Cell-free production of geranyl pyrophosphate from glycerol in a cell-free production system

Abstract

Geranyl pyrophosphate (GPP) is a key intermediate molecule in the bioproduction of thousands of natural products. Currently, natural products are either cultivated from plants, synthesized by complex chemical synthesis strategies, or synthesized through cell-based factories (also known as biofoundries). However, to replicate the process in a cell-free environment, a large number of enzymes and cofactors must be utilized, making this approach expensive and unfeasible. To make this process feasible, a novel approach using fewer enzymes and cofactors was needed. As described herein, the present invention shows that it is possible to produce GPP from glycerol through a short and concise biosynthetic pathway outside of the cell.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 127,836, filed December 18, 2020, and U.S. Provisional Application No. 63 / 127,758, filed December 18, 2020, the specifications of which are incorporated by reference in their entireties herein.

[0002] (REFERENCE TO SEQUENCE LISTING) The Applicant declares that the information recorded in the form of Annex C / ST.25 text file entitled "DEBUT_20_03_PCT_Sequence_Listing_ST25" submitted under Rule 13ter.1(a) is identical to the information forming part of the international application as filed, the contents of which are incorporated herein by reference in their entirety.

[0003] FIELD OF THEINVENTION The present invention features methods for producing geranyl pyrophosphate (GPP) from glycerol, and in particular, the present invention features cell-free production methods. [Background technology]

[0004] BACKGROUND OF THEINVENTION Geranyl pyrophosphate (GPP) is a key intermediate molecule in the bioproduction of thousands of natural products, which are molecules formed through multi-step enzymatic pathways in organisms such as plants, animals, and bacteria. Currently, natural products are either cultivated from plants, synthesized through complex chemical synthesis strategies, or synthesized through cell-based factories (also known as biofoundries).

[0005] Previously, Valliere et al. (2019) produced GPP in a cell-free environment using glucose as the starting material. This exact replica of the native enzyme system uses 22 enzymes and 40 cofactor equivalents (12 adenosine triphosphate (ATP), 12 adenosine diphosphate (ADP), and 4 nicotinamide adenine dinucleotide phosphate (NADP)). + ), two nicotinamide adenine dinucleotides (NAD + ), six acetyl coenzyme A (CoA), and four nicotinamide adenine dinucleotide phosphate (NADPH). The number of cofactors required by the process described in the literature means that this approach is always expensive and unfeasible ( FIG. 1B ). To make this process feasible, novel approaches are needed that use fewer enzymes and cofactors and improve reliability. As described herein, the present invention demonstrates that it is possible to produce GPP through a short and concise biosynthetic pathway outside of cells (known as cell-free biosynthesis). Summary of the Invention [Means for solving the problem]

[0006] BRIEF SUMMARY OF THEINVENTION It is an object of the present invention to provide a cell-free method that allows the production of geranyl pyrophosphate (GPP) and additional secondary metabolites starting from glycerol by a cell-free biosynthetic platform, as specified in the independent claims. Embodiments of the present invention are provided in the dependent claims. The embodiments of the present invention may be freely combined with one another, if they are not mutually exclusive.

[0007] The production of natural products by culture, chemical synthesis, or in cells suffers from many problems that limit the commercial viability of high-value chemical production. First, culture is often economically unfeasible, requiring large amounts of land / energy / water, and plants can only produce high-value substances in small quantities. Second, chemical synthesis requires extensive, elaborate, expensive, toxic, and inefficient multi-step chemical reactions to produce natural products that are often too complex to produce in the laboratory. Finally, biofoundry (the use of whole cells) suffers from product toxicity, carbon flux redirection, diffusion problems through cell walls, and the production of toxic by-products. To circumvent these above problems, cell-free manufacturing emerges as a viable option.

[0008] In cell-free systems, the key components of cells, i.e., cofactors and enzymes, are used in chemical reactions without cells. The same enzymes found in plants, animals, and bacteria are produced in vivo (typically through protein overexpression in a host such as bacteria), isolated by chromatography, and then added to a bioreactor containing a substrate (starting material). The enzymes convert the substrate in the same manner as occurs in plants, animals, and bacteria, but without the complexity of living organisms. In this manner, natural products can begin to be produced without plants, cells, or chemical synthesis.

[0009] The present invention creates an enzymatic pathway (FIG. 1A) that eliminates glycolysis (glucose to pyruvate) to allow for a shorter, more affordable, and simpler process for converting glycerol to GPP. The process uses 12 enzymatic steps, eliminating 24 cofactor equivalents and 10 enzymes (FIG. 1B). Furthermore, enzyme immobilization is used to improve the conversion to GPP by avoiding enzyme precipitation, inactivation, and the unreliability of non-immobilized systems. The present invention also provides evidence showing that without enzyme immobilization, enzyme precipitation and destruction of the enzymatic pathway are observed. Finally, the process starts with glycerol, not glucose. Glycerol is an abundant natural substance that is not used in the production of GPP. The present invention allows this natural industrial waste product to be utilized as a viable alternative to glucose.

[0010] In some embodiments, this features a method for converting glycerol to geranyl pyrophosphate (GPP) and additional secondary metabolites. In some embodiments, the method includes adding glycerol to a reaction mixture. In some embodiments, the method includes adding a plurality of enzymes to the reaction mixture. In some embodiments, the enzymes are selected from the group consisting of alditol oxidase (Aldo), dihydroxy acid dehydratase (DHAD), pyruvate oxidase (PyOx), phosphate acetyltransferase (PTA), acetyl-CoA acetyltransferase (PhaA), HMG-CoA synthase A110G (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate kinase (MDC), isopentyl-PP isomerase (IDI), and farnesyl-PP synthase S82F (FPPS). In some embodiments, the method includes removing the supernatant from the reaction mixture. In some embodiments, the method includes isolating or purifying GPP. In some embodiments, the enzymes can be added to the reaction mixture asynchronously. In other embodiments, the enzymes can be added to the reaction mixture simultaneously.

[0011] In other embodiments, the invention features a method for converting glycerol to geranyl phosphate (GPP) and additional secondary metabolites. In some embodiments, the method includes adding glycerol and alditol oxidase (Aldo) to a reaction mixture. In some embodiments, the method further includes adding dihydroxy acid dehydratase (DHAD) to the reaction mixture. In some embodiments, the method includes removing a supernatant of the reaction mixture and adding pyruvate oxidase (PyOx) to the supernatant of the reaction mixture. In some embodiments, the method includes removing the supernatant of the reaction mixture, and adding at least two enzymes selected from the group consisting of phosphate acetyltransferase (PTA), acetyl-CoA acetyltransferase (PhaA), HMG-CoA synthase A110G (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate kinase (MDC), isopentyl-PP isomerase (IDI), and farnesyl-PP synthase S82F (FPPS) to the supernatant of the reaction mixture. In some embodiments, the method includes removing the supernatant from the reaction mixture, and producing GPP. In some embodiments, the enzymes can be added to the reaction mixture asynchronously. In other embodiments, the enzymes can be added to the reaction mixture simultaneously.

[0012] One of the unique and progressive technical features of the present invention is the use of a cell-free system for the production of geranyl pyrophosphate (GPP) and additional secondary metabolites starting from glycerol. Without wishing to limit the present invention to any theory or mechanism, it is believed that the technical features of the present invention advantageously provide higher reaction concentrations, no cell walls to fight for diffusion of products and substrates, no competition within cells for carbon flux and by-product formation, no cell death due to the formation of toxic compounds (no cells), and increased flexibility as a platform solution for the production of multiple compounds compared to cells that must be reprogrammed each time. In this approach, novel chemical entities can be produced by simply changing the enzymes in the pathway.

[0013] Moreover, prior literature teaches away from the present invention. For example, Valliere et al. (2019) used a more complex cell-free system to produce GPP as an intermediate in the production of cannabidiol (CBD) from glucose. The present pathway eliminates 60% of the expensive cofactors, 10 enzymes, and elaborate purge valves, and uses alternative starting materials. Although the present invention shares 10 enzymes with Valliere et al.'s approach, the present invention significantly improved the activity, lifespan, and reliability of the enzymes by immobilizing and optimizing all 10 enzymes. Notably, Valliere et al. include data showing precipitation of the enzymes after incubation of all enzymes in the reaction mixture. Precipitation of these enzymes was also observed when repeating the work after only 16 hours or so of combination in the reaction mixture, severely limiting the above work. A key success was immobilizing each of the 12 enzymes, eliminating precipitation, allowing the enzymes to remain active in the reaction mixture for extended periods of time.

[0014] Furthermore, the inventive technical features of the present invention contributed to the surprising results. The first module in the pathway of the present invention converts glycerol to pyruvate by combining the activities of alditol oxidase (ALDO) and dihydroxy acid dehydratase (DHAD) as reported in Gao et al. (2015). Surprisingly, the present invention had to make several modifications to this published work in order to successfully produce pyruvate from glycerol. First, a maltose binding protein was added to the N-terminus of Streptomyces ALDO to aid in solubility and stability while maintaining the activity of the enzyme (SEQ ID NO: 1). Second, it was found that MBP-ALDO required oxygenation for optimal activity. Third, the DHAD of Sulfolobus solfataricus reported in Gao et al. (2015) did not function. Many DHAD orthologues were screened and the DHAD of Thermosynechococcus vulcanus (SEQ ID NO: 2) was found to be the most active. Fourth, it was found that DHAD required deoxygenation and a very specific pH range for optimal activity.

[0015] Any feature or combination of features described herein is included within the scope of the present invention, unless the features included in any such combination are mutually inconsistent, as would be apparent from the context, the specification, and the knowledge of one of ordinary skill in the art. Further advantages and aspects of the present invention are apparent in the following detailed description and claims.

[0016] (Brief description of some figures of the drawing) The features and advantages of the present invention will become apparent from the following detailed description given in conjunction with the accompanying drawings. [Brief description of the drawings]

[0017] [Figure 1]Figures 1A and 1B show the pathway used for the production of geranyl pyrophosphate (GPP) (left, Figure 1A) in contrast to a previous attempt to make GPP from glucose (right, Figure 1B). As shown, the pathway on the left (Figure 1A) has 70% fewer cofactors and 10 fewer enzymes.

[0018] [Diagram 2] Figures 2A and 2B show HPLC traces of cannabigerolic acid (CBGA) for both the CBGA standard (top, Figure 2A) and the immobilized enzyme batch reaction (bottom, Figure 2B). A retention time of 3.68 minutes at 228 nm is observed for both reactions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention Before the present compounds, compositions and methods are disclosed and described, it is to be understood that the invention is not limited to specific synthetic methods or specific compositions, as these may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0020] Furthermore, although the embodiments of the present disclosure have been described in detail, certain variations and modifications, including embodiments that do not provide all of the features and advantages described herein, will be apparent to those skilled in the art. It will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative or additional embodiments and / or uses and their obvious modifications and equivalents. Furthermore, although many variations have been shown and described with various details, other modifications that are within the scope of the present disclosure will be readily apparent to those skilled in the art based on the present disclosure. It is also contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments may be made and still be within the scope of the present disclosure. Thus, it should be understood that the various features and aspects of the disclosed embodiments may be combined with or substituted for each other to form various forms of the present disclosure. Thus, it is intended that the scope of the disclosure disclosed herein should not be limited by the specific disclosed embodiments described herein.

[0021] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0022] As used herein, a "reaction solution" may refer to all components necessary for an enzyme-based chemical conversion, typically but not limited to buffers, salts, cofactors, and substrates (i.e., starting materials).

[0023] As used herein, a "reaction mixture" may refer to all components from a "reaction solution" plus enzymes and / or reaction products. In some embodiments, a "reaction mixture" may refer to only the reaction solution, without any enzymes or reaction products.

[0024] In some embodiments, the terms "reaction solution" and "reaction mixture" may be used interchangeably.

[0025] As used herein, "buffering agent" may refer to a chemical added to a water-based solution that resists pH changes through the action of acid-base conjugate components.

[0026] As used herein, "supernatant" may refer to the soluble liquid fraction of a sample.

[0027] As used herein, a "batch reaction" may refer to a chemical or biochemical reaction carried out in a closed system, such as a fermenter or a typical reaction flask.

[0028] As used herein, "cofactor" may refer to a non-protein compound that can bind to protein and assist biological chemical reactions. Cofactors may be metal ions, organic compounds, or other chemicals. Non-limiting examples of cofactors may include, but are not limited to, ATP and NADPH.

[0029] As used herein, "cofactor recycling" can refer to the regeneration of a functional cofactor that can participate in an enzyme-catalyzed reaction. A non-limiting example of this regeneration is a separate reaction that acts on an altered cofactor generated by a primary enzymatic reaction, such as the enzymatic conversion of ADP back to ATP.

[0030] Referring now to Figures 1A, 1B, 2A, and 2B, the present invention features a method for producing geranyl pyrophosphate (GPP) and additional secondary metabolites from glycerol.

[0031] This features a method for converting glycerol to geranyl pyrophosphate (GPP) and additional secondary metabolites. In some embodiments, the method includes adding glycerol to a reaction mixture. In some embodiments, the method includes adding a plurality of enzymes to the reaction mixture. In some embodiments, the enzymes are selected from the group consisting of alditol oxidase (Aldo), dihydroxy acid dehydratase (DHAD), pyruvate oxidase (PyOx), phosphate acetyltransferase (PTA), acetyl-CoA acetyltransferase (PhaA), HMG-CoA synthase A110G (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate kinase (MDC), isopentyl-PP isomerase (IDI), and farnesyl-PP synthase S82F (FPPS). In some embodiments, the method includes removing the supernatant from the reaction mixture. In some embodiments, the method includes isolating or purifying GPP.

[0032] The invention also features a method for converting glycerol to geranyl phosphate (GPP) and additional secondary metabolites. In some embodiments, the method includes adding glycerol and alditol oxidase (Aldo) to a reaction mixture. In some embodiments, the method further includes adding dihydroxy acid dehydratase (DHAD) to the reaction mixture. In some embodiments, the method includes removing a supernatant of the reaction mixture and adding pyruvate oxidase (PyOx) to the supernatant of the reaction mixture. In some embodiments, the method includes removing the supernatant of the reaction mixture and adding to the supernatant of the reaction mixture at least two enzymes selected from the group consisting of phosphate acetyltransferase (PTA), acetyl-CoA acetyltransferase (PhaA), HMG-CoA synthase A110G (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate kinase (MDC), isopentyl-PP isomerase (IDI), and farnesyl-PP synthase S82F (FPPS). In some embodiments, the method includes removing the supernatant from the reaction mixture and producing or isolating GPP.

[0033] In some embodiments, at least one enzyme is added to the reaction mixture. In some embodiments, at least two enzymes are added to the reaction mixture. In some embodiments, at least three enzymes are added to the reaction mixture. In some embodiments, at least four enzymes are added to the reaction mixture. In some embodiments, at least five enzymes are added to the reaction mixture. In some embodiments, at least six enzymes are added to the reaction mixture. In some embodiments, at least seven enzymes are added to the reaction mixture. In some embodiments, at least eight enzymes are added to the reaction mixture. In some embodiments, at least nine enzymes are added to the reaction mixture. In some embodiments, at least ten enzymes are added to the reaction mixture. In some embodiments, at least eleven enzymes are added to the reaction mixture. In some embodiments, at least twelve enzymes are added to the reaction mixture.

[0034] In some embodiments, the enzymes may be added to the reaction mixture asynchronously, while in other embodiments, the enzymes may be added to the reaction mixture simultaneously.

[0035] In some embodiments, the method further comprises adding NphB enzyme to convert GPP to cannabigerolic acid (CBGA) before final removal of the supernatant from the reaction mixture. In some embodiments, CBGA is used to determine the amount of GPP produced in the method. In some embodiments, the production of CBGA is used as an analytical tool. In some embodiments, the production of CBGA from GPP by NphB is used as a detection method. In some embodiments, the production of CBGA by NphB is used to detect the amount of GPP. In some embodiments, the amount of CBGA produced from the conversion of GPP by NphB enzyme is 1:1.

[0036] In some embodiments, the reaction mixture described herein includes a cofactor. In some embodiments, the cofactor is adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), or the like. + ), Nicotinamide adenine dinucleotide phosphate (NADP + ), or a combination thereof. In some embodiments, the methods described herein utilize cofactor recycling. In some embodiments, the cofactor is recycled. In some embodiments, the method further comprises adding glucose dehydrogenase (GDH) and polyphosphate kinase 2 (PPK2) to the reaction mixture. In some embodiments, glucose dehydrogenase (GDH) and polyphosphate kinase 2 (PPK2) are added to the reaction mixture to recycle the cofactor.

[0037] In some embodiments, the temperature of the reaction may range from about 22° C. to about 50° C. In some embodiments, the temperature of the reaction is about 20° C. In some embodiments, the temperature of the reaction is about 25° C. In some embodiments, the temperature of the reaction is about 30° C. In some embodiments, the temperature of the reaction is about 35° C. In some embodiments, the temperature of the reaction is about 40° C. In some embodiments, the temperature of the reaction is about 45° C. In some embodiments, the temperature of the reaction is about 50° C. In some embodiments, the temperature of the reaction is about 55° C.

[0038] In some embodiments, the pH of the reaction may range from about 6.5 to about 9.0. In some embodiments, the pH of the reaction is about 5.0. In some embodiments, the pH of the reaction is about 5.5. In some embodiments, the pH of the reaction is about 6.0. In some embodiments, the pH of the reaction is about 6.5. In some embodiments, the pH of the reaction is about 7.0. In some embodiments, the pH of the reaction is about 8.5. In some embodiments, the pH of the reaction is about 9.0. In some embodiments, the pH of the reaction is about 9.5. In some embodiments, the pH of the reaction is about 10.0.

[0039] In some embodiments, the time to carry out the reaction may range from about 2 hours to about 32 hours. In some embodiments, the time to carry out the reaction is about 0.5 hours. In some embodiments, the time to carry out the reaction is about 1 hour. In some embodiments, the time to carry out the reaction is about 2 hours. In some embodiments, the time to carry out the reaction is about 5 hours. In some embodiments, the time to carry out the reaction is about 10 hours. In some embodiments, the time to carry out the reaction is about 15 hours. In some embodiments, the time to carry out the reaction is about 20 hours. In some embodiments, the time to carry out the reaction is about 25 hours. In some embodiments, the time to carry out the reaction is about 30 hours. In some embodiments, the time to carry out the reaction is about 35 hours. In some embodiments, the time to carry out the reaction is about 40 hours. In some embodiments, the time to carry out the reaction is about 45 hours. In some embodiments, the time to carry out the reaction is greater than 45 hours.

[0040] In some embodiments, the enzyme is immobilized. In some embodiments, the immobilized enzyme is immobilized on a solid support. Non-limiting examples of solid supports may include, but are not limited to, epoxy methacrylate, amino C6 methacrylate, or microporous polymethacrylate. In further embodiments, various surface chemistries may be used to link the immobilized enzyme to the solid surface, including, but are not limited to, covalent, adsorption, ionic, affinity, encapsulation, or entrapment. In other embodiments, the enzyme is not immobilized.

[0041] In some embodiments, one or more of the enzymes are immobilized. In other embodiments, all of the enzymes are immobilized. In some embodiments, one or more of the enzymes are not immobilized. In other embodiments, all of the enzymes are not immobilized. In some embodiments, multiple enzymes are immobilized. In other embodiments, multiple enzymes are not immobilized.

[0042] In some embodiments, various reaction conditions may be altered to ensure a functional enzyme, including, but not limited to, reaction time, oxygenation / deoxygenation, pH, buffering agents, and reaction temperature.

[0043] In some embodiments, the methods described herein teach a departure from previously described methods because they utilized fewer enzymes and cofactors to generate GPP. In certain embodiments, the methods described herein do not use the 24 cofactor equivalents (or 60% of the cofactors) used by Valliere et al. In certain embodiments, only 10 of the enzymes taught by Valliere et al. are used in the present methods. EXAMPLES

[0044] (Example)

[0045] The following are non-limiting examples of the present invention. It should be understood that the examples are not intended to limit the present invention in any manner. Equivalents or substitutes are within the scope of the present invention.

[0046] Enzyme expression and purification: All genes were synthesized and cloned into expression plasmids and then transformed into E. coli for expression. Cells were grown at 37°C and 200 rpm in TB medium supplemented with 50 μg / mL kanamycin sulfate. 600 =0.6. Cells were cooled to 18°C, expression was induced, and grown for an additional 18 hours. Cell pellets were collected by centrifugation, frozen, and then resuspended in 5 mL of lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 1 mM PMSF) per gram of cell paste. Cell lysates were prepared by sonication, and cell debris was removed by centrifugation. The clarified lysate was loaded onto a GE XK series column containing IMAC-Nickel resin. Proteins were eluted using a 15 CV gradient from Buffer A (50 mM Tris pH 8.0, 300 mM NaCl, 10% glycerol) to 70% Buffer B (2 M imidazole, 50 mM Tris pH 8.0, 300 mM NaCl, 10% glycerol). Fractions containing the protein of interest were pooled and transferred into Buffer A (above) using a GE HiPrep 26 / 10 desalting column, with the exception of MBP-Aldo, which was stored in 50 mM Tris (pH 8.0), 500 mM NaCl, 0.1% Triton® X-100.

[0047] (1.0 Production of GPP via an Unfixed Cell-Free Pathway): As described herein, this pathway first converts glycerol to glycerate using alditol oxidase (Aldo, EC 1.1.3.41) and then to pyruvate using dihydroxyacid dehydratase (DHAD, EC 4.2.1.9, FIG. 1A). Although the first two enzymes were described, several significant advances were required to make this pathway functional, since previously reported work could not be replicated.

[0048] To ensure a functional enzyme, specific reaction conditions such as timed reaction oxygenation, deoxygenation, pH change, specific buffer, and reaction temperature needed to be found. First, the reported Aldo is unstable and inactive. To overcome this, we created a modified Aldo (SEQ ID NO: 1) containing a fused maltose binding protein (MBP) tag to improve solubility and stability in solution. This MBP-fused Aldo converted glycerol to glyceric acid (100%, 1.75 g / L). Second, the published reaction with DHAD from Sulfolobus solfataricus was also not reproducible. To overcome this, we screened many DHAD enzyme orthologues and found that DHAD from Thermosynechococcus vulcanus converted 100% of glyceric acid to pyruvate (1.32 g / L).

[0049] Using these novel enzymes, a one-pot reaction containing both Aldo (11 μM) and DHAD (16 μM) converted glycerol to pyruvate (100% conversion, yield 1.23 g / L). Compared to previous studies starting from glucose, this improved system provided 14 mM pyruvate in 24 h, without the use of cofactors, and with 9 fewer enzymes, a significant improvement. It should also be noted that previous attempts from glucose required many additional enzymes, cofactors, and reaction production complications (protein precipitation, etc.). Removing these constraints would enable a commercially viable approach to GPP from inexpensive carbon sources.

[0050] After optimization of Aldo and DHAD, the remaining enzymes in the pathway had to be generated and optimized, which convert pyruvate to geranyl pyrophosphate (GPP, Figure 1A and Figure 1B). After optimization of the individual enzymes, GPP was obtained (43 mg / L in 120 hours). The results validated this shorter, improved pathway, which uses much fewer cofactors for GPP production, but protein precipitation was observed after several days, which meant that this approach would not be suitable for commercial production. To overcome this limitation, each enzyme in the pathway was immobilized on a solid support to ensure that the protein remained active and usable. This required extensive optimization and understanding to create the individual enzyme-solid support complexes.

[0051] (2.0 Protein Immobilization and Optimization): Each of the purified enzymes in the pathway was immobilized on a solid support to increase stability, lifetime, and catalysis. Various commercially available support materials were routinely screened for product and substrate retention, enzyme retention, and activity of the immobilized enzyme. The support collection included a variety of surface chemistries for the following types of linkage: covalent, adsorption, ionic, affinity, encapsulation, and entrapment. Typically, 50 mg of resin was mixed with 4.0 mg of enzyme in desalting buffer for 16-24 hours at room temperature. The amount of immobilized enzyme was quantified by measuring the protein concentration in the solution before and after immobilization by either the BCA assay or the Bradford assay. All immobilized enzymes were screened for optimum values ​​for resin type, substrate concentration (5 mM-250 mM), pH (5.0-9.0), temperature (20°C-50°C), buffer (Tris, HEPES, PO4), and time (1 hour-36 hours). The optimal reaction conditions and results for each enzyme are as follows (Table 1A).

[0052] [Table 1A]

[0053] The yield percentages in the above table are presented for illustrative purposes. In some embodiments, each step of the GPP biomanufacturing process may have a yield percentage of up to 99%, up to 99.5%, up to 99.9%, or up to 100%. In some embodiments, the yield percentage at each step of the GPP biomanufacturing process may have a value that falls within the ranges in the following table (Table 1B).

[0054] [Table 1B]

[0055] 2.1 Optimization of Alditol Oxidase (Aldo): MBP-Aldo was immobilized on activated amino C6 methacrylate resin. The immobilized enzyme was used to convert glycerol to glyceric acid. The reaction solution (50 mM Tris (pH 9), 2.5 mM MgCl2, 20 mM glycerol) was mixed with 50 μM of immobilized enzyme at 37° C. for 21 h. The immobilized MBP-Aldo converted 100% of 20 mM glycerol to 20 mM (1.75 g / L) glyceric acid. For sampling, the reaction mixture was analyzed by high performance liquid chromatography (HPLC). The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a 30 cm Aminex HPX-87H column with a Microguard Cation H refill cartridge. The column was heated to 55° C. and the sample block was maintained at 25° C. For each sample, 1 μL was injected and a mobile phase consisting of 100% sulfuric acid (10 mM) was used. The total sample time was 45 min, with glyceric acid eluting at 17.2 min and glycerol eluting at 21.0 min. After a 2-h equilibration period to generate a stable baseline, a Refractive Index Detector (RID, Agilent) was used.

[0056] (2.2 Optimization of dihydroxydehydratase (DHAD): DHAD was mixed with activated amino C6 methacrylate resin and the immobilized enzyme was used to convert glycerate to pyruvate. The reaction solution (50 mM Tris (pH 8.5), 2.5 mM MgCl2, 20 mM glycerate) was mixed with 50 μM immobilized enzyme at 45° C. for 16 hours. The immobilized DHAD could convert 99% of 20 mM glycerate to pyruvate with a yield of 15 mM (1.32 g / L, 75%). For sampling, the reaction mixture was checked on an HPLC system to check the amount of glycerol and glycerate. The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a 30 cm Aminex HPX-87H column with a Microguard Cation H refill cartridge. The column was heated to 55° C. and the sample block was maintained at 25° C. For each sample, 1 μL was injected and an isocratic gradient consisting of 100% sulfuric acid (10 mM) was used as the mobile phase. The total sample time was 45 min, with pyruvate eluting at 16.0 min and glycerate eluting at 17.2 min. After a 2-h equilibration period to generate a stable baseline, a Refractive Index Detector (Agilent) was used.

[0057] (2.3 Optimization of pyruvate oxidase (PyOx): PyOx was mixed with activated amino C6 methacrylate resin, and the immobilized enzyme was used to convert pyruvate to acetyl phosphate. The reaction solution (10 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4) was mixed with 10 mM ethyl 1,2-dihydropyridine (1H2O4), ... 4、A 5 mM NaCl solution (pH 6.5, 5.0 mM MgCl2, 100 mM NaCl, 20 mM pyruvate, 20 mM thiamine pyrophosphate) was mixed with 3.85 μM of immobilized enzyme for 16 hours at 37° C. The immobilized PyOx was able to convert 91% of the 5 mM pyruvate for a yield of 4.55 mM (837 mg / L) acetyl phosphate. For sampling, the reaction was examined in an HPLC system to determine the amount of pyruvate and acetyl phosphate. The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a 30 cm Aminex HPX-87H column with a Microguard Cation H refill cartridge. The column was heated to 55° C. and the sample block was maintained at 25° C. The HPLC method consisted of a 5 μl sample injection volume and used an isocratic gradient consisting of 100% sulfuric acid (10 mM) as the mobile phase. The total run time was 25 min with acetyl phosphate eluting at 23.6 min and pyruvate eluting at 16.0 min. After a 2-h equilibration period to generate a stable baseline, a Refractive Index Detector (Agilent) was used.

[0058] (2.4 Optimization of phosphate acetyltransferase (PTA): PTA was mixed with epoxy methacrylate resin, and the immobilized enzyme was used to convert acetyl phosphate to acetyl-coenzyme A (acetyl-CoA). The reaction solution (10 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4) was mixed with 10 mM ethyl phosphate (10 mM TRIS), 50 mM KHPO4, 50 mM KHPO4, and 50 mM KHPO4. 4、pH 8.0, 5.0 mM MgCl2, 100 mM NaCl, 3.2 mM acetyl phosphate, 3.2 mM CoA) was mixed with the immobilized enzyme for 8 hours at 32°C. The immobilized PTA (38.4 μM) was able to convert 60% of the 3.2 mM acetyl phosphate to yield 1.92 mM (1.7 g / L) acetyl CoA. For sampling, the reaction solution was checked by HPLC system to check the amount of acetyl phosphate and acetyl CoA. The HPLC method was as follows: Agilent 1200 HPLC was equipped with a HYPERSIL ODS COLUMN 150 mm x 3 mm with a BetaSil C18 20 mm x 2.1 mm guard column. The column was heated to 25°C and the sample block was kept at 4°C. The HPLC method consisted of a 5 μl sample injection volume and an isocratic mobile phase consisting of 75 mM CH3COONa (sodium acetate) and 100 mM NaH2PO4 (sodium dihydrogen phosphate) mixed with acetonitrile (ACN) in a ratio of 94:6. The total run time was 12 min, with acetyl-CoA eluting at 8.5 min and coenzyme A (CoA) eluting at 3.9 min. A diode array detector (Agilent) was used for detection of the molecules of interest at 259 nm.

[0059] (2.5 Optimization of Acetyl-Coenzyme A C-Acetyltransferase (PhaA): PhaA was mixed with epoxy methacrylate resin, and the immobilized enzyme was used to convert acetyl-CoA to acetoacetyl-CoA. The reaction solution (50 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4) was added to 100 mM ethanol. 4、pH 8.0, 5.0 mM MgCl2, 100 mM NaCl, 2.5 mM acetyl-CoA) was mixed with the immobilized enzyme at 32°C for 8 hours. Immobilized PhaA (20 μM) could convert 44% of 2.5 mM acetyl-CoA to yield 1.1 mM (1.1 g / L) acetoacetyl-CoA. The retention times of AcCoA and acetoacetyl-CoA are consistent. Therefore, PhaA activity was measured based on the amount of CoA produced in this reaction. The reason is that CoA and acetoacetyl-CoA are produced in equimolar amounts. For sampling, the reaction solution was checked with an HPLC system to check the amount of AcCoA and CoA. The HPLC method was as follows: Agilent 1200 HPLC was equipped with a HYPERSIL ODS COLUMN 150 mm×3 mm with a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to 25°C and the sample block was maintained at 4°C. The HPLC method consisted of a 5 μl sample injection volume and an isocratic mobile phase consisting of 75 mM CH3COONa and 100 mM NaH2PO4 mixed with ACN in a 94:6 ratio. The total run time was 12 min with acetyl-CoA eluting at 8.5 min and coenzyme A eluting at 3.9 min. A diode array detector (Agilent) was used for detection of the molecules of interest at 259 nm.

[0060] (2.6 Optimization of Hydroxymethylglutaryl-CoA Synthase (HMGS): HMGS was mixed with epoxy methacrylate resin and the immobilized enzyme was used to convert acetoacetyl-CoA to HMG-CoA. The reaction solution (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 7.5, 5 mM acetoacetyl-CoA) was mixed with 0.5 μM of the immobilized enzyme at 32°C for 2 hours. After 2 hours, the reaction solution was incubated with 20.7 μM of HMGR and 5 mM of NADPH. HMGR is used to convert NADPH to NADP+. Thus, the reaction performance can be monitored at 340 nm. This coupled reaction could convert 54% of the starting material to mevalonic acid (2.7 mM or 416 mg / L). The activity of HMGR was measured by monitoring the loss of NADPH at 340 nm using a spectrophotometer.

[0061] (2.7 Optimization of Hydroxymethylglutaryl-CoA Reductase (HMGR): HMGR was mixed with epoxy methacrylate resin and the immobilized enzyme was used to convert NADPH to NADP+. The reaction solution (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 7.0, 5 mM NADPH) was mixed with 0.4 μM of immobilized enzyme for 2 hours at 32°C. The immobilized HMGR was able to convert 98% of 5 mM nicotinamide adenine dinucleotide phosphate (NADPH) to a yield of 4.9 mM NADP+, which is equimolar to the mevalonic acid (4.9 mM or 755 mg / L) produced in the reaction. The activity of HMGR was measured by monitoring the loss of NADPH at 340 nm using a spectrophotometer.

[0062] (2.8 Optimization of mevalonate kinase (MVK): MVK was mixed with macroporous polymethacrylate resin and the immobilized enzyme was used to convert mevalonate to mevalonate-5-phosphate. The reaction solution (50 mM Tris, 5 mM MgCl2, pH 8, 4 mM ATP, 4 mM mevalonate) was mixed with 133 μM of immobilized enzyme at 37 °C for 8 h. The immobilized MVK could convert 79% of 4 mM ATP to yield 3.16 mM (1.68 g / L) ADP. For sampling, the reaction mixture was examined by HPLC system to check the amount of adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm x 3 mm with a BetaSil C18 20 mm x 2.1 mm guard column. The column was heated to 25°C and the sample block was maintained at 4°C. The HPLC method consisted of a 5 μl sample injection volume and an isocratic mobile phase consisting of 100 mM KH2PO4 (potassium dihydrogen phosphate), 8 mM TBAHS (tetrabutylammonium hydrogen sulfate), pH 6.0, 20% methanol (vol / vol). The total run time was 10 min, with ATP eluting at 5.7 min and ADP eluting at 4.6 min. A diode array detector (Agilent) was used for detection of the molecules of interest at 254 nm.

[0063] (2.9 Optimization of phosphomevalonate kinase (PMVK): PMVK was mixed with amino C6 methacrylate resin and the immobilized enzyme was used to convert mevalonate-5-phosphate to mevalonate-5-pyrophosphate. The reaction solution (50 mM Tris, 5 mM MgCl2, pH 8, 4 mM ATP, 4 mM mevalonate-5-phosphate) was mixed with 160 μM of immobilized enzyme at 37 °C for 32 h. The immobilized MVK could convert 96% of 4 mM ATP to ADP with a yield of 3.84 mM (1.79 g / L). For sampling, the reaction solution was examined by HPLC system to check the amount of ATP and ADP. The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm x 3 mm with a BetaSil C18 20 mm x 2.1 mm guard column. The column was heated to 25°C and the sample block was maintained at 4°C. The HPLC method consisted of a 5 μl sample injection volume and an isocratic mobile phase consisting of 100 mM KH2PO4, 8 mM TBAHS, pH 6.0, 20% methanol (vol / vol). The total run time was 10 min with ATP eluting at 5.7 min and ADP eluting at 4.6 min. A diode array detector (Agilent) was used for detection of the molecules of interest at 254 nm.

[0064] (2.10 Optimization of diphosphomevalonate kinase (MDC): MDC was mixed with epoxy methacrylate resin and the immobilized enzyme was used to convert mevalonate-5-pyrophosphate to isopentenyl pyrophosphate. The reaction solution (50 mM Tris, 5 mM MgCl2, pH 8, 4 mM ATP, 4 mM mevalonate-5-pyrophosphate) was mixed with 160 μM of immobilized enzyme at 37° C. for 32 hours. The immobilized MVK could convert 94% of 2 mM ATP to ADP with a yield of 1.8 mM (839 mg / L). For sampling, the reaction solution was examined by HPLC system to check the amount of ATP and ADP. The HPLC method was as follows: Agilent 1200 HPLC was equipped with a HYPERSIL ODS COLUMN 150 mm×3 mm with a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to 25°C and the sample block was maintained at 4°C. The HPLC method consisted of a 5 μl sample injection volume with an isocratic mobile phase consisting of 100 mM KH2PO4, 8 mM TBAHS, pH 6.0, 20% methanol (vol / vol). The total run time was 10 min with ATP eluting at 5.7 min and ADP eluting at 4.6 min. A diode array detector (DAD) was used for detection of the molecules of interest at 254 nm.

[0065] (2.11 Optimization of Isopentenyl Diphosphate Delta Isomerase (IDI): IDI was mixed with macroporous polymethacrylate resin and the immobilized enzyme was used to convert isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP). The reaction solution (50 mM Tris (pH 8), 5 mM MgCl2, 10 mM NaCl, 0.24 mM IPP) was mixed with 86 μM of immobilized enzyme for 2 hours at 25 °C. The reaction mixture was then incubated with 0.24 mM olivetolic acid, 85 μM NphB, and 29.7 μM FPPS for 2 hours. The completed reaction was extracted three times with ethyl acetate, evaporated, and then resuspended in methanol for analysis on an HPLC system to determine the amount of CBGA present in the reaction mixture. This coupled reaction was capable of converting 28% of the starting material to 21.3 mg / L of product. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a 250 mm x 4.6 mm 5 μm liChrospher RP8 column with a guard column. The column was heated to 30° C. and the sample block was maintained at 25° C. The HPLC method consisted of a 5 μl sample injection volume with an isocratic mobile phase consisting of 25% Buffer A (water, 0.1% formic acid, 5 mM ammonium formate) and 75% Buffer B (acetonitrile, 0.1% formic acid, 5 mM ammonium formate). CBGA produced in the reaction was measured using a DAD at 228 nm. The run time was 10 minutes total with CBGA eluting at 3.68 minutes.

[0066] (2.12 Optimization of polyprenyl synthetase family protein (FPPS): FPPS was mixed with macroporous polymethacrylate resin and the immobilized enzyme was used to convert isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to geranyl pyrophosphate (GPP). The reaction solution (50 mM Tris (pH 8), 5 mM MgCl2, 10 mM NaCl, 0.24 mM IPP, 0.24 mM DMAPP, 0.24 mM olivetolic acid (OA)) and 120 μM NphB protein were mixed with 86 μM immobilized enzyme at 25 °C for 4 h. The immobilized FPPS could convert 81% of 240 μM IPP and 240 μM DMAPP to yield 195 μM GPP. Analysis of GPP production is linked to the activity of prenyltransferase (NphB) which combines GPP with olivetolic acid to produce CBGA. The completed reaction was extracted three times with ethyl acetate, evaporated, and resuspended in methanol for analysis on an HPLC system to determine the amount of CBGA present in the reaction mixture. The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a 250 mm x 4.6 mm 5 μm liChrospher RP8 column with a guard column. The column was heated to 30°C and the sample block was maintained at 25°C. The HPLC method consisted of a 5 μl sample injection volume with an isocratic mobile phase consisting of 25% buffer A (water, 0.1% formic acid, 5 mM ammonium formate) and 75% buffer B (acetonitrile, 0.1% formic acid, 5 mM ammonium formate). This coupled reaction produced cannabigerolic acid (CBGA) at 129.6 μM or 41 mg / L. The CBGA produced in this reaction was measured using a DAD at 228 nm. The run time was 10 minutes total, with CBGA eluting at 3.68 minutes.

[0067] (2.13 Optimization of polyphosphate kinase 2 (PPK2): PPK2 was mixed with epoxy methacrylate resin and the immobilized enzyme was used to convert ADP to ATP and recycle the cofactor. The reaction solution (10 mM Tris (pH 9), 10 mM MgCl2, 10 mM NaCl, 5.0 mM polyphosphate, 5.0 mM ADP) was mixed with 95 μM of immobilized enzyme at 37° C. for 1 h. The immobilized PPK2 was able to convert 5.0 mM ADP for a yield of 5.0 mM ATP (100%, 2.5 g / L). For sampling, the reaction solution was examined by HPLC system to check the amount of ATP and ADP present in the reaction mixture. The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a HYPERSIL ODS Column 150mm x 3mm with a BetaSil C18 20mm x 2.1mm guard column. The column was heated to 25°C and the sample block was maintained at 4°C. For each sample, 5μl was injected and the isocratic mobile phase consisted of 100mM KH2PO4, 8mM TBAHS, pH 6.0, 20% methanol. The total run time was 10 minutes with ATP eluting at 5.7 minutes and ADP eluting at 4.6 minutes. A diode array detector (DAD) was used for detection of the molecules of interest at 254nm.

[0068] (2.14 Optimization of glucose dehydrogenase (GDH): GDH was mixed with epoxy methacrylate resin and the immobilized enzyme was used to convert NADP+ to NADPH to recycle this essential cofactor. The reaction solution (50 mM Tris (pH 9), 20 mM glucose, 5.0 mM NADP+) was mixed with 200 μM of immobilized enzyme at 22° C. for 15 min. The immobilized GDH was able to convert 5.0 mM NADP+ to produce 5.0 mM NADPH (100%, 3.7 g / L). The activity of GDH was detected by measuring the NADPH concentration of the reaction solution at 340 nm with a plate reader.

[0069] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] *Note: Pyruvate oxidase (PyOx, Aerococcus viridans) was purchased from AG Scientific (product P-1600).

[0070] 3.0 Use of all immobilized enzymes to produce GPP from glycerol: After demonstrating the production of GPP from glycerol using free enzymes and also showing that all of the individual enzymes required are active when immobilized, the next goal was to produce GPP from glycerol using immobilized enzymes. Each enzyme was immobilized to 10 mg of the resin listed in Table 3. The purified enzymes were mixed with the resin for 18 hours at 21° C. and the immobilized enzymes were pooled in a single tube.

[0071] [Table 3]

[0072] For this multi-step reaction, the first three enzymes (Aldo, DHAD, and PyOX) were first added sequentially to a batch reactor. First, immobilized MBP-Aldo was added to the reaction solution (50 mM Tris (pH 9), 2.5 mM MgCl2, 10 mM glycerol) for 18 hours at 37°C. Then, immobilized DHAD was added to the reaction solution and incubated at 45°C for 18 hours. The supernatant was removed from the immobilized enzyme, and the reaction solution was adjusted to contain 50 mM NaCl, 20 mM potassium phosphate (pH 6.5), 10 mM thiamine pyrophosphate, and finally, the pH of the reaction solution was adjusted to pH 6.0. Then, immobilized PyOx was added to the reaction solution for 16 hours at 37°C. The supernatant was then removed from the immobilized enzymes and the reaction solution was adjusted to contain 50 mM Tris, 20 mM potassium phosphate, 2.5 mM MgCl2, 50 mM NaCl, 10 mM NADPH, 10 mM ATP, 10 mM CoA, 4 mM olivetolic acid, and pH 8.0 for a final volume of 1.0 mL. The remaining 10 immobilized enzymes in the pathway were added to the reaction mixture. The reaction was run at 37° C. for 5 days, then extracted with ethyl acetate (2×200 μL), evaporated under reduced pressure, and resuspended in methanol (1 mL) for analysis on an HPLC system to determine the amount of CBGA present in the reaction mixture. The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a 250 mm×4.6 mm 5 μm liChrospher RP8 column with a guard column. The column was heated to 30°C and the sample block was maintained at 25°C. The HPLC method consisted of a 5 μl sample injection volume and used an isocratic gradient consisting of 25% Buffer A (water, 0.1% formic acid, 5 mM ammonium formate) and 75% Buffer B (acetonitrile, 0.1% formic acid, 5 mM ammonium formate) as the mobile phase. The reaction yielded cannabigerolic acid (CBGA) at 155 μM or 49 mg / L (Figures 2A and 2B). The CBGA produced in the reaction was measured using a DAD at 228 nm. The run time was 10 minutes total with CBGA eluting at 3.68 minutes.

[0073] 4.0 Use of immobilized enzymes to generate GPP from glycerol with cofactors (ATP and NADPH recycling): In addition to demonstrating the success of an immobilized glycerol to GPP pathway, recycling of two common cofactors (ATP and NADPH) in the reaction was achieved. The immobilized enzyme compositions shown in Table 4 were used in the reaction.

[0074] [Table 4]

[0075] For this multi-step reaction, the first three enzymes (Aldo, DHAD, and PyOX) were first added sequentially to a batch reactor. First, immobilized MBP-Aldo was added to the reaction solution (50 mM Tris (pH 9), 2.5 mM MgCl2, 10 mM glycerol) for 18 hours at 37°C. Then, immobilized DHAD was added to the reaction solution and incubated at 45°C for 18 hours. The supernatant was removed from the immobilized enzyme, and the reaction solution was adjusted to contain 50 mM NaCl, 20 mM potassium phosphate (pH 6.5), 10 mM thiamine pyrophosphate, and finally, the pH of the reaction solution was adjusted to pH 6.0. Then, immobilized PyOx was added to the reaction solution for 16 hours at 37°C. The supernatant was then removed from the immobilized enzymes and the reaction solution was adjusted to contain 50 mM Tris, 20 mM potassium phosphate, 2.5 mM MgCl2, 50 mM NaCl, 10 mM NADPH, 10 mM ATP, 10 mM CoA, 4 mM olivetolic acid, and pH 8.0 for a final volume of 1.0 mL. The remaining 12 enzymes (as shown in Table 4) were added to a reaction solution of 50 mM Tris, 20 mM potassium phosphate, 2.5 mM MgCl2, 50 mM NaCl, 3.3 mM NADPH, 3.3 mM ATP, 5.0 mM polyphosphate, 5.0 mM glucose, 10 mM CoA, 4 mM olivetolic acid, and pH 8.0 in a final volume of 1.0 mL. The batch reaction was allowed to continue for 5 days at 37° C., then extracted with ethyl acetate (3×200 μL), evaporated under reduced pressure, and resuspended in methanol (1 mL) for analysis on an HPLC system to determine the amount of CBGA present in the reaction mixture. The HPLC method was as follows: an Agilent 1200 HPLC was fitted with a 250 mm×4.6 mm 5 μm liChrospher RP8 column with a guard column. The column was heated to 30° C. and the sample block was maintained at 25° C. The HPLC method consisted of a 5 μl sample injection volume and a mobile phase consisting of 25% Buffer A (water, 0.1% formic acid, 5 mM ammonium formate) and 75% Buffer B (acetonitrile, 0.1% formic acid, 5 mM ammonium formate).The reaction yielded cannabigerolic acid (CBGA) at 114 μM or 36 mg / L. The CBGA produced in this reaction was measured using a DAD at 228 nm. The run time was 10 minutes total, with CBGA eluting at 3.68 minutes.

[0076] As used herein, the term "about" refers to the referenced number ±10%.

[0077] Although preferred embodiments of the present invention have been shown and described, it is readily apparent to one skilled in the art that modifications may be made thereto that do not exceed the scope of the appended claims. Thus, the scope of the present invention should be limited only by the appended claims. In some embodiments, the drawings presented in this application are to scale, including angles, dimensional ratios, etc. In some embodiments, the drawings are merely illustrative, and the claims are not limited by the dimensions of the drawings. In some embodiments, the description of the invention described herein using the phrase "comprising" includes embodiments that could be described as "consisting essentially of" or "consisting of", and thus the description requirement for claiming one or more embodiments of the invention using the phrase "consisting essentially of" or "consisting of" is met.

Claims

1. 1. A method for converting glycerol to geranyl pyrophosphate (GPP) and additional secondary metabolites, comprising: a) adding glycerol to the reaction mixture; b) adding a plurality of enzymes to the reaction mixture from (a), the plurality of enzymes including an enzyme comprising a maltose binding protein alditol oxidase (MBP-ALDO) of SEQ ID NO: 1 or an optimized form thereof, an enzyme comprising a dihydroxyacid dehydratase (DHAD) of SEQ ID NO: 2 or an optimized form thereof, an enzyme comprising a phosphate acetyltransferase (PTA) of SEQ ID NO: 3 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PTA) of SEQ ID NO: 4 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 5 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 6 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 7 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 8 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 9 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 10 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 11 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 12 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 13 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 14 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 15 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 16 or an optimized form thereof, an enzyme comprising a phosphodiesterase (PHA) of SEQ ID NO: 17 or an optimized form thereof, an enzyme comprising a phosphodi one or more enzymes selected from the group consisting of an enzyme comprising SEQ ID NO:6 (HMG-CoA reductase (HMGR)) or an optimized form thereof, an enzyme comprising SEQ ID NO:7 (mevalonate kinase (MVK)) or an optimized form thereof, an enzyme comprising SEQ ID NO:8 (phosphomevalonate kinase (PMVK)) or an optimized form thereof, an enzyme comprising SEQ ID NO:9 (diphosphomevalonate kinase (MDC)) or an optimized form thereof, and an enzyme comprising SEQ ID NO:10 (isopentyl-PP isomerase (IDI)) or an optimized form thereof; c) removing the supernatant from the reaction mixture from (b); and d) isolating GPP; A method comprising:

2. The method of claim 1 , wherein at least three enzymes are added to the reaction mixture.

3. 3. The method of claim 1 or claim 2, wherein at least five enzymes are added to the reaction mixture.

4. The method according to any one of claims 1 to 3, wherein at least 10 enzymes are added to the reaction mixture.

5. 5. The method of any one of claims 1 to 4, further comprising adding NphB enzyme to step (b) to convert GPP to (cannabigerolic acid) CBGA.

6. The method of claim 5 , wherein the conversion of GPP to CBGA is used to determine the amount of GPP produced from the method.

7. The method of any one of claims 1 to 6, wherein the reaction mixture comprises a cofactor.

8. The cofactors include adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD ＋ ), nicotinamide adenine dinucleotide phosphate (NADP ＋ 8. The method of claim 7, wherein said first and second aryl groups are selected from the group consisting of aryl, aryloxy ...

9. 9. The method of claim 7 or claim 8, wherein the cofactor is recycled.

10. 10. The method of claim 9, wherein glucose dehydrogenase (GDH) and polyphosphate kinase 2 (PPK2) are added to the reaction mixture to recycle the cofactors.

11. The method according to any one of claims 1 to 10, wherein one or more of the enzymes are immobilized.

12. The method according to any one of claims 1 to 10, wherein the plurality of enzymes is immobilized.

13. The method of any one of claims 1 to 10, wherein one or more of the enzymes is not immobilized.

14. The method of any one of claims 1 to 10, wherein the plurality of enzymes is not immobilized.

15. 1. A method for converting glycerol to geranyl pyrophosphate (GPP) and additional secondary metabolites, comprising: a) adding glycerol and an enzyme comprising SEQ ID NO:1 (Maltose binding protein alditol oxidase (MBP-ALDO)) or an optimized form thereof to a reaction mixture; b) adding dihydroxy acid dehydratase (DHAD) to the reaction mixture from (a); c) removing the supernatant of the reaction mixture from (b); d) adding pyruvate oxidase (PyOx) to the supernatant of the reaction mixture from (c); e) removing the supernatant of the reaction mixture from (d); f) adding at least two enzymes selected from the group consisting of phosphate acetyltransferase (PTA), acetyl-CoA acetyltransferase (PhaA), HMG-CoA synthase A110G (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate kinase (MDC), isopentyl-PP isomerase (IDI), and farnesyl-PP synthase S82F (FPPS) to the supernatant of the reaction mixture from (e); g) removing the supernatant from the reaction mixture from (f); and h) isolating GPP; A method comprising:

16. 16. The method of claim 15, further comprising adding NphB enzyme to step (f) to convert GPP to (cannabigerolic acid) CBGA.

17. 17. The method of claim 16, wherein the conversion of GPP to CBGA is used to determine the amount of GPP produced from the method.

18. The method of any one of claims 15 to 17, wherein the reaction mixture comprises a cofactor.

19. The cofactors include adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD ＋ ), nicotinamide adenine dinucleotide phosphate (NADP ＋ ), or a combination thereof.

20. 19. The method of claim 17 or claim 18, wherein the cofactor is recycled.

21. 20. The method of claim 19, wherein glucose dehydrogenase (GDH), and polyphosphate kinase 2 (PPK2) are added to the reaction mixture to recycle the cofactors.

22. The method according to any one of claims 15 to 21, wherein one or more of the enzymes are immobilized.

23. The method according to any one of claims 15 to 21, wherein all of the enzymes are immobilized.

24. The method of any one of claims 15 to 21, wherein one or more of the enzymes is not immobilized.

25. The method of any one of claims 15 to 21, wherein all of the enzymes are non-immobilized.

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