Inventors: JIADONG ZHOU

CROSS-REFERENCE TO RELATED APPLICATIONS

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

SEQUENCE LISTING

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

FIELD

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.

BACKGROUND

Acrylic acid (AA) is a commercially important chemical. By 2025, the production capacity of AA is expected to reach 9 million tons, and its market size $20 billion. Its current production depends on a petrochemical process, which is unsustainable and environmentally unfriendly. There has been a great amount of interest to produce AA from renewable non-food biomass.

Combining bio- and chemo-catalytic processes has been attempted. In one approach, lactic acid (LA) was first produced microbially. But the subsequent AA production by the dehydration of LA proved difficult due to its hydrolysis resistance. More recently, AA production by dehydration of 3-hydroxypropionic acid (3-HP) produced by microbial fermentation was achieved. This combined process does not offer substantial benefits over the petroleum-based production, due to its high-energy consumption and additional costs from catalysts, purification, and separation steps.

To develop economically attractive processes for production of bulk chemicals from renewable resources, three features are considered: high product yields, high productivity, and high product titers. The latter property is important in order to minimize capital equipment and downstream separation costs for product purification. Titers of bulk chemicals in economical fermentation processes often exceed 100 g/L.

SUMMARY

Some embodiments include a method of preparing a 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 a yeast, acrylic acid, and a yeast growth media.

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

Some embodiments include a yeast that is tolerant to acrylic acid, comprising a yeast with a non-naturally occurring genetic mutation or modification, wherein the yeast has the property that, when the yeast has an optical density of 0.2 at 600 nm in a reference acrylic acid composition, the yeast propagates so 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 1.8 g/L of acrylic acid, 20 g/L of glucose, 5 g/L of (NH ₄ ) ₂ SO ₄ , 3 g/L of KH ₂ PO ₄ , 0.5 g/L of MgSO ₄ «7H ₂ O, 50 pg/L of d-Biotin, 1 mg/L of D-pantothenic acid hemicalcium salt, 1 mg/L of Thiamin-HCI, 1 mg/L of Pyridoxin-HCI, 1 mg/L of nicotinic acid, 0.2 mg/L of 4-aminobenzoic acid, 25 mg/L of m-inositol, 3 mg/L of FeSO ₄ -7H ₂ O, 4.5 mg/L of ZnSO ₄ -7H ₂ O, 4.5 mg/L of CaCI ₂ -2H ₂ O, 1 mg/L of MnCI ₂ -4H ₂ O, 0.3 mg/L of COCI ₂ -6H ₂ O, 0.3 mg/L of CuSO ₄ -5H ₂ O, 0.4 mg/L of Na ₂ MoO ₄ -2H ₂ O, 1 mg/L of H3BO3, 0.1 mg/L of KI, and 19 mg/L of Na ₂ EDTA-2H ₂ O.

Some embodiments include a genetically modified Saccharomyces cerevisiae (5. cerevisiae) having a modification to the genome of the wild-type that results in the 5. cerevisiae synthesizing a modified ACH1, WAR1, NHA1, TRK1, ALD3, PMA1, GLG2, MIX23, ATG26, SYT1, SNF3, ARR3, TRS33, GPR1, OCTI, TP01, MET2, RPC82, or a combination thereof.

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

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot depicting the growth of wild-type and evolved 5. cerevisiae strains in 1.8 g/L of acrylic acid.

DETAILED DESCRIPTION

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

The yeast comprises any type of yeast in need of improved tolerance in the presence of acrylic acid, such as 5. cerevisiae.

A yeast growth media may contain a yeast extract, a carbohydrate, a peptide, and antibiotic, a phosphate salt, a sulfate salt, or a combination thereof. In some embodiments, the yeast growth media comprises a yeast extract, a carbohydrate, a peptide, and antibiotic, a phosphate salt, and a sulfate salt.

A yeast growth media may include any suitable yeast extract, e.g. the cell contents of yeast without the cell walls.

A yeast growth media may include any suitable carbohydrate including a monosaccharide (such as glucose), a disaccharide (such as sucrose), a polysaccharide (such as a starch), etc. In some embodiments, the carbohydrate includes agar, glucose, or a combination thereof. In some embodiments, the carbohydrate includes agar. In some embodiments, the carbohydrate includes glucose.

A yeast growth media may include any suitable peptide, such as a peptone, e.g. a water- soluble product of partial hydrolysis of a protein.

A yeast growth media may include any suitable antibiotic, such as G418 disulfate, ampicillin sodium, or a combination thereof.

A yeast growth media may include any suitable phosphate salt, such as KH2PO4. A yeast growth media may include any suitable sulfate salt, such as (NHzihSC , MgSO4-7H2O, or a combination thereof.

A yeast growth media may include a vitamin solution, e.g. at a concentration of about 0.1-10 mL/L, about 0.1-2 mL/L, about 2-4 mL/L, about 4-6 mL/L, about 6-8 mL/L, about 8-10 mL/L, or about 1 mL/L of a vitamin solution.

A suitable vitamin solution may contain d-Biotin (e.g., in an amount so that the yeast growth media contains about 1-100 pg/L, about 40-60 pg/L, or about 50 pg/L of d-Biotin), D- pantothenic acid (e.g., in an amount so that the yeast growth media contains about 0.1-10 mg/L, about 0.5-1.5 mg/L, or about 1 mg/L of a hemicalcium salt, or a molar equivalent amount of another form), Thiamin (e.g., in an amount so that the yeast growth media contains about 0.1-10 mg/L, about 0.5-1.5 mg/L, or about 1 mg/L of an HCI salt, or a molar equivalent amount of another form), Pyridoxin (e.g., in an amount so that the yeast growth media contains about 0.1-10 mg/L, about 0.5-1.5 mg/L, or about 1 mg/L of an HCI salt, or a molar equivalent amount of another form), 1 mg/L of nicotinic acid (e.g., in an amount so that the yeast growth media contains about 0.1-10 mg/L, about 0.5-1.5 mg/L, or about 1 mg/L of the acid form, or a molar equivalent amount of a salt form), 4-aminobenzoic acid (e.g., in an amount so that the yeast growth media contains about 0.05-0.5 mg/L, about 0.1-0.3 mg/L, or about 0.2 mg/L of the acid form, or a molar equivalent amount of a salt form), m-inositol (e.g., in an amount so that the yeast growth media contains about 5-200 mg/L, about 20-30 mg/L, or about 25 mg/L), or a combination thereof.

Some embodiments include d-Biotin (e.g., in an amount so that the yeast growth media contains 50 pg/L), D-pantothenic acid hemicalcium salt (e.g., in an amount so that the yeast growth media contains 1 mg/L), Thiamin-HCI (e.g., in an amount so that the yeast growth media contains 1 mg/L), Pyridoxin-HCI (e.g., in an amount so that the yeast growth media contains 1 mg/L), nicotinic acid (e.g., in an amount so that the yeast growth media contains 1 mg/L), 4- aminobenzoic acid (e.g., in an amount so that the yeast growth media contains 0.2 mg/L), and m-inositol (e.g., in an amount so that the yeast growth media contains 25 mg/L). A yeast growth media may include a trace metal solution, e.g. at a concentration of about 0.1-10 mL/L, about 0.1-2 mL/L, about 2-4 mL/L, about 4-6 mL/L, about 6-8 mL/L, about 8- 10 mL/L, or about 1 mL/L of trace metal solution.

A suitable trace metal solution may contain FeSC (e.g., in an amount so that the yeast growth media contains 0.5-10 mg/L, about 2-4 mg/L, or about 3 mg/L of the -7H2O form, or a molar equivalent amount of another form), ZnSC (e.g., in an amount so that the yeast growth media contains about 1-10 mg/L, about 4-5 mg/L, or about 4.5 mg/L of the -7H2O form, or a molar equivalent amount of another form), CaC (e.g., in an amount so that the yeast growth media contains about 1-10 mg/L, about 4-5 mg/L, or about 4.5 mg/L of the -2H2O form, or a molar equivalent amount of another form), MnC (e.g., in an amount so that the yeast growth media contains about 0.1-10 mg/L, about 0.5-1.5 mg/L, or about 1 mg/L of the H2O form, or a molar equivalent amount of another form), C0CI2 (e.g., in an amount so that the yeast growth media contains about 0.05-6 mg/L, about 0.2-0.4 mg/L, or about 0.3 mg/L of the -61-120 form, or a molar equivalent amount of another form), CUSO4 (e.g., in an amount so that the yeast growth media contains about 0.05-6 mg/L, about 0.2-0.4 mg/L, or about 0.3 mg/L of the -5H2O form, or a molar equivalent amount of another form), Na2MoO4 (e.g., in an amount so that the yeast growth media contains about 0.05-8 mg/L, about 0.3-0.5 mg/L, or about 0.4 mg/L of the •2H2O form, or a molar equivalent amount of another form), H3BO3 (e.g., in an amount so that the yeast growth media contains about 0.1-10 mg/L, about 0.5-1.5 mg/L, or about 1 mg/L of H3BO3, or molar equivalent amount of a salt thereof), KI (e.g., in an amount so that the yeast growth media contains about 0.01-1 mg/L, about 0.05-0.15 mg/L, or about 0.1 mg/L of KI, or a molar equivalent amount of another iodide salt), and Na2EDTA-2H2O (e.g., in an amount so that the yeast growth media contains about 1-40 mg/L, about 10-30 mg/L, or about 19 mg/L of the •2H2O form, or a molar equivalent amount of another form), or a combination thereof.

In some embodiments, the trace metal solution contains FeSC (e.g., in an amount so that the yeast growth media contains 0.5-10 mg/L, about 2-4 mg/L, or about 3 mg/L of the •7H2O form, or a molar equivalent amount of another form), ZnSC (e.g., in an amount so that the yeast growth media contains about 1-10 mg/L, about 4-5 mg/L, or about 4.5 mg/L of the •7H2O form, or a molar equivalent amount of another form), CaC (e.g., in an amount so that the yeast growth media contains about 1-10 mg/L, about 4-5 mg/L, or about 4.5 mg/L of the •2H2O form, or a molar equivalent amount of another form), MnC (e.g., in an amount so that the yeast growth media contains about 0.1-10 mg/L, about 0.5-1.5 mg/L, or about 1 mg/L of the •4H2O form, or a molar equivalent amount of another form), C0CI2 (e.g., in an amount so that the yeast growth media contains about 0.05-6 mg/L, about 0.2-0.4 mg/L, or about 0.3 mg/L of the -6H2O form, or a molar equivalent amount of another form), CuSC (e.g., in an amount so that the yeast growth media contains about 0.05-6 mg/L, about 0.2-0.4 mg/L, or about 0.3 mg/L of the -5H2O form, or a molar equivalent amount of another form), Na2MoO4 (e.g., in an amount so that the yeast growth media contains about 0.05-8 mg/L, about 0.3-0.5 mg/L, or about 0.4 mg/L of the -2H2O form, or a molar equivalent amount of another form), H3BO3 (e.g., in an amount so that the yeast growth media contains about 0.1-10 mg/L, about 0.5-1.5 mg/L, or about 1 mg/L), KI (e.g., in an amount so that the yeast growth media contains about 0.01-1 mg/L, about 0.05-0.15 mg/L, or about 0.1 mg/L), and Na2EDTA-2H2O (e.g., in an amount so that the yeast growth media contains about 1-40 mg/L, about 10-30 mg/L, or about 19 mg/L of the •2H2O form, or a molar equivalent amount of another form).

Some embodiments include FeSO4-7H2O (e.g., in an amount so that the yeast growth media contains 3 mg/L), ZnSO4-7H2O (e.g., in an amount so that the yeast growth media contains 4.5 mg/L), CaCl2-2H2O (e.g., in an amount so that the yeast growth media contains 4.5 mg/L), MnCl2-4H2O (e.g., in an amount so that the yeast growth media contains 1 mg/L), CoCl2-6H2O (e.g., in an amount so that the yeast growth media contains 0.3 mg/L), CuSG -SFhO (e.g., in an amount so that the yeast growth media contains 0.3 mg/L), Na2MoO4-2H2O (e.g., in an amount so that the yeast growth media contains 0.4 mg/L), H3BO3 (e.g., in an amount so that the yeast growth media contains 1 mg/L ), KI (e.g., in an amount so that the yeast growth media contains 0.1 mg/L), Na2EDTA-2H2O (e.g., in an amount so that the yeast growth media contains 19 mg/L), or a combination thereof.

Some embodiments include FeSO4-7H2O (e.g., in an amount so that the yeast growth media contains 3 mg/L), ZnSO4-7H2O (e.g., in an amount so that the yeast growth media contains 4.5 mg/L), CaCh-ZF O (e.g., in an amount so that the yeast growth media contains 4.5 mg/L), MnCl2-4H2O (e.g., in an amount so that the yeast growth media contains 1 mg/L), CoC -GHzO (e.g., in an amount so that the yeast growth media contains 0.3 mg/L), CuSO4-5H2O (e.g., in an amount so that the yeast growth media contains 0.3 mg/L), Na2MoO4-2H2O (e.g., in an amount so that the yeast growth media contains 0.4 mg/L), H3BO3 (e.g., in an amount so that the yeast growth media contains 1 mg/L ), KI (e.g., in an amount so that the yeast growth media contains 0.1 mg/L), and Na2EDTA-2H2O (e.g., in an amount so that the yeast growth media contains 19 mg/L).

The pH of a composition for ALE may be adjusted to about 4-6, such as about 5 with KOH or another base, and buffered, e.g., with a citric acid and/or phosphate buffer, such as 97 mL/L 0.5 M Citric Acid and 103 mL/L of IM Na ₂ HPO ₄ .

To prepare a yeast with improved tolerance to acrylic acid, the yeast in a composition for ALE is allowed to propagate in the presence of acrylic acid. Generally, the ALE process starts with a lower concentration of acrylic acid, and the concentration of acrylic acid is gradually increased over time to encourage evolution of new yeast organisms with increased tolerance toward acrylic acid.

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

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

During the ALE process, the yeast in the composition for ALE is allowed to propagate. This may occur at any suitable temperature, such as at a temperature of about 1-200 °C, about 5-100 °C, about 100-150 °C, about 150-200 °C, about 1-10 °C, about 10-20 °C, about 20-30 °C, about 25-35°C, about 30-40 °C, about 40-60 °C, about 60-80 °C, or about 80-100 °C. The increase in the acrylic acid may be carried out by a gradual, continuous process, e.g. continuous additions of a small amount of acrylic acid over time, or by a stepwise process, e.g. addition of increased amounts of acrylic acid at discreet intervals, although in the stepwise process, the interval and the amount of acrylic acid could vary from one addition to another.

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

For continuous addition of acrylic acid, the rate of increase of acrylic acid concentration may be, for example, 58-5787 ng-L ^_1 -s ^-1 , about 58-116 ng-L ^-1 -s ^-1 , about 116-231 ng-L ^-1 -s ^-1 , about 231-347 ng-L^-s ¹ , about 347-463 ng-L^-s ¹ , about 463-579 ng-L^-s ¹ , about 579-694 ng-L^-s ¹ , about 694-810 ng-L^-s ¹ , about 810-926 ng-L^-s ¹ , about 926-1042 ng-L^-s ¹ , about 1042-1157 ng-L ^-1 -s ^-1 , about 1157-2315 ng-L ^-1 -s ^-1 , about 2315-3472 ng-L ^-1 -s ^-1 , about 3472-4630 ng-L ^-1 -s ^-1 , or about 4630-5787 ng-L^-s ¹ .

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

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

A stepwise addition of acrylic acid may be carried out in a series of propagation cycles. A propagation cycle comprises increasing the concentration of acrylic acid, adding additional yeast growth media, and allowing the yeast to propagate again. Any suitable amount of propagation cycles may be carried out, such as an additional 1-100, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 propagation cycles. An additional propagation cycle is the first increase in the concentration of acrylic acid, addition of additional yeast growth media, and allowing the yeast to propagate after the yeast had been allowed to propagate for an interval at the initial conditions. A propagation cycle may be carried out over any suitable period of time, such as at least 1 hour, about 1 hour to about 4 weeks, about 1-200 hours, about 1-8 hours, about 8-16 hours, about 16-24 hours, about 24-30 hours, about 30-36 hours, about 36-48 hours, about 48-72 hours, about 72-96 hours, about 1-7 days, about 7-14 days, about 2-4 weeks, about 20-30 hours, or about 24 hours.

While a propagation cycle may be carried out in a number of ways, in some embodiments, a volume of the ALE composition is diluted, e.g. by transfer, into a fresh media with a higher acrylic acid concentration, such as an increased amount of acrylic acid corresponding to a rate of increase in acrylic acid concentration recited in a paragraph above. The dilution may be done to achieve a certain yeast level, such as a yeast level associated with an OD600 of about 0.01-0.2, about 0.01-0.05, about 0.05-0.1, about 0.1-0.15, about 0.15-2, about 0.08-0.12 or about 0.1.

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

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

The methods described herein may be used to prepare an altered yeast, such as a yeast (e.g., 5. cerevisiae) that is tolerant to acrylic acid. In some embodiments, such a yeast comprises a yeast with a genetic mutation or modification that is not naturally occurring, wherein the yeast has the property that, when the yeast has an optical density of 0.2 at 600 nm in a reference acrylic acid composition, the yeast propagates so 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 1.8 g/L of acrylic acid, 20 g/L of glucose, 5 g/L of (NF hSC , 3 g/L of KH2PO4, 0.5 g/L of MgSO4*7H2O, 50 pg/L of d-Biotin, 1 mg/L of D-pantothenic acid hemicalcium salt, 1 mg/L of Thiamin-HCI, 1 mg/L of Pyridoxin-HCI, 1 mg/L of nicotinic acid, 0.2 mg/L of 4-aminobenzoic acid, 25 mg/L of m-inositol, 3 mg/L of FeSC - FhO, 4.5 mg/L of ZnSC - FhO, 4.5 mg/L of CaCl2-2H2O, 1 mg/L of MnCl2-4H ₂ O, 0.3 mg/L of CoCI ₂ -6H ₂ O, 0.3 mg/L of CuSO ₄ -5H ₂ O, 0.4 mg/L of Na ₂ MoO ₄ -2H ₂ O, 1 mg/L of H3BO3, 0.1 mg/L of KI, and 19 mg/L of Na ₂ EDTA-2H ₂ O. The yeast described herein may be useful for preparing acrylic acid, such as by fermenting hydroxypropionic acid in the presence of the yeast.

In some embodiments, the genetically modified or mutated yeast does not produce an acyl-CoA compound of formula R-(C=O)-COA, wherein R is a carbon chain of 5 atoms or fewer. In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of one of the proteins or peptides in Table 1 below.

Table 1. In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1, such as: R334G, A367T, T383K, V353F, G393E, fsN137, H439Y, Q275K, or a combination thereof; or a modification to the amino acid sequence of WAR1, such as W936L.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of NHA1, such as: H164D, E290K, or combination thereof; or a modification to the amino acid sequence of TRK1, such as: S144F, R8975, N750K, P11635, L764F, or a combination thereof.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ALD3, such as F295L; a modification to the amino acid sequence of PMAl, such as D591V; a modification to the amino acid sequence of GLG2, such as Q144P; a modification to the amino acid sequence of MIX23, such as T17K; a modification to the amino acid sequence of ATG26, such as Y599C; a modification to the amino acid sequence of SYT1, such as E126V; or a modification to the amino acid sequence of SNF3, such as V509I.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ARR3, such as N401K; a modification to the amino acid sequence of TRS33, such as T178K; a modification to the amino acid sequence of GPR1, such as A622E or A622P; a modification to the amino acid sequence of OCTI, such as R90P.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of TPO1, such as V256F; or a modification to the amino acid sequence of MET2, such as Q343K, H372L, or a combination thereof.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of RPC82, such as D84G. In the above modifications to the amino acid sequences, the notation indicates the position of the amino acid change and which amino acid replaces the amino acid in the original peptide. For example, the modification R334G to ACH1, means that in ACH1, the 334 ^th amino acid is changed from arginine (R) in the wild-type to glycine (G) in the modified yeast. The symbols for the amino acids are shown in the Table 2 below. The modification fsN137 to ACH1 means that the 137 ^th amino acid, which is asparagine (N), is deleted from the peptide sequence.

Table 2.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1 or WAR1; and a modification to the amino acid sequence of NHA1 or TRK1, such as: S144F, R8975, N750K, P11635, L764F, or a combination thereof.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1 and NHA1.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1 and ALD3.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1, PMAl, and ARR3.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1 and TRK1.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1, TRK1, GLG2, and TR533.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1, NHA1, MIX23, GPR1, TPO1, and RPC82.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1, TRK1, and ATG26. In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1, TRK1, ATG26, and GPR1.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1, NHA1, and SYT1.

In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of WAR1 and TRK1. In some embodiments, a yeast having increased tolerance to acrylic acid may be a 5. cerevisiae having a modification its genome that results in a modification to the amino acid sequence of ACH1, TRK1, SNF3, OCTI, and MET2.

In the above embodiments, other than the modifications described, the modified 5. cerevisiae may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at or least 99% homology to the wild-type 5. cerevisiae, up to 100% homology.

Table 3. Sequence Information for Nucleic Acids and Protein Modifications to 5. cerevisiae

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

The following embodiments are contemplated: Embodiment 1. A composition comprising a yeast, acrylic acid, and a yeast growth media.

Embodiment 2. The composition of embodiment 1, wherein the yeast growth media comprises a yeast extract.

Embodiment s. The composition of embodiment 1 or 2, wherein the yeast growth media comprises a carbohydrate. Embodiment 4. The composition of embodiment 1, 2, or 3, wherein the yeast growth media comprises a peptide.

Embodiment s. The composition of embodiment 1, 2, 3, or 4, wherein the yeast growth media comprises an antibiotic.

Embodiment 6. The composition of embodiment 1, 2, 3, 4, or 5, wherein the yeast growth media comprises a phosphate or a sulfate salt.

Embodiment 7. The composition of embodiment 1, 2, 3, 4, 5, or 6, wherein the yeast growth media comprises a buffer. Embodiment s. A method of preparing a yeast with improved tolerance to acrylic acid, comprising allowing a yeast to propagate in the presence of acrylic acid.

Embodiment 9. The method of embodiment 8, wherein the yeast is allowed to propagate in the presence of at least 0.05 g/L of acrylic acid.

Embodiment 10. The method of embodiment 8 or 9, wherein the concentration of the 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 a propagation cycle comprises: increasing the concentration of acrylic acid, adding additional yeast growth media, and allowing the yeast to propagate again.

Embodiment 12. The method of embodiment 11, wherein an additional 1 to 100 propagation cycles are performed.

Embodiment 13. The method of embodiment 12, wherein an additional 30 to 40 propagation cycles are performed.

Embodiment 14. The method of embodiment 8, 9, 10, 11, 12, or 13, wherein the yeast is allowed to propagate 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 allowed to propagate for about 1 hour to about 200 hours in each propagation cycle.

Embodiment 16. The method of embodiment 15, wherein the yeast is allowed to propagate for about 20 hour to about 30 hours in each propagation cycle.

Embodiment 17. The method of embodiment 11, 12, 13, 14, 15, or 16, wherein the propagation cycles are 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 that is tolerant to acrylic acid, comprising a yeast with a non-naturally occurring genetic mutation or modification, wherein the yeast has the property that, when the yeast has an optical density of 0.2 at 600 nm in a reference acrylic acid composition, the yeast propagates so 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 1.8 g/L of acrylic acid, 20 g/L of glucose, 5 g/L of (NH ₄ ) ₂ SO ₄ , 3 g/L of KH ₂ PO ₄ , 0.5 g/L of MgSO ₄ «7H ₂ O, 50 pg/L of d-Biotin, 1 mg/L of D-pantothenic acid hemicalcium salt, 1 mg/L of Thiamin-HCI, 1 mg/L of Pyridoxin-HCI, 1 mg/L of nicotinic acid, 0.2 mg/L of 4-aminobenzoic acid, 25 mg/L of m-inositol, 3 mg/L of FeSO ₄ -7H ₂ O, 4.5 mg/L of ZnSO ₄ -7H ₂ O, 4.5 mg/L of CaCI ₂ -2H ₂ O, 1 mg/L of MnCI ₂ -4H ₂ O, 0.3 mg/L of COCI ₂ -6H ₂ O, 0.3 mg/L of CuSO ₄ -5H ₂ O, 0.4 mg/L of Na ₂ MoO ₄ -2H ₂ O, 1 mg/L of H3BO3, 0.1 mg/L of KI, and 19 mg/L of Na ₂ EDTA-2H ₂ O.

Embodiment 20. A genetically modified 5. cerevisiae having a modification to the genome of the wild-type that results in the 5. cerevisiae synthesizing a modified ACH1, WAR1, NHA1, TRK1, ALD3, PMA1, GLG2, MIX23, ATG26, SYT1, SNF3, ARR3, TRS33, GPR1, OCTI, TPO1, MET2, RPC82, or a combination thereof.

Embodiment 21. The genetically modified S. cerevisiae of embodiment 20, having a modification to the genome of the wild-type that results in the 5. cerevisiae synthesizing a modified ACH1.

Embodiment 22. The genetically modified 5. cerevisiae of embodiment 21, wherein the modified ACH1 comprises a change that is R334G, A367T, T383K, V353F, G393E, fsN137, H439Y, Q275K, or a combination thereof.

Embodiment 23. The genetically modified S. cerevisiae of embodiment 20, 21, or 22, having a modification to the genome of the wild-type that results in the 5. cerevisiae synthesizing a modified WAR1.

Embodiment 24. The genetically modified 5. cerevisiae of embodiment 23, wherein the modified WAR1 comprises a change that is W936L.

Embodiment 25. The genetically modified S. cerevisiae of embodiment 20, 21, 22, 23, or 24, having a modification to the genome of the wild-type that results in the 5. cerevisiae synthesizing a modified NHA1.

Embodiment 26. The genetically modified 5. cerevisiae of embodiment 25, wherein the modified ACH1 comprises a change that is H164D, E290K, or combination thereof. Embodiment 27. The genetically modified S. cerevisiae of embodiment 20, 21, 22, 23, 24, 25, or 26, having a modification to the genome of the wild-type that results in the 5. cerevisiae synthesizing a modified TRK1.

Embodiment 28. The genetically modified 5. cerevisiae of embodiment 27, wherein the modified ACH1 comprises a change that is S144F, R8975, N750K, P11635, L764F, or a combination thereof.

Embodiment 29. A method of preparing acrylic acid, comprising dehydrating hydroxypropionic acid in the presence of a yeast of embodiment 18 or 19, or the genetically modified S. cerevisiae of embodiment 20, 21, 22, 23, 24, 25, 26, 27, or 28.

EXAMPLES

EXAMPLE 1

Methods

Strains, DNA handling and media

The strains 5. cerevisiae CEN.PK113-7D (MATa MAL2-8c SUC2), CEN.PK113-5D (), 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 the ALE experiments. Escherichia coli DH5a was used for plasmid 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 with respective kits from Qiagen.

Selection and maintenance of plasmids in E. coli was performed in LB medium containing 10 g/L of peptone, 10 g/L of NaCI, 5 g/L of yeast extract and supplemented with 100 mg/L of ampicillin sodium salt. Solid LB also included 16 g/L of agar.

For selection of 5. cerevisiae strains with the KANMX marker, YPD medium containing 10 g/L yeast extract, 20 g/L of peptone, 20 g/L of glucose and supplemented with 200 mg/L G418 disulphate (SigmaAldrich) was used. For selection with Ura marker, standard minimal media was made without the addition of uracil. Solid versions of either medium were prepared by adding 20 g/L of agar.

All growth and evolution experiments were performed in the minimal medium described by (Verduyn et al., 1992) containing 20 g/L of glucose, 5 g/L of (NFUhSC , 3 g/L of KH2PO4, 0.5 g/L of MgSO4-7H2O, 1 mL/L of vitamin solution and 1 mL/L of 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 of IM Na ₂ HPO ₄ .

AA was adjusted to pH 5 using KH2PO4, and made to a stock concentration of 0.1 g/mL.

Growth screening

All growth tests were performed in a Growth profiler 960 (Enzyscreen) using 96-half- deepwell microplates with a culture volume of 250 pL, agitation at 250 rpm, temperature controlled at 30 °C and initial OD600 of 0.05. Maximum specific growth rates for each strain were calculated by finding the highest slope in plots of the natural logarithm of the OD600 versus time.

Adaptive laboratory evolution (ALE)

The strain GL01 was evolved using serial cultivations in media containing acrylic acid, in concentrations that increased from 0.3 g/L to 2 g/L. Six independent cultures were evolved. As 5. cerevisiae propagates, it initially uses glucose as food. This is referred to as the glucose phase. When the glucose is consumed, the 5. cerevisiae uses ethanol as food. This is referred to as the ethanol phase. In this experiment, two initial AA concentrations were chosen, so that, in 24 hours, three of the six cultures would be at the ethanol phase, and the other three would be at the glucose phase. The cultures were serially propagated in 30 mL flasks in a 30 C shaker at 200 rpm.

Every 24 hours, OD600 of each culture was measured, and the volume of the old culture to be transferred to the fresh media was calculated so that the starting OD600 of the new culture would be 0.1. AA concentration in the new culture was adjusted so that it would reach the designated growth phase (ethanol or glucose) in twenty-four hours. Ending OD600 was recorded for each flask, and a glycerol stock of each culture was kept in -80 freezer. The evolution experiment was stopped when there was no observable increase in the growth rate (as reflected by the increase in ending OD600) during a two-week period.

DNA extraction and sequencing

Clones isolated from the evolved populations were cultivated overnight in YPD medium. The Blood & Cell Culture DNA Mini Kit (Qiagen, location) was used to extract genomic DNA from 3 mL of the overnight yeast culture (~5xl08 cells) using the protocol recommended by the manufacturer. Pair-end sequencing were performed on Illumina® sequencing platform, with the read length of PE150 bp at each end, and a genome coverage of lOOx per sample.

Sequencing data analysis

The effective sequencing data were aligned with the reference sequences (S. cerevisiae S288c; R64-2-1) through BWA software. SNPs and InDeis were detected using SAMtools. SVs were detected using BreakDancer. CNVs were detected using CNVnator. All variants were annotated using ANNOVAR. A custom tool was used to subtract wild-type (GL01) control background.

Results

Growth of evolved yeast strains

After 35 passages of ALE, evolved strains (35E and 35G) were tested in minimal media (pH 5) at various AA concentrations, with wild-type strain GL01 as control.

Table 2. Comparing growth rate between evolved strains and the wild-type

Sequencing of evolved yeast strains

Variants detected in AA evolved strains with glucose phase transfer are presented in

Table 3. Strain name starts with passage number, with G indicating glucose phase transfer, and E indicating ethanol phase transfer. For the number after the letter, the first digit is the culture number (1-3), and the second digit is the isolate number (1-8).

Table 3. Examples of detected variants

EXAMPLE 2

Reverse engineered strains with point mutations or single nucleotide insertion/deletion may be constructed using the single gRNA method described in (Laughery et al., 2015). Web based resources are available for help selecting the best gRNA site, according to the proximity to nucleotide to be substituted and the possibility of inactivating the PAM site using a synonymous substitution. The repair template may be designed to introduce the desired mutation and inactivating the PAM site, and synthesized as two complementary oligonucleotides. Desired 20 bp gRNA target sequence (without PAM site) flanked by two 50 bp sequences homologous to the gRNA site on available plasmid can be synthesized as two complementary 120 bp oligonucleotides. 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 to cool down at room temperature. The double stranded gRNA insert can be cloned into suitable plasmid capable of both Cas9 expression and gRNA expression, using standard methods such as Gibson Assembly. 100 ng of each gRNA+Cas9 plasmid can be co-transformed with 2 ug of double stranded repair fragment for the respective gene into a suitable strain, depending on the selection marker on the plasmid, using high-efficiency protocol such as (Gietz and Woods, 2006). The transformation mixture may be spread on plates containing suitable selection drug depending on the strain and the plasmid. Individual colonies can be picked, and PCR amplification of the modified region can be carried out. Sequencing of the PCR product can allow the confirmation that each mutation is successfully introduced.

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 W02002042418

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 expressing quantities of ingredients, properties such as molecular weight, reaction conditions, etc., used in herein 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, the numerical parameters may be modified according to the desired properties sought to be achieved, and should, therefore, be considered as part of the disclosure. At the very least, the examples shown herein are for illustration only, not as an attempt to limit the scope of the disclosure.

The terms "a," "an," "the" and similar referents used in the context of describing embodiments of the present disclosure (especially in the context of the following embodiments) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein may 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 (e.g., "such as") provided herein is intended merely to better illustrate embodiments of the present disclosure and does not pose a limitation on the scope of any embodiment. No language in the specification should be construed as indicating any non-embodied element essential to the practice of the embodiments of the present disclosure. Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member may be referred to and embodied individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability.

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

In closing, it is to be understood that the embodiments disclosed herein are illustrative of the principles of the embodiments. Other modifications that may be employed are within the scope of the embodiments. Thus, by way of example, but not of limitation, alternative embodiments may be utilized in accordance with the teachings herein. Accordingly, the embodiments are not limited to embodiments precisely as shown and described.