Recombinant host cell for producing malonic acid

Abstract

Provided herein are systems and methods for producing malonic acid or a salt thereof in a recombinant host cell.

Description

[0001] This application is a divisional application of CN202080073740.1.

[0002] Priority claim

[0003] This application claims priority to U.S. Provisional Application No. 62 / 894,548, filed on August 30, 2019, the contents of which are incorporated herein by reference in their entirety.

[0004] Government interests

[0005] This invention was made with U.S. Government support under U.S. Department of Energy Grant Nos. DE-SC0006469 and DE-EE0006113 and National Science Foundation Grant No. IIB-1456081. The U.S. Government has certain rights in this invention.

[0006] Reference to a sequence listing

[0007] This application contains a sequence listing submitted via EFS-web, which is incorporated herein by reference in its entirety for all purposes. The ASCII copy created on August 31, 2020 is named LYGOS_0030_01_WO_ST25.txt and is 120KB in size. Background of the Invention

[0009] Long-term economic and environmental concerns associated with the petrochemical industry have provided an impetus for increasing research, development, and commercialization of methods for converting carbon feedstocks into chemicals that can replace chemicals derived from petroleum feedstocks. One approach is to develop biorefining processes that convert renewable feedstocks into products that can replace petroleum-derived chemicals. Two common goals of improving biorefining processes include achieving lower production costs and reducing carbon dioxide emissions.

[0010] Malonic acid (propanedioic acid / "malonate", CAS No. 141-82-2) is currently produced from non-renewable petroleum feedstocks. One or both carboxylic acid moieties of malonic acid are monoesterified or diesterified with an alcohol (such as methanol or ethanol) to give monoalkyl malonate and dialkyl malonate, respectively. Alkane-4,6-diones ("Meldrum's acid", CAS No. 2033-24-1) are produced from malonic acid using acetone in acetic anhydride or using isopropenyl acetate in the acid.

[0011] Chemical synthesis is currently the preferred approach for synthesizing malonic acid and malonic acid derivative compounds. For example, dialkyl malonate is produced by hydrogen cyanide or carbon monoxide method. In the hydrogen cyanide method, sodium cyanide is reacted with sodium chloroacetate at high temperature to produce sodium cyanoacetate, which is then reacted with an alcohol / mineral acid mixture to produce dialkyl malonate. Hildbrand et al. reported a yield of 75-85% (see "Malonic acid and Derivatives", Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCH, Weinheim, New York (2002)). In the carbon monoxide method, dialkyl malonate (also referred to herein as diester malonate) is produced by chloroacetic acid and carbon monoxide in the presence of alcohol at high temperature and high pressure, and alkoxycarbonylation reaction catalyzed by cobalt is carried out.

[0012] Existing petrochemical-based production pathways for obtaining malonate and malonate-derived compounds are low-yielding, environmentally damaging, non-renewable feedstock dependent, and require expensive treatment of wastewater and exhaust gases. Thus, there remains a need for methods and materials for biocatalytic conversion of renewable feedstocks to malonate, purification of biosynthesized malonate, and subsequent preparation of downstream chemicals and products.

[0013] Public content overview

[0014] The present disclosure provides recombinant host cells, materials and methods for biological production of malonate; methods for detecting the presence of malonate and determining the content of malonate (referred to herein as "sensing malonate") in malonate-producing host cells; and methods for screening host cells in which malonate production is increased. In addition, the present disclosure provides methods for purifying biologically produced malonate and methods for converting malonate into other industrially important chemicals.

[0015] In a first aspect, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding an acyl-CoA hydrolase that catalyzes the conversion of malonyl-CoA to malonate, as described herein:

[0016]

[0017] These recombinant host cells produce more malonate than corresponding cells that do not contain the heterologous hydrolase. In various embodiments, the host cell can produce at least 10g / L malonate under appropriate fermentation conditions, and in various embodiments, the production level can be as high as 50g / L to 100g / L or higher. In some embodiments, the heterologous nucleic acid encodes an endogenously expressed enzyme in a mutant form; therefore, the present disclosure provides a variety of mutant acyl-CoA hydrolases, nucleic acids encoding them, and recombinant expression vectors comprising those nucleic acids. In other embodiments, the heterologous nucleic acid encodes an endogenous enzyme that is overexpressed. In addition, in some embodiments, the heterologous nucleic acid encodes a wild-type or mutant enzyme of a host cell heterologous (not naturally expressed in the host cell) acyl-CoA hydrolase. In some embodiments, the host cell is a yeast cell. In other embodiments, the host cell is a bacterial cell.

[0018] Thus, in various embodiments, the heterologous nucleic acids provided by the present disclosure encode a wild-type or mutant form of an acyl-CoA hydrolase. Non-limiting examples of acyl-CoA hydrolases encoded by the nucleic acids provided by the present disclosure and suitable for malonyl-CoA hydrolysis include wild-type and modified enzymes selected from the group consisting of 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4), 3-hydroxypropionyl-CoA hydrolase (EC 3.1.2.4), acetoacetyl-CoA hydrolase (EC 3.1.2.11), methylmalonyl-CoA hydrolase (EC 3.1.2.17), propionyl-CoA hydrolase (EC 3.1.2.18), succinyl-CoA hydrolase (EC 3.1.2.3), and malonyl-CoA:ACP transacylase (EC 2.3.1.39) mutated to have malonyl-CoA hydrolase activity as provided herein.

[0019] In various embodiments, the present disclosure provides a malonyl-CoA hydrolase that is a mutant of a 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4). Suitable 3-hydroxyisobutyryl-CoA hydrolases can be obtained from both eukaryotic and prokaryotic organisms, including both Gram-positive and Gram-negative organisms. In various embodiments, the 3-hydroxyisobutyryl-CoA hydrolase is obtained from a yeast strain, a Bacillus species, and a Pseudomonas species.

[0020] In other embodiments, the disclosure provides a malonyl-CoA hydrolase that is a malonyl-CoA:ACP transacylase (EC 2.3.1.39) encoded by a prokaryotic organism that is mutated to have malonyl-CoA hydrolase activity as provided herein. In various embodiments, the prokaryotic organism is a Gram-negative bacterium. In various embodiments of the disclosure, the Gram-negative bacterium is Escherichia.

[0021] In a second aspect, the present disclosure provides a recombinant expression vector encoding a wild-type or mutant acyl-CoA hydrolase that catalyzes the conversion of malonyl-CoA to malonate. In some embodiments, the expression vector is a yeast expression vector; in other embodiments, the expression vector is a bacterial expression vector. In various embodiments, the bacterial expression vector is an Escherichia coli expression vector.

[0022] In a third aspect, the disclosure provides a recombinant host cell suitable for biosynthesizing and producing malonate at a level that allows malonate to be separated and used as a starting material for other useful products of chemical synthesis. In some embodiments, the host cell is a eukaryotic organism. In some embodiments, the host cell is a yeast cell. In various embodiments, yeast is a Candida, Cryptococcus, Hansenula, Issatchenkia, Kluyveromyces, Komagataella, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces or Yarrowia species. In some embodiments, the eukaryotic host cell is a fungus. In some embodiments, the cell host is an algae.

[0023] In other embodiments, host cell is a bacterial cell. In various embodiments, host cell is a bacterial cell selected from the group consisting of: Bacillus, Clostridium, Corynebacterium, Escherichia, Pseudomonas and Streptomyces. In some embodiments, host cell is an Escherichia coli cell.

[0024] Typically, the recombinant host cells of the present disclosure have been genetically modified to improve malonate yield, titer, and / or productivity. In various embodiments, the host cell has been modified to increase malonate biosynthesis by one or more host cell modifications selected from the group consisting of the following modifications: modifications that result in increased acetyl-CoA biosynthesis, increased malonyl-CoA biosynthesis, reduced malonyl-CoA utilization, reduced malonate catabolism, increased secretion of malonate into the fermentation broth, increased host cell tolerance to malonate in the fermentation broth, and / or increased host cell catabolism of carbon sources (e.g., acetate, alginate, ethanol, fatty acids, lignocellulosic biomass, methanol, pentoses, and syngas).

[0025] In a fourth aspect, the disclosure provides a method for producing malonate in a recombinant host cell, the method generally comprising culturing the recombinant host cell in a fermentation broth under conditions that enable the recombinant host cell to produce malonate. In some embodiments, the host cell has been engineered to express more or less endogenous enzymes that cause more malonate to be produced than the corresponding cells that have not yet been so engineered. In some embodiments, the method includes culturing a recombinant host cell that expresses a heterologous (exogenous or non-natural) enzyme that causes malonate production to be increased. In some embodiments, the host cell used in the method comprises one or more expression vectors comprising a heterologous malonyl-CoA hydrolase. In some embodiments of these methods, the fermentation broth is supplemented with a carbon source that promotes malonate production and the carbon source is selected from the group consisting of: cellodextrins, 5-carbon sugars, 6-carbon sugars, carbon dioxide, ethanol, methanol, glycerol, acetate, and / or fatty acids.

[0026] In a fifth aspect, the disclosure provides a biosensor comprising a malonate transcription factor and a promoter operatively connected to a marker gene that is responsive to the transcription factor. The disclosure also provides a method for "sensing" malonate, malonate production and malonate productive host cells and a method for screening a host cell that is increased in malonate production. In various embodiments, the method includes culturing a host cell that expresses a malonate transcription factor and contains a promoter operatively connected to a marker gene that is responsive to the transcription factor, and selecting a host cell that is improved in malonate production by screening for the expression of the marker gene product and selecting those host cells that express a higher level of the marker gene product. In some embodiments, malonate is produced in a host cell, and the fermentation broth from the first cell is contacted with a second cell that comprises a malonate transcription factor and a promoter operatively connected to a marker gene that is responsive to the transcription factor (added to a culture medium containing the second cell), and the host cell that is improved in malonate production is identified by identifying the cell with the highest expression level of the marker gene product. In other embodiments, malonate is produced in a host cell comprising a malonate transcription factor and a promoter operatively connected to a marker gene that is responsive to malonate, and the host cell whose malonate production is increased is screened for malonate production by screening and identifying cells expressing the highest level of the marker gene product. In some embodiments, the transcription factor can bind malonate, which causes the transcription factor to bind a homologous promoter and activate a marker gene operatively connected to the promoter. In some embodiments, the transcription factor is an MdcY transcription factor. In some embodiments, the method is implemented to screen or select a genetically modified host cell whose malonate production is improved relative to a control cell.

[0027] In a sixth aspect, the disclosure provides purified malonate separated from the fermentation broth of a host cell (optionally a host cell of the disclosure) producing malonate. The disclosure also provides a method for purifying malonate from the fermentation broth of a host cell producing malonate, the method generally comprising culturing the host cell in a fermentation broth under conditions that enable the host cell to produce malonate, and purifying the malonate from the fermentation broth. In some embodiments of the disclosure, the malonate concentration in the fermentation broth is increased by dehydrating the fermentation broth during the purification process. In various embodiments of the disclosure, dehydration is achieved by reverse osmosis treatment, evaporation, or a combination of the two. In various embodiments, purification is achieved by adding one or more of the following: divalent cations, monovalent cations, ammonium, monosubstituted amines, disubstituted amines, trisubstituted amines, cationic purification resins or acids. In various embodiments of the disclosure, these reagents are added together with one or more organic solvents. In some embodiments of the disclosure, a hydrophobic solvent is used for liquid-liquid extraction of the fermentation broth. In other embodiments, malonate is purified from the fermentation broth by reactive extraction or distillation with an acid catalyst and an alcohol.

[0028] In a seventh aspect, the present disclosure provides methods for preparing compounds derived from malonic acid and compounds produced by the methods. The methods generally include reacting malonic acid with one or more substrates to produce compounds. In some embodiments of these methods, chemicals obtained from malonic acid using established synthetic pathways are produced using biogenic malonic acid. In other embodiments of these methods, new synthetic pathways for producing useful chemicals suitable for use with synthetic or biogenic malonic acid are provided. In some embodiments, monoalkyl malonate is synthesized from biogenic malonic acid. In other embodiments, dialkyl malonate is synthesized from biogenic malonic acid. In some embodiments, acrylate is synthesized from malonic acid or malonic acid. In other embodiments, acrylate is synthesized from malonic acid monoesters or diesters. In other embodiments, dicarboxylic acids are produced from malonic acid. Illustrative dicarboxylic acids that can be produced according to the methods of the present disclosure include dicarboxylic acids selected from the group consisting of glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, and the corresponding monoalkyl and dialkyl esters of each. In other embodiments of the present disclosure, the dicarboxylic acids are produced from malonic acid derived compounds. In other embodiments of the present disclosure, ε-caprolactam is produced from malonic acid. In other embodiments of the present disclosure, δ-valerolactam is produced from malonic acid.

[0029] In one embodiment, the present invention provides a recombinant Pichia kudriavzevii host cell. In one embodiment, the recombinant Pichia kudriavzevii host cell comprises a heterologous mutant Pseudomonas fulva (strain 12-X) 3-hydroxyisobutyryl-CoA hydrolase (also referred to herein as malonyl-CoA hydrolase) protein having UniProt ID: F6AA82, wherein the mutant Pseudomonas fulva protein

[0030] (a) contains one or more of the following mutations: E95N, V152A, F304R and Q348A, or

[0031] (b) has at least 75% sequence identity to SEQ ID NO:9 and comprises one or more mutations of the following amino acids: K107, L148, G170 and E367, or

[0032] (c) having at least 75%, at least 85%, at least 90% and at least 95% sequence identity to the sequence of SEQ ID NO:57, 58, 59, 60 or 61.

[0033] In one embodiment, the malonyl-CoA hydrolase has UniProt ID: F6AA82 and comprises one or more of the following mutations: E95N, V152A, F304R, and Q348A. In one embodiment, the malonyl-CoA hydrolase has UniProt ID: F6AA82 and comprises mutations: E95N, V152A, F304R, and Q348A. In another embodiment, the malonyl-CoA hydrolase has UniProt ID: F6AA82 and comprises mutations: E95N, V152A, F304R, and Q348A. In another embodiment, the malonyl-CoA hydrolase has UniProt ID: F6AA82 and has at least 75% sequence identity to SEQ ID NO: 9 and comprises one or more mutations of the following amino acids: K107, L148, G170, and E367. In another embodiment, the malonyl-CoA hydrolase has UniProt ID: F6AA82 and has at least 75% sequence identity to the sequence SEQ ID NO: 57. In one embodiment, the recombinant Pichia kudriavzevii host cell further comprises a heterologous S. cerevisiae aldehyde dehydrogenase, which is ALD6, which has UniProt ID P54115. In another embodiment, the recombinant Pichia kudriavzevii host cell further comprises a heterologous Yarrowia lipolytica acetyl-CoA synthetase, which is UniProt ID: Q6C2Q5. In another embodiment, the recombinant Pichia kudriavzevii host cell further comprises a heterologous Saccharomyces cerevisiae pyruvate decarboxylase PDC1, which is UniProt ID: P06169. In another embodiment, the recombinant Pichia kudriavzevii host cell further comprises a heterologous S. cerevisiae pyruvate decarboxylase PDC5, which is UniProt ID: P16467. In another embodiment, the recombinant Pichia kudriavzevii host cell further comprises a heterologous Saccharomyces cerevisiae pyruvate decarboxylase PDC6, which is UniProt ID: P26263. In another embodiment, the recombinant Pichia kudriavzevii host cell comprises a native acetyl-CoA carboxylase ACC1, which is UniProt ID: A0A099P5S6. In another embodiment, the recombinant Pichia kudriavzevii host cell comprises an Escherichia coli acetyl-CoA carboxylase. In another embodiment, the recombinant Pichia kudriavzevii host cell comprises an Escherichia coli pyruvate decarboxylase. In another embodiment, the recombinant Pichia kudriavzevii host cell comprises an Escherichia coli pyruvate dehydrogenase complex.

[0034] In another embodiment, provided herein is a method of identifying a host cell with increased malonate production, comprising the steps of:

[0035] fermenting the recombinant Pichia kudriavzevii host cells provided herein to provide a fermentation broth;

[0036] contacting the fermentation broth with 4-diazonobenzenesulfonate (DBS) and optionally a base; and

[0037] Cells with increased malonate production were identified based on increased absorbance levels at approximately 530 nm.

[0038] Methods for using DBS for other purposes are reported in Nishimoto et al., Analytical Biochemistry, 216, 159-164, 1994, and can be adapted by those skilled in the art based on the present disclosure. In another embodiment, a method for producing malonate is provided herein, comprising culturing a Pichia kudriavzevii host cell provided herein under conditions leading to the production of malonate.

[0039] In another embodiment, provided herein is a method of producing malonate, comprising culturing the identified host cell provided herein under conditions that result in the production of malonate.

[0040] In another embodiment, the present invention provides a method for preparing a malonate solution from a host cell fermentation broth, wherein the method comprises the following steps:

[0041] Cultivating a recombinant Pichia kudriavzevii host cell provided herein in a fermentation broth comprising a base under conditions that result in the production of malonate; and

[0042] The malonate solution was separated.

[0043] In another embodiment, the present invention provides a method for isolating dialkyl malonate, preferably di-C1-C6 alkyl malonate, from a host cell fermentation broth, comprising the following steps:

[0044] Cultivating a recombinant host cell provided herein or an identified recombinant host cell provided herein under conditions that result in production of malonate;

[0045] separating host cells from the fermentation broth;

[0046] adding a fatty alcohol and an acid catalyst to the fermentation broth to form a dialkyl malonate; and

[0047] The resulting dialkyl malonate is separated from the fermentation broth to provide isolated dialkyl malonate.

[0048] In one embodiment, the formation of the dialkyl malonate further comprises contacting with an ion exchange resin. In one embodiment, the fatty alcohol is ethanol. In one embodiment, the fatty alcohol is methanol.

[0049] These and other aspects and embodiments of the present disclosure are illustrated in the accompanying drawings and described in more detail below.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Provided are examples of the present disclosure using promoter P MdcL Dose-response curve of the E. coli MdcY malonate biosensor.

[0052] Figure 2 An HPLC chromatogram trace is provided illustrating the separation and detection of acrylic acid produced from malonic acid according to the methods of the present disclosure as described in Example 28. The X-axis shows acrylic acid eluting at about 17.5 minutes, while the Y-axis shows arbitrary units obtained by detecting acrylic acid by a UV detector monitoring 210 nm. Acrylic acid produced from malonic acid according to the methods of the present disclosure (dashed line) exhibits the same retention time as an authentic acrylic acid standard (solid line).

[0053] Details

[0054] The present disclosure provides recombinant host cells, materials, and methods for bioproducing malonate, screening for malonate-producing host cells with improved malonate production, purifying bioproduced malonate, and converting malonate synthesis into industrially important chemicals.

[0055] Although the present disclosure is described herein with respect to its aspects and specific embodiments, it will be appreciated by those skilled in the art that various changes may be made and that equivalents may be substituted without departing from the present disclosure. The present disclosure is not limited to specific nucleic acids, expression vectors, enzymes, host microorganisms or methods, because the various things may change. The terms used herein are only for the purpose of describing specific aspects and embodiments, and should not be interpreted as having a restrictive nature. In addition, many modifications may be made according to the present disclosure to adapt to specific circumstances, materials, compositions of matter, methods, one or more process steps. All of the modifications are within the scope of the claims attached hereto.

[0056] Section 1: Definitions

[0057] Throughout this specification and in the claims that follow, reference will be made to a number of terms that will be defined to have the following meanings.

[0058] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an expression vector" includes a single expression vector as well as multiple expression vectors that are the same (e.g., the same operon) or different; reference to "a cell" includes a single cell as well as multiple cells; etc.

[0059] The amino acid in the protein coding sequence is identified by the following abbreviations and symbols in this article. Specific amino acids are identified by the following single-letter abbreviations: A is alanine, R is arginine, N is asparagine, D is aspartic acid, C is cysteine, Q is glutamine, E is glutamic acid, G is glycine, H is histidine, L is leucine, I is isoleucine, K is lysine, M is methionine, F is phenylalanine, P is proline, S is serine, T is threonine, W is tryptophan, Y is tyrosine, and V is valine. The dash (-) in the consensus sequence indicates that there is no amino acid at the specified position. The plus sign (+) in the consensus sequence indicates that any amino acid can be present at the specified position. Therefore, the plus sign in the consensus sequence herein indicates that the amino acid is usually a non-conserved position at this place; the homologous enzyme sequence can have any amino acid at the indicated "+" position when compared with the consensus sequence. At positions in the consensus sequence where one of a subset of amino acids may be present, the following abbreviations are used: B indicates that one of the amino acids R, K, or H is present at the indicated position; J indicates that one of the amino acids D or E is present at the indicated position; O indicates that one of the amino acids I, L, or V is present at the indicated position; U indicates that one of the amino acids S or T is present at the indicated position; X indicates any amino acid, such as any naturally occurring amino acid, and X1 indicates that one of the amino acids A, D, R, H, K, S, T, N, Q, or Y (or a subset of those amino acids) is present at the indicated position. Illustrative subsets of X1 include X1 is A, D, K, S, T, N, or Y and X1 is S or N. Specific amino acids in a protein coding sequence are identified by their corresponding single letter abbreviation followed by the amino acid position in the protein coding sequence, where 1 corresponds to the amino acid at the N-terminus of the protein (usually methionine). For example, E124 in S. cerevisiae wild-type EHD3 refers to glutamic acid at position 124 starting from the N-terminal methionine (i.e., M1) of EHD3. Amino acid substitutions (i.e., point mutations) are indicated by identifying the mutant (i.e., progeny) amino acid followed by the single letter symbol and number in the parent protein coding sequence; for example, E124A in S. cerevisiae EHD3 refers to a substitution of alanine for glutamic acid at position 124 in the EHD3 protein coding sequence. Mutations may also be identified in parentheses, e.g., EHD3(E124A). Multiple point mutations in a protein coding sequence are separated by a backslash ( / ); for example, EHD3 E124A / Y125A indicates that mutations E124A and Y125A are both present in the EHD3 protein coding sequence. The number of mutations introduced in some examples has been annotated by a dash followed by the number of mutations before the mutation is identified in parentheses (e.g., A5W8H3-1(E95Q)). Uniprot IDs with and without a dash and number are used interchangeably herein (i.e., A5W8H3-1(E95Q)=A5W8H3(E95Q)).

[0060] As used herein, the term "expression" when used in connection with a nucleic acid encoding an enzyme or the enzyme itself in a cell refers to the production of the enzyme, which may be an endogenous or exogenous (heterologous) enzyme, in the cell. In these cases, the term "overexpression" refers to the production of an enzyme at a higher level, i.e., an increase in the level of enzyme, in the case of an endogenous enzyme compared to the wild type. It is understood by those skilled in the art that overexpression of an enzyme can be achieved by increasing the strength of the promoter used to drive expression of the coding sequence or changing the type of the promoter, increasing the strength of the ribosome binding site or Kozak sequence, increasing the stability of the mRNA transcript, changing the codon usage, increasing the stability of the enzyme, etc.

[0061] The term "expression vector" or "vector" refers to a nucleic acid and / or composition that can be introduced into a host cell, for example by transduction, transformation or infection, so that the cell then produces ("expresses") a nucleic acid that is different from the nucleic acid and / or protein that is naturally present in the cell, or in a sense not naturally present in the cell, contained in or encoded by the nucleic acid so introduced. Thus, an "expression vector" contains a nucleic acid (usually DNA) to be expressed by a host cell. Optionally, the expression vector may be contained in materials that facilitate entry of the nucleic acid into the host cell, such as materials associated with viruses, liposomes, protein coatings, etc. Expression vectors suitable for use in the various aspects and embodiments of the present disclosure include expression vectors into which a nucleic acid sequence can be or has been inserted, together with any preferred or desired operating elements. Thus, the expression vector can be transferred into a host cell and typically replicated therein (although in some embodiments, non-replicable vectors that provide "transient" expression may also be employed). In some embodiments, expression vectors integrated into chromosomal, mitochondrial or plasmid DNA are employed. In other embodiments, expression vectors that replicate extrachromosomally are employed. Typical expression vectors include plasmids, and expression vectors generally contain the operational elements required for transcribing the nucleic acid in the vector. The plasmids and other expression vectors are described herein or are well known to those of ordinary skill in the art.

[0062] The terms "ferment," "fermentative," and "fermentation" are used herein to describe the cultivation of microorganisms under conditions to produce a useful chemical, including but not limited to aerobic or anaerobic conditions under which microbial growth occurs.

[0063] As used herein, the term "heterologous" refers to a substance that is not naturally present in a cell. For example, a nucleic acid is heterologous to a cell and is therefore a "heterologous nucleic acid" with respect to the cell if at least one of the following is true: (a) the nucleic acid does not naturally occur in the cell (that is, it is an "exogenous" nucleic acid); (b) the nucleic acid naturally occurs in a given host cell (that is, "endogenous"), but the nucleic acid or the RNA or protein produced by transcription and translation of this nucleic acid is produced or present in a non-natural (e.g., greater than or less than naturally occurring) amount in the host cell; (c) the nucleic acid comprises a nucleotide sequence that encodes a protein endogenous to the host cell, but differs in sequence from an endogenous nucleotide sequence encoding the same protein (having the same or substantially the same amino acid sequence), thereby generally resulting in the protein being produced in greater amounts in the cell, or in the case of an enzyme, a mutant form having an altered (e.g., higher or lower or different) activity; and / or (d) the nucleic acid comprises two or more nucleotide sequences that are not present in the cell in the same relationship to each other. As another example, a protein is heterologous to the host cell if it is produced by translating RNA or the corresponding RNA is produced by transcribing a heterologous nucleic acid; the protein is also heterologous to the host cell if it is a mutated form of an endogenous protein and the mutation was introduced by genetic engineering.

[0064] The terms "host cell" and "host microorganism" are used interchangeably herein to refer to living cells that can (or have been) transformed by insertion of an expression vector. As described herein, a host microorganism or cell can be a prokaryotic cell (e.g., a microorganism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by those skilled in the art, prokaryotic cells lack a membrane-bound nucleus, while eukaryotic cells have a membrane-bound nucleus.

[0065] The terms "isolated" or "purified" 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 from components that normally accompany it in its native state, e.g., the state in which it exists in nature or the state in which it exists when it is originally produced.

[0066] Depending on the structure, pH and the ions present, the carboxylic acids described herein may be salts, acids, bases or derivatives. The terms "malonate" and "malonic acid" are used interchangeably herein. Malonic acid is also known as propanedioic acid (C3H4O4; CAS No. 141-82-2).

[0067] The term "malonic acid derivative compound" as used herein refers to monoalkyl malonate, including, for example and without limitation, monomethyl malonate (also known as monomethyl malonate, CAS No. 16695-14-0), monoethyl malonate (also known as monoethyl malonate, CAS No. 1071-46-1), monopropyl malonate, monobutyl malonate, monotert-butyl malonate (CAS No. 40052-13-9), etc.; dialkyl malonate, for example and without limitation, dimethyl malonate (CAS No. 108-59-8), diethyl malonate (CAS No. 105-53-3), dipropyl malonate (CAS No. 1117-19-7), dibutyl malonate (CAS No. 1190-39-2), etc., and Meldrum's acid (CAS No. 2033-24-1). Malonate-derived compounds can be produced synthetically from malonic acid and are valuable compounds themselves, but are also useful substrates in the chemical synthesis of many other valuable compounds.

[0068] As used herein, the term "nucleic acid" and its variations will be common to polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and polyribonucleotides (containing D-ribose). "Nucleic acid" may also refer to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, as well as other polymers containing a non-nucleotide backbone, provided that the polymer contains nucleoside bases in a configuration that allows base pairing and base stacking as found in DNA and RNA. As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9: 4022, 1970). "Nucleic acid" may also be referred to herein with respect to its sequence (i.e., the order in which the different nucleotides appear in the nucleic acid), since the sequence of nucleotides in a nucleic acid generally defines its biological activity, for example as in the sequence of a coding region, i.e., a nucleic acid in a gene that mainly consists of a promoter and a coding region, the coding region encoding the product of the gene, which may be RNA (e.g., rRNA, tRNA or mRNA) or a protein (when a gene encodes a protein, both the mRNA and the protein are the "gene products" of the gene).

[0069] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, ribosome binding site and transcription terminator) and a second nucleic acid sequence, coding sequence or coding region, wherein the expression control sequence directs or otherwise regulates the transcription and / or translation of the coding sequence.

[0070] As used herein, the term "optional" or "optionally" means that the subsequently described features or structures may or may not exist, or the subsequently described event or matter may or may not occur, and that the description includes instances where the particular features or structures exist and instances where the features or structures are not present, or instances where the event or matter occurs and instances where it does not occur.

[0071] As used herein, "recombination" refers to the alteration of genetic material by human intervention. Generally, recombination refers to the manipulation of DNA or RNA in cells or viruses or expression vectors by molecular biology (recombinant DNA technology) methods, including cloning and recombination. Recombination may also refer to the manipulation of DNA or RNA in cells or viruses by random or directed mutagenesis. A "recombinant" cell or nucleic acid can be generally described in terms of how it differs from its naturally occurring counterpart ("wild type"). In addition, any reference to "engineered" or "modified" cells or nucleic acids and variations of those terms is intended to refer to recombinant cells or nucleic acids.

[0072] As used herein, the term "transcription factor biosensor" refers to a system for detecting a substance such as malonate by activating the expression of a "marker" or "reporter" gene, wherein the reporter gene expression is mediated by a transcription factor that is able to bind to a promoter and activate transcription upon binding of that substance such as malonate. For example, malonate can bind a transcription factor (e.g., MdcY) and activate a promoter (e.g., P MdcL ) transcription. A "malonate transcription factor" is a transcription factor that can activate a promoter when bound to malonate. Thus, MdcY is a malonate transcription factor.

[0073] As used herein, the terms "transduction", "transformation", "transfection" and variations thereof refer to the introduction of one or more nucleic acids into a cell. For practical purposes, for a cell to be referred to as a "transduced", "transformed" or "transfected" cell, the nucleic acid must be stably maintained or replicated by the cell for a period sufficient to allow the function or product it encodes to be expressed. As will be appreciated by those skilled in the art, stable maintenance or replication of the nucleic acid can be achieved by incorporating the sequence of the nucleic acid into the chromosomal DNA (e.g., genome) of the cell (e.g., as occurs by chromosomal integration), or by extrachromosomal replication (e.g., as occurs with a freely replicating plasmid). When a virus is "infectious", it can be stably maintained or replicated: when it transduces a host microorganism, it replicates and (without the use of any complementary virus or vector) spreads a sub-generation expression vector to other microorganisms, such as a virus of the same type as the original transducing expression vector, wherein the sub-generation expression vector has the same reproductive capacity.

[0074] Section 2: Malonyl-CoA hydrolase

[0075] According to one aspect of the present disclosure, malonate is produced by the action of malonyl-CoA hydrolase catalyzing the conversion of malonyl-CoA into malonate. To date, no wild-type malonyl-CoA hydrolase gene has been identified, but the presence of small amounts of malonate in the fermentation medium of non-engineered strains indicates that a wild-type enzyme with this activity may be present. The present disclosure provides a number of genes that have been mutated to confer malonyl-CoA hydrolase activity as counterparts to the wild-type gene. The host cell for preparing the malonyl-CoA hydrolase is a recombinant host cell; in many embodiments, the host cell has been genetically modified to contain a heterologous nucleic acid encoding a malonyl-CoA hydrolase that catalyzes the hydrolysis of malonyl-CoA into malonate. In some embodiments, the recombinant host cell is a eukaryotic organism. In various embodiments, the eukaryotic organism is a yeast strain selected from the following non-limiting example genus: Candida, Cryptococcus, Hansenula, Issaffine, Kluyveromyces, Komagataella, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces or Yarrowia. Those skilled in the art will recognize that these genera fully encompass yeast, including yeasts that are distinguished as lipomyces. In some embodiments, the host cell is Saccharomyces cerevisiae. In other embodiments, the host cell is Pichia kudriavzevii. In other embodiments of the present disclosure, the eukaryotic host cell is a fungus or algae. In other embodiments, the recombinant host cell is a prokaryotic organism selected from the following non-limiting example genus: Bacillus, Clostridium, Corynebacterium, Escherichia, Pseudomonas, Rhodobacter (Rhodobacter) and Streptomyces. In some embodiments, the host cell is Escherichia coli.

[0076] The present disclosure arises, in part, from the discovery that various acyl-CoA hydrolases and transacylases can be engineered to have malonyl-CoA hydrolase activity and thus be used to bioproduce malonate. Non-limiting examples of acyl-CoA hydrolases suitable for modification to perform malonyl-CoA hydrolysis include any of those acyl-CoA hydrolases from the group consisting of 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4), 3-hydroxypropionyl-CoA hydrolase (EC 3.1.2.4), acetoacetyl-CoA hydrolase (EC 3.1.2.11), methylmalonyl-CoA hydrolase (EC 3.1.2.17), propionyl-CoA hydrolase (EC 3.1.2.18), succinyl-CoA hydrolase (EC 3.1.2.3), and malonyl-CoA:ACP transacylase (EC 2.3.1.39) mutated to have malonyl-CoA hydrolase activity as provided herein.

[0077] In some embodiments, the malonyl-CoA hydrolase used to produce malonate according to the present disclosure is a mutant Saccharomyces cerevisiae EHD3 acyl-CoA hydrolase (see SEQ ID NO: 1 for the wild-type EHD3 amino acid sequence). One such mutant with altered substrate specificity is the E124V mutant (see Rouhier, "Characterization of YDR036C from Saccharomyces cerevisiae." Dissertation, Miami University, Miami University and OhioLINK (2011)), but it has not been reported to have malonyl-CoA hydrolase activity, although it has been previously reported. In some embodiments of the present disclosure, E. coli host cells expressing the E124V mutant are used to produce malonate, which is then purified from the cells or fermentation broth. In other embodiments of the present disclosure, yeast cells expressing the E124V mutant are used to produce malonate according to the present disclosure. In another embodiment, an oleaginous yeast cell expressing the E124V mutant is used to produce malonate according to the present disclosure.

[0078] Previous efforts to produce the E124A mutant of EHD3 resulted in cell death after inducing protein expression from a pET28a expression vector; the protein could not be purified (see Rouhier, supra). The present disclosure provides expression vectors of the E124A mutant that can be used in E. coli host cells to render them capable of producing malonate. These E. coli expression vectors are characterized in that the E124A mutant is produced at lower non-toxic levels relative to the pET28a vector of Rouhier. This is achieved, for example, by employing an expression vector having a promoter with a copy number lower than that used by Rouhier or a promoter weaker than that used by Rouhier. Those skilled in the art will also appreciate that translation can be regulated by the affinity of the ribosome binding site (RBS) or Kozak sequence for the ribosome. Therefore, weaker RBS or Kozak sequences can also be used to reduce gene expression. Examples of expression vectors with lower copy numbers include, but are not limited to, pSC101 origin expression vectors, p15a origin expression vectors, and expression vectors integrated into chromosomal DNA. Examples of promoters weaker than the T7 promoter used by Rouhier include, but are not limited to, P LacO1 , P TRC and P BAD In some embodiments, the vector has a pSC101 origin of replication. In other embodiments, the promoter used to express the EHD3 E124A mutant coding sequence is P lacO1Promoter. In addition, the present disclosure provides vectors for expression of the E124A mutant encoding yeast host cells. The genetically modified Saccharomyces cerevisiae EHD3E124A expression vectors of the present disclosure can be used in vivo to produce malonate in Escherichia coli and Saccharomyces cerevisiae, and the methods of the present disclosure provide means for subsequent purification of malonate from fermentation broths of these strains and for conversion of malonate synthesis into derivative small molecule compounds.

[0079] The present disclosure also provides an E124S mutant of EHD3 that functions as a malonyl-CoA hydrolase, vectors for expressing this mutant, and host cells that express this mutant and produce malonate (see Example 31). Wild-type S. cerevisiae EHD3 catalyzes the hydrolysis of 3-hydroxypropionyl-CoA (3HPA-CoA) and 3-hydroxyisobutyryl-CoA (3HIBA-CoA), and while the present disclosure is not limited by theory, it is predicted that E124 interacts with the terminal hydroxyl moiety on 3HPA-CoA, thereby stabilizing the substrate in the EHD3 active site (see Rouhier, supra). Certain aspects of the present disclosure arise from the discovery that specific E124 point mutations increase the enzymatic hydrolysis of malonyl-CoA to produce malonate. Mutation of E124 to a nucleophilic amino acid (e.g., S or T), a basic amino acid (e.g., H, K or R), or an amide amino acid (e.g., N or Q) improves the binding of malonyl-CoA over 3-hydroxypropionyl-CoA in the EHD3 active site and increases malonate production (relative to the unmutated counterpart enzyme). The E124S, E124T, E124N, E124Q, E124H, E124K, and E124R mutations also reduce the production of byproducts (e.g., acetate, propionate, isobutyrate, and succinate) due to reduced hydrolysis of endogenous host cell acyl-CoA molecules. The E124S point mutation places the hydroxyl moiety in a position that promotes hydrogen bonding between the serine residue and the terminal carboxylate group of malonyl-CoA. The E124Q point mutation places the glutamine amide group in a position close to the terminal carboxylate group of malonyl-CoA. The E124K point mutation places the lysine amine group in a position that promotes hydrogen bonding between the lysine residue and the terminal carboxylate group of malonyl-CoA. Unlike the nucleophilic, amide, and basic E124 point mutations described above, mutations E124A and E124V remove the charged amino acid present at position 124; these mutations eliminate hydrogen bonding between the terminal carboxylate on malonate and the EHD3 124 amino acid side chain and open the EHD3 active site to promiscuous activity, thereby increasing undesirable byproduct formation and reducing malonate production.

[0080] In some embodiments of the present disclosure, an E. coli host cell expressing an E124S mutant is used to produce malonate. In other embodiments of the present disclosure, a yeast host cell expressing an E124S mutant is used to produce malonate. In other embodiments, an oil yeast host cell expressing an E124S mutant is used to produce malonate. In some embodiments of the present disclosure, an E. coli host cell expressing an E124Q mutant is used to produce malonate. In other embodiments of the present disclosure, a yeast host cell expressing an E124Q mutant is used to produce malonate. In other embodiments, an oil yeast host cell expressing an E124Q mutant is used to produce malonate. In some embodiments of the present disclosure, an E. coli host cell expressing an E124K mutant is used to produce malonate. In other embodiments of the present disclosure, a yeast host cell expressing an E124K mutant is used to produce malonate. In other embodiments, an oil yeast host cell expressing an E124K mutant is used to produce malonate. In some embodiments of the present disclosure, an E. coli host cell expressing an E124H mutant is used to produce malonate. In other embodiments of the present disclosure, yeast host cells expressing the E124H mutant are used to produce malonate. In other embodiments of the present disclosure, oleaginous yeast host cells expressing the E124H mutant are used to produce malonate. In some embodiments of the present disclosure, Escherichia coli host cells expressing the E124R mutant are used to produce malonate. In other embodiments of the present disclosure, yeast host cells expressing the E124R mutant are used to produce malonate. In other embodiments of the present disclosure, oleaginous yeast host cells expressing the E124R mutant are used to produce malonate. In other embodiments, recombinant host cells expressing EHD3 E124 nucleophilic amino acid point mutations (i.e., E124S or E124T) are used to produce malonate. In other embodiments, recombinant host cells expressing EHD3 E124 basic amino acid point mutations (i.e., E124H, E124K or E124R) are used to produce malonate. In other embodiments, recombinant host cells expressing an EHD3 E124 amide amino acid point mutation (ie, E124N or E124Q) are used to produce malonate.

[0081] The present disclosure also provides a mutant EHD3 comprising a mutant active site, a vector for expressing the mutant, and a host cell expressing the mutant and producing malonate. Certain aspects of the present disclosure arise in part from the discovery that specific amino acids (i.e., F121 and F177) are important for acyl-CoA substrate binding, and that the introduction of specific point mutations increases malonyl-CoA hydrolysis and malonate production. The introduction of mutations F121I or F121L increases the entry of malonyl-CoA into the active site. Similarly, the introduction of mutations F177I or F177L increases the entry of malonyl-CoA into the active site. One or more point mutations at amino acid positions F121 or F177 can be introduced alone or together with the E124 point mutation. In various embodiments, the F121 and / or F177 point mutations are introduced together with the E124 point mutation. In some embodiments, a recombinant host cell expressing an EHD3 F121I or F121L mutant is used to produce malonate. In other embodiments, recombinant host cells expressing EHD3 F177I or F178L mutants are used to produce malonate. In these embodiments, the recombinant host cells can be, without limitation, Escherichia coli or yeast (including but not limited to Saccharomyces cerevisiae or other yeast) host cells.

[0082] The present disclosure also provides mutant EHD3 comprising a mutant mitochondrial targeting sequence, a vector for expressing the mutant, and a host cell expressing the mutant and producing malonate. In a Saccharomyces cerevisiae host, wild-type EHD3 is localized in the mitochondria. Malonyl-CoA is present in both the mitochondria and the cytoplasm; EHD3 catalysis of cytoplasmic malonyl-CoA hydrolysis requires that EHD3 be localized in the cytoplasm. Certain aspects of the present disclosure arise from the discovery that mutations in the EHD3 mitochondrial targeting sequence can increase malonate production. EHD3 amino acids important for mitochondrial targeting include R3, K7, K14, K18, and R22, and mutating one or more of these basic amino acids to a hydrophobic amino acid (i.e., A or V) eliminates mitochondrial targeting. In some embodiments, a recombinant host comprising EHD3 consisting of one or more A or V mutations at an amino acid selected from the group consisting of R3, K7, K14, K18, and R22 is used to produce malonate. In some embodiments, the recombinant host is a yeast strain. In other embodiments, the host is Saccharomyces cerevisiae. In other embodiments, the recombinant host cell contains one or more copies of EHD3 with an unchanged mitochondrial targeting sequence (i.e., wild type) and one or more copies of EHD3 with a mutated mitochondrial targeting sequence. Other examples of mitochondrial targeting sequences for use in this aspect of the disclosure are: WTCOX4, SynA1, SynA2, Syn B1, and Syn B2, as outlined by Allison and Schatz (Allison and Schatz (1986) PNAS 83:9011-9015) for other applications. In other embodiments of the disclosure, peroxisomal targeting signals (e.g., PTS1, PTS2), which most often contain a Ser-Lys-Leu motif, are fused to the C-terminus of malonyl-CoA hydrolase to localize this protein to peroxisomes.

[0083] Thus, in one aspect of the present disclosure, a recombinant host cell comprises a heterologous nucleic acid encoding a mutant Saccharomyces cerevisiae EHD3 that results in increased malonate production relative to a host cell that does not comprise the mutant EHD3. In some embodiments, the mutant EHD3 is heterologously expressed in E. coli. In other embodiments, the mutant EHD3 is heterologously expressed in Saccharomyces cerevisiae. In other embodiments, the mutant EHD3 is heterologously expressed in an oleaginous yeast cell. In some embodiments, the mutant EHD3 contains a point mutation at position E124. In some embodiments, the point mutation at residue E124 is E124A or E124V. In some embodiments, the point mutation at E124 is E124S or E124T. In some embodiments, the point mutation at E124 is E124S. In some embodiments, the point mutation at E124 is a basic amino acid selected from the group consisting of E124H, E124K, and E124R. In some embodiments, the point mutation at E124 is E124H. In some embodiments, the point mutation at residue E124 is E124K. In some embodiments, the point mutation at residue E124 is E124R. In some embodiments, the point mutation at residue E124 is E124N or E124Q. In some embodiments, the point mutation at residue E124 is E124Q. In some embodiments, one or more EHD3 amino acids selected from the group consisting of F121 and F177 are mutated to I or L. In some embodiments, one or more EHD3 amino acids selected from the group consisting of R3, K7, K14, K18, and R22 are mutated to A or V.

[0084] In another aspect of the disclosure, enzymes other than or in addition to EHD3 are also used as malonyl-CoA hydrolases to produce malonate according to the disclosure. In some embodiments, Haemophilus influenzae YciA is heterologously expressed in a heterologous host to produce malonate according to the disclosure (see Zhuang et al., Biochemistry 47:2789-2796 (2008)). In other embodiments, the malonyl-CoA hydrolase is an endogenous acyl-CoA hydrolase of Rattus norvegicus (see Kovachy et al., J. Biol. Chem. 258:11415-11421 (1983)). In other embodiments, the malonyl-CoA hydrolase is an acyl-CoA hydrolase from the mitochondrial protein fraction of brown adipose tissue of golden rats (Mesocricetus auratus) (see Alexson et al., J. Biol. Chem. 263:13564-13571 (1988)).

[0085] Thus, according to the present disclosure, acyl-CoA hydrolases other than or in addition to EHD3 (from Saccharomyces cerevisiae or homologous enzymes from other organisms) can also be used to biosynthesize malonate in a recombinant host. In some embodiments, the recombinant host is Saccharomyces cerevisiae. In other embodiments, the recombinant host is Escherichia coli. In other embodiments, the recombinant host is a yeast other than Saccharomyces cerevisiae as described in further detail below. In various embodiments, the host is modified to express a mutant enzyme selected from the group consisting of: S. albicans EHD3, H. sapiens HIBCH (UniProt: Q6NVY1), A. thaliana CHY1 (UniProt: Q9LKJ1), R. norvegicus HIBCH (UniProt: Q5XIE6), M. musculus HIBCH (UniProt: Q8QZS1), G. gallus HIBCH (UniProt: Q5ZJ60), cattle (B.taurus) HIBCH (UniProt: Q2HJ73), zebrafish (D.rerio) HIBCH (UniProt: Q58EB4), Bacillus cereus (B.cereus) Bch, Pseudomonas aeruginosa (P.aeruginosa) Hich, Escherichia coli YciA, Haemophilus influenzae (H.influenzae) YciA, House mouse ACOT4, House mouse ACOT8, Salmonella enterica (S.enterica) SARI_01218, A.pernix K1, C.hutchinsoniiChut02003666, S.solfataricusP2SS02287, S.acidocaldariusDSM 639Saci_0145, P.aerophilumstr.IM2PAE3404, D.melanogasterCG1635, P.carbinolicusDSM 2380Pcar_1366, A.dehalogenans2CP-C 110, Gallus gallusACOT9 and X.laevisMGC114623.

[0086] Those skilled in the art will appreciate that one or more suitably mutated acyl-CoA hydrolases can be used in accordance with the present disclosure to convert malonyl-CoA to malonate in a host cell. In addition, acyl-CoA hydrolases other than those explicitly disclosed herein can be utilized in mutant or heterologously expressed form, and it will be well understood by those skilled in the art in light of the present disclosure how other appropriate enzymes can be identified, modified, and expressed to obtain the desired malonyl-CoA hydrolase activity as disclosed herein.

[0087] consensus sequence

[0088] The malonyl-CoA hydrolase of the present disclosure includes malonyl-CoA hydrolases homologous to the consensus sequences provided by the present disclosure. As indicated above, any enzyme substantially homologous to the enzymes specifically described herein can be used in the host cells of the present disclosure. When an enzyme exhibits the same target activity, it is homologous to another enzyme ("reference enzyme") and can be used for substantially similar purposes. Typically, homologous enzymes share substantial sequence identity. Each group of homologous enzymes typically has one or more specific amino acids that are conserved between all members of the consensus sequence protein class.

[0089] The percentage of sequence identity of an enzyme relative to a consensus sequence is determined by aligning the enzyme sequence against the consensus sequence. Those skilled in the art will recognize that various sequence alignment algorithms are suitable for aligning an enzyme with a consensus sequence. See, for example, Needleman, SB, et al., "A general method applicable to the search for similarities in the amino acid sequence of two proteins." Journal of Molecular Biology 48(3): 443-53(1970). After aligning the enzyme sequence relative to the consensus sequence, the percentage of the position where the enzyme has a certain amino acid (or underline) described by the same position in the consensus sequence determines the percentage of sequence identity. When a degenerate amino acid (i.e., B, J, O, U, "+") is present in the consensus sequence, any amino acid described by the degenerate amino acid may be present in the enzyme alignment position in the enzyme to be identical to the consensus sequence at the alignment position. When a underline is present in the consensus sequence, the enzyme need not have an amino acid present in the enzyme alignment position to be identical to the consensus sequence at the alignment position.

[0090] The present disclosure provides consensus sequences for identifying and constructing malonyl-CoA hydrolases of the present disclosure. In various embodiments, these malonyl-CoA hydrolase consensus sequences contain active site amino acid residues believed to be necessary for the formation of an oxygen ion pore (although the present disclosure is not limited by any theory of mechanism of action), which is responsible for stabilizing the enolate anion intermediate generated from the malonyl-CoA substrate and amino acid residues important for malonyl-CoA binding as described below. In terms of the ability to hydrolyze malonyl-CoA, the malonyl-CoA hydrolases covered by the consensus sequences provided herein have an enzymatic activity that is the same, or substantially the same, or at least substantially similar to the enzymatic activity of one of the enzymes exemplified herein. The malonyl-CoA hydrolase may exist in nature, or more typically, is an engineered mutant of a wild-type enzyme modified according to the present disclosure to have malonyl-CoA hydrolase activity. A malonyl-CoA hydrolase can be identified or constructed from another enzyme by mutating the sequence of the other enzyme to produce a sequence encompassed by the consensus sequence herein; if an enzyme shares substantial homology with the consensus sequence herein, but has suboptimal (including no) malonyl-CoA hydrolase activity, then according to the present disclosure, it is mutated to conform to the consensus sequence provided herein to provide a malonyl-CoA hydrolase of the present disclosure.

[0091] The present disclosure provides four malonyl-CoA hydrolase consensus sequences: (i) a malonyl-CoA hydrolase based on EHD3 EC 3.1.2.4 (SEQ ID NO:7), (ii) a malonyl-CoA hydrolase based on Bacillus EC 3.1.2.4 (SEQ ID NO:8), (iii) a malonyl-CoA hydrolase based on Pseudomonas EC 3.1.2.4 (SEQ ID NO:9), and (iv) a malonyl-CoA hydrolase based on both Bacillus EC 3.1.2.4 and Pseudomonas EC 3.1.2.4 (SEQ ID NO:10). The consensus sequences provide amino acid sequences with each position identifying an amino acid (if a specific amino acid is identified) or a subset of amino acids that are most likely to be present at a given position in that class of malonyl-CoA hydrolases (if a position is identified as variable). Those skilled in the art will recognize that the fixed and conserved amino acids in these consensus sequences are identical (in the case of fixed amino acids) or identical (in the case of conserved amino acids) to the wild-type sequence on which the consensus sequence is based. The underlining in the consensus sequence indicates that an enzyme suitable for mutation according to the present disclosure may exist in nature, which enzyme may have another amino acid at the underlined position in the sequence, but generally no amino acid is present at the underlined position.

[0092] Consensus sequence of malonyl-CoA hydrolase based on EHD3 EC 3.1.2.4 enzymes

[0093] The present disclosure provides a malonyl-CoA hydrolase consensus sequence (SEQ ID NO:7) based on the EHD3 EC 3.1.2.4 enzyme, and in various embodiments, the malonyl-CoA hydrolase suitable for use in the methods of the present disclosure has at least 63% identity to this malonyl-CoA hydrolase consensus sequence. In various embodiments, the enzyme suitable for mutating the key glutamate residue to X1 to confer malonyl-CoA hydrolase activity according to the methods of the present disclosure has 65%, 70%, 80%, 90% or 95% or more identity to SEQ ID NO:7. Proteins with significant homology to this consensus sequence include UniProt ID: C5DE94 (63% identity), UniProt ID: Q6CJH2 (64% identity), UniProt ID: G2WAE2 (66% identity), UniProt ID: J8Q6P9 (66% identity), UniProt ID: G8C0H0 (68% identity), UniProt ID: C5DX08 (68% identity), UniProt ID: P28817 (69% identity), UniProt ID: A7TTD5 (69% identity), UniProt ID: J7S9J9 (70% identity), UniProt ID: Q6FM09 (71% identity), UniProt ID: I2H4L2 (71% identity), UniProt ID: H2AME2 (73% identity), UniProt ID: G8ZTJ4 (77% identity), UniProt ID: G0W4I8 (77% identity), UniProt ID: G0V818 (78% identity) and UniProt ID: J5S5X3 (79% identity). In some embodiments, a malonyl-CoA hydrolase that is identical to or has greater than 63% identity to the consensus sequence SEQ ID NO: 7 is expressed in a recombinant host cell and used to produce malonate according to the present disclosure.

[0094] Among mutants and wild-type enzymes homologous to this consensus sequence (SEQ ID NO:7), highly conserved amino acids are V101, R110, L114, R116, K119, L120, N121, A122, L123, L135, E137, Y138, K140, S141, S151, R156, C159, G161, G162, D163, V164, A168 , F185, E188, Y189, S190, N192, A196, T197, K200, M206, G208, I209, T210, M211, G 212, G213, G214, V215, G216, H220, P222, F223, R224, T227, E228, T230, M234, P235 , E236, D238, I239, G240, F242, P243, D244, V245, F249, P252, Q263, Y267, L268, T 271, G272, G277, G284, S287, H288, Y289, L298, R301, L302, E304, E333, F334, L352 , V354, I355, F359, L374, F391, L399, K402, S403, S406, N417, D429, L430, T432, A433, E449, F450, K457, L458, K461, W468, L494, T502, Y506, P507, L514, P515 and K561. In various embodiments, the malonyl-CoA hydrolase homologous to this consensus sequence (SEQ ID NO: 7) contains at least 25% of these conserved amino acids, often contains the majority (greater than 50%) of these conserved amino acids, and sometimes contains all of these conserved amino acids.

[0095] Some amino acids in this consensus sequence (SEQ ID NO:7) are essential for activity and are conserved among all members of the class. Malonyl-CoA hydrolases encompassed by the consensus sequence based on EHD3 EC 3.1.2.4 contain six active site residues important for hydrolase activity: (i) three active site amino acid residues (G161, G162, G213) in the consensus sequence are believed to be necessary for the formation of the oxyanion pore (although the present disclosure is not limited by any theory of mechanism of action) that is responsible for stabilizing the enolate anion intermediate generated from the malonyl-CoA substrate; (ii) two amino acid residues (E236, D244) of the consensus sequence are essential for acyl-CoA hydrolysis; and (iii) the amino acid residue at position 188 (of SEQ ID NO:7) is believed to be essential for malonyl-CoA substrate binding. Then, of these six residues, five are present in the consensus sequence (SEQ ID NO: 7) and in all malonyl-CoA hydrolases encompassed by that sequence, and the sixth residue at position 188 (amino acid X1 in the consensus sequence) is selected from the group consisting of polar or positively charged amino acids (R, H, K, S, T, N, Q, Y) and A and D to provide the malonyl-CoA hydrolase of the present disclosure capable of producing malonate in a recombinant host cell. The six essential residues from the consensus sequence (G161, G162, G213, E236, D244, X1188) correspond to G99, G100, G149, E172, D180 and E124 (typically mutated to X1) in Saccharomyces cerevisiae EHD3 used to illustrate the present disclosure in Example 31, respectively.

[0096] Consensus sequence of malonyl-CoA hydrolase based on Bacillus EC 3.1.2.4 enzymes

[0097] The present disclosure provides a malonyl-CoA hydrolase consensus sequence (SEQ ID NO: 8) based on the Bacillus EC 3.1.2.4 enzyme, and in various embodiments, the malonyl-CoA hydrolase suitable for use in the methods of the present disclosure has at least 86% identity to this malonyl-CoA hydrolase consensus sequence. In various embodiments, the enzyme suitable for mutating the key glutamic acid residue to X1 to confer malonyl-CoA hydrolase activity according to the methods of the present disclosure has 90% or 95% or more identity to SEQ ID NO: 8. Proteins with significant homology to this consensus sequence include UniProt ID: C2TX63 (92% identity), UniProt ID: C2UV40 (91% identity), UniProt ID: C2QBT2 (93% identity), UniProt ID: C2XTU0 (93% identity), UniProt ID: C2PVQ0 (93% identity), UniProt ID: C3A5N3 (93% identity), UniProt ID: C2SJV4 (93% identity), UniProt ID: C2Z7U1 (92% identity), UniProt ID: C2VTI4 (97% identity), UniProt ID: B3Z9Y3 (97% identity), UniProt ID: B7JNH7 (97% identity), UniProt ID: Q63BK8 (97% identity), UniProt ID: B0Q3Q4 (97% identity), UniProt ID: B0AQX0 (97% identity), UniProt ID: B3YSW2 (97% identity), UniProt ID: C2NHG5 (97% identity), UniProt ID: B3ZIZ8 (97% identity), UniProt ID: C2QSV2 (97% identity), UniProt ID: C3C255 (97% identity), UniProt ID: B5UZZ1 (96% identity), UniProt ID: C2MKL7 (95% identity), UniProt ID: B9IZZ9 (95% identity), UniProt ID: F0PNG8 (95% identity), UniProt ID: Q738L0 (97% identity), UniProt ID: C2PEV7 (95% identity), UniProt ID: C2YRH7 (96% identity), UniProt ID: Q4MU30 (95% identity), UniProt ID: Q81DR3 (96% identity), UniProt ID: C2W7W8 (89% identity) and UniProt ID: A7GPH6 (86% identity).In various embodiments, a malonyl-CoA hydrolase that is identical or has greater than 86% identity to the consensus sequence SEQ ID NO: 8 is expressed in a recombinant host cell and used to produce malonate according to the present disclosure. The sequences of B9IZZ9 (SEQ ID NO: 46), C3ALI3 (SEQ ID NO: 47), F0PNG8 (SEQ ID NO: 49), Q63BK8 (SEQ ID NO: 51), and Q81DR3 (SEQ ID NO: 52) containing X1 at the position of the key glutamate residue mutated according to the present disclosure to confer malonyl-CoA hydrolase activity are provided in the sequence listing.

[0098] In the presence of this consensus sequence (SEQ ID NO:8) Among the homologous mutants and wild-type enzymes, the highly conserved amino acids are M1, T2, E3, V5, L6, F7, S8, G13, V14, A15, I17, T18, L19, N20, R21, P22, K23, A24, L25, N26, S27, L28, S29, Y30, M32, L33, I36, G37, K39, L40, K41, E42, W43, E44, I49, I52, V53, L54, K55, G56, A57, G58, K60, G61, F62, C63, A64, G65, G66, D67, I68, K69, T70, L71, Y72, E73, A74 , R75, S76, N77, E78, A80, L81, Q82, A84, E85, F87, F88, E90, E91, Y92, I94, D95, T96, Y99, Y101, K103, P104, I105, I106, A107, C108, L109, D110, G111, I112, V113, M114, G115, G116, G117, V118, G119, L120, T121, N122, G123, A 124, R127, I128, V129, T130, T133, K134, W135, A136, M137, P138, E139, M14 0. N141, I142, G143, F144, F145, P146, D147, V148, G149, A150, A151, Y152 , F153, L154, N155, A157, P158, G159, G162, V165, A166, L167, A169, L172, K 173, A174, D176, V177, L178, I180, A182, A183, D184, L192, F195, L196, W2 04. V210, L214, K215, L231, E236, H241, F242, E248, I250, I251, S253, L254 , E255, F261, L269, L270, S271, K272, S273, P274, S276, L277, K278, V279, T280, L281, K282, Q283, G287, K290, S291, E293, C295, F296, A297, T298, D2 99. L300, L302, A303, K304, N305, F306, M307, R308, H309, D311, F312, F31 3. E314, G315, V316, R317, S318, V320, D322, K323, D324, Q325, N326, P327,Y329, K330, Y331, D336, V337, V342, N343, F345, F346, L348 and L349. In various embodiments, malonyl-CoA hydrolases homologous to this consensus sequence (SEQ ID NO: 8) contain at least 25% of these conserved amino acids, often contain a majority (greater than 50%) of these conserved amino acids, and sometimes contain all of these conserved amino acids.

[0099] Some amino acids in this consensus sequence (SEQ ID NO:8) are essential for activity and are conserved among all members of the class. The malonyl-CoA hydrolase encompassed by the consensus sequence based on Bacillus EC 3.1.2.4 contains six active site residues important for hydrolase activity: (i) three active site amino acid residues (G65, G66, G116) in the consensus sequence are believed to be necessary for the formation of the oxyanion pore (although the present disclosure is not limited by any theory of mechanism of action), which is responsible for stabilizing the enolate anion intermediate generated from the malonyl-CoA substrate; (ii) two amino acid residues (E139, D147) of the consensus sequence are essential for acyl-CoA hydrolysis; and (iii) the mutant amino acid (X191) (of SEQ ID NO:8) is believed to be necessary for malonyl-CoA substrate binding. Of these six residues, then, five are present in the consensus sequence (SEQ ID NO: 8) and in all malonyl-CoA hydrolases encompassed by that sequence, and the sixth residue, X191, is essential to provide the malonyl-CoA hydrolases of the present disclosure that are capable of producing malonate in a recombinant host cell. The six essential residues (G65, G66, G116, E139, D147, X191) from the consensus sequence correspond to G65, G66, G116, E139, D147, and E91 (usually mutated to X1) in Bacillus thuringiensis subsp. finitimus (strain YBT-020) F0PNG8, respectively, used to illustrate the present disclosure in Example 31 (see SEQ ID NO: 49 containing the mutation E91S).

[0100] Non-limiting examples of enzymes suitable for malonyl-CoA hydrolysis that are homologous to the consensus sequence (SEQ ID NO: 8) and encoded by cloned or synthetic nucleic acids provided by the present disclosure include mutant enzymes containing at least one mutation illustrated by the mutant enzymes of the group consisting of: Bacillus cereus (strain Q1) B9IZZ9 (E91S), B9IZZ9 (E91A), B9IZZ9 (E91H), B9IZZ9 (E91K), B9IZZ9 (E91R), B9IZZ9 (E91Q), B9IZZ9 (E91T), B9IZZ9 (E91N), B9IZZ9 (E91Y), B9IZZ9 (E91D); Bacillus thuringiensis subsp. T-020)F0PNG8(E91S), F0PNG8(E91A), F0PNG8(E91H), F0PNG8(E91K), F0PNG8(E91R), F0PNG8(E 91Q), F0PNG8(E91T), F0PNG8(E91N), F0PNG8(E91Y), F0PNG8(E91D); Bacillus cereus (strain ATCC14579 / DSM 31)Q81DR3, Q81DR3(E91S), Q81DR3(E91A), Q81DR3(E91H), Q81DR3(E91K), Q81DR3(E91R ), Q81DR3(E91Q), Q81DR3(E91T), Q81DR3(E91N), Q81DR3(E91Y), Q81DR3(E91D); Bacillus cereus ( Strain ZK / E33L) Q63BK8, Q63BK8(E91S), Q63BK8(E91A), Q63BK8(E91H), Q63BK8(E91K), Q63B K8(E91R), Q63BK8(E91Q), Q63BK8(E91T), Q63BK8(E91N), Q63BK8(E91Y), Q63BK8(E91D).

[0101] Consensus sequence of malonyl-CoA hydrolase based on Pseudomonas EC 3.1.2.4 enzyme

[0102] The present disclosure provides a malonyl-CoA hydrolase consensus sequence (SEQ ID NO: 9) based on the Pseudomonas EC 3.1.2.4 enzyme. In various embodiments, a malonyl-CoA hydrolase suitable for use in the methods of the present disclosure has at least 75% identity to this malonyl-CoA hydrolase consensus sequence SEQ ID NO: 9. In various embodiments, an enzyme suitable for mutating the key glutamic acid residue to X1 to confer malonyl-CoA hydrolase activity according to the present disclosure has 80%, 90% or 95% or more identity to SEQ ID NO: 9. Proteins with significant homology to this consensus sequence include: UniProt ID: F5KBQ4 (80% identity), UniProt ID: A6VAN3 (81% identity), UniProt ID: A4XS22 (81% identity), UniProt ID: F6AA82 (75% identity), UniProt ID: E2XN63 (84% identity), UniProt ID: F2KE35 (85% identity), UniProt ID: C3KDS5 (83% identity), UniProt ID: F8G3B7 (86% identity), UniProt ID: G8PYD2 (85% identity), UniProt ID: Q4KGS1 (82% identity), UniProt ID: Q3KGL5 (85% identity), UniProt ID: B0KV51 (86% identity), UniProt ID: B1J4J2 (86% identity), UniProt ID: A5W8H3 (86% identity), UniProt ID: Q88N06 (86% identity), UniProt ID: Q1I5T5 (84% identity), UniProt ID: F8H1A4 (77% identity), UniProt ID: A4VIV7 (77% identity), and UniProt ID: Q9I5I5 (81% identity). In some embodiments, a malonyl-CoA hydrolase that is identical to or has greater than 75% identity to the consensus sequence SEQ ID NO: 9 is expressed in a recombinant host cell and used to produce malonate according to the present disclosure. Included in the sequence listing are the sequences of A4XS22 (SEQ ID NO: 45), F6AA82 (SEQ ID NO: 50), and E2XN63 (SEQ ID NO: 48), each of which contains an X1 at the position of a key glutamic acid residue mutated according to the present disclosure.

[0103] The highly conserved amino acids in this consensus sequence (SEQ ID NO:9) are M1, E6, G13, R15, I16, A19, L21, D22, A23, L27, N28, A29, L30, L32, P33, M34, I35, L38, W45, A46, C53, V54, L56, R57, G58, N59, G60, K62, A63, F64, C65, A66, G67, G68, V70, L73, C77, P81, G82, P85, L87, A88, F91, F92, Y96, R97, L98, H103, P106, K107, P108, C111, W112, H114, G115, V117, G119, G120, G121, M122, G123, L124, Q126, R1 31. I132, V133, T134, P135, R138, L139, M141, P142, E143, I146, G147, L148, D151, V152, G153, S155, F157, L158, R1 60. P162, G163, L165, G166, L167, F168, L171, N177, D180, A181, D183, L184, L186, A187, D188, R189, Q195, Q196, L 199, L203, Q205, N207, W208, E210, Q215, L216, S218, L219, A222, P232, L237, R239, R240, D244, L247, D248, A258, D 328, A329, R330, D332, P335, W337, W339, P346, A352, H353, and F354. In various embodiments, malonyl-CoA hydrolases homologous to this consensus sequence (SEQ ID NO:9) contain at least 25%, often contain a majority (greater than 50%), and sometimes contain all of these conserved amino acids.

[0104] Some amino acids in this consensus sequence (SEQ ID NO:9) are essential for activity and are conserved among all members of the class. The malonyl-CoA hydrolase encompassed by the consensus sequence based on Pseudomonas EC 3.1.2.4 contains six conserved active site residues essential for hydrolase activity: (i) three active site amino acid residues (G67, G68, G120) in the consensus sequence are believed to be essential for the formation of the oxyanion pore (although the present disclosure is not limited by any theory of mechanism of action), which is responsible for stabilizing the enolate anion intermediate generated from the acyl-CoA substrate; (ii) two amino acid residues (E143, D151) of the consensus sequence are believed to be essential for acyl-CoA hydrolysis; and (iii) amino acid X195 (of SEQ ID NO:9) is believed to be essential for malonyl-CoA substrate binding. Of these six residues, then, five are present in the consensus sequence (SEQ ID NO: 9) and in all malonyl-CoA hydrolases encompassed by that sequence, and the sixth residue, X195, is necessary to provide a malonyl-CoA hydrolase capable of producing malonate in a recombinant host cell of the present disclosure. In various embodiments of the present disclosure, the key wild-type glutamate residue (E95) is (has been) mutated to a polar or positively charged amino acid (i.e., R, H, K, S, T, N, Q, Y) or A or D to produce X195 and provide a malonyl-CoA hydrolase capable of producing malonate in a recombinant host cell of the present disclosure. In some embodiments of the present disclosure, the amino acid E95 is (has been mutated to) an amino acid selected from the group consisting of K, S, T, N, Y, A and D. In some embodiments of the present disclosure, the amino acid E95 is S or N. The six essential residues from the consensus sequence (G67, G68, G120, E143, D151, X195) correspond to G67, G68, G120, E143, D151 and E95 (generally mutated to X1) in Pseudomonas fulva (12-X) F6AA82-2 used to illustrate the present disclosure in Example 31 (see SEQ ID NO:50 containing mutation E95S / Q348A).

[0105] Non-limiting examples of enzymes suitable for malonyl-CoA hydrolysis that are homologous to the consensus sequence (SEQ ID NO:9) and encoded by cloned or synthetic nucleic acids provided by the present disclosure include mutant enzymes containing at least one mutation illustrated by the mutant enzymes consisting of the group consisting of: Pseudomonas flavescentis (strain 12-X) F6AA82(E95S), F6AA82(E95N), F6AA82(E95A), F6AA82(E95H), F6AA82(E95K), F6AA82(E95R), F6AA82(E95Q), F6AA82(E95D), F6AA82(E95T), F6AA82(E95Y, as illustrated in Example 43; Pseudomonas fluorescens WH6 E2XN63(E95S), E2XN63(E95N), E2XN63(E95A), E2XN63(E95H), E2XN63(E95K), E2XN63(E95R), E2XN63(E95Q), E2XN63(E95D), E2XN63(E95T), E2XN63(E95Y); Pseudomonas mendocinosae mendocina) (strain ymp) A4XS22(E95S), A4XS22(E95N), A4XS22(E95A), A4XS22(E95H), A4XS22(E95K), A4X S22(E95R), A4XS22(E95Q), A4XS22(E95D), A4XS22(E95T), E2XN63(E95Y) and F6A-6(E95N, V152A and F304).

[0106] In one embodiment, the malonyl-CoA hydrolase is a Pseudomonas fruticosa (strain 12-X) UniProt ID: F6AA82 protein comprising one or more of the following mutations: E95N, V152A, F304R, and Q348A. In another embodiment, the malonyl-CoA hydrolase is a Pseudomonas fruticosa (strain 12-X) UniProt ID: F6AA82 protein comprising one or more mutations of the following amino acids: E89R, D99C, and D99E. In various embodiments of the present disclosure, the malonyl-CoA hydrolase is F6AA82 (E95S) from Pseudomonas fruticosa (strain 12-X), E2XN63 (E95S) from Pseudomonas fluorescens WH6, A4XS22 (E95S) from Pseudomonas mendocina (strain ymp), as described in Example 31.

[0107] As described in Example 43, the F6AA82 E95X1 mutation produces malonyl-CoA hydrolase activity. F6AA82 proteins containing mutations E95S, E95Y, E95T, E95N, E96K, E95A and E95D produce significantly more malonate (t-test, p<0.05) than wild-type F6AA82 proteins. F6AA82 proteins containing these mutations are suitable for use as malonyl-CoA hydrolases and for producing malonate. In F6AA82 enzymes containing X1 mutations that confer malonyl-CoA hydrolase activity, mutations E95S or E95N are preferred. In F6AA82 enzymes containing X1 mutations that confer malonyl-CoA hydrolase activity, mutations E95A, E95T, E95K, E95Y and E95D are suitable. In various embodiments of the present disclosure, the malonyl-CoA hydrolase is F6AA82(E95S). In other embodiments of the present disclosure, the malonyl-CoA hydrolase is F6AA82(E95N). F6AA82 proteins containing mutations E95H, E95Q or E95R did not result in increased malonate production under the test conditions employed as described in the Examples.

[0108] When an enzyme having substantial homology to the consensus sequence herein has suboptimal or no malonyl-CoA hydrolase activity, then according to the present disclosure, it can be mutated to conform to the consensus sequence provided herein to provide a malonyl-CoA hydrolase of the present disclosure. For example, protein A5W8H3 shows 86% identity to malonyl-CoA hydrolase consensus SEQ ID NO: 9, but does not exhibit malonyl-CoA hydrolase activity (Example 31). One or more amino acids that differ between A5W8H3 and consensus SEQ ID NO: 9 can be mutated to introduce malonyl-CoA hydrolase activity. Specifically, the A5W8H3 mutations T2N, C5F, V7J, L8B, G10U, D12B, P24J, A26U, N31U, Q41B, A72B, A74O, Q75J, S83J, S90B, A94J, F100B, A101B, N129U, A159U, F169O, P175B, G185J, G192B, A198J, A213J, N21 7B, Q224J, C228B, A229J, W236O, H241Q, E242B, Q245J, A252O, R261A, Q264J, D272B, G274A, Q275B, Y297B, Q302J, Q305B, M310O, N333B, A347J, A355J, A357O and / or G368U can be used to confer malonyl-CoA hydrolase activity.

[0109] In some embodiments, the malonyl-CoA hydrolase has at least 75% identity to the malonyl-CoA hydrolase consensus sequence SEQ ID NO: 57, wherein X represents any naturally occurring amino acid present at the specified position. In some embodiments, X is X1, which represents any amino acid selected from A, D, R, H, K, S, T, N, Q or Y. The consensus sequence is based on the Pseudomonas EC 3.1.2.4 enzyme (such as the malonyl-CoA hydrolase consensus sequence SEQ ID NO: 9). Without being bound by theory, certain factors in generating SEQ ID NO: 57 include (1) amino acid residues predicted to enhance protein stability; (2) alignment of the protein structure with binding substrates similar to malonyl-CoA. The consensus sequence SEQ ID The conserved amino acids in NO:57 are M1, E6, R8, H12, G13, R15, I16, G17, I18, A19, S20, L21, A23, P24, L27, N28, A29, L30, L32, P33, M34, I35, L38, L42, W45, A46, D48, I51, A52, C53, V54, L56, R57, G58, G60, K62, A63, F64, C65, A66, G67 ,D69,V70,V71,L73,A74,K76,C77,L78,P81,G82,A84,P85,L87,A88,F91,F92,A93,Y97,R98,L102,H103, T104,Y105,P106,K107,P108,L109,I110,C111,W112,A113,H114,G115,H116,V117,L118,G119,G120,G1 21,M122,G123,L124,L125,Q126,G127,A128,R131,I132,V133,T134,P135,S136,S137,R138,L139,A140 ,M141,P142,E143,I144,I146,G147,L148,F149,P150,G153,G154,S155,H156,F157,L158,R160,L161,P 162,G163,K164,L165,G166,L167,F168,F169,G170,L171,T172,A173,S174,N177,A178,R179,D180,A18 1,L182,D183,L184,N185,L186,A187,D188,R189,F190,L191,L192,D193,Q195,Q196,L199,I200,D201m G202,L203,I204,Q205,L206,N207,W208,R209,E210,P212,Q215,L216,H217,S218,L219,L220,K221,A222,L223,Q226,A227,E230,L23 1,P232,A233,A234,Q235,W236,L237,P238,R239,R240,L243,D244,L246,L247,D248,Q 249,T251,S255,W256,A258,L259,L262,D265,D267,L269,A271,A273,A274,K275,T276 ,M277,G280,S281,P282,L283,T284,G285,L287,V288,W289,G290,Q291,I292,R294,A2 95,R296,H297,L298,S299,L300,A301,Q302,V303,X304,Q305,M306,E307,Y308,M310, L312,N313,C314,C315,R316,H317,P318,E319,F320,A321,E322,G323,V324,R325,A32 6, R327, L328, I329, D330, K331, D332, P335, W337, H338, W339, P340, D341, V342, V345, P346, Q348, V349, I350, A352, H353, F354, P356, D358, D359, H360, L362, A363, D364 and L365. In some embodiments, the malonyl-CoA hydrolase provided or utilized herein has at least 80% identity to the consensus sequence SEQ ID NO: 57. In one embodiment, the malonyl-CoA hydrolase provided or utilized herein has at least 80% identity to the consensus sequence SEQ ID NO: 57 and comprises the following sequence SEQ ID NO: 58:,

[0110] MXVXFEERAS XHGXRIGIAS LDAPASLNAL SLPMIXALXD XLXXWAXDAX

[0111] IACVLLRGNG SKAFCAGGDV VXLAXKCLXX PGXAPXLAXX FFAXXYRLXX

[0112] XLHTYPKPLI CWAHGHVLGG GMGLLQGAXX RIVTPSSRLA MPEIXIGLFP

[0113] XXGGSHFLXR LPGKLGLFFG LTASXXNARD ALDLNLADRF LLDXQQXXLI

[0114] DGLIQLNWRE XPXXQLHSLL KALXXQAXXE LPAAQWLPRR XXLDXLLDQX

[0115] TXXXSWXALX XLXXDXDXLX AXAAKTMXXG SPLTGXLVWG QIXRARHLSL

[0116] AQVXQMEYXM XLNCCRHPEF AEGVRARLID KDXXPXWHWP DVXXVPXQVI

[0117] AAHFXPLDDH XLADLA.

[0118] In some embodiments, the malonyl-CoA hydrolase provided or utilized herein has at least 85% identity to the consensus sequence SEQ ID NO: 57. In one embodiment, the malonyl-CoA hydrolase provided or utilized herein has at least 85% identity to the consensus sequence SEQ ID NO: 57 and comprises the following sequence, SEQ ID NO: 59:

[0119] MXVXFEERAS XHGXRIGIAS LDAPASLNAL SLPMIXALXD XLXXWAXDAX

[0120] IACVLLRGNG SKAFCAGGDV VXLAXKCLXX PGXAPXLAXX FFAXXYRLXX

[0121] XLHTYPKPLI CWAHGHVLGG GMGLLQGAXX RIVTPSSRLA MPEIXIGLFP

[0122] DVGGSHFLSR LPGKLGLFFG LTASXLNARD ALDLNLADRF LLDXQQXALI

[0123] DGLIQLNWRE QPXXQLHSLL KALEQQAXXE LPAAQWLPRR XXLDXLLDQA

[0124] TLXXSWQALX XLXXDXDALL AXAAKTMXGG SPLTGHLVWG QIXRARHLSL

[0125] AQVXQMEYXM SLNCCRHPEF AEGVRARLID KDHAPHWHWP DVXXVPXQVI

[0126] AAHFXPLDDH XLADLA.

[0127] In some embodiments, the malonyl-CoA hydrolase provided or utilized herein has at least 90% identity to the consensus sequence SEQ ID NO: 57. In some embodiments, the malonyl-CoA hydrolase provided or utilized herein has at least 90% identity to the consensus sequence SEQ ID NO: 57 and comprises the following sequence, SEQ ID NO: 60:

[0128] MXVTFEERAS XHGYRIGIAS LDAPASLNAL SLPMIXALXD XLXXWAXDAX IACVLLRGNG

[0129] SKAFCAGGDV VXLAXKCLAS PGEAPXLAXX FFARXYRLDH XLHTYPKPLI CWAHGHVLGG

[0130] GMGLLQGAXI RIVTPSSRLA MPEISIGLFP DVGGSHFLSR LPGKLGLFFG LTASXLNARD

[0131] ALDLNLADRF LLDXQQXALIDGLIQLNWRE QPXXQLHSLL KALEQQAXXE LPAAQWLPRR

[0132] XXLDXLLDQA TLXLSWQALA XLXXDXDALL AXAAKTMXGG SPLTGHLVWG QIRRARHLSL

[0133] AQVFQMEYXM SLNCCRHPEF AEGVRARLID KDHAPHWHWP DVNXVPEQVIAAHFAPLDDHPLADLA.

[0134] In some embodiments, the malonyl-CoA hydrolase provided or utilized herein has at least 95% identity to the consensus sequence SEQ ID NO: 57. In some embodiments, the malonyl-CoA hydrolase provided or utilized herein has at least 95% identity to the consensus sequence SEQ ID NO: 57 and comprises the following sequence SEQ ID NO: 61:

[0135] MNVTFEERAS XHGYRIGIAS LDAPASLNAL SLPMIXALXD RLXAWAEDAX

[0136] IACVLLRGNG SKAFCAGGDV VQLAKKCLAS PGEAPELAER FFARXYRLDH

[0137] XLHTYPKPLI CWAHGHVLGG GMGLLQGAXI RIVTPSSRLA MPEISIGLFP

[0138] DVGGSHFLSR LPGKLGLFFG LTASPLNARD ALDLNLADRF LLDTQQDALI

[0139] DGLIQLNWRE QPXXQLHSLL KALEQQAXSE LPAAQWLPRR XXLDALLDQA

[0140] TLPLSWQALA SLXXDXDALL AKAAKTMXGG SPLTGHLVWG QIRRARHLSL

[0141] AQVFQMEYXM SLNCCRHPEF AEGVRARLID KDHAPHWHWP DVNQVPEQVIAAHFAPLDDHPLADLA.

[0142] Consensus sequence of malonyl-CoA hydrolase based on bacterial EC 3.1.2.4 enzymes

[0143] Although Bacillus and Pseudomonas are evolutionarily distant (i.e., Bacillus is Gram-positive and Pseudomonas is Gram-negative), surprisingly, there is significant sequence conservation between the Bacillus EC 3.1.2.4 enzyme and the Pseudomonas EC 3.1.2.4 enzyme. The present disclosure provides a malonyl-CoA hydrolase consensus sequence (SEQ ID NO: 10) based on these bacterial EC 3.1.2.4 acyl-CoA hydrolases. Malonyl-CoA hydrolases encompassed by this consensus sequence typically have at least 25% (or most or all) of the highly conserved amino acids from this sequence selected from the group consisting of: L53, L59, N60, L62, M66, L88, F97, C98, A99, G100, G101, F124, F125, Y129, K140, P141, G148, G152, G153, G154, G156, L157, T167, M174, P175, E176, 1179, G180, D184, V185, G186, L191, L210, D219, A226, P333, N364, F375, E377, D385, and P390. Suitable malonyl-CoA hydrolases of the present disclosure that are homologous to this consensus sequence include the active site amino acids (G100, G101, G153, E176 and D184) of the consensus sequence that are necessary for malonyl-CoA hydrolysis, as well as X1128, wherein the key wild-type glutamate residue (E128) is mutated to a polar or charged amino acid (i.e., R, H, K, S, T, N, Q, Y) or D or A, and are capable of producing malonate in a recombinant host cell.

[0144] Malonyl-CoA hydrolase based on malonyl-CoA:ACP transacylase

[0145] In other embodiments of the present disclosure, the malonyl-CoA hydrolase is a mutant malonyl-CoA:ACP transacylase (EC 2.3.1.39). The present disclosure provides an E. coli FabD sequence (SEQ ID NO: 53), and in various embodiments, the malonyl-CoA hydrolase suitable for use in the methods of the present disclosure has at least 50%, 60%, 70%, 80%, 90%, 95% or more sequence identity when aligned relative to SEQ ID NO: 53 and contains one or more of the following amino acid mutations at the aligned positions: S92C, H201N, R117D, R117E, R117N, R117Y, R117G, R117H, Q11D, Q11E, Q11N, Q11Y, Q11G, Q11H, L93A, L93V, L93I, L93F, L93S, L93G.

[0146] In some embodiments of the present disclosure, the malonyl-CoA hydrolase is a mutant E. coli FabD malonyl-CoA:ACP transacylase (for the wild-type sequence, see SEQ ID NO:53) having one or more mutations selected from the group consisting of S92C, H201N, R117D, R117E, R117N, R117Y, R117G, R117H, Q11D, Q11E, Q11N, Q11Y, Q11G, Q11H, L93A, L93V, L93I, L93F, L93S, L93G. Example 35 illustrates recombinant yeast cells that express a mutant E. coli FabD enzyme containing one of the following mutation combinations and produce malonate at a level higher than wild-type yeast without the mutant FabD enzyme: S92C / L91V / R117H, L91I / R117Y / A246E, Q80L / L91S / R117G and L91I / R117Y.

[0147] Section 3: Expression Vectors

[0148] In various aspects of the disclosure, recombinant host cells have been modified by "genetic engineering" to produce recombinant malonyl-CoA hydrolase and malonate. The host cell is typically engineered by recombinant DNA technology to express a heterologous nucleic acid encoding a malonyl-CoA hydrolase, a mutated form of a naturally occurring acyl-CoA hydrolase or transacylase, or a non-naturally occurring malonyl-CoA hydrolase prepared according to a consensus sequence provided herein, or a naturally occurring acyl-CoA hydrolase having malonyl-CoA hydrolase activity that is overexpressed in a cell in which it naturally occurs or heterologously expressed in a cell in which it does not naturally occur.

[0149] The nucleic acid constructs of the present disclosure include expression vectors containing nucleic acids encoding one or more malonyl-CoA hydrolases. The nucleic acid encoding the enzyme is operably linked to a promoter and optionally other control sequences so that when cultured under suitable conditions, the subject enzyme is expressed in a host cell containing the expression vector. The promoter and control sequences employed depend on the host cell selected for the production of malonate. Therefore, the present disclosure not only provides expression vectors, but also provides nucleic acid constructs for constructing expression vectors. Methods for designing and preparing nucleic acid constructs and expression vectors are generally well known to those skilled in the art and are therefore only briefly reviewed herein.

[0150] Nucleic acids encoding malonyl-CoA hydrolase can be prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis and cloning. In addition, nucleic acid sequences used in the present disclosure can be obtained from commercial suppliers that provide de novo synthesis of nucleic acids.

[0151] Nucleic acids encoding required enzymes can be incorporated into expression vectors by known methods, including, for example, using restriction enzymes to cleave specific sites in expression vectors such as plasmids, thereby producing expression vectors of the present disclosure. Some restriction enzymes produce single-stranded ends, which can be annealed to obtain nucleic acid sequences with sequences complementary to the ends of the cracked expression vectors, or synthesized to have sequences complementary to the ends of the cracked expression vectors. The ends are then covalently linked using appropriate enzymes such as DNA ligase. DNA connectors can be used to promote the connection of nucleic acid sequences to expression vectors.

[0152] It is also possible to combine a group of individual nucleic acid sequences by utilizing methods based on polymerase chain reaction (PCR) known to those skilled in the art. For example, each desired nucleic acid sequence can be initially produced in a separate PCR. Thereafter, specific primers are designed so that the ends of the PCR products contain complementary sequences. When mixing, denaturing and reannealing PCR products, each chain with a matching sequence at their 3' ends overlaps and can serve as primers for each other. Extending this overlap by DNA polymerase will produce molecules that the original sequence is "spliced" together. In this way, a series of individual nucleic acid sequences can be joined and subsequently transduced into a host cell simultaneously. Therefore, the respective expression of multiple nucleic acid sequences is achieved.

[0153] Typical expression vectors contain a desired nucleic acid sequence preceded by one or more control sequences or regulatory regions and optionally followed by one or more control sequences or regulatory regions, including a promoter and (when the gene product is a protein) a ribosome binding site, such as a nucleotide sequence that is usually 3-9 nucleotides in length and is usually located 3-11 nucleotides upstream of the start codon preceding the coding sequence, which is followed by a transcription terminator in the case of E. coli or other prokaryotic hosts. See Shine et al., Nature 254:34 (1975) and Steitz, Biological Regulation and Development: Gene Expression (RF Goldberger, ed.), Vol. 1, p. 349 (1979) Plenum Publishing, NY. In the case of eukaryotic hosts such as yeast, typical expression vectors contain a desired nucleic acid coding sequence preceded by one or more regulatory regions and a Kozak sequence for initiating translation and followed by a terminator. See Kozak, Nature 308:241-246 (1984).

[0154] Regulatory regions or control sequences include, for example, those containing promoters and operators. The promoter is operably linked to the desired nucleic acid coding sequence, thereby initiating transcription of the nucleic acid sequence by RNA polymerase. The operator is a nucleic acid sequence adjacent to the promoter, which contains a protein binding domain in which a transcription factor can bind. Transcription factors activate or repress transcription from the promoter. In this way, control of transcription is based on the specific regulatory region used and the presence or absence of the corresponding transcription factor. Non-limiting examples for prokaryotic expression include lactose promoters (when contacted with lactose, LacI repressor protein changes conformation, thereby preventing LacI repressor protein from binding to the operator) and tryptophan promoters (when complexed with tryptophan, TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, TrpR repressor protein has a conformation that does not bind to the operator). Non-limiting examples of promoters for eukaryotic expression include pTDH3, pTEF1 (as described in Example 31), pTEF2, pRNR2, pRPL18B, pREV1, pGAL1, pGAL10, pGAPDH, pCUP1, pMET3, pPGK1, pPYK1, pHXT7, pPDC1, pFBA1, pTDH2, pPGI1, pPDC1, pTPI1, pENO2, pADH1, and pADH2. As described in Example 44, promoters for genes HSP150, PGK1, PHO5, SCT1, PRB1, TPI1, ACH1, HXK2, ACO1, JEN1, MDH2, POX1, CIT1, ALD4, ADH1, TDH3, ADH2, and SDH1 from Saccharomyces cerevisiae strain BY4741 were demonstrated for production of malonate according to the present disclosure. As will be appreciated by those of ordinary skill in the art, a variety of expression vectors and components thereof may be used in the present disclosure.

[0155] Although any suitable expression vector can be used and has the desired sequence, readily available expression vectors include, without limitation: plasmids such as pESC, pTEF, p414CYC1, p414GALS, pSC101, pBR322, pBBR1MCS-3, pUR, pEX, pMR100, pCR4, pBAD24, pUC19, pRS series; and bacteriophages such as M13 phage and lambda phage. Of course, the expression vectors may only be suitable for specific host cells or for expressing specific malonyl-CoA hydrolases. However, one of ordinary skill in the art can easily determine by routine experimentation whether any particular expression vector is suitable for any given host cell or protein. For example, an expression vector can be introduced into a host cell, followed by monitoring the viability of the host cell and the expression of the sequences contained in the vector. In addition, reference can be made to the text and literature describing expression vectors and their suitability for any specific host cell. In addition to using expression vectors, strains in which expression cassettes are directly integrated into the host genome are also constructed.

[0156] The expression vector is introduced or transferred into the host cell, for example, by transduction, transfection or transformation. The methods for introducing the expression vector into the host cell are well known to those of ordinary skill in the art. For example, one method for transforming E. coli with the expression vector involves calcium chloride treatment, wherein the expression vector is introduced by calcium precipitation.

[0157] For identifying whether a nucleic acid has been successfully introduced into or entered a host cell, a variety of methods are available. For example, a culture of a potentially transformed host cell can be separated into individual cells using a suitable dilution method and thereafter grown and tested individually for expression of the desired gene product of the gene contained in the introduced nucleic acid. For example, commonly used conventions involve selecting cells based on antibiotic resistance conferred by antibiotic resistance conferring genes in expression vectors, such as beta lactamase (amp), aminoglycoside phosphotransferase (neo), and hygromycin phosphotransferase (hyg, hph, hpt) genes.

[0158] Typically, the host cells of the present disclosure will have been transformed with at least one expression vector. When only a single expression vector is used, the vector will typically contain a malonyl-CoA hydrolase gene. Once the host cell has been transformed with the expression vector, the host cell is cultured in a suitable medium containing a carbon source such as a sugar (e.g., glucose). While the host cell is being cultured, expression of an enzyme for producing malonate occurs. Once expressed, the enzyme catalyzes the hydrolysis of the thioester bond of malonyl-CoA, thereby releasing malonate and CoA.

[0159] If the host cell of the present disclosure will include more than one heterologous gene, then the multiple genes can be expressed from one or more vectors. For example, a single expression vector may contain one, two or more genes encoding one, two or more malonyl-CoA hydrolase and / or other proteins that provide a useful function (e.g., malonate yield, titer and / or productivity improvement). The heterologous genes may be contained in a vector that replicates in an episomal form or in a vector that is integrated into the host cell genome, and when more than one vector is employed, then all vectors may replicate in an episomal form (outside the chromosome), or all vectors may integrate or some vectors may integrate and some vectors may replicate in an episomal form. Chromosomal integration is typically used for cells that will undergo continued proliferation, such as cells used to produce malonate for industrial applications. Example 45 illustrates the benefits of genomic integration of a malonyl-CoA hydrolase gene provided by the present disclosure; in that example, a single integrated copy of the gene was shown to result in a malonate titer that is higher than the titer obtained when the same gene is expressed from a plasmid. This example is also used to demonstrate that regulating the malonate production provided by the present disclosure in a manner that increases the copy number of the malonyl-CoA hydrolase encoding gene per cell results in an increase in malonate in the fermentation medium. Although a "gene" is typically composed primarily of a single promoter and a single coding sequence, in certain host cells, two or more coding sequences may be controlled by one promoter in an operon. In some embodiments, a two-operon or three-operon system is used.

[0160] In some embodiments, relative to a certain reference sequence, the coding sequence used has been modified to reflect the codon preference of the selected host cell. Codon usage tables of numerous organisms are readily available and can be used to guide sequence design. The use of universal codons of a given host organism will generally improve the translation of the target sequence in the host cell. As a non-limiting example, in some embodiments, the subject nucleic acid sequence will be modified for yeast codon preference (see, e.g., Bennetzen et al., J.Biol.Chem.257:3026-3031 (1982)). In some embodiments, the nucleotide sequence will be modified for Escherichia coli codon preference (see, e.g., Nakamura et al., Nucleic Acids Res.28:292 (2000)). In other embodiments, the nucleotide sequence is modified to include codons optimized for Saccharomyces cerevisiae codon preference (see Example 42).

[0161] Nucleic acid can be prepared by various conventional recombinant techniques. In short, the subject nucleic acid can be prepared from all genomic DNA fragments, cDNA and RNA directly extracted from cells or recombined by various amplification methods including but not limited to PCR and rt-PCR. The subject nucleic acid can also be prepared by direct chemical synthesis.

[0162] Numerous technologies can be used to increase (or reduce) the level of nucleic acid transcription in the host microorganism.For example, the copy number of nucleic acid can be increased by using an expression vector comprising a nucleic acid sequence of a higher copy number, or by making multiple copies of the desired nucleic acid be integrated into the genome of the host microorganism, as described in Example 45. The limiting examples of making the desired nucleic acid sequence integrated on the host chromosome include recA-mediated recombination, lambda phage recombinase-mediated recombination and transposon insertion. The nucleic acid transcript level can be increased by changing the order of the coding region on the polycistronic mRNA, or the polycistronic operon can be decomposed into multiple polycistronic or monocistronic operons with its own promoter each. The RNA level can be increased (or reduced) by increasing the strength of the promoter operably connected to the protein coding region. The illustrative techniques for providing malonate production for plasmid design and assembly are provided in Examples 1, 3, 31 and 35.

[0163] The translation level of the desired polypeptide sequence in the host microorganism can also be increased in many ways. Non-limiting examples include increasing mRNA stability, modifying ribosome binding site (or Kozak) sequence, improving the distance or sequence between the start codon of the ribosome binding site (or Kozak sequence) and the nucleic acid sequence encoding the desired polypeptide, modifying the 5' intercistronic region of the start codon of the nucleic acid sequence encoding the desired polypeptide, stabilizing the 3' end of the mRNA transcript, improving the codon usage of the polypeptide, and changing the expression of the low usage / rare codon tRNA used in the biosynthesis of the polypeptide. Preferred codons and low usage / rare codon tRNA can be determined based on sequence analysis of the gene derived from the host microorganism.

[0164] The half-life or stability of the polypeptide can be increased by mutating the nucleic acid sequence encoding the desired polypeptide, thereby resulting in the modification of the desired polypeptide sequence relative to the control polypeptide sequence. When the modified polypeptide is an enzyme, the activity of the enzyme in the host can be changed due to the following factors: increased solubility in the host cell; improved function at the desired pH; removal of domains that inhibit enzyme activity; improved kinetic parameters for the desired substrate (lower Km or higher Kcat value); removal of allosteric regulation by intracellular metabolites, etc. The altered / modified enzyme can also be isolated by inducing random mutations in the enzyme so that the altered / modified enzyme can be expressed from a free vector or from a recombinant gene integrated into the genome of the host microorganism.

[0165] Section 4: Recombinant Host Cells

[0166] In one aspect, the present disclosure provides a recombinant host cell suitable for biological production of malonate. Any suitable host cell can be used to implement the method of the present disclosure. In some embodiments, the host cell is a recombinant host microorganism, wherein the nucleic acid molecule has been inserted, deleted or modified (i.e., mutated; for example, by the insertion, deletion, substitution and / or inversion of nucleotides) to produce malonate, or to increase the yield, titer and / or productivity of malonate relative to a "control cell" or "reference cell". "Control cells" can be used for comparison purposes, and are typically wild-type or recombinant parent cells that do not contain one or more modifications performed on the target host cell.

[0167] In an important embodiment, the present disclosure provides a recombinant yeast cell suitable for producing malonate at a level sufficient for subsequent purification and use as described herein. Yeast host cells are excellent host cells for constructing recombinant metabolic pathways comprising heterologous enzymes that catalyze the production of small molecule products. There are established molecular biology techniques and nucleic acids encoding genetic elements necessary for constructing yeast expression vectors, the genetic elements including but not limited to promoters, replication origins, antibiotic resistance markers, nutrient deficiency markers, terminators, etc. Secondly, the technology for integrating nucleic acids into yeast chromosomes has been clearly established. Yeast also provides many advantages as an industrial fermentation host. Yeast can tolerate high concentrations of organic acids and maintain cell viability at low pH and can grow under both aerobic and anaerobic culture conditions, and there are established fermentation broths and fermentation schemes. The ability of strains to proliferate and / or produce desired products at low pH provides many advantages about the present disclosure. First, this feature provides tolerance to the environment created by the production of malonate. Second, from a process point of view, the ability to maintain a low pH environment will limit the number of organisms that can contaminate and destroy batches. Third, this feature also eliminates or at least reduces the need to add additional acid to aid in the purification of malonic acid by some of the methods provided by the present disclosure (see Example 37).

[0168] In some embodiments of the present disclosure, the recombinant host cell comprising a heterologous nucleic acid encoding a malonyl-CoA hydrolase is a eukaryotic organism. In various embodiments, the eukaryotic organism is a yeast selected from a non-limiting list of genera: Candida, Cryptococcus, Hansenula, Ississa, Kluyveromyces, Komagataella, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, or Yarrowia species.In various embodiments, the yeast is of a species selected from the group consisting of Candida albicans, Candida ethanolica, Candida krusei, Candida methanosorbosa, Candida sonorensis, Candida tropicalis, Cryptococcus curvatus, Hansenula polymorpha, Issatchenkia orientalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Komagataella pastoris, Lipomyces starkeyi, Pichia angusta, Pichia aserticola, Pichia galeiformis, Pichia kudamae, Pichia kudriazevii, Pichia membranaefaciens, Pichia methanolica, Pichia pastoris, Pichia salictaria, Pichia stipitis, Pichia athermotolerans, Pichia trehalophila, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodotorula graminis, Saccharomyces bayanus, Saccharomyces boulardi, Saccharomyces cerevisiae, Saccharomyces kluyveri, and Yarrowia lipolytica. One skilled in the art will recognize that this list is intended to broadly encompass yeast, including both lipidic and non-lipidic strains.Examples 46 and 47 illustrate the use of Pichia kudriavzevii strain Y-134 in accordance with the present disclosure.

[0169] The present disclosure provides alternative recombinant host cells for biological production of malonate. Illustrative examples include eukaryotic, prokaryotic and archaeal cells. Illustrative examples of eukaryotic cells include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Crypthecodinium cohnii, Cunninghamella japonica, Entomophthora coronata, Mortierella alpina, Mucor circinelloides, Neurosporacrassa, Pythium ultimum, Schizochytrium limacinum, Thraustochytrium aureum, Trichoderma reesei and Xanthophyllomyces dendrorhous. In general, if eukaryotic cells are used, non-pathogenic strains are employed. Illustrative examples of non-pathogenic strains include, but are not limited to, Pichia pastoris and Saccharomyces cerevisiae. In addition, certain strains, including Saccharomyces cerevisiae, have been designated by the U.S. Food and Drug Administration as generally recognized as safe (or GRAS) and therefore may be conveniently used in various embodiments of the methods of the present disclosure.

[0170] Illustrative examples of recombinant prokaryotic host cells provided by the present disclosure include, but are not limited to, Bacillus subtilis, Brevibacterium ammoniagenes, Clostridium beigerinckii, Enterobacter sakazakii, Lactobacillus acidophilus, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter capsulatus, Rhodobacter sphaeroides, Salmonella enterica, Salmonella typhi, Salmonella typhimurium. typhimurium, Shigella flexneri, Staphylococcus aureus, Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces aureus, Streptomyces fungicidicus, Streptomyces griseochromogenes, Streptomyces griseus, Streptomyces lividans, Streptomyces olivogriseus, Streptomyces rameus, Streptomyces tanashiensis, and Streptomyces vinaceus. Certain of these cells, including Bacillus subtilis, Lactobacillus acidophilus, have been designated as Generally Regarded as Safe (or GRAS) by the U.S. Food and Drug Administration and thus can be used in various embodiments of the methods of the present disclosure.Although desirable from a public safety and regulatory standpoint, GRAS status does not affect the ability of a host strain to be used to practice the present disclosure; therefore, non-GRAS and even pathogenic organisms are included in this list of illustrative host strains suitable for use in practicing the present disclosure.

[0171] E. coli is also an excellent prokaryotic host cell for metabolic pathway construction, and E. coli is also well used in industrial fermentation of small molecule products. Unlike most wild-type yeast strains, wild-type E. coli can metabolize both pentoses and hexoses as carbon sources. Relative to yeast, E. coli also has a shorter doubling time, so that experiments can be carried out more quickly. The present disclosure provides a wide variety of E. coli host cells suitable for producing malonate as described herein. In various embodiments of the method of the present disclosure, the recombinant host cell comprising a heterologous nucleic acid encoding malonyl-CoA hydrolase is an E. coli cell.

[0172] Section 5: Other Modifications and Fermentation Conditions for Achieving Improved Malonate Production In other aspects of the present disclosure, increased malonate yield, titer, and / or productivity is achieved by employing host cells provided by the present disclosure that have been genetically modified in a manner other than or in addition to the introduction of a heterologous malonyl-CoA hydrolase and / or employing fermentation conditions provided by certain methods of the present disclosure. Briefly, the recombinant host cells of the present disclosure comprise genetic modifications that increase acetyl-CoA biosynthesis, increase malonyl-CoA biosynthesis, decrease malonate catabolism, increase malonate secretion from the host cell, increase host cell tolerance to malonate, and increase catabolism of various carbon sources.

[0173] Genetic modifications and fermentation conditions to increase acetyl-CoA biosynthesis

[0174] According to the present disclosure, increased malonate titer, yield and / or productivity can be achieved by increasing genetic modifications that increase acetyl-CoA biosynthesis, and the present disclosure provides enzymes that increase acetyl-CoA biosynthesis, vectors for expressing enzymes that increase acetyl-CoA biosynthesis, host cells that express enzymes that increase acetyl-CoA biosynthesis and increase malonate titer, yield and / or productivity, and methods related thereto. As described above, malonate is produced by hydrolysis of malonyl-CoA that can be produced from acetyl-CoA; therefore, increased acetyl-CoA biosynthesis can improve malonate production.

[0175] One pathway by which acetyl-CoA is produced is through acetyl-CoA synthetase (EC 6.2.1.1), which catalyzes the formation of acetyl-CoA from acetate and coenzyme A (CoA). The present disclosure provides recombinant host cells suitable for producing malonate according to the methods of the present disclosure, the recombinant host cells comprising one or more heterologous acetyl-CoA synthetases (ACS) that increase malonate titer, yield and / or productivity relative to host cells that do not comprise heterologous acetyl-CoA synthetases. Non-limiting examples of suitable ACS enzymes are Saccharomyces cerevisiae ACS1 (GenBank: AAC04979.1) and ACS2 (GenBank: CAA97725.1). In one embodiment, the ACS enzyme is Yarrowia lipolytica acetyl-CoA synthetase YlACS (GenBank: XM_505057.1; UniProt ID: Q6C2Q5).

[0176] In some embodiments, recombinant host cells comprising Yarrowia lipolytica acetyl-CoA synthetase or Saccharomyces cerevisiae acetyl-CoA synthetase ACS1 and / or ACS2 are used to increase malonate titer, yield and / or productivity. In other embodiments, recombinant host cells comprising an acetyl-CoA synthetase selected from the group consisting of Salmonella enterica Acs, Escherichia coli AcsA and Bacillus subtilis AcsA are used to increase malonate yield, titer and / or productivity. It is understood by those skilled in the art that other acetyl-CoA synthetases can be expressed in recombinant host cells producing malonate according to the present disclosure to increase malonate yield, titer and / or productivity. This modification is illustrated in Examples 9 and 46.

[0177] In some embodiments, the recombinant host cell comprising a heterologous acetyl-CoA synthetase, such as Saccharomyces cerevisiae ACS1 or Saccharomyces cerevisiae ACS2, further comprises a Saccharomyces cerevisiae pyruvate decarboxylase (EC 4.1.1.43, 4.1.1.72 or 4.1.1.74). In some embodiments, the Saccharomyces cerevisiae pyruvate decarboxylase is selected from ScPDC1 (UniProt ID P06169), ScPDC5 (UniProt ID P16467) and ScPDC6 (UniProt ID P26263). In some embodiments, the Saccharomyces cerevisiae pyruvate decarboxylase is ScPDC1. In some embodiments, the Saccharomyces cerevisiae pyruvate decarboxylase is ScPDC5. In some embodiments, the Saccharomyces cerevisiae pyruvate decarboxylase is ScPDC6.

[0178] A second pathway by which acetyl-CoA is produced is achieved by a pyruvate dehydrogenase complex that catalyzes the formation of acetyl-CoA from pyruvate. The present disclosure provides recombinant host cells suitable for producing malonate according to the methods of the present disclosure, the recombinant host cells comprising one or more heterologous pyruvate dehydrogenase complex enzymes that increase malonate titer, yield and / or productivity relative to host cells that do not comprise heterologous pyruvate dehydrogenase complex enzymes. Non-limiting examples of suitable pyruvate dehydrogenase complex enzymes include Saccharomyces cerevisiae PDA1, PDB1, LAT1, LPD1, and PDX1.

[0179] In some embodiments of the present disclosure, the malonate yield, titer and / or productivity in the recombinant host cells for the production of malonate is increased by expressing one or more pyruvate dehydrogenases selected from the group consisting of Saccharomyces cerevisiae PDA1, PDB1, LAT1, LPD1 and PDX1. It will be appreciated by those skilled in the art that other pyruvate dehydrogenases can be expressed in the recombinant host cells for the production of malonate to increase the malonate yield, titer and / or productivity according to the present disclosure. This modification is illustrated in Example 10.

[0180] The third pathway through which acetyl-CoA is produced is achieved by a heterologous ethanol catabolic pathway comprising an enzyme that catalyzes the conversion of ethanol into acetyl-CoA. Ethanol is a relatively inexpensive chemical compared to malonate, and a host cell that produces malonate and expresses the ethanol catabolic pathway can convert ethanol into malonate. Alcohol dehydrogenase (EC 1.1.1.1) catalyzes the conversion of ethanol into acetaldehyde. Non-limiting examples of suitable alcohol dehydrogenases include alcohol dehydrogenases selected from the group consisting of Saccharomyces cerevisiae ADH2, Escherichia coli AdhP, Homo sapiens ADH1A, Homo sapiens ADH1B, and Homo sapiens ADH1C. In addition to alcohol dehydrogenases, the ethanol catabolic pathway also comprises acetaldehyde dehydrogenase (acylation; EC 1.2.1.10) or aldehyde dehydrogenase (EC 1.2.1.3) and acetyl-CoA synthetase (EC 6.2.1.1). Acetaldehyde dehydrogenase (acylation) catalyzes the conversion of acetaldehyde to acetyl-CoA, aldehyde dehydrogenase catalyzes the conversion of acetaldehyde to acetic acid, and acetyl-CoA synthase (synthase) as described above catalyzes the formation of acetyl-CoA from acetic acid and CoA. Non-limiting examples of suitable acetaldehyde dehydrogenase (acylation) include acetaldehyde dehydrogenases selected from the group consisting of E. coli MhpF, E. coli AdhE, Pseudomonas sp. CF600DmpF, and Pseudomonas putida TodL. Non-limiting examples of aldehyde dehydrogenases include Saccharomyces cerevisiae ALD2, ALD3, ALD4, ALD5, and ALD6; and Homo sapiens ALD1, ALD2, ALD4, and ALD10. Non-limiting examples of acetyl-CoA synthetases include Saccharomyces cerevisiae ACS1, Saccharomyces cerevisiae ACS2, and E. coli Acs.

[0181] The present disclosure provides recombinant host cells suitable for producing malonate according to the methods of the present disclosure, the recombinant host cells comprising one or more heterologous ethanol catabolic pathway enzymes that increase malonate yield, titer and / or productivity relative to host cells that do not comprise heterologous ethanol catabolic pathway enzymes. In some embodiments, the heterologous ethanol catabolic pathway enzymes are ethanol dehydrogenase and acetaldehyde dehydrogenase (acylation). In some embodiments, the heterologous ethanol catabolic pathway enzymes are Saccharomyces cerevisiae ADH2 ethanol dehydrogenase and Escherichia coli MhpF acetaldehyde dehydrogenase (acylation). In some embodiments, the heterologous Saccharomyces cerevisiae ADH2 and Escherichia coli MhpF are expressed in recombinant Escherichia coli expressing a heterologous Saccharomyces cerevisiae EHD3 malonyl-CoA hydrolase. In other embodiments, the heterologous Saccharomyces cerevisiae ADH2 and Escherichia coli MhpF are expressed in recombinant Saccharomyces cerevisiae expressing a heterologous Saccharomyces cerevisiae EHD3 malonyl-CoA hydrolase. In other embodiments, heterologous S. cerevisiae ADH2 and E. coli MhpF are expressed in recombinant oleaginous yeast expressing heterologous S. cerevisiae EHD3 malonyl-CoA hydrolase. In other embodiments, the heterologous ethanol catabolic pathway enzymes are S. cerevisiae ADH2 ethanol dehydrogenase and Pseudomonas species CF600 DmpF acetaldehyde dehydrogenase (acylating). In some embodiments, heterologous S. cerevisiae ADH2 and Pseudomonas species CF600 DmpF are expressed in recombinant E. coli expressing heterologous S. cerevisiae EHD3 malonyl-CoA hydrolase. In some embodiments, heterologous S. cerevisiae ADH2 and Pseudomonas species CF600 DmpF are expressed in recombinant S. cerevisiae expressing S. cerevisiae EHD3 malonyl-CoA hydrolase.

[0182] In some embodiments, heterologous S. cerevisiae ADH2 and a species of Pseudomonas CF600 DmpF are expressed in a recombinant oleaginous yeast expressing a heterologous S. cerevisiae EHD3 malonyl-CoA hydrolase. In other embodiments, the heterologous ethanol catabolic pathway enzymes are S. cerevisiae ADH2 ethanol dehydrogenase and Pseudomonas putida TodL ​​acetaldehyde dehydrogenase (acylation). In some embodiments, heterologous S. cerevisiae ADH2 and Pseudomonas putida TodL ​​are expressed in recombinant E. coli expressing a heterologous S. cerevisiae EHD3 malonyl-CoA hydrolase. In some embodiments, heterologous S. cerevisiae ADH2 and Pseudomonas putida TodL ​​are expressed in a recombinant S. cerevisiae expressing a heterologous S. cerevisiae EHD3 malonyl-CoA hydrolase. In some embodiments, heterologous S. cerevisiae ADH2 and Pseudomonas putida TodL ​​are expressed in a recombinant oleaginous yeast expressing a heterologous S. cerevisiae EHD3 malonyl-CoA hydrolase. In other embodiments, the heterologous ethanol catabolic pathway enzymes are one or more alcohol dehydrogenases selected from the group comprising Saccharomyces cerevisiae ADH2, Escherichia coli AdhP, Homo sapiens ADH1A, Homo sapiens ADH1B and / or Homo sapiens ADH1C and one or more acetaldehyde dehydrogenases (acylating) selected from the group comprising Escherichia coli MhpF, Escherichia coli AdhE, Pseudomonas sp. CF600DmpF and Pseudomonas putida TodL. It is understood by those skilled in the art that other alcohol dehydrogenases and acetaldehyde dehydrogenases (acylating) can be expressed in a recombinant host cell suitable for producing malonate according to the methods of the present disclosure to increase malonate yield, titer and / or productivity.

[0183] In other embodiments, the heterologous ethanol catabolic pathway enzymes are alcohol dehydrogenase, aldehyde dehydrogenase, and acetyl-CoA synthetase. In some embodiments, the heterologous ethanol catabolic pathway enzymes are Saccharomyces cerevisiae ALD2 alcohol dehydrogenase, Saccharomyces cerevisiae ALD2 aldehyde dehydrogenase, and Saccharomyces cerevisiae ACS1 acetyl-CoA synthetase. In other embodiments, the heterologous ethanol catabolic pathway enzymes are Saccharomyces cerevisiae ALD2 alcohol dehydrogenase, Saccharomyces cerevisiae ALD2 aldehyde dehydrogenase, and Saccharomyces cerevisiae ACS2 acetyl-CoA synthetase. In some embodiments, the ethanol catabolic pathway enzyme is Saccharomyces cerevisiae ALD6 alcohol dehydrogenase (UniProt ID: P54115). In some embodiments, the ethanol catabolic pathway enzymes are Saccharomyces cerevisiae ALD6 alcohol dehydrogenase (UniProt ID: P54115) and Yarrowia lipolytica ACS (UniProt ID: Q6C2Q5). In other embodiments, the heterologous ethanol catabolic pathway enzymes are Saccharomyces cerevisiae ALD2 alcohol dehydrogenase, Saccharomyces cerevisiae ALD6 aldehyde dehydrogenase, and Saccharomyces cerevisiae ACS1 acetyl-CoA synthetase. In other embodiments, the heterologous ethanol catabolic pathway enzymes are Saccharomyces cerevisiae ALD2 alcohol dehydrogenase, Saccharomyces cerevisiae ALD6 aldehyde dehydrogenase, and Saccharomyces cerevisiae ACS2 acetyl-CoA synthetase. In other embodiments, the heterologous ethanol catabolic pathway enzymes are one or more alcohol dehydrogenases selected from the group containing Saccharomyces cerevisiae ADH2, E. coli AdhP, Homo sapiens ADH1A, Homo sapiens ADH1B, and / or Homo sapiens ADH1C, one or more aldehyde dehydrogenases selected from the group containing Saccharomyces cerevisiae ALD2, Saccharomyces cerevisiae ALD3, Saccharomyces cerevisiae ALD4, Saccharomyces cerevisiae ALD5, Saccharomyces cerevisiae ALD6, Homo sapiens ALD1, Homo sapiens ALD2, Homo sapiens ALD4, and / or Homo sapiens ALD10, and one or more acetyl-CoA synthetases selected from the group containing Saccharomyces cerevisiae ACS1, Saccharomyces cerevisiae ACS2, and / or E. coli Acs.

[0184] In some embodiments, the recombinant host cell suitable for producing malonate according to the method of the present disclosure comprises a heterologous ethanol catabolic pathway enzyme and converts the endogenously produced ethanol into acetyl-CoA and increases malonate yield, titer and / or productivity. In other embodiments, ethanol is exogenously added to the fermentation broth and the recombinant host cell suitable for producing malonate according to the method of the present disclosure comprises a heterologous ethanol catabolic pathway enzyme and converts the exogenously added ethanol into acetyl-CoA and increases malonate yield, titer and / or productivity. When exogenously added to the fermentation broth, ethanol is added to obtain a minimum concentration of 1% ethanol volume / volume, and is typically added to the fermentation broth to obtain a concentration of 1-15% volume / volume. The ethanol catabolic metabolism for improving malonate production according to the present disclosure is described in Example 11.

[0185] Increasing the cytoplasmic pool of acetyl-CoA is the fourth way to increase malonate biosynthesis; in many plant and animal cells, but not in Saccharomyces cerevisiae, ATP citrate lyase (EC 2.3.3.8) is the main enzyme responsible for the biosynthesis of cytoplasmic acetyl-CoA. In more detail, acetyl-CoA in mitochondria is condensed with oxaloacetate by the activity of citrate synthase to form citric acid. Subsequently, citric acid is transported from mitochondria to the cytoplasm, where ATP citrate lyase catalyzes the formation of acetyl-CoA, oxaloacetate and ADP. Although Saccharomyces cerevisiae does not contain natural ATP citrate lyase, suitable heterologous ATP citrate lyases in oleaginous yeast strains have been described (see, e.g., Boulton et al., J. Gen. Microbiol. 127: 169-176 (1981)). The present disclosure provides recombinant host cells comprising one or more heterologous nuclear oleaginous yeast ATP citrate lyases. The non-limiting examples of lipogenic yeast ATP citrate lyase include citrate lyases selected from the group of lipogenic yeasts consisting of: Candida curvata, Cryptococcus albidus, Lipomyces lipofer, Rhodospiridium toruloides, Rhodotorula glutanis, Trichosporon cutaneum, Yarrowia lipolytica etc. In various embodiments, the recombinant host cell comprises a heterologous nucleic acid encoding ATP citrate lyase. In various embodiments, the ATP citrate lyase is from an organism selected from the group consisting of Candida curvata, Cryptococcus albidus, Lipomyces lipofer, Rhodospiridium toruloides, Rhodotorula glutanis, Trichosporon cutaneum, Yarrowia lipolytica etc. This modification is described in Example 12.

[0186] The biosynthesis of acetyl-CoA can also be increased according to the present disclosure by changing the expression of one or more nucleic acids encoding proteins that affect fatty acid storage or catabolism. The present disclosure provides a host cell comprising a genetic modification of one or more nucleic acids encoding proteins that affect fatty acid storage and catabolism. In Saccharomyces cerevisiae, these proteins include SNF2, IRA2, PRE9, PHO90, SPT21, POX1, ANT1, FOX3, PAS1, PAS3, ARE1, ARE2, DGA1, LRO1, ACL1, MAE1, GLC3, GLG1, GLG2, PAT1 and PEX11. This modification is illustrated in Example 13.

[0187] In some embodiments of the present disclosure, the host cell comprises a genetic modification that affects the expression and / or activity of proteins involved in fatty acid catabolism. For example, most host cells will naturally degrade fatty acids, hydroxy fatty acids, and many diacids by the β-oxidation pathway. In most cases, β-oxidation occurs by activating free fatty acid groups to CoA thioesters with acyl-CoA ligases. Acyl-CoA intermediates are further oxidized and degraded-by 2,3 enoyl-CoA, 3-hydroxyacyl-CoA, and 3-ketoacyl-CoA-and then cleavage leads to the production of acetyl-CoA and acyl-CoA that is shortened by two carbons relative to the initial substrate. The enzyme activity required for β-oxidation is known. The present disclosure provides host cells with increased activity of the catabolism pathway for medium-chain (C4-C8) and long-chain (>C8) fatty acids, hydroxy fatty acids, and diacids compared to control host cells. For example, in yeast (e.g., Saccharomyces cerevisiae), β-oxidation occurs in peroxisomes; exemplary non-limiting nucleic acid products that affect peroxisomal β-oxidation are Saccharomyces cerevisiae PAT1 and PEX11. In some embodiments of the present disclosure, a host cell modified to achieve increased expression of PAT1 and / or PEX11 for use in the methods herein for producing malonate is provided. This modification is described in Example 14.

[0188] Genetic modifications and fermentation conditions to increase malonyl-CoA biosynthesis

[0189] According to the present disclosure, malonate titer, yield and / or productivity increase can be achieved by increasing malonyl-CoA biosynthesis, and the present disclosure provides host cells, vectors, enzymes and methods related thereto. Malonyl-CoA is produced in a host cell by the activity of acetyl-CoA carboxylase (EC 6.4.1.2) catalyzing the formation of malonyl-CoA from acetyl-CoA and carbon dioxide. The present disclosure provides a recombinant host cell expressing heterologous acetyl-CoA carboxylase (ACC) for the production of malonate. In some embodiments, ACC is Pichia kudriavzevii ACC1 (abbreviated as PkACC1; UniProt ID: A0A099P5S6). In some embodiments, the host cell is Pichia kudriavzevii and ACC is PkACC1 (UniProt ID: A0A099P5S6). In some embodiments, the host cell is Saccharomyces cerevisiae and ACC is PkACC1 (UniProt ID: A0A099P5S6). In some embodiments, PkACC1 comprises one or more of the following mutations: S644A, S671A, and S1142A. In some embodiments, ACC is Saccharomyces cerevisiae ACC1 (abbreviated as ScACC1; UniProt ID: Q00955). In some embodiments, the host cell is Pichia kudriavzevii and ACC is ScACC1 (UniProt ID: Q00955). In some embodiments, ScACC1 comprises one or more of the following mutations: S659A and S1157A. In some embodiments, ACC is Yarrowia lipolytica ACC (UniProt ID: A0A371C979). In some embodiments, ACC is Escherichia coli ACC. Escherichia coli ACC comprises proteins expressed by genes ACCA (UniProt ID: P0ABD5), ACCB (UniProt ID: P0ABD8), ACCC (UniProt ID: P24182), and ACCD (UniProt ID: P0A9Q5). The E. coli enzyme complex contains two catalytic units and a carrier protein, encoded by four different genes. The catalytic unit is biotin carboxylase (BC), which is a homodimer encoded by the accC gene, and acetyl-CoA carboxylase complex (ACCT), which is an α2β2 tetramer encoded by the accA and accD genes. The carrier protein is biotin carboxyl carrier protein (dimer) (BCCP), which is a homodimer encoded by the accB gene. See, for example, https: / / ecocyc.org / ECOLI / NEW-IMAGE? type=ENZYME&object=ACETYL-COA-CARBOXYLMULTI-CPLX.In some embodiments, ACC is Arabidopsis ACC (abbreviated as AtACC), such as AtACC1 (UniProt ID: Q38970), AtACC2 (UniProt ID: F4I1L3) and AtACCA (UniProt ID: Q9LD43). In some embodiments, ACC is AtACCA with the chloroplast signal peptide removed. In some embodiments, the host cell is a Saccharomyces cerevisiae cell, which contains a heterologous Saccharomyces cerevisiae acetyl-CoA carboxylase ACC1 or an enzyme homologous thereto. In some embodiments, the host cell modified to heterologously express ACC such as Saccharomyces cerevisiae ACC1 is further modified to eliminate ACC1 post-translational regulation by genetically modifying Saccharomyces cerevisiae SNF1 protein kinase or an enzyme homologous thereto. The present disclosure also provides a recombinant host cell suitable for producing malonate according to the present disclosure, which is an E. coli cell containing a heterologous nucleic acid encoding the expression of the E. coli acetyl-CoA carboxylase complex protein AccA, AccB, AccC and AccD or one or more enzymes homologous thereto. According to the present disclosure, other acetyl-CoA carboxylases can be heterologously expressed to increase malonyl-CoA and malonate biosynthesis. This modification is illustrated in Example 15. In some embodiments, ACC is fused to carbonic anhydrase (abbreviated as CAH; EC4.2.1.1). In some embodiments, CAH is Porphyromonas gingivalis carbonic anhydrase PgCAH (UniProt ID: T2NA71). In some embodiments, CAH is Pisum sativum carbonic anhydrase PsCAH (UniProt ID: P17067). In some embodiments, CAH is Mycobacterium tuberculosis carbonic anhydrase MtCAH (UniProt ID: P9WPJ9). In some embodiments, CAH is Homo sapiens carbonic anhydrase HsCAH (UniProt ID: P00918). In some embodiments, ACC is covalently linked to CAH. Any combination of connecting the N and C termini of ACC and CAH is contemplated, i.e., NN, NC, CN, CC. In some embodiments, the N terminus of ACC is connected to the C terminus of CAH. In some embodiments, the C terminus of ACC is connected to the N terminus of CAH. In some embodiments, ACC is connected to CAH via a fusion peptide. Examples of suitable fusion peptides include, but are not limited to, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56. In some embodiments, the fusion peptide is SEQ ID NO: 54. In some embodiments, the fusion peptide is SEQ ID NO: 55. In some embodiments, the fusion peptide is SEQ ID NO: 56.

[0190] In various embodiments of the present disclosure, the expression of BirA (i.e., biotin-[acetyl-CoA carboxylase] holoenzyme synthetase) is co-expressed with the E. coli acetyl-CoA carboxylase complex proteins AccA, AccB, AccC, and AccD to enhance the activity of the ACC complex and result in increased production of malonyl-CoA and malonate. In various embodiments of the present disclosure, the yeast cerevisiae ACC1 is further modified to eliminate ACC1 post-translational regulation by introducing serine to alanine mutations at any, all, or any combination of the following residues: S10, S233, S430, S1114, S1145, S1148, S1157, S1159, S1162, S1163, S1169. In some embodiments of the present disclosure, the acetyl-CoA carboxylase used is from Yarrowia lipolytica CLIB122, referred to herein as Y1ACC. Example 42 illustrates how including this enzyme in a malonate-producing host strain provided by the disclosure results in a doubling of the titer of malonate from that host after 120 hours of fermentation relative to the same host fermented without this enzyme. In other embodiments of the disclosure, this enzyme is co-expressed with the biotin-[acetyl-CoA carboxylase] holoenzyme synthetase (BPL1) also derived from this organism. In other embodiments of the disclosure, the acetyl-CoA carboxylase and biotin-[acetyl-CoA carboxylase] holoenzyme synthetase encoding genes are dtsR1 accBC and derived from Corynebacterium glutamicum. In other embodiments of the disclosure, these genes are derived from yeast strains including, but not limited to, yeast strains of the following genera: Candida, Pichia, or any other yeast herein. In various embodiments of the disclosure, the host cell producing malonate expresses any combination of these acetyl-CoA carboxylases and biotin-[acetyl-CoA carboxylase] holoenzyme synthetase.

[0191] In some embodiments of the present disclosure, a host cell suitable for producing malonate according to the method of the present disclosure comprises a genetic modification that affects the expression and / or activity of proteins involved in fatty acid biosynthesis. Malonyl-CoA is a natural substrate in the biosynthesis of fatty acids, and turning malonyl-CoA to fatty acid production reduces the ability of the host cell to produce malonate. The present disclosure provides a recombinant host cell expressing a heterologous fatty acid synthase (FAS) multienzyme complex for producing malonate. The temperature-sensitive mutation of the yeast cerevisiae fatty acid synthase complex is known (see Knobling et al., Eur. J. Biochem., 59: 415-421 (1975)). The expression of the heterologous temperature-sensitive fatty acid synthase complex can turn malonyl-CoA to fatty acid biosynthesis controlled by the temperature in which the host cell is cultured. In some embodiments, the host cell is a yeast cerevisiae cell comprising yeast cerevisiae fatty acid synthases FAS1 and FAS2 or an enzyme homologous thereto. In some embodiments of the present disclosure, the FAS1 and FAS2 enzymes are temperature-sensitive FAS1 or FAS2 enzymes.

[0192] In addition to the genetic modification of the host cell, fatty acid biosynthesis can be reduced by adding a FAS inhibitor to the cell culture medium. For example, the FAS inhibitor cerulenin forms a covalent bond with the active site cysteine ​​C1305 in the ketoacyl synthase domain of the Saccharomyces cerevisiae of the FAS complex, thereby inhibiting enzyme activity (Johansson et al., PNAS, 105: 12803-12808 (2008)). Cerulenin not only effectively inhibits Saccharomyces cerevisiae FAS activity, but is also typically an inhibitor of the FAS complex containing Cys-His-His or Cys-His-Asn catalytic triads in the ketoacyl synthase domain. In some embodiments, cerulenin is added to the fermentation broth to obtain a final concentration of 5 mg / l to 100 mg / l to inhibit fatty acid biosynthesis and increase the production of malonate in the recombinant host cell producing malonate according to the method of the present disclosure. In various embodiments of the method of the present disclosure, a FAS inhibitor is added to the fermentation broth containing the recombinant host cell producing malonate. In some embodiments of the methods of the present disclosure, the FAS inhibitor is cerulenin as described in Example 16. In some embodiments of the methods of the present disclosure, cerulenin is supplemented in the fermentation broth at a concentration of 5 mg / l to 100 mg / l. In other embodiments of the methods of the present disclosure, the fatty acid synthase complex inhibitor is selected from the group consisting of platensimycin, thiolactomycin and triclosan.

[0193] One of the substrates of acetyl-CoA carboxylase is carbon dioxide, and increasing the carbon dioxide partial pressure in the fermentation broth promotes the formation of malonyl-CoA. The fermentation broth should contain a minimum dissolved carbon dioxide pressure of 0.01 atmosphere, and it is desirable to increase the dissolved carbon dioxide partial pressure above this threshold. The fermentation broth should generally contain a dissolved carbon dioxide partial pressure of 0.1 atmosphere to 1 atmosphere. The dissolved carbon dioxide partial pressure in the fermentation broth can be increased to above saturation conditions, or above 1 atmosphere of dissolved carbon dioxide. In some embodiments of the method of the present disclosure, the dissolved carbon dioxide partial pressure in the fermentation broth is increased to 0.1 atmosphere to 1 atmosphere. In some embodiments of the method of the present disclosure, the carbon dioxide partial pressure is increased by adding carbonate or bicarbonate to the fermentation broth. For example and without limitation, calcium carbonate can be added to the fermentation broth to increase the dissolved carbon dioxide partial pressure. In other embodiments of the method of the present disclosure, the fermentation is run in a pressurized container containing carbon dioxide at a pressure higher than atmospheric pressure. In other embodiments of the method of the present disclosure, carbon dioxide gas is sprayed into the fermentation broth. The sparged gas mixture may contain other gases if the added components do not interfere with host cell growth or malonate production. It may be advantageous to co-locate a source of carbon dioxide gas with the malonate fermentation. For example and without limitation, gaseous carbon dioxide produced by various fermentation processes (e.g., ethanol, isobutanol, 3-hydroxypropionic acid, etc.), chemical processes (e.g., downstream malonate synthesis chemistry), or energy generation (e.g., coal or natural gas power plants) may be pumped into the fermentation broth from malonate-producing host cells to increase the carbon dioxide partial pressure, as described in Example 17.

[0194] Genetic modification to reduce malonate catabolism

[0195] According to the present disclosure, malonate titer, yield and / or productivity increase can be achieved by reducing malonate catabolism, and the present disclosure provides host cells, vectors, enzymes and methods related thereto. One metabolic pathway through which malonate is catabolized in a host cell is achieved by the activity of acyl-CoA synthetase catalyzing the conversion of malonate and coenzyme A into malonyl-CoA. In some embodiments of the present disclosure, a recombinant host cell suitable for producing malonate according to the method of the present disclosure comprises a genetic modification that results in the deletion, attenuation or modification of one or more nucleic acids encoding acyl-CoA synthetases. In some embodiments of the present disclosure, the recombinant host cell is yeast and one or more acyl-CoA synthetases are selected from the group consisting of FAA1, FAA2, FAA3, FAA4, LSC1 and LSC2. In other embodiments of the present disclosure, the recombinant host cell is Escherichia coli and one or more acyl-CoA synthetases are selected from the group consisting of FadD, FadK, FadI, SucC, SucD and YahF. This aspect of the disclosure is illustrated in Example 18.

[0196] Genetic modification to increase malonate secretion from host cells

[0197] According to the present disclosure, malonate titer, yield and / or productivity increase can be achieved by increasing malonate transport into the fermentation broth, and the present disclosure provides host cells, materials and methods related thereto. In some embodiments of the present disclosure, a recombinant host cell suitable for use in the methods of the present disclosure is a Saccharomyces cerevisiae cell comprising a heterologous nucleic acid encoding the expression of a Saccharomyces cerevisiae transporter selected from the group consisting of PDR5, PDR10, PDR11, PDR12, PDR15 and PDR18. In some embodiments of the present disclosure, a recombinant host cell suitable for producing malonate according to the methods of the present disclosure is an E. coli cell comprising a heterologous nucleic acid encoding the expression of E. coli DcuC. This aspect of the present disclosure is illustrated in Example 19.

[0198] Genetic modification to increase host cell tolerance to malonate

[0199] According to the present disclosure, malonate titer, yield and / or productivity increase can be achieved by increasing the tolerance of host cells to malonate, and the present disclosure provides host cells, materials and methods related thereto. High concentrations of malonate can competitively inhibit succinate dehydrogenase (EC 1.3.5.1) activity (see Slater, Methods Enzymol.10:48-57 (1967)). The present disclosure is based in part on the discovery that mutant succinate dehydrogenase exhibits a lower competitive inhibition by malonate. For example, Saccharomyces cerevisiae succinate dehydrogenase SDH1 residues E300, R331 and R442 are important for substrate (e.g., succinate) recognition. Increasing the size of the SDH1 active site can reduce the competitive inhibition performed by malonate, while still allowing the enzyme to maintain activity to the natural substrate succinate. Specifically, the introduction of one or more mutations selected from the group consisting of E300D, R331K or R331H, and R442K and R442H reduces the competitive inhibition of SDH1 by malonate. In some embodiments, the recombinant host cell expressing SDH1 with the point mutation R300D is used to produce malonate according to the present disclosure. In other embodiments, the recombinant host cell expressing SDH1 with the point mutation R331K or R331H is used to produce malonate according to the present disclosure. In other embodiments, the recombinant host cell expressing SDH1 with the point mutation R442K or R442H is used to produce malonate according to the present disclosure. This aspect of the present disclosure is described in Example 20.

[0200] Genetic modifications to increase the catabolism of various carbon sources

[0201] In the method of the present disclosure, carbon raw materials are used to produce malonate. Suitable carbon sources include, without limitation, carbon sources selected from the group consisting of: purified sugars (e.g., dextrose, sucrose, xylose, arabinose, lactose, etc.); plant-derived mixed sugars (e.g., sugar cane, sweet sorghum, molasses, corn starch, potato starch, beet sugar, wheat, etc.), vegetable oils, fatty acids, glycerol, cellulosic biomass, alginate, ethanol, carbon dioxide, methanol, and synthetic gas ("syngas"). A given host cell may efficiently or ineffectively degrade a specific raw material. If the host cell ineffectively degrades the raw material, the host cell may be modified to enhance or create a decomposition pathway for that raw material. For example, Example 47 shows the use of Pichia kudriavzevii as a host strain to produce malonate from many different carbon sources according to the present disclosure. Although this organism does not degrade sucrose under the conditions tested, the present disclosure provides for the introduction of a sucrose invertase (β-fructofuranosidase, EC3.2.1.26) to facilitate the use of this carbon source. Example 30 also illustrates the use of multiple carbon sources, according to the methods of the present disclosure to produce malonate. Other embodiments of the present disclosure include the use of methanol catabolic host strains. In some embodiments, the host is a yeast strain. In some embodiments, the host is selected from Komagataella pastoris, Pichia methanolica or Pichia pastoris.

[0202] The present disclosure provides a host cell comprising a genetic modification that increases malonate titer, yield and / or productivity by increasing the ability to decompose non-natural carbon sources. Wild-type saccharomyces cerevisiae cells cannot decompose pentoses, lignocellulosic biomass or alginate raw materials. In some embodiments, the present disclosure provides a saccharomyces cerevisiae cell comprising a heterologous nucleic acid encoding an enzyme that enables decomposition of pentoses for producing malonate as described herein. In other embodiments, the heterologous nucleic acid encoding enables the enzyme that decomposes lignocellulosic raw materials. In other embodiments of the present disclosure, the heterologous nucleic acid encoding increases the catabolism enzyme of alginate raw materials.

[0203] Those skilled in the art will recognize that individual manipulations to increase malonate production from a given host cell can be used in virtually unlimited combinations and often confer a boost that results in malonate output much higher than the sum of the individual manipulations, as well as in many cases the sum of the individual manipulations. Thus, the present disclosure encompasses not only the individual manipulations described herein, but it is also embodied by any combination of these perturbations that result in increased malonate production by the host strain.

[0204] Section 6: Testing malonate-producing host cells and screening for host cells with improved malonate production

[0205] The present disclosure also provides a transcription factor biosensor system that can be used to accurately sense malonate in liquid culture medium. In many applications, this system is used to detect malonate produced in host cells by sensing the presence of malonate in a fermentation medium containing malonate-producing host cells. "Accurate sensing" refers to detecting the presence of malonate and / or directly or indirectly determining the concentration of malonate; therefore, by accurately sensing malonate, this aspect of the present disclosure can be applied to strain improvement, i.e., for increasing malonate production in host cells. In this system, malonate binds to a protein portion present on a transcription factor. Malonate binding to the malonate-binding portion causes the transcription factor to bind to a promoter activated by the transcription factor, or in some embodiments, causes the promoter to be de-repressed by the transcription factor bound by malonate.

[0206] Many transcription factors that bind malonate, or are activated to bind a promoter in response to a signal generated by a malonate binding moiety, are suitable for use in this system.

[0207] In some embodiments, the transcription factor can bind malonate, which causes the transcription factor to bind to a cognate promoter and activate a gene operably linked to the promoter. Non-limiting examples of transcription factors that bind malonate include the transcription factor Acinetobacter calcoaceticus MdcY (SEQ ID NO: 3), Rhizobium leguminosarum MatR (SEQ ID NO: 4), Klebsiella pneumoniae MauR (SEQ ID NO: 5), and homologs thereof.

[0208] In some embodiments, the transcription factor used in the present disclosure is the MdcY transcription factor. The MdcY transcription factor can directly bind to malonate and regulate the expression of proteins such as P MdcL MdcY and MdcY-responsive promoters are known in the art (see, e.g., Koo et al., J Bacteriol. 182:6382-6390 (2000)).

[0209] An example of an MdcY polypeptide sequence from Acinetobacter calcoaceticus is provided in SEQ ID NO:3. MdcY polypeptides that can be employed in accordance with the present disclosure include variants and homologs of the MdcY polypeptide sequence set forth in SEQ ID NO:3. Thus, the MdcY transcription factor polypeptide can have an amino acid sequence that is at least 60% identical, typically at least 75%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO:3, preferably over a region of at least 100 or more amino acids, or at least 200 or more amino acids, or over the length of the entire polypeptide. It will be appreciated by those skilled in the art in light of the present disclosure that variants can also be employed, for example using known sequences as a guide for selecting amino acid substitutions that will not result in loss of function.

[0210] In some embodiments, the MdcY transcription factor used in the present disclosure is naturally present in a host cell. In other embodiments, the host cell is genetically modified to express the foreign transcription factor by introducing a heterologous nucleic acid encoding the malonate transcription factor into the host cell. In some embodiments, the genetically modified host cell comprising the heterologous nucleic acid encoding the malonate transcription factor is an Escherichia coli host cell.

[0211] The MdcY transcription factor can bind to many promoters and activate the expression of genes operably linked to the promoter. Examples of MdcY responsive promoters suitable for use in accordance with the present disclosure are provided in SEQ ID NO:6. In some embodiments, the MdcY responsive promoter used in the present disclosure typically comprises the operator sequence ATTGTATACAAT. In some embodiments, the promoter is at least 75% identical to the promoter sequence shown in SEQ ID NO:6. In some embodiments, the promoter comprises a subsequence of SEQ ID NO:6, comprising 10, 20, 25, 30, 35 or more consecutive nucleotides of SEQ ID NO:6.

[0212] Therefore, in one aspect, the present disclosure provides a method for accurately sensing malonate. In some embodiments, the sensing step includes detecting (e.g., measuring) the amount of the gene product of a reporter gene or a marker gene (e.g., a fluorescent reporter gene). In some embodiments, the gene product of the reporter gene affects the growth rate of the host cell of the component of the malonate transcription factor biosensor comprising the present disclosure. In some embodiments, the gene product of the reporter gene causes the modified host cell to become resistant or sensitive to a compound. For example, in some embodiments, the reporter gene is an antibiotic resistance gene (e.g., a tetA gene), wherein the presence of malonate in the culture medium induces antibiotic resistance, so that when the antibiotic exists, the host cell exhibits growth improvement in the presence of malonate, as described in Example 21. In some embodiments, the host cell of the component of the transcription factor biosensor comprising the present disclosure is a host cell capable of producing malonate. Example 36 illustrates the use of antibiotic resistance (tetA) and colorimetric signal (lacZ) as outputs associated with the malonate concentration produced according to other aspects of the present disclosure in the fermentation medium to implement this aspect of the present disclosure.

[0213] Generally, then, the present disclosure provides a method for screening or selecting host cells that produce malonate, comprising: (a) providing a modified host cell of the present disclosure, (b) culturing the host cell, and (c) screening or selecting the host cell based on the expression of a reporter gene by the host cell.

[0214] In some embodiments of the present disclosure, the method for screening or selecting a host cell producing malonate comprises: (a) providing a plurality of modified host cells of the present disclosure, wherein the modified host cells with different modifications are cultured separately, (b) culturing each separate culture of the host cell, (c) screening or selecting the host cell based on the expression of a reporter gene by the host cell, and (d) comparing the expression of the reporter gene of the separate culture. In some embodiments of the present disclosure, step (d) comprises identifying one or more cultures and / or corresponding host cells that have increased expression of a gene product of the reporter gene. It will be recognized by those skilled in the art that these cultures are produced by a single transformant and represent a clonal colony.

[0215] In some embodiments, the method of the present disclosure is a method for selecting a host cell producing malonate, wherein the selection is a positive selection. In a positive selection, the selection step selects a host cell that has a higher expression of a reporter gene that maintains vigor and a probability of doubling, and therefore maintains a higher probability of vigor and doubling. For example, a host cell producing malonate will maintain vigor and proliferate at a rate faster than a host cell that does not produce malonate. Similarly, a host cell whose malonate production is increased compared to a control strain will proliferate at a rate faster than a control strain.

[0216] In some embodiments of the present disclosure, a method for selecting an E. coli host cell that produces malonate comprises: (a) providing a plurality of modified E. coli host cells of the present disclosure, wherein the modified host cells having different modifications are cultured separately, (b) culturing each separate culture of the host cells, (c) positively selecting the host cells based on the expression of a reporter gene by the host cells, and (d) comparing the expression of the reporter gene of the separate cultures.

[0217] In other embodiments of the present disclosure, methods for selecting E. coli host cells that produce malonate include: (a) providing a plurality of modified E. coli host cells of the present disclosure, wherein the modified host cells having different modifications are cultured simultaneously, (b) culturing the heterogeneous mixture of modified host cells in a growth medium containing a positive selection agent so that host cells that exhibit higher production of malonate than the plurality of host cells will proliferate at a faster rate than host cells that exhibit lower production of malonate, and (d) isolating the host cells that exhibit the highest production of malonate.

[0218] Section 7: Malonic Acid Purification

[0219] In a sixth aspect, the disclosure provides a method for purifying malonate from a fermentation broth, the method comprising: (a) culturing a host cell under conditions suitable for producing malonate, (b) and recovering (i.e., purifying) malonate from the fermentation broth. The disclosure also provides purified malonate produced according to the method of the disclosure. Biosynthesized malonate can be produced intracellularly and / or secreted into a culture medium. The malonate produced intracellularly is typically secreted into the culture medium using a membrane transporter as described above. Unsecreted malonate can be removed from the host cell by chemical, enzymatic or mechanical cell lysis. Malonate can be recovered from cells, fermentation broth, or both. If the cell is engineered to secrete malonate, then it can be selected to recover malonate only from the fermentation broth or it can be selected to recover it from both the culture medium and the cell (i.e., by causing the cell to dissolve). If the cell is not engineered to secrete malonate, then the host cell can be dissolved to separate the malonate therein.

[0220] The present disclosure provides methods for separating biologically produced malonate. As used herein, "separation", "purification" and "recovery" are used to refer to separating malonate from other substances present. "Separation", "purification" or "recovery" as used in this context is intended to express the preparation of malonate rich in malonate, but may or may not be substantially (i.e., more than 50%) pure cells or fermentation broth relative to the production of malonate on a weight / weight (w / w) basis. Separation of malonate according to these methods involves separating at least a portion or all of the malonate produced from the fermentation medium, host cells and their respective parts that produce malonate. Malonic acid can be purified from fermentation broth and / or productive cells according to the present disclosure, i.e., to a purity of more than 50% based on w / w, wherein any naturally occurring or recombinant host cell (e.g., Escherichia coli, Saccharomyces cerevisiae, oleaginous yeast, etc.) producing malonate is grown, i.e., the host cell is not limited to the recombinant host cell of the present disclosure. The separated malonate may be free of or substantially free of impurities from the host cell. Malonic acid is separated or purified to such an extent that any impurities present do not interfere with the subsequent use of the malonic acid. For example, if the subsequent use is as an industrial chemical (such as a chemical to be used in a polymerization reaction), the malonic acid should be substantially free of impurities, and any remaining impurities will not interfere with the use of the malonic acid in the polymerization reaction. Typically, the malonic acid used for the polymerization reaction has a purity of at least 95% w / w or more. If the malonic acid is to be used as a fuel (such as a fuel to be used in a combustion reaction), the compound should be substantially free of impurities, and any remaining impurities will not interfere with the use of the malonic acid as a fuel. If the malonic acid is used as an animal feed, the malonic acid should be substantially free of impurities, and any remaining impurities will not interfere with the use of the substance as an animal feed. When the malonic acid is used as an animal feed, it is possible to choose to recover the biomass containing the malonic acid from the fermentation broth and use the biomass as an animal feed.

[0221] In some embodiments of the purification methods of the present disclosure, the fermentation broth is concentrated to increase the working concentration of malonate and reduce the volume of liquid that needs to be processed. In various embodiments of the purification methods of the present disclosure, this concentration is achieved by evaporation (including vacuum and heating), reverse osmosis, "high-pass" membrane dehydration, and / or thin film evaporation.

[0222] In some embodiments, the purification method of the present disclosure includes a step of recovering the produced malonate, wherein the recovery step is simultaneous with the culturing step or after the culturing step. In some embodiments, malonate is purified from fermentation broth and host cells. In other embodiments, host cells are separated from fermentation broth, dissolved, and then malonate is recovered from host cells. In other embodiments, host cells in the fermentation broth are dissolved and malonate is recovered from dissolved cells and fermentation broth. A method for recovering malonate from fermentation broth provided by the present disclosure is to precipitate malonate with cations. In some embodiments, this is a monovalent cation, and in other embodiments, it is a divalent cation. Typically, cations are added to the fermentation broth (or lysate) in the form of salts. For example, according to the present disclosure, calcium malonate is precipitated from an aqueous solution containing malonate, which may be a fermentation broth or a cell lysate or a mixture of both, by adding a calcium salt to achieve (Weiner, Org. Synth. 18: 50 (1938); Weiner, Org. Synth. Coll. 2: 376 (1943)). Various calcium salts (eg, calcium hydroxide, calcium carbonate, calcium chloride) can be used in accordance with the present disclosure to precipitate malonate from the fermentation broth.

[0223] When the two malonic acid carboxylic acids are not protonated and calcium is present in the fermentation broth, the calcium malonate salt is formed. Calcium malonate is insoluble in aqueous solutions such as the fermentation broth, will precipitate, and can then be recovered from the fermentation broth. The pK of the two malonic acid carboxylic acid moieties is aThe values ​​are 2.83 and 5.69; therefore, when the fermentation broth pH is lower than 5.7, calcium malonate is not formed and malonic acid is still dissolved in the fermentation broth. When the fermentation broth pH is higher than 5.7, calcium malonate can be formed and will precipitate from the fermentation broth. Carboxylic acids are weak acids, and the accumulation of malonic acid in the fermentation broth will reduce the pH, and if the pH drops below 5.7, the addition of calcium cations does not result in the formation of calcium malonate. A method for purifying malonic acid from the fermentation broth of the present disclosure is to add a calcium salt, wherein the calcium salt anion is a base (i.e., calcium carbonate and calcium hydroxide) and the addition of a calcium salt will increase and / or maintain the fermentation broth pH above 5.7. In some embodiments of the method of the present disclosure, calcium hydroxide is added to the fermentation broth to reach / maintain a pH of 5.69 to 7.5 and calcium malonate is precipitated. In a second embodiment of the method of the present disclosure, calcium carbonate is added to the fermentation medium to reach / maintain a pH of 5.69 to 7.5 and calcium malonate is precipitated. In a third embodiment of the present disclosure, calcium chloride is added to the fermentation broth and calcium malonate is precipitated when the fermentation broth pH is 5.69 to 7.5. Other calcium salts may be added to the fermentation broth to precipitate malonic acid according to the methods of the present disclosure. Adding calcium carbonate has the additional advantage of increasing the carbon dioxide partial pressure in the fermentation broth, thereby promoting the formation of malonyl-CoA by the activity of the above-mentioned acetyl-CoA carboxylase. Example 23 illustrates this aspect of the present disclosure by describing the purification of malonate using calcium salts.

[0224] Calcium salts can be added at the beginning of fermentation (i.e., before a large amount of malonate has been accumulated), during fermentation, or at the end of fermentation. In some embodiments, calcium carbonate or calcium hydroxide is added to the fermentation broth before the host cell has begun to produce malonate. In other embodiments, calcium carbonate or calcium hydroxide is added to the fermentation broth before the malonate concentration exceeds 5, 10, 15, 20, 25, 30, 40, or 50 g / l. In other embodiments, calcium carbonate or calcium hydroxide is added to the fermentation broth at the end of fermentation. It can be selected to recover the calcium malonate precipitate from the fermentation broth simultaneously with the fermentation or after the fermentation.

[0225] Because the solubility of calcium malonate monohydrate decreases with increasing temperature, the recovery of malonate from the fermentation broth can be increased by increasing the temperature to 50°C to 100°C during the recovery step. In some embodiments of the methods of the present disclosure, a calcium salt (i.e., calcium carbonate, calcium hydroxide, and / or calcium chloride) is added to the fermentation broth, the temperature is increased to 50°C to 100°C, and malonate is recovered from the fermentation broth. In other embodiments of the present disclosure, the temperature is increased to above 100°C. However, thermal decomposition of malonate may occur at temperatures of about 100°C, and therefore, increasing the temperature to 100°C or above 100°C may negatively affect the yield.

[0226] In some embodiments of the present disclosure, malonate is purified from the production medium by precipitation with monovalent cations. Typically, the monovalent cation is added to the fermentation broth (or lysate) in the form of a salt. For example, precipitation of sodium malonate from an aqueous solution containing malonate, which may be a fermentation broth or a cell lysate or a mixture of both, is achieved by adding a sodium salt according to the present disclosure. Various sodium salts (e.g., sodium hydroxide, sodium carbonate, sodium chloride) may be used to precipitate malonate from the fermentation broth according to the present disclosure. When the two malonic acid carboxylic acids are not protonated and sodium is present in the fermentation broth, a sodium malonate salt is formed. Sodium malonate is soluble in aqueous solutions such as fermentation broths, but the solution may be pressed out by increasing the pH of the culture medium to a higher pKa (5.69) than malonate; see Example 32. Additional precipitation of sodium malonate is achieved by evaporating the solvent (water) to increase the concentration of the ions. As with aspects of the disclosure relating to calcium malonate precipitation, addition of a sodium salt containing base (e.g., sodium hydroxide or sodium carbonate) can be used to maintain the pH of the fermentation broth above 5.7, thereby promoting precipitation of the sodium salt. Separation of the precipitate from the fermentation broth according to the disclosure provides purified malonate.

[0227] Another method for separating malonic acid from fermentation broth of the present disclosure is to achieve by reactive extraction with aliphatic primary amines, secondary amines or tertiary amines or primary, secondary or tertiary alcohols. This method is partly due to the discovery that both primary alcohols and tertiary amines are highly effective reagents for selectively removing malonic acid from fermentation broth, as described in Examples 24-26, 36-41. In some embodiments of the present disclosure, malonic acid is purified from fermentation broth by reactive extraction in an organic solvent with trioctylamine. In other embodiments of the present disclosure, malonic acid is purified from fermentation broth using a tertiary amine selected from the group consisting of: triethylamine, tripropylamine, tributylamine, tripentylamine, trihexylamine, triheptylamine, trinonylamine and tridecylamine. In other embodiments of the present disclosure, the amine compound is ammonia. In other embodiments, it is a dialkylamine, including but not limited to diethylamine, dipropylamine, diisopropylamine, dibutylamine, dipentylamine, dihexylamine, diheptylamine, dioctylamine, dinonylamine, didecylamine. Embodiment 33 explanation uses diethylamine to separate malonic acid from fermentation broth.In various embodiments of the present disclosure, amide is to add together with including but not limited to following organic solvent or mixture of solvent: amyl alcohol, hexanol, enanthol, octanol, nonanol, decanol, hexane, decane, kerosene, ether, ethyl acetate or its any combination.In various embodiments, solvent is branched or unbranched alkane, alcohol, glycol, ester, ether, diether or lactone containing 4 to 20 carbons.In various embodiments, adding solvent helps amide to be transferred in aqueous phase and amine coordinated malonic acid is transferred in organic phase, thus improves purification.

[0228] Many parameters can be adjusted according to the present disclosure to improve malonic acid extraction. The length of the alkyl group, the ionic strength of the fermentation broth, pH, the molar equivalent of the trialkylamine, temperature and co-solvent each have an effect on the extraction efficiency of the trialkylamine. The effect of alkyl length is illustrated in Example 36. Tripropylamine, trihexylamine and trioctylamine each can separate malonic acid from the aqueous phase and dissolve it into the 1-octanol organic phase according to the present disclosure. The extraction efficiency trioctylamine>trihexylamine>tripropylamine indicates that these longer chain lengths are preferred embodiments of the present disclosure (for high yields) at low pH and in the 1-octanol co-solvent.

[0229] As with salt precipitation, pH has an effect on the extraction of malonic acid from the aqueous phase when using trialkylamines. Trialkylamine extraction has the highest efficiency when both acid moieties are carboxylated. Example 37 shows the extraction efficiency of trioctylamine over a range of pH values. The highest extraction level occurs at pH 1.5 and decreases as the pH increases. When above the second pKa of malonic acid (5.69), there is virtually no extraction. Although the present disclosure provides for the extraction of malonic acid from the aqueous phase using trialkylamines without limiting pH, embodiments at or below pH 2.0 are preferred for maximum efficiency. In some embodiments of the present disclosure, host cell growth and malonic acid production at low pH as illustrated by Example 48 contribute to protonation of malonic acid to enhance extraction with trialkylamines. In other embodiments of the present disclosure, exogenous acid is added to assist in the protonation.

[0230] In various embodiments of the present disclosure, different concentrations of trialkylamine are used. As reported in Example 38, a linear relationship between extraction efficiency and trioctylamine:malonic acid mole fraction was observed; specifically, a linear relationship existed for trioctylamine:malonic acid mole fractions of 0 to 1. At a molar ratio above 1, 100% of the malonic acid was extracted into the organic phase. Therefore, to maximize the extraction efficiency, the amount of trialkylamine in the organic phase must be equimolar to the amount of malonic acid in the aqueous phase (e.g., fermentation broth). Ideally, a greater than equimolar amount of trialkylamine would be added to the organic phase to compensate for the reduced extraction efficiency due to other organic acids and anions in the fermentation broth.

[0231] As demonstrated in Example 39, the extraction efficiency of malonic acid by trialkylamines is negatively affected by increasing the ionic strength (salt concentration) of the aqueous phase. Desalination of various ions can be achieved by a number of methods including the addition of other reagents to produce insoluble salts, pH adjustment, and / or passage through various membranes.

[0232] In various embodiments of the present disclosure, reactive extraction is that malonic acid is subjected to Fischer esterification with alcohol at one or two carboxylic acids to form monoalkyl malonate and dialkyl malonate respectively in the presence of an acid catalyst. For example, diethyl malonate can be produced by using ethanol to subject malonic acid to Fischer esterification. Monoalkyl malonate and dialkyl malonate have low solubility and low volatility in aqueous solution, thereby enabling malonate to be separated from other volatile components in water and fermentation broth by distillation. In various embodiments of the present disclosure, the separation of esters is achieved by phase distribution in an organic solvent. In general, primary or secondary aliphatic alcohols are suitable for Fischer esterification with malonic acid; by way of example and without limitation, the alcohol may be selected from the group consisting of methanol (CAS No. 67-56-1), ethanol (CAS No. 64-17-5), 1-propanol (CAS No. 71-23-8), 1-butanol (CAS No. 71-36-3), isopropanol (CAS No. 67-64-0), and isobutanol (CAS No. 78-83-1). Many acid catalysts are suitable for Fischer esterification of alcohols with malonic acid; by way of example and without limitation, the acid catalyst may be selected from the group consisting of sulfuric acid, p-toluenesulfonic acid, scandium (III) trifluoromethanesulfonate, N,N'-dicyclohexylcarbodiimide, and tetrabutylammonium tribromide. In various embodiments of the present disclosure, malonic acid is purified from a fermentation broth by reactive distillation using ethanol to convert malonic acid to diethyl malonate. In other embodiments of the present disclosure, malonate is purified from the fermentation broth by reactive distillation using methanol to convert malonate to dimethyl malonate.In various embodiments of the present disclosure, the acid catalyst is sulfuric acid.

[0233] As illustrated in Example 41, Fischer esterification with ethanol using sulfuric acid as a catalyst can result in >99% purification of malonic acid from the aqueous phase. In various embodiments of the present disclosure, the excess ethanol used in this process is recovered under reduced pressure and recycled for use in subsequent fermentation batches. In various other embodiments of the present disclosure, the catalyst is substrate-based (e.g., amberlite resin) and is recovered, reloaded, and reused in subsequent batches.

[0234] The economics of purifying malonate by Fischer esterification can be improved in accordance with the present disclosure by co-locating malonate biorefining with alcohol biorefining or chemical refining (used herein to refer to the production of alcohols from renewable or petroleum feedstocks using synthetic chemistry rather than fermentation processes). In some embodiments of the present disclosure, malonate biorefining is co-located with alcohol biorefining or chemical refining to reduce the cost of monoalkyl malonate or dialkyl malonate synthesis. In some embodiments of the present disclosure, the alcohol produced by biorefining or chemical refining has a purity lower than that of alcohols commonly sold and distributed for fuel and / or chemical applications.

[0235] In other embodiments, the methods of the present disclosure for isolating malonate from a fermentation broth involve liquid-liquid extraction using an organic solvent. The pK values ​​of the two malonate carboxylic acid moieties are a The values ​​are 2.83 and 5.69; therefore, when the pH of the culture medium is reduced to below 2.8, more than 50% of the molecules are not ionized. These substances have a higher affinity for organic solvents than for aqueous fermentation broth. These molecules are separated into the organic phase for driving the equilibrium toward protonated substances and thus gradually entering the organic solvent. In some embodiments of the present disclosure, the acidic pH of the fermentation broth is a result of the fermentation conditions. In other embodiments of the present disclosure, the pH is reduced to a suitable acidity by adding an acid. In various embodiments of the present disclosure, the solvent is selected from, but not limited to, the group consisting of: ethyl acetate, dichloromethane, dichloroethane, decane, dodecane, hexane, octanol, pentanol, or a mixture thereof. In some embodiments of the present disclosure, host cell growth and malonate production at low pH help to protonate malonate to enhance the solubility of malonate in organic solvents. In other embodiments of the present disclosure, exogenous acid is added to help this protonation. In some embodiments of the present disclosure, the solvent is removed (completely or partially) by vacuum distillation. In various embodiments of the present disclosure, concentrated malonic acid is esterified with an alcohol, as described elsewhere, and the ester is purified by distillation.

[0236] In other embodiments of the present disclosure, malonate is removed from the fermentation broth by binding to an ion exchange resin. In various embodiments of the present disclosure, the resin is selected from, but not limited to, the following: VP OC 1065, MP-64 chloride ion type, 66 free alkali, IRA-67 free base, IRA-96 free base, 4200 Chloride ion type, MonoPlusM 500 Chloride ion type, 1X8 chloride type, Amberlyst A26 hydroxide type, IRA958 chloride form. In various embodiments of the present disclosure, malonic acid is eluted from the resin using a pH or salt gradient. In other embodiments of the present disclosure, one or more of these resins are used to remove impurities from concentrated malonic acid.

[0237] Although the purification methods of the present disclosure can be used as a single-step purification method for purifying malonate from fermentation broth, two or more purification methods can be used in succession according to the present disclosure. For example and without limitation, precipitation of malonate with calcium hydroxide can be followed by Fischer esterification of malonate with ethanol and further purification of the resulting diethyl malonate using distillation.

[0238] Section 8: Chemistry of other chemicals using malonic acid

[0239] Malonic acid is a chemical precursor in many important industrial reactions. The present disclosure enables the use of biogenic malonic acid in these reactions for the first time. In any known reaction utilizing malonic acid as a reactant, biogenic malonic acid can be used. In any reaction utilizing monoalkyl malonates, dialkyl malonates, or Meldrum's acid, biogenic malonic acid can be used after conversion to an appropriate reaction substrate. In addition, the present disclosure provides novel chemical synthesis methods utilizing malonic acid as a starting material. For these reactions, malonic acid produced by any means including bioproduction of malonic acid by enzymatic oxidation of malonic semialdehyde, synthetic oxidation of biologically or synthetically derived 3-hydroxypropionic acid, or synthetic oxidation of biologically or synthetically derived 1,3-propanediol can be used.

[0240] Thus, in addition to providing bioderived malonate for use in known chemical reactions, the present disclosure provides novel methods for using malonate and malonate-derived compounds to produce other useful compounds.

[0241] The present disclosure is due in part to the fact that malonic acid and malonic acid derivative compounds can be used as substrates in the synthesis of acrylic esters (i.e., acrylate, methyl acrylate, ethyl acrylate, propyl acrylate, and butyl acrylate) by reaction with formaldehyde. Acrylic esters account for billions of dollars per year in terms of sales and are used in products ranging from diapers to shampoos to films and coatings. The present disclosure provides two alkaline syntheses for producing acrylic esters by reacting malonic acid or malonic acid diesters with formaldehyde. In one method, a malonic acid diester is reacted with formaldehyde in the presence of a suitable base (e.g., diethylamine) to produce a diester of 2-methylenemalonic acid. Subsequent ester saponification and heating result in the production of 1 part acrylic acid, 1 part CO2, and 2 parts alcohol. The composition, base, and solvent of the ester portion of the malonic acid diester used can vary widely. For example and without limitation, dimethyl ester, diethyl ester, dipropyl ester, dibutyl ester, diisopropyl ester, or dihexyl ester can be utilized. Similarly, the base used can be one of many bases. In various embodiments, the base is a trisubstituted amine. Non-limiting examples of trisubstituted amines suitable for use according to the method of the present disclosure include trisubstituted amines selected from the group containing piperidine, trimethylamine, triethylamine, tripropylamine, triisopropylamine, tributylamine or a longer chain trialkylamine. In some embodiments of this aspect of the present disclosure, diethyl malonate is reacted with formaldehyde in pyrrolidine to form acrylate and subsequently saponified and heated to produce 1 part of acrylic acid, 1 part of CO and 2 parts of ethanol in a Knoevenagel condensation. In other embodiments of the method of the present disclosure, dimethyl malonate is reacted with formaldehyde in diethylamine to form 2-methylene dimethyl malonate, which is subsequently converted into acrylic acid, CO and methanol.

[0242] In other embodiments, a magnesium salt catalyzes the condensation of a malonate diester with formaldehyde to form a 2-methylene malonate diester, which is then treated in the manner described. For example and without limitation, magnesium silicate is a magnesium salt suitable for this reaction. In some embodiments, diethyl malonate is reacted with formaldehyde in the presence of a magnesium silicate catalyst to form diethyl 2-methylene malonate. In other embodiments, diisopropyl malonate is reacted with formaldehyde in the presence of a magnesium silicate catalyst to form diisopropyl 2-methylene malonate. A general process using a monoester of malonate is described herein:

[0243]

[0244] In this case, R1 is selected from the non-limiting examples of H, CH3, CH3CH2, CH3(CH2)2, CH3(CH2)3, (CH3)2CH2 or (CH3)2CH2CH2.

[0245] In another embodiment, the Doebner modification of the Knovenagel condensation is employed and malonic acid is reacted with formaldehyde in pyridine to produce acrylate. Example 28 illustrates the production of acrylate from malonic acid according to this embodiment of the present disclosure. In other embodiments, monomethyl malonate is reacted with formaldehyde in pyridine to form methyl acrylate. In the process described herein, the monomethyl malonate serves as the starting material in the Doebner modification of the Knovenagel condensation.

[0246]

[0247] R1 is selected from the non-limiting examples of H, CH3, CH3CH2, CH3(CH2)2, CH3(CH2)3, (CH3)2CH2, or (CH3)2CH2CH2. In other embodiments, monoethyl malonate is reacted with formaldehyde in pyridine to form ethyl acrylate.

[0248] In other embodiments of the present disclosure, malonic acid and malonic acid derivative compounds can be used as substrates in the synthesis of unsaturated dicarboxylic acids by reaction with various substrates, including but not limited to aldehydes, alkyl halides, dialdehydes, alkyl dihalides, terminal olefins, and combinations thereof (i.e., bifunctional substrates). In the case of reaction of malonic acid or malonic acid derivative compounds with aldehydes, alkenes are formed, which can subsequently be hydrogenated to produce saturated products. In general, malonic acid and malonic acid derivative compounds can be used to synthesize C5-C12 straight-chain, saturated or unsaturated diacids (i.e., glutaric acid (CAS110-94-1), adipic acid (CAS124-04-9), pimelic acid (CAS111-16-0), suberic acid (CAS505-48-6), azelaic acid (CAS123-99-9), sebacic acid (CAS111-20-6), undecanedioic acid (CAS1852-04-6), dodecanedioic acid (CAS 693-23-2)) and their corresponding dialkyl esters. In various embodiments of the present disclosure, malonic acid is used as a substrate in the chemical synthesis of C5-C12 straight-chain saturated diacids. In various embodiments of the present disclosure, malonic acid or malonic acid derivative compounds are used as substrates in the chemical synthesis of glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid or dodecanedioic acid.

[0249] In other embodiments of the present disclosure, compounds derived from malonic acid provided by or without the present disclosure are used as substrates in the chemical synthesis of glutaric acid. Glutaric acid can be formed according to the present disclosure by synthesizing diethyl malonate and formaldehyde (see Ahluwalia et al., "Organic reaction mechanisms," 2nd edition Alpha Science International: Harrow (2005) pp. 340-341); synthesizing diethyl malonate and dichloromethane (see Perkin et al., J Am Chem Soc 59:990-995 (1891)); and synthesizing Meldrum acid and formaldehyde (see Hedge et al., J. Org. Chem. 26:3166-3170 (1961)).

[0250] In other embodiments of the present disclosure, malonic acid or malonic acid derivative compounds are used as substrates in the chemical synthesis of adipic acid. Hexane dicarboxylic acid is one of the two compounds used to produce nylon 6,6 and accounts for billions of dollars in raw material sales each year. Nylon 6,6 is used in a wide range of durable consumer goods, including carpets, automobile air bags and ropes. Adipic acid can be formed according to the present disclosure by the reaction of magnesium-chelated malonic acid monoesters and 1,2-dichloroethane. Alkylation of dichloroethane and dimethyl malonate has been described (U.S. Patent No. 6,262,298); however, the undesired product cyclopropane-1,1-dicarboxylic acid dimethyl ester is the only product reported at 99.3% purity and 83% theoretical yield. The formation of cyclopropane-1,1-dicarboxylic acid ester is caused by intramolecular alkylation after the initial alkylation of 1,2-dichloroethane. In contrast, the present disclosure provides materials and methods using magnesium-chelated malonate monoesters that do not allow for undesired intramolecular alkylation of cyclopropane. The magnesium-chelated malonate monoester is reacted with 1,2-dichloroethane via decarboxylation-promoted enol production; a second intramolecular reaction (i.e., formation of cyclopropane dicarboxylate) cannot proceed after decarboxylation. An exemplary catalyst for use according to this embodiment of the present disclosure is magnesium silicate. It is understood by those skilled in the art that various dihaloalkanes (e.g., 1,3-dihalopropane, 1,2-dihalopropane) can also be used with magnesium-chelated malonate monoesters to form dicarboxylic acid products and that those halide groups can be I, Cl, Br, or F. By way of example and without limitation, 1,3-dihalopropane can be reacted with magnesium-chelated malonate monoesters to produce pimelic acid.

[0251] The present disclosure also provides a method for producing adipic acid by the reaction of malonic acid and malonic acid derivative compounds with glyoxal. It is known that olefinic acid is formed by the Knovenagel reaction of malonic acid diesters and aliphatic monoaldehydes (see, for example, Rao et al., J Am Oil Chem Soc, 70: 297-299 (1993); and Zhang et al., Synth Comm, 40: 3093-3100 (2010)). The present disclosure is partly due to the discovery that dialdehydes can be reacted with malonic acid derivative compounds to produce corresponding 1,3-diene-1,1,4,4-tetracarboxylic acid tetraesters. For example, diethyl malonate reacts with glyoxal to produce tetraethyl butane-1,3-diene-1,1,4,4-tetracarboxylate; similarly, malonic acid reacts with glyoxal (oxalaldehyde) to produce hexa-2,4-dienedioic acid. When necessary, ester hydrolysis is performed by adding acid to the reaction mixture. Heat-induced decarboxylation to form an unsaturated dicarboxylic acid (i.e., hexa-2,4-dienedioic acid), and the unsaturated dicarboxylic acid is subsequently hydrogenated to produce adipic acid. In some embodiments of the present disclosure, diethyl malonate and glyoxal are condensed to produce dialkylated ethyl dimalonate; followed by saponification to hydrolyze the ester group, followed by heat-induced decarboxylation to produce the unsaturated dicarboxylic acid. Hydrogenation of the unsaturated dicarboxylic acid produces adipic acid. This method is summarized in the following reaction scheme, wherein the alkyl groups (R1 and R2) are selected from non-limiting examples: H, CH3, CH3CH2, CH3(CH2)2, CH3(CH2)3, (CH3)2CH2, or (CH3)2CH2CH2.

[0252]

[0253] It is understood by those skilled in the art that the Knovenagel condensation of various dialdehydes and malonate derivative compounds can be used to synthesize saturated and unsaturated (where olefins are at the 2-position and n-2-position) dicarboxylic acids according to the methods of the present disclosure. For example and without limitation, malondialdehyde can be used to synthesize heptanedicarboxylic acid, butane-1-4-dialdehyde can be used to synthesize octanedicarboxylic acid, and pentane-1,5-dialdehyde can be used to synthesize nonanedicarboxylic acid.

[0254] In other embodiments of the present disclosure, malonic acid or malonic acid derivative compounds are used as substrates in the chemical synthesis of pimelic acid. In addition to the above-mentioned dihaloalkane and dialdehyde pathways for obtaining pimelic acid, the present disclosure also provides a method for synthesizing pimelic acid by the reaction of propanedial (acrolein) and malonic acid that may or may not be derived from malonic acid as provided by the present disclosure. The Michael addition of malonic acid to propanedial produces 2-(3-oxopropyl) malonic acid, and then condenses with the aldehyde moiety with another malonic acid molecule to produce hex-1-ene-1,1,6,6-tetracarboxylic acid. Heat-induced decarboxylation followed by hydrogenation produces pimelic acid.

[0255] In another method of the present disclosure, malonic acid is used as a substrate in the chemical synthesis of lactam or lactone. In one embodiment, the synthesis of diethyl malonate is converted into delta-valerolactam by the following steps: diethyl malonate is produced by the Fisher esterification of malonic acid and ethanol. The Michael addition of diethyl malonate to acrylonitrile produces 2-(2-cyanoethyl) diethyl malonate. Hydrogenation of the nitrile part of 2-(2-cyanoethyl) diethyl malonate can produce lactamization under mild conditions to produce the amino acid of 2-oxopiperidin-3-ethyl formate. The ester part of 2-oxopiperidin-3-ethyl formate is saponified, followed by heating to cause decarboxylation to obtain delta-valerolactam. This non-limiting example is illustrated here:

[0256]

[0257] In another method of the present disclosure, ethyl malonate is used as a substrate in the chemical synthesis of ethyl acetate. Heating ethyl malonate causes heat-induced decarboxylation to form ethyl acetate. In some embodiments, ethyl malonate is used for the synthesis of ethyl acetate. Those skilled in the art will recognize that there are many established methods for preparing monoalkyl malonate from the malonic acid provided by the present disclosure.

[0258] In another method of the present disclosure, a monoalkyl malonate (e.g., monomethyl malonate, monoethyl malonate, monopropyl malonate, monobutyl malonate, monoisobutyl malonate) is used as a substrate in the chemical synthesis of 3-hydroxypropionic acid or its related alkyl esters. Borane will chemically selectively reduce the free carboxylic acid portion of the monoalkyl malonate and produce the corresponding 3-hydroxypropionic acid alkyl ester. The 3-hydroxypropionic acid alkyl ester itself is a valuable product. The ester can also be hydrolyzed to produce 3-hydroxypropionic acid. 3-Hydroxypropionic acid can be dehydrated to generate acrylic acid as a valuable bulk chemical described in more detail herein. In some embodiments, the monoalkyl malonate is used to synthesize 3-hydroxypropionic acid. In some embodiments, the monoalkyl malonate is monomethyl malonate. In some embodiments, the monoalkyl malonate is monoethyl malonate.

[0259] In another method of the present disclosure, malonic acid is used to synthesize dialkylated 1,3-dicarbonyl compounds. In various embodiments, malonic acid is used as a substrate in the chemical synthesis of a compound selected from the group consisting of: 2,2-dimethyl diethyl malonate, 2,2-diethyl diethyl malonate, 2,2-dipropyl diethyl malonate, 2,2-dibutyl diethyl malonate, 2,2-dimethyl dimethyl malonate, 2,2-diethyl dimethyl malonate, 2,2-dipropyl dimethyl malonate and 2,2-dibutyl dimethyl malonate. The dialkylated 1,3-dicarbonyl compounds synthesized by synthesizing dialkyl malonates obtained from alkyl halides can be formed according to the present disclosure (see Gatterman et al., "The practical methods of organic chemistry" 3rd edition, The Macmillan Company: New York, pp. 189-191 (1916)).

[0260] In other embodiments of the present disclosure, malonic acid or malonic acid derivative compounds provided by the present disclosure are reacted with urea to form barbituric acid, the core structure of approximately 50 related pharmaceuticals currently on the market, including phenobarbital.

[0261] In some embodiments of the synthesis methods of the present disclosure, highly purified malonate preparations are used for synthesis reactions and / or related downstream applications; in other embodiments, less pure malonate is used. In other cases, crude lysates or other relatively unpurified malonate can be used for synthesis reactions and / or downstream applications. For most synthetic chemical reactions, malonate purity greater than 90% w / w is generally required. In the case where malonate is used for polymerization reactions, even greater purity (i.e., 95% w / w or more) may be required. However, in other applications, malonate preparations of much less purity may be used. For example, malonate is used as animal feed, and for this purpose, productive cells themselves or their crude lysates may be used.

[0262] In another embodiment of the present disclosure, malonic acid (produced by any means) is reacted with glutaric semialdehyde (which can also be produced from malonic acid) to produce heptanedioic acid. This is another example of a Knovenagel condensation and can be performed with a wide range of solvents and catalytic reagents associated with this reaction. Heptanedioic acid is then reduced using catalytic hydrogenation to produce heptanedioic acid (pimelic acid). Pimelic acid has a wide variety of uses, including as a monomer in nylon 5,7; as a fermentation medium supplement for enhancing biotin biosynthesis; and as a plasticizer. Prior art methods for the synthesis of pimelic acid are expensive and low-yielding. The present disclosure provides a new chemical synthesis method for this important compound that can be derived from malonic acid as provided by the present disclosure.

[0263] In other embodiments of the present disclosure, steam or liquid phase hydrogenation is used to convert dialkyl malonate to 1,3-propylene glycol. Due to the reactive nature of carbon No. 2 of malonic acid and malonic acid esters, polysubstituted derivatives of various compounds can be produced using malonic acid provided by the present disclosure. In some embodiments, neopentyl glycol (2,2-dimethylpropane-1,3-diol), as an important component of some polyesters, is produced by first producing 2,2-dimethyl-dialkyl malonate. This compound can be produced using a variety of bases as catalysts and methyl halides as a source of methyl groups. Subsequent hydrogenation of the ester group results in the production of neopentyl glycol and related alcohols from the ester group. This represents a novel approach to obtain this important industrial compound. In addition, due to the highly reactive nature of carbon No. 2 of malonic acid and malonic acid-derived compounds, other alkyl halides can be used to produce novel compounds with novel properties potential when incorporated into polymers.

[0264] The disclosure, having been described in detail, is illustrated by the following examples, which should not be construed as limiting the disclosure in view of its various aspects, embodiments and applications. Example

[0265] Example 1: Construction of recombinant nucleic acids and expression vectors encoding wild-type EHD3 and EHD3 mutants E124S, E124A, E124A / E308V, and E124V malonyl-CoA hydrolases for producing malonate in Escherichia coli

[0266] The present disclosure provides methods for producing malonate in an E. coli host, and E. coli host cells that produce malonate and express EHD3 mutant enzymes, including but not limited to E124S, E124A, and E124V. This example describes the construction of protein coding sequences for EHD3 and mutant EHD3 proteins used in the present disclosure, expression vectors containing those coding sequences, and host cells containing those expression vectors. Nucleic acids encoding wild-type Saccharomyces cerevisiae EHD3 were amplified by PCR from Baker's yeast using primer pair A93 / A94. The point mutation E124A was introduced using primer pairs A93 / A96 and A95 / A94, the mutation E124V was introduced using primer pairs A93 / A98 and A97 / A94, and the mutation E124S was introduced using primer pairs A93 / A100 and A99 / A94. The resulting nucleic acids were cloned into a plasmid containing a pSC101 origin of replication, a chloramphenicol resistance cassette, and a P LacO1 The obtained vector was transformed into E. coli DH10b host and plated in a medium containing 50 μg / ml chloramphenicol (Cm 50 ) and 2% w / v glucose on Luria-Bertani (LB) agar plates. Individual colonies were then inoculated into 3 ml LB medium in 48-well plates; after 6 hours of growth, plasmids were isolated and the EHD3 protein coding region was sequenced. When the plasmid of the EHD3-derived clone (E124A) was sequenced, it was found that the second point mutation E308V stabilized the EHD3 (124A) clone; the presence of uncharged valine residues may stabilize protein folding. The EHD3 (E124A, E308V) strain was also used to produce malonate (see Example 2). Primers A93-A100 are SEQ ID NOs: 13-20, respectively.

[0267] Example 2: In vivo production of malonate in E. coli using EHD3 mutants E124S and E124A / E308V

[0268] This example describes host cells and culture conditions that lead to in vivo production of malonate in E. coli host cells using a heterologous EHD3 malonyl-CoA hydrolase. E. coli strain K12 was transformed with vectors containing wild-type EHD3, EHD3(E124A), EHD2(E124V), EHD3(E124S), EHD3(E124A, E308V), or an empty vector negative control. Plates were plated on LB agar plates (Cm 50 After overnight growth at 37°C, individual colonies were inoculated into 3 ml LB (Cm 50The cultures were incubated at 37°C on a plate shaker for 6 hours, at which time each culture was seeded at 1% v / v into 48-well plates supplemented with Cm 50 and mixed carbon sources (0.5% glycerol, 0.05% glucose, 0.2% lactose) in M9 minimal medium. The cultures were incubated at 30°C on a plate shaker and 500 μL of the fermentation broth samples were removed after 48 hours of incubation for analysis.

[0269] Centrifuge (x6000g, 1 minute) sample and analyze supernatant to quantify malonic acid.Chemical standards were prepared in 20 μM water.Malonate was separated on a Shimadzu Prominence XR UPLC connected to a refractive index detector and a UV detector (210nm monitoring).Product separation was performed on a Bio-Rad Aminex HPX-87h fermentation monitoring column.UPLC was programmed to run isocratically, using 5mM H2SO4 as eluent, and the flow rate was 600 μL per minute.Each sample was injected with 10 μL, and the sample plate temperature was maintained at 4 ° C.Malonate standards began to elute at approximately 19.8 minutes.Adding standards to samples containing malonic acid showed that the malonic acid peak area increased in proportion, thereby confirming the production of malonic acid.Calculate malonic acid concentration (mg / l) by comparing with the standard curve prepared by authoritative malonic acid.

[0270] For cultures with empty vector controls, no peak was observed at the same retention time as the malonate standard, and the integrated peak area was below the detection limit of the instrument (i.e., no malonate production was observed). Malonate production was observed in samples with wild-type EHD3, EHD3(E124A), EHD3(E124V), EHD3(E124S), and EHD3(E124A, E308V); malonate concentrations were (mean ± standard deviation; n = 3): wild-type EHD3, 6.0 ± 0.2 mg / l; EHD3(E124A), 7.6 ± 0.7 mg / l; EHD3(E124V), 0.28 ± 0.03 mg / l; EHD3(E124S), 82.3 ± 7.8 mg / l; and EHD3(E124A / E308V), 8.35 ± 2.5 mg / l. EHD3(E124V) resulted in reduced malonate production relative to the wild-type protein, likely due to both poor malonyl-CoA substrate binding and high promiscuous activity toward other endogenous acyl-CoA molecules. Similarly, EHD3(E124A) resulted in only a small increase in malonate production relative to wild-type EHD3. The EHD3 mutant E124S resulted in a significant (p<0.05, t-test) increase in malonate production over wild-type EHD3, EHD3(E124A), and EHD(E124V), indicating that E124S is important in increasing malonyl-CoA substrate binding and malonate production.

[0271] Example 3: Construction of other EHD3E124 mutants and expression vectors for in vivo production of malonate in E. coli

[0272] Example 1 describes the construction of E. coli expression vectors for wild-type EHD3 and a subset of E124 mutations (specifically E124A, E124V, E124S, and E124A / E308V), and Example 2 describes their use for production of malonate in E. coli cells. This example describes the construction of E. coli expression vectors for all EHD3 E124 point mutations, namely E124G, E124T, E124C, E124L, E124I, E124M, E124P, E124Y, E124W, E124D, E124N, E124Q, E124H, E124K, E124R, and E124F. Expression vectors were constructed using the pSC101 origin and p124F vectors as described in Example 1. LacO1The E. coli vector with a replication origin was used to construct these EHD3 mutants. The forward PCR primer contained a nucleic acid sequence (5'-aattttttactgatNNNtattctttgaattttcaaatagc-3'), wherein the sequence "NNN" is three nucleotides encoding the desired E124 amino acid point mutation; similarly, the reverse PCR primer contained a nucleic acid sequence (5'-ttcaaagaataaNNNatcagtaaaaaatttgatggacttg-3'), wherein the sequence "NNN" is complementary to the three nucleotides encoding the desired E124 amino acid point mutation. The forward PCR primer was used together with PCR primer A94 (SEQ ID NO: 14), and the reverse PCR primer was used together with PCR primer A93 (SEQ ID NO: 13) to generate two overlapping EHD3 gene fragments containing the desired point mutation. The two EHD3 gene fragments were amplified with primers A93 and A94 to generate a full-length EHD3 gene containing the desired point mutation. Expression vectors were constructed using standard cloning protocols and transformed into E. coli DH10b and subsequently isolated as described in Example 1 and verified by sequencing.

[0273] Example 4: In vivo production of malonate in recombinant E. coli using the EHD3 E124 mutant

[0274] This example describes the fermentation of a panel of E. coli host cells, each member of which contains one of 19 possible EHD3 E124 amino acid substitutions, and determination of the level of malonate produced. Specific E124 point mutations should theoretically improve malonyl-CoA binding and malonate production relative to wild type; specifically, point mutations E124T, E124N, E124Q, E124H, E124K, and E124R should improve malonate production, theoretically due to the introduction of amino acids containing functional groups that improve interaction with the terminal carboxylic acid moiety of malonate. Point mutations E124S, E124Q, and E124K have side chains located in the EHD3 binding pocket in a position that should theoretically best cooperate with malonate binding. Malonate production and quantification were performed substantially as described in Example 2.

[0275] Example 5: Construction of a YciA malonyl-CoA hydrolase expression vector for in vivo production of malonate in Escherichia coli

[0276] This example describes the production of malonate in E. coli using E. coli YciA according to the present disclosure. Wild-type E. coli acyl-CoA YciA was PCR amplified from the host genome using primers A120 (SEQ ID NO: 21) and A121 (SEQ ID NO: 22). The resulting nucleic acid was cloned into a pSClO1 E. coli expression plasmid containing the pSC101 replication origin and the ampicillin resistance gene.LacO1 The control vector included a LacO1 An empty vector of the yciA gene was inserted after the promoter. Individual colonies were grown, their plasmids were isolated, and the coding region of the YciA gene insert was sequenced as described in Example 1.

[0277] Example 6: In vivo production of malonate in recombinant E. coli using heterologous YciA

[0278] This example describes host cells and culture conditions that lead to in vivo production of malonate in E. coli host cells using an expression vector encoding a heterologous YciA malonyl-CoA hydrolase as described in Example 5. Wild-type E. coli strain K12 was transformed with a vector containing E. coli YciA; wild-type E. coli with an empty vector served as a negative control. Malonate production and quantification were performed as described in Example 2.

[0279] Example 7: Construction of an engineered EHD3 malonyl-CoA hydrolase expression vector for in vivo production of malonate in yeast

[0280] The present disclosure also provides expression vectors, host cells and methods for in vivo production of malonate in yeast cells.In general, yeast cells can tolerate higher concentrations of organic acids in fermentation broths than E. coli and have better established industrial fermentation protocols.

[0281] The yeast expression vector described in this example was generated in part by using the E. coli expression vector described in Examples 1 and 3 as a PCR template for the EHD3 gene. The yeast expression vector contains a 2-micron replication origin, a ura3 auxotrophic marker, and a TEF promoter; the vector also contains a puc replication origin and an ampicillin or chloramphenicol resistance cassette to enable amplification of the vector in E. coli. The plasmid was transformed into the Saccharomyces cerevisiae BY4741 or BY4742 background, two derivatives of the S288C parent strain.

[0282] Other mutations can be introduced into the EHD3 coding sequence to abolish mitochondrial targeting. The basic amino acids R3, K7, K14, K18, and R22 in the EHD3 coding sequence are important for mitochondrial targeting, and mutating any one or more of them to A or V will reduce mitochondrial EHD3 expression and increase cytoplasmic EHD3 expression. Mutations are introduced by PCR amplification of the EHD3 template with forward and reverse primers containing the desired point mutations. Mutations are introduced using primers that match the EHD3 gene except that the nucleotide sequence at the position of the desired amino acid point mutation is changed to "gyt" (where y is a cytosine or thymine nucleotide in a mixed population of oligonucleotide PCR primers). The gene fragment is first cloned into a pSC101 replication origin, pLacO1 coli expression vector containing the promoter and chloramphenicol resistance marker, and the vector sequence was confirmed as described in Example 1; after isolating the desired mutant, the EHD3 gene was amplified and cloned into a yeast expression vector.

[0283] Example 8: In vivo production of malonate in recombinant yeast using engineered EHD3 malonyl-CoA hydrolase

[0284] Saccharomyces cerevisiae BY4742 host cells were transformed using standard protocols with yeast expression vectors with heterologous EHD3 or empty vector negative control plasmids prepared substantially as described in Example 7. Transformants were streaked on synthetic complete omission medium (SD) agar plates lacking uracil and cultured at 30°C; individual colonies were grown overnight at 30°C in 3 mL SD medium and subsequently diluted at 1% v / v into 3 ml SD lacking uracil. Strains were cultured at 30°C for 72 hours; 500 μl aliquots were sampled at 24, 48, and 72 hour time points to quantify malonate production and OD 600 The TEF promoter is a constitutive promoter.

[0285] Example 9: Increasing malonate biosynthesis in engineered yeast by expressing heterologous acetyl-CoA synthetase

[0286] In addition to the methods, vectors, and host cells for expressing heterologous malonyl-CoA hydrolases and producing malonate in vivo as described in Examples 1-8, the present disclosure also provides methods and host cells for improving the titer, yield, and / or productivity of malonate. In one aspect, malonate production is improved by increasing the biosynthesis of acetyl-CoA. This example describes heterologous expression of acetyl-CoA synthetase in a recombinant Saccharomyces cerevisiae host comprising a malonyl-CoA hydrolase pathway and the resulting improvements in malonate production. The five acetyl-CoA synthetase proteins described are Saccharomyces cerevisiae ACS1 and ACS2, Escherichia coli AcsA, Salmonella enterica Acs, and Bacillus subtilis AcsA. All genes were PCR amplified from their respective hosts and cloned into a yeast expression vector with a 2 micron replication origin, a ura3 auxotrophic marker, and a TEF promoter; the vector also contained a puc replication origin and an ampicillin resistance cassette to achieve amplification of the vector in E. coli.

[0287] Example 10: Increasing malonate biosynthesis in engineered yeast by expressing heterologous pyruvate dehydrogenase

[0288] While Example 9 describes increasing acetyl-CoA biosynthesis by expressing heterologous acetyl-CoA synthetase, this example describes increasing acetyl-CoA biosynthesis by expressing heterologous pyruvate dehydrogenase. Specifically, Saccharomyces cerevisiae pyruvate dehydrogenases PDA1, PDB1, LAT1, LPD1, and PDX1 were heterologously expressed in a recombinant Saccharomyces cerevisiae comprising a malonyl-CoA hydrolase pathway. The genes were all PCR amplified from the Saccharomyces cerevisiae chromosome and cloned into a yeast expression vector with a 2-micron replication origin, a ura3 auxotrophic marker, and a TEF promoter; the vector also contained a puc replication origin and an ampicillin resistance cassette to enable amplification of the vector in E. coli.

[0289] Example 11: Increasing malonate biosynthesis in Escherichia coli and Saccharomyces cerevisiae by heterologous expression of the ethanol catabolic pathway

[0290] This example describes a third approach to increase acetyl-CoA biosynthesis: heterologous expression of an ethanol catabolic pathway. The ethanol catabolic pathway comprises two or three enzymes. Alcohol dehydrogenase and acetaldehyde dehydrogenase (acylating), or alcohol dehydrogenase, acetaldehyde dehydrogenase (non-acylating), and acetyl-CoA synthetase. Alcohol dehydrogenases Saccharomyces cerevisiae ADH2, Escherichia coli AdhP, and Homo sapiens ADH1A, Homo sapiens ADH1B, and Homo sapiens ADH1C are cloned in combination with acetaldehyde dehydrogenase (acylating) or aldehyde dehydrogenase and acetyl-CoA synthetase. Acetaldehyde dehydrogenase (acylating) Escherichia coli MhpF, Escherichia coli AdhE, a species of Pseudomonas CF600 DmpF, and Pseudomonas putida TodL ​​are also cloned in combination. In the ethanol catabolic pathway utilizing acetaldehyde dehydrogenase (non-acylating), Saccharomyces cerevisiae ALD2, ALD3, ALD4, ALD5, and ALD6 are used. The acetyl-CoA synthetases used were Saccharomyces cerevisiae ACS1 and ACS2 and Escherichia coli Acs. All genes were PCR amplified from genomic DNA.

[0291] For E. coli host cells, a p15a replication origin, an ampicillin resistance marker, and a P lacO1 The vector backbone of the promoter expressed the ethanol catabolic pathway. All combinations of all two-gene and three-gene pathways were constructed into a single operon. E. coli K12 was co-transformed with the EHD3 expression plasmid and the ethanol catabolic pathway plasmid and plated on LB agar plates (Cm 50 , Cb 50 , 2% w / v glucose). The control strain had an empty vector. Production culture and analysis were performed as described in Example 2, with the notable exception of the addition of a second antibiotic required for the maintenance of the second plasmid.

[0292] Saccharomyces cerevisiae fermentations can be performed using the same ethanol concentrations as for the E. coli experiments. The yeast expression vector has a 2 micron origin, a ura3 auxotrophic marker, and a CUP promoter, and all combinations of the ethanol catabolic pathway are constructed on this vector backbone; the plasmid without the ethanol catabolic pathway serves as a negative control. The ethanol catabolic pathway plasmid was transformed into recombinant Saccharomyces cerevisiae BY4742 containing an engineered EHD3 malonyl-CoA hydrolase pathway on the yeast chromosome. All fermentations were performed at 30°C in SD medium without uracil. After 12 or 24 hours of growth, ethanol catabolic pathway expression was induced with 100 μM copper sulfate. 500 μl aliquots were sampled at 24, 48, and 72 hours to quantify ethanol, acetaldehyde, acetic acid, and malonate concentrations; the cell density OD at each time point was also recorded. 600 Measurement results. In addition to the malonate titer, ethanol consumption was also calculated.

[0293] Example 12: Increasing malonate biosynthesis in Saccharomyces cerevisiae by heterologous expression of ATP citrate lyase

[0294] This embodiment describes a fourth method for increasing acetyl-CoA biosynthesis and improving malonate production in recombinant Saccharomyces cerevisiae. ATP citrate lyase (EC 2.3.3.8) catalyzes the formation of acetyl-CoA, oxaloacetate and ADP from citric acid in the cytoplasm. ATP citrate lyase from oleaginous yeast Candida bentii, Cryptococcus albus, oleaginous oleaginous yeast, Rhodotorula toro, Rhodotorula glutinosus, Trichosporon dermatophyte, Yarrowia lipolytica was amplified from genomic DNA PCR and cloned into the CUP promoter in a yeast expression vector; the expression vector contains a 2 micron origin and a leu2d auxotrophic marker. The plasmid without ATP citrate lyase serves as a negative control.

[0295] The ATP citrate lyase pathway plasmid was transformed into recombinant Saccharomyces cerevisiae BY4742 containing an engineered EHD3 malonyl-CoA hydrolase pathway on the yeast chromosome. All experiments were performed at 30°C in SD medium without uracil. After 12 or 24 hours of growth, ATP citrate lyase pathway expression was induced with 100 μM copper sulfate. Some cultures were also supplemented with 0.5, 1, 2.5 or 5 g / l citrate to provide additional evidence of pathway activity. 500 μl aliquots were sampled at 24, 48 and 72 hours to quantify citric acid (when applicable) and malonate concentrations; cell density OD at each time point was also recorded. 600 Measurement Results. In addition to the malonate titer, the citrate consumption can be calculated.

[0296] Example 13: Increasing malonate biosynthesis in recombinant yeast by improving host cell fatty acid storage

[0297] This example describes a fifth method for increasing acetyl-CoA biosynthesis and improving malonate production in recombinant Saccharomyces cerevisiae. The fatty acid biosynthetic pathway competes with malonate production for acetyl-CoA and malonyl-CoA, and altering host cell fatty acid anabolism can increase malonate production. The present disclosure provides host cells comprising genetic modifications to one or more nucleic acids encoding proteins that affect fatty acid storage and catabolism. In Saccharomyces cerevisiae, proteins SNF2, IRA2, PRE9, PHO90, SPT21, POX1, ANT1, FOX3, EHD3, PAS1, PAS3, ARE1, ARE2, DGA1, LRO1, ACL1, MAE1, GLC3, GLG1, GLG2, PAT1, and PEX11 were knocked out individually and in combination and the resulting strains were cultured to produce malonate. All Saccharomyces cerevisiae strains constructed contained an engineered EHD3 malonyl-CoA hydrolase pathway for producing malonate. Fermentation was performed as described in Example 8 and malonate was quantified as described in Example 2.

[0298] Example 14: Increasing malonate biosynthesis in recombinant yeast by increasing β-oxidation enzyme activity

[0299] This example describes a sixth method for increasing acetyl-CoA biosynthesis and improving malonate production in recombinant Saccharomyces cerevisiae. In addition to reducing host cell fatty acid anabolism, increasing host cell fatty acid catabolism can also increase malonate production. The present disclosure provides host cells modified to increase expression of PAT1 and / or PEX11. PAT1 and PEX11 were PCR amplified from genomic Saccharomyces cerevisiae DNA and cloned behind the CUP promoter in a yeast expression vector; the expression vector contained a 2 micron origin and a leu2d auxotrophic marker. A plasmid without β-peroxidase served as a negative control.

[0300] The β-oxidation enzyme pathway plasmid was transformed into recombinant Saccharomyces cerevisiae BY4742 containing an engineered EHD3 malonyl-CoA hydrolase pathway on the yeast chromosome. The engineered malonyl-CoA hydrolase was integrated into the chromosome using standard recombinant methods. The resulting strain served as a basis for testing subsequent modifications and their effects on malonate production. All experiments were performed at 30°C in SD medium without uracil. After 12 or 24 hours of growth, pathway expression was induced with 100 μM copper sulfate. Some cultures were also supplemented with 0.5, 1, 2.5 or 5 g / l palmitic acid to provide additional evidence of pathway activity. 500 μl aliquots were sampled at 24, 48 and 72 hours to quantify palmitic acid (when applicable) and malonate concentrations; cell density OD at each time point was also recorded. 600 Measurement results. In addition to the malonic acid titer, the palmitic acid consumption can be calculated.

[0301] Example 15: Improving malonate biosynthesis in engineered yeast by increasing acetyl-CoA carboxylase activity

[0302] In addition to methods, vectors and host cells for expressing heterologous malonyl-CoA hydrolases and producing malonate in vivo, the disclosure also provides methods and host cells for improving the titer, yield and / or productivity of malonate. In one aspect, malonate production is improved by increasing the biosynthesis of malonyl-CoA. Malonyl-CoA is the penultimate intermediate in the biosynthesis of malonate from acetyl-CoA, and in Saccharomyces cerevisiae, this reaction is catalyzed by acetyl-CoA carboxylase (ACC1).

[0303] Malonyl-CoA biosynthesis was increased by overexpressing Saccharomyces cerevisiae ACC1. To this end, the ACC1 gene was cloned behind the CUP promoter on a Saccharomyces cerevisiae expression plasmid containing a 2 micron replication origin and the ura3 auxotrophic marker using standard methods. The control vector included an empty vector. The Saccharomyces cerevisiae host strain was engineered with chromosome deletions of the ACC1 and SNF1 protein kinases responsible for ACC1 phosphate regulation; chromosome deletions were constructed in both independent and combined ways. Host cells with expression plasmids or control plasmids were grown as described in Example 8 and malonate production was quantitatively produced as described in Example 2.

[0304] Example 16: Improving malonate biosynthesis in host cells by supplementing the fermentation broth with cerulenin

[0305] In this example, malonate production in recombinant host cells expressing an EHD3-derived malonyl-CoA hydrolase was improved by supplementing the fermentation broth with cerulenin. LacO1 The malonate-producing plasmid A4 of S. cerevisiae EHD3(E124S) under the control of the promoter was transformed into the E. coli K12 host.

[0306] Individual colonies were inoculated into 3 ml LB medium (Cm 50 , 2% w / v glucose) and cultured at 37°C on a plate shaker for 6 hours. The strain was then subcultured to 3 mL M9 minimal medium (Cm 50 , 0.5% w / v glycerol, 0.05% w / v glucose, 0.2% w / v lactose) and cultured on a plate shaker at 30°C. After 6 hours of growth, half of the culture was supplemented with 10 mg / l cerulenin. After 48 hours of growth, the malonate concentration in the supernatant was measured as described in Example 2.

[0307] Example 17: Improving Malonate Biosynthesis in Engineered Yeast by Supplementing Fermentation Broth with Carbon Dioxide

[0308] In this example, fermentation conditions were modified to increase the biosynthesis of malonyl-CoA. The enzymatic conversion of acetyl-CoA to malonyl-CoA by the enzyme acetyl-CoA carboxylase requires a stoichiometric amount of carbon dioxide, and supplementing the growth medium with carbon dioxide increases malonate production. Carbon dioxide is added to the growth medium in the form of solid calcium carbonate or gaseous carbon dioxide.

[0309] Recombinant yeast cells with a malonyl-CoA biosynthetic pathway were grown in a defined minimal medium supplemented with 0.1-10 g / l of calcium carbonate. Control cultures were not supplemented with calcium carbonate. Malonic acid production was quantified as described in Example 2 over a 48 hour growth period.

[0310] Example 18: Improving malonate biosynthesis in engineered yeast by reducing malonate catabolism

[0311] In this example, malonate production is increased by eliminating endogenous malonate catabolism in the host cell. Saccharomyces cerevisiae contains multiple acyl-CoA synthetases, including FAA1, FAA2, FAA3, FAA4, LSC1, and LSC2; by deleting or modifying the nucleic acids encoding these proteins on the host genome, the catabolism of malonate in the growth medium can be reduced.

[0312] Malonyl-CoA knockout strains were constructed with each yeast acyl-CoA synthetase. The resulting strains were then cultured in defined medium supplemented with 1-5 g / l sodium malonate. Malonic acid concentrations in the fermentation broth were monitored over the course of 48 hours and quantified as described in Example 2. Similar procedures can be followed to construct strains with multiple gene knockouts.

[0313] Example 19: Improving malonate biosynthesis in engineered yeast by improving malonate secretion from host cells

[0314] In this example, malonate production was improved by increasing the secretion of malonate from the host cell. This was achieved by overexpressing one or more of the various Saccharomyces cerevisiae pleiotropic resistance pumps (i.e., PDR5, PDR10, PDR11, PDR12, PDR15, and PDR18).

[0315] Example 20: Improving malonate biosynthesis in engineered yeast by reducing competitive inhibition of succinate dehydrogenase by malonate

[0316] In this embodiment, the competitive inhibition of yeast saccharomyces cerevisiae succinate dehydrogenase SDH1 is reduced, thereby making it possible to obtain a higher malonate titer. First, standard method genetic modification yeast saccharomyces cerevisiae is used to lack the natural chromosome copy of succinate dehydrogenase (SDH1). The resulting bacterial strain is grown anaerobically to promote the growth in the absence of SDH1 protein. Subsequently, the SDH1 deletion strain is transformed with a carrier of a genetically modified SDH expression cassette, the expression cassette containing E300D, R331K, R331H, R442K, R442H sudden change or the combination of these sudden changes. These sudden change SDH genes are cloned after the constitutive TEF promoter on the yeast main chain with 2 micron starting points and ura3 auxotrophic markers. Subsequently, the carrier is transformed into the yeast saccharomyces cerevisiae strain encoding malonyl-CoA hydrolase on the chromosome and the resulting bacterial strain is grown in the SD substratum lacking uracil. As described in Example 8, the bacterial strain transformed is cultivated to produce malonate and as described in Example 2, quantitative malonate with the impact of these SDH sudden changes on malonate production.

[0317] Example 21: MdcY malonate transcription factor biosensor using exogenously added malonate.

[0318] Plasmid S14 was used to demonstrate the response of the biosensor to exogenously added malonate in E. coli host cells. S14 uses the malonate-responsive transcription factor MdcY (SEQ ID NO: 3) from Acinetobacter calcoaceticus and the MdcY-responsive promoter P MdcL (SEQ ID NO:6). The biosensor of the present disclosure was constructed using an E. coli vector backbone with an ampicillin resistance marker and a ColE1 replication origin; the tetA tetracycline resistance gene was placed in the P MdcL Transformation of plasmid S14 into an E. coli host produced a strain that expressed the tetA gene product after supplementation of the fermentation broth with malonate and exhibited a malonate-dependent increase in tetracycline resistance.

[0319] Synthesize and produce the gene products encoding MdcY and TetA, P MdcL The biosensor vector was then constructed by PCR amplification of the nucleic acid and subsequent cloning into the E. coli vector backbone. The plasmid was transformed into chemically competent E. coli DH10b and the resulting clones were plated in a 50 μg / ml culture medium containing carbenicillin (Cb 50 ) on LB agar plates. Individual colonies were grown overnight in 3 ml LB medium supplemented with antibiotics and the sequence of the purified plasmids was verified.

[0320] E. coli strain K12 was co-transformed with plasmid S14 and plated on LB agar plates (Cb 50) to separate individual colonies. 50 ) until the optical density at 600 nm (OD 600 ) was about 0.50, at which point a cell stock was prepared and stored at -80°C; the cell stock was 0.5 ml of cell culture and 0.5 ml of 50% v / v glycerol solution.

[0321] All biosensor assays were performed with malonic acid. An aliquot of the biosensor cell stock was thawed and used to inoculate 50 ml of LB medium in a 250 ml baffled Erlenmeyer flask (Cb 50 The culture was incubated at 37°C for 2 hours; subsequently, 0.6 ml of the biosensor culture was added to 2.3 ml of LB medium (Cb 50 ) were prepared in 48-well plates (n=4). Each plate was then grown at 30°C on an orbital titer plate shaker. After 12 hours of incubation, 200 μl samples were taken to measure the OD 600 .

[0322] Biosensor cultures with S14 showed a malonate dose-dependent response ( Figure 1 ).exist Figure 1 In the figure, the X-axis is the concentration of exogenous malonate added to the fermentation broth; the Y-axis is the cell culture density (OD) after 12 h of growth in a medium containing 25 μg / ml tetracycline. 600 ). Use included in P MdcL E. coli transformed with plasmid S14 containing the MdcY transcription factor and tetA gene under the control of the promoter produced the tetracycline resistance protein TetA upon exogenous addition of malonate. The biosensor showed a malonate-dependent increase in tetracycline resistance as indicated by an increase in OD with increasing concentrations of exogenously added malonate. 600 The dynamic range measured (maximum OD between fully induced samples and those without malonate supplementation) was 600 The difference in value) is 1.2OD 600 Unit, thus indicating the MdcY-P MdcL The biosensor was highly reactive to exogenously added malonate. OD 600 increases, thereby providing a suitable range within which malonate can be quantified using this method.

[0323] Example 22. MdcY Malonate Transcription Factor Biosensor for Detecting Bioproduced Malonate in Fermentation Broth

[0324] In this example, a malonate transcription factor biosensor is used to detect the production of malonate from a yeast strain engineered as described in other aspects of the disclosure.

[0325] Genetically engineered yeast strains are used to produce malonate as follows. Saccharomyces cerevisiae BY4741 yeast cells with a vector comprising a CYC1 terminator, an ampicillin resistance cassette, a PMB1 origin of replication, a CEN / ARS origin of replication, and a URA3 selectable marker for expressing malonyl-CoA hydrolase are used for fermentation. F0PNG8-1 malonyl-CoA hydrolase (from Bacillus thuringiensis subsp. fascicularis strain YBT-020; UniProt ID F0PNG8, with E91S mutation) and F6AA82-2 malonyl-CoA hydrolase (from Pseudomonas flavescentis strain 12-X; UniProt ID F6AA82, with E95S and Q348A mutations) are each expressed from this plasmid under the control of the TEF1 promoter. The culture medium described in Example 30 is used together with 20g / L glucose as a carbon source. Produce as follows. 20 μ l starter cultures of the production strains were inoculated into 2 ml of culture medium in 48-well plates in quadruplicate. The plates were covered with a breathable membrane, incubated on a plate shaker at 30 ° C, and sampled after 142 hours of growth to carry out HPLC and biosensor analysis of the accumulated products. Cells and cell debris were removed from the culture medium by centrifugation and 0.45 micron membrane filtration, and analyzed by HPLC or biosensor subsequently.

[0326] With malonic acid reactive P mdcL The plasmid pS14 encoding the tetracycline resistance gene (tetA) under the expression control of the promoter, or the malonate-responsive P mdcL E. coli cells of plasmid pS27 encoding the lacZ gene under the expression control of the promoter were used as biosensor indicator strains. Vector pS27 was constructed in the same manner as described for pS14 in Example 21, in which the lacZ gene encoding β-galactosidase was inserted instead of the tetA gene. Biosensor strains were prepared as described in Example 21.

[0327] Yeast spent medium from the 96-well production plate was added to the 96-well plate containing 120 ul of biosensor cell culture. For the TetA (pS14) biosensor, 10 ul of tetracycline stock solution (to provide a range of 20-35 ug / ml) was added to each well. For both the TetA (pS14) biosensor and the lacZ (pS27) biosensor, the remaining volume of each well was replaced with LB medium (Cb 50) was filled to a final volume of 600ul and grown as previously described. After 2 hours (for S27 cultures) and after 5-8 hours (for S14 cultures), samples (200ul) were collected in 96-well plates and OD600 was measured. Samples from S27 (β-galactosidase reporter gene) biosensor plates were assayed for o-nitrophenyl-β-galactoside (ONPG) as follows. Cells were diluted 1:4 in 25ul lysis buffer and then 90ul ONPG stock solution (10mg / ml in deionized water) was added to each well. The contents of each well were thoroughly mixed and kept at 30°C for 4-16 hours. Optical density was measured at 420nm.

[0328] A dose-dependent response to malonate was observed. Specific malonate concentrations were measured by HPLC and quantified by comparison with a standard curve, as also described in Example 2. Linear regression analysis between the quantifiable output of the biosensor, OD420 (for pS27) or OD600 (for pS14), and the specific malonate concentration measured by HPLC was calculated to obtain the coefficient of determination (R 2 )0.88492 (37 OD420 samples plotted) and 0.89755 (18 OD600 samples plotted).

[0329] Those skilled in the art will recognize that these extremely high coefficients of determination indicate that there is a correlation between the biosensor output and the malonate concentration in the culture medium. This aspect of the present disclosure provides a huge advantage in terms of cost and time for screening the differential output in biological malonate production. Dilution of the culture medium used to excite the biosensor can help the dynamic response range of the sensor to extend from zero to complete solution saturation of malonate. Compared to HPLC analysis of each sample requiring 2-20 minutes or more, using plate-based screening enables screening of 96 samples in a few minutes. By limiting or completely replacing HPLC, the savings in capital investment and solvent usage and the resulting emissions are also substantial.

[0330] Example 23. Precipitation of malonic acid from fermentation broth by addition of calcium hydroxide, calcium carbonate and calcium chloride

[0331] In this embodiment, malonic acid is purified from fermentation broth by being precipitated with divalent cations (specifically calcium).Exogenous addition to the synthetic metabolites in the fermentation broth is used to illustrate the purification process.The yeast culture of Saccharomyces cerevisiae BY4741 was grown 72 hours in 0.5L synthetic complete medium at 30 ℃, 200rpm.After 72 hours, the 25ml aliquots of whole cell fermentation broth were used to dissolve the malonic acid of 0,0.5,1,5,10,25,50,75,100g / l equivalent.Each 25ml sample was divided into five 5ml aliquots, and the pH of an aliquot of each concentration was adjusted to 5.5,6.0,6.5,7.0 or 7.5.Then centrifugal (x6000 rcf, 5 minutes, 25 ℃) all samples, and supernatant was transferred in a separate tube.HPLC analysis shows that only malonic acid exists in the supernatant part. Next, a representative divalent cation calcium chloride was used to partially precipitate malonic acid from the clarified supernatant by adding at 25°C in an equivalent amount equimolar to 0.5, 1, 5, 10, 25, 50, 75, 100 g / l malonic acid of each sample. The concentration of malonic acid remaining in the supernatant and in the precipitate was then measured by HPLC as described in Example 2. At concentrations below 5 g / L, the extraction efficiency was 10% or less. Once the pH was reduced to 5.5, the extraction efficiency was also negligible. However, at higher concentrations and pH values ​​of 6-7.5, this method was very effective in purifying malonic acid. The corresponding extraction efficiencies (percent separated from the fermentation broth) of this method at 100 g / L, 75 g / L, 50 g / l, 25 g / L and 10 g / L are as follows: pH 7.5 = 89.8%, 88.5%, 83.3%, 70.1% and 66.4%; pH 7.0 = 88.0%, 86.9%, 81.8%, 71.5% and 63.9%; pH 6.5 = 80.5%, 79.0%, 75.8%, 65.0% and 59.0%; pH 6.0 = 54.4%, 55.0%, 52.8%, 43.0%, 34.9%.

[0332] These results demonstrate that this method of the present disclosure allows the purification of malonate from a fermentation broth, thereby separating the malonate from the yeast cells and other dissolved chemicals.

[0333] Example 24: Purification of biogenic malonate from fermentation broth by reactive extraction with ethanol and methanol

[0334] In this embodiment, the malonic acid produced endogenously from the culture of the saccharomyces cerevisiae with the malonic acid biosynthetic pathway is extracted from the fermented liquid by reaction. First, the host cells are removed from 50 mL of the fermented liquid by centrifugation (x6000 rcf, 5 minutes). Use from: Gatterman L. and Babsinian VS. " The practical methods of organic chemistry " the 3rd edition, The Macmillan Company: New York, the 161-162 pages (1916) method of reorganization is carried out to form diethyl ester and dimethyl ester in the fermented liquid. In short, sodium chloride is added in the fermented liquid; Subsequently, ethanol and sulfuric acid are added, thereby causing the formation of diethyl malonate.

[0335] Example 25: Reactive extraction of malonic acid from fermentation broth using tertiary amines

[0336] Yeast fermentation broth was prepared as described in Example 23. Malonic acid was added exogenously to the fermentation broth to reach a final concentration of 50 g / l. The pH of the solution was adjusted to a value of <4.0 by adding the acid.

[0337] Three tertiary amine (TA) solutions were prepared at 0.25, 0.5 and 0.75 mol-TA / kg 1-octanol using 1-octanol as diluent. The tertiary amines used were triethylamine, tripropylamine, tributylamine, tripentylamine, trihexylamine, triheptylamine, trinonylamine and tridecylamine.

[0338] Make equal volume of organic solvent containing amine and diluent and fermentation broth containing malonate be mixed.Stir the reaction at 1000rpm, 25 ℃ for 2 hours; Then, separate two phases by centrifugation (x6000 rcf, 10 minutes).The malonate concentration in each phase can be measured by HPLC as described in Example 2.

[0339] Example 26: Reactive distillation of malonic acid from fermentation broth using methanol and ethanol

[0340] This example describes the reactive distillation of malonic acid from fermentation broth using methanol and ethanol; cation exchange resin Amberlyst-15 is used as a solid catalyst. Prior to use, the resin was dried in a vacuum oven at 70°C for 6 hours. The assembly consists of a glass column filled with ceramic connected to a collection container. Esterification occurs in a reactor connected to the bottom of the column. A condenser is placed at the top of the column to condense low volatility vapors. The esterification reactor is fed with fermentation broth and ion exchange resin Amberlyst-15 (2% w / w) is added as a catalyst. Sufficient heat is applied to the reactor to evaporate the reaction mixture, and once the desired temperature is obtained, methanol or ethanol is added to the reactor. Samples are taken out of the discharge valves of the esterification reactor and the collection container. After the reaction is completed, the volume and mass of the final reaction mixture from the esterification reactor and the collection container are measured. Malonic acid consumption and product formation are measured as described in Example 2.

[0341] Example 27: Malonic acid synthesis and conversion into dimethyl malonate and diethyl malonate

[0342] Biogenic malonate is produced from recombinant Saccharomyces cerevisiae in 1L culture flasks as follows: the strain is streaked on a synthetic complete omitting medium (SD) agar plate lacking uracil and cultured at 30°C; individual colonies are grown overnight in 3mL SD medium at 30°C and subsequently diluted to 500ml SD lacking uracil with 1% v / v. The strain is cultured at 30°C for 72 hours. Centrifugal (x6000g, 10 minutes) the resulting fermentation broth and the supernatant is separated from the cell centrifugal block. Calcium chloride is used to precipitate malonate from the fermentation broth (see Example 23). The resulting calcium malonate is then converted into diethyl malonate by adding excess ethanol and equimolar sulfuric acid to catalyze Fisher esterification.

[0343] Example 28: Production of Acrylic Esters from Malonic Acid and Formaldehyde by Doebner-Modification of Knovenagel Condensation

[0344] Acrylic esters are produced according to the present disclosure by condensation of malonic acid with paraformaldehyde in pyridine. The reaction is carried out in a 3-necked round-bottom flask with a magnetic stirrer. 15 ml of pyridine and 15 ml of toluene are added to the flask, and 10 g of powdered malonic acid is added in 5 equal portions; subsequently, 1.1 equivalents (3.2 g) of paraformaldehyde are added to the reaction vessel over a period of 30 minutes. The mixture is stirred vigorously to promote dissolution of the components. The reaction temperature is started at 0°C and then increased to 50°C over the course of several hours until carbon dioxide formation is observed as evidenced by the formation of bubbles. After heating at 50°C for 2 hours, the flask is returned to room temperature and an aliquot of the reaction is diluted 100-fold in water and analyzed by HPLC using the method described in Example 2. A sample of malonic acid-derived acrylic acid is co-eluted with an authentic standard at an elution time of about 17.5 minutes (see Figure 2 ).

[0345] Although the Dobner-modified form of the Knovenagel reaction has been used to produce many compounds, it is novel and important for use in the production of acrylates. More than one billion kilograms of acrylic acid are used to prepare a wide range of products from diapers to films and coatings each year. It is currently mainly derived from petroleum and is produced using the methods described in the present disclosure, providing a partial or optional fully renewable approach to this commodity chemical depending on the source of paraformaldehyde. In addition, the malonic acid produced by the method of the present disclosure will be substantially cheaper than its petroleum-derived counterpart. This helps to produce sustainable or partially sustainable acrylic acid at a cost that can compete with the petrochemical approach in use.

[0346] Example 29: Production of glutaric acid from malonic acid and formaldehyde

[0347] As described in Example 8, biogenic malonate was produced from recombinant yeast saccharomyces cerevisiae in 1L culture flasks. Centrifugal (x6000g, 10 minutes) obtained fermentation broth and supernatant was separated from cell centrifugal block. Calcium chloride was used to precipitate malonate from fermentation broth and convert it into diethyl malonate. Then, biogenic diethyl malonate was reacted with formaldehyde in pyridine and processed into required acid (Hedge et al. (1961) JOC 26:3166-3170) according to literature method.

[0348] Example 30: Biocatalytic production of malonate from various carbon sources.

[0349] According to the present disclosure, Saccharomyces cerevisiae BY4741 yeast cells having a malonyl-CoA hydrolase expression vector comprising a CYC1 terminator, an ampicillin resistance gene, a PMB1 replication origin, a CEN / ARS replication origin, and a URA3 selection marker are grown in a yeast fermentation medium comprising 5 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 g / L sodium chloride, 0.1 g / L calcium chloride, 2 mg / L inositol, 0.5 mg / L boron. Acid, 0.4mg / L calcium pantothenate, 0.4mg / L niacin, 0.4mg / L pyridoxine hydrochloride, 0.4mg / L thiamine hydrochloride, 0.4mg / L zinc sulfate, 0.4mg / L manganese sulfate, 0.2mg / L p-aminobenzoic acid, 0.2mg / L riboflavin, 0.2mg / L sodium molybdate, 0.2mg / L ferric chloride, 0.1mg / L potassium iodide, 40μg / L copper sulfate, 2μg / L folic acid, 2μg / L biotin, 10mg / L adenine, 50mg / L L-arginine hydrochloride, 80mg / L L-aspartic acid, 20mg / L L-histidine hydrochloride, 50mg / L L-isoleucine, 100mg / L L-leucine, 50mg / L L-lysine hydrochloride, 20mg / L methionine, 50mg / L L-phenylalanine, 100mg / L L-threonine, 50mg / L L-tryptophan, 50mg / L L-tyrosine and 140mg / L L-valine (basal medium). In different fermentations, the following were used as the sole carbon source respectively: 20g / L glucose, 2% v / v ethanol or 2% v / v glycerol.

[0350] In this example, F0PNG8-1 malonyl-CoA hydrolase (from Bacillus thuringiensis subsp. thuringiensis strain YBT-020; UniProt IDF0PNG8, with E91S mutation) was used under the control of the TEF promoter. 50 μl of a saturated culture of the production strain was used to inoculate 1.5 ml of basal medium supplemented with 2% carbon source in a 48-well plate in triplicate. The culture plates were covered with a gas permeable membrane, incubated on a plate shaker at 30°C, and samples were taken after 138 hours of growth for HPLC analysis of product accumulation.

[0351] HPLC analysis of malonate accumulation was performed as described in Example 2. The results are as follows: glucose as carbon source: 4.8 mM + / - 0.2 mM (standard deviation) malonate; ethanol as carbon source: 7.5 mM + / - 0.8 mM malonate; and glycerol as carbon source: 1.7 mM + / - 0.1 mM malonate. These results show that the carbon sources tested are suitable for producing malonate according to the present disclosure.

[0352] Example 31: Construction and expression of recombinant plasmid vectors encoding various malonyl-CoA hydrolases and their use in producing malonate in yeast

[0353] Nucleic acids encoding various malonyl-CoA hydrolases provided by the present disclosure were amplified by PCR from plasmids using the primers listed below: EHD3 (E124S) primers Y1-11_A13-R (SEQ ID NO: 23) / Y1-11_A13-F (SEQ ID NO: 24); B9IZZ9-1 primers YO012 (SEQ ID NO: 25) / YO013 (SEQ ID NO: 26); F0PNG8-1 primers YO014 (SEQ ID NO: 27) / YO015 (SEQ ID NO: 28); C3ALI3-1 primers YO018 (SEQ ID NO: 29) / YO019 (SEQ ID NO: 30); Q81DR3-1 primers YO020 (SEQ ID NO: 31) / YO021 (SEQ ID NO: 32); NO:32); A4XS22-1 primers YO024 (SEQ ID NO:33) / YO025 (SEQ ID NO:34); E2XN63-1 primers YO026 (SEQ ID NO:35) / YO027 (SEQ ID NO:36); A5W8H3-1 primers YO028 (SEQ ID NO:37) / YO029 (SEQ ID NO:38); and F6AA82-1 primers YO030 (SEQ ID NO:39) / YO031 (SEQ ID NO:40). The purified PCR product was cloned downstream of the TEF1 promoter and upstream of the CYC1 terminator in a shuttle vector containing an ampicillin resistance cassette, a PMB1 replication origin, a CEN / ARS replication origin, and a URA3 selection marker. The resulting plasmid was transformed into E. coli competent host cells and incubated in a culture medium containing Cb 50 After overnight incubation at 37°C, individual colonies were inoculated into 2 ml LB-Cb in a 48-well plate. 50 The construct was cultured in 4% paraformaldehyde and grown on a shaker at 37°C for 5 hours, after which the plasmid was isolated and confirmed by sequencing. After sequencing the construct containing the protein F6AA82(E95S), the expected point mutation Q348A was found to be absent; this mutation was attributed to an error during PCR amplification. The resulting protein F6AA82(E95S / Q348A) is also referred to herein as F6AA82-2. The Q348A point mutation was not demonstrated to be necessary for protein activity.

[0354] Saccharomyces cerevisiae BY4741 cells were used as hosts for vectors expressing various malonyl-CoA hydrolases. Plasmid vectors were individually introduced into yeast host cells using standard procedures. Transformants were selected on agar plates with the medium described in Example 30 containing 2% glucose as a carbon source.

[0355] Eight hydrolase-expressing S. cerevisiae strains were cultured as described in Example 22 and analyzed by HPLC as described in Example 2. The relative malonate concentrations in the fermentation medium were as follows (expressed as integrated area under the malonate peak; mean ± SD; n = 4): S. cerevisiae BY4741 (negative control) 48,865 ± 9,345; EHD3 (E124S) 94,721 ± 8,115; B9IZZ9 (E91S) 261,717 ± 38,012; F0PNG8 (E91S) 216,654 ± 31 ,145; F6AA82 (E95S / Q348A) 212,096 ± 29,338; E2XN63 (E95S) 198,046 ± 35,084; Q81DR3 (E91S) 193,665 ± 37,898; Q63BK8 (E91S) 167,477 ± 8,110; and A5W8H3 (E95S) 52,047 ± 9,042. The identifier is the Uniprot ID (http: / / www.uniprot.org / ) followed by the mutation that results in malonate production provided by the present disclosure. Malonic acid was not detected in samples consisting of medium not inoculated with yeast cells.

[0356] In other embodiments, C3ALI3 from Bacillus mycoides and A4XS22 from Pseudomonas mendocinae (strain ymp), containing E101S and E95S mutations, respectively, were used as malonyl-CoA hydrolases. Because the media conditions were slightly changed due to buffering to pH 4.0, F0PNG8-1 and F6AA82-1 were included for comparison; all other fermentation, sampling and analysis conditions were as described above. The results were as follows: C3ALI3 (E101S) 10±1 mM, A4XS22 (E95S) 7±1 mM, F0PNG8 (E91S) 11±2 mM, and F6AA82 (E95S / Q348A) 23±2 mM malonate. In the absence of malonyl-CoA hydrolase protein, S. cerevisiae cells did not produce detectable concentrations of malonate.

[0357] This example illustrates that, according to the present disclosure, malonate can be produced in yeast host cells expressing enzymes containing an X1 mutation that confers malonyl-CoA hydrolase activity. The E to S active site mutation common to all mutant hydrolases used in this example can be used in other members of these enzyme classes to provide similar results.

[0358] Example 32: Precipitation of Malonic Acid from Cells and Fermentation Broth Using Monovalent Cations

[0359] In this embodiment, malonate is purified from fermentation broth by precipitation with monovalent cations. The monovalent cation is sodium. The purification method is illustrated using synthetic malonate added to the fermentation broth from an exogenous source. A culture of Saccharomyces cerevisiae BY4741 was grown, prepared, and inoculated with malonate as described in Example 23. Then, the representative monovalent cation sodium chloride was used to precipitate malonate from the clarified supernatant portion. At 25°C, 2 or 4 molar equivalents (compared to the malonate concentration) of sodium chloride were added to the supernatant portion of each sample. The malonate concentration remaining in the supernatant and in the precipitate was then measured by HPLC as described in Example 2. The concentration of malonate remaining in the culture medium inoculated with 100 g / L malonate and treated with 2 molar equivalents of sodium chloride varied as follows according to pH: pH 5.5=103%, pH 6.0=96%, pH 6.5=71%, pH 7.0=74%, pH7.5=86%. The concentration of malonate remaining in the medium inoculated with 100 g / L malonate and treated with 4 molar equivalents of sodium chloride varied as a function of pH as follows: pH 5.5 = 92%, pH 6.0 = 86%, pH 6.5 = 66%, pH 7.0 = 81%, pH 7.5 = 86%.

[0360] Example 33: Purification of Malonic Acid from Cells and Fermentation Broth Using Diethylamine

[0361] In this embodiment, malonate is purified from fermentation broth by adding diethylamine. The purification method is illustrated using commercially available malonate added to the fermentation broth from an exogenous source. Yeast fermentation medium is prepared and a known amount of malonate is added as described in the previous embodiment. Representative disubstituted amine diethylamine is used to purify malonate from the clarified supernatant portion. 0.5 to 100 g / l of diethylamine is added to the supernatant portion of each sample at 25 ° C. The metabolite concentration remaining in the supernatant and in the precipitate is then measured by HPLC as described previously as described in Example 2. The concentration of malonate remaining in the culture medium treated with 4 equivalents of diethylamine per equivalent of malonate inoculated with 100 g / L malonate varies as follows according to pH: pH 5.5=100%, pH 6.0=86%, pH 6.5=65%, pH 7.0 67%, pH 7.5=57%.

[0362] Example 34: Synthesis of Pimelic Acid

[0363] This example describes the synthesis of pimelic acid by condensation of glutaric semialdehyde with malonic acid and catalytic hydrogenation. In a 250 ml round-bottom flask, 10 grams of glutaric semialdehyde were dissolved in 40 ml of pyridine. 9 grams of malonic acid were added and the mixture was heated at 80 ° C for 5 hours under stirring. Pyridine was removed using rotary evaporation, and the resulting material containing the intermediate hept-2-ene-1-7-dioic acid was redissolved in hexanol. Pd / C catalyst was added and the resulting mixture was stirred under a hydrogen atmosphere for 24 hours. The catalyst was removed by filtering through diatomaceous earth; the solvent was removed under reduced pressure; and the resulting pimelic acid was purified using silica gel flash chromatography.

[0364] Pimelic acid is a key component of nylon 5,7, which is used in fermentation to compensate for biotin auxotrophy and for the production of several plastics. Prior art methods for pimelic acid synthesis are expensive and low yielding. The present disclosure provides a new chemical synthesis for this important compound that can be derived from malonate as provided by the present disclosure.

[0365] Example 35: Construction and expression of recombinant vectors encoding other malonyl-CoA hydrolases and production of malonate in yeast

[0366] In this example, E. coli malonyl-CoA:ACP transacylase FabD (SEQ ID NO:53) was mutated to contain one or more of the following amino acid changes at the indicated positions: S92C, H201N, R117D, R117E, R117N, R117Y, R117G, R117H, Q11D, Q11E, Q11N, Q11Y, Q11G, Q11H, L93A, L93V, L93I, L93F, L93S, L93G, and used as a malonyl-CoA hydrolase in Saccharomyces cerevisiae. Nucleic acid encoding E. coli FabD was PCR amplified from E. coli strain K12 using primers F1 (5'-ATGACGCAATTTGCATTTGTGTTCCC-3') and F2 (5'-TTAAAGCTCGAGCGCCGCT-3'). The amplified gene was then mutated using standard methods and inserted into a shuttle expression plasmid under the control of the TEF1 promoter and upstream of the CYC1 terminator. This vector contains an ampicillin resistance cassette, a PMB1 origin of replication, a CEN / ARS origin of replication, and a URA3 selection marker. The individual mutation combinations determined are listed below with the results.

[0367] Individual colonies were inoculated into 1 ml of the medium described in Example 30 containing 2% glucose as the carbon source. The cultures were incubated on a shaker at 30°C for 24 hours, and 20 μl of these cultures were used to inoculate 2 ml of production cultures of the same medium. The production cultures were covered with a gas permeable membrane, incubated at 30°C with shaking, and samples were taken after 96 hours and 168 hours of growth for HPLC analysis of product accumulation.

[0368] Malonate was not detected in samples consisting of medium not inoculated with yeast cells. Wild-type yeast produced less than 0.1 mM malonate after 168 hours of fermentation. Expression of any of the four FabD variants produced higher levels of malonate than cells not expressing these proteins. Malonate accumulation after 96 and 168 hours of fermentation using various engineered FabD malonyl-CoA-ACP transacylases expressed in S. cerevisiae was as follows: FabDS92C / L93V / R117H 96 hours = 1.01 mM, 168 hours = 2.49 mM; FabDL93I / R117Y 96 hours = 1.47 mM, 168 hours = 2.48 mM; FabDL93S / R117G 96 hours = 1.11 mM, 168 hours = 2.89 mM; FabD L93I / R117Y 96 hours = 1.64 mM, 168 hours = 3.47 mM.

[0369] Example 36: Reactive extraction of malonic acid from water using 1-octanol containing a trialkylamine.

[0370] In this example, malonic acid is purified from water by reactive extraction with 3 trialkylamines (tripropylamine, trihexylamine and trioctylamine). The purification method is illustrated using authentic malonic acid added to distilled water. Malonic acid is added to water to reach a final concentration of 100 g / l; the pH of the solution is about 1.5.

[0371] 250ul of malonic acid aqueous solution was mixed with 250ul of organic phase consisting of 25% v / v trialkylamine and 75% v / v 1-octanol. A sample was prepared without the addition of organic phase; this sample provides a measurement of the initial malonic acid concentration in each sample. The samples were mixed by inversion for 18 hours, centrifuged (x18,000g) for 1 minute, and the aqueous phase was sampled for analysis of malonic acid concentration by HPLC.

[0372] For HPLC analysis of malonic acid in aqueous phase, we adopt the Shimadzu XR HPLC system equipped with UV detector.5 μ l of each sample is injected into the system and separated with Aminex HPX-87h fermentation monitoring column (Bio-Rad, Hercules, CA).Mobile phase is deionized water (being adjusted to pH 1.95 with sulfuric acid), and flow velocity is 0.6 ml / minute, and oven temperature is 50 ℃, and UV detector is monitored under 210 nm.The malonic acid washout that monitoring sample contains in about 10 minutes after injection.

[0373] 33±2.6% malonic acid was extracted by tripropylamine, 73±4.4% malonic acid was extracted by trihexylamine, and 89±11.9% malonic acid was extracted by trioctylamine (n=3). Therefore, long chain length trialkylamines are preferred over short chain length trialkylamines to increase extraction efficiency. Among the trialkylamines used in this example, trioctylamine is preferred over shorter chain length trihexylamine and tripropylamine.

[0374] Example 37: Lowering the pH of the aqueous phase to increase the reactive extraction of malonic acid using trialkylamines.

[0375] In this example, malonic acid was purified from water by reactive extraction with 1-octanol containing trioctylamine at different pH values. A stock solution of 100 g / l malonic acid was first prepared in water. The stock solution was then divided into working samples adjusted to the desired pH. Because the addition of base dilutes the malonic acid concentration at each pH value, the malonic acid concentration was obtained both before and after the reactive extraction. Calculation of the difference in malonic acid concentration between the pre-extraction sample and the post-extraction sample provides the yield percentage at each pH value tested.

[0376] Reactive extraction was performed as follows. 250ul of malonic acid aqueous solution was mixed with 250ul of an organic phase consisting of 25% v / v trioctylamine and 75% v / v 1-octanol. All samples were mixed by inversion for 18 hours, centrifuged (x18,000g) for 1 minute, and the aqueous phase was sampled to analyze the malonic acid concentration by HPLC as described in Example #36.

[0377] The extraction efficiency at each pH was as follows: pH 1.5, 70%; pH 2.26, 57%; pH 2.93, 45%; pH 4.05, 30%; pH 4.62, 23%; pH 5.0, 15%; pH 5.5, 5%; pH 6.0, 0%; and pH 7.0, 3%.

[0378] The extraction efficiency decreases with increasing pH, and the highest extraction efficiency is obtained at pH 1.5. The extraction efficiency is negligible above pH 6.0. Therefore, it is preferred that the pH of the fermentation broth is below 2.0 when using trialkylamines to extract malonate.

[0379] Example 38: Increasing the trialkylamine concentration in the organic phase to increase the reactive extraction of malonic acid from aqueous solution.

[0380] In this example, malonic acid is purified from water using a trialkylamine / 1-octanol overlying organic phase containing varying concentrations of trioctylamine. The purification process is illustrated using synthetic malonic acid added to distilled water to a final concentration of 100 g / l; the pH of the solution is about 1.5.

[0381] 250 ul of malonic acid in water was mixed with 250 ul of an organic phase consisting of 1-octanol containing the indicated amount of trialkylamine (expressed as a mole fraction relative to the malonic acid in the aqueous phase). One sample was prepared without the addition of organic phase and was used to provide a measurement of the initial malonic acid concentration in the aqueous phase.

[0382] The samples were mixed by inversion for 18 hours, centrifuged (x18,000 g) for 1 minute, and the aqueous phase was sampled for analysis of malonate concentration by HPLC as described in Example #36.

[0383] A linear relationship was observed between the extraction efficiency and the trioctylamine:malonic acid mole ratio; specifically, the linear relationship existed for a trioctylamine:malonic acid mole ratio of 0 to 1. When the molar ratio was above 1, 100% of the malonic acid was extracted into the organic phase. Therefore, the amount of trialkylamine in the organic phase must be equimolar to the amount of malonic acid in the aqueous phase (e.g., fermentation broth) to maximize the extraction yield. Ideally, a greater than equimolar amount of trialkylamine would be added to the organic phase to compensate for the reduced extraction efficiency due to other organic acids and anions in the fermentation broth.

[0384] Example 39: Increasing the yield of reactive extraction of malonic acid from aqueous solutions at different ionic strengths by using equimolar amounts of trioctylamine in 1-octanol.

[0385] In this example, malonic acid was purified from water at different ionic strengths by reactive extraction with trioctylamine; by increasing the molar ratio of trioctylamine in the organic phase to malonic acid in the aqueous phase to 1:1, we were able to improve the malonic acid extraction efficiency.

[0386] Malonic acid was added to water to reach a final concentration of 100 g / l. The ionic strength was adjusted to the indicated concentrations by adding sodium chloride. After the addition of malonic acid and sodium chloride, the final pH of all samples was about pH 1.5.

[0387] 250ul of malonic acid aqueous solution was mixed with 250ul of organic phase consisting of 1-octanol containing trioctylamine. The volume of trioctylamine was calculated so that the molar ratio of trioctylamine:malonic acid was as indicated; the remaining volume was 1-octanol. A sample was prepared without the addition of organic phase; this sample provides the measurement of the initial malonic acid concentration. The sample was mixed at 18°C ​​for 18 hours by inversion, centrifuged (x18,000g) for 1 minute, and the aqueous phase was sampled for analysis of malonic acid concentration by HPLC as described in Example 36.

[0388] Using 0.59, 1.07, 1.61, and 2.14 molar equivalents of trioctylamine, the ionic strength (mM concentration) affected the extraction of malonic acid as follows: at an ionic strength of 0, 63.5%, 98.6%, 99.5%, and 99.4% of malonic acid were recovered, respectively; at an ionic strength of 75, 60.3%, 91.3%, 93.7%, and 93.6% of malonic acid were recovered, respectively; at an ionic strength of 150, 55.9%, 85.8%, 89.5%, and 89.1% of malonic acid were recovered, respectively; at an ionic strength of 225, 52.4%, 83.6%, 89.7%, and 89.8% of malonic acid were recovered, respectively. %, 83.5%, 86.0% and 85.9% of malonic acid were recovered respectively; at an ionic strength of 300, 46.5%, 79.4%, 83.1% and 82.6% of malonic acid were recovered respectively; at an ionic strength of 375, 44.3%, 76.7%, 79.9% and 80.1% of malonic acid were recovered respectively; at an ionic strength of 450, 43.3%, 72.8%, 77.0% and 78.1% of malonic acid were recovered respectively; at an ionic strength of 500, 43.1%, 72.2%, 75.1% and 76.6% of malonic acid were recovered respectively.

[0389] Increasing the trioctylamine:malonic acid molar ratio to 1:1 increased the efficiency of extracting malonic acid from aqueous solution at all ion concentrations tested. No meaningful improvement in malonic acid extraction efficiency was obtained by increasing the trioctylamine:malonic acid molar ratio above 1:1. When extracting malonic acid from aqueous solution, it is preferred to use a trioctylamine:malonic acid molar ratio of at least 1:1, and if no other organic acids are present, a molar ratio of exactly 1:1 is preferred. If contaminating organic acids are present, a molar ratio above 1:1 may be preferred to improve malonic acid extraction efficiency.

[0390] Example 40: Increasing the yield of malonic acid extraction from a trialkylamine / 1-octanol organic phase by increasing the ionic strength of the aqueous phase.

[0391] In this example, we demonstrate the extraction of malonic acid into an aqueous phase using an organic phase consisting of 1-octanol containing trioctylamine.

[0392] Malonic acid was added to water to a final concentration of 100 g / l and extracted into an organic phase consisting of trioctylamine (used at a molar ratio of 1:1 to malonic acid) dissolved in 1-octanol. 10 ml of the aqueous solution was extracted into 10 ml of the organic phase at 18 °C for 18 hours. The organic phase was then separated by centrifugation (x4000 g, 5 minutes). The aqueous phase was also sampled to quantify the unextracted malonic acid concentration.

[0393] Then 250ul of the organic phase was mixed with 250ul of an aqueous solution at a given ionic strength of sodium chloride or sodium hydroxide. The samples were mixed at 18°C ​​for 18 hours by inversion. The samples were then centrifuged (x18,000g, 1 minute) and the aqueous phase was sampled to analyze the malonic acid concentration by HPLC as described for Example 36.

[0394] The data presented below are normalized to the concentration of malonic acid back-extracted into distilled water. Adding 0.5 molar sodium chloride or sodium hydroxide increases the yield of the back-extraction reaction by more than 25 times. Adding sodium chloride above 1.5 M reduces the back-extraction yield; however, the sodium hydroxide back-extraction yield continues to improve with further addition of sodium hydroxide. The back-extraction yield will be further increased by operating the back-extraction reaction at high temperature (i.e., above 18° C.). Millimolar concentrations of NaCl and improvement factors: 0 mM, 1; 0.5 mM, 27.3; 1 mM, 43.1; 1.5 mM, 74.5; 2 mM, 63.4; 2.5 mM, 66.5; 3 mM, 69.4; 3.5 mM, 63.7; 4 mM, 57.8; 4.5 mM, 53.3; and 5 mM, 45.2. Millimolar concentration of NaOH and improvement factor: 0mM, 1; 0.5mM, 33.6; 1mM, 46.5; 1.5mM, 57.5; 2mM, 52.7; 2.5mM, 56.4; 3mM, 62.6; 3.5mM, 58.2; 4mM, 68.5; 4.5mM, 67; 5mM, 74.2.

[0395] Example 41: Purification of malonic acid from aqueous solution by esterification and subsequent phase separation

[0396] In this example, we demonstrate the esterification of malonic acid and ethanol to form diethyl malonate and the subsequent phase separation of the diethyl malonate into a hexane organic phase.

[0397] Prepare 500 μl of an aqueous solution containing 100 g / l concentration of malonic acid, indicated concentration of ethanol and indicated concentration of sulfuric acid. Malonic acid and ethanol are substrates for forming diethyl malonate; sulfuric acid is added as a catalyst. Add 250 μl of an organic overlay of hexane to each sample, mix them at 25 ° C and atmospheric pressure for 18 hours by inversion, centrifuge (x18,000 g) for 1 minute, and sample the aqueous phase to analyze the malonic acid concentration by HPLC as described in Example 36.

[0398] Consumption of malonic acid was measured by the decrease in malonic acid concentration in the aqueous phase.The baseline malonic acid concentration in the aqueous phase (ie 100% unreacted malonic acid) was determined from a sample containing 0% v / v ethanol and 0% v / v sulfuric acid.

[0399] Addition of both ethanol and sulfuric acid to the reaction mixture is necessary to catalyze the esterification of malonic acid. The most preferred reaction conditions, as measured by the percent consumption of malonic acid, are obtained at high ethanol and sulfuric acid concentrations. That is, greater than 40% v / v ethanol (a molar ratio to malonic acid of 7.14) and greater than 10% v / v sulfuric acid (a molar ratio to malonic acid of 1.94) are necessary to catalyze nearly complete consumption of malonic acid. The results of this illustration are as follows: Esterification reactions were performed with 5, 10, 20, 30, 40, and 50% (v / v) ethanol. The remaining malonic acid in the aqueous phase was as follows: 5% (V / V) sulfuric acid, 78.9%, 61.6%, 43.2%, 43.2%, 28.2% and 16.6%; 10% (V / V) sulfuric acid, 83.5%, 70.3%, 49.5%, 28.6%, 16.6% and 8.5%; 5% (V / V) sulfuric acid, 71.8%, 53.2%, 28.1%, 11.8%, 4.4% and 0.6%. The no acid control showed that 95.1%, 93.2%, 85.8%, 69.1%, 63.0% and 87.3% malonic acid remained in the corresponding solutions with ethanol. Addition of the acid in the absence of ethanol resulted in the following concentrations of malonic acid remaining in the aqueous phase: 96.1%, 92.7% and 84.4% for 5, 10 and 20% (v / v) sulfuric acid, respectively.

[0400] Example 42: Improving malonate biosynthesis in engineered yeast by increasing acetyl-CoA carboxylase activity

[0401] In addition to methods, vectors and host cells for expressing heterologous malonyl-CoA hydrolases and producing malonate in vivo, the disclosure also provides methods and host cells for improving the titer, yield and / or productivity of malonate. In one aspect, malonate production is improved by increasing the biosynthesis of malonyl-CoA. Malonyl-CoA is the penultimate intermediate in the biosynthesis of malonate from acetyl-CoA, and in Saccharomyces cerevisiae, this reaction is catalyzed by acetyl-CoA carboxylase (ACC1).

[0402] In this example, the ACC from yeast Yarrowia lipolytica CLIB122 (NCBI reference sequence: XP_501721) was back-translated into a DNA sequence using a standard codon optimization table for expression in Saccharomyces cerevisiae. The result of the YlACC DNA sequence is included below as SEQ ID NO: 11.

[0403] DNA encoding 50 nucleotides of upstream Saccharomyces cerevisiae FAA1 homolog, 300 base pairs of Saccharomyces cerevisiae TEF1 promoter sequence, YlACC sequence (SEQ ID NO: 11) and finally 50 nucleotides of downstream Saccharomyces cerevisiae FAA1 was synthesized in a single DNA format using a standard protocol and amplified by PCR. The resulting flanking YlACC PCR product and a linear DNA construct containing a selectable URA3 gene and flanking FAA1 homologous sequences to directly recombine into the FAA1 site on the chromosome were co-transformed into BY4741 (strain LYM004) containing 2 copies of malonyl-CoA hydrolase F6AA82-2 provided by the present disclosure, and selected by plating on synthetic defined medium (SD) plates containing 2% glucose and lacking uracil (-Ura). Clones and corresponding genomic DNA were isolated and verified by PCR to contain YlACC integrated into FAA1.

[0404] For in vivo malonate production, an individual bacterium colony of each transformant selected was inoculated in a 50 μ l aliquot of SD-Ura culture medium in a 96-well plate. LYM004 was grown to compare the yeast lacking the expression of heterologous YlACC. At 30 ℃, on a shaker, the plate was incubated for about 4 hours and 25 μ l of these pre-cultures were used to inoculate the fermentation plate. In this embodiment, 2 ml SD culture medium in a 48-well plate was used for fermentation. The fermentation plate was covered with a breathable membrane, incubated on a plate shaker at 30 ℃, and sampled after fermentation 72 and 120 hours to perform HPLC analysis on product accumulation. As described in Example 36, HPLC analysis was performed on the malonate accumulation in the fermentation broth.

[0405] Malonate was not detected in samples consisting of medium alone. LYM004 expressing YlACC produced 1.94 and 2.43 times more malonate at 72 and 120 hours, respectively, than LYM004 alone. The highest malonate concentration measured in the fermentation medium was observed in samples collected after 120 hours of fermentation.

[0406] Example 43: Production of Malonate in Saccharomyces cerevisiae Using the Engineered F6AA82 Malonyl-CoA Hydrolase

[0407] In this example, all 20 protein gene amino acid point mutations were introduced at position E95 in protein F6AA82 (Q348A). It is believed that amino acid position 95 interacts with the end of malonyl-CoA, and mutation of E95 will introduce malonyl-CoA hydrolase activity into the protein.

[0408] F6AA82 (Q348A) mutated at position 95 (i.e., all amino acids) was constructed using standard methods and cloned into a yeast plasmid containing a uracil auxotrophic marker, a CEN / ARS replication origin, a TEF promoter, and a CYC terminator. The F6AA82 mutant was cloned behind the TEF promoter and directly upstream of the CYC terminator. All plasmids contained an ampicillin resistance marker and a ColE1 origin for plasmid propagation in E. coli.

[0409] The plasmid was transformed into Saccharomyces cerevisiae using the lithium acetate procedure and transformants were selected on SD-uracil agar plates at 30°C. 12 colonies of each mutant were inoculated into a pre-culture of 500 μl SD-Ura medium in a 96-well plate and incubated at 30°C under shaking for about 16 hours. 5 μl aliquots of these pre-cultures were used to inoculate a 96-well production plate containing 500 μl RD4-Ura (1X YNB, 3XSC supplement, 2% glucose, 75mM succinate buffer (pH 4.0), uracil omitted) medium. The production plate was incubated at 30°C under shaking for 3 days, and the fermentation broth was subsequently sampled. The sample was clarified by centrifugation and filtered on a 0.45 μm membrane, followed by HPLC analysis. HPLC analysis was performed on the malonate accumulation in the fermentation broth as described in Example 36.

[0410] The yields of the various F6AA82 mutants (which contained the mutation Q348A in addition to the E95 mutation) under these conditions were as follows: F6AA82 (E95N) 8.03 ± 0.14 mM, F6AA82 (E95S) 4.18 ± 0.61 mM, F6AA82 (E95Y) 3.87 ± 0.30 mM, F6AA82 (E95A) 2.33 ± 0.50 mM, F6AA82 (E95K) 1.65 ± 0.23 mM, F6AA82 (E95T) 1.16 ± 0.62 mM, F6AA82 (E95D) 0.75 ± 0.27 mM, F6AA82 (E95F) 0.17 ± 0.13 mM, F6AA82 (E95V) 0.11 ± 0.32 mM, F6AA82 ( E95L)0.11±0.12mM, F6AA82(E95G)0.00±0.01mM, F6AA82(E95P)-0.31±0.78mM, F6AA82(E95R)-0.32±0.69mM, F6AA82(E95)-0.32±0.40mM, F6AA82(E95W)-0.34± 0.73 mM, F6AA82 (E95Q) -0.37 ± 0.74 mM, F6AA82 (E95H) -0.53 ± 0.70 mM, F6AA82 (E95C) -0.57 ± 0.69 mM, F6AA82 (E95I) -0.64 ± 0.81 mM, and F6AA82 (E95M) -0.77 ± 0.79 mM. Negative concentrations of malonate are due to a drop in production titers below the detectable level by HPLC and indicate the absence of malonate production.

[0411] The F6AA82 (Q348A) proteins containing mutations E95S, E95Y, E95T, E95N, E96K, E95V and E95D produced significantly (t-test, p<0.05) more malonate than the F6AA82 (Q348A) protein. The F6AA82 proteins containing these mutations were suitable for hydrolyzing malonyl-CoA and producing malonate. The F6AA82 mutations E95S and E95N were preferred for hydrolyzing malonyl-CoA and producing malonate.

[0412] E95Y, E95T, E95K, E95V and E95D produced significantly (t-test, p<0.05) more malonate than the wild-type F6AA82 protein. The F6AA82 protein containing these mutations was suitable for hydrolyzing malonyl-CoA and producing malonate.

[0413] Example 44: Effect of Promoter Selection on Malonate Production in Saccharomyces cerevisiae

[0414] In this embodiment, the expression plasmid backbone containing CEN / ARS replication origin, PMB1 replication origin, ampicillin resistance marker, URA3 marker and hph marker was amplified from plasmid Y20. Malonyl-CoA hydrolase F6AA82-2 flanked by CYC1 terminator was amplified from plasmid Y1-F6AA82-2 by PCR and assembled with the backbone using standard techniques. The resulting plasmid pPLIB0-Q9I was used as the backbone for cloning different selected promoters.

[0415] 96 reference genes were selected from the yeast strain BY4741. The promoter sequences of these genes were generated by PCR amplification of a nucleic acid fragment corresponding to about 750 base pairs using the yeast strain BY4741 genomic DNA as a template, which was immediately upstream of the open reading frame of the gene, including the start codon of the target gene. These promoter sequences were cloned immediately upstream of F6AA82-2 (start codon at 3739 in SEQ ID NO: 12) in pPLIB0-Q9I using standard cloning techniques. The resulting plasmid was propagated in E. coli and the desired promoter was verified by sequencing.

[0416] The above plasmids were used to transform Saccharomyces cerevisiae BY4741 using a standard lithium acetate procedure. Transformants were selected on agar plates of CSM medium without uracil at 30°C. One transformant with each plasmid was inoculated in a pre-culture plate consisting of 300ulRD-Ura medium (2X YNB, 3XSC-U, 2% glucose) in a 96-well plate. The pre-pre-culture plate was incubated under shaking for about 20 hours at 30°C, and 30μl of these pre-cultures were used to inoculate a production plate (each pre-culture corresponds to 1 production well) consisting of 1.8mlRD4-Ura medium (2X YNB, 3X SC-U, 2% glucose, 75mM succinate buffer (pH 4.0)) in a 48-well culture plate. Each plate was incubated under shaking at 30°C for 144 hours, and then the fermentation broth was sampled. The sample was clarified by centrifugation and filtered on a 0.45μm membrane, followed by HPLC analysis.

[0417] HPLC analysis of malonate accumulation in the fermentation broth was performed as described in Example 36. After 144 h of fermentation with different promoters driving the expression of F6AA82-2, the millimolar concentration of malonate in the fermentation broth was as follows: HSP1508.8; PGK1 8.6; PHO5 8.5; SCT17.8; PRB1 7.6; TPI1 7.1; ACH1 7; HXK2 6.9; ACO1 6.9; JEN16.9; MDH2 6.8; POX1 6.6; CIT1 6.6; ALD4 6.6; ADH1 6.5; TDH3 6.4; ADH2 6.4; SDH1 6.4; TDH1 6.1; MLS1 6.1; RPN6 6; GLK1 5.9; POT15.8; HSP26 5.8; FBA1 5.7; LPD1 5.7; CYC15.5; COX5a 5.5; TEF1 5.5; SHH4 5.5; GND2 5.5; TPS1 5.5; MDH1 5.4; PDC1 5.4; HXK1 5.3; TDH2 5.3; IDH2 5.3; DDR2 5.2; SLT2 5.2; ENO2 5.1; COX6 5; CHO14.9; PHO3 4.9; PFK14.9; ACS1 4.9; GUT2 4.8; PHM7 4.8; CIT2 4.7; ACS2 4.7; ALD2 4.5; IDH1 4.5; IDP2 4.5; FBP1 4.3; PHO12 4.3; PDC54.2; PFY1 4.1; GDH1 4.1; PEX13 4.1; ICT1 4.1; YSA1 4; KGD23.9; GIM5 3.9; GAP1 3.9; DBP5 3.8; STE5 3.7; BIO2 3.7; PDC6 3.6; HXT53.6; REG1 3.6; TPS3 3.5; BIO5 3.3; PHO8 3.3; IRC7 3.2; GPM1 3.2; ALA1 3.1; KGD1 3.1; MIG1 3.1; YKT62.9; SNO4 2.8; ARA1 2.8; PDR10 2.7; YBR139W 2.3; ERR2 2.2; CRC1 1.9; TSL1 1.3;ENO11.2;PFK2 1.1; RPL6A 1.1. Because many of these promoters are unnamed, the alphanumeric code preceding each numerical value indicates the gene immediately downstream of the cloned promoter in S. cerevisiae strain BY4741.

[0418] This example demonstrates that a wide variety of promoters can be used to control the expression of malonyl-CoA hydrolase to produce malonate in vivo in yeast. Furthermore, in this experiment, 40 different promoters produced titers of at least 5 mM malonate, and 7 promoters resulted in the production of at least 7 mM.

[0419] Example 45: Integrating the gene encoding malonyl-CoA hydrolase into the genome of Saccharomyces cerevisiae to produce malonate.

[0420] In this example, Saccharomyces cerevisiae strain BY4741 was used as a host strain for genomic integration of a malonyl-CoA hydrolase provided by the present disclosure. Three sites on the Saccharomyces cerevisiae genome were selected for integration of synthetic nucleic acid constructs in this strain and the three linear nucleic acid cassettes to be integrated each carried a unique homology site for targeted integration and a unique selectable marker encoding upstream of the malonyl-CoA hydrolase. In all three cases, a nucleic acid encoding the malonyl-CoA hydrolase F6AA82 (E95S / Q348A) flanked by the TEF1 promoter and the CYC1 terminator was used as the expression portion of the cassette.

[0421] In two cases, individual long terminal repeats YPRCδ15 and YORWδ22, which are non-coding DNA of the chromosome, were targeted for integration. In the third case, the malonyl-CoA hydrolase cassette (F6AA82 (E95S / Q348A)) provided by the present disclosure was integrated in place of the BUD21 gene. The sequences of these linear insertion cassettes are included below as SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43. The unique integration site and selectable markers are YPRCδ15 (HIS3), YORWδ22 (LEU2), and BUD21 (URA3), respectively.

[0422] These nucleic acid cassettes were constructed using standard protocols and transformed into Saccharomyces cerevisiae BY4741. Transformants were selected on agar plates made from SC medium omitting the appropriate amino acids at 30°C. Several transformants of each construction were re-streaked on selective agar plates. Transformants were then screened and verified by the production of malonate and by PCR. The following strains were generated from these integrations: LYM002 contains 1 copy of F6AA82 (E95S / Q348A) integrated at YPRCδ15 (HIS3); LYM004 carries 2 copies of F6AA82 (E95S / Q348A), 1 at YPRCδ15 (HIS3) and the second at YORWδ22 (LEU2); LYM007 is derived from LYM004 and has a third copy of F6AA82 (E95S / Q348A) integrated at the BUD21 locus.

[0423] For fermentation and malonate production, the saccharomyces cerevisiae BY4741 carrying plasmid Y1-F6AA82 (E95S / Q348A) serves as a positive control. At 30°C, under shaking, incubate the pre-culture of 500 μl RD4 substratum in 96-well plates for 16-20 hours. 5 μl aliquots of these pre-cultures are used to inoculate the 96-well production plates containing 500 μl RD4 substratum (1X YNB, 3X SC supplement, 2% glucose, 75mM succinic acid buffer (pH 4.0)). At 30°C, under shaking, incubate the production plates for 120 hours, then sample the fermented liquid. Prepare samples as described in Example 36 and analyze by HPLC.

[0424] A standard curve established with an authentic standard was used to determine the malonate concentration in the fermentation broth. The malonate production levels obtained from these fermentations were as follows: S. cerevisiae BY4741+Y1-F6AA82 (E95S / Q348A), 2.6±0.4 mM; LYM002, 3.2±0.2 mM; LYM004, 4.6±0.2 mM; LYM007, 7.7±0.3 mM.

[0425] This example illustrates the benefit of genomic integration of the malonyl-CoA hydrolase encoding gene provided by the present disclosure, as a single integrated copy of the gene produces a higher titer of malonate than that obtained by expressing the same gene from a plasmid. This example also serves to illustrate the regulation of malonate by the present disclosure through the number of malonyl-CoA hydrolase encoding genes present in the cell.

[0426] Example 46: Expression of acetyl-CoA synthetase to increase malonate production from engineered host cells.

[0427] Acetyl-CoA synthetases (ACS) are common to many organisms. These enzymes use acetate and adenosine triphosphate (ATP) as substrates to produce acetyl-CoA. According to the present disclosure, these enzymes can be used to convert acetate from any source, including endogenous metabolism, feedstock hydrolysis, or feedstock, into acetyl-CoA, the penultimate precursor in malonyl-CoA biosynthesis. By increasing acetyl-CoA levels, malonyl-CoA can be increased, and thus malonate in the host cells provided by the present disclosure can be increased.

[0428] An illustrative ACS suitable for this purpose can be obtained from the bacterium Salmonella enterica. To express the Salmonella enterica ACS (SeACS) in Saccharomyces cerevisiae, the amino acid sequence was back-translated into a DNA sequence using a codon optimization table, and one residue determined to be involved in reducing enzyme activity was mutated (L641P). The resulting codon-optimized DNA sequence provided by the present disclosure is SEQ ID NO: 44.

[0429] Nucleic acids encoding SeACS1 (L641P) were resynthesized from oligonucleotides and inserted into yeast plasmid Y1 under the control of the TEF1 promoter from Saccharomyces cerevisiae using standard protocols. This plasmid also contains the CEN / ARS origin and a gene that confers uracil prototrophy. Clones of LYM004 containing this plasmid were selected by plating on SD-uracil plates and verified by sequencing to contain the SeACS1 (L641P) gene.

[0430] LYM004 is used as a host for expressing SeACS1 (L641P) acetyl-CoA synthetase. For in vivo malonate production, an individual colony of each selected transformant is inoculated in a 50 μl aliquot of SD-Ura medium in a 96-well plate. LYM004 is grown to compare the expression of a heterologous SeACS1 (L641P) lacking, but containing yeast with an empty Y1 vector. The plate is incubated on a shaker at 30°C for approximately 48 hours, and 50 μl of these pre-cultures are used to inoculate the fermentation plate. In this embodiment, 500uL RD4U medium in a 96-well plate is used for fermentation. The fermentation plate is covered with a breathable membrane, incubated on a plate shaker at 30°C, and sampled after 120 hours of fermentation to perform HPLC analysis on product accumulation. HPLC analysis is performed as described in Example 36.

[0431] Malonate was not detected in samples consisting of medium not inoculated with yeast cells. Under the conditions tested, LYM004 expressing SeACS1 produced 1.86-fold more malonate at 120 hours (5.5 ± 0.62 mM) than LYM004 with an empty Y1 control alone, thereby illustrating the improvement in malonate production provided by this embodiment of the present disclosure.

[0432] Example 47: Production of malonate using different carbon sources using modified Pichia kudriavzevii strains.

[0433] Strain LPK3003 was derived from Pichia kudriavzevii strain Y-134 (obtained from USDA Agricultural Research Services, Peoria, IL) by genomic integration of nucleic acid cassettes encoding hph hygromycin phosphotransferase driven by the PkTEF1 promoter (conferring resistance to hygromycin B), and F6AA82 (E95S / Q348A) malonyl-CoA hydrolase driven by the PkTDH1 promoter.

[0434] A seed culture of LPK3003 was grown in YPD medium at 30°C with shaking for 16-20 hours. This seed culture was used to inoculate 1.1X YNB, which was then aliquoted in 96-well plates at 450 μl per well. 50 μl of a solution of various carbon sources (glucose, sucrose, ethanol, glycerol, or acetic acid from sodium acetate) was added to each well (in triplicate) to reach a final concentration of 2% (w / v). The plates were incubated at 30°C with shaking for 115 hours and the fermentation broth was sampled. Samples were prepared as described in Example 36 and analyzed by HPLC.

[0435] In this embodiment, for sucrose or acetate carbon source, it is observed that there is a little growth or there is no growth, and the malonate accumulation in these samples is negligible. As mentioned above, the host cell can be modified to impart or enhance sucrose and / or acetate catabolism by introducing sucrose invertase and / or acetyl-CoA synthase (ACS) respectively. Other carbon sources tested in this embodiment cause malonate accumulation (average replicates ± SD): 4.6 ± 0.5 mM (from glucose), 5.38 ± 0.05 mM (from glycerol) and 3.7 ± 0.3 mM (from ethanol).

[0436] These results demonstrate that a variety of carbon sources can be used to produce malonate from engineered Pichia kudriavzevii strains expressing malonate-CoA hydrolases. Notably, glycerol provided the highest titers in this example.

[0437] Example 48: Bioreactor-based malonate production.

[0438] In this embodiment, yeast strain LYM004 (for construction details, see Example 45) is grown in a 0.5L bioreactor in a fed batch control mode. A single colony of LYM004 is separated from a SC plate and cultured in 5mL RD4 medium (for formulation, see Example 43). At 30°C, the culture is maintained overnight under shaking at 200rpm. 4mL culture is used to inoculate 50mL fresh RD4 medium in a 250mL non-baffled culture bottle and grown overnight at 30°C, 200rpm. The OD 600nm absorbance at time zero is 0.304. After overnight growth (16 hours), this culture is used to inoculate 1L RD4 medium. This culture is divided into 2 separate 500mL aliquots and added to two separate bioreactors. Both fermentations are maintained at 30°C, with a single impeller running at 400rpm, and the injection rate achieved using compressed air is 1 container volume per minute (VVM). The culture was grown overnight (21 hours) to reach glucose consumption before the initial fed-batch phase. Feed (formulation below) was delivered for 2 seconds every 980 seconds. 0.5 mL sample was obtained every day and the production of malonic acid was analyzed. After 4 days, the culture had accumulated 34 mM malonic acid and reached OD 600 nm of 16.2. After 9 days, the culture had accumulated 116 mM malonic acid and reached OD 600 nm of 52.1.

[0439] Fed-batch medium consisted of 17 g / L Difco YNB; 50 g / L ammonium sulfate; 49.8 g / L synthetic complete (SC) supplement lacking histidine, methionine, and leucine; 2.57 g / L methionine; 8.85 g / L succinate and 20 g / L glucose; pH was adjusted to 4.0.

[0440] This scheme is repeated for the bacterial strain LYM007 (see Example 45) expressing 3 copies of malonyl-CoA hydrolase. In this embodiment, a single impeller is operated at 700 rpm and the composition of the feed is as follows: 68 g / L YNB (Sigma), 16.6 g / L SC-his-met-leu (Sunrise Science), 1.284 g / L methionine, 75 mM succinate buffer (pH 4.0), 400 g / L glucose. The feed rate used is 5 seconds per 980 seconds of circulation. 0.2 mL samples are obtained every day and the generation of malonate, acetate, succinate, pyruvic acid is analyzed by HPLC. Growth is monitored by measuring the optical density (OD600) under 600 nm. The OD600 and malonate concentration at each time point were as follows: 18 hours, OD = 6.9, 3.2 mM; 45 hours, OD = 13.8, 14.0 mM; 69 hours, OD = 16.6, 24.2 mM; 88.5 hours, OD = 24.4, 31.1 mM; 111.8 hours, OD = 36.9, 47.8 mM.

[0441] In a comparative example, LPK3003 (see Example 47) was grown in a 0.5L fermentor in a fed-batch controlled manner. A single colony of LPK3003 was isolated from a YPD plate and cultured in a 5mL YPD medium. The culture was grown overnight at 30°C with shaking at 200rpm. 5mL of the culture was used to inoculate 50mL of fresh minimal medium containing 6.8g / L YNB (Difco) and 2g / L glucose and grown overnight at 30°C, 200rpm. This culture was then used to inoculate 500mL of 6.8g / LYNB (Difco) medium, 2g / L glucose and grown in a 1 liter fermentor. The temperature was maintained at 30°C, a single impeller was operated at 700rpm, and compressed air was used, the injection rate was set to 1 container volume per minute (VVM). The culture was grown overnight for 21 hours so that glucose consumption was achieved before the start of the fed-batch phase. A feed containing 68 g / L YNB and 400 g / L glucose was started by setting the fermentor feed to cycle for 5 seconds every 980 seconds. 200 uL samples were taken daily and analyzed for growth (OD600) and the production of malonate (as described in Example 36). Foaming was controlled by adding 200 uL of defoamer at each sampling interval. pH was measured at each sampling interval and adjusted to 4.5 with 10 N NaOH. Malonic acid continued to be produced for more than 111 hours, thereby accumulating 36.4 mM malonate. Importantly, this is the first time that production has been shown to be achieved without the addition of amino acids, uracil or adenine. The OD600, malonic acid concentration and pH at each time point are as follows: 18 hours, OD = 2.1, 0.7 mM, pH = 2.3; 45 hours, OD = 12.2, 8.8 mM, pH = 2.0; 69 hours, OD = 12.8, 18.1 mM, pH = 2.0; 88.5 hours, OD = 27.6, 22.9 mM, pH = 2.7; 111.8 hours, OD = 40.1, 36.4 mM, pH = 2.4; 194 hours, OD = 60.6, 94 mM, pH = 3.3.

[0442] Despite the higher titer of malonate from LYM007, the conditions under which LPK3003 can grow (minimal medium, low pH) and the fact that it contains only a single copy of the F6AA82-2 malonyl-CoA hydrolase make this strain a superior strain for further engineering attempts.

[0443] Example 49: Construction of recombinant Pichia kudriavzevii strains LPK152352, LPK1511264, LPK153531, and LPK1511260 comprising genetic modifications of the present disclosure that increase production of acetyl-CoA, malonyl-CoA, and malonate.

[0444] Example 49 describes the construction of recombinant Pichia kudriavzevii host cells LPK152352, LPK1511264, LPK153531 and LPK1511260 of the present disclosure, each of which comprises heterologous nucleic acids encoding F6A-6 malonyl-CoA hydrolase (including UniProt ID: F6AA82 with E95N, V152A and F304), ScALD6 (UniProt ID: P54115), YlACS (UniProt ID: Q6C2Q5) and PkACC1 (UniProt ID: A0A099P5S6). LPK153531 and LPK1511260 each further comprise heterologous nucleic acids encoding ScPDC6 (UniProt ID P26263).

[0445] The heterologous nucleic acid used in this embodiment has been codon optimized and synthesized for yeast. Each gene is cloned into its own entry vector pEV together with an upstream transcription promoter and a downstream transcription terminator. The transcription promoter in front of each gene (5') is constitutive and comes from Pichia kudriavzewei. The transcription terminator behind each gene (3') comes from Saccharomyces cerevisiae. The promoter and terminator of F6A-6 are Pichia kudriavzewei TDH1 promoter (pPkTDH1) and Saccharomyces cerevisiae TEF1 terminator (tScTEF1) respectively. The promoter and terminator of PkACC1 are Pichia kudriavzewei TDH1 promoter (pPkTDH1) and Saccharomyces cerevisiae TEF1 terminator (tScTEF1) respectively. The promoter and terminator of ScALD6 are Pichia kudriavzewei FBA1 promoter (pPkFBA1) and Saccharomyces cerevisiae GRE3 terminator (tScGRE3) respectively. The promoter and terminator of YlACS are the Pichia kudriavzevii PGK1 promoter (pPkPGK1) and the Saccharomyces cerevisiae HXT1 terminator (tScHXT1), respectively. The promoter and terminator of ScPDC6 are the Pichia kudriavzevii FBA1 promoter (pPkFBA1) and the Saccharomyces cerevisiae TEF1 terminator (tScTEF1), respectively. F6A-6 was integrated into the URA3 locus of Pichia kudriavzevii. ScALD6, YlACS, and PkACC1 were integrated into the ADH1 locus of Pichia kudriavzevii. ScPDC6 was integrated into the YAT1 locus of Pichia kudriavzevii. In addition, a HIS3 marker—comprising the Pichia kudriavzevii TEF1 promoter (pPkTEF1), the HIS3 coding region, and the Saccharomyces cerevisiae TDH3 terminator (tScTDH3)—was included in the heterologous expression cassette to complement the histidine auxotrophy defect in the parental strain.

[0446] Each gene is amplified from its respective pEV vector using primers that provide upstream and downstream regions (about 25bp to 700bp each) homologous to adjacent segments of the target integration site in the genomic site. For example, the 5' and 3' ends of the F6A-6 expression cassette are homologous to the upstream and downstream segments of the URA3 locus of Pichia kudriavzevii, and the expression cassette is integrated into the URA3 locus. Therefore, one or two copies of the PkURA3 gene are deleted from the host genome. In another sample, the 5' and 3' ends of the ScALD6, YlACS, and PkACC1 expression cassettes are homologous to the upstream and downstream segments of the ADH1 locus of Pichia kudriavzevii, and the expression cassette is integrated into the ADH1 locus. Therefore, one or two copies of the PkADH1 gene are deleted from the host genome. In another sample, the 5' and 3' ends of the ScPDC6 expression cassette were homologous to segments upstream and downstream of the Pichia kudriavzevii YAT1 locus, and the expression cassette integrated into the YAT1 locus. Thus, one or both copies of the PkYAT1 gene were deleted from the host genome.

[0447] All PCR products were purified and provided to Pichia kudriavzevii as exogenous nucleic acids. Transformations were performed sequentially. For LPK152352 and LPK1511264, Pichia kudriavzevii was first transformed with the F6A-6 expression cassette. Transformants were selected on CSM-His medium. Successful integration of the heterologous nucleic acid was confirmed by sequencing the target locus and flanking regions. The integrated HIS3 marker was looped out, so the strain was ready for the next round of transformation and selection. The next round of transformation included the ScALD6, YlACS and PkACC1 expression cassettes. The transformation, selection and looping out steps were repeated. To construct LPK153531 and LPK1511260, the above transformation, selection and looping out steps were performed a third time using the ScPDC6 expression cassette.

[0448] Example 50: Cultivation of recombinant Pichia kudriavzevii strains LPK152352, LPK1511264, LPK153531 and LPK1511260 to produce malonate

[0449] This example describes the cultivation of recombinant Pichia kudriavzevii strains LPK152352, LPK1511264, LPK153531 and LPK1511260 (constructed as disclosed in Example 49) in the presence of malonate. Single colonies of strains LPK152352, LPK1511264, LPK153531 and LPK1511260 were picked and inoculated into 0.5 ml of production medium (i.e., 125 g / L glucose, 0.3 g / L MgSO4-7H2O, 0.8 g / L K2SO4, 0.12 h / L Na2SO4, 0.5 g / L KH2PO4, 4.3 g / L urea, standard trace metal solution for yeast, standard vitamin solution for yeast, 0.25 g / L uracil and 0.1 M MES pH 6.5) in a 96-well plate and incubated at 30° C. and shaking at 400 rpm. After 72 hours, samples from the culture broth were collected, spin filtered and stored frozen. Samples were analyzed by HPLC within 48 hours of harvest.

[0450] Example 51: HPLC analysis of culture broths of recombinant Pichia kudriavzevii strains LPK152352, LPK1511264, LPK153531 and LPK1511260 for the presence of malonate

[0451] Example 50 describes HPLC analysis of recombinant Pichia kudriavzevii strains LPK152352, LPK1511264, LPK153531, and LPK1511260 (constructed as disclosed in Example 49 and cultured under the conditions described in Example 50) for the presence of malonate.

[0452] For HPLC analysis, each stored sample of the culture broth from Example 50 was thawed and treated with o-phthalaldehyde (OPA) for derivatization as recommended by Agilent for an automated pre-column derivatization protocol integrated with HPLC analysis using an Agilent Zorbax Eclipse-AAA column (4.6 mm x 75 mm, 3.5 μm). UV 338 nm measurements were collected within 15 minutes.

[0453] For the detection of malonate, sugars and other organic acids by HPLC, the filtered samples were diluted 6-fold with 0.5M HCl before analysis by HPLC, usually within 48 hours after harvest. The diluted samples were analyzed by HPLC using a Bio-Rad Micro-GuardCation H+Guard column and a Bio-Rad Aminex 87H column (300x7.8 mm) installed in series, with an isocratic flow rate of 0.8 ml / min using 5.5 mM aqueous sulfuric acid and a column temperature of 35°C. Refractive index and UV 210nm measurements were collected within 18.5 minutes.

[0454] The dry cell weight (DCW) and malonate concentration of each strain were as follows: LPK152352: 1.93 + 0.40g-DCW / L,7.5 + 0.6g-malonic acid / L; LPK1511264:7.32 + 1.04g-DCW / L,14.7 + 2.8g-malonic acid / L; LPK153531:1.82 + 0.58g-DCW / L,9.4 + 0.1g-malonic acid / L; and LPK1511260:3.96 + 1.44g-DCW / L,11.8 + 2.2g-malonic acid / L.

[0455] Example 52: Identification of Pichia kudriavzevii strains with increased malonate production by colorimetry

[0456] Example 52 describes a DBS-based colorimetric assay that can be used for high-throughput screening of Pichia kudriavzevii strains with increased malonate production. The assay described herein identifies strain mutants from a library that have enhanced malonate production compared to non-mutated strains. The assay is used as a step prior to strain analysis by HPLC.

[0457] First, colonies from the library were picked from the plate and used to inoculate cultures in 96-well plates with deep wells. As a positive control, LPK1513359, which contains the F6A-6 malonyl-CoA hydrolase, was also grown. The cells were cultured at 30°C and shaking at 950 rpm. After 72 hours, two measurements were taken: (1) malonate concentration in the culture broth; and (2) OD 600nm For OD 600nm, the samples were diluted 20 times with water and measured in a 96-well plate using a plate reader. For malonate concentration, each culture was filtered using a Corning #3504 filter plate and the filtrate was diluted 10 times with water. The malonate concentration in the diluted filtrate was measured using DBS colorimetry.

[0458] The DBS assay was performed according to the method described by Satoru et al. in Anal. Biochem. 216, 159-164 (1994) with slight modifications. In brief, malonate purified from our recombinant Pichia kudriavzevii was used as the malonate standard for the assay. DBS was obtained from Carbosynth # FD61408. DBS was resuspended in 6.4 mM NaNO2 / 2M sodium / ammonium acetate pH 4.6 to a final concentration of 8.5 mM DBS solution. A reaction stop solution of 34% (w / v) NaOH / 0.16 M EDTA stop solution was also prepared. Malonate standards were prepared by serial dilution to produce concentrations of 0.4 mM to 12.5 mM, and 100 μl of each standard was pipetted into a standard transparent 96-well plate. The culture broth samples were diluted 10 to 20 times, and 100 μl of each sample was pipetted into the wells adjacent to the malonate standards. 6.1 μl of DBS solution was added to each well along w...

Claims

1. A recombinant Pichia kudriavzevii host cell comprising a heterologous malonyl-CoA hydrolase, wherein the heterologous malonyl-CoA hydrolase is a mutant Pseudomonas fulva (strain 12-X) protein of UniProt ID: F6AA82, wherein the mutant Pseudomonas fulva protein: (a) contains one or more of the following mutations: E95N, V152A, F304R and Q348A, or (b) has at least 75% sequence identity to SEQ ID NO:9 and comprises one or more mutations of the following amino acids: K107, L148, G170 and E367, or (c) having at least 75%, at least 85%, at least 90% and at least 95% sequence identity to the sequence of SEQ ID NO:57, 58, 59, 60 or 61.

2. The recombinant host cell according to claim 1, further comprising a heterologous S. cerevisiae aldehyde dehydrogenase, which is ALD6. 3 . The recombinant host cell according to claim 1 , further comprising a heterologous Yarrowia lipolytica CLIB122 acetyl-CoA synthetase, which is UniProt ID: Q6C2Q5.

4. The recombinant host cell of any one of claims 1-3, further comprising a heterologous Saccharomyces cerevisiae pyruvate decarboxylase, which is PDC6. 5 . The recombinant host cell according to claim 1 , wherein the host cell comprises native acetyl-CoA carboxylase ACC1 having UniProt ID: A0A099P5S6.

6. The recombinant host cell of any one of claims 1-4, wherein the host cell comprises acetyl-CoA carboxylase fused to carbonic anhydrase.

7. A method for identifying a host cell having increased production of malonate or a salt thereof, comprising the steps of: Fermenting the recombinant host cell of any one of claims 1 to 6 to provide a fermentation broth; contacting the fermentation broth with 4-diazonobenzenesulfonate (DBS) and optionally a base; and Cells with increased malonate production were identified based on increased absorbance levels at approximately 530 nm.

8. A method for producing malonate or a salt thereof, comprising culturing the host cell of any one of claims 1 to 6 under conditions resulting in the production of malonate.

9. A method for producing malonate or a salt thereof, comprising culturing the identified host cell of claim 7 under conditions that result in the production of malonate.

10. A method for preparing a malonate solution from a host cell fermentation broth, wherein the method comprises the following steps: Cultivating the recombinant host cell of any one of claims 1 to 6 or the identified recombinant host cell of claim 7 in a fermentation broth comprising a base under conditions that result in the production of malonate; and The malonate solution was separated.

11. A method for separating dialkyl malonate, preferably di-C1-C6 alkyl malonate, from a host cell fermentation broth, comprising the following steps: Cultivating the recombinant host cell of any one of claims 1 to 6 or the identified recombinant host cell of claim 7 under conditions that result in the production of malonate; separating host cells from the fermentation broth; adding a fatty alcohol and an acid catalyst to the fermentation broth to form a dialkyl malonate; and The resulting dialkyl malonate is separated from the fermentation broth to provide isolated dialkyl malonate.

12. The method of claim 11, wherein the dialkyl malonate formation further comprises contacting with an ion exchange resin.

13. The method according to claim 11 or 12, wherein the fatty alcohol is ethanol or methanol.

Images