In vitro synthesis of coenzyme a and acetyl-COA for cell-free biocatalysis

Abstract

The present disclosure relates to multi-enzyme pathways for the in vitro cell-free biosynthesis of the cofactor, coenzyme A ("CoA") from lower-cost substrates, such as D-pantothenate, L-cysteine, and ATP, and the use of these pathways in CoA-dependent cell-free systems for the biobased production of chemicals, such as acetyl-CoA, hexanoyl-CoA, malonyl-CoA, and cannabigerolic acid (CBGA).

Description

IN VITRO SYNTHESIS OF COENZYME A AND ACETYL-COA FOR CELL-FREE BIOCATALYSISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit to U.S. Provisional Application No. 63 / 556,767, filed February 22, 2024, which is hereby incorporated by reference herein.FIELD

[0002] The present disclosure relates to a multi-enzyme pathway for the in situ generation of the cofactor Coenzyme A (CoA) from lower-cost reagents, such as D-pantothenate, L-Cysteine, and Adenosine Triphosphate (ATP), and the use of this pathway in CoA-dependent cell-free systems for the biobased production of compounds, such as cannabigerolic acid (CBGA).STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] The inventions disclosed herein were made with government support under grant award number DE-AR0001507 from the Advanced Research Projects Agency - Energy (ARPA-E) of the U.S. Department of Energy. The government has certain rights in the inventions.REFERENCE TO SEQUENCE LISTING

[0004] The official copy of the Sequence Listing is submitted concurrently with the specification as an WIPO Standard ST.26 formatted XML file with file name “15041-005PV1 .xml”, a creation date of February 21 , 2024, and a size of 61 ,378 bytes.BACKGROUND

[0005] Coenzyme A (“CoA”) and acetyl-CoA (and malonyl-CoA) are central cofactors (CoA) and metabolites (acetyl-CoA, malonyl-CoA) utilized in all domains of life. The cofactor CoA and its acylated thioester derivatives (e.g., acetyl-CoA, malonyl-CoA, etc.) are involved in myriad primary and secondary metabolic pathways that are important for the production of commercially valuable compounds (e.g., fatty acids, polyketides, isoprenoids, cannabinoids, alkaloids, etc.). The isolated cofactor, CoA, is difficult to produce in large quantities either synthetically or in vivo, due to tight regulation in cells. CoA, therefore, is an expensive reagent that can add greatly to the cost of a biosynthetic pathway. As a result, most complex in vitro pathways to natural products use alternative routes that are not CoA-dependent (e.g., a simple pathway starting from isoprenol rather than from a mevalonate pathway).

[0006] A full CoA biosynthesis pathway of A. thaliana has been reconstituted in vitro (see e.g., 10.1074 / jbc.M306321200). Additionally, feedback insensitive type III PanK (CoaX) has been used to produce up to 1 mM coenzyme A from D-pantetheine but not from D-pantothenate (see e.g., 10.1128 / AEM.00541-21).

[0007] An efficient, low-cost, biosynthesis pathway for in situ CoA production, however, has not been achieved due to a number of factors: the feedback inhibition of the initial enzyme, PanK; the high ATP cofactor demand; and the CTP dependence of bacterial CoaBC homologues.

[0008] Accordingly, there remains a need for an improved biosynthetic process and compositions for efficient, low-cost in situ production of CoA.SUMMARY

[0009] The present disclosure relates generally to processes and compositions that utilize alternative lower-cost, multi-enzymatic, cell-free biosynthesis pathways for the in situ production of Coenzyme A (“CoA”). This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0010] In at least one embodiment, the present disclosure provides a process for synthesis of Coenzyme A (“CoA”) comprising contacting in a cell-free solution under suitable reaction conditions the following substrates and enzymes:(a) substrates:D-pantothenate, L-cysteine, and ATP and / or CTP;(b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), and dephospho-CoA kinase (2.7.1.24) (“DPCK”).

[0011] In at least one embodiment of the process, the substrates do not include CTP.

[0012] In at least one embodiment of the process, the substrates include ATP and CTP.

[0013] In at least one embodiment of the process, the substrates further comprise polyphosphate.

[0014] In at least one embodiment of the process, the process further comprises: (a) an enzyme capable of regenerating ATP from ADP or from AMP; (b) an enzyme capable of regenerating ADP from AMP; (c) ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); optionally, wherein the PPK enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26; and / or (d) PPK and polyphosphate AMP phosphotransferase (“PAP").

[0015] In at least one embodiment of the process, the enzymes further comprise inorganic pyrophosphatase (3.6.1.1) ("PPase").

[0016] In at least one embodiment of the process, the enzymes CoaB or CoaC are not CTP- dependent.

[0017] In at least one embodiment of the process, the enzymes CoaB or CoaC are derived from a eukaryotic source organism.

[0018] In at least one embodiment of the process, the enzymes CoaB and CoaC are from A thaliana or H. sapiens.

[0019] In at least one embodiment of the process, the enzyme CoaB comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 14, and 16.

[0020] In at least one embodiment of the process, the amino acid sequence of the enzyme CoaB further comprises an N-terminal tag and / or a C-terminal tag.

[0021] In at least one embodiment of the process, the enzyme CoaC comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 18, and 20.

[0022] In at least one embodiment of the process, the amino acid sequence of the enzyme CoaC further comprises an N-terminal tag and / or a C-terminal tag.

[0023] In at least one embodiment of the process, the enzyme PanK is selected from a Type I PanK or a Type II PanK.

[0024] In at least one embodiment of the process, the enzyme is a Type I PanK selected from EcCoaA and CaCoaA.

[0025] In at least one embodiment of the process, the amino acid sequence of the Type I PanK enzyme comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 2, and 4.

[0026] In at least one embodiment of the process, the enzyme is a Type II PanK selected from SaCoaW and SpCoaW.

[0027] In at least one embodiment of the process, the amino acid sequence of the Type II PanK enzyme comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 6, 8, or 10.

[0028] In at least one embodiment of the process, the amino acid sequence of the enzyme PanK further comprises an N-terminal tag and / or a C-terminal tag.

[0029] In another embodiment, the present disclosure provides a composition comprising a cell-free solution of: (a) D-pantothenate, L-cysteine, and ATP or CTP; and (b) pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), and phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”).

[0030] In at least one embodiment of the composition, the cell-free solution does not comprise CTP.

[0031] In at least one embodiment of the composition, the PanK: (a) is a Type I PanK enzyme comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 2, and 4; or (b) is a Type II PanK enzyme comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 6, 8, or 10.

[0032] In at least one embodiment of the composition, CoaB comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 14, and 16.

[0033] In at least one embodiment of the composition, CoaC comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 18, and 20.

[0034] In at least one embodiment of the composition, the cell-free solution further comprise polyphosphate.

[0035] In at least one embodiment of the composition, the cell-free solution further comprises: (a) ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); optionally, wherein the PPK enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26; (b) PPK and polyphosphate AMP phosphotransferase (“PAP"); (c) an enzyme capable of regenerating ATP from ADP or from AMP; and / or (d) an enzyme capable of regenerating ADP from AMP.

[0036] In at least one embodiment of the composition, the cell-free solution further comprises: (a) ethanol; (b) an alcohol oxidase (1.1.3.13) (“AOX”), optionally, wherein the AOX comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 34; (c) a catalase (1.11.1.6) (“CAT”), optionally, wherein the CAT comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 36 and 38; (d) an alcohol dehydrogenase (1 .1 .1 .1) (“ADH”), optionally, wherein the ADH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32; and (e) an aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

[0037] In at least one embodiment of the composition, the cell-free solution further comprises: (a) ethanol; (b) NAD+; (c) an NADH oxidase (1 .6.3.4) (“NOX”), optionally, wherein the NOX comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 28 and 30; (d) an alcohol dehydrogenase (1.1.1.1) (“ADH”), optionally, wherein the ADH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32; and (e) an aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

[0038] In at least one embodiment of the composition, the cell-free solution further comprises a fatty acid or diacid and a CoA-ester synthase.

[0039] In at least one embodiment of the composition, cell-free solution further comprises a fatty acid comprising from 1 to 30 carbons.

[0040] In at least one embodiment of the composition, the fatty acid is a saturated fatty acid selected from butyric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid.

[0041] In at least one embodiment of the composition, the fatty acid is an unsaturated fatty acid selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[0042] In at least one embodiment of the composition, the fatty acid is hexanoic acid and the CoA-ester synthase is an acyl-activating enzyme (6.2.1.1) (“AAE”).

[0043] In at least one embodiment of the composition, cell-free solution further comprises a dicarboxylic acid comprising from 1 to 30 carbons.

[0044] In at least one embodiment of the composition, the dicarboxylic acid is a saturated dicarboxylic acid selected from malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, hexadecanedioic acid, heneicosanedioic acid, docosanedioic acid, and 1 ,4- cyclohexanedicarboxylic acid.

[0045] In at least one embodiment of the composition, the dicarboxylic acid is an unsaturated dicarboxylic acid selected from maleic acid, fumaric acid, acetylenedicarboxylic acid, glutaconic acid, traumatic acid, muconic acid, glutinic acid, citraconic acid, mesaconic acid, and itaconic acid.

[0046] In yet another embodiment, the present disclosure provides a first process for the synthesis of acetyl-CoA comprising contacting in a cell-free solution under suitable reaction conditions the following substrates and enzymes:(a) substrates: D-pantothenate, L-cysteine, polyphosphate, ATP and / or CTP, and ethanol;(b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), dephospho-CoA kinase (2.7.1.24) (“DPCK”), inorganic pyrophosphatase (3.6.1.1) ("PPase"), alcohol oxidase (1.1.3.13) (“AOX”), a catalase (1.11.1.6) (“CAT”), alcohol dehydrogenase (1.1.1.1) (“ADH”), and aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

[0047] In yet another embodiment, the present disclosure provides a second process for the synthesis of acetyl-CoA comprising contacting in a cell-free solution under suitable reaction conditions the following substrates and enzymes:(a) substrates: D-pantothenate, L-cysteine, polyphosphate, ATP or CTP, ethanol, and NAD+;(b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), dephospho-CoA kinase (2.7.1.24) (“DPCK”), inorganic pyrophosphatase (3.6.1.1) ("PPase"), NADH oxidase (1 .6.3.4) (“NOX”), alcohol dehydrogenase (1.1.1.1) (“ADH”), and aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

[0048] In yet another embodiment, the present disclosure provides a process for the synthesis of hexanoyl-CoA comprising contacting in a cell-free solution under suitable reaction conditions the following substrates and enzymes:(a) substrates: D-pantothenate, L-cysteine, polyphosphate, ATP or CTP, and hexanoic acid;(b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), dephospho-CoA kinase (2.7.1.24) (“DPCK”), inorganic pyrophosphatase (3.6.1.1) ("PPase"), and acyl-activating enzyme (6.2.1.1) (“AAE”).

[0049] In at least one embodiment of the processes for synthesis hexanoyl-CoA further comprising contact the hexanoyl-CoA synthesized in a cell-free solution under suitable reaction conditions with the following substrates and enzymes:(a) substrates: malonate, and isoprenol;(b) enzymes: Malonyl CoA synthetase (MatB), Hydroxyethylthiazole kinase (ThiM), Isopentyl diphosphate isomerase (IDI), Isopentenyl kinase (IPK), Farnesyl-PP synthase (FPPS), Olivetol synthase (OLS), Olivetolic acid cyclase (OAC), and Soluble prenyltransferase (NphB); thereby, resulting in the synthesis of the cannabinoid, cannabigerolic acid (CBGA).

[0050] In at least one embodiment of the processes for synthesis of acetyl-CoA or hexanoyl- CoA, the process further comprises: (a) an enzyme capable of regenerating ATP from ADP or from AMP; (b) an enzyme capable of regenerating ADP from AMP; (c) ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); optionally, wherein the PPK enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26; and / or (d) PPK and polyphosphate AMP phosphotransferase (“PAP").

[0051] In at least one embodiment of the processes for synthesis of acetyl-CoA or hexanoyl- CoA, the PanK: (a) is a Type I PanK enzyme comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 2, and 4; or (b) is a Type II PanK enzyme comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 6, 8, or 10.

[0052] In at least one embodiment of the processes for synthesis of acetyl-CoA or hexanoyl- CoA, the CoaB comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 14, and 16.

[0053] In at least one embodiment of the processes for synthesis of acetyl-CoA or hexanoyl- CoA, the CoaC comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 18, and 20.

[0054] In at least one embodiment of the processes for synthesis of acetyl-CoA or hexanoyl- CoA: (a) PPAT comprises an amino acid sequence having at least 90% identity to a sequence of SEQ ID NO: 22; (b) DPCK comprises an amino acid sequence having at least 90% identity to a sequence of SEQ ID NO: 24; (c) NOX comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 28, and 30; and / or (d) ADH comprisesan amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 32, and 34.BRIEF DESCRIPTION OF THE DRAWINGS

[0055] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0056] FIG. 1A depicts an exemplary Coenzyme A (“CoA”) in vitro cell-free biosynthetic reaction scheme of the present disclosure. CoA is produced biosynthetically from the substrates, D-Pantothenate, L-Cysteine, and ATP, using the following enzymes: ATP- polyphosphate phosphotransferase (2.7.4.1) (“PPK”); pantothenate kinase (2.7.1.33) (“PanK”); phosphopantothenate-cysteine ligase (6.3.2.5 for CTP, or 6.3.2.51 for ATP) (“CoaB”); phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”); phosphopantetheine adenylyltransferase (2.7.7.3) (“PPAT”); dephospho-CoA kinase (2.7.1.24) (“DPCK”); and inorganic pyrophosphatase (3.6.1.1) (“Ppase”).

[0057] FIG. 1B depicts plots of screening data (obtained as described in Example 1) showing the production of CoA in a reaction scheme of FIG. 1 A when using a combination of an ATP- dependent eukaryotic CoaB from Homo sapiens (“HsCoaB”) and a “stand-alone” decarboxylase CoaC from Homo sapiens (“HsCoaC”), or a single CTP-dependent bacterial CoaBC enzyme from E. coli (“EcCoaBC”). Type I, type II, and type III PanK enzymes were also screened: “CaCoaA” is a type I PanK, “SpCoaW” is a type II PanK, and “TmCoaX” is a type III PanK. As shown by the results in FIG. 1B (and further described in Example 1), the combination of the ATP-dependent HsCoaB enzyme and the standalone HsCoaC enzyme together with a type I or type II PanK resulted in greatly increased CoA production.

[0058] FIG. 1C depicts plots of screening data (obtained as described in Example 1) showing the production of CoA in a reaction scheme of FIG. 1 A when using various different type I, type II, and type III PanK enzymes. As shown by the results in FIG. 1C (and further described in Example 1), two type I PanK enzymes (“EcCoaA” and “CaCoaA”) and two type II PanK enzymes (“SpCoaW” and “SaCoaW”) resulted in greatly increased CoA production.

[0059] FIG. 2A depicts an exemplary reaction schemes for the in vitro cell-free biosynthesis of acetyl-CoA from the substrates, D-pantothenate, L-cysteine, ATP, and ethanol. The reaction schemes utilize CoA produce via the de novo biosynthesis scheme of FIG. 1A. The substrate ethanol can be oxidized to the intermediate acetaldehyde via either of two pathways: (1) NOX- ADH (“Option 1”); or (2) AOX-CAT (“Option 2"). The reaction schemes use the following enzymes: NADH oxidase (1 .6.3.4) (“NOX"); alcohol dehydrogenase (1.1.1.1) (“ADH”); alcohol oxidase (1.1.3.13) (“AOX”); aldehyde dehydrogenase (1 .2.1.3) (“AIDH”).

[0060] FIG. 2B depicts plots of data (obtained as in Example 2) comparing the “Option 1” and “Option 2” pathways of FIG 2A in the in vitro production of acetyl-CoA. Option 1 slightly outperformed Option 2.

[0061] FIG. 2C depicts plots of data (obtained as in Example 2) comparing the in vitro cell-free production of acetyl-CoA according to the pathway of FIG. 2A Option 1 , when the Type I and Type II PanK enzymes, SpCoaW and CaCoaA, are used to produce CoA via FIG. 1A pathway at ATP concentrations of 2.5 mM, 5 mM, and 10 mM. The type II PanK, SpCoaW slightly outperforms the type I PanK, CaCoaW, and nearly goes to completion at all ATP concentrations.

[0062] FIG. 2D depicts plots of data (obtained as in Example 2) showing the time course of the FIG. 2A “Option 1” reaction using the type II PanK, SpCoaW at an ATP concentration of 20mM. As shown by comparison of the time plots of CoA and acetyl-CoA production, the in situ biosynthesis of CoA via the reaction scheme of FIG. 1 A outperforms the ethanol upgrading “Option 1” reaction of FIG. 2A at the enzyme loads used.

[0063] FIG. 3A depicts a two-pot reaction scheme for de novo synthesis of hexanoyl-CoA in a first reaction (“RXN 1”), and then conversion of the hexanoyl-CoA to CBGA in a second reaction (“RXN 2”), as described further in Example 3. RXN1 produces CoA via the reaction scheme of FIG. 1 A and then further converts the in situ produced CoA and added substrate, hexanoic acid to hexanoyl-CoA using an acyl-activating enzyme (“AAE3’’). RXN2 converts CoA produced in situ via the reaction scheme of FIG. 1A, and the added substrates, hexanoyl-CoA (produced in RXN1), malonate, and isoprenol, to the cannabinoid product, CBGA. The biosynthetic reactions of RXN2 that produce CBGA requires at least the following eight enzymes: MatB, ThiM, IDI, IPK, FPPS, OLS, OAC, and NphB.

[0064] FIG. 3B depicts an HPLC trace showing hexanoyl-CoA produced by RXN1 at 10 mM concentration (and trace amounts of CoA) as described in Example 3.

[0065] FIG. 3C depicts an HPLC trace showing CBGA production by RXN2 at 237 mg / mL concentration starting from 4 mM hexanoyl-CoA produced in RXN1 as described in Example 3.DETAILED DESCRIPTION

[0066] For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use theterm “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.”

[0067] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.

[0068] Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011 , and now available in journal format online as Current Protocols in Molecular Biology, Vols. 00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter “Ausubel”).

[0069] All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.

[0070] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. For purposes of interpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa.

[0071] Definitions

[0072] “Conversion” as used herein refers to the enzymatic conversion of the substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the“enzymatic activity’’ or “activity” of an enzymatic conversion can be expressed as “percent conversion” of the substrate to the product.

[0073] “Product” as used herein in the context of an enzyme mediated process refers to the compound or molecule resulting from the activity of the enzyme. .

[0074] “Substrate” as used herein in the context of an enzyme mediated process refers to the compound or molecule acted on by the enzyme. .

[0075] “ATP-dependent enzyme” as used herein refers to an enzyme that uses adenine triphosphate (ATP) as a coenzyme for the conversion of a substrate to a product.

[0076] “CTP-dependent enzyme” as used herein refers to an enzyme that uses cytidine triphosphate (CTP) as a coenzyme for the conversion of a substrate to a product.

[0077] “CoA precursor compound” or “CoA precursor substrate” as used herein refers a compound or molecule acted on by an enzyme in a biosynthetic step for producing CoA as described in FIG. 1A and elsewhere herein. Exemplary CoA precursor compounds are provided in Table 1.

[0078] TABLE 1: Exemplary CoA precursor substrate compounds

[0079] “Pantothenate kinase” or "PanK” as used herein refer to an enzyme that, in the presence of ATP, phosphorylates D-pantothenate to produce the CoA precursor, D-4’- phosphopantothenate, and includes those enzymes of classification EC 2.7.1 .33. Exemplary PanK enzymes useful in the compositions and methods of the present disclosure include but are not limited to those Type I PanK and Type II PanK enzymes listed in Table 2 below.

[0080] “Phosphopantothenate-cysteine ligase” or“CoaB” as used herein refer to an enzyme that, in the presence of ATP, ligates an L-cysteine to the CoA precursor, D-4’- phosphopantothenate, to produce D-4’-phosphopantothenoyl-L-cysteine, and includes those CTP-dependent enzymes of classification EC 6.3.2.5, and the ATP-dependent enzymes of classification EC 6.3.2.51 . Exemplary CoaB enzymes useful in the compositions and methods of the present disclosure include but are not limited to those CoaB enzymes listed in Table 2 below.

[0081] “Phosphopantothenoylcysteine decarboxylase” or“CoaC” as used herein refer to an enzyme that decarboxylates the CoA precursor, D-4’-phosphopantothenoyl-L-cysteine, to produce D-4’-phosphopantetheine, and includes those enzymes of classification EC 4.1.1.36.Exemplary CoaC enzymes useful in the compositions and methods of the present disclosure include but are not limited to those CoaC enzymes listed in Table 2 below.

[0082] “Phosphopantetheine adenylyltransferase” or“PPAT” as used herein refer to an enzyme that, in the presence of ATP, transfers an adenylyl group to the CoA precursor, D-4’- phosphopantetheine, to produce the CoA precursor, dephospho-coenzyme A, and includes those enzymes of classification EC 2.7.7.3. Exemplary PPAT enzymes useful in the compositions and methods of the present disclosure include but are not limited to those PPAT enzymes listed in Table 2 below.

[0083] “Dephospho-CoA kinase” or“DPCK” as used herein refer to an enzyme that, in the presence of ATP, phosphorylates dephospho-coenzyme A to produce CoA, and includes those enzymes of classification EC 2.7.1 .24. Exemplary DPCK enzymes useful in the compositions and methods of the present disclosure include but are not limited to those DPCK enzymes listed in Table 2 below.

[0084] “ATP-polyphosphate phosphotransferase” or “PPK” as used herein refer to an enzyme that phosphorylates ADP to produce ATP and includes those enzymes of classification EC 2.7.4.1.

[0085] “Inorganic pyrophosphatase” or “Ppase” as used herein refer to an enzyme that reversibly hydrolyzes the phosphoanhydride bond in pyrophosphate (PPi) to produce two phosphate molecules and includes those enzymes of classification EC 3.6.1.1.

[0086] “Host cell” as used herein refers to a cell capable of being functionally modified with recombinant nucleic acids and functioning to express recombinant products, including polypeptides and compounds produced by activity of the polypeptides.

[0087] “Nucleic acid,” or “polynucleotide” as used herein interchangeably to refer to two or more nucleosides that are covalently linked together. The nucleic acid may be wholly comprised ribonucleosides (e.g., RNA), wholly comprised of 2'-deoxyribonucleotides (e.g., DNA) or mixtures of ribo- and 2'-deoxyribonucleosides. The nucleoside units of the nucleic acid can be linked together via phosphodiester linkages (e.g., as in naturally occurring nucleic acids), or the nucleic acid can include one or more non-natural linkages (e.g., phosphorothioester linkage). Nucleic acid or polynucleotide is intended to include singlestranded or double-stranded molecules, or molecules having both single-stranded regions and double-stranded regions. Nucleic acid or polynucleotide is intended to include molecules composed of the naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), or molecules comprising that include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.

[0088] “Protein,” “polypeptide,” and “peptide” are used herein interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). As used herein “protein” or “polypeptide” or “peptide” polymer can include D- and L-amino acids, and mixtures of D- and L-amino acids.

[0089] “Naturally-occurring” or “wild-type” as used herein refers to the form as found in nature. For example, a naturally occurring nucleic acid sequence is the sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0090] “Recombinant,” “engineered,” or “non-naturally occurring” when used herein with reference to, e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0091] “Nucleic acid derived from” as used herein refers to a nucleic acid having a sequence at least substantially identical to a sequence of found in naturally in an organism. For example, cDNA molecules prepared by reverse transcription of mRNA isolated from an organism, or nucleic acid molecules prepared synthetically to have a sequence at least substantially identical to, or which hybridizes to a sequence at least substantially identical to a nucleic sequence found in an organism.

[0092] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0093] “Heterologous nucleic acid” as used herein refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0094] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the imine reductase enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0095] “Preferred, optimal, high codon usage bias codons” refers to codons that are used at higher frequency in the protein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see for example, Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella,"1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p. 2047-2066. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001 ; Uberbacher, E. C., 1996, Methods Enzymol. 266:259-281 ; Tiwari et al.,1997, Comput. Appl. Biosci. 13:263-270).

[0096] “Control sequence” as used herein refers to all sequences, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide as used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding a polypeptide. Such control sequences include, but are not limited to, a leader, a promoter, a polyadenylation sequence, a pro-peptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences typically include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0097] “Operably linked” as used herein refers to a configuration in which a control sequence is appropriately placed (e.g., in a functional relationship) at a position relative to a polynucleotide sequence or polypeptide sequence of interest such that the control sequence directs or regulates the expression of the sequence of interest.

[0098] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of apolynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0099] “Percentage of sequence identity,” “percent sequence identity,” “percentage homology,” or “percent homology” are used interchangeably herein to refer to values quantifying comparisons of the sequences of polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (or gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage values may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res.3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.

[0100] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length nucleic acid or polypeptide sequence. A reference sequence typically is at least 20 nucleotide or amino acid residue units in length, but can also be the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (or gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

[0101] “Corresponding to,” “reference to,” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence ratherthan by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered imine reductase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0102] “Isolated” as used herein in reference to a molecule means that the molecule (e.g., cannabinoid, polynucleotide, polypeptide) is substantially separated from other compounds that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces nucleic acids which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis).

[0103] “Substantially pure” refers to a composition in which a desired molecule is the predominant species present (i.e. , on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition) and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight.

[0104] “Recovered” as used herein in relation to an enzyme, protein, or cannabinoid compound, refers to a more or less pure form of the enzyme, protein, or cannabinoid.

[0105] In situ CoA Production for Use in Cell-Free CoA-Dependent Biosynthesis Systems

[0106] In situ production of coenzyme A, acetyl-CoA (and other acyl-CoAs) in a cell-free, one- pot or two-pot system, as described herein, drastically reduces the costs for cell-free biosynthesis of any compound that uses a CoA-dependent pathway. For example, the in situ production of CoA, as described herein, can provide for the cell-free biomanufacturing of fatty acids, esters, polyketides, isoprenoids, cannabinoids, alkaloids, and other natural and nonnatural products. Besides application to enzymatic natural product / pharmaceutical biosynthesis, in situ CoA biosynthesis could also be applied to upgrading of renewable, CO2 neutral (or negative) ethanol into industrially relevant building blocks (i.e. 3-hydroxybutanoic acid, hexanoic acid, hexanol, ethyl butyrate, ethylhexanoate), potentially displacing current processes that rely on fossil fuels to produce these commodity chemicals.

[0107] CoA is an essential coenzyme used as a substrate in the cell-free biosynthesis pathways of biobased chemical compounds, such as cannabinoids (e.g., CBGA). CoA, however, is a relatively expensive reagent that adds significant costs affecting the commercial viability of any biobased chemical production that uses CoA as a substrate. The present disclosure describes various alternative sets of enzymes and CoA precursor substrates that can be used in cell-free solutions to biosynthesize CoA in situ. This in situ produced CoA can then be used as a substrate in biosynthetic enzyme pathways that require CoA also present in the solution. Accordingly, the present disclosure includes cell-free solution compositionscontaining sets of reagents and enzymes, as well as processes of using these composition in cell-free biosynthesis systems that require CoA.

[0108] As described elsewhere herein, including the Examples, the present disclosure provides a cell-free biosynthetic process, and associated compositions, useful for the production of the coenzyme, CoA. CoA is an essential substrate used in the biosynthesis of a wide range of biobased chemical compound, such as fatty acids, esters, polyketides, isoprenoids, cannabinoids, alkaloids. For example, CoA is essential to biosynthesis of acetyl-CoA, one of the most used central metabolites in cellular metabolism. Acetyl-CoA is a starting block substrate for the production of myriad commercially valuable compounds via biosynthesis. The cell-free processes, and associated compositions, include a combination of reagents and enzymes that are capable of in situ production of CoA, which can then be used to fuel CoA dependent enzymatic reactions in the biosynthetic process. Generally, the processes comprise contacting a cell-free solution composition that contains a set of enzymes and CoA precursor reagents, under suitable reaction conditions. Notably, there is no need to add the CoA to this cell-free solution containing the enzymes and reagents. The CoA is produced in situ from its precursors and can utilized directly as a substrate in an enzyme pathway to a desired downstream biosynthesis product. That is, the CoA is produced in the cell-free solution and then used as a substrate in a further enzymatic conversion reaction to obtain a further desired compound (e.g., CBGA) or a precursor to that compound (e.g., hexanoyl-CoA).

[0109] Briefly, the in situ cell-free biosynthetic production of CoA is carried out via an enzymatic reaction scheme as illustrated in FIG. 1A. The reaction scheme utilizes the CoA precursor compounds, D-pantothenate, L-cysteine, and ATP as substrates for a series (or pathway) of enzymes in a cell-free solution. The series of enzymes includes: ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); pantothenate kinase (2.7.1.33) (“PanK”); phosphopantothenate-cysteine ligase (6.3.2.5 for CTP, or 6.3.2.51 for ATP) (“CoaB”); phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”); phosphopantetheine adenylyltransferase (2.7.7.3) (“PPAT”); dephospho-CoA kinase (2.7.1.24) (“DPCK”); and inorganic pyrophosphatase (3.6.1.1) (“Ppase”).

[0110] As shown in FIG. 1A, the in situ biosynthetic pathway starts with a PanK enzyme acting on the substrate, D-pantothenate resulting in the intermediate compound, D-4’- phosphopantathenate, which is then converted by a CoaB enzyme, along with the cosubstrates, ATP, and L-cysteine, into the intermediate compound, D-4’-phosphopantothenoyl-L- cysteine. The FIG. 1A biosynthesis pathway continues with the enzyme CoaC converting D-4’- phosphopantothenoyl-L-cysteine to D-4’-phosphopantetheine, the enzyme PPAT converting D- 4’-phosphopantetheine to dephospho-coenzyme A, and finally, the enzyme DPCK converting dephospho-coenzyme A to coenzyme A.

[0111] Amino acid sequences of exemplary enzymes (and exemplary encoding DNA sequences) useful for the cell-free in situ biosynthesis pathway of FIG. 1 A (and otherbiosynthetic processes described elsewhere herein) are summarized in Table 2 below and the accompanying Sequence Listing.

[0112] TABLE 2

[0113] Table 2 provides exemplary enzymes in each of the enzyme classes useful in the processes and compositions for cell-free CoA production described herein. This includes enzymes in the following enzyme classes: ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); pantothenate kinase (2.7.1.33) (“PanK”); phosphopantothenate-cysteine ligase (6.3.2.5 for CTP, or 6.3.2.51 for ATP) (“CoaB”); phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”); phosphopantetheine adenylyltransferase (2.7.7.3) (“PPAT”); dephospho-CoA kinase (2.7.1.24) (“DPCK”); and inorganic pyrophosphatase (3.6.1.1) (“Ppase”). One of ordinary skill however, will recognize that the biosynthetic processes of the present disclosure can be carried using alternative to the exemplary enzymes of Table 2 in each of the various classes.

[0114] In at least one embodiment, the biosynthetic process for the production of CoA that uses an enzymatic pathway comprising a Type II PanK (e.g., CoaW), a eukaryotic ATP- dependent CoaB, a eukaryotic ATP-dependent CoaC, and a PPK that catalyzes ATP regeneration. In at least one embodiment, the biosynthesis process is capable of producing CoA at a titer above 5 mM (3.8 g / L) starting from the inputs D-pantothenate, L-cysteine, ATP, and polyphosphate substrates.

[0115] Further exemplary combinations of the enzymes described in Table 2, and CoA precursor compounds (described in Table 1) useful as substrates in the compositions and processes of the present disclosure for the in situ biosynthesis of CoA are described in greater detail in the Examples below.

[0116] Additionally, one of ordinary skill will recognize that the exemplary enzymes of Table 2 include N-terminal and / or C-terminal tags in their amino acid sequences. These histidine tags facilitate purification of the enzymes but may not be required for the enzymatic activity necessary for use in the cell-free biosynthesis process. Accordingly, the His tags are intended to be viewed as optional or variable portions of the amino acid sequences. For example, it is contemplated that the N-term and C-term His tags can be replaced with alternative purificationtags in the amino acid sequences and the resulting enzymes can be used in the processes and compositions of the present disclosure. Accordingly, in at least one embodiment, it is contemplated that the biosynthetic processes of the present disclosure can be carried out in a cell-free solution using any of the enzymes listed in Table 2, except wherein the amino acid sequence does not include the N-terminal and / or C-terminal His tag(s).

[0117] Additionally, one of ordinary skill in the art will recognize that sequence homologs or engineered versions of exemplary enzymes of Table 2 can be used in the biosynthetic processes and compositions of the present disclosure. Such sequence homologs, can be identified from annotated sequence databases, prepared, and screened for the necessary activity. Similarly, engineered versions of the enzymes having the amino acid sequences of even-numbered SEQ ID NOs: 2-38 listed in Table 2 but with e.g., 1-5, 1-10, 1-15, 1-20, 1-25, or more amino acid substitutions can be designed (e.g., based on known homolog sequences), prepared, and screened for the necessary activity. Accordingly, in at least one embodiment, it is contemplated that the biosynthetic processes and compositions of the present disclosure using any of the enzymes listed in Table 2, can also use an active homolog or engineered version an enzyme listed in Table 2. Thus, in at least one embodiment, any of the enzymes having amino acid sequences of even numbered SEQ ID NOs: 2-38, that are listed in Table 2 for use in a process or composition of the present disclosure, can be substituted in the process or composition with an active enzyme having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater, percentage identity to the sequence listed in Table 2.

[0118] For example, the PanK enzyme used in the biosynthetic process of preparing CoA of the present disclosure (as in FIG. 1A) can be a Type II PanK enzyme comprising an amino acid sequence of SEQ ID NO: 6, 8, or 10, or comprising an amino acid sequence of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater identity to a sequence selected from SEQ ID NO: 6, 8, or 10.

[0119] In at least one embodiment, the in situ biosynthesis of CoA biosynthesis can be coupled to the further biosynthesis of an acylated CoA derivative, such as acetyl-CoA, malonyl-CoA, hexanoyl-CoA. Two exemplary schemes using ethanol for the further biosynthesis of acetyl- CoA starting are illustrated in FIG. 2A as “Option 1” and “Option 2." Further details of these biosynthetic processes are described in Example 2.

[0120] In at least one embodiment, the biosynthesis process of the present disclosure that produces CoA and then acetyl-CoA, produces acetyl-CoA at 16 mM (13 g / L) starting from the inputs D-pantothenate, L-cysteine, ATP, and polyphosphate substrates.

[0121] In another embodiment, the in situ biosynthesis of CoA can be coupled to the further biosynthesis of a cannabinoid compound, such as CBGA. For example, CBGA can be produced in a cell-free biosynthesis process that utilizes CoA, hexanoyl-CoA and malonyl-CoA,produced via in situ biosynthesis, and the added substrate, isoprenol. This exemplary process for the cell-free biosynthesis of CBGA is illustrated in FIG. 3A.

[0122] Briefly, the CBGA biosynthesis process of FIG. 3A uses the previously described processes and enzymes for the in situ production of CoA as part of a two-pot biosynthesis. The two-pot process begins by first producing CoA and then hexanoyl-CoA in a first one-pot process (FIG. 3A, “RXN1”). As shown in FIG. 3A, the RXN1 synthesis of hexanoyl-CoA combining the following substrates and enzymes in a cell-free solution under suitable reaction conditions: (a) substrates: D-pantothenate, L-cysteine, polyphosphate, ATP or CTP, and hexanoic acid; (b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenatecysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), dephospho-CoA kinase (2.7.1.24) (“DPCK”), inorganic pyrophosphatase (3.6.1.1) ("PPase"), and acyl-activating enzyme (6.2.1.1) (“AAE”).

[0123] The hexanoyl-CoA is then used in a second, one-pot multi-enzyme process with malonic acid to produce CBGA (FIG. 3A “RXN2”).

[0124] The multi-enzyme “RXN2” process that produces CBGA from the hexanoyl-CoA, malonic acid, and isoprenol, uses a pathway of the following 10 enzymes: (i) Acetyl-phosphate transferase (PT A); (ii) Malonate decarboxylase alpha subunit (mdcA); (iii) Acyl activating enzyme 3 (AAE3); (iv) Olivetol synthase (OLS); (v) Olivetolic acid cyclase (OAC); (vi) Hydroxyethylthiazole kinase (ThiM); (vii) Isopentenyl kinase (IPK); (viii) Isopentyl diphosphate isomerase (I DI) ; (ix) Diphosphomevalonate decarboxylase alpha subunit (MDCa); and / or (x) Geranyl-PP synthase (GPPS) or Farnesyl-PP synthase mutant S82F (FPPS S82F). The one- pot, multi-enzyme processes for producing CBGA are described in e.g., PCT application publications W02020028722A1 , WO2021134024A1 , and WO2022251285A1 , each of which is hereby incorporated by reference herein.

[0125] Similar to the production of acetyl-CoA described above and in Example 2, as shown in RXN2 of FIG. 3A, the coenzyme malonyl-CoA is produced in situ by contacting the CoA produced in RXN1 with the co-substrate, malonic acid (or malonate) in the presence of the malonyl CoA synthetase enzyme MatB. Further details of the biosynthetic processes for producing hexanoyl-CoA, malonyl-CoA, and CBGA according to RXN1 and RXN2 of FIG. 3A are described in Example 3.

[0126] Thus, using the combinations of CoA precursor compounds and enzymes as disclosed herein, a cell-free solution that uses CoA as an enzyme substrate (i.e., is “CoA dependent”) in a process for the biosynthesis of a biobased compound does not need CoA added as a reagent. Instead, the required CoA is produced in situ by the activity of the added enzymes on the CoA precursor compounds. Accordingly, a cell-free solution for CoA-dependent biosynthesis of a compound need not include any CoA prior to contacting the solution with the CoA precursorsubstrates and enzymes in the combination of the present disclosure that results in production of CoA in situ (e.g., process of FIG. 1 A).

[0127] For example, a cell-free process for CoA-dependent biosynthesis of a compound (e.g., CBGA) can be carried out by contacting in a cell-free solution under suitable reaction conditions the CoA precursors and enzymes of FIG. 1 A. It is not necessary to add CoA to the reaction mixture, and the necessary CoA is produced in the solution (i.e., in situ) where it can be used by the various enzymes also present in the solution that need CoA as a co-substrate for the conversion of the substrate to the compound or to a downstream precursor of the final compound. That is, the cell-free solution does not need to include any added CoA prior to contacting with any of the CoA producing enzymes and CoA precursor compound combinations.

[0128] It is contemplated by that processes and compositions comprising the combinations of enzymes and CoA precursor reagents of the present disclosure can be incorporated (or “dropped in”) to any cell free biosynthetic process that utilizes CoA as a substrate, simply by adding the enzymes and CoA precursor reagents to the reaction mixture. A range of such cell- free biosynthetic processes are known in the art, for cell-free production of compounds such as fatty acids (and derivatives thereof), polyketides, isoprenoids (and terpenes thereof), flavonoids, alkaloids. For example, a cell-free CBGA biosynthesis system is disclosed in US Patent Application Publication 2023 / 0348866 A1 , published November 2, 2023, which is hereby incorporated herein by reference.

[0129] Generally, the cell free solutions comprising a combination of reagents and enzymes of the present disclosure can include additional reagents and enzymes that may be required to carry out the particular CoA-dependent biosynthesis reaction. For example, the cell-free solution can further comprise a precursor substrate that can be converted to another substrate required for the CoA-dependent enzyme conversion. Additionally, other enzymes capable of converting that precursor substrate to the desired substrate. In at least one embodiment, the cell-free solution can include a precursor substrate that is a sugar, such as a C6 sugar (e.g., xylose), a C5 sugar (e.g., ribose), or a mixture of a C6 and C5 sugar. Various pathways of enzymes leading from precursor substrates (e.g., sugars) to substrates that can be converted by an CoA-dependent enzyme reaction to a desired biobased chemical compound in a cell free process are known in the art.

[0130] It is also contemplated that the processes using combinations of CoA precursor compounds and enzymes to produce CoA in situ can be incorporated in biosynthetic processes that further comprise additional chemical or biocatalytic steps carried out on the desired product compound, including product compound work-up, extraction, isolation, purification, and / or crystallization, each of which can be carried out under a range of conditions.

[0131] Generally, the suitable reaction conditions for cell free biosynthesis processes described herein, or known in the art, can be readily optimized by routine experimentation that includes,but is not limited to, contacting the relevant combinations of enzyme(s) and substrate(s) under experimental reaction conditions of concentration, pH, temperature, solvent conditions, and detecting the production of the desired compound(s), for example, using the methods described in the Examples provided herein. For example, it is contemplated that the reaction conditions suitable for the cell-free biosynthesis of cannabinoids using precursor substrates with CoA (see e.g., Valliere et al. 2020; W02020028722A1 ; or US Patent Application Publication 2023 / 0348866 A1) can be used as-is or readily adapted for use in the processes of the present disclosure.

[0132] The present disclosure contemplates ranges of suitable reaction conditions that can be used in the processes using combinations of CoA precursor compounds and enzymes to produce CoA in situ for use in cell free biosynthesis of compounds as disclosed herein, including but not limited to ranges of pH, temperature, buffer, solvent system, substrate loading, polypeptide loading, co-substrate or co-factor loading, atmosphere, and reaction time.Exemplary suitable reaction conditions including substrate loading, enzyme concentrations, pH, buffering, solvents, temperature, and reaction times are provided in the Examples below.EXAMPLES

[0133] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.Example 1: Cell-free Biosynthetic Production of Coenzyme A

[0134] This example illustrates a study showing that coenzyme A (“CoA”) can be produced via the in vitro cell-free biosynthesis pathway of FIG. 1A. In this example, the biosynthesis is carried out using substrates, D-pantothenate, L-cysteine, ATP and polyphosphate, and the following enzymes: ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); pantothenate kinase (2.7.1.33) (“PanK”); phosphopantothenate-cysteine ligase (6.3.2.5 for CTP, or 6.3.2.51 for ATP) (“CoaB”); phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”); phosphopantetheine adenylyltransferase (2.7.7.3) (“PPAT”); dephospho-CoA kinase (2.7.1.24) (“DPCK”); and inorganic pyrophosphatase (3.6.1.1) (“Ppase”).

[0135] A. Materials and Methods

[0136] An initial in vitro production of CoA using the reaction scheme of FIG. 1A was carried out using the following enzymes from E.coli: EcCoaA, EcCoaBC, EcCoaD, EcCoaE.

[0137] A. Results

[0138] The enzyme, EcCoaD was found to be insoluble with an N-terminal His tag but was soluble and could be purified with a C-terminal tag. Regardless, the biosynthesis of CoA using these E. coli enzymes required the stoichiometric addition of CTP, as no regeneration pathway for CTP is available.

[0139] B. Materials and Methods

[0140] In order to increase the efficiency of the CoA biosynthesis reaction of FIG. 1A, the CTP dependent, single bifunctional enzyme EcCoaBC was replaced with two homologous eukaryotic enzymes, CoaB and CoaC. Reactions contained 200 mM TrisHCI pH 9, 5 mM MgCI2, 10 mM KCI, 2 mM ATP (and 2 mM CTP for EcCoaBC), 16 mg / mL hexametaphosphate, 10 mM D- pantothenic acid, 20 mM L-cysteine, 12 pM CoaA., 10 pM EcCoaBC or HsCoaB+HsCoaC, 10 pM EcCoaD, 10 pM EcCoaE, 3 pM AaPPK, 2 pM CaAAE3, 2.5 pM GsPPase. Reactions were run over night, quenched using methanol and analyzed by HPLC.

[0141] B. Results

[0142] As shown by the results in FIG. 1B, the pair of CoaB and CoaC from H. sapiens, HsCoaB and HsCoaC, produced significantly increased amounts of CoA relative to the EcCoaBC from E. coli. CoaB and CoaC homologues from A. thaliana were also tested and found to work equally well as the H. sapiens homologues in producing CoA. The H. sapiens enzymes purified at a higher concentration, however, and therefore were selected for further pathway optimizations.

[0143] C. Materials and Methods

[0144] In an effort to further improve CoA production using the biosynthetic scheme of FIG. 1A, nine different PanK enzymes of Type I, Type II, and Type III were screened for their ability to mediate increased production of CoA. Reactions contained 200 mM TrisHCI pH 9, 5 mM MgCI2, 10 mM KCI, 2 mM ATP, 16mg / ml_ hexametaphosphate, 10 mM D-pantothenic acid, 20 mM L- cysteine, 6 pM CoaA, 3 pM HsCoaB, 3 pM HsCoaC, 5 pM EcCoaD, 5 pM EcCoaE, 1.8 pM AaPPK, 1 .5 pM GsPPase. Reactions were run over night, quenched using methanol and analyzed by HPLC.

[0145] C. Results

[0146] As shown by the results depicted in FIG. 1C, both the two type I PanK enzymes, EcCoaA and CaCoaA, and the two type II PanK enzymes, SpCoaW and SaCoaW, worked well in the reaction scheme of FIG. 1A, and produced CoA at a concentration greater than 1 mM. It was also found that C-terminally tagging of the two type II PanK enzymes, SpCoaW and SaCoaW, was necessary for these enzymes to be functional in the production of CoA.Example 2: Cell-free Biosynthesis of Acetyl-CoA Using in situ Produced CoA

[0147] This example illustrates a study showing that acetyl-CoA can be produced biosynthetically via an in vitro cell-free reaction scheme summarized in FIG. 2A. Acetyl-CoA isone of the most used central metabolites in cellular metabolism and therefore is used as a starting block for the production of a myriad of commercially valuable compounds (e.g., fatty acids, esters, polyketides, isoprenoids, cannabinoids, alkaloids, etc.). The reaction scheme of FIG. 2A uses the intermediate substrate, CoA, which is produced in situ using the cell-free biosynthesis reaction of FIG. 1A. Accordingly, the cell-free biosynthetic production of acetyl- CoA incorporates the substrates and enzymes used to produce CoA as described in Example 1. As shown in FIG. 2A, the production of acetyl-CoA uses ethanol as an additional substrate. The scheme of FIG. 2A provides two options for ethanol “upgrading” to acetaldehyde: “option 1” relies on an alcohol dehydrogenase (1 .1 .1 .1) (“ADH”) and cofactor recycling via a NADH oxidase (1.6.3.4) (“NOX”); “option 2” relies instead on a NAD+ independent peroxide forming alcohol oxidase (1 .1 .3.13) (“AOX”) and a detoxifying catalase (“CAT”) to remove H2O2. We tested both pathways in parallel in the same reaction as the de novo CoA synthesis. The conversion of acetaldehyde and CoA is achieved by the enzymes, NOX, and aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

[0148] A. Materials and Methods

[0149] The cell-free CoA biosynthesis described in Example 1 was modified further with a pathway of enzymes for converting ethanol to acetaldehyde, and then producing acetyl-CoA, as shown in FIG. 2A. The starting substrates used in the biosynthetic reaction are D- pantothenate, L-cysteine, ATP, polyphosphate, ethanol, and a catalytic amount of NAD+. The theoretical maximum yield of the reaction was 20 mM as fixed by the ATP added to the reaction. A comparison of cell-free reactions using either the enzymes of option 1 or option 2 was carried out. Reactions contained 200 mM TrisHCI pH 9, 5 mM MgCI2, 10 mM KCI, 10 mM / 5 mM / 2.5 mM ATP, 16 mg / mL hexametaphosphate, 10 mM D-pantothenic acid, 20 mM L- cysteine, 4 mM NAD+, 100 mM ethanol, 12 pM CoaA., 10 pM HsCoaB, 10 pM HsCoaC, 10 pM EcCoaD, 10 pM EcCoaE, 1.8 pM AaPPK, 1.5 pM GsPPase, 2.5 pM GsADH, 2.5 pM LINOX, 2.5 pM TtPduP. Reactions were run over night, quenched using methanol and analyzed by HPLC.

[0150] A. Results

[0151] As shown by the results in FIG. 2B, the option 1 was found to outperform option 2 and produce 15.6 mM (12.6 g / L) of acetyl-CoA.

[0152] B. Materials and Methods

[0153] As ATP is the most expensive substrate used in the biosynthesis, a further experiment was carried out to test the performance of option 1 under conditions of limited ATP as described above in A.

[0154] B. Results

[0155] As shown by the results in FIG. 2C, the option 1 pathway run under limiting amount of ATP substrate was able to produce almost stoichiometric amounts of acetyl-CoA when SpCoaW was used as the PanK enzyme.

[0156] C. Materials and Methods

[0157] To better understand the temporal dynamics of the biosynthetic system of FIG. 2A, a time-course of the reaction was monitored under the most optimal conditions as determined above. Reactions contained 200 mM TrisHCI pH 9, 5 mM MgCI2, 10 mM KCI, 40 mM ATP, 16 mg / mL hexametaphosphate, 40 mM D-pantothenic acid, 40 mM L-cysteine, 4 mM NAD+, 100 mM ethanol, 6 pM SpCoaW, 3 pM HsCoaB, 3 pM HsCoaC, 5 pM EcCoaD, 5 pM EcCoaE, 1.8 pM AaPPK, 1 .5 pM GsPPase, 5 pM GsADH, 5 pM LINOX, 5 pM TtPduP. Reactions were run over night, quenched using methanol and analyzed by HPLC.

[0158] C. Results

[0159] As shown by the results depicted in the plots of FIG. 2D, about 5 mM CoA was produced within the first 4h of the reaction, while only minimal amounts of acetyl-CoA were produced in this time span. This indicates that the in situ production of CoA outpaces the ethanol upgrading to acetyl-CoA at the current enzyme loading. Further experiments are planned to optimize the enzyme loading conditions.Example 3: Cell-free Biosynthesis of CBGA with in situ Produced CoA

[0160] This example illustrates a study showing that the cannabinoid compound, cannabigerolic acid (CBGA) can be produced in a pair of cell-free biosynthesis reactions as summarized in FIG. 3A. The intermediate substrate, CoA, used in the two reactions of FIG. 3A is produced in situ during the reaction using the cell-free biosynthesis reaction of FIG. 1A illustrated in Example 1 .

[0161] A. Materials and Methods

[0162] As shown in RXN1 of FIG. 3A, an optimized in situ CoA biosynthesis system (as in FIG. 1A) comprising the enzymes PanK, CoaB, CoaC, PPAT, and DPCK, and the substrates D- pantothenate, L-cysteine, and ATP, was further modified with the acyl-activating enzyme, AAE3, and the substrate hexanoic acid. Reactions contained 200 mM TrisHCI pH 9, 5 mM MgCI2, 10 mM KCI, 30 mM ATP, 16 mg / mL hexametaphosphate, 20 mM D-pantothenic acid, 20 mM L-cysteine, 20 mM hexanoic acid, 12 pM CoaA, 6 pM HsCoaB, 6 pM HsCoaC, 10 pM EcCoaD, 10 pM EcCoaE, 1.8 pM AaPPK, 1.5 pM GsPPase, 5 mM CsAAE3. Reactions were run over night, quenched using methanol and analyzed by HPLC. Unquenched reaction mix was used in

[0111] ,

[0163] A. Results

[0164] As shown by the HPLC plot of FIG. 3B, this RXN1 biosynthesis system produces hexanoyl-CoA at 10mM concentration, and trace amounts of CoA.

[0165] B. Materials and Methods

[0166] As shown in RXN2 of FIG. 3A, the hexanoyl-CoA produced in RXN1 can be an input substrate along with the co-substrates, malonate and isoprenol, in a biosynthesis system comprising the enzymes MatB, ThiM, IDI, IPK, FPPS, OLS, OAC, and NphB, and that producesthe cannabinoid compound, CBGA. Reactions contained 200 mM TrisHCI pH 9, 5 mM MgCI2, 10 mM KOI, 5 mM ATP, 16mg / ml_ hexametaphosphate, 25% v / v RXN1 mix, 20 pM CsOAC, 5 pM CsOLS, 2.5 pM GsPPase, 20 pM RpMatB, 3.8 pM EcThiM, 1.7 pM EclDI, 1.1 pM MtIPK, 5 pM GsFPPS S82F, 8 pM NphB M31 , 1.2 pM AaPPK. Reactions were run over night, quenched using methanol and analyzed by HPLC.

[0167] B. Results

[0168] As shown by the HPLC plot of FIG. 3C, this RXN2 biosynthesis system produced 237 mg / L of CBGA (and smaller amounts of the side-products, HTAL, PDAL, and olivetol) starting from 4 mM hexanoyl-CoA from RXN1.

[0169] In summary, as demonstrated by Examples 1-3, the in situ CoA biosynthesis system of FIG. 1 A as described herein, can produce amounts of CoA in situ that are sufficient other CoA dependent biosynthetic pathways. Typically, less than 1 mM of in situ CoA production is needed to support such CoA-dependent biosynthetic pathways. This in situ CoA production allows biosynthesis of acetyl-CoA synthesis from ethanol. This pathway for “upgrading” ethanol to acetyl-CoA can be further modified to allow more efficient biosynthetic production of short and medium chain fatty acids, alcohols, and fatty acid ethyl esters. Moreover, in the case of fatty acid ethyl esters, only catalytic amounts of coenzyme A will be needed, thus lowering the required input of ATP.

[0170] While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the appended claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the appended claims.

[0171] Additional embodiments of the invention are set forth in the following claims.

[0172] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or other document were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.

Claims

CLAIMSWhat is claimed is:

1. A process for synthesis of Coenzyme A (“CoA”) comprising contacting in a cell-free solution under suitable reaction conditions the following substrates and enzymes:(a) substrates:D-pantothenate, L-cysteine, and ATP and / or CTP;(b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB’’), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), and dephospho-CoA kinase (2.7.1.24) (“DPCK”).

2. The process of claim 1 , wherein the substrates do not include CTP.

3. The process of claim 1 , wherein the substrates include ATP and CTP.

4. The process of any one of claims 1 -3, wherein the substrates further comprise polyphosphate.

5. The process of any one of claims 1 -4, wherein the process further comprises:(a) an enzyme capable of regenerating ATP from ADP or from AMP;(b) an enzyme capable of regenerating ADP from AMP;(c) ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); optionally, wherein the PPK enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26; and / or(d) PPK and polyphosphate AMP phosphotransferase (“PAP").

6. The process of any one of claims 1 -5, wherein the enzymes further comprise inorganic pyrophosphatase (3.6.1.1) ("PPase").

7. The process of any one of claims 1 -6, wherein the enzymes CoaB or CoaC are not CTP- dependent.

8. The process of any one of claims 1 -7, wherein the enzymes CoaB or CoaC are derived from a eukaryotic source organism.

9. The process of any one of claims 1 -8, wherein the enzymes CoaB and CoaC are from A thaliana or H. sapiens.

10. The process of any one of claims 1-9, wherein the enzyme CoaB comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 14, and 16.11 . The process of claim 10, wherein the amino acid sequence of the enzyme CoaB further comprises an N-terminal tag and / or a C-terminal tag.

12. The process of any one of claims 1-11 , wherein the enzyme CoaC comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 18, and 20.

13. The process of claim 12, wherein the amino acid sequence of the enzyme CoaC further comprises an N-terminal tag and / or a C-terminal tag.

14. The process of any one of claims 1-13, wherein the enzyme PanK is selected from a Type I PanK or a Type II PanK.

15. The process of claim 14, wherein the enzyme is a Type I PanK selected from EcCoaA and CaCoaA.

16. The process of claim 15, wherein the amino acid sequence of the Type I PanK enzyme comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 2, and 4.

17. The process of claim 14, wherein the enzyme is a Type II PanK selected from SaCoaW and SpCoaW.

18. The process of claim 17, wherein the amino acid sequence of the Type II PanK enzyme comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 6, 8, or 10.

19. The process of any one of claims 14-18, wherein the amino acid sequence of the enzyme PanK further comprises an N-terminal tag and / or a C-terminal tag.

20. A composition comprising a cell-free solution of:(a) D-pantothenate, L-cysteine, and ATP and / or CTP; and(b) pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase(6.3.2.5, or 6.3.2.51) (“CoaB”), and phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”).21 . The composition of claim 20, wherein the cell-free solution does not comprise CTP.

22. The composition of any one of claims 20-21 , wherein the PanK:(a) is a Type I PanK enzyme comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 2, and 4; or(b) is a Type II PanK enzyme comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 6, 8, or 10.

23. The composition of any one of claims 20-22, wherein CoaB comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 14, and 16.

24. The composition of any one of claims 20-23, wherein CoaC comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 18, and 20.

25. The composition of any one of claims 20-24, wherein the cell-free solution further comprise polyphosphate.

26. The composition of any one of claims 20-25, wherein the cell-free solution further comprises:(a) ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); optionally, wherein the PPK enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26;(b) PPK and polyphosphate AMP phosphotransferase (“PAP");(c) an enzyme capable of regenerating ATP from ADP or from AMP; and / or(d) an enzyme capable of regenerating ADP from AMP.

27. The composition of any one of claims 20-26, wherein the cell-free solution further comprises:(a) ethanol;(b) an alcohol oxidase (1.1.3.13) (“AOX”), optionally, wherein the AOX comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 34;(c) a catalase (1.11.1.6) (“CAT”), optionally, wherein the CAT comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 36 and 38;(d) an alcohol dehydrogenase (1.1.1.1) (“ADH”), optionally, wherein the ADH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:32; and(e) an aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

28. The composition of any one of claims 20-26, wherein the cell-free solution further comprises:(a) ethanol;(b) NAD+;(c) an NADH oxidase (1 .6.3.4) (“NOX”), optionally, wherein the NOX comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NO: 28 and 30,(d) an alcohol dehydrogenase (1.1.1.1) (“ADH”), optionally, wherein the ADH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 32; and(e) an aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

29. The composition of any one of claims 20-28, wherein the cell-free solution further comprises a fatty acid or diacid and a CoA-ester synthase.

30. The composition of claim 29, wherein cell-free solution further comprises a fatty acid comprising from 1 to 30 carbons.31 . The composition of claim 30, wherein the fatty acid is a saturated fatty acid selected from butyric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid.

32. The composition of claim 30, wherein the fatty acid is an unsaturated fatty acid selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

33. The composition of claim 30, wherein the fatty acid is hexanoic acid and the CoA-ester synthase is an acyl-activating enzyme (6.2.1.1) (“AAE”).

34. The composition of claim 30, wherein cell-free solution further comprises a dicarboxylic acid comprising from 1 to 30 carbons.

35. The composition of claim 34, wherein the dicarboxylic acid is a saturated dicarboxylic acid selected from malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, hexadecanedioic acid, heneicosanedioic acid, docosanedioic acid, and 1 ,4- cyclohexanedicarboxylic acid.

36. The composition of claim 34, wherein the dicarboxylic acid is an unsaturated dicarboxylic acid selected from maleic acid, fumaric acid, acetylenedicarboxylic acid, glutaconic acid, traumatic acid, muconic acid, glutinic acid, citraconic acid, mesaconic acid, and itaconic acid.

37. A process for the synthesis of acetyl-CoA comprising contacting in a cell-free solution under suitable reaction conditions the following substrates and enzymes:(a) substrates:D-pantothenate, L-cysteine, polyphosphate, ATP and / or CTP, and ethanol;(b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), dephospho-CoA kinase (2.7.1.24) (“DPCK”), inorganic pyrophosphatase (3.6.1.1) ("PPase"), alcohol oxidase (1.1.3.13) (“AOX”), a catalase (1.11.1.6) (“CAT”), alcohol dehydrogenase (1.1.1.1) (“ADH”), and aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

38. A process for the synthesis of acetyl-CoA comprising contacting in a cell-free solution under suitable reaction conditions the following substrates and enzymes:(a) substrates:D-pantothenate, L-cysteine, polyphosphate,ATP or CTP; ethanol, and NAD+;(b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), dephospho-CoA kinase (2.7.1.24) (“DPCK”), inorganic pyrophosphatase (3.6.1.1) ("PPase"), NADH oxidase (1.6.3.4) (“NOX”), alcohol dehydrogenase (1.1.1.1) (“ADH”), and aldehyde dehydrogenase (1.2.1.3) (“AIDH”).

39. A process for the synthesis of hexanoyl-CoA comprising contacting in a cell-free solution under suitable reaction conditions the following substrates and enzymes:(a) substrates:D-pantothenate, L-cysteine, polyphosphate, ATP or CTP, and hexanoic acid;(b) enzymes: pantothenate kinase (2.7.1.33) (“PanK”), phosphopantothenate-cysteine ligase (6.3.2.5, or 6.3.2.51) (“CoaB”), phosphopantothenoylcysteine decarboxylase (4.1.1.36) (“CoaC”), phosphopantetheine adenyltransferase (2.7.7.3) (“PPAT”), dephospho-CoA kinase (2.7.1.24) (“DPCK”), inorganic pyrophosphatase (3.6.1.1) ("PPase"), and acyl-activating enzyme (6.2.1.1) (“AAE”).

40. The process of claim 39, further comprising contact the hexanoyl-CoA synthesized in a cell- free solution under suitable reaction conditions with the following substrates and enzymes:(a) substrates: malonate, and isoprenol;(b) enzymes:Malonyl CoA synthetase (MatB),Hydroxyethylthiazole kinase (ThiM),Isopentyl diphosphate isomerase (IDI) ,Isopentenyl kinase (IPK),Farnesyl-PP synthase (FPPS),Olivetol synthase (OLS),Olivetolic acid cyclase (OAC), andSoluble prenyltransferase (NphB); thereby, resulting in the synthesis of the cannabinoid, cannabigerolic acid (CBGA).41 . The process of any one of claims 37-40, wherein the process further comprises:(a) an enzyme capable of regenerating ATP from ADP or from AMP;(b) an enzyme capable of regenerating ADP from AMP;(c) ATP-polyphosphate phosphotransferase (2.7.4.1) (“PPK”); optionally, wherein the PPK enzyme comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26; and / or(d) PPK and polyphosphate AMP phosphotransferase (“PAP").

42. The process of any one of claims 37-41 , wherein the PanK:(a) is a Type I PanK enzyme comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 2, and 4; or(b) is a Type II PanK enzyme comprising an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 6, 8, or 10.

43. The process of any one of claims 37-42, wherein CoaB comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 14, and 16.

44. The process of any one of claims 37-43, wherein CoaC comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 18, and 20.

45. The process of any one of claims 37-44, wherein:(a) PPAT comprises an amino acid sequence having at least 90% identity to a sequence of SEQ ID NO: 22;(b) DPCK comprises an amino acid sequence having at least 90% identity to a sequence of SEQ ID NO: 24;(c) NOX comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 28, and 30; and / or(d) ADH comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NO: 32, and 34.

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