RECOVERY OF MALONIC ACID AND ITS ESTERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority under 35 U.S.C. 119(e) and Article 2 of the Paris Convention for the Protection of Industrial Property (1883) to U.S. provisional application serial number 62/549,164, filed 23 August 2017, the entire contents of which are incorporated herein by this reference.

BACKGROUND

[002] The long-term economic and environmental concerns associated with the petrochemical industry have provided the impetus for increased research, development, and commercialization of processes for conversion of carbon feedstocks into chemicals that can replace those petroleum feedstocks. One approach is the development of biorefining processes to convert renewable feedstocks into products that can replace petroleum-derived chemicals. Two common goals in improving a biorefining process include achieving a lower cost of production and reducing detrimental effects on the environment.

[003] Malonic acid or propanedioic acid (CAS No. 141-82-2) and its esters (most typically dimethyl and diethyl malonate, but also monoesters) are important building block chemicals that are used in a wide range of industries and applications including flavors and fragrances, electronics, polymer & materials as well as a precursor in pharmaceutical synthesis. The diverse set of applications derive from specific properties of the malonic acid family of chemicals including strong chelation of metals, high water solubility, low temperature cross linking, two acid functional groups combined with reactive center carbons in addition to being an acetyl donor upon decarboxylation.

[004] Most malonic acid is currently produced from non-renewable, petroleum feedstocks via reaction of chloroacetic acid with sodium cyanide. Both of these key starting materials are very toxic and present significant safety risks to workers and surrounding communities. Typically, the chloroacetic acid is first reacted with NaHC03 to convert to the salt form. Subsequent reaction with sodium cyanide generates the nitrile with concomitant release of sodium chloride salts as a by-product. The nitrile group is then hydrolyzed with a strong base such as NaOH to form sodium malonate with release of ammonia. The ammonia is typically captured as ammonium sulfate via absorption and reaction with a sulfuric acid solution. The current process consumes more than 3 kg of reagents per kg pure malonic acid produced and produces significant amounts of undesired co-products, including more than 1.2 kg combined sodium chloride and ammonium sulfate waste salts. The sodium malonate produced is not the main product of commerce and typically is converted to diesters of malonate, such as diethyl or dimethyl malonate, using sulfuric acid in a stoichiometric ratio resulting in an additional 1.4 kg sodium sulfate byproduct per kg malonic acid. The inherent inefficiency of the current process, combined with use of toxic chemicals and generation of significant amounts of waste and byproducts, clearly demonstrates the need for a more efficient and environmentally benign approach.

[005] Methods to produce malonic acid from renewable resources have been developed by direct fermentation from sugars (U.S. Patent Application No. 14/386,272), oxidation of 3-hydroxypropionic acid (U.S. Patent No. 5,817,870), or oxidation of 1,3 -propanediol. The direct microbial route to malonic acid from glucose is particularly attractive with a high theoretical yield of at least 2 mole malonic acid per mole glucose catabolized by the microbe. Malonic acid can be recovered from fermentation broth as an insoluble calcium salt (e.g., calcium malonate dihydrate) that is subsequently converted to free malonic acid by reaction with sulfuric acid that also generates gypsum co-product.

SUMMARY

[006] The present disclosure provides improved methods for higher fermentation yields and productivities in the production of malonic acid and soluble malonic acid salts. The present disclosure also provides methods for conversion of soluble malonic acid salts into high quality malonic diesters and free malonic acid.

[007] According to one aspect, a process to produce malonic acid diesters and free malonic acid is presented, the process comprising:

1. fermenting a renewable feedstock to produce malonic acid, with partial or complete neutralization, using an alkali metal base or ammonium base to produce a soluble malonic acid salt;

2. recovering the malonic acid salt from fermentation broth;

3. forming a malonate diester by esterifying the recovered malonic acid salt with an alcohol using supercritical carbon dioxide (sCC^) or sulfuric acid as a catalyst; and,

4. distilling the malonate diester to isolate a purified malonate diester.

[008] A renewable feedstock is one produced from renewable resources that are capable of naturally replenishing in a short amount of time, with or without human intervention, with minimal generation of harmful by-products. Use of a renewable feedstock therefore helps to overcome reliance upon non-renewable energy sources and serves to overcome resource depletion caused by an excess of human consumption.

[009] In some embodiments, the alkali metal is sodium or potassium. In many embodiments, the alkali metal base is an alkali metal bicarbonate, for example sodium bicarbonate. In other embodiments, the alkali metal base is an alkali metal hydroxide, for example sodium hydroxide and/or potassium hydroxide. In those embodiments where an ammonium base is used, the ammonium base is selected from the group consisting of ammonia, ammonium hydroxide, and ammonium carbonate. In some

embodiments, the ammonium base is ammonia. In other embodiments, the ammonium base is ammonium hydroxide. In still other embodiments, the ammonium base is ammonium carbonate.

[0010] In some embodiments, an alkali metal carbonate resulting from esterification of the malonic acid salt is isolated and used to partially or completely neutralize malonic acid in the fermentation step. In other embodiments, an ammonium base is used to partially or completely neutralize malonic acid in the fermentation step.

[0011] In some embodiments, sodium sulfate (NaaSO/i) or ammonium sulfate is isolated as a co-product from (F^SC -catalyzed esterification of malonic acid (MA) salts.

[0012] In some embodiments, the malonate diesters produced from the esterification are hydrolyzed to produce free malonic acid. In that regard, the present disclosure provides a process to produce free malonic acid from diesters, comprising:

1. hydrolyzing purified malonate diesters with an acid to produce free malonic acid, wherein

hydrolysis temperatures fall within a range that precludes malonic acid decarboxylation;

2. crystallizing the malonic acid; and

3. purifying the malonic acid crystals.

[0013] In some embodiments, the esterification alcohols are selected from methanol, ethanol, 1- propanol, 1-butanol, or isobutanol. In some embodiments, the esterification alcohol is ethanol. In some embodiments, the esterification alcohol is methanol.

[0014] With respect to the foregoing, the inventors have surprisingly found that the elimination of insoluble calcium base in favor of the disclosed soluble alkali metal bases results in improved processes with increased malonic acid titers, yields, and/or productivities. Without wishing to be bound by theory, the inventors have observed that insoluble calcium base can precipitate other salts from the fermentation broth in addition to malonic acid such as insoluble calcium phosphate and sulfate salts that may result in depletion of essential nutrients resulting in a less productive fermentation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Those skilled in the art will understand that the drawings described herein are for illustration purposes only. The drawings are not intended to limit the scope of the present disclosure.

[0016] FIG. 1 is a block diagram of the process for making diethyl malonate (DEM), and optionally malonic acid, from fermentation broth containing disodium malonate (Na ₂ M) by esterification of isolated Na2M with supercritical carbon dioxide (SCO2) and ethanol. [0017] FIG. 2 is a block diagram of the process for making DEM, and optionally MA, from fermentation broth containing Na ₂ M by esterification of isolated Na ₂ M with sulfuric acid and ethanol.

DETAILED DESCRIPTION

[0018] The present disclosure provides materials and methods for the production and purification of malonic acid and its diesters, e.g. , dimethyl malonate and diethyl malonate, from fermentation broths containing a soluble alkali metal salt of malonic acid and/or an ammonium salt of malonic acid. In some embodiments, the present disclosure provides recombinant host cells, materials, methods and process flows for the production of disodium malonate. In some embodiments, the present disclosure provides recombinant host cells, materials, methods and process flows for the production of diammonium malonate. The disodium malonate and the diammonium malonate produced by the disclosed methods can be transformed to various commodity chemicals, including, for example, diethyl malonate, dimethyl malonate, and malonic acid. The present disclosure also provides methods and process flows for the conversion of disodium malonate or diammonium malonate to malonate diesters and/or malonic acid, as well as methods and process flows for their recovery and purification.

[0019] The present disclosure also provides materials and methods for the production and purification of malonic acid and its monoesters, e.g. , monomethyl malonate and monoethyl malonate, from fermentation broths containing a soluble alkali metal salt of malonic acid and/or an ammonium salt of malonic acid. In some embodiments, the present disclosure provides recombinant host cells, materials, methods and process flows for the production of disodium malonate. In some embodiments, the present disclosure provides recombinant host cells, materials, methods and process flows for the production of diammonium malonate. The disodium malonate and the diammonium malonate produced by the disclosed methods can be transformed to various commodity chemicals, including, for example, diethyl malonate, monoethyl malonate, dimethyl malonate, monomethyl malonate, and malonic acid. The present disclosure also provides methods and process flows for the conversion of disodium malonate or diammonium malonate to malonate monoesters and/or malonic acid, as well as methods and process flows for their recovery and purification.

[0020] While the present disclosure describes aspects and specific embodiments, those skilled in the art will recognize that various changes may be made and equivalents may be substituted without departing from the scope of the present disclosure. The present disclosure is not limited to particular nucleic acids, expression vectors, enzymes, biosynthetic pathways, host microorganisms, or processes, as such may vary. The terminology used herein is for the purposes of describing particular aspects and embodiments only, and is not to be construed as limiting. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process steps or process flows, in accordance with the present disclosure. All such modifications are within the scope of the claims appended hereto.

Section 1: Definitions

[0021] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0022] The terms "ferment", "fermentative", and "fermentation" are used herein to describe culturing microbes under conditions to produce useful chemicals, including but not limited to conditions under which microbial growth, be it aerobic or anaerobic, occurs.

[0023] The terms "host cell" and "host microorganism" are used interchangeably herein to refer to a living cell that can be, or has been, transformed via insertion of an expression vector. A host cell or microorganism as described herein may be a prokaryotic cell (e.g., a microorganism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane -bound nucleus.

[0024] The terms "isolated" or "pure" refer to material that is substantially, e.g., greater than 50% or greater than 75%, or essentially, e.g., greater than 90%, 95%, 98% or 99%, free of components that normally accompany it in its native state, e.g., the state in which it is naturally found or the state in which it exists when it is first produced. Additionally, any reference to a "purified" material is intended to refer to an isolated or pure material.

[0025] As used herein, "recombinant" refers to the alteration of genetic material by human intervention. Typically, recombinant refers to the manipulation of DNA or R A in a cell or virus or expression vector by molecular biology (recombinant DNA technology) methods, including cloning and recombination. Recombinant can also refer to manipulation of DNA or RNA in a cell or virus by random or directed mutagenesis. A "recombinant" cell or nucleic acid can typically be described with reference to how it differs from a naturally occurring counterpart (the "wild-type"). In addition, any reference to a cell or nucleic acid that has been "engineered" or "modified" and variations of those terms, is intended to refer to a recombinant cell or nucleic acid.

[0026] As used herein, "malonic acid" means the molecule having the chemical formula

CH ₂ (COOH) ₂ and a molecular mass of 104.06 g/mol (CAS # 141-82-2). The terms "malonic acid," "MA," "free malonic acid," and "propanedioic acid" are used interchangeably in the present disclosure, and practitioners skilled in the art understand that these terms are synonyms.

[0027] As used herein, "monoethyl malonate" means monoethyl-substituted malonic acid, that is the molecule having the chemical formula C5H8O4 and a molecular mass of 132.11 g/mol (CAS # 1071-46-1). The terms "monoethyl malonate," "MEM," "ethyl malonate," "monoethyl hydrogen malonate," "(ethoxycarbonyl)acetic acid," "3-thoxy-3-oxopropanoic acid," "ethyl hydrogen malonate" are used interchangeably in the present disclosure, and practitioners skilled in the art understand that these terms are synonyms. MEM is an example of a MA monoester.

[0028] As used herein, "diethyl malonate" means diethyl-substituted malonic acid, that is the molecule having the chemical formula and a molecular mass of 160.17 g/mol (CAS # 105-53-3). The terms "diethyl malonate," "DEM," "malonic acid diethyl ester" and "diethyl

propanedioate" are used interchangeably in the present disclosure, and practitioners skilled in the art understand that these terms are synonyms. DEM is an example of a MA diester.

[0029] As used herein, "disodium malonate" means a disodium malonate salt, that is the molecule having the chemical formula CH ₂ (C0 ₂ Na) ₂ and a molecular mass of 148.03 g/mol (CAS # 141-95-7). The terms "disodium malonate," "sodium malonate," "Na ₂ M," "sodium malonate dibasic" and "malonic acid disodium salt" are used interchangeably in the present disclosure, and practitioners skilled in the art understand that these terms are synonyms.

[0030] As used herein, "dipotassium malonate" means a dipotassium malonate salt, that is the molecule having the chemical formula C3H2K2O4 and a molecular mass of 180.242 g/mol. The terms "dipotassium malonate," "potassium malonate," "K2M," and "malonic acid dipotassium salt" are used interchangeably in the present disclosure, and practitioners skilled in the art understand that these terms are synonyms.

[0031] As used herein, "diammonium malonate" means a diammonium malonate salt, that is the molecule having the chemical formula C3H10N2O4 and a molecular mass of 138.12 g/mol. The terms "diammonium malonate," "ammonium malonate," and "malonic acid diammonium salt" are used interchangeably in the present disclosure, and practitioners skilled in the art understand that these terms are synonyms.

[0032] As used herein, "ammonium base" means ammonia (CAS # 14798-03-9), ammonium hydroxide (CAS # 1336-21-6), ammonium carbonate (CAS # 506-87-6) and, in some embodiments, combinations of the foregoing.

[0033] As used herein, a "malonic acid salt" (MA salt) means a molecule that is formed when ionized malonic acid reacts with an alkali metal cation or an ammonium cation. Non-limiting examples of MA salts include Na ₂ M, (NH ₄ ) ₂ M, and K ₂ M.

[0034] Malonic acid salts provided by the present disclosure may be prepared e.g. in crystalline form and may be solvated or hydrated. Suitable solvates include commercially acceptable solvates, such as hydrates, and may further include both stoichiometric solvates and non-stoichiometric solvates. Solvate refers to forms of the disclosed malonic acid salts that are associated with a solvent or with water (referred to as "hydrate"). This physical association typically includes hydrogen bonding. Conventional solvents include water, ethanol, methanol, acetic acid and the like. Malonic acid salts of the present disclosure can crystallize in various states of hydration. For example, Na2M can form Na2M-monohydrate crystals, wherein a single molecule of water crystallizes with a single molecule of Na2M. Na2M crystals that lack water are anhydrous. Similarly, K2M can form K^M-monohydrate crystals as well as anhydrous K2M crystals. In some embodiments, the hydrates will be capable of isolation, for example when one or more water molecules are incorporated in the crystal lattice of the crystalline solid.

[0035] As used herein, "sodium malonate dibasic monohydrate" means the molecule having the chemical formula CitiCC Na^-itO and a molecular mass of 166.04 g/mol (CAS # 26522-85-0). The terms "sodium malonate dibasic monohydrate," "sodium malonate monohydrate," "Na ₂ M-monohydrate," "malonic acid disodium salt monohydrate" and "propanedioic acid disodium salt monohydrate" are used interchangeably in the present disclosure, and practitioners skilled in the art understand that these terms are synonyms.

[0036] In various aspects provided by the present disclosure, malonic acid and its derivatives are synthesized from biologically produced organic components by a fermenting microorganism. For example, MA is synthesized from the fermentation of sugars by recombinant host cells of the present disclosure. Similarly, the synthetic processes that produce Na2M, K2M, DEM and MEM start with the fermentation of sugars by recombinant host cells of the present disclosure. The disclosed production methods are therefore renewable and produce bio-based MA and its derivatives. Practitioners skilled in the art understand that the prefix "bio-" or the adjective "bio-based" may be used to distinguish these biologically produced MA, Na ₂ M, K ₂ M, Na ₂ M-monohydrate, (NH^M, DEM and MEM compounds from those that are derived from petroleum feedstocks. As used herein, "MA," "Na2M," "K ₂ M," "Na ₂ M-monohydrate," (NH ₄ ) ₂ M, "DEM" and "MEM" are "bio-based MA," "bio-based Na ₂ M," "bio-based K ₂ M," "bio-based Na ₂ M-monohydrate," bio-based (NH^M, "bio-based DEM" and "bio- based MEM."

Section 2: Recombinant host cells for production of disodium malonate

[0037] The recombinant host cells of the present disclosure are capable of producing MA. In some embodiments, the recombinant host cells comprise one or more heterologous nucleic acids encoding a malonyl-CoA hydrolase. In such embodiments, MA is produced through the action of a malonyl-CoA hydrolase catalyzing the conversion of malonyl-CoA to MA. The host cell making the malonyl-CoA hydrolase is a recombinant host cell that has been genetically modified to comprise heterologous nucleic acid(s) encoding malonyl-CoA hydrolase enzyme(s) catalyzing hydrolysis of malonyl-CoA to malonate.

[0038] In various aspects, the recombinant host cells disclosed herein have been genetically engineered to produce a recombinant malonyl-CoA hydrolase enzyme and therefore MA. The host cell can be engineered via recombinant DNA technology to express heterologous nucleic acids that encode a malonyl-CoA hydrolase, which is either a mutated version of a naturally occurring acyl-CoA hydrolase or transacylase, a non-naturally occurring malonyl-CoA hydrolase, or a naturally occurring acyl-CoA hydrolase with malonyl-CoA hydrolase activity. In various embodiments, the heterologous nucleic acids are either overexpressed in cells in which they otherwise naturally occur, or are expressed in cells in which they do not naturally occur.

[0039] Nucleic acid constructs provided by the present disclosure include expression vectors that comprise nucleic acids encoding one or more malonyl-CoA hydrolase enzymes. The nucleic acids encoding the enzymes are operably linked to promoters and optionally other control sequences such that the subject enzymes are expressed in a host cell containing the expression vector when cultured under suitable conditions. The promoters and control sequences employed depend on the host cell selected for the production of malonate. Methods for designing and making nucleic acid constructs and expression vectors generally are well known.

[0040] Nucleic acids encoding the malonyl-CoA hydrolase enzymes can be prepared by any suitable method including, for example, direct chemical synthesis and cloning. Further, nucleic acid sequences for use in the disclosed methods can be obtained from commercial vendors that provide de novo synthesis of the nucleic acids.

[0041] A nucleic acid encoding the desired enzyme can be incorporated into an expression vector by known methods that include, for example, the use of restriction enzymes to cleave specific sites in an expression vector (e.g. , plasmid), thereby producing an expression vector. Some restriction enzymes produce single stranded ends that may be annealed to a nucleic acid sequence having, or that is synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. The ends are then covalently linked using an appropriate enzyme (e.g. , DNA ligase). DNA linkers may be used to facilitate linking of nucleic acids sequences into an expression vector.

[0042] A set of individual nucleic acid sequences can also be combined by utilizing polymerase chain reaction (PCR)-based methods. For example, each of the desired nucleic acid sequences can be initially generated in its own PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences. When the PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and can act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are "spliced" together. In this way, a series of individual nucleic acid sequences may be joined and subsequently transduced into a host cell simultaneously. Thus, expression of each of the plurality of nucleic acid sequences is effected. [0043] A typical expression vector contains the desired nucleic acid sequence preceded and optionally followed by one or more control sequences or regulatory regions, including a promoter and, when the gene product is a protein, ribosome binding site, e.g., a nucleotide sequence that is generally 3-9 nucleotides in length and generally located 3-11 nucleotides upstream of the initiation codon that precedes the coding sequence, which is followed by a transcription terminator in the case of E. coli or other prokaryotic hosts. See, e.g. , Shine et al, Nature. 254:34 (1975) and Steitz, Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), vol. 1, p. 349 (1979) Plenum Publishing, N.Y. In the case of eukaryotic hosts like yeast a typical expression vector contains the desired nucleic acid coding sequence preceded by one or more regulatory regions, along with a Kozak sequence to initiate translation and followed by a terminator. See, e.g. , Kozak, Nature 308:241-246 (1984).

[0044] Regulatory regions or control sequences include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired nucleic acid coding sequence, thereby initiating transcription of the nucleic acid sequence via an RNA polymerase. An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein-binding domain where a transcription factor can bind. Transcription factors activate or repress transcription initiation from a promoter. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding transcription factor. Non-limiting examples for prokaryotic expression include lactose promoters (Lacl repressor protein changes conformation when contacted with lactose, thereby preventing the Lacl repressor protein from binding to the operator) and tryptophan promoters (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator). Non-limiting examples of promoters to use for eukaryotic expression include pTDH3, pTEFl, pTEF2, pRNR2, pRPL18B, pREVl, pGALl, pGALlO, pGAPDH, pCUPl, pMET3, pPGKl, pPYKl, pHXT7, pPDCl, pFBAl, pTDH2, pPGIl, pPDCl, pTPIl, pEN02, pADHl, and pADH2.

[0045] Examples of expression vectors suitable for use with embodiments provided by the present disclosure include, without limitation: plasmids, such as pESC, pTEF, p414CYCl, p414GALS, pSClOl, pBR322, pBBRlMCS-3, pUR, pEX, pMRlOO, pCR4, pBAD24, pUC19, pRS series; and bacteriophages, such as Ml 3 phage and λ phage. Of course, such expression vectors may only be suitable for particular host cells or for expression of particular malonyl-CoA hydrolases. Determining which expression vector to use can occur by, for example, introducing the expression vector into a host cell and monitoring the host cell for viability and expression of the sequences contained in the vector. In addition, reference may be made to the relevant texts and literature, which describe expression vectors and their suitability to any particular host cell. In addition to the use of expression vectors, strains are built where expression cassettes are directly integrated into the host genome.

[0046] The expression vectors are introduced or transferred, e.g. by transduction, transfection, or transformation, into the host cell. Such methods for introducing expression vectors into host cells are well known. For example, one method for transforming E. coli with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate.

[0047] In some embodiments, the malonyl-CoA hydrolase is a mutated 3-hydroxyisobutyryl-CoA hydrolase derived from Pseudomonas fulva (UniProt ID F6AA82) {see, e.g. , PCT application no.

PCT/US2013/029441). This malonyl-CoA hydrolase enzyme is a 366 amino acid protein that is capable of catalyzing the conversion of malonyl-CoA to MA. In some embodiments, the malonyl-CoA hydrolase is a mutated 3-hydroxyisobutyryl-CoA hydrolase derived from Pseudomonas fulva (UniProt ID F6AA82- 2) containing the mutations E95S and Q348A (see, e.g. , PCT application no. PCT/US2013/029441).

[0048] Without wishing to be bound by any theory, it is presently believed that all Pseudomonas malonyl CoA hydrolase enzymes contain six conserved active site residues necessary for hydrolase activity: (i) three active site amino acid residues (G67, G68, G120) believed to be necessary for the formation of an oxyanion hole responsible for stabilizing the enolate anion intermediate derived from an acyl-CoA substrate; (ii) two amino acid residues (E143, D 151) believed to be necessary for acyl-CoA hydrolysis; and (iii) a sixth amino acid at position 95 that is believed to be necessary for malonyl-CoA substrate binding. Of these six residues, the sixth, at position 95, is useful to provide a malonyl-CoA hydrolase capable of producing MA in a recombinant host cell.

[0049] In the wild-type Pseudomonas malonyl CoA hydrolase enzymes, position 95 is a glutamic acid residue, E95. In some embodiments, E95 is mutated to a polar or positively charged amino acid (i.e. , H, K, N, Q, R, S, T, Y), to an alanine residue (A), or to an aspartic acid residue (D), to produce a malonyl-CoA hydrolase capable of producing malonate in a recombinant host cell. In some embodiments, amino acid E95 is mutated to an amino acid selected from the group consisting of A, D, H, K, N, Q, R, S, T and Y. In some embodiments, amino acid E95 is mutated to an amino acid selected from the group consisting of A, D, K, N, S, T and Y. In some embodiments, amino acid E95 is N or S. In some embodiments, amino acid E95 is N, the mutation in such embodiments being referred to as E95N.

[0050] In the wild-type Pseudomonas malonyl CoA hydrolase enzymes, position 304 is a phenylalanine residue, F304. In some embodiments, F304 is mutated to an amino acid residue having a basic side chain (i.e. , H, K, R) to produce a malonyl-CoA hydrolase capable of producing malonate in a recombinant host cell. In some embodiments, amino acid F304 is mutated to an amino acid selected from the group consisting of H, K and R. In some embodiments, amino acid F304 is R, the mutation in such embodiments being referred to as F304R. [0051] In the wild-type Pseudomonas malonyl CoA hydrolase enzymes, position 348 is a glutamine residue, Q348. In some embodiments, Q348 is mutated to an amino acid residue having an aliphatic side chain (i.e. , A, G, I, L, V) to produce a malonyl-CoA hydrolase capable of producing malonate in a recombinant host cell. In some embodiments, amino acid Q348 is mutated to an amino acid selected from the group consisting of A, G, I, L and V. In some embodiments, amino acid Q348 is A, the mutation in such embodiments being referred to as Q348A.

[0052] In some embodiments, the recombinant host cell comprises an integrating plasmid containing an expression cassette consisting of the AOX 1 promoter common to methanol catabolizing yeasts followed by a malonyl-CoA hydrolase gene encoding the F6AA82(3) enzyme, which is a mutated malonyl-CoA hydrolase containing three amino acid mutations (E95N/Q348A/F304R) (see, e.g. , Example 1 of PCT Application No. PCT/US2014/047645).

[0053] In some embodiments, the recombinant host cell comprises a vector for expression of a mutated malonyl-CoA hydrolase, the vector comprising a CYC1 terminator, an ampicillin resistance cassette, a PMB 1 origin of replication, a CEN/ARS origin of replication, a URA3 selection marker and a TEF1 promoter. In these embodiments, the mutated malonyl-CoA hydrolase is the malonyl-CoA hydrolase from Pseudomonas fulva strain 12-X; UniProt ID F6AA82-2, with a mutation selected from E95S and E95N (see, e.g. , Example 22 of PCT Application No. PCT/US2013/029441).

[0054] The recombinant host cells produce MA at high titers, yields and productivities. In some embodiments, the recombinant host cells are capable of producing MA under aerobic conditions. In some embodiments, the recombination host cells are capable of producing MA under anaerobic conditions.

[0055] Any suitable host cell may be used in practice of the methods of the present disclosure. Examples of host cells useful in the compositions and methods provided herein include archaeal, prokaryotic, or eukaryotic cells. In some embodiments of the present disclosure, the recombinant host cell is a yeast strain selected from the genera Candida, Cryptococcus, Hansenula, Issatchenkia, luyveromyces, omagataelia, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, and Yarrowia. In some embodiments, the recombinant host cell is selected from Saccharomyces cerevisiae and Pichia kudriavzevii . In some embodiments, the host cell is Pichia kudriavzevii . In some embodiments, the recombinant host cell is a prokaryote selected from the genera Bacillus, Clostridium, Corynebacterium, Escherichia, Pseudomonas, Rhodobacter, and Streptomyces. In some embodiments, the host cell is Escherichia coli.

[0056] In some embodiments, the recombinant host cell is Pichia kudriavzevii that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising one or more mutations selected from the group consisting of E95N, F304R, Q348A, and combinations thereof. In some embodiments, the recombinant host cell is Pichia kudriavzevii that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising the mutation E95N. In some embodiments, the recombinant host cell is Pichia kudriavzevii that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising the mutations E95N and F304R. In some embodiments, the recombinant host cell is Pichia kudriavzevii that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising the mutations E95N and Q348A. In some embodiments, the recombinant host cell is Pichia kudriavzevii that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising the mutations E95N, F304R and Q348A.

[0057] In some embodiments, the recombinant host cell is Escherichia coli that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising one or more mutations selected from the group consisting of E95N, F304R, Q348A, and combinations thereof. In some embodiments, the recombinant host cell is Escherichia coli that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated

Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising the mutation E95N. In some embodiments, the recombinant host cell is Escherichia coli that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising the mutations E95N and F304R. In some embodiments, the recombinant host cell is Escherichia coli that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising the mutations E95N and Q348A. In some embodiments, the recombinant host cell is Escherichia coli that has been genetically modified to comprise a heterologous nucleic acid encoding a mutated Pseudomonas fulva malonyl-CoA hydrolase enzyme comprising the mutations E95N, F304R and Q348A.

[0058] Methods of construction and genotypes of these recombinant host cells, including the P. kudriavzevii strain, are described in PCT Application Nos. PCT/US2015/037530 and

PCT/US2013/029441, the entire contents of which are incorporated herein in their entirety by this reference.

Section 3. Methods of producing a malonic acid salt

[0059] Methods are provided herein for producing a MA salt from the recombinant host cells provided by the present disclosure. In certain embodiments, the methods comprise:

(1) culturing a recombinant host cell as provided by the present disclosure in a fermentation broth and producing MA; (2) converting MA to a MA salt; and

(3) recovering the MA salt from the fermentation broth.

In some embodiments the fermentation broth can also comprise at least one carbon source and one nitrogen source. In some embodiments, the culturing is performed under aerobic conditions. In some embodiments, the recombinant host cells comprise one or more heterologous nucleic acids encoding a malonyl-CoA hydrolase capable of catalyzing the conversion of malonyl-CoA to MA, such that MA is produced by such cells.

3.1 Fermentative production of a malonic acid salt by recombinant host cells

[0060] Any of the recombinant host cells of the present disclosure can be cultured to produce and/or secrete MA. MA is highly soluble in water, requiring evaporation of a large amount of water before MA crystallization can begin. In aqueous solutions, MA is unstable and prone to decarboxylation at high temperatures, especially at temperatures greater than 80°C. Conversion of MA to a salt in aqueous solution (i. e., a MA salt) can facilitate more easeful and efficient purification processes. MA salts are very stable and do not readily decarboxylate, even at temperatures greater than 80°C. Stability of MA salts at higher temperatures facilitates concentration, crystallization, and isolation of MA salts, contributing to improvements in downstream process efficiency and greater product yield. As disclosed herein, once purified the MA salts can be esterified and distilled to generate a purified ester. In some embodiments, small and/or more manageable volumes of the purified ester can later be converted back to a highly purified form of bio-MA.

[0061] In certain embodiments, recombinant host cells produce MA for the biosynthetic production of a MA salt (e.g., Na ₂ M, K ₂ M, (NH ₄ )2M, or Na ₂ M-monohydrate) and/or DEM according to methods of the present disclosure. In certain embodiments, MA is isolated and subjected to further treatments for chemical synthesis of Na ₂ M, K ₂ M, (NH ₄ ) ₂ M, Na ₂ M-monohydrate, DEM and/or MA. In certain embodiments, a MA salt is isolated and subjected to further treatments for chemical synthesis of Na ₂ M- monohydrate, DEM and/or MA.

[0062] Materials and methods for the maintenance and growth of microbes, as well as fermentation conditions, are well known to practitioners of ordinary skill in the art. The skilled artisan will understand that consideration must be given to appropriate culture medium, pH, temperature, revival of frozen stocks, growth of seed cultures and seed trains, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the host cells, the fermentation, and process flows.

[0063] The methods of producing a MA salt provided herein may be performed in a suitable fermentation broth in a suitable bioreactor such as a fermentation vessel, including but not limited to a culture plate, a flask, or a fermenter. Further, the methods can be performed at any scale of fermentation known in the art to support microbial production of small-molecules on an industrial scale. Any suitable fermenter may be used including, for example, a stirred tank fermenter, an airlift fermenter, a bubble column fermenter, a fixed bed bioreactor, or any combination thereof.

[0064] In some embodiments, the fermentation broth is any fermentation broth in which a recombinant host cell capable of producing MA (described in the present disclosure) can subsist (maintain growth, viability, and/or catabolize glucose or other carbon source). In some embodiments, the fermentation broth is an aqueous medium comprising assimilable carbon, nitrogen, and phosphate sources. Such a medium can also include appropriate salts, minerals, metals, and other nutrients. In some embodiments, the carbon source and each of the essential cell nutrients are provided to the fermentation broth incrementally or continuously, and each essential cell nutrient is maintained at essentially the minimum level required for efficient assimilation by growing cells. Examples of cell growth procedures include batch fermentation, fed-batch fermentation with batch separation, fed-batch fermentation with continuous separation, and continuous fermentation with continuous separation. These procedures are well known to practitioners of ordinary skill in the art.

[0065] In some embodiments, the culturing of recombinant cells to produce MA, Na ₂ M, K ₂ M, (NH- 4)2M, Na2M-monohydrate, and/or DEM may be divided up into phases. For example, the cell culture process may be divided up into a growth phase, a production phase, and/or a recovery phase. The following paragraphs provide examples of specific conditions that may be used for these phases. One skilled in the art will recognize that these conditions may vary based on the recombinant host cells used, the product(s) desired (MA, Na2M, (NFL^M, Na2M-monohydrate, and/or DEM) yield, titer, productivity and/or other factors.

[0066] Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of MA and/or MA salt. It will be understood by persons having ordinary skill in the art that fermentation procedures can be scaled up for manufacturing MA and/or MA salt. Examples of fermentation procedures include, for example, fed-batch fermentation and batch product separation; fed- batch fermentation and continuous product separation; batch fermentation and batch product separation; and continuous fermentation and continuous product separation.

3.1.1 Carbon source

[0067] The carbon source provided to the fermentation can be any carbon source that can be fermented by the recombinant host cell. Suitable carbon sources include, but are not limited to, monosaccharides, disaccharides, polysaccharides, glycerol, acetate, ethanol, methanol, methane, or combinations thereof. Examples of monosaccharides suitable for use in the methods of the present disclosure include, but are not limited to, dextrose (glucose), fructose, galactose, xylose, arabinose, and any combination thereof. Examples of disaccharides suitable for use in the methods of the present disclosure include, but are not limited to, sucrose, lactose, maltose, trehalose, cellobiose, and any combination thereof. Examples of polysaccharides suitable for use in the methods of the present disclosure include, but are not limited to, starch, glycogen, cellulose, and combinations thereof. In some embodiments, the carbon source is dextrose. In other embodiments, the carbon source is sucrose.

3.1.2 pH

[0068] The pH of the fermentation broth can be controlled by the addition of acid or base to the culture medium. In some embodiments, the pH is maintained in the range of 3-8, in some embodiments in the range of 4-8. Non-limiting examples of suitable acids include aspartic acid, acetic acid, hydrochloric acid, and sulfuric acid. Non-limiting examples of suitable bases include sodium bicarbonate (NaHCC ), sodium hydroxide (NaOH), potassium bicarbonate (KHCO3), potassium hydroxide (KOH), calcium hydroxide (Ca(OH) ₂ ), calcium carbonate (CaCC ), ammonium bases (i.e., ammonia, ammonium hydroxide, or ammonium carbonate), and diammonium phosphate. In some embodiments, a concentrated acid or concentrated base is used to limit dilution of the fermentation broth.

[0069] In some embodiments, NaHCC is the base used for modulating fermentation pH. In other embodiments, NaOH is the base used for modulating fermentation pH. NaHCC and NaOH are inexpensive and readily available. Sodium ions and malonate ions react to form Na2M in fermentation broths. In some embodiments, KHCO3 is the base used for modulating fermentation pH. In other embodiments, KOH is the base used for modulating fermentation pH. Potassium ions and malonate ions react to form K2M in fermentation broths. In other embodiments, an ammonium base is used as the base for modulating fermentation pH. In some of these embodiments, the ammonium base is ammonia. In some of these embodiments, the ammonium base is ammonium hydroxide. In some of these

embodiments, the ammonium base is ammonium carbonate. In some embodiments, the ammonium base is selected from ammonia, ammonium hydroxide, ammonium carbonate and combinations thereof.

3.1.3 Temperature

[0070] The temperature of the fermentation broth can be any temperature suitable for growth of the recombinant host cells and/or production of MA or MA salts. Preferably, during MA or MA salt production, the fermentation broth is maintained at a temperature in the range of from about 20°C to about 45°C, in some embodiment in the range of from about 25°C to about 37°C, and in some embodiments in the range from about 28°C to about 32°C. At the end of fermentation, decreasing fermentation broth temperature decreases the solubility of MA salts, facilitating their crystallization. Alternatively, increasing fermentation broth temperature aids crystallization of MA salts by evaporating solute, thereby concentrating the MA salt in the fermentation broth.

3.1.4 Oxygen

[0071] Generally speaking, microbial production of MA from glucose results in the formation of NADH and/or NADPH, redox cofactors that must be converted back to NAD+ and NADP+ in order to maintain catabolism of glucose. Under aerobic conditions, microbes will commonly use molecular oxygen as an electron acceptor to reoxidize these cofactors. If the fermentation is not appropriately oxygenated, MA production will decrease. In several embodiments, during cultivation, aeration and agitation conditions are selected to produce an oxygen uptake rate (OUR) that results in MA formation. In some embodiments, fermentation conditions are selected to produce an OUR of greater than 10 mmol/l/hr. In some embodiments, fermentation conditions are selected to produce an OUR of greater than 20 mmol/l/hr, greater than 30 mmol/l/hr, greater than 40 mmol/l/hr, greater than 50 mmol/l/hr, greater than 75 mmol/l/hr, greater than 100 mmol/l/hr, greater than 125 mmol/l/hr, greater than 150 mmol/l/hr, greater than 175 mmol/l/hr, or greater than 200 mmol/l/hr. OUR as used herein refers to the volumetric rate at which oxygen is consumed during the fermentation. Inlet and outlet oxygen concentrations can be measured by exhaust gas analysis, for example by mass spectrometers. OUR can be calculated by one of ordinary skill in the art using the Direct Method described in Bioreaction Engineering Principles 3 ^rd Edition, 2011, Spring Science + Business Media, p. 449. The recombinant host cells of the present disclosure are able to produce MA and/or MA salts under a wide range of oxygen concentrations. In some embodiments, the recombinant host cells produce MA and/or MA salts under aerobic conditions.

3.1.5 Yields and titers

[0072] A high yield of either MA from the provided carbon source(s) is desirable to decrease the production cost. As used herein, yield is calculated as the percentage of the mass of carbon source catabolized by the cells of the present disclosure and used to produce MA. In some cases, only a fraction of the carbon source provided to a fermentation is catabolized by the cells, and the remainder is found unconsumed in the fermentation broth or is consumed by contaminating microbes in the fermentation. Thus, it is advantageous to ensure that fermentation is both substantially pure of contaminating microbes and that the concentration of unconsumed carbon source at the completion of the fermentation is measured. For example, if 100 grams of glucose is fed into the fermentation, and at the end of the fermentation 25 grams of MA are produced and there remains 10 grams of glucose, the MA yield is 27.7% {i.e., 25 grams MA from 90 grams glucose). In certain embodiments, the final yield of MA on the carbon source is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or greater than 50%. In certain embodiments, the recombinant host cells provided herein are capable of producing at least 80%, at least 85%, or at least 90% by weight of carbon source to MA. Those skilled in the art will recognize that when a MA salt is found in the fermentation broth the MA yield can be determined by calculating the mols of MA salt present and adjusting for the molecular weight difference between the MA salt and MA.

[0073] In addition to yield, the titer (or concentration), of MA produced in the fermentation is another useful metric for production. Assuming all other metrics are equal, a higher titer is preferred to a lower titer. Generally speaking, titer is provided as grams of product (e.g., MA) per liter of fermentation broth (i. e., g/1). In some embodiments, the MA titer is at least 1 g/1, at least 5 g/1, at least 10 g/1, at least 15 g/1, at least 20 g/1, at least 25 g/1, at least 30 g/1, at least 40 g/1, at least 50 g/1, at least 60 g/1, at least 70 g/1, at least 80 g/1, at least 90 g/1, at least 100 g/1, or greater than 100 g/1 at some point during the fermentation, and in some embodiments at the conclusion of the fermentation. As with yield calculations, those skilled in the art will recognize that a MA titer can be calculated from the MA salt titer by adjusting for molecular weight differences between the MA salt and MA.

[0074] Further, efficient productivity, or the rate of product (i.e., MA) formation, aids in decreasing production cost, and, assuming all other metrics are equal, a higher productivity is preferred over a lower productivity. Generally speaking, productivity is provided as grams product produced per liter of fermentation broth per hour (i. e., g/l/hr). In some embodiments, MA productivity is at least 0.1 g/1, at least 0.25 g/1, at least 0.5 g/1, at least 0.75 g/1, at least 1.0 g/1, at least 1.25 g/1, at least 1.25g/l, at least 1.5 g/1, or greater than 1.5 g/1 over some time period during the fermentation.

[0075] Decreasing byproduct formation also aids in decreasing production cost, and, generally speaking, the lower the byproduct concentration the lower the production cost. Additionally, the occurrence of specific fermentation byproducts can hinder downstream purification processes.

Byproducts that can accumulate during MA and/or MA salt production, in accordance with the methods of the present disclosure, include glycerol, ethanol, acetate, citrate, pyruvate and succinate, among others. In certain embodiments, the recombinant host cells produce pyruvate at a low titer from the provided carbon source at the conclusion of the fermentation. In certain embodiments, pyruvate is produced at a titer of 10 g/1 or less, 5 g/1 or less, or 2.5 g/1 or less at the conclusion of the fermentation. In certain embodiments, the recombinant host cells produce glycerol at a low titer from the provided carbon source at the conclusion of the fermentation. In certain embodiments, glycerol is produced at a titer of 10 g/1 or less, 5 g/1 or less, or 2.5 g/1 or less at the conclusion of the fermentation.

[0076] Additionally, succinic acid byproduct is converted to one or more esters during conversion of the malonate salt to its esters (MEM, DEM). Succinic and malonic esters have close boiling points, resulting in more costly separation and host cells that produce very low levels of, and/or no, succinic acid facilitate lower cost recovery of high purity malonic acid esters. In some embodiments, the recombinant host cells produce succinic acid at a low titer from the provided carbon source at the conclusion of the fermentation. In some embodiments, succinic acid is produced at a titer of 10 g/1 or less, 5 g/1 or less, or 2.5 g/1 or less at the conclusion of the fermentation. Certain impurities present in the carbon source(s) that are not consumed during fermentation can also interfere with recovery operations and subsequent esterification. For instance, maltose is a typical impurity present in glucose carbohydrate sources that can accumulate during fermentation if not consumed and interfere with subsequent crystallization of malonic acid salts. It is therefore desirable to provide recombinant host cells with the capability to fully consume such impurities and convert them to malonic acid during fermentation. In certain embodiments, the recombinant host cells consume maltose present in the provided carbon source such that a very low, or no, concentration of maltose remains at the conclusion of the fermentation. In certain embodiments, maltose is consumed with a residual concentration of 10 g/1 or less, 5 g/1 or less, or 2.5 g/1 or less at the conclusion of fermentation.

[0077] Practitioners of ordinary skill in the art understand that HPLC is an appropriate method to determine the amount of MA and/or MA salts produced, the amount of many byproducts produced (e.g. , organic acids and alcohols), and the amount of unconsumed glucose and other carbohydrates such as maltose left in the fermentation broth. Aliquots of fermentation broth can be isolated for analysis at any time during fermentation, as well as at the end of fermentation. Briefly, molecules in the fermentation broth are first separated by liquid chromatography (LC); then, specific liquid fractions are selected for analysis using an appropriate method of detection (e.g., UV-VIS, refractive index, and/or photodiode array detectors). In some embodiments, Na2M is the fermentative product present in the fermentation broth. Practitioners in the art understand that Na2M is acidified before or during HPLC analysis, producing MA. Hence, the MA concentration calculated by HPLC analysis can be used to calculate the Na ₂ M titer in the broth by adjusting for the difference in molecular weight between the two compounds. Section 4. Purification of malonic acid salts

[0078] The present disclosure describes methods for purifying MA salts (i.e., Na2M,

Na2M-monohydrate or K2M) produced by the recombinant host cells of the present disclosure. In various aspects, this purification comprises isolating the MA salt(s) from the fermentation broth prior to further manipulation (if any). The isolation/purification methods comprise: (1) separating soluble MA salts from fermentation broth, cells, cell debris and soluble impurities; (2) converting MA salts from soluble form to crystalline form; and (3) isolating crystalline malonic salts.

[0079] At the end of fermentation, the fermentation broth can contain soluble MA salts together with biomass and soluble impurities that include other salts, proteins, unconverted sugars, and other impurities including color bodies. The MA salts are isolated from the biomass and soluble impurities via one or more purification steps. In certain embodiments, purification steps may include centrifugation, microfiltration, ultrafiltration, nanofiltration, diafiltration, ion exchange, crystallization, and any combination thereof. In some of these embodiments, ion exchange resins and nanofiltration membranes are used in combination with one or more of the steps referenced in the previous sentence as polishing steps, performed after the preceding step(s), to remove trace amounts of soluble impurities, unconverted sugars and color bodies.

4.1 Removal of cells and cell debris [0080] In some embodiments, a process of purifying MA salts comprises separating a liquid fraction containing MA salts from a solid fraction that contains cells and cell debris. For this separation, any amount of fermentation broth can be processed, including the entirety of the fermentation broth. One skilled in the art will recognize the amount of fermentation broth processed can depend on the type of fermentation process used, such as batch or continuous fermentation processes. In various embodiments, removal of cells and cell debris can be accomplished, for example, via centrifugation using specific g- forces and residence times, and/or filtration using molecular weight cutoffs that are suitable for efficiently separating the liquid fraction containing MA salts from the solid fraction that contains cells and cell debris. In some embodiments, removal of cells and cell debris is repeated at least once at one or in more than one step in the methods provided herein.

[0081] In some embodiments, centrifugation is used to provide a liquid fraction comprising MA salts that is substantially free of cells. Many types of centrifuges useful for the removal of cells and solids from fermentation broth are known to those skilled in the art, including disc-stack and decanter centrifuges. Centrifuges are well suited for separating solids with a particle size of between 0.5 μπι to 500 μπι and density greater than that of the liquid phase (ca. 1.0 g/ml). Yeast cells, as a non-limiting example of a MA producing microbe, typically have a particle size between 4-6 μπι and a density of around 1.1 g/ml; therefore, centrifugation is well suited for removing yeast cells from fermentation broth.

[0082] In some embodiments, a disc-stack centrifuge is used to provide a liquid fraction comprising MA salts that substantially free of cells. A disc stack centrifuge separates solids, which are discharged intermittently during operation, from liquids, typically in a continuous process. A disc-stack centrifuge is well suited for separating soft, non-abrasive solids, including cells. In some embodiments, a decanter centrifuge is used to provide a liquid fraction comprising MA salts that is substantially free of cells. A decanter centrifuge can typically process larger amounts of solids and is often preferred over a disc-stack centrifuge for processing fermentation broth when the cell mass and other solids exceeds about 3% w/w.

[0083] Other methods can be used in addition to, or alone, with the above centrifugation processes. For example, microfiltration is also an effective means to remove cells from fermentation broth.

Microfiltration includes filtering the fermentation broth through a membrane having pore sizes from about 0.5 μπι to about 5 μπι. In some embodiments, microfiltration is used to provide a liquid fraction comprising MA salts that is substantially free of cells.

[0084] In some embodiments, cells removed by centrifugation and/or microfiltration are recycled back into the fermentation. One skilled in the art will recognize recycling cells back into the fermentation can increase MA yield since less carbon source (e.g., glucose) must be used to generate new cells.

Additionally, recycling cells back into the fermentation can also increase MA productivity since the concentration of cells producing MA in the fermenter can be increased. [0085] While suitable for removing cells, centrifugation and microfiltration may not be effective at removing cell debris, proteins, DNA and other smaller molecular weight compounds from the fermentation broth, depending on the compositions of impurities present in the fermentation broth.

Ultrafiltration is a process similar to microfiltration, but the membrane has pore sizes ranging from about 0.005 μπι to 0.1 μπι. This pore size equates to a molecular weight cut-off (the size of macromolecule that will be ca. 90% retained by the membrane) from about 1,000 Daltons to about 200,000 Daltons. The ultrafiltration permeate will contain low -molecular weight compounds, including MA salts and various other soluble salts while the ultrafiltration retentate will contain the majority of residual cell debris, DNA, and proteins. In some embodiments, ultrafiltration is used to provide a liquid fraction comprising MA salts that is substantially free of cell debris and proteins.

4.2 Nanofiltration and ion exchange polishing of clarified fermentation broth containing malonic acid salts

[0086] In some embodiments, nanofiltration is used to separate out certain contaminating salts, sugars, color forming bodies, and other organic compounds present in clarified fermentation broth containing MA salts. In nanofiltration, the clarified fermentation broth (i. e., the fermentation broth resulting from the centrifugation, microfiltration, ultrafiltration, nanofiltration, diafiltration, ion exchange, crystallization, and/or any combination thereof steps described above) is filtered through a membrane having pore sizes ranging from 0.0005 μπι to 0.005 μπι, equating to a molecular weight cut-off of about 100 Daltons to about 2,000 Daltons. Nanofiltration can be useful for removing divalent and multivalent ions, maltose and other disaccharides (e.g., sucrose), polysaccharides, and other complex molecules with a molecular weight larger than Na ₂ M (148 g/mol) or ¾M ( 180 g/mol). Non-limiting examples of nanofiltration materials include ceramic membranes, metal membranes, polymer membranes, and composite membranes.

[0087] In some embodiments, ion exchange is used to remove specific contaminating salts present in clarified fermentation broth containing MA salts. Ion exchange elements can take the form of resin beads as well as membranes. Frequently, the resins are cast in the form of porous beads. The resins can be cross- linked polymers having active groups in the form of electrically charged sites. At these sites, ions of opposite charge are attracted but may be replaced by other ions depending on their relative concentrations and affinities for the sites. Ion exchangers can be cationic or anionic. Factors that determine the efficiency of a given ion exchange resin include the favorability for a given ion, and the number of active sites available.

[0088] In some embodiments, a combination of nanofiltration and ion exchange steps can be combined to produce a purified solution of MA salts from clarified fermentation broth.

4.3 Crystallization of malonic acid salts [0089] Malonic acid salts purified as described thus far can be crystallized to further remove water and any remaining trace, water-soluble impurities, in some embodiments, the isolated/purified MA salts produced by the aforementioned steps are then crystallized. In some embodiments, the majority of the MA salt(s) is recovered in the insoluble, crystallized form, leaving a minor amount of MA salt remaining in the mother liquor.

[0090] In some embodiments, the temperature of the mother liquor is changed to facilitate MA salt crystallization. For example, Na2M is soluble in water at about 148 g/1 at 20°C. In some embodiments, the mother liquor is cooled to a temperature below 20°C to decrease Na ₂ M solubility. In these embodiments, the mother liquor is heated to evaporate excess water. In some of these embodiments, evaporative crystallization is preferred as it offers a high yield of Na ₂ M and prevents the formation of stable gels, which may occur if temperature is reduced below the gelling point of concentrated Na ₂ M solutions. In some of these embodiments, Na ₂ M crystallization is achieved by combining various heating and cooling steps. In some of these embodiments, supersaturation is achieved by evaporative crystallization wherein the solute is more concentrated in a bulk solvent that is normally possible under given conditions of temperature and pressure; increased supersaturation of MA salts in the mother liquor causes the MA salts to crystallize. Non-limiting examples of crystallizers include forced circulation crystallizers,

turbulence/draft tube and baffle crystallizers, induced circulation crystallizers and Oslo-type crystallizers.

[0091] In some embodiments, the aforementioned heating, cooling and change in pH are combined in various ways to crystallize MA salts, and modified as needed, as apparent to practitioners skilled in the art.

[0092] Malonic acid salt crystals can be isolated from the mother liquor by any technique apparent to those of skill in the art. In some embodiments, MA salt crystals are isolated based on size, weight, density, or combinations thereof. MA salt crystal isolation based on size can be accomplished, for example, via filtration, using a filter with a specific particle size cutoff. MA salt crystal isolation based on weight or density can be accomplished, for example, via gravitational settling or centrifugation, using, for example, a settler, decanter centrifuge, disc-stack centrifuge, basket centrifuge, or hydrocyclone wherein suitable g-forces and settling or centrifugation times can be determined using methods known in the art. In some embodiments, MA salt crystals are isolated from the mother liquor via settling for from 30 minutes to 2 hours at a g-force of 1. In other embodiments, MA salt crystals are isolated from the fermentation broth via centrifugation for 20 seconds to 60 seconds at a g-force of from 275 x-g to 1,000 x-g-

[0093] Following isolation from the mother liquor, MA salt crystals are often wet with residual mother liquor that coats the crystals. Thus, it is useful to wash the MA salt crystals to remove these trace impurities that may be in the mother liquor. In some embodiments, the wash may be performed with water. When washing MA salt crystals, it is useful to minimize the dissolution of isolated crystals in the wash water; for this reason, in some embodiments a cold wash (around 4°C) is used. Additionally, it is useful to minimize the amount of wash used to minimize crystal dissolution. In many embodiments, less than 10% w/w wash water is used to wash the MA salt crystals.

[0094] In some embodiments, the methods further comprise removing impurities from MA salt crystals. Impurities may react with MA salt crystals and reduce final yields, or contribute to MA salt crystals of lesser purity that limits industrial utility. Non-limiting examples of impurities include acetic acid, succinic acid, malic acid, ethanol, glycerol, citric acid, and propionic acid. In some embodiments, removal of such impurities is accomplished by dissolving the isolated MA salt crystals into an aqueous solution and recrystallizing the MA salts. A non-limiting example of dissolving and recrystallizing MA salt crystals can include dissolving the MA salt in water, evaporating the resulting aqueous solution (as mentioned above), and re-isolating the MA salt crystals by filtration and/or centrifugation. None, one, or more than one cycle of MA salt recrystallization may be used to produce crystalline MA salts for subsequent esterification. In some embodiments, no MA salt recrystallizations are performed. In other embodiments, one MA salt recrystallization is performed. In still further embodiments, two or more MA salt recrystallizations are performed.

[0095] In some embodiments, MA salt crystals are dewatered using a combination of screening and drying methods. In some of these embodiments, crystal dewatering steps are selected from centrifugation, belt drying, filtration, application of vacuum, and combinations thereof. In some embodiments, vacuum is applied at 20 mm of Hg below atmospheric pressure. Suitable devices for crystal dewatering may include a Horizontal Vacuum Belt Filter (HVBF) or a Rotary Drum Vacuum Filter (RDVF). Na ₂ M or (NH ₄ ) ₂ M isolation based on size can be accomplished, for example, via filtration, using, for example, a filter press, candlestick filter, or other industrially used filtration system with appropriate molecular weight cutoff. Na ₂ M or (NH/i) ₂ M isolation based on weight or density can be accomplished, for example, via gravitational settling or centrifugation, using, for example, a settler, decanter centrifuge, disc-stack centrifuge, basket centrifuge, or hydrocyclone, at suitable g-forces and settling or centrifugation times.

[0096] In some embodiments, MA salts are crystallized in the fermentation broth prior to removal of cells, cell debris, contaminating salts and various soluble impurities. In many of these embodiments, the MA salt crystals are separated from fermentation broth, cells, cell debris, contaminating salts and various soluble impurities by sedimentation, centrifugation, ultrafiltration, nanofiltration, ion exchange, or any combination thereof.

[0097] The result of any one of the foregoing isolation/purification steps, and/or any combination thereof, results in the production of one or more isolated, crystalline MA salts, free, or substantially free, of fermentation products and byproducts and ready for either further processing (e.g., esterification) or use as bio-based MA in any number of commercial processes.

Section 5. Esterification of disodium malonate and recovery of esterification products

5.1 Esterification of disodium malonate with supercritical carbon dioxide for diethyl malonate production

[0098] In some embodiments, a dried, crystalline MA salt is esterified using supercritical carbon dioxide (sCC ) and alcohol to produce a MA diester. In some embodiments, a dried, crystalline MA salt such as Na ₂ M is esterified with SCO ₂ and ethanol (EtOH) to produce DEM and a NaHCC co-product. One molecule of Na ₂ M reacts with 2 molecules of EtOH and 2 molecules of CO ₂ to produce 1 molecule of DEM and 2 molecules of NaHC03. This reaction is irreversible. Sufficient amounts, e.g.,

stoichiometric amounts, of each reaction component are used. Because Na ₂ M has low solubility in EtOH, a co-solvent can help accelerate Na ₂ M dissolution and subsequent esterification. Suitable co-solvents include diethyl carbonate, MA, MEM, and DEM. In some embodiments, the co-solvent(s) is recovered from the downstream distillation step and reused in the esterification step. In some embodiments of the present disclosure, dried (NH^M is esterified using supercritical carbon dioxide (SCO2) and alcohol to produce a MA diester. In some embodiments, (NH^M is esterified with SCO2 and ethanol (EtOH) to produce DEM and a (NH^COs co-product.

[0099] Supercritical carbon dioxide is a fluid state of CO2 that is held at or above the temperature and pressure at which it would behave as a gas. Carbon dioxide is a gas at standard temperature and pressure and is a solid when frozen. When the temperature and pressure are elevated above standard temperature and pressure to, or just above, the critical point for carbon dioxide (temperature: 304.25 K, 31.10 °C, 87.98 °F; pressure: 72.9 atm, 7.39 MPa, 1,071 psi), it adopts behaviors that are both gas- and fluid-like, such that it will fill a container as if it was a gas, but will have a density more like that of a fluid. The toxicity of SCO2 is low and, because the temperature at which it exists is still relatively low, it does not damage the crystalline MS salts during esterification.

[00100] During the dissolution and esterification processes, the reaction mixture is stirred. Further, temperature is increased in an incremental and controlled manner to prevent decarboxylation, which can lower product yield and generate unwanted acetic acid and ethyl acetate byproducts. The reaction mixture is filtered to enable recovery of solid NaHC03 or (NH^COs, which can be reused in fermentation to neutralize MA during fermentation. The reaction can be carried out in a distillation column, a jacketed glass reactor with a condenser, a Parr stirred reactor system, or other appropriate reaction vessel as deemed appropriate by skilled practitioners in the art. [00101] In some embodiments, the esterification alcohol used in the sCC catalyzed esterification of the MA salt is EtOH. In some embodiments, the esterification alcohol used in the sCC catalyzed esterification of the MA salt is methanol (MeOH).

[00102] In some embodiments, the esterification conditions comprise a temperature within the range of 150°C to 250°C, depending on the pressure.

[00103] In some embodiments, the esterification conditions comprise a pressure within the range of 500 psi to 2,000 psi, depending on the temperature.

5.2 Esterification of disodium malonate or diammonium malonate with sulfuric acid for diethyl malonate production

[00104] In some embodiments, a dried, crystalline MA salt is esterified using sulfuric acid (H ₂ SO ₄ ) as a catalyst. In some embodiments, a dried, crystalline MA salt such as Na2M is esterified using H ₂ SO ₄ as a catalyst. One molecule of Na2M reacts with 2 molecules of EtOH to produce 1 molecule of DEM and 2 molecules of water. This reaction produces Na2S04 as a co-product which, unlike NaHC03 (which is a co-product of Na2M esterification with SCO2), cannot be recycled to fermentation for pH control purposes. Na2S04 forms a decahydrate under suitable conditions (i.e., at temperatures less than 80°C in water) and can serve as an effective dehydrating agent that drives esterification to completion in a single step. The irreversible co-production of Na2S04 drives the reaction forward at relatively low temperatures that prevent decarboxylation and ethyl acetate formation. At the end of the reaction, the mixture is filtered to remove the Na2S04 salt byproduct. The reaction can be carried out in a distillation column, a jacketed glass reactor with a condenser, a Parr stirred reactor system, or other appropriate reaction vessel.

[00105] In other embodiments, dried (N¾)2M is esterified using sulfuric acid (H2SO4) as a catalyst. One molecule of reacts with 2 molecules of EtOH to produce 1 molecule of DEM and 2 molecules of water. This reaction produces (NH4)2S04 as a co-product that can be recovered as a commercial byproduct for use as a fertilizer. In some embodiments, the reaction temperature is less than 100°C. In some embodiments, the reaction temperature is less than 80°C. In some embodiments, the reaction temperature is less than 60°C. Several reaction parameters can be modified to increase DEM yield, such as EtOH concentration, H ₂ SO ₄ concentration, or improved removal of water from the reaction. At the end of the reaction, the mixture is filtered to remove the (NH ₄ )2S04 salt byproduct. The reaction can be carried out in a distillation column, a jacketed glass reactor with a condenser, a Parr stirred reactor system, or other appropriate reaction vessel.

[00106] In some embodiments, the esterification alcohol used in the sulfuric acid catalyzed esterification of the MA salt is EtOH. In some embodiments, the esterification alcohol used in the sulfuric acid catalyzed esterification of the MA salt is methanol (MeOH). [00107] In some embodiments, the esterification is conducted at a temperature within the range of 25°C to 300°C, depending on the pressure. In some embodiments, the reaction temperature is maintained within the range of 75°C to 100°C. In some embodiments, the reaction temperature is held at 200°C. In some embodiments, the esterification is conducted at a temperature that does not exceed the boiling point of EtOH. In some embodiments, the esterification is conducted at a temperature greater than 25°C.

[00108] In some embodiments, the esterification is conducted at a pressure within the range of 300 psi to 2,000 psi, depending on the temperature. In some embodiments, the reactor pressure is maintained within the range of 300 psi to 800 psi.

[00109] In some embodiments, Na ₂ M is reacted with H ₂ SO ₄ to form both a monoester of MA and a diester of MA. In some embodiments, the monoester is the predominant reaction product. In other embodiments, is reacted with H ₂ SO ₄ to form a monoester of MA and a diester of MA. In some embodiments, the monoester is the predominant reaction product. In some embodiments, the reaction temperature is less than 80°C. In some embodiments, the reaction temperature is less than 60°C. In some embodiments, the reaction temperature is less than 40°C. In some embodiments, the reaction temperature is ambient or room temperature.

[00110] In the foregoing reactions, solid Na2SC>4 or solid (NH4)2S04 is produced and readily separated from the reaction mixture to yield a solution comprising the MA ester(s) (for example, MEM), catalyst (for example, H2SO4) and excess alcohol (for example, EtOH or MeOH). As noted above, in some embodiments (of both reactions), the monoester is the predominant reaction product. The monoester thus formed is very stable and allows one to raise the reaction temperature to drive high conversion of monoester to diester (for example, DEM) without risk of decarboxylation and byproduct formation. In that regard, in some embodiments, production of a diester proceeds via a two-stage process, as outlined above. Such a process can reduce overall reaction time, reduce or eliminate formation of undesirable byproducts, and increase yield and purity of the final diester product.

5.3 Distillation of diethyl malonate

[00111] In various embodiments, once formed, DEM is purified via distillation from esterification reaction mixtures. In some embodiments, the reactions mixtures are filtered prior to distillation. The reaction mixtures can be distilled according to any one or more of several known methods to separate unconverted alcohol and partially converted monoester from the diester.

[00112] In some embodiments, distillation temperature is maintained within the range of 50°C to 300°C, depending on the pressure. In some embodiments, distillation temperature is maintained within the range of 50°C to 100°C. In various embodiments, distillation temperature refers to the temperature measured at the bottom of the distillation vessel. [00113] In some embodiments, the distillation pressure is maintained within the range of 0 Torr to 100 Torr, depending on the temperature. In some embodiments, distillation pressure is maintained within the range of 20 Torr to 40 Torr.

[00114] Purified DEM can be analyzed by a variety of methods including HPLC (high-performance liquid chromatography) and GC-MS (gas chromatography mass spectrometry). Chromatography methods are useful for reporting the percentage product purity as a percentage of total measured organic compounds. In some embodiments, DEM purity as a percentage of total organic compounds is greater than 95%. In some embodiments, DEM purity as a percentage of total organic compounds is greater than 98%. In some embodiments, DEM purity as a percentage of total organic compounds is greater than 99%. In some embodiments, DEM purity as a percentage of total organic compounds is greater than 99.5%. In some embodiments, DEM purity as a percentage of total organic compounds is greater than 99.8%.

[00115] In addition to chromatographic measurement of purity, a useful measure of product quality is water content. Karl Fischer titration can be used to determine the water content of purified DEM. In some embodiments, the amount of amount of water present in the purified DEM solution is less than 0.1%. In some embodiments, the amount of amount of water present in the purified DEM solution is less than 0.05%.

[00116] Another useful measure of product purity is color. The presence or absence of color in purified DEM solution can be measured at the end of distillation, and over a period of time when the diester product is in storage. Color measurements can be made using a colorimeter such as a HunterLab colorimeter. For near white solutions, the presence or absence of color can be reported according to the Yellowness Index (YI) with calculations according to the ASTM E313 standard. A change in YI over time is a measure of color formation over time. In some embodiments of the present disclosure, YI of purified DEM at the end of distillation is very low. In some embodiments, YI of purified DEM remains substantially the same over time. In some embodiments, YI of purified DEM does not change significantly over time. In some embodiments, YI of purified DEM reflects desirable visual and/or optical/photonic properties.

5.4 Hydrolysis of diethyl malonate to produce malonic acid

[00117] In some embodiments, a diester reaction product is hydrolyzed to produce MA. In this reaction, an acid or a base is used to hydrolyze the ester moieties, generating MA. If a base is used, a MA salt is produced that can be converted to free MA using a strong acid ion exchange resin in the H ⁺ form. The free MA product thus produced can be further purified as described below. The reaction is kept below 75°C to prevent production of acetic acid via decarboxylation of MA.

5.5 Purification of malonic acid [001 18] In some embodiments, the methods provided herein further comprise the step of removing impurities from the MA solution. Impurities may react with MA and reduce final yields, or contribute to MA being of lower purity and having more limited industrial utility. Non-limiting examples of impurities include colored bodies, hydrophobic compounds, excess cations, volatile compounds (such as odorants), chloride ions and uncatabolized sugars. Impurities can be removed by nanofiltration, diafiltration, chromatography, stream stripping, or any combination thereof, among other purification technologies. Non-limiting examples of nanofiltration materials include ceramic membranes, metal membranes, polymer membranes, and composite membranes.

5.6 Crystallization of malonic acid

[001 19] The methods provided herein further comprise the step of converting soluble MA to crystalline form. For such conversion, in some embodiments a MA solution is brought to saturation, whereby the MA solution is concentrated at temperatures below decarboxylation temperature and may include one or more evaporation and/or cooling steps. In some embodiments, MA solution is heated to evaporate excess water at temperatures less than 80°C at atmospheric pressure. In some such

embodiments, evaporation is carried out at 65 °C until MA concentration is greater than 70% (w/w), which is near the solubility limit of MA at 65 °C at atmospheric pressure. In other embodiments, evaporation is carried out at lower than atmospheric pressure (for example, using a wiped film evaporator or falling film evaporator) and a temperature of less than 80°C.

[00120] Alternatively, or in addition to concentration by evaporation, in some embodiments the solubility of MA can be decreased by lowering the solution temperature.

[00121] In some embodiments, evaporative crystallization is used to crystallize MA. In other embodiments, cooling crystallization is used to crystallize MA. In some embodiments, multiple crystallization schemes are employed, comprising a series of various heating and cooling steps.

[00122] Crystallized MA can be isolated from the mother liquor (crystallization solution) by any one of several known techniques. In some embodiments, MA crystals are dewatered using a combination of screening and drying methods. In some embodiments, crystal dewatering steps (as described herein for Na ₂ M crystals) comprise centrifugation, belt drying, filtration, application of vacuum, or a combination thereof. In some of these embodiments, vacuum is applied at 20 mm of Hg below atmospheric pressure. Suitable devices for crystal dewatering may include a HVBF or a RDVF. MA isolation based on size can be accomplished, for example, via filtration, using, for example, a filter press, candlestick filter, or other industrially used filtration system with appropriate molecular weight cutoff. MA isolation based on weight or density can be accomplished, for example, via gravitational settling or centrifugation, using, for example, a settler, low g-force decanter centrifuge, or hydrocyclone, wherein suitable g-forces and settling or centrifugation times can be determined using methods known in the art. Section 7. Examples

Example 1: A recombinant P. kudriavzevii strain with increased malonate titer

[00123] In this example, the P. kudriavzevii strain described in PCT Application No.

PCT/US2015/037530 (the entire contents of which are incorporated herein by this reference) is used to produce MA and/or MA salts. Methods on strain construction and culture requirements are also disclosed in this PCT application. Fermentation conditions for the production of MA and MA salts by this strain are described in Example 2 below. Practitioners in the art understand that other host cells may be considered for malonate production and that the recombinant P. kudriavzevii strain described here is a non-limiting example.

Example 2: Fermentative production of malonic acid by recombinant P. kudriavzevii

[00124] Consideration is preferably given to appropriate culture medium depending on the specific requirements of recombinant host cells, fermentation process and downstream purification processes. The media recipes disclosed herein are examples and can be modified as needed to suit individual fermentation goals and needs. More details on media recipes and fermentation conditions are described in PCT Application No. PCT US2015/037530.

[00125] V01 solution comprised myo-inositol, thiamin hydrochloride, pyridoxial hydrochloride, nicotinic acid, calcium pantothenate, biotin, folic acid, p-aminobenzoic acid, and riboflavin.

[00126] T02 solution comprised citric acid monohydrate, H3BO3, CuSO/t-SFhO, FeC -ethO, MnCh, molybdate, and ZnSO/t-TFhO.

[00127] T05 solution comprised citric acid monohydrate, H3BO3, CuSO/t-SFhO, FeC -ethO, MnCh, sodium molybdate, and ZnSO/t-TFhO.

[00128] S21 solution comprised KH ₂ P0 ₄ , urea, and MgS0 ₄ -7H ₂ 0.

[00129] S24 solution comprised KH2PO4, and urea.

[00130] DE95 solution comprised approximately 70% (w/w) glucose solution. DE95 is a corn syrup equivalent that is commonly used in the industry.

[00131] HM PSA 24 medium comprised appropriate amounts of S21 solution, T05 solution, DE95 solution, and V01 solution.

[00132] HM PSA 25 medium comprised appropriate amounts of S21 solution, T05 solution, glucose, maltose, and V01 solution.

[00133] HF 22 medium comprised DE95 solution, KH2PO4, urea, T02 solution, and V01 solution.

[00134] HF 24 medium comprised glucose, maltose, KH2PO4, urea, T02 solution, and V01 solution.

[00135] In this example, recombinant P. kudriavzevii was used to produce MA according to the materials and methods of the present disclosure. Each fermentation run was seeded from a single colony of recombinant .P. kudriavzevii . Three separate, fed batch, fermentation runs were carried out - the Run IDs are 170707_S3, 170707_S2 and 170316 S3; PSA 24 medium or PSA 25 medium was used as the batch medium, and HF 22 medium or HF 24 medium was used as the feed medium. The following parameters were common to all three fermentation runs: (1) 30°C run temperature; (2) an impeller or agitator stir rate of 900-1,100 rpm (in some examples, the OUR was around 100-130 mmol/l/hr); (3) sterile air was blown into the fermenters at 1 1/min; (4) antifoam at manufacturer's recommended working concentrations; (5) run pH was maintained at around pH 5; (6) 5-10 M NaOH was used to maintain fermentation pH at around pH 5; and (7) fermentation runs were at least 53 hours long.

Example 3: Analysis of fermentative production of malonic acid by recombinant P. kudriavzevii

[00136] In this example, fermentation runs 170707_S3, 170707_S2 and 170316 S3 were analyzed by HPLC to determine the amount of MA produced, possible byproducts produced, and the amount of unconsumed glucose left in the broth. Well-known methods and calculations were used for determination of fermentation titers. All three fermentation runs achieved titers of greater than 80 g/1 of MA. (MA titers, as described above, reflect the titers of Na2M on a molar basis.) Specifically, measured titers (expressed as g/1 MA) were 85, 92, and 93 g/1 for fermentation runs 170707_S3, 170707_S2, and 170316 S3, respectively. For all three runs, the concentrations of acetate, citrate, ethanol, glucose, glycerol, pyruvate, and succinate present in the fermentation broth were less than about 3 g/1. All three runs produced sufficient Na2M for downstream processing to produce DEM, which include Na2M polishing and crystallization, esterification of pure Na2M to DEM, and distillation of DEM.

[00137] Thus, this example demonstrated the production of Na2M in fermentations at high titers, productivities, and low byproduct concentrations. High Na2M titers are useful for maximizing downstream yields and low byproduct concentrations are useful for increasing process yields and the purity of the isolated Na2M, DEM, and MA in the downstream steps.

Example 4: Crystallization of Na ₂ M-monohydrate

[00138] In this example, 2,845 g of crystallized Na2M-monohydrate was produced according to methods of the present disclosure. For this example, a mock fermentation broth was generated by reacting MA with NaOH in water.

[00139] Prior to Na2M-monohydrate crystallization, excess water was evaporated from the pure Na2M solution to concentrate the dissolved Na2M. Evaporation was carried out in a vessel with a jacket for temperature control - the jacket temperature was held at 100°C while the Na2M solution temperature was maintained at around 87°C. The evaporation temperature did not exceed 100°C to prevent formation of anhydrous Na2M instead of Na2M-monohydrate. Evaporated water was collected using a condenser so that the amount of water removed was recorded throughout the crystallization process (Table 1). [00140] Na2M-monohydrate began to crystallize as water was removed and the concentration Na2M increased to its solubility limit. Na2M-monohydrate crystals were observed to form at a Na2M

concentration of 65% (w/w) (Table 1). Following crystallization, the Na2M-monohydrate crystals were dewatered by centrifugation.

[00141] At concentrations higher than 75 % Na2M-monohydrate, the Na2M-monohydrate slurry appeared viscous and dewatering of crystals proved challenging. 2,033 g of Na2M-monohydrate crystals were collected from an estimated 2,845 g of Na ₂ M in solution, resulting in a yield of 71% (w/w).

Table 1 : Crystallization of Na2M-monohydrate

Example 5: Esterification of disodium malonate with concentrated sulfuric acid for diethyl malonate production

[00142] This example demonstrated, according to the methods of the present disclosure, esterification of Na2M with ethanol using sulfuric acid as the catalyst, resulting in production of DEM. Purified, dried Na2M-monohydrate crystals were first dissolved in water, and then mixed with H2SO4 and EtOH. The H2SO4 was added slowly (over 15 minutes to 45 minutes) and the reaction time was at least 5 hours. The reaction mixture appeared cloudy due to Na2S04 precipitation. In example reaction 170801-1, the reaction comprised 1.1 molar equivalents of concentrated H2SO4, 30 molar equivalents of EtOH, and a reaction temperature of 25°C; 43% of the Na2M was converted to DEM in this reaction. In example reaction 170802-2, the reaction comprised 1.1 molar equivalents of concentrated H2SO4, 30 molar equivalents of EtOH, and a reaction temperature of 78°C; 95% of the Na2M was converted to DEM in this reaction. In example reaction 170803-3, the reaction comprised 1.2 molar equivalents of concentrated H2SO4, 30 molar equivalents of EtOH, and a reaction temperature of 78°C; 98% of the Na2M was converted to DEM in this reaction. At the end of each of the three reactions, the mixtures were taken through: (1) filtration to remove NaaSO/t; and (2) distillation to purify DEM.

[00143] As demonstrated in this example, the esterification of Na2M with ethanol was pushed to near completion (98% conversion) by use of greater than 1.1 molar equivalents of sulfuric acid as the catalyst. Example 6: Distillation of diethyl malonate

[00144] In this example, a portion of the DEM produced in Example 5 was purified from the esterification reaction mixture by a 2-step distillation process. First, the distillation reactor pressure was set at 20 Torr and the reaction was slowly heated up to 75 °C to remove EtOH, water and ethyl acetate. Then, distillation was carried out at 100°C at 20 Torr for DEM recovery. EtOH was collected from distillation for reuse in future esterification reactions. Purified DEM was analyzed by HPLC - purified DEM was at least 99% pure and contained less than 1% MEM, ethyl acetate, or MA. The water content was measured at less than 0.01% water.

[00145] Therefore, this example demonstrated, according to the methods provided by the present disclosure, the production of purified DEM that was substantially free of impurities, including water, unreacted Na2M, partially converted DEM, as well as the decarboxylation byproducts, acetic acid and ethyl acetate.

Example 7: Using ammonium hydroxide to control fermentation pH increases malonic acid volumetric productivity as compared to calcium hydroxide

[00146] In this example, aerobic, fed-batch fermentations of a P. kudriavzevii strain, LPK151290, engineered to produce malonic acid were performed using either calcium hydroxide or ammonium hydroxide as the based used to control fermentation pH. Higher malonic acid productivities were achieved using ammonium hydroxide as compared to calcium hydroxide as the fermentation base.

[00147] Two 72 -hour fermentations were performed using ammonium hydroxide as the base and three fermentations were performed using calcium hydroxide as the base. All fermentations first comprised a batch phase followed by a fed-batch phase; the fed-batch phase was triggered by depletion of all the glucose in the batch phase. A median oxygen transfer rate of about 95 mmol/l/hr was achieved by sparging air into the fermenter throughout the run. Temperature was maintained at 30°C and pH was maintained at pH 5.0 for the entirety of the run. The fermentation base was either 5M ammonium hydroxide (ammonium hydroxide runs) or 3M calcium hydroxide (calcium hydroxide runs); addition of either ammonium hydroxide or calcium hydroxide occurred automatically and was triggered by a pH probe in the broth.

[00148] It was observed that use of calcium hydroxide as the base resulted in the formation of a white calcium malonate precipitate during the fermentation; in contrast, no precipitate was observed when using ammonium hydroxide as the base. [00149] The batch medium comprised glucose (62 g/1), monopotassium phosphate (5.38 g/1), urea (9.6 g/1), magnesium sulfate heptahydrate (2.82 g/1), boric acid (1.4 mg/1), copper (II) sulfate pentahydrate (1.6 mg/1), Iron (III) chloride hexahydrate (22.8 mg/1), manganese (II) chloride (0.94 mg/1), sodium molybdate (0.56 mg/1), zinc sulfate heptahydrate (13.2 mg/1), and citric acid monohydrate (0.4 g/1). Following depletion of glucose in the batch medium, as indicated by a spike in dissolved oxygen concentration, the fed-batch phase of each fermentation began. In the fed-batch phase, additional medium was pulsed into the fermentation. Each pulse was triggered by an increase in dissolved oxygen concentration. The fed- batch medium comprised glucose (715 g/1), urea (9 g/1), boric acid (2 mg/1), copper (II) sulfate pentahydrate (1.27 mg/1), Iron (III) chloride hexahydrate (22.42 mg/1), manganese (II) chloride (1.33 mg/1), sodium molybdate (0.8 mg/1), zinc sulfate heptahydrate (10.76 mg/1), and citric acid monohydrate (0.44 g/1).

[00150] The fermentations were stopped after 72-hours, and malonic acid concentrations were measured by high-performance liquid chromatography. Malonic acid fermentation volumetric productivities were then calculated by dividing the malonic acid titer by the fermentation run time.

[00151] When 3M calcium hydroxide was used to control fermentation pH, the malonic acid volumetric productivities were 0.80, 0.81, and 0.83 g/l/hr. When 5M ammonium hydroxide was used to control fermentation pH, the malonic acid volumetric productivities were 0.87 and 0.89 g/l/hr, or between 5-11% higher than when using calcium hydroxide.

[00152] This result was particularly surprising given that, despite the ammonium hydroxide base being more concentrated, a larger volume of base is required to neutralize malonic acid when using ammonium hydroxide as compared to calcium hydroxide (/ ^' . e. , two mols of ammonium cation are required to neutralize malonic acid while only one mol of calcium cation is required). Using ammonium hydroxide dilutes the malonic acid in the fermentation and it would be expected to decrease volumetric

productivities; however, the opposite was observed, and malonic acid fermentation productivities were increased using ammonium hydroxide as compared to calcium hydroxide. Thus, this example demonstrated that production of a soluble malonate salt (diammonium malonate) resulted in improved fermentation performance as compared to an insoluble malonate salt (calcium malonate).

Example 8: Using ammonium hydroxide to control fermentation pH increases the concentration phosphorous in fermentation broth as compared to calcium hydroxide

[00153] The fermentations in this Example were substantially similar to those described in Example 7 with the exception that at the conclusion of 72-hour run the concentration of phosphorous in the fermentation broth was measured. Four fermentations using 5M ammonium hydroxide were performed and three fermentations using 3M calcium hydroxide were performed. At the conclusion of each fermentation, samples of fermentation broth were first centrifuged to separate the clarified broth from cells, cell debris, salt precipitates, and other solid materials. The clarified broth samples were then analyzed by inductively coupled plasma mass spectrometry (ICP-MS; University of Nebraska-Lincoln) to determine the concentration phosphorous in the fermentation broth.

[00154] When 3M calcium hydroxide was used to control fermentation pH, the concentration of phosphorous in the clarified broth samples was 9.6 +/- 8.4 ppm (avg+/-stdev; n=3). In contrast, when 5M ammonium hydroxide was used, the concentration phosphorous was 162 +/- 81 ppm (avg+/-stdev; n=4). Thereby, using ammonium hydroxide resulted in an over 16-fold increase in available phosphorous in the fermentation broth at the conclusion of the fermentation.

[00155] Since phosphorous is an elemental nutrient required for cellular growth, maintenance, and metabolism it is very useful for phosphorous to be available to the cell. Use of calcium bases (including calcium hydroxide) are undesirable since in addition to precipitating the malonic acid they also precipitate phosphate salts from the broth, thereby decreasing the phosphorous available to the cell and negatively affecting malonic acid biosynthesis.

[00156] Various publications were referenced in this application. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this disclosure pertains.

It should be noted that there are alternative ways of implementing the embodiments disclosed herein. Accordingly, the present embodiments are to be considered as illustrative and not restrictive; various modifications can be made without departing from the spirit of this disclosure. Furthermore, the claims are not to be limited to the details given herein, and are entitled their full scope and equivalents thereof.