RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.

61/871,940, filed on August 30, 2013. The entire teachings of this application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Isoprenoids belong to the largest group of natural products found in living organisms. These lipids have highly diverse, complex and multicyclic structures and some have therapeutic value for antibacterial, antineoplastic, and other pharmaceutical uses.

However, the supply of isoprenoids is limited by the scarce plant resources from which they were originally extracted and by the difficulty in total chemical synthesis due to their structural complexity. Despite the structural diversity of isoprenoids, they are largely derived from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), and the ability of cells to synthesize IPP and DMAPP largely determines the amount of isoprenoids that can be produced.

[0003] For example, artemisin is a clinically useful isoprenoid natural product derived from IPP and DMAPP, and is a key ingredient in a potent treatment to malaria [2], a contagious disease that claims millions of lives annually and continues to infect more than 0.5% of the global population, especially in less developed nations [1]. However, traditional supply of artemisinin depends on extraction of artemisinin from the leaves of the sweet wormwood plant Artemisia annua [3]. Since the growth of crops is slow and .

seasonal, this method inevitably results in supply fluctuation of artemisinin [4]. Efforts in metabolic engineering and synthetic biology have made some promises to even out the supply cycle by engineering fast growing microbes to produce artemisinin and its precursor, artemisinic acid [5] . In addition, a multistep semi-synthesis of artemisinin has been reported in which yeast cells were used to produce precursors of artemisinin for further chemical conversions [6]. Invariably, the complex cellular environment renders any optimization process a challenge to control and maximize productivity [7]. [0004] Thus, there is a need for an efficient and reliable way to synthesize isoprenoids that is not limited by the ability of a cell.

SUMMARY OF THE INVENTION

[0005] The invention described herein relates to systems and methods for producing an isoprenoid precursor or an isoprenoid in vitro.

[0006] One embodiment is a method comprising providing a multienzyme mixture comprising at least a first isolated enzyme, a second isolated enzyme and a third isolated enzyme from the mevalonate pathway, wherein the at leasf a first, a second and a third isolated enzymes are consecutive enzymes in the mevalonate pathway and the first isolated enzyme is the first consecutive enzyme of the at least a first, a second and a third isolated enzymes in the mevalonate pathway present in the multienzyme mixture; and treating a substrate of the first isolated enzyme with the multienzyme mixture in a reaction medium for a sufficient period of time to convert the substrate into the isoprenoid precursor or the isoprenoid, thereby producing the isoprenoid precursor or the isoprenoid.

[0007] Another embodiment is a method of producing amorpha-4,11-diene, comprising providing a multienzyme mixture comprising at least isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase, isolated isopentenyl pyrophosphate isomerase, isolated farnesyl pyrophosphate synthase and isolated amorpha-4,11-diene synthase; and treating a substrate of an isolated enzyme in the multienzyme mixture with the multienzyme mixture in a reaction medium for a sufficient period of time to convert the substrate into amorpha-4, 11 -diene.

[0008] Yet another embodiment is a method for producing dihydroartemisinic acid, comprising providing a multienzyme mixture comprising isolated alcohol dehydrogenase, isolated double bond reductase, isolated aldehyde dehydrogenase and cytochrome P450; and treating amorpha-4,11-diene with the multienzyme mixture in a reaction medium for a sufficient period of time to convert amorpha-4,11-diene into dihydroartemisinic acid.

[0009] Another embodiment is a system comprising a multienzyme mixture comprising at least a first isolated enzyme, a second isolated enzyme and a third isolated enzyme from the mevalonate pathway, wherein the at least a first, a second and a third isolated enzymes are consecutive enzymes in the mevalonate pathway and the first isolated enzyme is the first consecutive enzyme of the at least a first, a second and a third isolated enzymes in the mevalonate pathway present in the multienzyme mixture.

[0010] Another embodiment is a system comprising a multienzyme mixture comprising at least isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase, isolated isopentenyl pyrophosphate isomerase, isolated famesyl pyrophosphate synthase and isolated amorpha-4, 11 -diene synthase.

[0011] Yet another embodiment is a system comprising a multienzyme mixture comprising isolated alcohol dehydrogenase, isolated double bond reductase, isolated aldehyde dehydrogenase and cytochrome P450.

[0012] The systems and methods disclosed herein provide complementary ways of producing valuable drug precursors, and enable the identification of limiting steps and the optimization of enzymatic flux in a metabolic pathway in an efficient manner.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing will be apparent from the following more particular description of example embodiments of the invention.

[0014] FIG. 1 A is a depiction of the mevalonate pathway.

[0015] FIG. IB is a depiction of a pathway for amorpha-4,11-diene production (Ergl2: mevalonate kinase, Erg8: phosphomevalonate kinase, Ergl9: diphosphomevalonate decarboxylase, Idi: isopentenyl pyrophosphate isomerase, IspA: famesyl pyrophosphate synthase, Ads: amorpha-4, 11 -diene synthase, Pi: phosphate, Ppi: pyrophosphate).

[0016] FIG. 2 is a graph of the amount of the indicated enzyme in the soluble and insoluble fractions.

[0017] FIG. 3 A shows the average values of each level of factors Erg 12, Erg8 and Idi on AD yield. This group of enzymes has a positive correlation with AD yield.

[0018] FIG. 3B shows the average values of each level of factors Erg 19 and IspA on

AD yield. This group of enzymes has little or no effect on AD yield.

[0019] FIG. 3C is a half-normal plot and indicates the significant factors on AD yield.

Factors A, B and D represent Ergl2, Erg8 and Idi, respectively.

[0020] FIGS. 4A-4C show the inhibitory effect of IspA and an analysis of the precipitates. A set of separate experiments was conducted to validate the inhibitory effect of IspA, which was attributed to the precipitation of famesyl pyrophosphate (FPP). FIG. 4A shows the fold change in amorpha-4,11-diene (AD) yield when increasing IspA and Idi concentrations while keeping other enzymes at reference level. Fold change in AD yield was calculated by normalizing against AD yield obtained by reference enzyme levels, as indicated by the arrows. Presented data are average of triplicates and standard errors are drawn on the plot. FIG. 4B shows the results of UPLC-(TOF)MS analysis of the

intermediates in the precipitates. Presented data are average of triplicates and standard errors are drawn on the plot. FIG. 4C is a SDS-PAGE analysis of enzymes in the precipitates. The molecular weight of the each band present in the protein marker is indicated.

[0021] FIG. 5 is a summary of optimization of amorpha-4,11-diene production. EA: equal activities of the enzyme, which their concentrations in terms of Taguchi coded levels are Ergl2(l), Erg8(l), Ergl9(l), Idi(l), IspA(l). TO A: optimized enzymatic activities by Taguchi orthogonal array method, which their concentrations in terms of Taguchi coded levels are Ergl2(4), Erg8(4), Ergl9(l), Idi(3), IspA(2). This combination of enzyme concentrations was used as the reference condition. RSM: response surface methodology suggested increasing Ads activity. The other five enzymes were kept at reference level.

[0022] FIGS. 6 A and 6B show the effects of monovalent ions. Monovalent ions were used to increase the specific activity of amorpha-4,11-diene synthase (Ads) and hence the specific amorpha-4,11-diene (AD) yield of the multienzyme synthesis reaction. FIG. 6 A is a titration of potassium chloride concentrations, and show its effects on Ads specific activity. Presented data are average of triplicates and standard errors are drawn on the plots. Student's t-Test with paired two samples for means was used to calculate the p-value in the statistical analysis. FIG. 6B is a titration of different monovalent ions concentrations, and shows their effects on AD yield by reference enzymatic levels. Fold change in AD yield was calculated by normalizing against AD yield obtained by reaction without addition of monovalent ions, as indicated by the arrow. Presented data are average of triplicates and standard errors are drawn on the plots.

[0023] FIG. 7 shows the optimization of buffer pH and magnesium concentration.

Varying buffer pH and magnesium concentrations was found to be helpful to enhance the specific amorpha-4,11-diene (AD) yield. Fold change in AD yield was calculated by normalizing against AD yield obtained with buffer pH 7.4 and 10 mM Mg ²⁺ , as indicated by the arrow. Presented data are average of triplicates and standard errors are drawn on the plot.

[0024] FIG. 8 is a depiction of a biochemical pathway to convert amorphadiene to dihydroartemisinic acid (DHAA) (CYP71AV1 : cytochrome P450, Adhl : alcohol dehydrogenase, Dbr: double bond reductase, Aldhl : aldehyde dehydrogenase).

[0025] FIG. 9A is a graph of Ads specific activity and FPP as a function of adenosine 5'-triphosphate (ATP) concentration, and shows that Ads activity was inhibited in the presence of ATP.

[0026] FIG. 9B is a graph of Ads specific activity and FPP as a function of PPi concentration, and shows that ADS specific activity was inhibited in the presence of PPi.

[0027] FIG. 9C is a graph of AD production as a function of time in the presence of the indicated species and enzymes, and shows that more than 90% of the starting material was converted to AD within 4 hours with the additional enzymes pyruvate kinase (PyfK) and pyrophosphatase (Ppa).

[0028] FIG. 1 OA is a schematic representation of immobilized multienzyme System I described in the Exemplification.

[0029] FIG. 10B is a schematic representation of immobilized multienzyme System II described in the Exemplification.

[0030] FIG. IOC is a schematic representation of immobilized multienzyme System III described in the Exemplification.

[0031] FIG. 10D is a bar graph of AD production and FPP concentration at 4 hours and 12 hours as a function of immobilized multienzyme system represented in FIGs. 10A, 10B and IOC, and shows that a significant improvement in AD yield was observed in the order of System III>System II>System I. The concentration of FPP inversely correlated with AD yield.

[0032] FIG. 10E is a bar graph of AD production in each of the immobilized multienzyme systems represented in FIGs. 10A, 10B and IOC as a function of reaction cycle, and shows that more than 60% AD yield was retained in System II and System III, while less than 10% AD yield remained after the seventh cycle of the reaction in System I.

[0033] FIG. 11 A is a schematic representation of an in vitro biosynthetic reaction for producing artemisinic acid (AA) and dihydroartemisinic acid (DHAA), comprising extracted amorphadiene, whole-cell CYP15 enzyme and cell lysate mixture containing Adhl, Aldhl and Dbr2.

[0034] FIG. 1 IB is a bar graph, and shows the product distribution of the in vitro reaction represented in FIG. 11 A using different combinations of enzymes. Approximately 80% AD is converted to downstream oxidized products, about half of which is DHAA (AD: amorpha-4,1 1-diene, AO: artemisinic alcohol, AA: artemisinic acid, DHAA:

dihydroartemisinic acid).

DETAILED DESCRIPTION OF THE INVENTION

[0035] A description of example embodiments of the invention follows.

[0036] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" can include a plurality of enzyme. Further, the plurality can comprise more than one of the same enzyme or a plurality of different enzymes.

[0037] Each embodiment includes a description of particular aspects of that

embodiment. Unless otherwise stated, the embodiments encompass any combination of the particular aspects described.

Methods

[0038] A first embodiment of the invention is a method of producing an isoprenoid precursor or an isoprenoid. The method comprises providing a multienzyme mixture comprising at least a first isolated enzyme, a second isolated enzyme and a third isolated enzyme from the mevalonate pathway, wherein the at least a first, a second and a third isolated enzymes are consecutive enzymes in the mevalonate pathway and the first isolated enzyme is the first consecutive enzyme of the at least a first, a second and a third isolated enzymes in the mevalonate pathway present in the multienzyme mixture; and treating a substrate of the at least a first, second and a third isolated enzymes (e.g., a substrate of the first isolated enzyme) with the multienzyme mixture in a reaction medium for a sufficient period of time to convert the substrate into the isoprenoid precursor or the isoprenoid, thereby producing the isoprenoid precursor or the isoprenoid.

[0039] "Isoprenoid" and "terpenoid," as used herein, refer to an organic compound made up of two or more structural units derived from isoprene. Many isoprenoids and terpenoids are naturally occurring compounds. Non-limiting examples of isoprenoids include geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), squalene, taxol, amorphadiene and artemisinin. A preferred example of an isoprenoid is amorpha-4,11- diene (AD). Other preferred examples of isoprenoids include GPP and FPP, which are useful intermediates in the synthesis of more complex isoprenoids, including AD. Other examples of isoprenoids include artemisinic alcohol, artemisimc aldehyde, artemisinic acid, dihydroartemisinic aldehyde, dihydroartemisinic acid and artemisinin. In some

embodiments herein, the isoprenoid is dihydroartemisinic acid.

[0040] "Isoprenoid precursor," as used herein, refers to a substrate, intermediate or product that can be transformed into an isoprenoid by metabolism, for example, the mevalonate pathway. Non-limiting examples of isoprenoid precursors include the substrates and products of the mevalonate pathway depicted in FIG. 1 A. Preferred examples of isoprenoid precursors include IPP and/or DMAPP.

[0041] The mevalonate pathway is depicted in FIG, 1 A and is one of two metabolic pathways associated with isoprenoid biosynthesis. As shown in FIG. 1 A, the mevalonate pathway consists of the metabolic processes responsible for transforming acetyl-CoA into dimethylallyl pyrophosphate (DMAPP), an isoprenoid precursor. The steps involved in the mevalonate pathway include the transformation of acetyl-CoA into acetoacetyl-CoA by thiolase; the transformation of acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA by HMG-CoA synthase; the transformation of 3-hydroxy-3-methylglutaryl-CoA into mevalonic acid by HMG-CoA reductase; the transformation of mevalonic acid into mevalonat-5-phosphate by mevalonate kinase (Ergl2); the transformation of mevalonate-5- phosphate into mevalonate-5-pyrophosphate by phosphomevalonate kinase (Erg8); the transformation of mevalonate-5 -pyrophosphate into isopentenyl-5 -pyrophosphate (IPP) by mevalonate-5 -pyrophosphate (or diphosphomevalonate) decarboxylase (Ergl9); and the transformation of IPP into dimethylallyl pyrophosphate (DMAPP) by isopentenyl-5- pyrophosphate (or isopentenyl pyrophosphate) isomerase (Idi).

[0042] "Isolated," as used herein, with respect to an enzyme, for example, in "isolated enzyme" refers to an enzyme that exists outside of a cell and catalyzes a chemical reaction. When the term "isolated" precedes a particular enzyme, as in "isolated isopentenyl pyrophosphate isomerase," it refers to isopentenyl pyrophosphate isomerase that exists outside of a cell and catalyzes a chemical reaction, for example, the reaction of IPP to DMAPP. Methods for obtaining an isolated enzyme are well-known in the art, and exemplary methods are described in the Exemplification section herein.

[0043] The molecule that an enzyme transforms during a chemical reaction is referred to herein as a "substrate" of the enzyme, and a molecule that results from a chemical reaction catalyzed by an enzyme is referred to herein as a "product" of the enzyme.

Because the terms "substrate" and "product" are used with respect to an enzyme, the same molecule can be both a substrate and a product. For example, diphosphomevalomc acid is a substrate of Ergl9 and a product of Erg8 {see FIG. 1A).

[0044] "Enzyme," as used herein, refers to a protein that acts as a catalyst to bring about a specific biochemical reaction. "Enzyme" includes both wild-type enzymes (generated biosynthetically, by cells, or generated using chemical techniques, e.g., in a laboratory) and mutants thereof, so long as the mutations present in the mutant do not alter or do not significantly alter the ability of the protein to bring about the specific biochemical reaction catalyzed by the wild-type protein. Methods of producing mutant enzymes are well-known in the art, and exemplary methods are described in the Exemplification herein. It is to be understood that any enzyme specified herein, except in the Figures or the Exemplification, includes the wild-type enzyme or a mutant thereof. Thus, in some embodiments, the specified enzyme is a wild-type enzyme. In some embodiments, the specified enzyme is a mutant of the specified wild-type enzyme. Typically, the specified enzyme is a wild-type enzyme. Exemplary enzymes include thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase and amorpha-4,11- diene synthase. Further exemplary enzymes include pyruvate kinase, pyrophosphatase, cytochrome P450, cytochrome P450 reductase, alcohol dehydrogenase, double bond reductase and aldehyde dehydrogenase.

[0045] "Consecutive enzymes," as used herein, refers to two or more enzymes that catalyze successive steps in the mevalonate pathway. For example, Erg 19 and Idi, and Erg8, Erg 19 and Idi are consecutive enzymes. Idi and Erg8 are not consecutive enzymes.

[0046] "First consecutive enzyme," as used herein, refers to the first occurring of the two or more enzymes in the mevalonate pathway depicted in FIG. 1 A. Thus, of the consecutive enzymes Ergl9 and Idi, Ergl9 is the first consecutive enzyme. [0047] In the method or system of producing an isoprenoid precursor or an isoprenoid, the first isolated enzyme is the first consecutive enzyme of the at least a first, a second and a third isolated enzymes in the mevalonate pathway present in the multienzyme mixture. For example, if a multienzyme mixture consists of Ergl2, Erg8, Ergl9 and Idi, the first isolated enzyme is Erg 12 because Erg 12 is the first consecutive enzyme in the mevalonate pathway of the enzymes present in the multienzyme mixture. It also follows that if the first isolated enzyme is Erg 12, the first substrate of the first isolated enzyme is mevalonic acid.

[0048] In some aspects of the first embodiment of the invention, the at least a first, a second and a third isolated enzymes are selected from the group consisting of thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl pyrophosphate isomerase. In some aspect of the first embodiment, the at least a first, a second and a third isolated enzymes are selected from the group consisting of mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl pyrophosphate isomerase.

[0049] In some aspects of the first embodiment, the at least a first, a second and a third enzymes comprise isolated isopentenyl pyrophosphate isomerase. In other aspects of the first embodiment, the at least a first, a second and a third enzymes comprise isolated mevalonate kinase, isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase and isolated isopentenyl pyrophosphate isomerase.

[0050] In some aspects of the first embodiment, the multienzyme mixture further comprises one or more isolated enzymes for transforming an isoprenoid precursor, for example, DMAPP, into an isoprenoid, such as geranyl pyrophosphate, farnesyl

pyrophosphate and/or amorpha-4,11-diene. For example, in some aspects of the first embodiment, the multienzyme mixture further comprises isolated farnesyl pyrophosphate synthase (IspA). In some aspects of the first embodiment, the multienzyme mixture further comprises isolated IspA and isolated amorpha-4,11-diene synthase (Ads). In some aspects of the first embodiment, the multienzyme mixture further comprises isolated ispA, isolated ADS and cytochrome P450 (e.g., isolated cytochrome P450). In some aspects of the first embodiment, the multienzyme mixture further comprises isolated ispA, isolated ADS, cytochrome P450 (e.g., isolated cytochrome P450) and isolated Adh. In some aspects of the first embodiment, the multienzyme mixture further comprises isolated ispA, isolated ADS, cytochrome P450 (e.g., isolated cytochrome P450), isolated Adh and isolated Aldh. In some aspects of the first embodiment, the multienzyme mixture further comprises isolated ispA, isolated ADS, cytochrome P450 (e.g., isolated cytochrome P450), isolated Adh, isolated Aldh and isolated Dbr.

[0051] A second embodiment of the invention is a method of producing amorpha-4,11- diene. The method comprises providing a multienzyme mixture comprising at least isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase, isolated isopentenyl pyrophosphate isomerase, isolated farnesyl pyrophosphate synthase and isolated amorpha-4,11-diene synthase; and treating a substrate of an isolated enzyme in the multienzyme mixture with the multienzyme mixture in a reaction medium for a sufficient period of time to convert the substrate into amorpha-4,11-diene. In preferred aspects of this embodiment, the substrate is the substrate of the first consecutive enzyme in the mevalonate pathway present in the multienzyme mixture (e.g., mevalonic acid or phosphomevalonic acid). A pathway for amorpha-4,11-diene production is depicted in FIG. IB.

[0052] In some aspects of the method of producing amorpha-4, 11 -diene, the

multienzyme mixture further comprises isolated mevalonate kinase.

[0053] In some aspects of the second embodiment, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Erg8:Ergl9:Idi:IspA:Ads of 50-150:0.5-5:20-30:1-10:1-5. In more specific aspects, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Erg8:Ergl9:Idi:IspA:Ads of 75-125:0.5-2.5:23-27:3-7:1-3. In yet more specific aspects of the second embodiment, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Erg8:Ergl9:Idi:IspA:Ads of about 100:about 1 :about 25:about 5: about 2, for example, 100: 1 :25:5:2.

[0054] In some aspects of the second embodiment in which the multienzyme mixture further comprises isolated mevalonate kinase, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Ergl2:Erg8:Ergl9:Idi:IspA:Ads of 50-150:50-150:0.5-5:20-30:1-10:1-5. In more specific aspects, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Ergl2:Erg8:Ergl9:Idi:IspA:Ads of 75- 125:75-125:0.5-2.5:23-27:3-7:1-3. In yet more specific aspects of the second embodiment, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Ergl2:Erg8:Ergl9:Idi:IspA:Ads of about 100:about 100:about l :about 25:about 5:about 2, for example, 100:100:1 :25:5:2.

[0055] Activity ratio is based on the activity of 1 OOmg/L Ads (lxAA). The activity of lOOmg/L Ads is approximately 0.08μΜ/8, based on the experimental K _cat value and its theoretical polypeptide molecular weight. Therefore, the activity of lOOx AA for Erg 12, for example, is approximately 8 μΜ/s, which calculates back to 75-80 mg/L. The

concentrations of the enzymes in the ratio of 100 Ergl2:100 Erg8:l Ergl9:25 Idi:5 IspA:2 Ads are Ergl2: 70-80mg/L, Erg8: 18-22mg/L, Ergl9: 1.4-1.6mg/L, Idi: 40-50mg/L, IspA: 8-lOmg/L, Ads: 100-200mg/L.

[0056] A third embodiment of the invention is a method of producing artemisinic acid or dihydroartemisinic acid, preferably dihydroartemisinic acid. The method comprises providing a multienzyme mixture comprising isolated alcohol dehydrogenase, isolated double bond reductase, isolated aldehyde dehydrogenase and cytochrome P450; and treating amorpha-4,11-diene with the multienzyme mixture in a reaction medium for a sufficient period of time to convert amorpha-4,11-diene into artemisinic acid or dihydroartemisinic acid, preferably dihydroartemisinic acid. It is to be understood that cytochrome P450 can be provided as an isolated enzyme or in vivo (e.g., by providing a yeast strain engineered to produce the enzyme).

[0057] In some aspects of the third embodiment, the method further comprises producing AD in vivo. Methods of producing AD in vivo are well-known in the art.

[0058] It will be understood that, although described independently of one another, the methods described in embodiments two and three can be performed together or in sequence (i.e., two then three) to prepare dihydroartemisinic acid. Thus, in some aspects of the third embodiment, the method further comprises producing AD in vitro. The method for producing AD in vitro is as described with respect to the second embodiment, or any aspect thereof.

[0059] In a particular aspect of the third embodiment, the method comprises:

providing a multienzyme mixture comprising at least isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase, isolated isopentenyl pyrophosphate isomerase, isolated farnesyl pyrophosphate synthase, isolated amorpha-4,11-diene synthase, isolated alcohol dehydrogenase, isolated double bond reductase, isolated aldehyde dehydrogenase and cytochrome P450 (e.g. isolated cytochrome P450); treating a substrate of an isolated enzyme in the multienzyme mixture with the multienzyme mixture in a reaction medium for a sufficient period of time to convert the substrate into dihydroartemisinic acid. In a more particular aspect of this aspect, the substrate is the substrate of the first consecutive enzyme in the mevalonate pathway present in the first multienzyme mixture.

[0060] In another particular aspect of the third embodiment, the method comprises: providing a first multienzyme mixture comprising at least isolated

phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase, isolated isopentenyl pyrophosphate isomerase, isolated farnesyl pyrophosphate synthase and isolated amorpha-4,l l-diene synthase;

treating a substrate of an isolated enzyme in the first multienzyme mixture with the first multienzyme mixture in a first reaction medium for a sufficient period of time to convert the substrate into amorpha-4,11-diene in vitro;

isolating amorpha-4,11-diene from the multienzyme mixture and the reaction medium;

providing a second multienzyme mixture comprising isolated alcohol

dehydrogenase, isolated double bond reductase, isolated aldehyde dehydrogenase and cytochrome P450; and

treating amorpha-4,1 1-diene with the second multienzyme mixture in a second reaction medium for a sufficient period of time to convert amorpha-4,11-diene into dihydroartemisinic acid. In a more particular aspect of this aspect, the substrate is the substrate of the first consecutive enzyme in the mevalonate pathway present in the first multienzyme mixture.

[0061] The methods described herein generally comprise treating a substrate with a multienzyme mixture in a reaction medium for a sufficient period of time to convert the substrate into an isoprenoid precursor or an isoprenoid.

[0062] In some aspects of the methods, the method further comprises isolating the isoprenoid precursor (e.g., isopentenyl pyrophosphate, dimethylallyl pyrophosphate) or the isoprenoid (e.g., AD, DHAA) from the multienzyme mixture and the reaction medium. Methods for isolating a product, such as AD, from a reaction mixture are well-known in the art and include solid-phase extraction, such as that described in the Exemplification, extractive work-up, distilling the reaction medium away from the product or otherwise drying the product, for example, by lyophilization, and chromatographic techniques, such as high performance liquid chromatography. In embodiments in which the product, such as AD, is isolated by solid-phase extraction, the product can be isolated by collecting the solid phase and rinsing the solid phase with a solvent, such as a non-polar and/or amphiphilic organic solvent (e.g., hexane), or a combination thereof.

[0063] In some aspects of the methods, the reaction medium comprises a buffer.

Exemplary buffers include Tris, HEPES, PIPES, MOPS, phosphate buffered-saline and phosphate buffers.

[0064] In some aspects of the methods described herein, the reaction medium is biphasic. For example, the reaction medium comprises an aqueous layer (e.g., a buffer) and an organic layer (e.g., an organic solvent) not miscible with the aqueous layer. The organic layer can advantageously be used to dissolve and capture an isoprenoid produced according to the methods of the invention, particularly in cases in which the isoprenoid produced is volatile and, as a consequence, readily evaporates. Exemplary organic solvents for use in the organic layer include hydrocarbon solvents, such as hexanes, cyclohexane and dodecane.

[0065] In some aspects of the methods described herein, the pH of the reaction medium is greater than about 6 to less than about 10. In preferred aspects, the pH of the reaction medium is about 7 to about 8.5.

[0066] In some aspects of the methods described herein, the reaction medium comprises an alkali or alkaline earth metal salt (e.g., magnesium chloride, potassium chloride) or an ammonium salt (e.g. , ammonium chloride). The alkali or alkaline earth metal salt or ammonium salt can be present in the reaction medium in a concentration of from about 25 ihM to about 500 mM, from about 25 mM to about 250 mM, from about 50 mM to about 200 mM, from about 75 mM to about 150 mM or about 100 mM. For example, in some aspects, the alkali or alkaline earth metal salt is a magnesium salt.

[0067] In some aspects of the methods described herein, the multienzyme mixture further comprises an isolated enzyme for regenerating ATP (e.g., isolated pyruvate kinase) or an isolated enzyme for metabolizing pyrophosphate (e.g., isolated pyrophosphatase) or a combination of the foregoing (i.e., an isolated enzyme for regenerating ATP and an isolated enzyme for metabolizing pyrophosphate). [0068] In some aspects of the methods described herein, each enzyme in the

multienzyme mixture is immobilized on a solid surface.

[0069] As used herein, "immobilized" refers to the attachment of an enzyme to a solid surface, typically, a resin. Attachment can be either covalent or non-covalent (e.g., can rely on a metal ion affinity, as in the Ni NTA-hexahistidine interaction; ion-exchange

chromatography, as in the carboxylmethylcellulose-polyarginine and diethylaminoethyl- cellulose-polyglytamic acid interaction; or substrate and recognition binding protein interactions, as in the biotin-streptavidin, amylose-maltose binding protein and glutathione- glutathione-S-transferase interactions). Methods of immobilizing proteins to solid surfaces are well-known in the art, and include metal ion affinity chromatography. In some aspects of this aspect, each enzyme is immobilized on a separate solid surface. Each solid surface, in these aspects, can be the same type of solid surface or can be independently different from the other solid surfaces or any combination of the foregoing (e.g., two specific enzymes can be immobilized separately on the same type of solid surface, while a third specific enzyme is immobilized on a different type of solid surface from the first two specific enzymes). In another aspect of this aspect, the enzymes are immobilized together on a solid surface.

[0070] Exemplary solid surfaces include Ni-NTA resin, negatively charged resin, such as carboxymethyl-cellulose, positively charged resin, such as diethylaminoethyl-cellulose, and substrate and recognition binding protein resin, such as biotin resin, amylose resin and glutathione resin.

[0071] Enzymes immobilized on a solid surface can effectively be reused for additional reaction cycles, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional reaction cycles, often without significantly affecting the yield of the desired isoprenoid precursor or isoprenoid. Thus, in some aspects of the methods disclosed herein, the method further comprises isolating the multienzyme mixture; and treating a substrate (e.g., amorpha-4,11-diene or a second substrate, which can be the same substrate or a substrate different from the original or first substrate, typically, the same substrate) of an enzyme (e.g., the first isolated enzyme, the first consecutive enzyme in the mevalonate pathway present in the multienzyme mixture) with the isolated multienzyme mixture in a reaction medium (e.g., a second reaction medium, which can be the same reaction medium or a reaction medium different from the original, or first, reaction medium, typically, the same reaction medium) for a - In sufficient period of time to convert the substrate into the isoprenoid precursor (e.g., IPP, DMAPP) or the isoprenoid (e.g., amorpha-4,1 1-diene, dihydroartemisinic acid). A multienzyme mixture comprising enzymes immobilized on a solid surface can be isolated by collecting the solid surface (e.g., by filtration, centrifugation) and, optionally, rinsing the surface to remove unbound or unattached species.

[0072] In some aspects, the enzymes immobilized on a solid surface maintain greater than about 60%, greater than about 75%, greater than about 90% or greater than about 95% of the original yield of the desired isoprenoid precursor or isoprenoid after 2 additional reaction cycles (3 reaction cycles total, wherein the original yield is calculated from reaction cycle 1). In some aspects, the enzymes immobilized on a solid surface maintain greater than about 35%, greater than about 50%, greater than about 60%, greater than about 75%, greater than about 90% or greater than about 95% of the original yield of the desired isoprenoid precursor or isoprenoid after 3 additional reaction cycles (4 reaction cycles total, wherein the original yield is calculated from reaction cycle 1). In some aspects, the enzymes immobilized on a solid surface maintain greater than about 25%, greater than about 50%, greater than about 60%, greater than about 75%, greater than about 90% or greater than about 95% of the original yield of the desired isoprenoid precursor or isoprenoid after 4 additional reaction cycles (5 reaction cycles total, wherein the original yield is calculated from reaction cycle 1).

[0073] In a particular aspect of the second embodiment, or any aspect thereof, isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase and, optionally, isolated mevalonate kinase are immobilized together on a first solid surface and isolated isopentenyl pyrophosphate isomerase, isolated farnesyl pyrophosphate synthase and isolated amorpha-4,11-diene synthase are immobilized together on a second solid surface. In aspects of the second embodiment wherein the multienzyme mixture further comprises an isolated enzyme for regenerating ATP (e.g., isolated pyruvate kinase) or an isolated enzyme for metabolizing pyrophosphate (e.g., isolated pyrophosphatase) or a combination of the foregoing, isolated enzyme for regenerating ATP, when present, is immobilized on the first solid surface (together with isolated phosphomevalonate kinase, isolated

diphosphomevalonate decarboxylase and, optionally, isolated mevalonate kinase) and isolated enzyme for metabolizing pyrophosphate, when present, is immobilized on the second solid surface (together with isolated isopentenyl pyrophosphate isomerase, isolated famesyl pyrophosphate synthase and isolated amorpha-4,11-diene synthase).

[0074] As used herein, "immobilized together" means that the specified enzymes are immobilized in a spatially localized manner, for example, on a particular resin bead. Thus, for example, when the first and second solid surfaces are each resins, the isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase and, optionally, isolated mevalonate kinase are immobilized on a first resin bead(s) while the isolated isopentenyl pyrophosphate isomerase, isolated famesyl pyrophosphate synthase and isolated amorpha-4,11-diene synthase are immobilized on a different resin bead(s). The first and second solid surfaces (e.g., first and second resins) can be the same type of solid surface or different types of solid surface.

[0075] In preferred aspects of the methods described herein, the method is a method for producing an isoprenoid precursor (e.g., IPP, DMAPP) or isoprenoid (e.g., amorpha-4,11- diene, dihydroartemisinic acid) in vitro. As used herein, "in vitro" refers to reactions, such as biochemical reactions, performed outside of a cell,- typically, though not exclusively, under cell-free conditions.

Systems

[0076] A fourth embodiment of the invention is a system for producing an isoprenoid precursor or an isoprenoid. The system comprises at least a first isolated enzyme, a second isolated enzyme and a third isolated enzyme from the mevalonate pathway, wherein the at least a first, a second and a third isolated enzymes are consecutive enzymes in the mevalonate pathway and the first isolated enzyme is the first consecutive enzyme of the at least a first, a second and a third isolated enzymes in the mevalonate pathway present in the multienzyme mixture.

[0077] In an aspect of the fourth embodiment, the system further comprises a substrate of the first isolated enzyme (e.g., mevalonic acid).

[0078] In some aspects of the fourth embodiment, the at least a first, a second and a third isolated enzymes are selected from the group consisting of thiolase, HMG-CoA synthase, HMG-Co A reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl pyrophosphate isomerase. In some aspects, the at least a first, a second and a third isolated enzymes are selected from the group consisting of mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl pyrophosphate isomerase.

[0079] In some aspects of the fourth embodiment, the at least a first, a second and a third isolated enzymes comprise isolated isopentenyl pyrophosphate isomerase. In other aspects of the fourth embodiment, the at least a first, a second and a third enzymes comprise isolated mevalonate kinase, isolated phosphomevalonate kinase, isolated

diphosphomevalonate decarboxylase and isolated isopentenyl pyrophosphate isomerase.

[0080] In some aspects of the fourth embodiment, the system further comprises one or more isolated enzymes for transforming an isoprenoid precursor, for example, DMAPP, into an isoprenoid, such as geranyl pyrophosphate, farnesyl pyrophosphate and/or amorpha- 4,11-diene. For example, in some aspects of the fourth embodiment, the multienzyme mixture further comprises isolated farnesyl pyrophosphate synthase (IspA). In some aspects, the multienzyme mixture further comprises isolated IspA and isolated amorpha- 4,11-diene synthase (Ads). In some aspects of the fourth embodiment, the multienzyme mixture further comprises isolated ispA, isolated ADS and cytochrome P450 (e.g., isolated cytochrome P450). In some aspects of the fourth embodiment, the multienzyme mixture further comprises isolated ispA, isolated ADS, cytochrome P450 (e.g., isolated cytochrome P450) and isolated Adh. In some aspects of the fourth embodiment, the multienzyme mixture further comprises isolated ispA, isolated ADS, cytochrome P450 (e.g., isolated cytochrome P450), isolated Adh and isolated Aldh. In some aspects of the fourth embodiment, the multienzyme mixture further comprises isolated ispA, isolated ADS, "cytochrome P450 (e.g., isolated cytochrome P450), isolated Adh, isolated Aldh and isolated Dbr.

[0081] In one aspect of the fourth embodiment, the at least a first, a second and a third enzymes comprise at least isolated phosphomevalonate kinase, isolated

diphosphomevalonate decarboxylase, isolated isopentenyl pyrophosphate isomerase, isolated farnesyl pyrophosphate synthase and isolated amorpha-4,11-diene synthase. In an aspect of this aspect of the system, the at least a first, a second and a third enzymes further comprise mevalonate kinase.

[0082] A fifth embodiment of the invention is a system for producing amorph-4,11- diene. The system comprises a multienzyme mixture comprising at least isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase, isolated isopentenyl pyrophosphate isomerase, isolated farnesyl pyrophosphate synthase and isolated amorpha-4,l l-diene synthase.

[0083] In an aspect of the fifth embodiment, the system further comprises a substrate of an isolated enzyme in the multienzyme mixture. In preferred aspects of this embodiment, the substrate is the substrate of the first consecutive enzyme in the mevalonate pathway present in the multienzyme mixture (e.g., mevalonic acid or phosphomevalonic acid).

[0084] In another aspect of the fifth embodiment, the multienzyme mixture further comprises isolated mevalonate kinase.

[0085] In some aspects of the fifth embodiment, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Erg8:Ergl9:Idi:IspA:Ads of 50-150:0.5-5:20-30:1-10:1-5. In more specific aspects, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Erg8:Ergl9:Idi:IspA:Ads of 75-125:0.5-2.5:23-27:3-7:1-3. In yet more specific aspects of the fifth embodiment, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Erg8:Ergl9:Idi:IspA:Ads of about 100:about l :about 25:about 5: about 2, for example, 100:1 :25:5:2.

[0086] In some aspects of the fifth embodiment in which the multienzyme mixture further comprises isolated mevalonate kinase, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Ergl2:Erg8:Ergl9:Idi:IspA:Ads of 50-150:50-150:0.5-5:20-30:1-10:1-5. In more specific aspects, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Ergl2:Erg8:Ergl9:Idi:IspA:Ads of 75- 125:75-125:0.5-2.5:23-27:3-7:1-3. In yet more specific aspects of the fifth embodiment, each isolated enzyme is present in the multienzyme mixture in an appropriate amount to achieve an enzymatic activity ratio for Ergl2:Erg8:Ergl9:Idi:IspA:Ads of about 100:about 100:about l :about 25:about 5:about 2, for example, 100:100:1 :25:5:2.

[0087] A sixth embodiment of the invention is a system for producing artemisinic acid or dihydroartemisinic acid, preferably dihydroartemisinic acid. The system comprises a multienzyme mixture comprising isolated alcohol dehydrogenase, isolated double bond reductase and isolated aldehyde dehydrogenase, and cytochrome P450. [0088] In some aspects of the sixth embodiment, the system further comprises amorph- 4,11-diene.

[0089] It will be understood that, although described independently of one another, the systems described in embodiments five and six can be combined. Thus, in some aspects of the sixth embodiment, the method further comprises a system accordingly to the fifth embodiment, or any aspect thereof.

[0090] In a particular aspect of the sixth embodiment, the multienzyme mixture further comprises isolated phosphomevalonate kinase, isolated diphosphomevalonate

decarboxylase, isolated isopentenyl pyrophosphate isomerase, isolated farnesyl

pyrophosphate synthase and isolated amorpha-4,11-diene synthase. In particular aspects of this aspect, the system further comprises a substrate of an isolated enzyme in the multienzyme mixture, preferably, the substrate of the first consecutive enzyme in the mevalonate pathway present in the multienzyme mixture.

[0091] In some aspects of the systems described herein, the multienzyme mixture further comprises an isolated enzyme for regenerating ATP (e.g., isolated pyruvate kinase) or an isolated enzyme for metabolizing pyrophosphate (e.g., isolated pyrophosphatase) or a combination of the foregoing.

[0092] In some aspects of the systems described herein, each enzyme in the

multienzyme mixture is immobilized on a solid surface. In some aspects of this aspect, each enzyme is immobilized on a separate solid surface. Each solid surface, in these aspects, can be the same type of solid surface or can be independently different from the other solid surfaces or any combination of the foregoing (e.g., two specific enzymes can be immobilized separately on the same type of solid surface, while a third specific enzyme is immobilized on a different type of solid surface from the first two specific enzymes). In another aspect of this aspect, the enzymes are immobilized together on a solid surface. Exemplary solid surfaces are as described above.

[0093] In some aspects, systems comprising enzymes immobilized on a solid surface maintain greater than about 60%, greater than about 75%, greater than about 90% or greater than about 95% of the original yield of the desired isoprenoid precursor or isoprenoid after 2 additional reaction cycles. In some aspects, systems comprising enzymes immobilized on a solid surface maintain greater than about 35%, greater than about 50%, greater than about 60%, greater than about 75%, greater than about 90% or greater than about 95%» of the original yield of the desired isoprenoid precursor or isoprenoid after 3 additional reaction cycles. In some aspects, systems comprising enzymes immobilized on a solid surface maintain greater than about 25%, greater than about 50%, greater than about 60%, greater than about 75%, greater than about 90% or greater than about 95% of the original yield of the desired isoprenoid precursor or isoprenoid after 4 additional reaction cycles.

[0094] In a particular aspect of the fifth embodiment, or any aspect thereof, isolated phosphomevalonate kinase, isolated diphosphomevalonate decarboxylase and, optionally, isolated mevalonate kinase are immobilized together on a first solid surface and isolated isopentenyl pyrophosphate isomerase, isolated famesyl pyrophosphate synthase and isolated amorpha-4,11-diene synthase are immobilized together on a second solid surface. In aspects of the fifth embodiment wherein the multienzyme mixture further comprises isolated enzyme for regenerating ATP (e.g., isolated pyruvate kinase) or an isolated enzyme for metabolizing pyrophosphate (e.g., isolated pyrophosphatase) or a combination of the foregoing, isolated enzyme for regenerating ATP, when present, is immobilized on the first solid surface (together with isolated phosphomevalonate kinase, isolated

diphosphomevalonate decarboxylase and, optionally, isolated mevalonate kinase) and isolated enzyme for metabolizing pyrophosphate, when present, is immobilized on the second solid surface (together with isolated isopentenyl pyrophosphate isomerase, isolated famesyl pyrophosphate synthase and isolated amorpha-4,11-diene synthase).

[0095] In some aspects of the fourth, fifth and sixth embodiments, the system further comprises a reaction medium. Exemplary reaction media are as described hereinabove with respect to the methods.

EXEMPLIFICATION

Abbreviations

AD Amorpha-4, 11 -diene

ADP Adenosine-5'-diphosphate

ADS Amorpha-4,11-diene synthase

ATP Adenosine 5 '-triphosphate

C0 ₂ Carbon dioxide

DMAPP Dimethylallyl pyrophosphate E. coli Escherichia coli

ERG 12 Mevalonate kinase

ERG8 Phosphomevalonate kinase

Erg 19 Diphosphomevalonate decarboxylase

FPP Farnesyl pyrophosphate

GPP Geranyl pyrophosphate

Idi Isopentenyl pyrophosphate isomerase

IPP Isopentenyl pyrophosphate

IspA Farnesyl pyrophosphate synthase

MVA Mevalonic acid

Pi Phosphate

PMVA Phosphomevalonic acid

Ppi Pyrophosphate

PPMVA Diphosphomevalonic acid

[0096] In vitro synthesis of chemicals and pharmaceuticals using enzymes is of considerable interest as these biocatalysts facilitate a wide variety of reactions under mild conditions with excellent regio-, chemo- and stereoselectivities. A significant challenge in a multi-enzymatic reaction is the need to optimize the various steps involved simultaneously so as to obtain high-yield of a product. In this study, statistical experimental design was used to guide the optimization of a total synthesis of amorpha-4,11-diene (AD) using multiple enzymes in the mevalonate pathway. A combinatorial approach guided by

Taguchi orthogonal array design identified the local optimum enzymatic activity ratio for Ergl2:Erg8:Ergl9:Idi:IspA to be 100:100:1 :25:5, with a constant concentration of amorpha- 4,11-diene synthase (Ads, 100 mg/L). The model also identified an unexpected inhibitory effect of farnesyl pyrophosphate synthase (IspA), where the activity was negatively correlated with AD yield. This was due to the precipitation of farnesyl pyrophosphate (FPP), the product of IspA. Response surface methodology was then used to optimize IspA and Ads activities simultaneously so as to minimize the accumulation of FPP, and the result showed Ads to be a critical factor. By increasing the concentration of Ads, a complete conversion (-100%) of mevalonic acid (Mva) to AD was achieved. Monovalent ions and pH were effective means of enhancing the specific Ads activity and specific AD yield significantly. The results from this study represent the first in vitro reconstitution of the mevalonate pathway for the production of an isoprenoid and the approaches developed herein may be used to produce other isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) based products.

[0097] In vitro multienzyme pathway assembly is a useful approach complementing in vivo metabolic engineering [8]. Cheng et al. demonstrated the feasibility of producing polyketide by enzymatic total synthesis [9]. Cheng et al. assembled 12 pathway enzymes from different production hosts and were able to achieve an overall yield of 25% from simple raw material. This bottom-up method successfully bypasses cellular barriers and allows a higher degree of freedom for pathway manipulation. At the same time, it ensures regioselectivity and enantioselectivity of the product. Moreover, enzymatic reactions involve fewer chemicals that can simplify purification and reduce the cost of downstream processing. In vitro multienzyme biosynthesis has been touted as a promising technology that may replace many chemical synthesis processes due to its high efficiency [10].

[0098] The production of artemisinin or its precursors in vitro is yet to be explored. The synthesis of amorpha-4,11-diene (AD), a key precursor to artemisinin, from mevalonic acid is demonstrated herein by assembling seven enzymatic steps in one-pot with two-phase reaction condition (FIG. IB). Previous in vivo analysis has shown that pathway balancing is critical to maximize the production of scarce therapeutic products [11,12]. Disclosed herein is the optimization of pathway productivity by means of Taguchi orthogonal array design in an attempt to balance the enzymatic levels under pre-determined reaction conditions. The information gained led to identification of an inhibitory step of farnesyl pyrophosphate synthase (IspA), the critical factor Ads and significantly improved the AD yield.

Materials and Methods

Bacteria strains and plasmids

[0099] Bacteria strains and plasmids used in this study are summarized in Table 1. The pET-1 la (Stratagene, CA) was modified by replacing the T7 promoter with Lacl promoter to facilitate the transfer of the plasmids among different strains. A 5' SacI site and a 3' Xhol site were introduced downstream from the 6xHis open reading frame. The mevalonate pathway enzymes, namely mevalonate kinase (Erg 12), phosphomevalonate kinase (Erg8) and pyrophosphomevalonate decarboxylase (Ergl9), were amplified from S. cerevisiae genomic DNA with forward and reverse primers that contain corresponding Sacl and Xhol sites. The PCR products were ligated into the modified pET-1 la vector (Stratagene, CA) and transformed into competent E. coli strain DH10B. Isopentenyl pyrophosphate isomerase (Idi) and IspA were from our previous study [13]. Ads gene was codon optimized and synthesized by Genescript with sequences encoding C-terminal 6xHis-tag, and subsequently cloned into a modified pBAD-B vector (Invitrogen, CA) using 5' Sacl site and 3' Xhol site. The primers used for amplification of the genes are listed in Table 2. All the plasmids were transformed and harboured from E. coli XLlO-gold (Stratagene, CA) and then transformed to strains for enzyme overexpression (Table 1).

Table 1.

Name Description Reference

E. coli BL21-

F ^" ompT hsdS (r _B m _B ) dcm ⁺ Tet ^r gal (DE3) endA Hte Stratagene Gold (DE3)

E. coli DH10B araD139 A(ara-leu)7697 fhuA lacX74 galK (Φ80 A(lacZ)M15) mcrA galU recAl endAl NEB nupG rpsL A(mrr-hsdRMS-mcrBC)

E. coli XL10- Tetr D(mcrA)183 D(mcrCB-hsdSMRmrr) 173 endAl supE44 thi-1 recAl gyrA96 relAl lac

Stratagene

Gold Hte [F9 proAB ladqZDM15 TnlO (Tetr) Tn5 (Kanr) Amy]

pTrc-His ₆ -Ergl2 Plasmid for overexpression of Ergl2 in E. coli DH10B This study pTrc-His ₆ -Erg8 Plasmid for overexpression of Erg8 in E. coli DH10B This study pTrc-His ₆ -Ergl Plasmid for overexpression of Ergl9 in E.coli DH10B This study pET-His ₆ -Idi Plasmid for overexpression of Idi in E. coli BL21-Gold (DE3) [13] pET-His ₆ -IspA Plasmid for overexpression of IspA in E.cColi BL21-Gold (DE3) [13] Name Description Reference pBAD-Ads-His ₆ Plasmid for overexpression of Ads in E. coli DH10B . This study

Table 2.

Primer Name Sequence SEQ ID NO

SacI-Sc.ERG12 Forward GCGAGCTCTCATTACCGTTCTTAACTTCTGC 1

Sc.ERG12-XhoI Reverse GCCTCGAGTTATGAAGTCCATGGTAAATTCG 2

SacI-Sc.ERG8 Forward GCGAGCTCTCAGAGTTGAGAGCCTTCAGT 3

Sc.ERG8-XhoI Reverse GCCTCGAGTTATTTATCAAGATAAGTTTCCGGA 4

SacI-Sc.Ergl9 Forward GCGAGCTCACCGTTTACACAGCATCCG 5

Sc.Ergl9-XhoI Reverse GCCTCGAGTTATTCCTTTGGTAGACCAGTCT 6

Sacl-Ec idi Forward GCTTAGAGCTCCAAACGGAACACGTCA 7

Ec idi-Xhol Reverse GTAACCTCGAGTTATTTAAGCTGGGTAAATGC 8

SacI-Ec ispA Forward GCTTAGAGCTCGACTTTCCGCAGCAACT 9

Ec ispA-XhoI Reverse GTAACCTCGAGTTATTTATTACGCTGGATGA 10

Sacl-Ads Forward GCGGAGCTCTCTCTGACTGAGGAAAAACCA 11

Ads-His6-XhoI Reverse CGCCTCGAGTCAGTGATGGTGATGATGATG 12

Expression and purification ofErgl2, Erg8, Erg 19, Idi arid IspA.

[00100] Newly transformed colonies were picked from the agar plate, inoculated into 2xPY medium (20 g/L Peptone, 10 g/L Yeast extract, and 10 g/L NaCl, pH=7) containing 100 mg/L ampicillin and grown until stationary phase overnight at 37 °C in an incubator- shaker (Shin Saeng Shaking Incubator, Finetech, Korea). The culture was then further transferred into fresh 2xPY medium (1% inoculation) with ampicillin for another 2.5 h at 37 °C, until optical density Α ₆₀₀ reached 0.6-1.0. The enzyme expression was induced with 0.1 mM isopropyl-l-thio- -D-galactopyranoside (IPTG). Temperature was reduced to 20 °C after induction for higher solubility of the enzymes [13]. The culture was grown for another 48 h and harvested by centrifugation. The cell pellets were stored at -20 °C until further use. To purify the enzymes, the frozen cell pellets were resuspended in B-PERII reagent (Pierce, IL,), according to the manufacturer's instructions, and vortexed at room temperature for 30 mins to completely lyse the cells. The soluble proteins were contained in the supernatant, which was diluted 15 times in NPI10 buffer (50 raM NaH ₂ P0 ₄ , 300 mM NaCl, 10 mM imidazole, pH=8) and incubated with 200 mg Ni-NTA resin (USB, Affymetrix, CA) at 4 °C for 2 h. The resin was washed with NPI10 buffer after discarding the binding supernatant, and the enzymes were eluted and collected with 400 μΐ. NPI400 (50 mM NaH ₂ P0 ₄ , 300 mM NaCl, 400 mM imidazole, pH=8). The enzymes were further concentrated by 3K Amicon ultra-0.5 mL centrifugal filter unit (Millipore, MA), and the protein concentrations were measured by Micro BCA protein assay kit (Thermo scientific, MA). The purified enzymes were further confirmed by sodium dodecyl sulfate- 12% polyacrylamide gel electrophoresis (Bio-Rad, CA).

Expression and purification of Ads.

[00101] Bacteria culture was grown in 2xPY medium at 20 °C until stationary phase after Ads expression was induced with 10 mM L-arabinose. The cells were harvested by high speed centrifugation and resuspended in phosphate-buffered saline (PBS). To purify Ads, cells were lysed by three rapid freeze-thaw cycles by -80 °C freezer and 37 °C incubator. The released enzyme was separated from cell debris by centrifuging at 3000 g for 15 min, and purified by Ni-NTA resin as described above.

Enzyme kinetics

[00102] The pathway enzyme kinetics were determined individually by initial rate measurements. In brief, the substrates and cofactors were added to 100 mM Tris/HCl reaction buffer (pH 7.4), and the reaction was initiated by adding pre-determined enzyme amount to ensure less than 10% substrate was consumed in 15 minutes at 30 °C. The substrate concentrations were varied in equal steps in reciprocal space from 0.1 to T mM. The reaction was terminated by adding equal volume of 1% ammonium hydroxide and diluted 10 times into cold methanol. After high speed centrifugation, the supernatant was subject to LTPLC-(TOF)MS analysis. Double-reciprocal plots of each enzymatic activity were constructed for the determination of K _m and K _cat values for the respective substrates. The calculated values for K _m and K _cat as well as enxyme yield are shown in Table 3. Table 3. Purification and characterizations of individual pathway enzymes from bacterial culture.

Enzyme

Enzyme Synonym EC No. MW** Yield*

/μΜ /s ' mg/L

Mevalonate 460±153

Ergl2 2.7.1.36 49524 5.5±1.6 2-8 kinase (MVA)

Phosphome

780±280

valonate Erg8 2.7.4.2 51520 22.0±7.0 1.5-2.5

(PMVA)

Kinase

Diphospho

mevalonate 190±52

Erg 19 4.1.1.33 45181 2.8±0.5 15-60 Decarboxyl (PPMVA)

ase

Isopentenyl

pyrophosph

Idi 5.3.3.2 21331 6.5-28 ate

isomerase

Farnesyl

200±92

pyrophosph 2.5.1.92 32982 1.5±0.6 0.75-2

(IPP)

ate synthase

Amorpha-

0.05±0.0

4,11-diene Ads 4.2.3.24 64624 43.72±10.4 0.3-1.6

13

synthase

The bracket contains the specific substrate that the K _m is measured for.

*The enzyme yield is defined as the final amount of enzyme obtained after purification from a liter of bacterial culture. The results have been repeated more than three times.

**The molecular weight (MW) of the enzyme was calculated based on its amino acid sequence.

Multienzyme reaction

[00103] The multienzyme reaction was carried out in a buffer (25 μΐ) that consisted of Tris/HCl (100 mM, pH7.4), MgCl ₂ (10 mM), (±)Mevalonic acid (10 mM), ATP (15 mM) and the purified enzymes. The reaction was performed at 30 °C with an overlay of dodecane phase that contained trans-caryophyllene (50 mg/L) as an internal standard. At the end of the reaction, the dodecane phase was diluted 10 times in ethyl acetate and subject to GCMS analysis. The (±)mevalonic acid was prepared by complete alkaline hydrolysis of 2 M (±)mevalonolactone (Sigma, MO) with equal volume of 2 M KOH at 37 °C for 1.5 h, and neutralized by adding 1 M hydrochloric acid to pH 7 [14].

Experimental design

[00104] Taguchi orthogonal array design and response surface methodology with central composite design were calculated using Design Expert® V8 Software (Stat-Ease, Inc). Taguchi L ₁₆ (4 ⁵ ) orthogonal array was constructed, which can accommodate five control factors corresponding to the five pathway enzymes, each varied at four levels of concentrations (Table 4). The four enzymatic levels were normalized against Ads activity (AA), ranging from IxAA, 5xAA, 25xAA and lOOxAA to achieve sufficient coverage. The lowest level was equalized enzymatic activity, whereas the highest level was comparable enzymatic concentrations. The level of Idi was varied according to IspA. 16 randomized experimental runs were conducted to maximize AD yield (Equation 1). The specific AD yield (Equation 2) was another indicator of the pathway productivity but was not considered in the design experiment. The two dimensionless readouts were calculated as follows:

Specific AD yield = Actual AD yield (mM)

Total enzymatic concentrations (mM) (2)

Variables (mg/L) 2 3 4

A: Ergl2 4 20 80

B: Erg8 1 5 20

C: Ergl9 7.5 37.5 150

D: Idi 9 45 180 Coded levels

Variables (mg/L) 1 2 3 4

E: IspA 1.8 9 45 180

[00105] Table 5 shows the details of the Tagushi L 16 (4 ⁵ ) orthogonal array design as well as the results of the array.

Table 5. Taguchi LI 6 (4 ) orthogonal array design and results.

Levels*

AD Specific

Runs A: B: C: D: E: Yield AD Yield

Ergl2 Erg8 Ergl9 Idi IspA

1 2 3 4 1 2 11% 34

2 2 4 3 2 1 12% 62

3 1 2 2 2 2 9% 63

4 1 4 4 4 4 18% 16

5 1 1 1 1 1 5% 49

6 1 3 3 3 3 10% 29

7 2 1 2 3 4 8% 15

8 4 2 3 1 4 14% 24

9 3 3 1 2 4 13% 27

10 4 3 2 4 1 24% 33

11 2 2 1 4 3 16% 23

12 3 4 2 1 3 9% 39

13 3 2 4 3 1 10% 22

14 4 4 1 3 2 26% 72

15 3 1 3 4 2 13% 19

16 4 1 4 2 3 16% 32

4 4 4 4 ,2 15% 17

Predicted Max AD yield

4 4 4 4 1 2% 2

Experimentally Max AD

4 4 1 3 2 20% 56 yield

Analysis of variance

Model p-value 0.0046

R ² 0.94

Adj-R ² 0.85

* Refer to Table 4 for the actual enzyme concentrations corresponding to the coded levels. [00106] Optimization of IspA and Ads activities was carried out by response surface methodology with central composite design, which involved the investigation of two factors (concentrations of IspA and Ads), each varied at five levels and four centre points for replication (Table 6). The AD production at 6 hours was taken as the response, before the reaction reached completion and any visible precipitation formed. The experimental data obtained were fitted based on the most suitable model suggested by the software.

Table 6. Coded level combinations for a five-level, two factor response surface

methodology with central composite design.

Levels

Run A: IspA B: Ads AD (mg/L)

1 1 5 15.9

2 3 3 8.1

3 3 3 10.4

4 5 5 15.6

5 1 1 3.6

6 0.17 3 11.9

7 5 1 3.8

8 3 0.17 0.3

9 5.8 3 9.9

10 3 3 10.7

11 3 5.8 24.9

12 3 3 9.5

Actual enzyme concentrations corresponding to the coded levels

Coded level variables

Variables (mg/L) ^ ^

A: ispA 36 180

B: ADS 36 180

Alpha (Rotatable) 1.41

Analysis of variance Model p-value <0.0001

R ² 0.93

Adj-R ² 0.91

UPLC-(TOF)MS analysis of mevalonate pathway intermediates

[00107] The analysis was done based on the method developed previously with slight modification [15]. In brief, 5 μΐ samples were injected into a UPLC C18 column (Waters CSH C18 1.7 μηι, 2.1 mm x 50 mm) connected to UPLC (Waters ACQUITY UPLC)- (TOF)MS (Bruker micrOTOF II, MA). Elution was carried out with a step change from 100% aqueous solution containing 15 mM acetic acid and 10 niM tributylamine (0.5 min) to 10% aqueous solution with 90% methanol for another 3.5 minutes. Electrospray ionization was used and mass spectrometry was operated to scan 50-800 m/z in negative mode with 2500 V end plate voltage and 3200 V capillary voltage. Nebulizer gas was provided in 2 bar, dry gas flow rate was 9 mL/min, and dry gas temperature was 200 °C. Under the assay conditions, all the intermediates were detected in the form [M-H]\ Retention time was subsequently determined for each intermediate with respective synthetic standards and the set m/z extraction range. The peak area was calculated and subsequently used to compute the intermediate concentrations with the software provided by the manufacturer. The calibration curves were constructed with synthetic standards prepared under similar reaction conditions without enzymes. Linearity of the assays were determined individually with . coefficients of determinants (R ² ) greater than 0.90.

GCMS analysis of amorpha-4, 11-diene

[00108] The analysis was carried out based on the modified method developed by Martin et al. by scanning three ions; the m/z values are 117, 189 and 204 [14,16]. 1 μΐ, sample was injected into HP-5 column (Agilent Technologies 7890A gas chromatograph-mass spectrometry, Agilent, CA) with a linear temperature increase of 50 °C/minute from 80 °C to 300 °C and hold at 300 °C for another minute. The peak area was calculated and subsequently used to compute the amorpha-4, 11-diene concentrations with the software provided by the manufacturer. Amorpha-4, 11-diene concentrations were determined relative to the internal standard trans-caryophyllene of known concentration.

Results Enzymatic purification and characterization

[00109] Individual enzyme was overexpressed in E. coli strains. Sodium dodecylsufate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the enzymes were expressed at high levels. However, the yield of purified individual enzymes obtained by immobilized metal affinity chromatography differed significantly (Table 3). This was mainly due to the differences in the solubility of the enzymes (FIG. 2) [13]. In particular, there was almost no detectable soluble(s) fraction of Ads. This led us to extensively optimize the strains, growth conditions and enzyme extraction methods for Ads. Repeated freeze-thaw method [17] was found to be effective in isolating Ads (1.6 mg/L) from cells with high purity as compared to detergent based lysis method. An initial attempt was made by mixing equal mass of the six enzymes in one pot with an overlay of dodecane phase where amorpha-4,11-diene was found to be produced in trace amounts (results not shown).

[00110] In order to better understand and optimize the in vitro system, steady-state kinetics of each enzyme was initially measured. The results are summarized in Table 3. The enzymatic concentrations were determined to ensure the measurement of the initial rate of reaction were linear in the first 15 minutes. From the results, Ads displayed a significantly lower turnover number, two orders of magnitude lower than the other five enzymes. It seemed to be an intrinsic property of terpene synthases, which was proposed to be limited by the release of the product [18-20]. Therefore, Ads was identified to be the bottleneck step in the multienzyme synthesis reaction.

Tuning enzymatic levels by Taguchi orthogonal array design

[00111] To balance the enzymatic flux and to analyze the contribution of the other five enzymes to the final yield of AD, a combinatorial approach was carried out assisted with Taguchi orthogonal array design [21,22]. The reaction conditions were fixed at pH7.4 and 30 °C, with a constant Ads concentration of 100 mg/L (1.5 μΜ). The results were summarized in Table 5. Remarkably, divergent AD yield with varying amounts of enzymes was observed, where the best enzymatic ratio (runl4) produced 5 fold more AD as compared to the lowest ratio (run 5) in which the enzymes had equal activities. To further examine the influence of each enzyme on AD yield, the average effects analysis was determined. FIGS. 3 A and 3B show the average values of each level of the five enzymes on the AD yield. The five enzymes could be classified into two main groups: A (Ergl2), B (Erg8) and D (Idi) positively enhanced AD yield when their activities were increased (FIG. 3 A), while varying the activities of Ergl9 and IspA did not have appreciable effect on AD yield (FIG. 3B). Moreover, the half normal plot (FIG. 3C) clearly indicates that Ergl2, Erg8 and Idi were three main factors that had stronger influence to maximize AD yield. Therefore, among the 16 runs, higher AD yield was obtained from combinations where all the three main enzymes were at higher activities (run 14 and run 12). The model predicted the maximum AD yield would be achieved when the first four enzymes were at their highest activities (Table 5). Attempts to validate this finding showed no significant improvement over AD yield (Table 5), suggesting that the activity ratio of

Ergl2:Erg8:Ergl9:Idi:IspA:Ads of 100:100:1 :25:5:1 (run 14) was near the local optimum. Further improvement of AD yield may require a change in the pre-determined reaction conditions. More importantly, this combination of enzymatic levels resulted in the highest specific AD yield among all the experimental runs designed by Taguchi method. Therefore, this enzymatic ratio was chosen as the reference condition to further optimize the multienzymatic synthesis system.

Optimize IspA and Ads levels to enhance AD yield

[00112] One notable conclusion drawn from the Taguchi model prediction was that, to maximize AD yield, the activities of the first four enzymes were required to be maximized while retaining the activity of the fifth enzyme IspA, at moderate levels (Table 5). This alluded to the possibility of an inhibitory effect of IspA enzyme, since intuitively the yield should increase with increasing enzyme concentration. This led us to conduct a set of separate experiments where IspA concentrations were optimized, while retaining the other four enzymes at their reference levels. FIG. 4A showed the fold change in AD yield, with respect to that obtained by the reference enzymatic levels, when either Idi or IspA concentrations were varied. Idi concentrations were optimized as a control as it displayed a positive correlation with AD yield (FIG. 4A). In contrast, a remarkable inhibitory effect was found when IspA activity was increased above its reference level (FIG. 4A). A critical lead at this point of the study was that precipitates were formed in the reaction when IspA activity was increased. This interesting observation led us to hypothesize that the inhibitory effect of IspA was correlated with the precipitation. Interestingly, LCMS analysis revealed that the precipitates contained FPP and Mva which are the product of IspA and the raw material, respectively (FIG. 4B). SDS-PAGE indicated that enzymes were co-precipitated, and therefore, exacerbating the overall productivity of the multienzyme reaction (FIG. 4C). To further identify which factor was the main reason that induced precipitation, the multienzyme reaction was analyzed stepwise. Precipitates were only visible when FPP was produced (Table 7). Therefore, it was hypothesized that the negative relationship of the increased activity of IspA and the overall productivity may be due to the accumulation of FPP in the context of the system examined.

Table 7.

Reaction Precipitation Estimated concentration of FPP

Ergl2-Erg8-Ergl9 No

Erg 12-Erg8-Ergl 9-Idi-IspA Yes 0.4mM

Ergl2-Erg8-Ergl9-Idi-IspA-Ads Yes 0.4mM

[00113] In order to test this hypothesis and further improve the AD yield of the system, response surface methodology was carried out to optimize the activities of IspA and Ads, so as to minimize the accumulation of FPP. The concentration ranges of IspA and Ads were chosen to be above and inclusive of the reference levels (Table 6). The experimental data obtained based on the design was fitted to a linear mathematical model (Equation 3). The Pv ² and adjusted R ² values were 0.93 and 0.91 respectively, which indicated that the model was suitable to represent adequately the real relationships between the factors used.

Interpretations from the model coefficients suggested a marked agreement with previous observation that IspA level was negatively correlated with AD yield. Moreover, the model implied a positive correlation between Ads level and AD yield, and Ads had a more pronounced effect on AD production. Thus, to validate the model, Ads activity was increased twice when compared to the reference enzymatic ratio. Unexpectedly, the conversion from MVA to AD was ~100%, which was ~5 fold improvement of AD yield as compared to the reference condition (FIG. 5). This further confirmed that Ads was the rate limiting step in the multienzyme synthesis reaction for AD production.

AD = -0.096 - 0.18 IspA + 3.68 κ Ads _m Enhancement Ads specific activity by buffer optimization

[00114] Next, an attempt was made to enhance AD specific yield by examining the contribution of ions in the buffer. Potassium ion has recently been shown to significantly improve the activity of a terpene synthase by interacting with the HI -a loop of the enzyme [20]. Interestingly, a similar structure was found in Ads. Thus, to test the effectiveness of monovalent ion, we supplemented the Ads reaction buffer with potassium ions. FIGS. 6 A and 6B show the change in Ads specific activity and the fold change in AD yield with respect to the reference condition respectively. As predicted, Ads specific activity was found to be enhanced approximately twice with 100 mM potassium ion (FIG. 6A). More interestingly, the overall AD yield by the reference enzymatic ratio was significantly improved about three times in the presence of 100 mM potassium (FIG. 6B). Other monovalent ions, sodium chloride and ammonium chloride, were also titrated into the multienzyme reaction. A similar trend was observed that, with either 100 mM sodium ion or 50 mM ammonium ion, there was a marked three-fold improvement of AD yield (FIG. 6B).

[00115] To further explore the effect of the buffer used, pH was varied from 6 to 9.1 and magnesium concentrations from 5 mM to 20 mM for AD production with reference enzymatic levels. FIG. 7 shows the fold change in AD yield with respect to the reference condition, when either pH or Mg concentrations was varied. Remarkably, AD yield increased 3 times when the pH increased from 7.4 to 8.2, and there was no amorpha-4,11- diene detected when the pH was reduced to 6. By keeping pH at 7.4, the optimum Mg ²⁺ concentration was found to be 15 mM, which resulted in a moderate 1.8 fold improvement of AD yield. However, no synergistic effect was observed at pH 8.2 and 15 mM Mg ²⁺ . The optimum condition found was at pH8.2 with 10 mM Mg , which significantly enhanced specific AD yield three times (FIG. 7).

Discussion

[00116] In metabolic engineering and synthetic biology, an essential process is the in vivo balancing of metabolic pathway flux to achieve optimal productivity. Early successes of controlling pathway flux in vivo have been mainly achieved by tuning the promoter strength, ribosomal binding site sequences and plasmid copy numbers [23]. Controlling enzymatic concentration and activities in vivo is a significant challenge where limitations in global cellular and biochemical mechanisms including the solubility of overexpressed enzymes are not easily predictable [13]. Hence, cell-free enzymatic reaction would be an enabling technology that offers an alternative to these challenges. Recently, multienzyme synthesis for therapeutic products has been successfully demonstrated [24-26]. In this study, the mevalonate pathway together with the downstream plant enzyme amorpha-4,11- diene synthase was individually purified and reconstituted in a single vessel and the overall biochemical reaction achieved an almost complete theoretical yield (340 mg/L) of amorpha- 4,11 -diene production.

[00117] One prerequisite for the system is the availability of functional and purified enzymes. Most of the recombinant enzymes produced in this study were insoluble and thus pose a challenge to obtaining sufficient amounts for in vitro reconstitution. When Ads was overexpressed in E. coli, most of the enzyme was found in inclusion bodies [27]. This is possibly due to the nature of the enzyme, which more than 30% of the amino acids of Ads containing hydrophobic structures, thus rendering the enzyme less soluble as compared to other pathway enzymes. Purification of terpene synthases is challenging; some active enzymes were only recovered from inclusion bodies by in vitro refolding [28-30]. For Ads, using commercially available detergent to lyse the cells, we were unable to recover any functional enzymes. Repeated freeze-thaw method was found to be an effective method to improve the recovery of functional and soluble Ads for in vitro reaction.

[00118] Although the mevalonate pathway has been extensively optimized in engineered microbes, the in vivo mechanistic interactions of the combinations of pathway enzymes affecting amorpha-4, 11 -diene production are currently unknown. One advantage of the in vitro multienzyme system is the ability to precisely control and modulate the enzymatic activities in the same reaction condition. This allows the identification of interacting components or factors which can then guide further optimization. The Taguchi orthogonal array design methodology was used in this study to identify the local optimal ratio of enzymatic activities and attempts to further manipulate the ratio did not result in significant improvement in the yield of AD. This approach was rather helpful in identifying the negative effect of IspA, which was due mainly to the accumulation and precipitation of FPP as well as the enzymes. Attempts were made to understand the involvement of MVA in the formation of precipitates. There was no obvious precipitates formation when MVA but not FPP was present in the reaction, suggesting a stochastic process that possibly resulted from the charge interaction between high concentration of MVA and Mg . Similarly, no precipitation was found when enzymes were present without FPP. Hence, it was likely that FPP accumulated to sufficiently high levels may then precipitate, an observation consistent with a previous report [31]. FPP contains a hydrophobic 15-carbon moiety and is involved in post-translational lipid modification by protein farnesyltransferases [32,33]. It is not unexpected that Mg counterions may shield the negative charges of the pyrophosphate moiety resulting in the precipitation of FPP along with the enzymes. By increasing Ads activity, the flux of FPP towards AD production would have increased, and hence minimizing the accumulation of FPP. A benefit from such an increase in metabolite flux is the remarkable increase in the conversion of mevalonic acid (-100%). It will be interesting to examine the effect of co-immobilizing IspA and Ads to improve the channeling of substrate by proximity effect.

[00119] By optimizing the buffer conditions, the specific AD yield was further enhanced, demonstrating the flexibility of the in vitro system conditions often intolerable to cells. Monovalent ions were found to be effective in improving both the specific activity of Ads and the specific AD yield by 2 and 3 fold, respectively. Monovalent ions are well known to be required for many enzymatic activities [34]. Whether the monovalent ions may act as an allosteric activator to Ads by binding to specific secondary structures, e.g. Hl-a loop of the enzyme [20], remains to be verified.

[00120] pH and magnesium ions play a critical role in moderating the enzymatic activities. Individual enzyme displayed vastly different kinetic properties under different pH and magnesium concentrations. Literature suggests that purified Ads displayed a higher catalytic efficiency when pH was increased in the presence of Mg ²⁺ [18]. Therefore, the increased productivity of the multienzymatic pathway with increased buffer pH would likely be due to the enhanced specific activity of Ads in a more alkaline medium and the hypothesis remained to be verified.

[00121] In summary, amorpha-4,11-diene was successfully produced and achieved quantitative conversion by a multienzyme, biphasic system. The utility of Taguchi method to efficiently identify the local optimum enzymatic ratio and an inhibitory effect of IspA, resulting in the accumulation of FPP, has been demonstrated. Further optimization of IspA and Ads activities by response surface methodology identified that Ads was a critical factor. By increasing the Ads activity, almost 100% conversion from raw materials to AD has been achieved. Further buffer optimization of monovalent ions, pH and magnesium ions, was able to enhance the specific AD yield 2-3 fold significantly. The work-flow demonstrated here will be valuable to produce other isoprenoids in an efficient manner.

[00122] One major hurdle to scale-up of the in vitro synthetic method described above is the use of expensive co-factors and enzymes. Described herein is the introduction of a co-factor regeneration enzyme and by-product removal enzyme to enhance the time-yield of AD using the in vitro synthetic multi-enzyme biocatalytic system described above by 3-fold. Moreover, immobilizing the multienzymes using the immobilized metal affinity chromatography (IMAC) method enabled in situ purification and localization of the enzymes, by which, the specific yield of AD was further increased by 6-fold. Taken together, 2.2g L of AD were produced in 4 days using the in vitro immobilized multi-biocatalytic synthesis system.

[00123] Cell-free synthesis complements in vivo biosynthesis, especially when the downstream compounds are cytotoxic. Many isoprenoids are secondary metabolites isolated from plants. Overexpression of plant enzymes in microorganisms can result in undue stress to the host cell. As a result, metabolite production can be compromised due to impaired cell growth. Coupled in vivo and in vitro biosynthesis represents a potential solution to this challenge. For example, small molecules can be produced in vivo and subsequently converted to more complex molecules via in vitro/semi-in vitro synthesis. Specifically, the in vitro synthesis method has been demonstrated with the downstream cytochrome enzyme, CYP71 AVI, that oxidizes AD to artemisinic acid (AA). The biochemical pathway to convert AD to AA and DHAA is depicted in FIG. 8.

Unraveling the regulatory behavior of in vitro reconstituted amorpha-4,ll-diene synthesis pathway by Lin-log approximation

[00124] A canonical modelling method, Lin-log approximation, is promising to predict the regulatory patterns of the biochemical pathway based on steady state fluxes [35]. It was employed to unravel the regulatory behavior of in vitro reconstituted AD synthesis pathway in order to increase the productivity of the pathway. The use of reconstituted pathway is advantageous, since it is highly reproducible and open to manipulation by changing

concentrations of its constituents [36]. Based on the elasticities estimated from Lin- log approximation, a novel interaction between ATP and Ads was identified. FIG. 9A shows a statistically significant decrease in Ads specific activity when 0.2mM of ATP was

supplemented to the reaction medium. Further structural interaction analysis revealed that it was the polyphosphate moiety in ATP that elicited the inhibitory effect. Since pyrophosphate is the by-product of Ads, another unrecognized product inhibitor of Ads was identified. PPi is a more potent inhibitor of Ads, as shown in FIG. 9B, which shows that a significant change in Ads specific activity was observed when 0.2mM Ppi was added. Subsequently, upon the addition of ATP recycling enzyme pyruvate kinase (Pyf ) and pyrophosphatase (Ppa), the time- yield of the pathway was further enhanced by more than 3-fold. More than 90% starting material was converted to AD within 4 hours (FIG. 9C).

Developing a recyclable platform to co-immobilize the multienzymes for AD production

[00125] The pathway enzymes for AD biosynthesis were engineered with a hexahistidine- tag and conveniently co-immobilized on Ni-NTA modified solid surface. Initial attempts to recycle the co-immobilized AD synthesis pathway did not yield satisfactory productivity and recyclability (FIG. 10A). As shown in FIG. 10D, approximately 40% AD yield was achieved after 12 hours incubation. Less than 10% AD yield was maintained after seventh cycle of reaction (FIG. 10E). This was likely due to the presence of high concentrations of ATP and pyrophosphate. When pyruvate kinase and inorganic pyrophosphatase were implemented and co-immobilized in a random fashion (FIG. 10B), both the rate of production and overall AD yield were improved by 3- and 2.5-fold, respectively (FIG. 10D and FIG. 10E). Further guided by the regulatory topology of the multienzyme pathway, a rationally designed, novel bi-modular system was implemented (FIG. IOC), which further improved AD yield. A 25% improvement in AD yield was observed and a near quantitative conversion of the substrate after 12 hours was achieved. The multienzymes can be effectively reused for more than seven reaction cycles. Taken together, approximately 2.2g/L of amorphadiene were produced within 4 days, which is a greater than 6-fold enhancement of AD specific yield as compared to the non-immobilized enzymatic system.

Coupled in vivo and in vitro biosynthetic platform for dihydroartemisinic acid (DHAA) production

[00126] Recent study suggested that the precursor of artemisinin is DHAA [37], Although a chemical route existed to convert artemisinic acid to DHAA, it is more desirable and environmentally friendly to use biocatalysts to produce DHAA. The biochemical route from AD to DHAA is depicted in FIG. 8. It involves the plant cytochrome enzyme, CYP71AV1, that performs the first oxidation reaction to convert inert hydrocarbon molecule AD to artemisinic alcohol (AOH). AOH is then converted to DHAA by the subsequent enzymes: alcohol dehydrogenase (ADH1), double bond reductase (DBR2), and aldehyde dehydrogenase

(ALDH2). The co-expression of CYP71 AVI and the mevalonate pathway in vivo jeopardized the cell growth and hence the productivity was severely affected. Therefore, coupled in vitro and in vivo production was demonstrated to produce AD and CYP71AV1 enzyme in separate host cells (FIG. 11A). Firstly, in vivo AD production was optimized. The secreted AD was extracted by solid phase CI 8 beads and then eluted using hexane. The hexane solvent was then removed by evaporation and the AD dissolved in DMSO, which was miscible with the aqueous reaction phase. Approximately 50% of the AD produced in vivo could be recovered by this method.

[00127] CYP71 AVI, isolated from Artemisia annua, has been engineered by replacing the first N-terminal 15 amino acid (CYP15) with a short leading sequence from bovine CYP450. Moreover, its electron-donating partner, cytochrome p450 reductase from Artemisia annua (CPRaa) was fused to CYP15 for more efficient electron transfer. This enzyme was then transformed into yeast with deleted cell-wall protein (dCWP2) (BY4741, MATa; his3A 1; leu2A 0; metl5A 0; ura3A 0; Y L096w-a::kanMX4) and overexpressed under the control of Gall promoter. When extracted AD was supplemented to the yeast whole cells with CYP15, approximately 60-70% of AD was converted to artemisinic acid (FIG. 1 IB). Interestingly, about 70% of the oxidized products were outside the cell. This observation enabled use of the subsequent pathway enzymes externally to further convert AOH to DHAA. The advantage is that all the enzymes could be individually overexpressed in separate hosts to their maximum yields. Therefore, by mixing the cell lysates with the dCWP2 cells expressing CYP15, 40% of DHAA was successfully produced (FIG. 1 IB). The characteristic MS peak of DHAA was identical to that previously reported.

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[00128] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[00129] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.