Engineered cells for production of malonate

Abstract

The present disclosure relates to methods, cells, and compositions useful for producing malonate.

Description

[0001] ENGINEERED CELLS FOR PRODUCTION OF MALONATE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 519,216, filed August 11, 2023, entitled “ENGINEERED CELLS FOR PRODUCTION OF MALONATE,” the entire disclosure of which is hereby incorporated by reference in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (G091970117WOOO-SEQ-KVC.xml; Size: 78,853 bytes; and Date of Creation: August 8, 2024) is herein incorporated by reference in its entirety.

[0006] FIELD

[0007] The present disclosure relates to methods, cells, and compositions useful for producing malonate.

[0008] BACKGROUND

[0009] Malonate is an ionized form of malonic acid or propanedioic acid, which is a dicarboxylic acid with the chemical formula CH2(COOH)2. Malonic acid or malonate is used in the manufacture of many polymers or other high value compounds including, for example, polyesters, gamma-nonalactone, and cinnamic acid. It is useful in a variety of applications including, for example, coatings, electronics, flavors and fragrances, solvents, polymer crosslinking, pharmaceuticals, and adhesives. Although malonic acid is naturally occurring in many fruits and vegetables, malonic acid and compounds produced from malonic acid are generally obtained commercially from petrochemical feedstocks and through chemical synthesis.

[0010] SUMMARY

[0011] Aspects of the present disclosure relate to a method for producing malonate comprising: culturing a host cell that expresses a heterologous polynucleotide encoding a malonyl-CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence of SEQ ID NO: 1, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an alanine (A) at a position corresponding to position 348 in SEQ ID NO: 1; and wherein the host cell is Yarrowia lipolytica (Y. lipolytica).

[0012] Aspects of the present disclosure relate to a method for producing malonate comprising: culturing a host cell that expresses a heterologous polynucleotide encoding a malonyl-CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence of SEQ ID NO: 1, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an alanine (A) at a position corresponding to position 348 in SEQ ID NO: 1; and wherein the host cell is Yarrowia lipolytica (Y. lipolytica) strain YB-392, Y-7320, YB-275, YB-387, Y-7754, Y-17536, Y-17622, YB-276, YB-420, YB-48157, Y-1095, YB- 11853, YB-271, or Y-5383.

[0013] In some embodiments, the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of SEQ ID NO: 3.

[0014] In some embodiments, the Y lipolytica strain is YB-392, Y-7320, YB-275, or YB- 387. In some embodiments, the Y. lipolytica strain is YB-387.

[0015] In some embodiments, the method produces at least 100 grams per liter of malonate. In some embodiments, the method produces at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, or at least 140 grams per liter of malonate.

[0016] In some embodiments, the method produces malonate in a cell culture. In some embodiments, the pH of the cell culture is between about 3.0 and about 5.0. In some embodiments, the cell culture further comprises a base. In some embodiments, the base is NaOH or NH4OH.

[0017] Aspects of the present disclosure relate to a host cell that comprises a heterologous polynucleotide encoding a malonyl-CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence of SEQ ID NO: 1 and comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an alanine (A) at a position corresponding to position 348 in SEQ ID NO: 1; and wherein the host cell is Yarrowia lipolytica (Y. lipolytica) strain YB-392, Y-7320, YB-275, YB-387, Y-7754, Y-17536, Y- 17622, YB-276, YB-420, YB-48157, Y-1095, YB-11853, YB-271, or Y-5383.

[0018] In some embodiments, the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of SEQ ID NO: 3.

[0019] In some embodiments, the Y lipolytica strain is YB-392, Y-7320, YB-275, or YB- 387. In some embodiments, the Y. lipolytica strain is YB-387.

[0020] In some embodiments, the host cell is capable of producing at least 100 grams per liter of mal onate. In some embodiments, the host cell is capable of producing at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, or at least 140 grams per liter of malonate.

[0021] Aspects of the present disclosure relate to a method of producing greater than 115 grams per liter of malonate, the method comprising: culturing a host cell that expresses a heterologous polynucleotide encoding a malonyl-CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence of SEQ ID NO: 1 and comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an alanine (A) at a position corresponding to position 348 in SEQ ID NO: 1; and wherein the host cell is a Yarrowia lipolytica (Y. lipolytica) strain.

[0022] In some embodiments, the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of SEQ ID NO: 3.

[0023] In some embodiments, the host cell is Y lipolytica strain YB-392, Y-7320, YB-275, YB-387, or YB-420.

[0024] Aspects of the present disclosure relate to a method for producing greater than 0.3 gram of malonate per gram of glucose comprising: culturing a host cell that expresses a heterologous polynucleotide encoding a malonyl-CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence of SEQ ID NO: 1 and wherein the amino acid sequence of the malonyl-CoA hydrolase comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an A at a position corresponding to position 348 in SEQ ID NO: 1; wherein the host cell is a Yarrowia lipolytica (Y. lipolytica) strain.

[0025] In some embodiments, the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of SEQ ID NO: 3.

[0026] In some embodiments, the Y. lipolytica strain is YB-392, Y-7320, YB-275, YB-387, Y-17536, YB-276, YB-420, YB-48157, YB-11853, or YB-271.

[0027] In some embodiments, the method produces greater than 0.4 gram of malonate per gram of glucose. In some embodiments, the host cell is Y. lipolytica strain YB-392, Y-7320, YB-275, YB-276, YB-420, or YB-387.

[0028] In some embodiments, the method produces greater than 0.5 gram of malonate per grams of glucose. In some embodiments, the host cell is Y. lipolytica strain YB-392.

[0029] Aspects of the present disclosure relate to a method of producing greater than 115 grams per liter of malonate, the method comprising: culturing a host cell as described herein under fermentation conditions that comprise a pH of about 3.0, about 4.0, or about 5.0. In some embodiments, the yield on glucose is about 0.2 to about 0.5 gram of malonate per gram of glucose.

[0030] Aspects of the present disclosure relate to a fermentation composition, comprising: a host cell as described herein, culture media comprising a pH of 3.0, 4.0, or 5.0 and 115 grams per liter of malonate.

[0031] Aspects of the present disclosure relate to a method for producing malonate comprising: culturing a host cell that expresses a heterologous polynucleotide encoding a malonyl-CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence of any one of SEQ ID NOs: 1, 3, or 7-28. In some embodiments, the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of any one of SEQ ID NOs: 3 or 7-28. In some embodiments, the host cell is Yarrowia lipolytica (Y. lipolytica). In some embodiments, the host cell is Yarrowia lipolytica (K lipolytica) strain YB-392, Y-7320, YB-275, YB-387, Y-7754, Y-17536, Y-17622, YB- 276, YB-420, YB-48157, Y-1095, YB-11853, YB-271, or Y-5383.

[0032] Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used in this disclosure is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations of thereof in this disclosure, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented in this disclosure. The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0035] FIG. 1 shows malonate production by three Yarrowia lipolytica (Y. lipolytica) strains: YB-392, Y-7320, and Y-48157 expressing the variant MCH enzyme of SEQ ID NO: 3.

[0036] FIG. 2 shows production of malonate by Y lipolytica strain YB-387 expressing the variant MCH enzyme of SEQ ID NO: 3 in a 30-liter fermenter.

[0037] FIG. 3 shows bar graphs illustrating the levels of malonate produced by Y lipolytica strains expressing MCH enzyme variant F6AA82 (SEQ ID NO: 3) and MCH enzyme variant A4XS22 (SEQ ID NO: 28).

[0038] DETAILED DESCRIPTION

[0039] Aspects of the disclosure relate to production of malonate in host cells that heterologously express a malonyl-CoA hydrolase (MCH) enzyme. As described in the Examples, it was surprisingly found that expression of a variant MCH in Y lipolytica strains led to high-level production of malonate. As used herein, a strain or culture exhibits high level production of malonate if it produces malonate at a higher titer, a higher yield, or a high productivity relative to a reference strain or culture. Due to the environmental impacts associated with the petrochemical industry, a renewable or bio-based commercial source of malonate as provided herein would be beneficial.

[0040] Malonyl-CoA Hydrolase

[0041] As used herein, the term “malonyl-CoA hydrolase” or “MCH” refers to an enzyme that catalyzes the conversion of malonyl-CoA to malonate.

[0042] Aspects of the present disclosure relate to genetically-modified host cells that express an MCH. In some embodiments, the MCH is a variant MCH. In some embodiments, a host cell that expresses a variant MCH produces more malonate relative to a corresponding reference cell (e.g., wherein the reference cell does not express the variant MCH). In some embodiments, the reference cell is of the same species as the host cell. In some embodiments, the reference cell does not comprise the same genetic modifications as the host cell. In some embodiments, the reference cell does not express the variant MCH. In some embodiments, the reference cell expresses a wild-type homologue of the MCH but not the variant MCH.

[0043] In some embodiments, the MCH expressed by the genetically-modified host cell is from the same species as the host cell. In other embodiments, the MCH expressed by the genetically-modified host cell is from a different species than the host cell.

[0044] Expression and / or activity of an MCH can be increased in a host cell by expressing in the host cell a heterologous polynucleotide encoding an MCH. The term “heterologous” with respect to a polynucleotide, such as a polynucleotide comprising a gene, is used interchangeably with the term “exogenous” or “recombinant” and refers to: a polynucleotide that has been artificially supplied to a biological system such as a cell; a polynucleotide that has been modified within a biological system; or a polynucleotide whose expression or regulation has been manipulated within a biological system. A heterologous polynucleotide that is introduced into or expressed in a host cell may be a synthetic polynucleotide, a polynucleotide that comes from a different organism or species from the host cell, or a polynucleotide that results from modification or selective editing within the host cell of a polynucleotide that was naturally occurring in the host cell or one that was introduced into the host cell. A polynucleotide that is naturally present in a host cell may be considered heterologous (ie., is a heterologous polynucleotide) when it is: situated non-naturally in the host cell; expressed recombinantly in the host cell, either stably or transiently; present in a copy number that differs from the naturally occurring copy number within the host cell; or expressed in a non-natural way or at a non-natural level, within the host cell, such as by manipulating regulatory regions that control expression of the polynucleotide. In some embodiments, a heterologous polynucleotide is a polynucleotide that is naturally present in a host cell but whose expression is driven by a promoter that does not naturally regulate expression of the polynucleotide. In other embodiments, a heterologous polynucleotide is a polynucleotide that is naturally present in a host cell and whose expression is driven by a promoter that does naturally regulate expression of the polynucleotide. In other embodiments, a heterologous polynucleotide is a polynucleotide that is naturally present in a host cell, but its expression is driven by a promoter that has been modified or its expression is regulated by another regulatory region that has been modified. In some embodiments, the promoter is recombinantly activated or repressed. For example, gene-editing based techniques may be used to regulate expression of a polynucleotide in a cell, including an endogenous polynucleotide, from a promoter, including an endogenous promoter. See, e.g., Chavez et al., Nat Methods. 2016 Jul; 13(7): 563-567. A heterologous polynucleotide may comprise a wild-type sequence or a mutant sequence as compared with a reference polynucleotide sequence.

[0045] MCH enzymes are known in the art. See for example, US 11248215 and US20220380779, which disclose a MCH consensus enzyme sequence based on Pseudomonas EC 3.1.2.4 enzymes. In some embodiments, the MCH is encoded by the 3- hydroxyisobutyryl-CoA hydrolase gene (F6AA82) from Pseudomonas fulva (P.fulva) (strain 12-X). In some embodiments, the MCH is encoded by the 3-hydroxyisobutyryl-CoA hydrolase gene (A4XS22) from Pseudomonas mendocina (P. mendocina).

[0046] In some embodiments, a MCH associated with the disclosure is a MCH from P.fulva. The amino acid sequence of a MCH from P.fulva is accessible through UniProtKB Accession No. F6AA82, and is provided here as SEQ ID NO: 1 : NO: 1)

[0047] A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 1 is provided as SEQ ID NO: 2:

[0048] In some embodiments, a MCH associated with the disclosure is a MCH from P. mendocina. The amino acid sequence of a MCH from P. mendocina is accessible through UniProtKB Accession No. A4XS22, and is provided here as SEQ ID NO: 27: NO: 27)

[0049] In some embodiments, a MCH associated with the disclosure is a variant MCH. A variant MCH can comprise an amino acid sequence as set forth in SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 corresponds to a variant of SEQ ID NO: 1. The variant has the following four point mutations relative to SEQ ID NO: 1 : E95N, V152A, F304R, and Q348A.

[0050] A non-limiting example of a variant MCH is provided here as SEQ ID NO: 3: NO: 3) A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 3 is provided as SEQ ID NO: 4:

[0051] A variant MCH can comprise an amino acid sequence as set forth in SEQ ID NO: 28. The amino acid sequence of SEQ ID NO: 28 corresponds to a variant of SEQ ID NO: 27. The variant has the following three point mutations relative to SEQ ID NO: 27: E95N, V152A, and F304R.

[0052] A non-limiting example of a variant MCH is provided here as SEQ ID NO: 28:

[0053] NO: 28)

[0054] A non-limiting example of a nucleotide sequence encoding SEQ ID NO: 28 is provided as SEQ ID NO: 49:

[0055] As shown in the Examples, it was surprisingly found that multiple K lipolytica strains expressing a variant MCH comprising the sequence of SEQ ID NO: 3 produced high yields of malonate.

[0056] In some embodiments, an MCH associated with the disclosure comprises an amino acid sequence that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NOs: 1, 3 or 7-28. In some embodiments, the amino acid sequence of an MCH comprises or consists of the sequence of SEQ ID NOs: 1, 3 or 7-28, or a conservatively substituted version thereof.

[0057] Host cells associated with the disclosure, and / or methods associated with the disclosure, may comprise an MCH that comprises an amino acid sequence that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NOs: 1, 3, or 7-28. In some embodiments, host cells associated with the disclosure, and / or methods associated with the disclosure, comprise an MCH, wherein the amino acid sequence of the MCH comprises or consists of the sequence of SEQ ID NOs: 1, 3, or 7-28, or a conservatively substituted version thereof. In some embodiments, the sequence of an MCH enzyme associated with the disclosure comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein at least one of the amino acid substitutions is at a position corresponding to position 8, 9, 10, 50, 88, 94, 95, 96, 152, 169, 194, 204, 218, 251, 255, 266, 285, 304, 309, 310, 343, 348, and / or 357 in SEQ ID NO: 1.

[0058] In some embodiments, an MCH enzyme comprises any one or more of the following amino acid substitutions: the amino acid isoleucine (I) at a residue corresponding to position 8 in the sequence of SEQ ID NO: 1 (e.g., 81); the amino acid leucine (L) at a residue corresponding to position 9 in the sequence of SEQ ID NO: 1; the amino acid alanine (A) at a residue corresponding to position 10 in the sequence of SEQ ID NO: 1; the amino acid alanine (A) at a residue corresponding to position 50 in the sequence of SEQ ID NO: 1; the amino acid serine (S) at a residue corresponding to position 50 in the sequence of SEQ ID NO: 1; the amino acid threonine (T) at a residue corresponding to position 88 in the sequence of SEQ ID NO: 1; the amino acid glycine (G) at a residue corresponding to position 94 in the sequence of SEQ ID NO: 1; the amino acid asparagine (N) at a residue corresponding to position 95 in the sequence of SEQ ID NO: 1; the amino acid phenylalanine (F) at a residue corresponding to position 96 in the sequence of SEQ ID NO: 1; the amino acid alanine (A) at a residue corresponding to position 152 in the sequence of SEQ ID NO: 1; the amino acid methionine (M) at a residue corresponding to position 169 in the sequence of SEQ ID NO: 1; the amino acid serine (S) at a residue corresponding to position 194 in the sequence of SEQ ID NO: 1; the amino acid threonine (T) at a residue corresponding to position 204 in the sequence of SEQ ID NO: 1; the amino acid asparagine (N) at a residue corresponding to position 218 in the sequence of SEQ ID NO: 1; the amino acid alanine (A) at a residue corresponding to position 251 in the sequence of SEQ ID NO: 1; the amino acid glutamate (E) at a residue corresponding to position 251 in the sequence of SEQ ID NO: 1; the amino acid alanine (A) at a residue corresponding to position 255 in the sequence of SEQ ID NO: 1; the amino acid methionine (M) at a residue corresponding to position 255 in the sequence of SEQ ID NO: 1; the amino acid glutamine (Q) at a residue corresponding to position 266 in the sequence of SEQ ID NO: 1; the amino acid tyrosine (Y) at a residue corresponding to position 285 in the sequence of SEQ ID NO: 1; the amino acid arginine (R) at a residue corresponding to position 304 in the sequence of SEQ ID NO: 1; the amino acid serine (S) at a residue corresponding to position 309 in the sequence of SEQ ID NO: 1; the amino acid isoleucine (I) at a residue corresponding to position 310 in the sequence of SEQ ID NO: 1; the amino acid glutamate (E) at a residue corresponding to position 343 in the sequence of SEQ ID NO: 1; the amino acid alanine (A) at a residue corresponding to position 348 in the sequence of SEQ ID NO: 1; the amino acid cysteine (C) at a residue corresponding to position 357 in the sequence of SEQ ID NO: 1; or any combination thereof.

[0059] In some embodiments, an MCH enzyme comprises amino acid substitutions at residues corresponding to the following positions in the amino acid sequence of SEQ ID NO: 1 : positions 95, 152, 304, and 348; positions 95, 152, and 251; positions 50, 95, and 152; positions 8, 95, and 152; positions 95, 152, and 255; positions 95 and 152; positions 95, 152, and 343; positions 10, 95, and 152; positions 95, 152, 266, and 310; positions 95, 152, 251, and 357; positions 95, 152, 194, and 309; positions 94, 95, and 152; positions 95, 152, and 218; positions 95, 152, 285, and 310; positions 95, 152, 266, and 310; positions 9, 95, 152, and 285; positions 95, 152, 204, and 251; positions 95, 96, and 152; positions 95, 152, and 169; or positions 88, 95, and 152.

[0060] In some embodiments, an MCH enzyme comprises the following amino acid substitutions relative to the sequence of SEQ ID NO: 1 : E95N [the amino acid Glutamate (E) at Position 95 has been replaced by Asparagine (N)], V152A, F304R, and Q348A; E95N, V152A, and T251E; D50S, E95N, and V152A; D50A, E95N, and V152A; R8I, E95N, and V152A; E95N, V152A, and S255A; E95N and V152A; E95N, V152A, and N343E; E95N, V152A, and S255M; S10A, E95N, and V152A; E95N, V152A, E266Q, and M3101; E95N, V152A, T251A, and L357C; E95N, V152A, T194S, and G309S; R94G, E95N, and V152A; E95N, V152A, and S218N; E95N, V152A, and G285Y, M3101; E95N, V152A, and E266Q, M310I; E95N, V152A, T251A, and L357C; R94G, E95N, and V152A; E95N, V152A, G285Y, and M310I; A9L, E95N, V152A, and G285Y; E95N, V152A, I204T, and T251A; E95N, Y96F, and V152A; E95N, V152A, and F169M; A88T, E95N, and V152A; or any other combination of mutations disclosed herein.

[0061] In some embodiments, the sequence of an MCH enzyme associated with the disclosure comprises one or more amino acid substitutions relative to SEQ ID NO: 27, wherein at least one of the amino acid substitutions is at a position corresponding to position 95, 152, and / or 304 in SEQ ID NO: 27.

[0062] In some embodiments, an MCH enzyme comprises any one or more of the following amino acid substitutions: the amino acid asparagine (N) at a residue corresponding to position 95 in the sequence of SEQ ID NO: 27 (e.g., 95N); the amino acid alanine (A) at a residue corresponding to position 152 in the sequence of SEQ ID NO: 27; the amino acid arginine (R) at a position corresponding to position 304 in the sequence of SEQ ID NO: 27; or any combination thereof. In some embodiments, an MCH enzyme comprises amino acid substitutions at residues corresponding to positions 95, 152, and 304 in the amino acid sequence of SEQ ID NO: 27. In some embodiments, an MCH enzyme comprises the following amino acid substitutions relative to the sequence of SEQ ID NO: 27: E95N, V152A, and F304R.

[0063] Polynucleotides Encoding Malonyl-CoA Hydrolase

[0064] Aspects of the present disclosure relate to recombinant enzymes, functional modifications and variants thereof, polynucleotides encoding said enzymes, as well as uses relating to any thereof. For example, the methods described in this application may be used to produce malonate.

[0065] A polynucleotide encoding any of the MCHs, or any other polypeptides associated with the disclosure, may be incorporated into any appropriate vector through any method known in the art. For example, the vector may be an expression vector, including but not limited to a viral vector (e.g., a lentiviral, retroviral, adenoviral, or adeno-associated viral vector), any vector suitable for transient expression, any vector suitable for constitutive expression, or any vector suitable for inducible expression (e.g., a galactose-inducible or doxycycline-inducible vector). The vector may be a cloning vector, such as a plasmid, fosmid, phagemid, virus genome or artificial chromosome.

[0066] As used in this application, the terms "expression vector" or "expression construct" refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular polynucleotide in a host cell, such as a yeast cell (e.g., a Y. lipolytica cell) or a bacterial cell. In some embodiments, a polynucleotide associated with the disclosure is inserted into an expression vector or expression construct such that it is operably joined to regulatory sequences and, in some embodiments, expressed as an RNA transcript. In some embodiments, the expression vector or expression construct contains one or more markers, such as a selectable marker, to identify cells transformed or transfected with the expression vector or expression construct. A polynucleotide encoding a polypeptide associated with the disclosure is “operably joined” or “operably linked” to a regulatory sequence when the polynucleotide and the regulatory sequence are covalently linked and the expression or transcription of the polynucleotide is under the influence or control of the regulatory sequence.

[0067] In some embodiments, a polynucleotide encoding any of the polypeptides described in this application is under the control of regulatory sequences (e.g., enhancer sequences). In some embodiments, a polynucleotide (e.g., a polynucleotide comprising a gene) is expressed under the control of a promoter. In some embodiments, the promoter is a native promoter, corresponding to the promoter of the gene in its endogenous context. In other embodiments, the promoter is not the native promoter of the gene, e.g., the promoter is different from the promoter of the gene in its endogenous context.

[0068] In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, GALI, GAL10, GAL7, GAL3, GAL2, MET3, MET25, HXT3, HXT7, ACT1, ADH1, ADH2, CUP1-1, EN02, and SOD1, as would be known to one of ordinary skill in the art (see, e.g., Addgene website: blog. addgene. org / plasmids-101-the-promoter- region). In some embodiments, the promoter is a prokaryotic promoter (e.g., bacteriophage or bacterial promoter). Non-limiting examples of bacteriophage promoters include Plslcon, T3, T7, SP6, and PL. Non-limiting examples of bacterial promoters include Pbad, PngrB, Ptrc2, Plac / ara, Ptac, and Pm.

[0069] In some embodiments, the promoter is an inducible promoter. As used in this application, an “inducible promoter” is a promoter controlled by the presence or absence of a molecule. Non-limiting examples of inducible promoters include chemically-regulated promoters and physically-regulated promoters. For chemically-regulated promoters, the transcriptional activity can be regulated by one or more compounds, such as alcohol, an antibiotic such as tetracycline, a carbon source such as galactose, a steroid, a metal, or other compounds. For physically-regulated promoters, transcriptional activity can be regulated by a phenomenon such as light or temperature. Non-limiting examples of tetracycline-regulated promoters include anhydrotetracycline (aTc)-responsive promoters and other tetracyclineresponsive promoter systems (e.g., a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)). Non-limiting examples of steroid-regulated promoters include promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily. Non-limiting examples of metal-regulated promoters include promoters derived from metallothionein (proteins that bind and sequester metal ions) genes. Non-limiting examples of pathogenesis-regulated promoters include promoters induced by salicylic acid, ethylene or benzothiadiazole (BTH). Non-limiting examples of temperature / heat-inducible promoters include heat shock promoters. Nonlimiting examples of light-regulated promoters include light responsive promoters from plant cells. In certain embodiments, the inducible promoter is a galactose-inducible promoter. In some embodiments, the inducible promoter is induced by one or more physiological conditions (e.g., pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, or concentration of one or more extrinsic or intrinsic inducing agents). Non-limiting examples of an extrinsic inducer or inducing agent include amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or any combination thereof.

[0070] In some embodiments, the promoter is a constitutive promoter. As used in this application, a “constitutive promoter” refers to an unregulated promoter that allows continuous transcription of a gene. Non-limiting examples of a constitutive promoter include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, HXT3, HXT7, ACT1, ADH1, ADH2, EN02, and SOD1. Other inducible promoters or constitutive promoters known to one of ordinary skill in the art are also contemplated.

[0071] In some embodiments, a polynucleotide sequence encoding a MCH is operably linked to a promoter.

[0072] In some embodiments, a promoter is a promoter known in the art, including but not limited to those described in and incorporated by reference from: WO2011148339A1, US20140147888A1, US8969049B2, US8906650B2, WO2012138612A1, US7202356B2, US8323960B2, WO2012135773A1, US7259255B2, W02005049805A3, W02006052754A3, WO1996041889A1, US6083717A, US4937189A, US61470539P0, US8735094B2, and US8609369B2.

[0073] In some embodiments, the promoter is GAPDH (pGAPDH).

[0074] A non-limiting example of a nucleotide sequence of a GAPDH promoter is provided by SEQ ID NO: 5:

[0075] Additional regulatory sequences can include transcriptional terminators (terminators).

[0076] In some embodiments, a terminator is a terminator known in the art, including but not limited to those described in and incorporated by reference from: US4937189A, WO2023044937A1, US20130149754A1, US20090093543A1, or US9908917B2.

[0077] In some embodiments, the terminator is EXP (tEXP). A non-limiting example of a nucleotide sequence of an EXP terminator is provided by SEQ ID NO: 6:

[0078] In some embodiments, introduction of a polynucleotide, such as a polynucleotide encoding a MCH or any other polypeptide associated with the disclosure, into a host cell results in genomic integration of the polynucleotide. In some embodiments, a host cell comprises at least 1 copy, at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, at least 8 copies, at least 9 copies, at least 10 copies, at least 11 copies, at least 12 copies, at least 13 copies, at least 14 copies, at least 15 copies, at least 16 copies, at least 17 copies, at least 18 copies, at least 19 copies, at least 20 copies, at least 21 copies, at least 22 copies, at least 23 copies, at least 24 copies, at least 25 copies, at least 26 copies, at least 27 copies, at least 28 copies, at least 29 copies, at least 30 copies, at least 31 copies, at least 32 copies, at least 33 copies, at least 34 copies, at least 35 copies, at least 36 copies, at least 37 copies, at least 38 copies, at least 39 copies, at least 40 copies, at least 41 copies, at least 42 copies, at least 43 copies, at least 44 copies, at least 45 copies, at least 46 copies, at least 47 copies, at least 48 copies, at least 49 copies, at least 50 copies, at least 60 copies, at least 70 copies, at least 80 copies, at least 90 copies, at least 100 copies, or more, including any values in between, of a polynucleotide sequence, such as a polynucleotide sequence encoding a MCH or any other polypeptide described in this application, in its genome. Said copies may be inserted into the same locus or into different loci of a recombinant host cell of the disclosure. In some embodiments, the sequence of a polynucleotide (e.g., a polynucleotide comprising a gene) is codon-optimized. Codon optimization may increase expression of a gene by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, including all values in between) relative to a reference sequence that is not codon-optimized.

[0079] In some embodiments, a polynucleotide encoding a MCH comprises a sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%, including all values in between) identical to a polynucleotide encoding SEQ ID NOs: 1, 3 or 7-28. In some embodiments, a polynucleotide encoding a MCH comprises a sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%, including all values in between) identical to SEQ ID NOs: 2, 4, or 29-49.

[0080] Aspects of the disclosure relate to polynucleotides encoding any of the polypeptides described, such as MCH and any other polypeptides associated with the disclosure. Variants of polynucleotide or polypeptide sequences described in this application are also encompassed by the present disclosure. A variant may share at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a reference sequence, including all values in between.

[0081] Unless otherwise noted, the term “sequence identity” refers to the relatedness of the sequences of two polypeptides or polynucleotides when the sequences are aligned, and the term “percent identity” refers to the percentage of residues (amino acids or nucleotides) that are identical when two or more polypeptide or polynucleotide sequences are aligned. In some embodiments, sequence identity and / or percent identity is determined across the entire length of a sequence, while in other embodiments, sequence identity and / or percent identity is determined over a region of a sequence. Percent identity of polypeptide or polynucleotide sequences can be calculated by any of the methods known to one of ordinary skill in the art. For example, percent identity can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264- 68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST® protein searches can be performed, for example, with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST® can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST® and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.

[0082] A second example of a local alignment technique is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147: 195-197). An example of a global alignment technique is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453), which is based on dynamic programming. A further example of a global alignment technique is the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA)

[0083] In some embodiments, the identity of two polypeptide sequences is determined by aligning the two amino acid sequences of the polypeptides, calculating the number of identical amino acids, and dividing by the length of one of the polypeptide sequences. In some embodiments, the identity of two polynucleotide sequences is determined by aligning the two nucleotide sequences of the polynucleotides, calculating the number of identical nucleotides and dividing by the length of one of the polynucleotide sequences.

[0084] For multiple sequence alignments, computer programs including Clustal Omega (Sievers et al., Mol SystBiol. 2011 Oct 11;7:539) may be used.

[0085] In preferred embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence, such as a sequence disclosed in this application and / or recited in the claims when sequence identity is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264- 68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993 (e.g., BLAST®, NBLAST®, XBLAST® or Gapped BLAST® programs, using default parameters of the respective programs).

[0086] In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence, such as a sequence disclosed in this application and / or recited in the claims when sequence identity is determined using the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147: 195-197) or the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443- 453).

[0087] In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence, such as a sequence disclosed in this application and / or recited in the claims when sequence identity is determined using a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).

[0088] In some embodiments, a sequence, including a nucleic acid or amino acid sequence, is found to have a specified percent identity to a reference sequence, such as a sequence disclosed in this application and / or recited in the claims when sequence identity is determined using Clustal Omega (Sievers et al., Mol SystBiol. 2011 Oct 11;7:539).

[0089] As used in this application, a residue (such as a nucleic acid residue or an amino acid residue) in sequence “X” is referred to as corresponding to a position or residue (such as a nucleic acid residue or an amino acid residue) “Z” in a different sequence “Y” when the residue in sequence “X” is at the counterpart position of “Z” in sequence “Y” when sequences X and Y are aligned using sequence alignment tools known in the art.

[0090] Variant sequences may be homologous sequences. As used in this application, homologous sequences are sequences (e.g., nucleic acid or amino acid sequences) that share a certain percent identity (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least

[0091] 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least

[0092] 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least

[0093] 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0094] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

[0095] 96%, at least 97%, at least 98%, at least 99%, or 100% percent identity, including all values in between) and include but are not limited to paralogous sequences, orthologous sequences, or sequences arising from convergent evolution. Paralogous sequences arise from duplication of a gene within a genome of a species, while orthologous sequences diverge after a speciation event. Two different species may have evolved independently but may each comprise a sequence that shares a certain percent identity with a sequence from the other species as a result of convergent evolution.

[0096] In some embodiments, a polypeptide variant (e.g., MCH variant or variant of any other polypeptide associated with the disclosure) comprises a domain that shares a secondary structure (e.g., alpha helix, beta sheet) with a reference polypeptide (e.g., a reference MCH, or any other polypeptide associated with the disclosure). In some embodiments, a polypeptide variant (e.g., MCH variant or variant of any other polypeptide associated with the disclosure) shares a tertiary structure with a reference polypeptide (e.g., MCH, or any other polypeptide associated with the disclosure). As a non-limiting example, a variant polypeptide may have low primary sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity) compared to a reference polypeptide, but share one or more secondary structures (e.g, including but not limited to loops, alpha helices, or beta sheets, or have the same tertiary structure as a reference polypeptide. For example, a loop may be located between a beta sheet and an alpha helix, between two alpha helices, or between two beta sheets. Homology modeling may be used to compare two or more tertiary structures.

[0097] Mutations can be made in a nucleotide sequence by any method known to one of ordinary skill in the art. For example, mutations can be made by gene editing tools, PCR, site-directed mutagenesis (e.g., according to Kunkel, Proc. Nat. Acad. Set. U.S.A. 82: 488- 492, 1985), chemical synthesis of a gene or polypeptide, or by insertions, such as insertion of a tag (e.g., a HIS tag or a GFP tag). Mutations can include, for example, substitutions, deletions, additions, insertions, fusions, and translocations, generated by any method known in the art.

[0098] In some embodiments, methods for producing variants include circular permutation (Yu and Lutz, Trends Biotechnol. 2011 Jan;29(l): 18-25). In circular permutation, the linear primary sequence of a polypeptide can be circularized (e.g, by joining the N-terminal and C- terminal ends of the sequence) and the polypeptide can be severed (“broken”) at a different location. Thus, the linear primary sequence of the new polypeptide may have low sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less or less than 5%, including all values in between) compared to the linear sequence of the polypeptide before it was circularized and severed as determined by linear sequence alignment methods (e.g, Clustal Omega or BLAST). Topological analysis of the two polypeptides, however, may reveal that the tertiary structure of the two polypeptides is similar or dissimilar. Without being bound by a particular theory, a variant polypeptide created through circular permutation of a reference polypeptide and with a similar tertiary structure as the reference polypeptide can share similar functional characteristics (e.g., enzymatic activity, enzyme kinetics, substrate specificity or product specificity). In some instances, circular permutation may alter the secondary structure, tertiary structure or quaternary structure and produce a polypeptide with different functional characteristics (e.g., increased or decreased enzymatic activity, different substrate specificity, or different product specificity). See, e.g., Yu and Lutz, Trends Biotechnol. 2011 Jan;29(l): 18-25.

[0099] It should be appreciated that in a polypeptide that has undergone circular permutation, the linear amino acid sequence of the polypeptide would differ from a reference polypeptide that has not undergone circular permutation. However, one of ordinary skill in the art would be able to determine which residues in the polypeptide that has undergone circular permutation correspond to residues in the reference polypeptide that has not undergone circular permutation by, for example, aligning the sequences and detecting conserved motifs, and / or by comparing the structures or predicted structures of the polypeptides, e.g., by homology modeling.

[0100] In some embodiments, an algorithm that determines the percent identity between a sequence of interest and a reference sequence described in this application accounts for the presence of circular permutation between the sequences. The presence of circular permutation may be detected using any method known in the art, including, for example, RASPODOM (Weiner et al., Bioinformatics . 2005 Apr l;21(7):932-7). In some embodiments, the presence of circulation permutation is corrected for (e.g., the domains in at least one sequence are rearranged) prior to calculation of the percent identity between a sequence of interest and a sequence described in this application. The claims of this application should be understood to encompass sequences for which percent identity to a reference sequence is calculated after taking into account potential circular permutation of the sequence.

[0101] Functional variants of MCH, and any other polypeptides disclosed in this application are also encompassed by the present disclosure. For example, functional variants may bind one or more of the same substrates or produce one or more of the same products. Functional variants may be identified using any method known in the art. For example, the algorithm of Karlin and Altschul Proc. Natl. Acad. Set. USA 87:2264-68, 1990 described above may be used to identify homologous proteins.

[0102] Putative functional variants may also be identified by searching for polypeptides with functionally annotated domains. Databases including Pfam (Sonnhammer et al., Proteins. 1997 Jul;28(3):405-20) may be used to identify polypeptides with a particular domain.

[0103] Homology modeling may also be used to identify amino acid residues that are amenable to mutation without affecting function. A non-limiting example of such a method may include use of position-specific scoring matrix (PSSM) and an energy minimization protocol. See, c.g., Storm o et al., Nucleic Acids Res. 1982 May 11;10(9):2997-3011.

[0104] PSSM may be paired with calculation of a Rosetta energy function, which determines the difference between the wild-type and a mutant, such as a point mutant. Without being bound by a particular theory, potentially stabilizing mutations can be desirable for protein engineering (e.g., production of functional homologs). In some embodiments, a potentially stabilizing mutation has a SAGcaic value of less than -0.1 (e.g., less than -0.2, less than -0.3, less than -0.35, less than -0.4, less than -0.45, less than -0.5, less than -0.55, less than -0.6, less than -0.65, less than -0.7, less than -0.75, less than -0.8, less than -0.85, less than -0.9, less than -0.95, or less than -1.0) Rosetta energy units (R.e.u.). See, e.g., Goldenzweig et al, Mol Cell. 2016 Jul 21;63(2):337-346. doi: 10.1016 / j.molcel.2016.06.012.

[0105] In some embodiments, a polynucleotide sequence encoding a MCH or a polynucleotide sequence encoding any other polypeptide associated with the disclosure comprises a mutation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

[0106] 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

[0107] 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,

[0108] 97, 98, 99, 100 or more than 100 nucleotide positions corresponding to a reference sequence.

[0109] In some embodiments, the polynucleotide sequence encoding the MCH or the polynucleotide sequence encoding any other polypeptide associated with the disclosure comprises a mutation in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

[0110] 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,

[0111] 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

[0112] 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more codons of a coding sequence relative to a reference coding sequence. As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code. In some embodiments, the one or more mutations in the coding sequence do not alter the amino acid sequence of the coding sequence relative to the amino acid sequence of a reference polypeptide.

[0113] In some embodiments, the one or more mutations in a polynucleotide sequence encoding an MCH or a polynucleotide sequence encoding any other polypeptide associated with the disclosure alter the amino acid sequence of the polypeptide relative to the amino acid sequence of a reference polypeptide. In some embodiments, the one or more mutations alter the amino acid sequence of the polypeptide relative to the amino acid sequence of a reference polypeptide and alter (enhance or reduce) an activity of the polypeptide relative to the reference polypeptide.

[0114] The activity, including specific activity, of any of the polypeptides described in this application may be measured using methods known in the art. As a non-limiting example, a polypeptide’s activity may be determined by measuring its substrate specificity, product(s) produced, the concentration of product(s) produced, or any combination thereof. As used in this application, “specific activity” of a polypeptide refers to the amount (e.g., concentration) of a particular product produced for a given amount (e.g., concentration) of the polypeptide per unit time.

[0115] Mutations in a polypeptide coding sequence may result in conservative amino acid substitutions. As used in this application, a “conservative amino acid substitution,” or “conservatively substituted amino acid” refers to an amino acid substitution that does not alter the relative charge or size characteristics or functional activity of the protein in which the amino acid substitution is made.

[0116] In some instances, an amino acid is characterized by its R group (see, e.g., Table 1). For example, an amino acid may comprise a nonpolar aliphatic R group, a positively charged R group, a negatively charged R group, a nonpolar aromatic R group, or a polar uncharged R group. Non-limiting examples of an amino acid comprising a nonpolar aliphatic R group include alanine, glycine, valine, leucine, methionine, and isoleucine. Non-limiting examples of an amino acid comprising a positively charged R group include lysine, arginine, and histidine. Non-limiting examples of an amino acid comprising a negatively charged R group include aspartate and glutamate. Non-limiting examples of an amino acid comprising a nonpolar, aromatic R group include phenylalanine, tyrosine, and tryptophan. Non-limiting examples of an amino acid comprising a polar uncharged R group include serine, threonine, cysteine, proline, asparagine, and glutamine. Functionally equivalent variants of polypeptides may include conservative amino acid substitutions. Non-limiting examples of conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Additional non-limiting examples of conservative amino acid substitutions are provided in Table 1.

[0117] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 residues can be changed when preparing variant polypeptides. In some embodiments, amino acids are replaced by conservative amino acid substitutions.

[0118] Table 1. Non-limiting examples of conservative amino acid substitutions

[0119] Amino acid substitutions in the amino acid sequence of a polypeptide to produce a polypeptide variant having a desired property and / or activity can be made by alteration of the coding sequence of the polypeptide. Similarly, conservative amino acid substitutions in the amino acid sequence of a polypeptide to produce functionally equivalent variants of the polypeptide typically are made by alteration of the coding sequence of the polypeptide (e.g., MCH, or any other polypeptide associated with the disclosure).

[0120] Host Cells

[0121] Any of the polynucleotides or polypeptides of the disclosure may be expressed in a host cell. As used in this application, the term “host cell” refers to a cell that can be used to express a polynucleotide, such as a polynucleotide that encodes a polypeptide such as a MCH.

[0122] Any suitable host cell may be used to express any of the recombinant polypeptides, including any MCH, and other polypeptides disclosed in this application, including eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, fungal cells (e.g., yeast cells), bacterial cells (e.g., E. coli cells), algal cells, plant cells, insect cells, and animal cells, including mammalian cells.

[0123] Suitable yeast host cells include, but are not limited to: Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia fmlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0124] In some embodiments, the yeast strain is a Yarrowia lipolytica yeast strain. In some embodiments, the Yarrowia lipolytica yeast strain is Y-1095, Y-7754, YB-437, Y-l 1854, YB-419, Y-17536, YB-387, Y-17622, Y-l 1853, Y-48157, YB-618, Y-48653, YB-271, Y- 63532, YB-432-3, YB-272, YB-421, YB-275, YB-392, YB-276, YB-423, YB-281, Y-5383, YB-420, Y-7149, YB-422, Y-7317, YB-523, Y-7320, or YB-566.

[0125] In some embodiments, a Yarrowia lipolytica yeast strain is a mutagenized derivative of a Yarrowia lipolytica yeast strain. In some embodiments, a Yarrowia lipolytica yeast strain is a mutagenized derivative of a Yarrowia lipolytica yeast strain that behaves like the parent yeast strain. For example, in some embodiments, the term “Y-1095” includes mutagenized derivatives of the parent Y-1095 strain that behave like the parent Y-1095 strain. In some embodiments, the term “Y-7754” includes mutagenized derivatives of the parent Y-7754 strain that behave like the parent Y-7754 strain. In some embodiments, the term “YB-437” includes mutagenized derivatives of the parent YB-437 strain that behave like the parent YB- 437 strain. In some embodiments, the term “Y-11854” includes mutagenized derivatives of the parent Y-11854 strain that behave like the parent Y-11854 strain. In some embodiments, the term “YEM 19” includes mutagenized derivatives of the parent YEM 19 strain that behave like the parent YB-419 strain. In some embodiments, the term “Y-17536” includes mutagenized derivatives of the parent Y-17536 strain that behave like the parent Y-17536 strain. In some embodiments, the term “YEM 87” includes mutagenized derivatives of the parent YB-387 strain that behave like the parent YEM 87 strain. In some embodiments, the term “Y-17622” includes mutagenized derivatives of the parent Y-17622 strain that behave like the parent Y-17622 strain. In some embodiments, the term “Y-l 1853” includes mutagenized derivatives of the parent Y-l 1853 strain that behave like the parent Y-l 1853 strain. In some embodiments, the term “Y-48157” includes mutagenized derivatives of the parent Y-48157 strain that behave like the parent Y-48157 strain. In some embodiments, the term “YB-618” includes mutagenized derivatives of the parent YB-618 strain that behave like the parent YB-618 strain. In some embodiments, the term “Y-48653” includes mutagenized derivatives of the parent Y-48653 strain that behave like the parent Y-48653 strain. In some embodiments, the term “YB-271” includes mutagenized derivatives of the parent YB-271 strain that behave like the parent YB-271 strain. In some embodiments, the term “Y-63532” includes mutagenized derivatives of the parent Y-63532 strain that behave like the parent Y- 63532 strain. In some embodiments, the term “YB-432-3” includes mutagenized derivatives of the parent YB-432-3 strain that behave like the parent YB-432-3 strain. In some embodiments, the term “YB-272” includes mutagenized derivatives of the parent YB-272 strain that behave like the parent YB-272 strain. In some embodiments, the term “YB-421” includes mutagenized derivatives of the parent YB-421 strain that behave like the parent YB- 421 strain. In some embodiments, the term “YB-275” includes mutagenized derivatives of the parent YB-275 strain that behave like the parent YB-275 strain. In some embodiments, the term “YB-392” includes mutagenized derivatives of the parent YB-392 strain that behave like the parent YB-392 strain. In some embodiments, the term “YB-276” includes mutagenized derivatives of the parent YB-276 strain that behave like the parent YB-276 strain. In some embodiments, the term “YB-423” includes mutagenized derivatives of the parent YB-423 strain that behave like the parent YB-423 strain. In some embodiments, the term “YB-281” includes mutagenized derivatives of the parent YB-281 strain that behave like the parent YB- 281 strain. In some embodiments, the term “Y-5383” includes mutagenized derivatives of the parent Y-5383 strain that behave like the parent Y-5383 strain. In some embodiments, the term “YB-420” includes mutagenized derivatives of the parent YB-420 strain that behave like the parent YB-420 strain. In some embodiments, the term “Y-7149” includes mutagenized derivatives of the parent Y-7149 strain that behave like the parent Y-7149 strain. In some embodiments, the term “YB-422” includes mutagenized derivatives of the parent YB-422 strain that behave like the parent YB-422 strain. In some embodiments, the term “Y-7317” includes mutagenized derivatives of the parent Y-7317 strain that behave like the parent Y- 7317 strain. In some embodiments, the term “YB-523” includes mutagenized derivatives of the parent YB-523 strain that behave like the parent YB-523 strain. In some embodiments, the term “Y-7320” includes mutagenized derivatives of the parent Y-7320 strain that behave like the parent Y-7320 strain. In some embodiments, the term “YB-566” includes mutagenized derivatives of the parent YB-566 that behave like the parent YB-566 strain.

[0126] In some embodiments, the yeast strain is an industrial polyploid yeast strain. Other non-limiting examples of fungal cells include cells obtained from Aspergillus spp., Penicillium spp. , Fusarium spp. , Rhizopus spp. , Acremonium spp. , Neurospora spp. , Sordaria spp. , Magnaporthe spp. , Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.

[0127] In certain embodiments, the host cell is an algal cell such as Chlamydomonas (e.g., C. Reinhardtii) and Phormidium (P. sp. ATCC29409).

[0128] In other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include gram positive, gram negative, and gram-variable bacterial cells. The host cell may be a species of, but not limited to: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azotobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Campylobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas. In some embodiments, the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable for the methods and compositions described in this application.

[0129] In some embodiments, the bacterial host cell is of the Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, A. riibi). the Arthrobacterspecies (e.g., A. aurescens, A. citreus, A. globformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparaffmus, A. sulfureus, A. ureafaciens), the Bacillus species (e.g., B. thuringiensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulars, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, B. amyloliquefaciens). In particular embodiments, the host cell will be an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus and B. amyloliquefaciens. In some embodiments, the host cell will be an industrial Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, C. beijerinckii). In some embodiments, the host cell will be an industrial Corynebacterium species (e.g., C. glutamicum, C. acetoacidophilum). In some embodiments, the host cell will be an industrial Escherichia species (e.g., E. coll). In some embodiments, the host cell will be an industrial Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, E. terreus). In some embodiments, the host cell will be an industrial Pantoea species (e.g., P. citrea, P. agglomerans). In some embodiments, the host cell will be an industrial Pseudomonas species, (e.g., P. putida, P. aeruginosa, P. mevalonii). In some embodiments, the host cell will be an industrial Streptococcus species (e.g., S. equisimiles, S. pyogenes, S. uberis). In some embodiments, the host cell will be an industrial Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fimgicidicus, S. griseus, S. lividans). In some embodiments, the host cell will be an industrial Zymomonas species (e.g., Z. mobilis, Z. lipolytica), and the like.

[0130] The present disclosure may also be suitable for use with a variety of animal cell types, including mammalian cells, for example, human (including 293, HeLa, WI38, PER.C6 and Bowes melanoma cells), mouse (including 3T3, NSO, NS1, Sp2 / 0), hamster (CHO, BHK), monkey (COS, FRhL, Vero), and hybridoma cell lines.

[0131] In various embodiments, cell types or strains that may be used in the practice of the disclosure, including both prokaryotic and eukaryotic cell or strains, are readily accessible to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). The present disclosure is also suitable for use with a variety of plant cell types.

[0132] The term “cell,” as used in this application, may refer to a single cell or a population of cells, such as a population of cells belonging to the same cell line or strain. Use of the singular term “cell” should not be construed to refer explicitly to a single cell rather than a population of cells. The host cell may comprise genetic modifications relative to a wild-type counterpart.

[0133] In some embodiments, the activity of a host cell is compared to that of a reference cell. In some embodiments, a reference cell is a cell of the same species as the genetically- modified host cell, but differs from the genetically-modified host cell in that the reference cell has not been similarly genetically modified.

[0134] In some embodiments, a host cell further comprises one or more additional heterologous nucleic acids encoding one or more heterologous genes which improve malonate production. In some embodiments, the one or more heterologous genes which improve malonate production include, but are not limited to, wild-type or mutants of any of: an additional acyl-CoA hydrolase or malonyl-CoA hydrolase, an aldehyde dehydrogenase, an acetyl-CoA synthetase, a pyruvate decarboxylase, and / or an acetyl-CoA carboxylase.

[0135] In some embodiments, an additional (e.g., a second or more) acyl-CoA hydrolase or malonyl-CoA hydrolase catalyzes conversion of malonyl-CoA to malonic acid. Non-limiting examples of acyl-CoA hydrolases include wild-type and modified enzymes selected from the group consisting of 3-hydroxyisobutyryl-CoA hydrolases (EC 3.1.2.4), 3-hydroxypropionyl- CoA hydrolases (EC 3.1.2.4), acetoacetyl-CoA hydrolases (EC 3.1.2.11), methylmalonyl- CoA hydrolases (EC 3.1.2.17), propionyl-CoA hydrolases (EC 3.1.2.18), succinyl-CoA hydrolases (EC 3.1.2.3), and malonyl-CoA:ACP transacylases (EC 2.3.1.39) mutated as provided herein to have malonyl-CoA hydrolase activity.

[0136] In some embodiments, an aldehyde dehydrogenase is, as non-limiting examples, S. cerevisiae aldehyde dehydrogenase, which is ALD6, or a homologue of aldehyde dehydrogenase derived from a different organism.

[0137] In some embodiments, an acetyl-CoA synthetase is, as non-limiting examples, a Yarrowia lipolytica acetyl-CoA synthetase, or a homologue of acetyl-CoA synthetase derived from a different organism. In some embodiments, a pyruvate decarboxylase is, as non-limiting examples, a Saccharomyces cerevisiae pyruvate decarboxylase PDC1, or an E. coli pyruvate dehydrogenase, or a homologue of pyruvate decarboxylase derived from a different organism.

[0138] In some embodiments, a pyruvate decarboxylase is, as non-limiting examples, a Saccharomyces cerevisiae pyruvate decarboxylase PDC5, or a homologue of pyruvate decarboxylase derived from a different organism.

[0139] In some embodiments, a pyruvate decarboxylase is, as non-limiting examples, a Saccharomyces cerevisiae pyruvate decarboxylase PDC6, or E. coli pyruvate decarboxylase, or a homologue of pyruvate decarboxylase derived from a different organism.

[0140] In some embodiments, an acetyl-CoA carboxylase is, as non-limiting examples, Saccharomyces cerevisiae ACC1 or E. coli acetyl-CoA carboxylase, or a homologue of acetyl-CoA carboxylase derived from a different organism.

[0141] Additional genetic modifications that can be made to a host cell to improve malonate production, as well as modifications to the growth medium and / or growth conditions that can improve malonate production, are described in the art, including in: US9816114 and US20220380779.

[0142] A vector or polynucleotide encoding any one or more of the polypeptides (e.g., a MCH) described in this application may be introduced into a suitable host cell using any method known in the art. Host cells may be cultured under any conditions suitable as would be understood by one of ordinary skill in the art. For example, any media, temperature, and incubation conditions known in the art may be used. For host cells carrying an inducible vector, cells may be cultured with an appropriate inducible agent to promote expression.

[0143] Any of the cells disclosed in this application can be cultured in media of any type (rich or minimal) and any composition prior to, during, and / or after contact and / or integration of a nucleic acid. The conditions of the culture or culturing process can be optimized through routine experimentation as would be understood by one of ordinary skill in the art. In some embodiments, the selected media is supplemented with various components. In some embodiments, the concentration and amount of a supplemental component is optimized. In some embodiments, other aspects of the media and growth conditions (e.g., pH, temperature, etc.) are optimized through routine experimentation. In some embodiments, the frequency that the media is supplemented with one or more supplemental components, and the amount of time that the cell is cultured, is optimized. In some embodiments, host cells disclosed in this application are cultured in a medium containing an initial glucose concentration of at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 g / L. In some embodiments, glucose concentration is maintained in a medium using an adaptive glucose feeding method. In some embodiments, glucose feed rate is based on the glucose uptake rate of cells in the medium.

[0144] In some embodiments, the pH of the medium is about 2.0, 3.0, 4.0, 5.0, or 6.0. In some embodiments, the methods described herein include a pH shift. A “pH shift,” as used herein, refers to a fermentation process that includes an initial phase at a first pH, then a shift to a second pH for a second phase. In some embodiments, the first pH is 2.0, 3.0, 4.0, or 5.0. In some embodiments, the second pH is 2.0, 3.0, 4.0, or 5.0. In some embodiments, the first pH is 5.0, then the pH is shifted to a second pH of 3.0. In some embodiments, pH is set using a base. In some embodiments, the pH is shifted using a base. In some embodiments, the base is NaOH. In some embodiments, the base is NH4OH. In some embodiments, the base is a 5 normality (5N) solution. In some embodiments, the base is a 9N solution.

[0145] In some embodiments, cells cultured in medium are grown under specific parameters. In some embodiments, the temperature of the medium is about 30°C, the pH is about 5.0, the base used to calibrate pH is NaOH or NH4OH, and the feed is about 50% w / v glucose. In some embodiments, the temperature of the medium is about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0146] In some embodiments, the medium contains one or more trace metals. In some embodiments, the one or more metal is FeSO4, ZnSO4, CaCh, MnCh, C0CI2, CuSO4, NaMoO4, H3BO3, KI, or Na2EDTA. In some embodiments, the one or more trace metals are in hydrate form. In some embodiments, the hydrate form is 2H2O, 3H2O, 4H2O, 5H2O, 6H2O, or 7H2O. For example, a FeSChin hydrate form is denoted as FeSCh * 7H2O. In some embodiments, the concentration of the one or more trace metal is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10.0, 15.0, 19.0, or 20.0 g / L.

[0147] In some embodiments, the medium contains one or more vitamins. In some embodiments, the one or more vitamins are biotin, calcium pantothenate, nicotinic acid, myoinositol, thiamine HC1, pyridoxal HC1, or p -aminobenzoic acid. In some embodiments, the concentration of the one or more vitamins is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10.0 g / L.

[0148] In some embodiments, the medium is a Yarrowia lipolytica production medium. In some embodiments, the Yarrowia lipolytica production medium comprises ammonium sulfate; KH2PO4; MgSCh * 7H2O, batch glucose, described herein; trace metals, described herein; and vitamins; described herein.

[0149] Culturing of the cells described in this application can be performed in culture vessels known and used in the art. In some embodiments, an aerated reaction vessel (e.g., a stirred tank reactor) is used to culture the cells. In some embodiments, a bioreactor or fermenter is used to culture the cells. Thus, in some embodiments, the cells are used in fermentation. As used in this application, the terms “bioreactor” and “fermenter” are interchangeably used and refer to an enclosure, or partial enclosure, in which a biological, biochemical and / or chemical reaction takes place, involving a living organism or part of a living organism. Any type of bioreactor or fermenter known in the art may be compatible with aspects of the disclosure.

[0150] In some embodiments, the method involves batch fermentation (e.g., shake flask fermentation). General considerations for batch fermentation (e.g., shake flask fermentation) include the level of oxygen and glucose. For example, batch fermentation (e.g., shake flask fermentation) may be oxygen and glucose limited, so in some embodiments, the capability of a strain to perform in a well-designed fed-batch fermentation is underestimated. Also, the final product (e.g., malonate) may display some differences from the substrate in terms of solubility, toxicity, cellular accumulation and secretion and in some embodiments can have different fermentation kinetics.

[0151] Any suitable host cell may be used to produce any of the recombinant polypeptides (e.g, MCH) disclosed in this application, including eukaryotic cells or prokaryotic cells.

[0152] Methods

[0153] In some aspects, the disclosure provides methods of using host cells for producing products of interest. In some embodiments, the product of interest is malonate. In some embodiments, the disclosure provides a method comprising culturing a host cell described in this application (e.g, a host cell comprising a heterologous polynucleotide encoding a MCH).

[0154] In some embodiments, the disclosure provides methods of using host cells for producing higher titers of malonate relative to a reference cell. In some embodiments, the host cell produces about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 250%, or 500% more malonate than a reference cell. In some embodiments, malonate titers are measured in grams per liter of malonate. In some embodiments, the host cell produces about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 grams of malonate per liter. In some embodiments, the host cell produces greater than 100 grams of mal onate per liter. In some embodiments, the host cell produces greater than 115 grams of mal onate per liter. In some embodiments, the host cell produces greater than 120 grams of mal onate per liter. In some embodiments, the host cell produces greater than 125 grams of mal onate per liter. In some embodiments, the host cell produces greater than 130 grams of mal onate per liter. In some embodiments, the host cell produces greater than 135 grams of mal onate per liter. In some embodiments, the host cell produces greater than 140 grams of mal onate per liter. In some embodiments, the host cell produces greater than 145 grams of mal onate per liter. In some embodiments, the host cell produces greater than 150 grams of mal onate per liter.

[0155] In some embodiments, malonate production is measured by malonate yield. In some embodiments, malonate yield is measured as gram of malonate per gram of glucose, wherein grams of glucose is the amount of glucose provided to a host cell. In some embodiments, the host cell yields greater than 0.3 gram of malonate per gram of glucose. In some embodiments, the host cell yields greater than 0.35 gram of malonate per gram of glucose. In some embodiments, the host cell yields greater than 0.4 gram of malonate per gram of glucose. In some embodiments, the host cell yields greater than 0.45 gram of malonate per gram of glucose. In some embodiments, the host cell yields greater than 0.5 gram of malonate per gram of glucose.

[0156] In some embodiments, malonate production is measured by productivity. In some embodiments, productivity is measured as grams of malonate per liter per hour. In some embodiments, the host cell malonate production productivity is greater than 1 gram of malonate per liter per hour. In some embodiments, the host cell yields greater than 1 gram of malonate per liter per hour. In some embodiments, the host cell yields greater than 1.2 grams of malonate per liter per hour. In some embodiments, the host cell yields greater than 1.4 , 1.5, 1.6 or 1.7 grams of malonate per liter per hour.

[0157] The present disclosure is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co pending patent applications) cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

[0158] Example 1. Construction of Yarrowia lipolytica expressing a heterologous malonyl-CoA hydrolase (MCH)

[0159] Y. lipolytica cells expressing a variant MCH enzyme (SEQ ID NO: 3) were generated as follows. DNA constructs for heterologous expression of MCH were synthesized and comprise a Y. lipolytica-co&on optimized nucleotide sequence (SEQ ID NO: 4) encoding the MCH enzyme. The codon-optimized nucleotide sequence (SEQ ID NO: 4) encoding the MCH enzyme was operably linked to a promoter (pGAPDH; SEQ ID NO: 5) and a terminator (tEXP; SEQ ID NO: 6).

[0160] The MCH-expression construct included an antibiotic resistance cassette that conferred resistance to nourseothricin, which was used as a selective marker.

[0161] Linearized DNA fragments of the MCH-expression construct were transformed into 30 Y lipolytica strains using a lithium acetate / single-stranded carrier DNA / polyethylene glycol method. This allowed for single copy integration of the MCH constructs into the genomes.

[0162] The 30 Y. lipolytica strains used in this experiment were obtained from the ARS culture collection (NRRL).

[0163] Example 2. Yarrowia lipolytica fermentation

[0164] Y lipolytica fermentation was performed using a glucose pulsed-feeding process known to those of skill in the art. Medium containing an initial 55 g / L of glucose was used as the fermentation media. Glucose levels were maintained during fermentation using an adaptive glucose feeding method in which the feed rate was based on the culture’s glucose uptake rate. The fermentation was performed at a pH of 2, 3, 4 or 5. Where a pH shift was employed during fermentation, an initial phase of fermentation was performed at pH 5.0 and then the pH of the media was allowed to shift to 3.0. The pH of the fermentation was set and / or maintained using NaOH or NH4OH (5N or 9N solution). End of fermentation was defined as 72hrs elapsed fermentation time.

[0165] Table 2. Representative fermentation parameters

[0166] Table 3. Representative Yarrowia lipolytica metals

[0167] Table 4. Representative Yarrowia lipolytica vitamins

[0168] Table 5. Representative Yarrowia lipolytica production medium

[0169] Example 3. Production of malonate by modified Y. lipolytica strains

[0170] Transformed Y. lipolytica cells generated as described in Example 1 were evaluated for production of malonate in a 96-deepwell plate and in Ambr250 fermentation.

[0171] For plate assays, Y. lipolytica cultures were grown at 28 °C for 48 hours in a 500 pL volume in 96 deepwell plates containing medium for plate assay, which corresponds to a modification of the fermentation medium described in Example 2.

[0172] For Ambr250 fermentation, Y. lipolytica cells were grown in a glucose pulsed-feeding process known to those of skill in the art. Medium containing an initial 55 g / L of glucose was used as the fermentation media (Example 2). Glucose levels were maintained during fermentation using an adaptive glucose feeding method in which the feed rate was based on the glucose uptake rate of the culture. The fermentation was performed at a pH of 2, 3, 4 or 5. Where a pH shift was employed during fermentation, an initial phase of fermentation was performed at pH 5.0 and then the pH of the media was allowed to shift to 3.0. The pH of the fermentation was set and / or maintained using NaOH or NH4OH (5N or 9N solution). End of fermentation was defined as 72hrs elapsed fermentation time.

[0173] Malonate in filtered supernatants was then analyzed using a colorimetric assay and HPLC-RID.

[0174] In the colorimetric assay, filtered supernatants were diluted 10-fold and analyzed for malonate using a 4-diazobenzenesulfonic acid (DBS) colorimetric assay, measuring absorbance at 530 nm. Such assays are known in the art as described in and incorporated by reference from, for example: W02021042058A3.

[0175] In the HPLC assay, an HPLC-RID method was used to detect malonate, acetate, glucose, citrate, ethanol, and glycerol in fermentation samples. Filtered supernatants were subject to HPLC-RID for malonate and other analytes including acetate, glucose, ethanol, and citric acid. Filtered supernatants were analyzed on a Bio-Rad Aminex HPX-87H column (300 x 7.8 mm) with a flow rate of 0.6 ml / minute using 5 mM sulfuric acid in water. 20 pL were injected per sample, the column temperature was 35°C with a total run time of 23 minutes per sample.

[0176] Results obtained showed that 30 K lipolytica strains transformed with the heterologous MCH expression construct described in Example 1 (expressing the MCH enzyme variant of SEQ ID NO: 3) produced between 4 and 23 g / L of malonate in the plate assay as measured by HPLC. These Y lipolytica transformants were derived from the following Y. lipolytica strains: Y-1095, Y-7754, YB-437, Y-11854, YB-419, Y-17536, YB- 387, Y-17622, Y-11853, Y-48157, YB-618, Y-48653, YB-271, Y-63532, YB-432-3, YB- 272, YB-421, YB-275, YB-392, YB-276, YB-423, YB-281, Y-5383, YB-420, Y-7149, YB- 422, Y-7317, YB-523, Y-7320, and YB-566.

[0177] Three MCH-expressing Y lipolytica strains producing the highest malonate titers (Y- 48157, YB-392 and Y-7320) were chosen for further evaluation of malonate production in Ambr250 fermentation. All Ambr250 fermentations used a Y. lipolytica-specific process and production medium as described above. The effect of low pH (2.0, 3.0, 4.0, and 5.0) on malonate production was also tested in a glucose pulsed-feeding fermentation process using NaOH as base. Results are shown in Table 6 and FIG. 1.

[0178] The highest malonate titers were observed at pH 4.0 under which strain YB-392 produced 125 g / L and strain Y-7320 produced 123 g / L. Malonate production was also observed for cultures at pH 3.0, under which the highest malonate titer of 112 g / L was produced by strain YB-392, and at pH 5.0 under which the highest malonate titer of 119 g / L was also produced by strain YB-392. The reported malonate titers were determined after 72 hours of fermentation. No malonate was detected in cultures at pH 2.0 cultures as the cells did not grow under these conditions.

[0179] Table 6 provides the yield of malonate produced by fermentation per gram of glucose determined at pH 3.0, 4.0 and 5.0 for Y. lipolytica strains YB-392, Y-7320, and Y-48157 expressing the variant MCH enzyme of SEQ ID NO. 3. Yield was calculated based on how much glucose was fed into the culture and how much malonate was produced per gram glucose.

[0180] Table 6. Malonate titer, rate, and yield for Y lipolytica strains expressing the variant MCH enzyme of SEQ ID NO. 3

[0181] Results indicated that K lipolytica strains expressing the MCH enzyme of SEQ ID NO: 3 produced significantly higher titers of mal onate than a Y. lipolytica strain that did not express an MCH enzyme, the latter producing <2 g / L malonate in Ambr250 fermentation.

[0182] The highest yield of malonate produced by Y. lipolytica strain YB-392 expressing the MCH enzyme of SEQ ID NO: 3 was 0.48 g of malonate per gram of glucose during fermentation at pH 4.0. The second highest yield of malonate produced by Y lipolytica strain YB-392 was 0.47 g of malonate per gram of glucose during fermentation at pH 5.0. These correspond to a 48 % and a 47 % yield per gram of glucose.

[0183] The highest yield of malonate produced by Y lipolytica strain Y-7320 expressing the MCH enzyme of SEQ ID NO: 3 was 0.40 g of malonate per gram of glucose during fermentation at pH 4.0. The second highest yield of malonate produced by Y lipolytica strain Y-7320 was 0.38 g of malonate per gram of glucose during fermentation at pH 5.0. These correspond to a 40 % and a 38 % yield per gram of glucose.

[0184] The highest yield of malonate produced by Y. lipolytica Y-48157 expressing the MCH enzyme of SEQ ID NO: 3 was 0.38 grams of malonate per gram of glucose during fermentation at pH 4.0. The second highest yield of malonate produced by Y lipolytica strain Y-48157 was 0.32 g of malonate per gram of glucose during fermentation at pH 3.0. These correspond to a 38 % and a 32 % yield per gram of glucose.

[0185] All three of the above Yarrowia lipolytica strains produced higher malonate titer, at a faster rate and better yield than a control Pichia kudriavzevii malonate production strain, which produced a titer of 72 g / L of malonate at a rate of 1.0 g / L / h'1and yield 0.27 grams of malonate per gram of glucose.

[0186] Furthermore, Y lipolytica strains produced higher malonate titers at pH 3.0 than the control P. kudriavzevii strain at pH 5.0. Better production performance at lower pH allowed for less base addition and less by-product formation (e.g, ammonium sulfate formation) when K lipolytica was used as a production host.

[0187] Example 4. Production of malonate by Yarrowia fermentation An additional twelve Y. lipolytica strains, specifically, Y-7754, Y-17536, Y-17622,

[0188] YB-275, YB-276, YB-420, YB-566, Y-1095, YB-387, Y-11853, YB-271 and Y-5383 were evaluated for malonate production in Ambr250 fermentation in a glucose pulsed-feeding process as described in Example 2. These Y. lipolytica host strains were engineered to express the variant MCH enzyme of SEQ ID NO: 3 as described above in Example 1. Fermentation was performed at a pH of 4 and NaOH was used as base to maintain pH. Fermentation was also performed with a pH shift from pH 5.0 to 3.0 at about 24 hours into the fermentation. Results are provided in Table 7.

[0189] Results showed that Y. lipolytica strain YB-392 expressing MCH of SEQ ID NO: 3 produced 129 g / L malonate during fermentation at pH 4.0 at 72 hours, a titer comparable to the 125 g / L obtained in Example 3.

[0190] Table 7. Malonate titer, rate, and yield of Y. lipolytica strains expressing a variant MCH

[0191] From the 12 additional K lipolytica strains, two strains resulted in higher malonate production than YB-392 at the pHs screened. Specifically, Y. lipolytica YB-275 produced malonate titers of 125 g / L in a fermentation at pH 4.0 and 137 g / L in a fermentation with a pH shift 5.0 to 3.0. And Y. lipolytica YB-387 produced malonate titers of 126 g / L in a fermentation at pH 4.0 and 148 g / L in a fermentation with a pH shift 5.0 to 3.0, respectively.

[0192] Example 5. Malonate production by Y. lipolytica fermentation using different neutralizing agents To test whether the NaOH base could be substituted for NH4OH to maintain the pH during fermentation, four of the best producing strains of Y lipolytica expressing a heterologous MCH enzyme (SEQ ID NO: 3) were evaluated. Specifically, Y lipolytica strains YB-392, YB-387, YB-275 and Y-7320 were tested in Ambr250, and fermentation pH was set or maintained using NaOH or NH4OH as base. Fermentation of Y lipolytica strains YB-387, YB-275 and Y-7320 was performed with a pH shift of 5 to 3. Fermentation of Y. lipolytica strain YB-392 was performed at a pH of 4.

[0193] Filtered supernatants were analyzed using HPLC as described in Example 3. Results in Table 8 below showed that malonate production was comparable in fermentations using NH4OH as base with the fermentations in which NaOH was used as base. The highest malonate titer among all conditions tested was 142 g / L, which was achieved by strain YB- 387, when fermentation pH was maintained using NaOH. This was 3.6 % higher than, i.e., substantially comparable to, the 138 g / L of malonate produced by the same strain when fermentation pH was maintained using NH4OH.

[0194] Table 8. Malonate titer, rate, and yield of Y. lipolytica strains expressing the variant

[0195] MCH enzyme of SEQ ID NO: 3

[0196] Example 6. Malonate production over 120 hours

[0197] To assess malonate production by fermentation beyond 72 hours, 120-hour Ambr250 fermentations were performed using Y. lipolytica YB-387 and YB-275 strains expressing a heterologous MCH enzyme (SEQ ID NO: 3). YB-387 reached 154 g / L (1480 mM) malonate at 72 hours. Malonate titer increased to

[0198] 180 g / L (1730 mM) through 96 hours with only a minor increase to 185 g / L (1778 mM) at 120 hours. The productivity of YB-387 at 96 hour was 1.9 g / L*h-1with a yield of 0.48 g / g glucose.

[0199] YB-275 reached 114 g / L (1096 mM) malonate at 72 hours. Malonate titer increased to 120 g / L (1153 mM) through 96 hours with only a minor increase to 125 g / L (1201 mM) at 120 hours. The productivity of YB-275 at 96 hour was 1.25 g / L*h-1with a yield of 0.32 g / g glucose.

[0200] In contrast, a control P. kudriavzevii production strain produced malonate at a titer of 72 g / L (692 mM), with a productivity of 1.0 g / L*h-1and a yield of 0.27 g / g glucose after 72 hours.

[0201] Thus, process optimizations led to improved malonate production in Yarrowia lipolytica fermentation as the best Y. lipolytica strain produced higher titer, at a better rate and yield, specifically, a malonate titer of 154 g / L with a productivity of 2.1 g / L*h-1and a yield of 0.48 g / glucose after 72h), easily surpassing the performance of the control P. kudriavzevii production strain in the Ambr run (titer: 72 g / L, productivity: 1.0 g / L*h_|, yield: 0.27 g / g glucose after 72h).

[0202] Example 7. Production of malonate by Y. lipolytica in a 30 L fermenter

[0203] Y. lipolytica strain YB-387 expressing a heterologous MCH enzyme (SEQ ID NO: 3) was evaluated in a 30-liter fermentation run for malonate production (FIG. 2). The fermentation process was similar to the Ambr250 fermentation process with a controlled feed rate based on the culture’s glucose uptake rate (adaptive feed rate). The end of fermentation titer, i.e., titer at 72 hours, for malonate was measured as 143 g / L with a yield of 0.46 g / g glucose.

[0204] These results indicated that the high malonate productivity by Y lipolytica in an Ambr250 fermentation process was scaled up to a 30L fermentation bioreactor without a decrease in malonate titer.

[0205] Example 8. Production of malonate by Y lipolytica strains expressing variant MCH enzymes

[0206] Y. lipolytica strains YB-387 and YB-392 were engineered as described in Example 1 to express additional variant MCH enzymes derived from the parent enzyme of SEQ ID NO. 1. The engineered cells were evaluated for production of malonate in a 96-deepwell plate and in Ambr250 fermentation as described in the examples above. Fermentation was performed at a pH of 5.0 or with a pH shift from pH 5.0 to 3.0 at about 24 hours into the fermentation.

[0207] The base used for pH adjustment in the fermentation was NH4OH.

[0208] Ambr250 fermentation results computed as summarized above are shown in Table 9.

[0209] Results show that K lipolytica strains with heterologous expression of MCH variants produced between 95 and 159 g / L mal onate. In contrast, strains without the MCH enzyme variants produced <2 g / L malonate in Ambr250 fermentation.

[0210] Table 9. Malonate titer, rate, and yield for Y. lipolytica strains expressing variant MCH enzymes

[0211]

[0212] Table 10. Nucleotide and amino acid sequences of variant MCH enzymes

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] Example 9. Production of malonate by Y. lipolytica strains expressing variant MCH enzyme

[0223] To identify additional variant enzymes capable of producing increased titers of malonate, variants of 10 homologs of the 3-hydroxyisobutyryl-CoA hydrolase from Pseudomonas fulva (SEQ ID NO: 1) were expressed in Y. lipolytica strains YB-387 and YB-

[0224] 392, which were engineered for expression of the variant homologs following the methods described in Example 1.

[0225] The engineered strains were evaluated for malonate production in a 96-deep well plate using the colorimetric assay described above. Of the strains tested, strains expressing SEQ ID NO: 28, which was derived from a parent 3-hydroxyisobutyryl-CoA hydrolase enzyme having the amino acid sequence set forth in SEQ ID NO: 27, were found to produce significant levels of malonate. FIG. 3 shows malonate levels as a colorimetric signal (top half) and malonate levels per biomass (bottom half) and demonstrates that Y. lipolytica strains expressing SEQ ID NO: 28 produced a colorimetric signal for malonate that was comparable to Y lipolytica strains expressing SEQ ID NO: 3. Wildtype Y lipolytica strains (strains that do not express an MCH enzyme), as well as strains expressing the other variants did not produce a colorimetric signal above background.

[0226] EQUIVALENTS

[0227] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in the present application. Such equivalents are intended to be encompassed by the following claims.

[0228] All references, including patent documents, are incorporated by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A method for producing mal onate comprising: culturing a host cell that expresses a heterologous polynucleotide encoding a malonyl- CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an alanine (A) at a position corresponding to position 348 in SEQ ID NO: 1; and wherein the host cell is Yarrowia lipolytica (Y. lipolytica) strain YB-392, Y-7320, YB-275, YB-387, Y-7754, Y-17536, Y-17622, YB-276, YB-420, YB-48157, Y-1095, YB- 11853, YB-271, or Y-5383.

2. The method of claim 1, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of SEQ ID NO: 3.

3. The method of claim 1 or 2, wherein the Y lipolytica strain is YB-392, Y-7320, YB- 275, or YB-387.

4. The method of any one of claims 1-3, wherein the Y. lipolytica strain is YB-387.

5. The method of any one of claims 1-4, wherein the method produces at least 100 grams per liter of malonate.

6. The method of any one of claims 1-5, wherein the method produces at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, or at least 140 grams per liter of malonate.

7. The method of any one of claims 1-6, wherein the method produces malonate in a cell culture.

8. The method of claim 7, wherein the pH of the cell culture is between about 3.0 and about 5.0.

9. The method of claim 7 or 8, wherein the cell culture further comprises a base.

10. The method of claim 9, wherein the base is NaOH or NH4OH.

11. A host cell that comprises a heterologous polynucleotide encoding a malonyl-CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1 and comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an alanine (A) at a position corresponding to position 348 in SEQ ID NO: 1; and wherein the host cell is Yarrowia lipolytica (Y. lipolytica) strain YB-392, Y-7320, YB-275, YB-387, Y-7754, Y-17536, Y-17622, YB-276, YB-420, YB-48157, Y-1095, YB-11853, YB- 271, or Y-5383.

12. The method of claim 11, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of SEQ ID NO: 3.

13. The host cell of claim 11 or 12, wherein the Y. lipolytica strain is YB-392, Y-7320, YB-275, or YB-387.

14. The host cell of any one of claims 11-13, wherein the Y. lipolytica strain is YB-387.

15. The host cell of any one of claims 11-14, wherein the host cell is capable of producing at least 100 grams per liter of mal onate.

16. The host cell of any one of claims 11-15, wherein the host cell is capable of producing at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, or at least 140 grams per liter of mal onate.

17. A method of producing greater than 115 grams per liter of mal onate, the method comprising: culturing a host cell that expresses a heterologous polynucleotide encoding a malonyl- CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1 and comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an alanine (A) at a position corresponding to position 348 in SEQ ID NO: 1; and wherein the host cell is a Yarrowia lipolytica (Y. lipolytica) strain.

18. The method of claim 17, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of SEQ ID NO: 3.

19. The method of claim 17 or 18, wherein the host cell is Y. lipolytica strain YB-392, Y- 7320, YB-275, YB-387, or YB-420.

20. A method for producing greater than 0.3 gram of malonate per gram of glucose comprising: culturing a host cell that expresses a heterologous polynucleotide encoding a malonyl- CoA hydrolase, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1 and wherein the amino acid sequence of the malonyl-CoA hydrolase comprises: an asparagine (N) at a position corresponding to position 95 in SEQ ID NO: 1, an alanine (A) at a position corresponding to position 152 in SEQ ID NO: 1, an arginine (R) at a position corresponding to position 304 in SEQ ID NO: 1, and an A at a position corresponding to position 348 in SEQ ID NO: 1; wherein the host cell is a Yarrowia lipolytica (Y. lipolytica) strain.

21. The method of claim 20, wherein the amino acid sequence of the malonyl-CoA hydrolase comprises the sequence of SEQ ID NO: 3.

22. The method of claim 20 or 21, wherein the K lipolytica strain is YB-392, Y-7320, YB-275, YB-387, Y-17536, YB-276, YB-420, YB-48157, YB-11853, or YB-271.

23. The method of any one of claims 20-22, wherein the method produces greater than 0.4 gram of malonate per gram of glucose.

24. The method of any one of claims 20-23, wherein the host cell is Y. lipolytica strain YB-392, Y-7320, YB-275, YB-276, YB-420, or YB-387.

25. The method of any one of claims 20-24, wherein the method produces greater than 0.5 gram of malonate per grams of glucose.

26. The method of any one of claims 20-25, wherein the host cell is Y. lipolytica strain YB-392.

27. A method of producing greater than 115 grams per liter of malonate, the method comprising: culturing a host cell according to any one of claims 11-16 under fermentation conditions that comprise a pH of about 3.0, about 4.0, or about 5.0.

28. The method of any one of the preceding claims, wherein the yield on glucose is about 0.2 to about 0.5 gram of malonate per gram of glucose.

29. A fermentation composition, comprising: a host cell according to any one of claims 11-16, culture media comprising a pH of 3.0, 4.0, or 5.0 and 115 grams per liter of malonate.

Images