequence of PMA1 Sequence modification, such as D591V; modification of the amino acid sequence of GLG2, such as Q144P; modification of the amino acid sequence of MIX23, such as T17K; modification of the amino acid sequence of ATG26, such as Y599C; modification of the amine of SYT1 Modification of the amino acid sequence, such as E126V; or modification of the amino acid sequence of SNF3, such as V509I.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequence of ARR3, such as N401K; to the amino acid sequence of TRS33 Modification of the sequence, such as T178K; modification of the amino acid sequence of GPR1, such as A622E or A622P; modification of the amino acid sequence of OCT1, such as R90P.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequence of TPO1, such as V256F; or to the amine group of MET2 Modification of acid sequences, such as Q343K, H372L or combinations thereof.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has a modification in its genome that results in a modification of the amino acid sequence of RPC82, such as D84G.

In the above modifications to the amino acid sequence, the numbers indicate the position of the amino acid change and which amino acid replaced the amino acid in the original peptide. For example, modifying R334G to ACH1 means that the 334th amino acid in ACH1 is changed from wild-type arginine (R) to glycine (G) in modified yeast. The symbols of the amino acids are shown in Table 2 below. Transforming fsN137 to ACH1 means deleting amino acid 137 (which is asparagine (N)) from the peptide sequence. Table 2. amino acids symbol alanine A Arginine R asparagine N aspartic acid D cysteine C Glutamine Q glutamate E glycine G Histidine H isoleucine I Leucine L lysine K methionine M Phenylalanine F proline P serine S threonine T Tryptophan W tyrosine Y Valine V

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae having modifications to its genome that result in modifications to the amino acid sequence of ACH1 or WAR1; and to amines of NHA1 or TRK1 Modification of amino acid sequences, such as: S144F, R8975, N750K, P11635, L764F or combinations thereof.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1 and NHA1.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1 and ALD3.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1, PMA1, and ARR3.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1 and TRK1.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1, TRK1, GLG2, and TR533.

In some embodiments, the yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae having modifications to its genome that result in modifications to the amino acid sequences of ACH1, NHA1, MIX23, GPR1, TPO1, and RPC82 .

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1, TRK1, and ATG26.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1, TRK1, ATG26, and GPR1.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1, NHA1, and SYT1.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of WAR1 and TRK1.

In some embodiments, a yeast with increased tolerance to acrylic acid may be Saccharomyces cerevisiae that has modifications to its genome that result in modifications to the amino acid sequences of ACH1, TRK1, SNF3, OCT1, and MET2.

In the above embodiments, in addition to the described modifications, the modified Saccharomyces cerevisiae may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least the same characteristics as wild-type Saccharomyces cerevisiae. 99% homology, up to 100% homology. Table 3. Sequence information for nucleic acid and protein modifications in Saccharomyces cerevisiae genome locus Transformation wild type protein Transformation Chr II, 194944 (Seq. ID 1) Wild type ACH1 (Seq. ID 2) Wild type Chr II, 194944 A1000G ACH1 R334G Chr II, 194944 G1099A ACH1 A367T Chr II, 194944 C1148A ACH1 T383K Chr II, 194944 G1057T ACH1 V353F Chr II, 194944 G1178A ACH1 G393E Chr II, 194944 C411 missing ACH1 fsN137 Chr II, 194944 C1315T ACH1 H439Y Chr II, 194944 C823A ACH1 Q275K Chr XIII, 112541 (Seq. ID 3) Wild type WAR1 (Seq. ID 4) Wild type Chr XIII, 112541 G2807T WAR1 W936L Chr XII, 419304 (Seq. ID 5) Wild type NHA1 (Seq. ID 6) Wild type Chr XII, 419304 C490G NHA1 H164D Chr XII, 419304 G868A NHA1 E290K Chr X, 173820 (Seq. ID 7) Wild type TRK1 (Seq. ID 8) Wild type Chr X, 173820 C431T TRK1 S144F Chr X, 173820 C2689A TRK1 R8975 Chr X, 173820 C2250G TRK1 N750K Chr X, 173820 C3487T TRK1 P11635 Chr X, 173820 A2292T TRK1 L764F

In Table 3, the engineering designations are similar to those used for peptides. For example, "A1000G" means that adenine (A) (the 1000th nucleotide) is replaced by guanine (G). The other nucleotides listed in the table above are cytosine (C) and thymine (T).

The following examples are covered: Example 1. A composition comprising yeast, acrylic acid, and yeast growth medium. Embodiment 2. The composition of embodiment 1, wherein the yeast growth medium comprises yeast extract. Embodiment 3. The composition of embodiment 1 or 2, wherein the yeast growth medium comprises carbohydrates. Embodiment 4. The composition of embodiment 1, 2 or 3, wherein the yeast growth medium comprises the peptide. Embodiment 5. The composition of embodiment 1, 2, 3 or 4, wherein the yeast growth medium contains antibiotics. Embodiment 6. The composition of embodiment 1, 2, 3, 4 or 5, wherein the yeast growth medium comprises phosphate or sulfate. Embodiment 7. The composition of embodiment 1, 2, 3, 4, 5 or 6, wherein the yeast growth medium comprises a buffer. Example 8. A method of preparing yeast with improved tolerance to acrylic acid, comprising allowing yeast to propagate in the presence of acrylic acid. Embodiment 9. The method of Embodiment 8, wherein the yeast is propagated in the presence of at least 0.05 g/L acrylic acid. Embodiment 10. The method of embodiment 8 or 9, wherein the concentration of acrylic acid is increased after the yeast has been allowed to propagate in the presence of acrylic acid for at least about 1 hour. Embodiment 11. The method of embodiment 8, 9 or 10, further comprising performing at least one additional propagation cycle, wherein the propagation cycle includes: increasing the concentration of acrylic acid, adding additional yeast growth medium and allowing the yeast to propagate again. Embodiment 12. The method of Embodiment 11, wherein an additional 1 to 100 breeding cycles are performed. Embodiment 13. The method of Embodiment 12, wherein an additional 30 to 40 breeding cycles are performed. Embodiment 14. The method of embodiment 8, 9, 10, 11, 12 or 13, wherein the yeast is propagated at a temperature of about 5°C to about 100°C. Embodiment 15. The method of embodiment 11, 12, 13 or 14, wherein the yeast is propagated in each propagation cycle for about 1 hour to about 200 hours. Embodiment 16. The method of Embodiment 15, wherein the yeast is propagated for about 20 hours to about 30 hours in each propagation cycle. Embodiment 17. The method of embodiment 11, 12, 13, 14, 15 or 16, wherein the reproduction cycle is stopped after there is no observable increase in growth rate during a two-week period. Embodiment 18. A yeast prepared by the method of embodiment 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. Embodiment 19. A yeast tolerant to acrylic acid, comprising a yeast having a non-naturally occurring genetic mutation or modification, wherein the yeast has an optical density of 0.2 at 600 nm in a reference acrylic acid composition. When the yeast is propagated so that the optical density of the yeast increases from 0.2 to 0.4 within 30 hours, the reference acrylic acid composition is composed of the following: 1.8 g/L acrylic acid, 20 g/L glucose, 5 g/L L (NH ₄ ) ₂ SO ₄ , 3 g/L KH ₂ PO ₄ , 0.5 g/L MgSO ₄ •7H ₂ O, 50 μg/L d-biotin, 1 mg/L D-pantothenate hemicalcium salt, 1 mg/L thiamine-HCl, 1 mg/L pyridoxine-HCl, 1 mg/L niacin, 0.2 mg/L 4-aminobenzoic acid, 25 mg/L m-inositol, 3 mg/ L FeSO ₄ ∙7H ₂ O, 4.5 mg/L ZnSO ₄ ∙7H ₂ O, 4.5 mg/L CaCl ₂ ∙2H ₂ O, 1 mg/L MnCl ₂ ∙4H ₂ O, 0.3 mg/L CoCl ₂ ∙6H ₂ O, 0.3 mg/L CuSO ₄ ∙5H ₂ O, 0.4 mg/L Na ₂ MoO ₄ ∙2H ₂ O, 1 mg/LH ₃ BO ₃ , 0.1 mg/L KI and 19 mg/L Na ₂ EDTA∙2H ₂ O. Embodiment 20. A genetically modified Saccharomyces cerevisiae having modifications to the wild-type genome that enable the Saccharomyces cerevisiae to synthesize the following: modified ACH1, WAR1, NHA1, TRK1, ALD3, PMA1, GLG2, MIX23, ATG26 , SYT1, SNF3, ARR3, TRS33, GPR1, OCT1, TPO1, MET2, RPC82, or combinations thereof. Embodiment 21. The genetically modified Saccharomyces cerevisiae of Embodiment 20, having modifications to the wild-type genome such that the Saccharomyces cerevisiae synthesizes modified ACH1. Embodiment 22. The genetically modified Saccharomyces cerevisiae of embodiment 21, wherein the modified ACH1 comprises changes to R334G, A367T, T383K, V353F, G393E, fsN137, H439Y, Q275K, or combinations thereof. Embodiment 23. The genetically modified Saccharomyces cerevisiae of embodiment 20, 21 or 22, having modifications to the wild-type genome such that the Saccharomyces cerevisiae synthesizes modified WAR1. Embodiment 24. The genetically modified Saccharomyces cerevisiae of embodiment 23, wherein the modified WAR1 comprises the change to W936L. Embodiment 25. The genetically modified Saccharomyces cerevisiae of embodiment 20, 21, 22, 23 or 24, which has modifications to the wild-type genome such that the Saccharomyces cerevisiae synthesizes modified NHA1. Embodiment 26. The genetically modified Saccharomyces cerevisiae of embodiment 25, wherein the modified ACH1 comprises changes to H164D, E290K, or a combination thereof. Embodiment 27. The genetically modified Saccharomyces cerevisiae of Embodiment 20, 21, 22, 23, 24, 25 or 26, which has modifications to the wild-type genome such that the Saccharomyces cerevisiae synthesizes the modified TRK1. Embodiment 28. The genetically modified Saccharomyces cerevisiae of embodiment 27, wherein the modified ACH1 comprises changes to S144F, R8975, N750K, P11635, L764F, or combinations thereof. Embodiment 29. A method for preparing acrylic acid, which is contained in the yeast of embodiment 18 or 19 or the genetically modified Saccharomyces cerevisiae of embodiment 20, 21, 22, 23, 24, 25, 26, 27 or 28. Hydroxypropionic acid is dehydrated in the presence of

Examples Example 1 Methods Strains, DNA manipulations and media Strains Saccharomyces cerevisiae CEN.PK113-7D (MATa MAL2-8c SUC2), CEN.PK113-5D (MATa MAL2-8c SUC2) and CEN.PK113-1A (MATa MAL2-8c SUC2) were obtained from Scientific Research and Development GmbH (Oberursel, Germany). A strain based on CEN.PK113-7D (GL01) was obtained from (Pereira et al., 2019) and used in ALE experiments. Escherichia coli DH5α was used for plastid isolation and maintenance.

Oligonucleotides were purchased from IDT or Genscript. PCR was performed using Platinum SuperFi II DNA polymerase from ThermoFisher. Plasmid extraction and PCR product purification were performed using respective kits from QIAGEN.

Selection and maintenance of plastids in E. coli were carried out in LB medium containing 10 g/L proteinaceous, 10 g/L NaCl, 5 g/L yeast extract and supplemented with 100 mg/L ampicillin sodium salt. Solid LB also includes 16 g/L agar.

For selection of S. cerevisiae strains with KANMX markers, YPD medium containing 10 g/L yeast extract, 20 g/L protease, 20 g/L glucose supplemented with 200 mg/L G418 bisulfate (SigmaAldrich) was used. For selection using Ura markers, standard minimal medium was prepared without the addition of uracil. Prepare a solid version of either medium by adding 20 g/L agar.

All growth and evolution experiments were performed in minimal medium as described by (Verduyn et al., 1992) containing the following: 20 g/L glucose, 5 g/L (NH ₄ ) ₂ SO ₄ , 3 g/L KH ₂ PO _4. 0.5 g/L MgSO ₄ ·7H ₂ O, 1 mL/L vitamin solution and 1 mL/L trace metal solution. The pH of the medium was adjusted to 5.0 with KOH and buffered at this pH by adding 97 mL/L 0.5 M citric acid and 103 mL/L 1 M Na ₂ HPO ₄ .

AA was adjusted to pH 5 using KH ₂ PO ₄ and made to a stock concentration of 0.1 g/mL.

Growth Screen All growth tests were performed in a Growth Mapper 960 (Enzyscreen) using 96 semi-deep well microplates with a culture volume of 250 μL, agitation at 250 rpm, temperature control at 30°C and an initial OD600 of 0.05. The maximum specific growth rate of each strain was calculated by finding the highest slope in the natural logarithm of OD600 versus time curve.

Adaptive laboratory evolution (ALE) evolved strain GL01 using continuous culture in media containing acrylic acid at increasing concentrations from 0.3 g/L to 2 g/L. Evolution of six independent cultures. As S. cerevisiae reproduces, it initially uses glucose as food. This is called the glucose phase. When glucose is depleted, S. cerevisiae uses ethanol as food. This is called the ethanol stage. In this experiment, two initial AA concentrations were chosen so that within 24 hours, three of the six cultures would be in the ethanol phase and the other three would be in the glucose phase. Cultures were continuously propagated in 30 mL flasks in a 30 °C shaker at 200 rpm.

The OD600 of each culture was measured every 24 hours and the volume of old culture to be transferred to fresh medium was calculated so that the starting OD600 of the new culture would be 0.1. Adjust the AA concentration in the new culture so that it will reach the designated growth phase (ethanol or glucose) within twenty-four hours. The end OD600 of each flask was recorded, and the glycerol stock solution of each culture was stored in a -80 freezer. The evolution experiment was stopped when there was no observable increase in growth rate during the two-week period (as reflected by an increase in termination OD600).

DNA Extraction and Sequencing Pure lines isolated from the evolving population were cultured overnight in YPD medium. Genes were extracted from 3 mL of overnight yeast culture (approximately 5 × 10 ⁸ cells) using the Blood & Cell Culture DNA Mini Kit (Qiagen, location) using the manufacturer's recommended protocol. body DNA. Pair-end sequencing was performed on an Illumina® sequencing platform with read length PE150 bp at each end and 100× genome coverage for each sample.

Sequencing data analysis The effective sequencing data was compared with the reference sequence (Saccharomyces cerevisiae S288c; R64-2-1) through BWA software. Use SAMtools to detect SNPs and InDels. Use BreakDancer to detect SV. Use CNVnator to detect CNV. Use ANNOVAR to annotate all variants. Custom tools were used to subtract wild-type (GL01) control background.

Results Growth of evolved yeast strains After 35 passages of ALE, the evolved strains (35E and 35G) were tested in minimal medium (pH 5) at various AA concentrations, with wild-type strain GLO1 as control. Table 2. Comparison of growth rates between evolved strains and wild type [AA] (g/L) strains μ _max (h ^-1 ) Lag time (h) 0 35E 0.43 ±0.01 1 35G 0.43 ± 0.00 1 GL01 0.41±0.00 1 0.7 35E 0.23 ± 0.04 3.3 ± 1.2 35G 0.23 ± 0.03 2.7±1.0 GL01 0.08±0.00 8.8±1.1 1.8 35E 0.10 ± 0.02 9.2 ± 3.1 35G 0.11±0.03 6.7 ± 2.8 GL01 0.025 ± 0.003 22±2.8

Sequencing of evolved yeast strains The variants detected in the AA evolved strain with glucose phase transfer are presented in Table 3. Strain names begin with the passage number, where G indicates glucose phase transfer and E indicates ethanol phase transfer. For numbers following letters, the first digit is the culture number (1 to 3) and the second digit is the isolation number (1 to 8). Table 3. Examples of detected variants locus Gene change 91G14 Chr I, 225859 PHO11 N134D (A-->G) Chr II, 194944 ACH1 Q275K (C-->A) Chr III, 270021 FIG2 T863R (C-->G) Chr V, 20192 DSF1 Q202X (C-->T) Chr XI, 68366 PTK1 S619P (T-->C) Chr VII, 1082465 COS6 V89F (G-->T) Chr XII, 419304 NHA1 E290K (G-->A) Chr XVI, 724341 SYT1 E126V (A-->T) 91G21 Chr I, 225859 PHO11 N134D (A-->G) Chr XIII, 112541 WAR1 W936L (G-->T) Chr III, 270021 FIG2 T863R (C-->G) Chr V, 20192 DSF1 Q202X (C-->T) Chr XI, 68366 PTK1 S619P (T-->C) Chr VII, 1082465 COS6 V89F (G-->T) Chr X, 173820 TRK1 P1163S (C-->T) 91G33 Chr I, 225859 PHO11 N134D (A-->G) Chr II, 194531 ACH1 N137fs (C411 missing) Chr IV, 113104 SNF3 V509I (G-->A) Chr XI, 191174 OCT1 R90P (G-->C) Chr XIV, 118375 MET2 Q343K (C-->A) Chr III, 270021 FIG2 T863R (C-->G) Chr V, 20125 DSF1 E179D (A-->T) Chr XI, 68366 PTK1 S619P (T-->C) Chr VII, 1082477 COS6 V85I (G-->A) Chr X, 175015 TRK1 L764F (A-->T)

Example 2 Reverse engineered strains with point mutations or single nucleotide insertions/deletions can be constructed using the single gRNA approach described in (Laughery et al., 2015). Web-based resources are available to help select optimal gRNA sites based on proximity to the nucleotide to be substituted and the likelihood of using synonymous substitutions to inactivate the PAM site. The repair template can be designed to introduce the desired mutation and render the PAM site inactive, and synthesized as two complementary oligonucleotides. The desired 20 bp gRNA target sequence (without PAM sites) flanked by two 50 bp sequences and the gRNA sites on the available plasmids can be synthesized into two complementary 120 bp oligonucleotides Same origin. The pair of complementary oligonucleotides can be mixed in equimolar amounts and annealed by heating the mixture to 95°C for 5 min and allowing it to cool at room temperature. Double-stranded gRNA inserts can be cloned into suitable plasmids capable of Cas9 expression and gRNA expression using standard methods such as Gibson Assembly. Depending on the selection marker on the plastid, 100 ng of each gRNA+Cas9 plastid can be co-transformed with 2 μg of the double-stranded repair fragment of the respective gene using a high-efficiency protocol such as (Gietz and Woods, 2006). Suitable for strains. Transformation mixtures can be spread on culture plates containing the appropriate drug of choice depending on the strain and plastids. Individual communities can be selected, and PCR amplification of the modified regions can be performed. Sequencing of PCR products allows confirmation of successful introduction of each mutation.

References: Xu, X., Williams, T. C., et al. 2019 Biotechnology for Biofuels 12: 97 Pereira, R., Wei, Y., et al. 2019 Metabolic Engineering 56: 130 Pereira, R., Mohamed, E. T., et al. 2020 PNAS 117: 27954 WO2002042418 Laughery, M.F., Hunter, T., et al. 2015 Yeast 32: 711 Gietz, R.D., Woods, R.A. 2006 Yeast Protocols. Humana Press, New Jersey, pp. 107-120

Unless otherwise indicated, all numbers used herein expressing amounts of ingredients, properties (such as molecular weight, reaction conditions, etc.) are to be understood as being modified in all instances by the term "about." Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless indicated to the contrary, numerical parameters may be modified according to the desired characteristics sought to be achieved, and therefore shall be considered a part of this invention. At the very least, the examples shown herein are for illustration only and are not intended to limit the scope of the invention.

Unless otherwise indicated herein or clearly contradicted by context, the terms "a/an", "the" and "an" are used in the context of describing embodiments of the invention (especially in the context of the following embodiments) Similar referents are to be construed to cover both the singular and the plural. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or representative language (eg, "such as") provided herein is intended merely to better illuminate embodiments of the invention and does not pose a limitation on the scope of any embodiment. No language in the specification should be construed as indicating any non-disclosed element necessary to practice the embodiments of the invention.

Alternative elements or groupings of embodiments disclosed herein should not be construed as limitations. Each group member may be referenced and embodied individually or in any combination with other members of the group or other elements found herein. It is contemplated that one or more members of a group may be included in or removed from the group for convenience and/or patentability reasons.

Certain embodiments are described herein, including the best mode known to the inventors for carrying out the embodiments. Of course, variations to the described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend for the embodiments of the invention to be practiced otherwise than as specifically described herein. Accordingly, the embodiments include all modifications and equivalents of the subject matter recited in the embodiments as permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations thereof is encompassed unless otherwise indicated herein or otherwise clearly contradicted by context.

In summary, it is to be understood that the embodiments disclosed herein illustrate the principles of the embodiments. Other modifications may be employed within the scope of the embodiments. Accordingly, by way of example and not limitation, alternative embodiments may be utilized in accordance with the teachings herein. Therefore, embodiments are not limited to those exactly as shown and described.

Cross-Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63/261,648, filed on September 24, 2021, which is incorporated herein by reference in its entirety.

Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on September 17, 2021 is named N3253_10150US01_SL.txt and is 46,904 bytes in size.

Figure 1 is a graph depicting the growth of wild-type and evolved Saccharomyces cerevisiae strains in 1.8 g/L acrylic acid.

TW202328430A_111136221_SEQL.xml

Claims (20)

A composition comprising yeast, acrylic acid and yeast growth medium. The composition of claim 1, wherein the yeast growth medium contains yeast extract. The composition of claim 1 or 2, wherein the yeast growth medium contains carbohydrates. The composition of claim 1, 2 or 3, wherein the yeast growth medium contains the peptide. The composition of claim 1, 2, 3 or 4, wherein the yeast growth medium contains antibiotics. The composition of claim 1, 2, 3, 4 or 5, wherein the yeast growth medium contains phosphate or sulfate. The composition of claim 1, 2, 3, 4, 5 or 6, wherein the yeast growth medium contains a buffer. A method of preparing yeast with improved tolerance to acrylic acid, comprising allowing the yeast to propagate in the presence of acrylic acid. The method of claim 8, wherein the yeast is propagated in the presence of at least 0.05 g/L acrylic acid. The method of claim 8 or 9, wherein the concentration of acrylic acid is increased after the yeast has been allowed to propagate in the presence of acrylic acid for at least about 1 hour. The method of claim 8, 9 or 10, further comprising performing at least one additional propagation cycle, wherein the propagation cycle includes: increasing the concentration of acrylic acid, adding additional yeast growth medium and allowing the yeast to propagate again. The method of claim 11, wherein an additional 30 to 40 breeding cycles are performed. The method of claim 11 or 12, wherein the yeast is allowed to propagate in each propagation cycle for about 20 hours to about 30 hours. A yeast prepared by the method of claim 8, 9, 10, 11, 12 or 13. A yeast that is tolerant to acrylic acid, comprising a yeast having a non-naturally occurring genetic mutation or modification, wherein the yeast has an optical density of 0.2 at 600 nm in a reference acrylic acid composition. The characteristic of propagating such that the optical density of the yeast increases from 0.2 to 0.4 within 30 hours, wherein the reference acrylic acid composition consists of the following components: 1.8 g/L acrylic acid, 20 g/L glucose, 5 g/L ( NH ₄ ) ₂ SO ₄ , 3 g/L KH ₂ PO ₄ , 0.5 g/L MgSO ₄ •7H ₂ O, 50 μg/L d-biotin, 1 mg/L D-pantothenic acid hemicalcium salt, 1 mg/ L Thiamine-HCl, 1 mg/L Pyridoxin-HCl (Pyridoxin-HCl), 1 mg/L niacin, 0.2 mg/L 4-aminobenzoic acid, 25 mg/L m-inositol, 3 mg/L FeSO ₄ ∙7H ₂ O, 4.5 mg/L ZnSO ₄ ∙7H ₂ O, 4.5 mg/L CaCl ₂ ∙2H ₂ O, 1 mg/L MnCl ₂ ∙4H ₂ O, 0.3 mg/L CoCl ₂ ∙6H ₂ O, 0.3 mg/L CuSO ₄ ∙5H ₂ O, 0.4 mg/L Na ₂ MoO ₄ ∙2H ₂ O, 1 mg/LH ₃ BO ₃ , 0.1 mg/L KI and 19 mg/L Na ₂ EDTA ∙2H ₂ O. A genetically modified Saccharomyces cerevisiae ( S. cerevisiae ), which has modifications to the wild-type genome that enable the S. cerevisiae to synthesize the following: modified ACH1, WAR1, NHA1, TRK1, ALD3, PMA1, GLG2, MIX23, ATG26, SYT1, SNF3, ARR3, TRS33, GPR1, OCT1, TPO1, MET2, RPC82, or combinations thereof. For example, the genetically modified Saccharomyces cerevisiae of claim 16 has modifications to the wild-type genome that enable the Saccharomyces cerevisiae to synthesize the modified ACH1. For example, the genetically modified Saccharomyces cerevisiae of Claim 16 or 17 has modifications to the wild-type genome that enable the Saccharomyces cerevisiae to synthesize the modified WAR1. For example, the genetically modified Saccharomyces cerevisiae of claim 16, 17 or 18 has modifications to the wild-type genome that enable the Saccharomyces cerevisiae to synthesize the modified NHA1. For example, the genetically modified Saccharomyces cerevisiae of claim 16, 17, 18 or 19 has modifications to the wild-type genome that enable the Saccharomyces cerevisiae to synthesize the modified TRK1.