A versatile continuous manufacturing platform for cell-free chemical production

Abstract

The present invention features a versatile continuous manufacturing platform for cell-free chemical production. In some embodiments, the present invention features a cell-free continuous manufacturing platform for chemical production. In some embodiments, the platform comprises one or more individual reactors and a pumping system adapted to flow a solution through the one or more individual reactors. In some embodiments, each of the one or more individual reactors comprises a cylindrical tube having a first end and a second end.

Description

[Technical field]

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

[0002] FIELD OF THEINVENTION The present invention relates to devices and methods for the production of chemicals in a cell-free continuous manufacturing platform. [Background technology]

[0003] BACKGROUND OF THEINVENTION Natural products are defined as molecules found in nature that are produced from natural processes. These broad classes of molecules find use as therapeutic agents, pesticides, or industrial starting materials. The natural processes that form these substances are typically multi-step enzymatic pathways. Such enzymatic pathways convert simple starting materials such as glycerol and glucose into complex substances through multi-step enzymatic reactions. Currently, the majority of natural products are either cultivated and extracted from plants, synthesized by complex chemical synthesis, or biomanufactured through cell-based factories (also known as biofoundries). The present invention details the working of a scalable continuous system that houses immobilized enzymes that mimics how nature produces a diverse range of natural products.

[0004] The production of natural products, either by cultivation or by chemical synthesis or the use of engineered cells, suffers from many problems that limit the commercial viability of biologically-based specialized chemical industrialization. First, cultivation requires 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, cells suffer from product toxicity, carbon flux redirection, diffusion problems through cell walls, and the production of toxic by-products. To overcome these problems, the use of enzymes in the present system offers a viable option. The work is called "cell-free" because the same enzymes as cells are used without the limitations of cells.

[0005] However, for cell-free manufacturing to be competitive, new technologies must be developed that allow each enzyme to experience its optimal reaction conditions to drive higher titers. 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 device and a method, which allows the production of chemicals in a cell-free manner, as specified in the independent claims. Embodiments of the invention are provided in the dependent claims. The embodiments of the invention may be freely combined with one another, if they are not mutually exclusive.

[0007] In a provisional patent application entitled "Cell-free production of geranyl pyrophosphate from glycerol in a cell-free manufacturing system," the inventors have demonstrated the conversion of glycerol to geranyl pyrophosphate (GPP) in a batch reaction using immobilized and non-immobilized enzymes in a prior provisional patent. Here, 12 individual enzymes were placed in a reactor and mixed for 5 days to produce 49 mg / L of GPP (Figure 1). However, using these different immobilized enzymes in a single batch operation limits the opportunities for the chemistry. The reaction efficiency would have been hindered because only one reaction temperature, time, and pH could be accommodated in a single reactor. Furthermore, to take advantage of the benefits of continuous manufacturing with this number of enzymes, the system would have to be engineered and optimized from the ground up.

[0008] The individual requirements for the 12 immobilized enzymes involved in the biochemical pathway that converts glycerol to geranyl pyrophosphate were first investigated (Table 1). Each enzyme has its own set of conditions necessary for optimal reactivity. Furthermore, deviation from the optimal conditions would result in loss of reactivity through protein precipitation and product and substrate degradation, factors that would make the process non-scalable and commercially unviable. Therefore, a system was needed that could provide these individual enzyme requirements, overcome protein precipitation, and provide multiple temperatures, multiple pHs, multiple reaction times, and different reaction solution oxygenation levels at any given time. In practice, it was not possible to satisfy all the individual enzyme requirements in the current production equipment. To overcome this limitation, the 12 immobilized enzymes were placed in a continuous production reactor that was tightly controlled by a set of external sensors and algorithmic feedback loops to generate optimal reaction conditions for these enzymes and more in the future.

[0009] Table 1: Optimal reaction conditions for each enzyme involved in the biochemical pathway that converts glycerol to GPP and then to CBGA through the use of a reporter enzyme system (NphB enzyme). In these reactions, between 10 mg and 100 mg of enzyme-resin complex were used and the reactions were monitored through the use of high performance liquid chromatography (HPLC) coupled to a refractive index detector (RID). [Table 1]

[0010] The inventors have created a versatile continuous manufacturing platform that allows for the scaling of cell-free biomanufacturing while providing the necessary conditions for enzymatic reactions to function. This patent application describes the manufacturing system and its use in important biomanufacturing approaches.

[0011] As described herein, cell-free systems and key reactor drivers (see Table 1) are used in cell-free chemical reactions (i.e., without the presence of cells). The desired enzyme is first produced in vivo (typically through protein overexpression), isolated by chromatography, and then added to a bioreactor containing a low-cost substrate. The enzyme converts the low-cost substrate into a product in exactly the same manner as occurs in plants, animals, and bacteria, but without the complexity of organisms. In this manner, pathways are utilized to produce natural molecules.

[0012] In some embodiments, the invention features a cell-free continuous manufacturing platform for chemical production. In some embodiments, the platform comprises one or more individual reactors and a pumping system adapted to flow a solution through the one or more individual reactors. In some embodiments, each of the one or more individual reactors comprises a cylindrical tube having a first end and a second end. In some embodiments, both the first end of the cylindrical tube and the second end of the cylindrical tube comprise fittings (i.e., stainless steel fittings). In some embodiments, the cylindrical tube inside of the individual reactor comprises a resin and an enzyme. In some embodiments, each of the one or more individual reactors comprises an input tubing connected to the first end of the cylindrical tube and an output tubing connected to the second end of the cylindrical tube to create a closed system. In some embodiments, the cylindrical tube inside of the individual reactor further comprises one or more sensors.

[0013] One of the unique and inventive technical features of the present invention is the use of cell-free production. 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 fight within cells for carbon flux and by-product formation, no cell death due to the formation of toxic compounds (because there are no cells), the cell-free system provides a platform solution for producing a large number of compounds, and the cells have to be reprogrammed each time, and the present invention simply changes the reactor column to another one containing different immobilized enzymes. None of the prior literature or research currently known has the unique and inventive technical features of the present invention.

[0014] Moreover, the prior art teaches away from the present invention. For example, the current use of immobilized enzymes typically uses a single reactor (batch or continuous), which allows only one set of reactor conditions (time, pH, temperature, etc.). The present invention allows the use of different reactor conditions between each individual reactor (or consecutive reactors) without intermediate isolation. Furthermore, the addition of gas via a controlled module allows enzymes that require oxygen (or the lack thereof) to be used in a continuous reactor, which was not possible with previous devices. Furthermore, the deoxygenation module allows "oxygen-loving" and "oxygen-refracting" enzymes to be used in series, again without intermediate purification.

[0015] Furthermore, the inventive technical features of the present invention contributed to the surprising results. For example, the device of the present invention can fit the same amount of enzyme into a 79 mL continuous reactor compared to a 1,000 L traditional fermenter, allowing for a higher reaction concentration. If a 45 L reactor is achieved, this is the equivalent of a 567,000 L traditional fermenter.

[0016] 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.

[0017] (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]

[0018] [Figure 1] Figure 1 shows the previously presented biochemical pathway to produce GPP from glycerol, in which glycerol is converted to GPP using 12 separate enzymes as described herein.

[0019] [Diagram 2] Figure 2 shows the optimum reaction temperature for DHAD, where 45°C is the optimum temperature and a significant decrease in yield is observed when moving away from 45°C.

[0020] [Diagram 3] FIG. 3 shows the reactor, with the heating / cooling Peltier elements attached and housed within a housing with supporting electronics.

[0021] [Figure 4] Figure 4 shows a Grafana data dashboard showing the temperature and electronic output data of the four reactors over time along with each reactor setpoint.

[0022] [Figure 5A] Figure 5A shows the pressure of the first reactor in the system over time (24 hours), 1 sample per second. Average = 0.092 PSI.

[0023] [Figure 5B] FIG. 5B shows the pressure observed by the pH adjustment pump between the first two reactors in the system over time (24 hours). Average = 0.789 PSI.

[0024] [Figure 6] FIG. 6 shows a screen capture of the Graphical User Interface (GUI).

[0025] [Figure 7A] Figure 7A shows a standard voltage divider circuit. As R2 varies, Vout varies.

[0026] [Figure 7B] FIG. 7B shows the standard equation for calculating the output voltage (Vout) based on the input voltage (Vin) and the resistance values ​​(R1, R2).

[0027] [Figure 8] FIG. 8 shows a simplified version of the Steinhart-Hart equation used to convert thermistor resistance to a temperature value.

[0028] [Figure 9A] 9A-9B show the circuit configuration for standard temperature control (FIG. 9A) or for temperature control when both heating and cooling are required without reorienting the Peltier element (FIG. 9B). [Figure 9B] 9A-9B show the circuit configuration for standard temperature control (FIG. 9A) or for temperature control when both heating and cooling are required without reorienting the Peltier element (FIG. 9B).

[0029] [Figure 10A] FIG. 10A shows a thermal image of the four reactors mounted and heated / cooled.

[0030] [Figure 10B] FIG. 10B shows a thermal image of the reactor cooled to 12°C.

[0031] [Figure 11] Figure 11 shows a graph of dissolved oxygen removal in liquid water as a function of time. The probe was placed in a reservoir of deionized water and then in the treated deoxygenated / degassed water and allowed to stir continuously. The dissolved oxygen went from 7.75 ppm to 4.07 ppm, a reduction of nearly 50%.

[0032] [Figure 12A] 12A-12D show a ¼ inch OD reactor housing and a ¾ inch OD reactor housing - the tapped side (FIG. 12A) or screw side (FIG. 12B) of the ¼ inch OD reactor housing and a ¾ inch OD reactor housing - the tapped side (FIG. 12C) or screw side (FIG. 12D). [Figure 12B] 12A-12D show a ¼ inch OD reactor housing and a ¾ inch OD reactor housing - the tapped side (FIG. 12A) or screw side (FIG. 12B) of the ¼ inch OD reactor housing and a ¾ inch OD reactor housing - the tapped side (FIG. 12C) or screw side (FIG. 12D). [Figure 12C] 12A-12D show a ¼ inch OD reactor housing and a ¾ inch OD reactor housing - the tapped side (FIG. 12A) or screw side (FIG. 12B) of the ¼ inch OD reactor housing and a ¾ inch OD reactor housing - the tapped side (FIG. 12C) or screw side (FIG. 12D). [Figure 12D] 12A-12D show a ¼ inch OD reactor housing and a ¾ inch OD reactor housing - the tapped side (FIG. 12A) or screw side (FIG. 12B) of the ¼ inch OD reactor housing and a ¾ inch OD reactor housing - the tapped side (FIG. 12C) or screw side (FIG. 12D).

[0033] [Figure 13] FIG. 13 shows the data flow from capture to display.

[0034] [Figure 14] FIG. 14 shows the reaction scheme for the reaction in Example 2.1.

[0035] [Figure 15] FIG. 15 shows the reaction setup for the reaction in Example 2.2.

[0036] [Figure 16] FIG. 16 shows the reaction scheme for the reaction in Example 2.3.

[0037] [Figure 17]FIG. 17 shows the reaction scheme for the reaction in Example 2.4.

[0038] [Figure 18] FIG. 18 shows the reaction setup for the reaction in Example 2.5.

[0039] [Figure 19] FIG. 19 shows the reaction scheme for the reaction in Example 2.6.

[0040] [Figure 20] FIG. 20 shows the reaction scheme for the reaction in Example 2.7.

[0041] [Figure 21] FIG. 21 shows the reaction scheme for the reaction in Example 2.8.

[0042] [Figure 22] FIG. 22 shows the reaction scheme for the reaction in Example 2.9.

[0043] [Figure 23] FIG. 23 shows the reaction scheme for the reaction in Example 2.10.

[0044] [Figure 24] FIG. 24 shows the reaction scheme for the reaction in Example 2.11.

[0045] [Figure 25A] Figure 25A shows the reaction setup for removal of oxygen from the reaction. A pump pushes the liquid through all four channels of the deoxygenation / degasser before entering the reactor.

[0046] [Figure 25B] FIG. 25B shows a diagram illustrating the deoxygenation and / or degassing module and the addition of nitrogen gas before it is pumped through the reactor.

[0047] [Figure 26] FIG. 26 shows the reaction setup for the reaction in Example 2.13.

[0048] [Figure 27] FIG. 27 shows a test setup to achieve equal throughput of two parallel reactors connected to one pump.

[0049] [Figure 28] FIG. 28 shows commercially available software for visualizing wavelength intensities across the spectrum of a volume flowing through a flow cell connected to a spectrometer.

[0050] [Figure 29] FIG. 29 shows the output of software written to capture and / or display the absorbance across the spectrum of a volume flowing through a flow cell connected to a spectrometer.

[0051] [Diagram 30] FIG. 30 shows a diagram of some of the individual reactors described herein.

[0052] [Figure 31A] Figures 31A and 31B show two-dimensional views of the cell-free manufacturing platform described herein. Figure 31A shows the cell-free manufacturing platform described herein with a pH module. Figure 31B shows the cell-free manufacturing platform described herein with a pressure sensor and a gas addition or degassing module. [Figure 31B] Figures 31A and 31B show two-dimensional views of the cell-free manufacturing platform described herein. Figure 31A shows the cell-free manufacturing platform described herein with a pH module. Figure 31B shows the cell-free manufacturing platform described herein with a pressure sensor and a gas addition or degassing module.

[0053] [Diagram 32] Figure 32 illustrates one embodiment of the cell-free manufacturing platform described herein. Figure 32 illustrates a three-dimensional cell-free manufacturing platform diagram with four individual reactors (i.e., four reactions) equipped with pH control modules.

[0054] [Diagram 33] FIG. 33 shows a circuit schematic that can be used to heat / cool a thermoelectric cooler or to heat an electric silicon heater.

[0055] [Diagram 34] Figures 34A and 34B show certain embodiments of the invention described herein: Figure 34A shows a two-dimensional view of an individual reactor of the cell-free manufacturing platform described herein, and Figure 34B shows a two-dimensional view of the cell-free manufacturing platform described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] 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.

[0057] 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.

[0058] 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."

[0059] As used herein, "reactor" or "individual reactor" may refer to a continuous reactor containing an enzyme-resin complex. An individual reactor means one reactor with an inlet and an outlet defined by the fluid entering or leaving the reactor, respectively. Furthermore, two or more individual reactors may be linked together to form a reactor system.

[0060] As used herein, a "control system" may refer to software and / or hardware implemented to receive parameters of conditions and respond by performing calculations and presenting data to a user, and / or by changing the state or configuration of the hardware in response to that data to achieve the required state.

[0061] As used herein, "pumping system" may refer to isocratic metering pumps or syringe pumps, or their equivalents, used to pump various fluids in a continuous manufacturing system.

[0062] As used herein, "pumping buffer" may refer to a water-based solution containing salts used to carry out enzyme-based reactions. Examples include, but are not limited to, protein buffered saline (PBS) or sodium acetate buffer.

[0063] The size of the individual reactors can vary in length, outer diameter, and inner diameter depending on the throughput desired. An exemplary embodiment can be an individual reactor having a length between 1.5 inches and 14.5 inches or greater, an outer diameter (OD) between 0.125 inches and 0.75 inches or greater, or an inner diameter (ID) between 0.055 inches and 0.652 inches or greater, or some combination thereof. Other embodiments are contemplated based on the desired throughput of the reactor. Individual reactors of other sizes can be used in accordance with the platforms described herein.

[0064] The reactor housing may be manufactured from any material having suitable properties (e.g., durability, strength, inertness to the reactor contents, etc.) In some embodiments, the housing of the individual reactors may be made from 6061 aluminum or a similar material.

[0065] The reactors may be manufactured from any material having suitable properties (e.g., durability, strength, inertness to the reactor contents, etc.) In some embodiments, the individual reactors may be made from 304 stainless steel cylindrical tubing or similar material.

[0066] As used herein, "reactor conditions" may refer to conditions that ensure that the enzyme-resin complex remains active and converts the substrate to the product, and / or conditions that ensure that the substrate and product are stable, and / or optimal conditions for the enzymatic reaction. Non-limiting examples of conditions that the reactor may control include, but are not limited to, temperature, pressure, throughput volume, solvent, pH, oxygen levels, other gas levels, or combinations thereof.

[0067] As used herein, "graphical user interface (GUI)" may refer to a visual method of interacting with and / or controlling a continuous manufacturing system from a computer, including, but not limited to, text boxes and / or buttons within a software application.

[0068] As used herein, "reaction medium" may refer to a solution flowing through a reactor that contains the chemicals necessary to carry out an enzyme-controlled reaction. This typically includes, but is not limited to, substrates (i.e., starting materials), cofactors, gases, buffer salts, and other solvents. The chemical reaction occurs in the reaction medium.

[0069] As used herein, "equilibration buffer" may refer to a buffer having the same composition as the "reactor buffer" or "substrate solution" but lacking (or nearly lacking) substrate.

[0070] As used herein, a "substrate solution" may refer to a solution flowed through a reactor that contains the chemicals necessary to carry out an enzyme-controlled reaction. This may include one or more of the following: one or more substrates (i.e., starting materials), one or more cofactors, one or more gases, one or more buffer salts, and one or more solvents.

[0071] In some embodiments, the time for conversion of substrate to product may be varied to optimize throughput and reaction yield. In some embodiments, conversion of substrate to product may proceed for 0.01 hours to 10 hours. In some embodiments, conversion of substrate to product may proceed for 0.01 hours to 100 hours, for example, about 10 hours to 100 hours. In some embodiments, conversion of substrate to product may proceed for about 0.01 hours to 1,000 hours, or about 10 hours to 1,000 hours, or about 100 hours to 1,000 hours. In some embodiments, conversion of substrate to product may proceed for more than 1,000 hours.

[0072] As used herein, "flow rate" may refer to the rate at which a fluid passes through a reactor and may be measured by flow meters inserted before and after the individual reactors.

[0073] In some embodiments, the flow rates of the individual reactors may be varied to optimize throughput and reaction yield. In some embodiments, the flow rates may range from about 0.1 μL / min to 1000 μL / min, or from about 0.1 mL / min to 100 mL / min, or from about 0.1 μL / min to 10 μL / min, or from about 0.1 μL / min to 1 μL / min, or from about 1 μL / min to 1000 μL / min, or from about 1 mL / min to 100 mL / min, or from about 1 μL / min to 1000 μL / min, or from about 10 mL / min to 100 mL / min, or from about 100 μL / min to 1000 μL / min. In some embodiments, the flow rates may range from about 10 μL / min to 100 μL / min. In other embodiments, the flow rates may range from about 100 μL / min to 1 mL / min. In further embodiments, the flow rate may range from about 1 mL / min to 10 mL / min. In some embodiments, the flow rate may range from about 1 mL / min to 1000 mL / min, or from about 1 mL / min to 100 mL / min, or from about 1 mL / min to 10 mL / min, or from 10 mL / min to 1000 mL / min, or from about 10 mL / min to 100 mL / min, or from about 100 mL / min to 1000 mL / min. In some embodiments, the flow rate may range from about 100 mL / min to 1 L / min. In some embodiments, the flow rate may range from about 1 L / min to 10 L / min. In some embodiments, the flow rate is between about 10 L / min and 100 L / min. In other embodiments, the flow rate may range from about 1 L / min and 1000 L / min, or from about 1 L / min and 100 L / min, or from about 1 L / min and 10 L / min, or from about 10 L / min and 1000 L / min, or from about 10 L / min and 100 L / min, or from about 100 L / min and 1000 L / min. In further embodiments, the flow rate may be greater than 100 L / min.

[0074] As used herein, "residence time" may refer to the length of time that a unit of fluid resides inside a reactor.

[0075] In other embodiments, the residence time may be varied to optimize throughput and reaction yield. In some embodiments, the residence time may range from about 0.1 min to 1 min, about 0.1 min to 10 min, or about 0.1 min to 100 min, or about 1.0 min to 100 min. In other embodiments, the residence time may range from about 0.5 min to 10 min, or about 0.5 min to 100 min, or about 0.5 min to 1000 min. In some embodiments, the residence time may range from about 1 min to 10 min, or about 1 min to 100 min, or about 1 min to 1000 min. In other embodiments, the residence time may range from about 10 min to 100 min, or about 10 min to 1000 min. In some embodiments, the residence time may range from about 100 min to 1000 min. In some embodiments, the residence time may be more than 100 min.

[0076] In some embodiments, the cell-free manufacturing platform comprises a cylindrical tube inside of an individual reactor containing resin. In some embodiments, the amount of resin loaded inside (i.e., inside the cylindrical tube) of the individual reactor can vary. In some embodiments, the individual reactor can have about 0.01 g and 1.0 g of resin loaded inside. In some embodiments, the individual reactor can have about 0.1 g and 1.0 g of resin loaded inside. In some embodiments, the individual reactor can have about 1.0 g and 10 g of resin loaded inside. In some embodiments, the individual reactor can have about 10 g and 100 g of resin loaded inside. In some embodiments, the individual reactor can have about 100 g and 1.0 kg of resin loaded inside. In some embodiments, the individual reactor can have about 1.0 kg and 10 kg of resin loaded inside. In some embodiments, the individual reactor can have about 10 kg and 100 kg of resin loaded inside. In some embodiments, an individual reactor may have more than 100 kg of resin loaded inside.

[0077] In some embodiments, the amount of resin loaded inside an individual reactor can be about 0.01 g to 1000 g, or about 0.01 g to 100 g, or about 0.01 g to 10 g, or about 0.01 g to 1 g, or about 0.01 g to 0.1 g, or about 0.1 g to 1000 g, or about 0.1 g to 100 g, or about 0.1 g to 10 g, or about 0.1 g to 1 g, or about 1 g to 1000 g, or about 1 g to 100 g, or about 1 g to 10 g, or about 10 g to 1000 g, or about 10 g to 100 g, or about 100 g to 1000 g. In other embodiments, the amount of resin loaded inside an individual reactor can be about 1 kg to 1000 kg, or about 1 kg to 100 kg, or about 1 kg to 10 kg, or about 10 kg to 1000 kg, or about 10 kg to 100 kg, or about 100 kg to 1000 kg.

[0078] In some embodiments, the cell-free manufacturing platform comprises a cylindrical tube inside of an individual reactor containing an enzyme. In some embodiments, the amount of enzyme inside the individual reactor (i.e., inside the cylindrical tube) can vary. In some embodiments, an individual reactor can have about 0.01 mg to 1000 mg of enzyme therein, or about 0.01 mg to 100 mg of enzyme therein, about 0.01 mg to 10 mg of enzyme therein, about 0.01 mg to 1 mg of enzyme therein, or about 0.01 mg to 0.1 mg of enzyme therein. In other embodiments, an individual reactor can have about 0.1 mg to 1000 mg of enzyme therein, or about 0.1 mg to 100 mg of enzyme therein, about 0.1 mg to 10 mg of enzyme therein, or about 0.1 mg to 1 mg of enzyme therein. In further embodiments, an individual reactor may have about 1 mg to 1000 mg of enzyme therein, or about 1 mg to 100 mg of enzyme therein, or about 1 mg to 10 mg of enzyme therein. In some embodiments, an individual reactor may have about 10 mg to 1000 mg of enzyme therein, or about 10 mg to 100 mg of enzyme therein. In other embodiments, an individual reactor may have about 100 mg to 1 g of enzyme therein. In some embodiments, an individual reactor may have about 1 g to 1000 g of enzyme therein, or about 1 g to 100 g of enzyme therein, or about 1 g to 10 g of enzyme therein. In other embodiments, an individual reactor may have about 10 g to 1000 g of enzyme therein, or about 10 g to 100 g of enzyme therein. In some embodiments, an individual reactor may have about 100 g to 1 kg of enzyme therein. In some embodiments, an individual reactor may have about 1 kg to 1000 kg of enzyme therein, or about 1 kg to 100 kg of enzyme therein, or about 1 kg to 10 kg of enzyme therein. In other embodiments, an individual reactor may have about 10 kg to 1000 kg of enzyme therein, or about 10 kg to 100 kg of enzyme therein. In some embodiments, an individual reactor may have about 100 kg to 1 Mg of enzyme therein. In some embodiments, an individual reactor may have about 1 Mg to 1000 Mg of enzyme therein, or about 1 Mg to 100 Mg of enzyme therein, or about 1 Mg to 10 Mg of enzyme therein.In other embodiments, an individual reactor may have between about 10 Mg and 1000 Mg of enzyme therein, or between about 10 Mg and 100 Mg of enzyme therein, in some embodiments, an individual reactor may have more than 100 Mg of enzyme therein.

[0079] In some embodiments, the temperature of the individual reactors may be altered to optimize throughput and reaction yield. In some embodiments, the temperature of the individual reactors is altered with a temperature altering element. In some embodiments, the temperature altering element is attached to the individual reactor housing or to the individual reactor. In other embodiments, the temperature altering element is attached to the outside of the cylindrical tube. In some embodiments, the temperature of the individual reactors may be about 10°C to 70°C, or about 10°C to 60°C, or about 10°C to 50°C, or about 10°C to 40°C, or about 10°C to 30°C, or about 10°C to 20°C. In other embodiments, the temperature of the individual reactors may be about 20°C to 70°C, or about 20°C to 60°C, or about 20°C to 50°C, or about 20°C to 40°C, or about 20°C to 30°C. In some embodiments, the temperature of the individual reactors may be about 30°C to 70°C, or about 30°C to 60°C, or about 30°C to 50°C, or about 30°C to 40°C. In other embodiments, the temperature of the individual reactors may be between about 40° C. and 70° C., or between about 40° C. and 60° C., or between about 40° C. and 50° C. In some embodiments, the temperature of the individual reactors may be between about 50° C. and 70° C., or between about 50° C. and 60° C. In some embodiments, the temperature of the individual reactors may be between 60° C. and 70° C. In some embodiments, the temperature of the individual reactors may be greater than 70° C.

[0080] The temperature in the individual reactors may be measured at various intervals. In some embodiments, the temperature in the individual reactors may be measured by a temperature sensor. In other embodiments, the temperature in the individual reactors may be measured by a temperature sensor inside the cylindrical tube. For example, the temperature of the individual reactors may be measured once per second. In some embodiments, the temperature of the individual reactors may be measured less than once per second. In other embodiments, the temperature of the individual reactors is measured more than once per second.

[0081] In some embodiments, the pH of the individual reactors (or of the solutions therein) may be altered to optimize throughput and reaction yield. In some embodiments, the pH of the individual reactors (or of the solutions therein) may be altered using a pH measurement module adapted to introduce an acid or base into the solution within the individual reactor. In some embodiments, the pH of the individual reactors may be about 4.0-10.0, or about 4.0-9.0, or about 4.0-8.0, or about 4.0-7.0, or about 4.0-6.0, or about 4.0-5.0. In some embodiments, the pH of the individual reactors may be about 5.0-10.0, or about 5.0-9.0, or about 5.0-8.0, or about 5.0-7.0, or about 5.0-6.0. In some embodiments, the pH of the individual reactors may be about 6.0-10, or about 6.0-9.0, or about 6.0-8.0, or about 6.0-7.0. In some embodiments, the pH of the individual reactors may be about 7.0-10.0, or about 7.0-9.0, or about 7.0-8.0. In some embodiments, the pH of the individual reactors may be about 8.0-10.0, or about 8.0-9.0. In some embodiments, the pH of the individual reactors may be about 9.0-10.0. In some embodiments, the pH may be less than 4.0. In some embodiments, the pH may be greater than 10.0.

[0082] The pH in the individual reactors (or of the solutions therein) may be measured at various intervals. In some embodiments, the pH in the individual reactors (or of the solutions therein) may be measured by a pH sensor. In other embodiments, the pH in the individual reactors (or of the solutions therein) may be measured by a pH sensor inside the cylindrical tube. In preferred embodiments, the pH of the individual reactors (or of the solutions therein) may be measured once per second. In some embodiments, the pH of the individual reactors (or of the solutions therein) may be measured less than once per second. In other embodiments, the pH of the individual reactors (or of the solutions therein) may be measured more than once per second. In some embodiments, the pH of the individual reactors (or of the solutions therein) may be changed through the addition of an acidic or basic solution.

[0083] In some embodiments, the pressure of an individual reactor may be varied to optimize throughput and reaction yield. In other embodiments, the pressure change of an individual reactor may be a by-product of introducing gas into the individual reactor. In further embodiments, the pressure change of an individual reactor may be a by-product of removing gas from the individual reactor. In some embodiments, the pressure may be from about 0 psig (pounds force per square inch) to 500 psig, or from about 0 psig to 250 psig, or from about 0 psig to 100 psig, or from about 0 psig to 50 psig, or from about 0 psig to 10 psig, or from about 0 psig to 1 psig, or from about 0 psig to 0.1 psig, or from about 0 psig to 0.01 psig. In other embodiments, the pressure can be from about 0.01 psig to 500 psig, or from about 0.01 psig to 250 psig, or from about 0.01 psig to 100 psig, or from about 0.01 psig to 50 psig, or from about 0.01 psig to 10 psig, or from about 0.01 psig to 1 psig, or from about 0.01 psig to 0.1 psig. In some embodiments, the pressure can be from about 0.1 psig to 500 psig, or from about 0.1 psig to 250 psig, or from about 0.1 psig to 100 psig, or from about 0.1 psig to 50 psig, or from about 0.1 psig to 10 psig, or from about 0.1 psig to 1 psig. In other embodiments, the pressure can be between about 10 psig and 500 psig, or between about 10 psig and 250 psig, or between about 10 psig and 100 psig, or between about 10 psig and 50 psig. In some embodiments, the pressure can be between about 50 psig and 500 psig, or between about 50 psig and 250 psig, or between about 50 psig and 100 psig. In some embodiments, the pressure can be between about 100 psig and 250 psig, or between about 100 psig and 500 psig. In other embodiments, the pressure can be between about 250 psig and 500 psig. In further embodiments, the pressure can be greater than 500 psig.

[0084] The pressure in the individual reactors may be measured at various intervals. In some embodiments, the pressure in the individual reactors may be measured by a pressure sensor. In other embodiments, the pressure in the individual reactors may be measured by a pressure sensor inside the cylindrical tube. In some embodiments, the pressure of the individual reactors may be measured once per second. In some embodiments, the pressure of the individual reactors may be measured less than once per second. In other embodiments, the pressure of the individual reactors may be measured more than once per second.

[0085] In some embodiments, the amount of dissolved oxygen in the individual reactors may be varied. In other embodiments, the amount of dissolved oxygen in the solution in the individual reactors may be varied. In some embodiments, the amount of dissolved oxygen may be altered using a gas addition module adapted to introduce gas into the solution in the individual reactors. In other embodiments, the amount of dissolved oxygen may be altered using a gas addition module adapted to introduce gas into the cylindrical tubing inside the individual reactors. In other embodiments, the amount of dissolved oxygen may be altered using a degassing module adapted to remove gas from the solution. In further embodiments, the amount of dissolved oxygen may be altered using a degassing module adapted to remove gas from the cylindrical tubing inside the individual reactors. In some embodiments, the amount of dissolved oxygen in an individual reactor can be from about 0.0 ppm (parts per million) to 10 ppm, or from about 0.0 ppm to 9.0 ppm, or from about 0.0 ppm or about 8.0 ppm, or from about 0.0 ppm to 7.0 ppm, or from about 0.0 ppm to 6.0 ppm, or from about 0.0 ppm to 5.0 ppm, or from about 0.0 ppm to 4.0 ppm, or from about 0.0 ppm to 3.0 ppm, or from about 0.0 ppm to 2.0 ppm, or from about 0.0 ppm to 1.0 ppm. In some embodiments, the amount of dissolved oxygen in an individual reactor can be about 1.0 ppm to 10 ppm, or about 1.0 ppm to 9.0 ppm, or about 1.0 ppm or about 8.0 ppm, or about 1.0 ppm to 7.0 ppm, or about 1.0 ppm to 6.0 ppm, or about 1.0 ppm to 5.0 ppm, or about 1.0 ppm to 4.0 ppm, or about 1.0 ppm to 3.0 ppm, or about 1.0 ppm to 2.0 ppm. In other embodiments, the amount of dissolved oxygen in an individual reactor can be between about 2.0 ppm and 10 ppm, or between about 2.0 ppm and 9.0 ppm, or between about 2.0 ppm or about 8.0 ppm, or between about 2.0 ppm and 7.0 ppm, or between about 2.0 ppm and 6.0 ppm, or between about 2.0 ppm and 5.0 ppm, or between about 2.0 ppm and 4.0 ppm, or between about 2.0 ppm and 3.0 ppm.In some embodiments, the amount of dissolved oxygen in the individual reactors can be about 3.0 ppm to 10 ppm, or about 3.0 ppm to 9.0 ppm, or about 3.0 ppm or about 8.0 ppm, or about 3.0 ppm to 7.0 ppm, or about 3.0 ppm to 6.0 ppm, or about 3.0 ppm to 5.0 ppm, or about 3.0 ppm to 4.0 ppm. In other embodiments, the amount of dissolved oxygen in the individual reactors can be about 4.0 ppm to 10 ppm, or about 4.0 ppm to 9.0 ppm, or about 4.0 ppm or about 8.0 ppm, or about 4.0 ppm to 7.0 ppm, or about 4.0 ppm to 6.0 ppm, or about 4.0 ppm to 5.0 ppm. In other embodiments, the amount of dissolved oxygen in the individual reactors may be about 5.0 ppm to 10 ppm, or about 5.0 ppm to 9.0 ppm, or about 5.0 ppm or about 8.0 ppm, or about 5.0 ppm to 7.0 ppm, or about 5.0 ppm to 6.0 ppm. In some embodiments, the amount of dissolved oxygen in the individual reactors may be about 6.0 ppm to 10 ppm, or about 6.0 ppm to 9.0 ppm, or about 6.0 ppm or about 8.0 ppm, or about 6.0 ppm to 7.0 ppm. In some embodiments, the amount of dissolved oxygen in the individual reactors may be about 7.0 ppm to 10 ppm, or about 7.0 ppm to 9.0 ppm, or about 7.0 ppm or about 8.0 ppm. In other embodiments, the amount of dissolved oxygen in the individual reactors may be 8.0 ppm to 10 ppm, or about 8.0 ppm to 9.0 ppm. In some embodiments, the amount of dissolved oxygen in the individual reactors may be between 9.0 ppm and 10.0 ppm, hi further embodiments, the amount of dissolved oxygen in the individual reactors may be greater than 10.0 ppm.

[0086] The amount of dissolved oxygen in the individual reactors (or in the solutions therein) may be measured at various intervals. In some embodiments, the amount of dissolved oxygen in the individual reactors (or in the solutions therein) may be measured by a dissolved oxygen (DO) sensor. In other embodiments, the amount of dissolved oxygen in the individual reactors (or in the solutions therein) may be measured by a dissolved oxygen (DO) sensor inside the cylindrical tube. In some embodiments, the amount of dissolved oxygen in the individual reactors (or in the solutions therein) may be measured once per second. In some embodiments, the amount of dissolved oxygen in the individual reactors (or in the solutions therein) may be measured less than once per second. In other embodiments, the amount of dissolved oxygen in the individual reactors (or in the solutions therein) may be measured more than once per second.

[0087] In some embodiments, dissolved oxygen may be removed by a deoxygenation / degasser (i.e., deoxygenation / degasser module). As used herein, a "deoxygenator" or "degasser" or "deoxygenation module" or "degasser module" may refer to a device that removes the amount of dissolved oxygen and / or other gases (e.g., nitrogen) in a fluid (i.e., solution) as the fluid is flowed through it. In other embodiments, nitrogen gas is introduced into a separate reactor to reduce oxygen levels.

[0088] As used herein, a "gas supply" or "gas supply module" may refer to a device that adds an amount of dissolved oxygen and / or other gases (e.g., nitrogen) to a fluid (i.e., a solution) as the fluid is flowed through it. In some embodiments, the cell manufacturing platform includes a gas addition module adapted to introduce gases into the solution. In some embodiments, oxygen, nitrogen, or a combination thereof may be added to an individual reactor. Other gases may be added to or removed from an individual reactor according to the platforms described herein.

[0089] As used herein, a "chemical stream" may refer to a solution that contains substrates, products, intermediates, cofactors, or another chemical.

[0090] 1-34B, the present invention features a cell-free manufacturing platform for continuous chemical production.

[0091] The present invention features a cell-free manufacturing platform for chemical production. In some embodiments, the platform comprises one or more individual reactors and a pumping system adapted to flow a solution through the one or more individual reactors. In some embodiments, each of the one or more individual reactors comprises a cylindrical tube having a first end and a second end. In some embodiments, both the first end of the cylindrical tube and the second end of the cylindrical tube comprise fittings (i.e., stainless steel fittings). In some embodiments, the cylindrical tube inside of the individual reactor comprises a resin and an enzyme. In some embodiments, each of the one or more individual reactors comprises an input tubing connected to the first end of the cylindrical tube and an output tubing connected to the second end of the cylindrical tube. In some embodiments, the cell-free manufacturing platform can automatically change each of the one or more reactors based on input from a sensor.

[0092] In some embodiments, the cell-free manufacturing platforms described herein are closed systems. In other embodiments, the cell-free manufacturing platforms described herein are open systems.

[0093] In some embodiments, each of the one or more individual reactors comprises an individual reactor housing. In some embodiments, the individual reactor housing surrounds and is fixed to the individual reactor. In embodiments, the cell-free manufacturing platform further comprises a temperature change element attached to the individual reactor housing or the individual reactor. In some embodiments, the temperature change element is a thermoelectric cooler (TEC). In some embodiments, the temperature change element is a flexible heating element.

[0094] In some embodiments, the cell-free manufacturing platform further comprises a spectrometer mounted in series with one or more individual reactors. In other embodiments, the cell-free manufacturing platform further comprises a degassing module adapted to remove gas from the solution. In some embodiments, the degassing module is a deoxygenation module. In some embodiments, the deoxygenation module is adapted to remove oxygen from the solution. In some embodiments, the cell-free manufacturing platform further comprises a gas addition module adapted to introduce a gas into the solution. In other embodiments, the cell-free manufacturing platform further comprises a pH module adapted to introduce an acid or base into the solution. In some embodiments, the cell-free manufacturing platform further comprises a graphical user interface (GUI) adapted to control the automation software and hardware.

[0095] In some embodiments, the invention features a cell-free manufacturing platform for chemical production. In some embodiments, the platform comprises one or more individual reactors. In some embodiments, each of the one or more reactors comprises a cylindrical tube with stainless steel fittings on both ends. In other embodiments, each of the one or more reactors comprises a resin, an enzyme, and one or more sensors. In some embodiments, the platform comprises an individual reactor housing. In some embodiments, the housing surrounds and is fixed to the individual reactor. In some embodiments, the platform comprises a temperature changing element (e.g., a thermoelectric cooler (TEC)) attached to the reactor housing or the individual reactor. In some embodiments, the platform comprises a pumping system adapted to flow a solution through the one or more reactors. In some embodiments, the platform comprises a degassing module (e.g., a deoxygenation module) adapted to remove gas from the solution. In some embodiments, the platform comprises a gas addition module adapted to introduce gas into the solution. In some embodiments, the platform comprises a spectrometer attached in series to the reactor. In some embodiments, the platform comprises a graphical user interface (GUI). In some embodiments, the platform comprises automation software and hardware. In some embodiments, the GUI is adapted to control the automation software and hardware. In some embodiments, the individual reactors comprise input and output tubing connected to each end of a cylindrical tube to create a closed system. In some embodiments, the platform can automatically change each of the conditions of one or more reactors based on input from sensors.

[0096] In other embodiments, the invention may feature a cell-free continuous manufacturing platform for chemical production. In some embodiments, the platform comprises an individual reactor comprising a cylindrical tube with stainless steel fittings at both ends of the reactor. In some embodiments, the platform comprises multiple reactors connected in series. In other embodiments, the platform comprises multiple reactors in parallel. In some embodiments, the reactor comprises a resin, an enzyme, and a sensor. In some embodiments, the platform comprises an individual or multiple reactor housing, the housing surrounding and fixed to the individual reactor. In some embodiments, the platform comprises a combination of a thermoelectric cooler (TEC) or other temperature changing element attached to the housing, a pumping system, a deoxygenation and / or degassing module, a gas addition module, a graphical user interface (GUI), and automation software and hardware to operate the device. In some embodiments, the individual reactors are sealed at each end of the cylindrical tube to create a closed system. In some embodiments, the platform can automatically change reactor conditions based on input from sensors. In some embodiments, the platform comprises a spectrometer for measuring the wavelength and absorption of the entering and leaving volumes. In other embodiments, the platform includes a spectrometer to measure the wavelength and / or absorption of the color of the output. In further embodiments, the platform can automatically change reactor conditions based on input from the sensors.

[0097] In some embodiments, one or more individual reactors are connected in parallel. In other embodiments, at least two of the one or more individual reactors are connected in parallel. In some embodiments, more than one reactor is connected in parallel. In further embodiments, one or more individual reactors connected in parallel are connected to one pump. In some embodiments, the parallel reactors are connected to one pump. In some embodiments, the flow through the parallel reactors is controlled via software and control valves.

[0098] In some embodiments, the enzyme is selected from the group consisting of MBP-Aldo (Aldo), dihydroxyacid dehydratase (TvDHAD), 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), farnesyl-PP synthase S82F (FPPS), and prenyltransferase (NphB). In some embodiments, the enzyme comprises MBP-Aldo (Aldo), dihydroxyacid dehydratase (TvDHAD), 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), farnesyl-PP synthase S82F (FPPS), prenyltransferase (NphB), or a combination thereof.

[0099] In some embodiments, the enzyme is immobilized. In other embodiments, the enzyme is not immobilized.

[0100] In some embodiments, the plurality of sensors includes a temperature sensor. In some embodiments, the plurality of sensors includes a pH sensor. In some embodiments, the plurality of sensors includes a pressure sensor. In some embodiments, the plurality of sensors includes a flow sensor. In some embodiments, the plurality of sensors includes a dissolved oxygen (DO) sensor. In some embodiments, the plurality of sensors includes a spectrometer. In some embodiments, the cylindrical tube inside of the individual reactor further comprises one or more sensors. In some embodiments, the one or more sensors include a temperature sensor, a pH sensor, a pressure sensor, a flow sensor, a dissolved oxygen (DO) sensor, a spectrometer, or a combination thereof.

[0101] In some embodiments, the individual reactors have a percent yield of about 10% to about 100%. In some embodiments, the individual reactors have a percent yield of about 10%. In some embodiments, the individual reactors have a percent yield of about 20%. In some embodiments, the individual reactors have a percent yield of about 30%. In some embodiments, the individual reactors have a percent yield of about 40%. In some embodiments, the individual reactors have a percent yield of about 50%. In some embodiments, the individual reactors have a percent yield of about 60%. In some embodiments, the individual reactors have a percent yield of about 70%. In some embodiments, the individual reactors have a percent yield of about 80%. In some embodiments, the individual reactors have a percent yield of about 90%. In some embodiments, the individual reactors have a percent yield of about 95%. In some embodiments, the individual reactors have a percent yield of about 98%. In some embodiments, the individual reactors have a percent yield of about 99%. In some embodiments, the individual reactors have a percent yield of about 100%.

[0102] In some embodiments, each individual reactor allows for an enzyme-resin complex to be contained within that reactor. In some embodiments, a reactor contains one enzyme-resin complex. In other embodiments, a reactor contains multiple enzyme-resin complexes.

[0103] In some embodiments, each individual reactor contains a fixed amount of enzyme-resin complex. In other embodiments, a fixed amount of enzyme-resin complex may allow an adjustable concentration of enzyme to be achieved in each individual reactor. For example, if more enzyme is desired, more enzyme-resin complex may be loaded into the individual reactor, and vice versa.

[0104] In some embodiments, each individual reactor provides the necessary reactor conditions to ensure that the enzyme-resin complex remains active to convert the substrate to a product. In some embodiments, the reactor conditions are precisely controlled to ensure the longevity of the enzyme. Non-limiting examples of conditions that a reactor may control include, but are not limited to, temperature or pH or oxygen levels.

[0105] In some embodiments, each individual reactor has customizable temperature control to ensure that each enzyme obtains its optimal reaction temperature. In some embodiments, each individual reactor has the ability to control reactor temperature to within ±0.1° C. by using a graphical user interface (GUI). There are no manual readjustments to the system, only algorithmic feedback and control after the user's temperature set point to the computer.

[0106] Although not wishing to limit the present invention to any theory or mechanism, it is believed that temperature is very important for any enzyme in the reaction. For example, Aldo (which converts glycerol to glyceric acid) requires an optimum temperature of 37°C (19.6 mM, 98% yield), while the next enzyme in the pathway (DHAD) requires 45°C to convert glyceric acid to pyruvate (6.4 mM, 32% yield, Table 1). If these reaction temperatures are not respected, the reaction yield and reaction rate will suffer. Figure 2 shows that 45°C is optimal for DHAD, and a significant shift from the optimum conversion is observed when the reaction temperature is reduced to 32°C or increased to 55°C.

[0107] In some embodiments, each individual reactor has reactor fluid pH adjustment and control to provide unique pH requirements for each enzyme (Table 1). In some embodiments, pH sensors and automated feedback loops ensure that the pH of the reaction medium can be altered between individual sequential reactors. In some embodiments, to alter the pH of the reaction medium, a specific volume of a specific pH solution is injected into the reactor system to alter and maintain a specific pH. This allows each reactor to have an optimal pH to avoid protein precipitation, loss of reactivity, or other degradation effects.

[0108] In some embodiments, the reactor system has the ability to add gas to individual reactors for enzymes that require gas. Non-limiting examples can include oxygen or nitrogen. As described herein, it has been demonstrated that adding oxygen and nitrogen to reactors ensures effective enzyme conversion. This is crucial, because without oxygen addition, immobilized ALDO provided 0% product. However, after introducing oxygen into the reactor, a 98% yield was obtained (19.6 mM).

[0109] In some embodiments, the reactor system can reduce the oxygen present in the reaction medium, if necessary. In some embodiments, oxygen is removed through the use of sonication and vacuum. In some embodiments, oxygen is removed through the addition of nitrogen.

[0110] As described herein, the DHAD enzyme evaluated required oxygen removal to increase reaction efficiency. A module was introduced for this system that removed the oxygen present in the fluid to 4.07 ppm through the use of ultrasonication and vacuum. This device allows pO2 removal from 7.50 ppm to 4.07 ppm in 10 minutes at room temperature. In addition to this module, nitrogen gas can be introduced to further reduce the amount of oxygen in the medium.

[0111] In some embodiments, each individual reactor allows the starting material (substrate solution) to be pumped through it using standard laboratory pumps. Substrate solution is injected into the reactor and the solution travels through the reactor, interacting with the enzyme to cause a chemical reaction. Once the chemical reaction is complete, the reacted fluid travels out of the reactor to a collection flask.

[0112] In some embodiments, the reactors may operate in a continuous mode, where the pump injecting fluid into an individual reactor is constantly pumping fluid. In other embodiments, the reactors may operate in a semi-continuous mode, where the pump injects fluid into an individual reactor for a specific amount of time and then stops to hold the fluid in that reactor for a specific amount of time. In some embodiments, after a set amount of time, the pump resumes pushing reacted fluid out of the reactor system. While not wishing to limit the invention to any theory or mechanism, it is believed that the semi-continuous mode allows longer enzyme reaction times to be achieved.

[0113] In some embodiments, the reactor system may allow additional chemical solutions to be added to the continuous system between the individual reactors. In some embodiments, computer-controlled pumps allow chemical solutions to be injected into the continuous manufacturing system when instructed. Non-limiting examples of chemical solutions may include, but are not limited to, buffers, cofactors, and chemical reagents. Examples of buffers may include, but are not limited to, 50 mM Tris (pH 12.0) for adjusting the pH of the process flow to pH 7.7 to pH 8.5, 100 mM Tris K2HPO4:KH2PO4 (1:1) (pH 6.33) for adjusting the pH of the process flow to pH 7.7 to pH 6.5, and 50 mM Tris (pH 12.85) for adjusting the pH of the process flow to pH 6.47 to pH 8.0.

[0114] In some embodiments, the platform is equipped with a wide range of sensors that allow for full system automation. These sensors include, but are not limited to, temperature sensors / thermistors, pressure sensors, flow meters, pH sensors / probes, dissolved oxygen sensors / probes, and spectrometers. Deviations from programmed optimum values ​​(temperature, flow, pH, pressure, etc.) in the GUI or code trigger corresponding changes in system adjustments and hardware states through algorithmic feedback loops to allow system conditions to reach the required state.

[0115] In some embodiments, the individual reactors are scalable. In some embodiments, the length, diameter, or number of individual reactors can be increased to achieve higher throughput. There is no theoretical limit to the size or number of these reactors. In some embodiments, the individual reactors can be of various sizes, shapes, and configurations.

[0116] In some embodiments, the cylindrical tubing of the individual reactors can be virtually any diameter tubing and any length that the user desires to allow for more or less resin / enzyme, or shorter / longer reaction times. In other embodiments, the fittings (e.g., stainless steel fittings) on the ends of the cylindrical tubing of the individual reactors can also be customized to allow for different sizes of input / output tubing to be used. In some embodiments, the material of the cylindrical tubing from which the individual reactors are manufactured can be altered to allow for more optimal temperature transfer or other parameters when allowing reactions to come into contact with that material.

[0117] In some embodiments, the reactor system includes a customizable user interface (commonly known as a GUI (Graphical User Interface)) that allows a user to control the reactor with the click of a mouse. EXAMPLES

[0118] (Example)

[0119] 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.

[0120] (1.1 Design of Individual Reactors): The individual reactors are fabricated from 304 stainless steel cylindrical tubing. The individual reactor housings are fabricated from 6061 aluminum. The individual reactor housings are used as a heat transfer medium to change the temperature of the reactors held inside by attaching a thermoelectric cooler (TEC) element to the housing (Figure 3). The temperature of the reactors can also be changed through attaching a flexible heating element to the outside of the reactor. The individual reactors can be heated and cooled from 12°C to 55°C. In comparison to conventional batch reactors, these reactors can reach the desired temperature in 11 minutes when tested at a set temperature of 45°C (Figure 4). Each individual reactor is fitted with Swagelok fittings at each end of the 304 stainless steel cylindrical tubing to seal the tubing and create the reactor. Additionally, Swagelok fittings allow inlet and outlet tubing to be attached to the reactors to introduce and remove fluids from the individual reactors. Notably, the volume, shape, size and material of the reactor can be varied; different materials such as plastic and different volumes including 1 g to 330 g have been tested.

[0121] To ensure that the individual reactors fit the pumping system, the reactor pressure was tested. The internal pressure of the individual reactors was monitored in real time over a 24-hour period by pumping a buffer solution (50 mM Tris, 20 mM glyceric acid, pH 7.7) through the first two reactors in the path. The buffer solution was pumped through the individual reactors containing the resin at a flow rate of 10 mL / min at a temperature of 21° C. (i.e., room temperature). After the first reactor, there is a junction where a buffer solution (50 mM Tris, pH 12.0) was pumped into the process flow along with the Tris solution to change its pH level. To monitor the pressure of the individual reactors in real time, a python program was written to collect the pressure level from both process pumps once per second. In this test system, the pumps are directly connected to the reactors through PFA tubing, so the pressure calculated by the pumps is a direct sampling of the internal pressure of the individual reactors.

[0122] The calculated pressures inside the individual reactors ranged from 0 psig to 70 psig. The average internal pressure of the first reactor in the system was <0.01 psig when tested over a 24 hour time frame (FIGS. 5A-5B). The average internal pressure of the second reactor in the system was <0.1 psig when tested over the same 24 hour time frame. This low pressure is necessary for reactor scale-up and also indicates that the system does not induce resin expansion through fluid entrapment. Finally, the pressure monitoring used in this system also allows the user to set pressure alert limits in the GUI for safety and control aspects. This feeds into the automation control of the continuous manufacturing system as a whole.

[0123] The amount of enzyme that a standard individual reactor can hold was also calculated. The individual reactor tested here can hold 57 g of enzyme-resin complex (<5.5 g of isolated enzyme). This means that a single reactor with a size of 14 inches long (L) x 0.652 inches inner diameter (ID) (total volume 79 mL, FIG. 6) can hold as much enzyme as a 1000 L or more fermenter when the enzyme expression level in the batch reactor is 5.5 mg / L or higher. This is just one example, and the comparison will change if the enzyme is expressed at different levels. However, this dramatic improvement means that the 1 L individual reactor described herein could hold the same amount of enzyme as a 12,600 L batch fermenter when the enzyme expression level in the batch reactor is 5.5 mg / L.

[0124] (1.2 Individual Reactor Temperature Control): Individual reactor temperature control is achieved through a proportional-integral-derivative (PID) algorithm and supporting code, such as contained in an Arduino Mega 2560 or STM32L476RG microcontroller. The temperature sensors used are NTC thermistors, which are activated by a change in electrical resistance when their temperature changes. This change in resistance is relayed to the microcontroller as a voltage through the use of a voltage divider circuit (Figure 7A-7B). The thermistor is placed in a recess in the reactor housing and held in place once the housing is tightly secured. The thermistor is in contact with both the reactor housing and the reactor itself. If the reactor is heated, such as with a flexible silicone heating element, then no reactor housing is used. In this case, the thermistor is inserted between the outside of the reactor and the heating element. The temperature difference between where the thermistor is placed and the inside of the reactor is measured, and this difference is accounted for in the software. Inside the PID algorithm software, the thermistor resistance is converted to an accurate temperature reading by converting the incoming voltage reading back to electrical resistance, which is then further calculated to degrees Kelvin by using a simplified version of the Steinhart-Hart formula (Figure 8). This value is then converted from Kelvin to Celsius. The temperature of the individual reactors is read once per second. The temperature reading is now fed into a temperature-responsive PID loop, which modifies the pulse-width modulation (PWM) duty cycle of the electrical output powering the heating element to change the temperature of the individual reactor to an accurate level. The circuitry can be seen in Figures 9A-9B. The PID software framework is licensed under the MIT Permissive License. The basics of this software were edited and extended to control the system described herein. This PID loop was fine-tuned to minimize the amount of temperature overshoot and equilibration time (Figure 4). The TECs that cause the temperature changes are attached to the individual reactor housings using thermally conductive adhesive.The adhesive keeps the TEC securely attached to the housing while allowing heat transfer to remain uninterrupted (Figure 3). Flexible silicone heating elements and the like have an adhesive on one side to allow for attachment to a variety of objects.

[0125] The temperature control system could also cool an individual reactor to 12°C (Figures 10A-10B). No change in performance was observed. Furthermore, the amount of power required to cool this temperature was 0.027 kW. This has a tremendous advantage compared to larger batch reactors that require large amounts of electricity to cool. Cooling can be achieved by flipping the TEC over so that its other side is the "cold" side. Alternatively, if there is an application where the reactor needs to be both heated and cooled, an alternate circuit can be implemented that allows switching between heating / cooling by inserting separate wires into the corresponding microcontroller digital pulse width modulation (PWM) pins in place of the wires used to activate the heating (Figure 9B). If cooling is required, it is necessary to attach a heat sink device to the Peltier element. This element works based on the temperature difference between each side of the plate, so if the cold side is being used to cool the reactor, the other side will be hot. Without a heat sink, the hot side would overwhelm the cold side until it reached an equilibrium temperature higher than the ambient temperature. The heat sink would keep the "hot" side cooler, which would allow the cold side to maintain a cooler temperature. A thermal image of this happening can be seen in Figure 10B.

[0126] (1.3 Individual reactors have strict pH control): Between each reactor there are injection points where acidic or basic solutions can be continuously injected into the process flow to provide in-line pH change that matches the pH requirements of the next reactor. The exact flow rate and pH level of the injectate required was determined through experimentation. In addition, a pH sensor was implemented that can measure the pH of the flowing solution in real time to provide accurate pH monitoring. The pH sensor was connected in-line and continuously sends the pH value to the control system once per second. This provides the user with additional data about the accuracy of the pH adjustment and automatically modifies the flow rate of the pH injection to maintain an accurate pH output.

[0127] (1.4 Gases may be added to individual reactors): The system is equipped with additional injection points for gas injection into the fluid flow before the individual reactors. The required gas tanks are connected to this system via mass flow controllers. Here, the user can set the flow rate of gas entering the individual reactors in a continuous manner. These mass flow controllers are controlled via voltage signals. These signals can be adjusted both in an analog manner (manual adjustment of a dial) using a voltage divider circuit with a potentiometer, or digitally via sending a digital signal through a digital-to-analog converter (DAC). These gases (compressed air, oxygen, and nitrogen) have been tested so far. Compressed air was used for the Aldo enzyme, which requires additional oxygen to react properly. Compressed nitrogen can be used to reduce the oxygen concentration of the fluid. The gas tanks are connected to the mass flow controllers via tubing and stainless steel fittings. The mass flow controllers are connected to the individual reactors via tubing and Swagelok fittings. The mass flow controllers are programmed to allow a specific volume of gas per minute to pass through the mass flow controllers and go to the individual reactors. The exact ratio of gas to liquid was experimentally tested and optimized, which is approximately a 1:1 volume ratio.

[0128] (1.5 Module to reduce oxygen content in fluid entering separate reactor): In addition to adding nitrogen gas to reduce oxygen, the system uses a deoxygenation and / or degassing device to reduce the amount of oxygen in the process flow. The deoxygenation / degassing module is placed in line with the flow. The fluid exiting the device has oxygen removed down to 3.68 ppm. A reduction in dissolved oxygen from 7.75 ppm to 4.07 ppm (nearly a 50% reduction) was demonstrated (Figure 11).

[0129] 1.6 Injection of additional chemical streams into individual reactors: In addition to the injection of solutions to adjust pH, there are injection points for adding chemical streams to the process flow. These chemical streams are specific to the reactor they are associated with and modify the chemical reactions required to produce the intended product.

[0130] (1.7 System Control and Automation for Individual Reactors): The system includes various sensors to monitor and / or adapt reaction conditions when communicated by the user or automatically. Sensors include, but are not limited to, temperature sensors / thermistors, flow meters, pressure sensors, pH probes, dissolved oxygen probes, mass flow controllers, and spectrometers. Temperature sensors for each individual reactor provide data to a microcontroller which then automatically adjusts the power output to a heating element which changes the temperature of that reactor. Flow meters monitor the rate at which fluids in the system flow through the reactor and pass that data to the microcontroller. If the process flow slows to a rate that is considered suboptimal, the microcontroller sends commands to gradually increase or decrease the flow rate of the pump until an acceptable flow rate is achieved. pH probes monitor the pH of the volume passing through them and adjust the amount of acidic or basic solution injected into the process flow to achieve the desired pH level. A dissolved oxygen probe measures the amount of oxygen in solution, and a microcontroller may adjust the rate at which oxygen or other gases are added to the process flow via a mass flow controller. A pressure sensor measures the pressure within the process flow. A spectrometer measures the wavelength of light in the volume leaving the reactor, reading intensity across the spectrum 340.6 nm to 1010 nm and absorbance levels across the same light spectrum.

[0131] 1.8 Use of Parallel Reactors with Equal Flow: The system may use reactors in a parallel fashion. Two individual reactors may be connected in parallel to one pump. The two reactors achieved equal flow through each by implementing control software with associated hardware. The associated hardware in this configuration is a flow meter and a control valve. The control valve is placed upstream from the reactor and the flow meter is placed in series downstream from the reactor. The software is connected to the flow meter and calculates the flow rate of the output of each reactor connected in parallel. The software maintains a total of the amount flowed through each reactor. The software then makes a decision based on the flow rate and amount flowed to temporarily stop or continue the flow in the individual reactors via solenoid valves to maintain a uniform total amount of solution flowing through each (Figure 27).

[0132] 1.9 Collection of System Output Color and Absorption: The system includes a spectrometer where the wavelength and absorption of the system output may be measured. A flow cell is connected directly to the system output through which the output flows. A light source is connected to the flow cell via a fiber optic cable. The flow cell is connected to the spectrometer via a fiber optic cable. The spectrometer readings may be collected in two ways. One way is by using a commercially available software package included with the spectrometer in conjunction with the spectrometer unit to read the wavelength of the output color or the absorption of the output in real time (Figure 28). The second way is by software written to communicate with the spectrometer to collect data via RS232. The software may collect the wavelength intensity of the sample across the spectrum 340.6 nm to 1010 nm. The software also performs the calculations to convert the sample intensity levels into absorbance values. The software has the ability to plot its findings on a graph and / or save its findings to a log file or spreadsheet document, etc. (Figure 29). The software was written in Python. Data from the color and absorbance of the samples can be further analyzed to make inferences about output concentration based on the color / spectral data.

[0133] (1.10 Reactors can be of various sizes, shapes, and configurations): The individual reactors can be of various diameters and lengths to allow for different residence times for the fluids flowing through the reactor. So far, three different sizes of reactors have been tried: 1. The first size reactor is 13.5 inches long with a 0.25 inch outer diameter (OD) and a 0.180 inch inner diameter (ID) giving a volume of 5.63 mL (Table 2). 2. The second size reactor is 14.5 inches long with 0.75 inch OD and 0.652 inch ID, giving a volume of 79.33 mL (Table 2). 3. The third size reactor is 5.5 inches long with 0.25 inch OD and 0.218 inch ID, giving a volume of 1.64 mL (Table 2).

[0134] [Table 2]

[0135] Two different sizes of reactor housings were made, one for a ¼ inch OD reactor and one for a ¾ inch OD reactor. Full dimensions can be seen in the views in Figures 12A-12D. To accommodate the shorter length of the third reactor, the reactor housing for the ¼ inch OD reactor was cut to length. The reactor sizes and their calculated residence times can be seen in Table 2.

[0136] (1.11 The reactor system includes a user interface for controlling the reactors): The system includes an optional user interface that allows the user to specify what temperature each reactor should be programmed to, start and stop the pumps in the system, manually check the pressure on each pump, and set upper pressure limits for the pumps (Figures 9A-B). The GUI was written in Python using the Kivy framework.

[0137] (1.12 The system provides a graphical dashboard for monitoring variables for each reactor in real time): In addition to the controls provided by the user interface, there is a monitoring dashboard that displays the temperature of each reactor, the flow rate provided by the flow meter, the pressure observed by the pump, and the PWM duty cycle applied to each Peltier element. Grafana is used to display the data and InfluxDB is used in conjunction with NodeRED to store the data in a database. The environment is powered and hosted by a raspberry pi. The data flow can be seen in Figure 13.

[0138] (1.13 Individual reactors can be linked in a sequence to provide a multi-stage enzyme reactor): Reactors can be connected in series via ¼ inch OD tubing to allow for a multi-stage enzyme reaction. The pathway requires 13 different enzymes, which are transferred to 13 different reactors. These reactors are connected in series with injection points for pH control and gas addition.

[0139] (Example 2.0: Experimental Example).

[0140] 2.1 Production of glyceric acid from glycerol using immobilized alditol oxidase (Aldo) (FIG. 14): A 13.5 inch long, 0.25 inch outer diameter and 0.180 inch inner diameter reactor containing immobilized Aldo enzyme (240 mg enzyme on 4.00 g resin) was heated to 37° C. and equilibration buffer (50 mM tris, pH 8.5) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (40 mL, 50 mM Tris, pH 8.5, 20 mM glycerol) was flowed through the reactor at a flow rate of 20 μL / min. Prior to entering the reactor, the substrate solution was mixed with an equal flow rate of compressed air (0.02 standard cubic centimeters per minute (sccm)) resulting in a total flow rate of 40 μL / min (18 minute residence time) through the reactor. After the solution passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through the separate reactors. The reaction was allowed to proceed for 72 hours with sampling performed every 24 hours. For sampling, 100 μl of the reaction fluid was tested on a high performance liquid chromatography (HPLC) system to determine the amount of glycerol and glyceric acid. The HPLC method was as follows: an Agilent 1200 HPLC was equipped with a 30 cm Aminex HPX-87H column and 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 mobile phase consisting of 100% sulfuric acid (10 mM) was used. The sample run time was 45 minutes in total with glyceric acid eluting at 17.2 minutes and glycerol eluting at 21.0 minutes. For detection, a RID detector (Agilent) was used after a 2 hour equilibration period to generate a stable baseline. Upon analysis, the following data was obtained: 24 h reaction time = 21% glyceric acid and 79% glycerol, 48 h reaction time = 76% glyceric acid and 24% glycerol, 72 h = 98% glyceric acid, 3% glycerol. At 72 h, the yield of the reaction converting glycerol to glyceric acid was 99% (19.6 mM, 2.06 g / L).Of note, the same reaction was run without the addition of compressed air and 0% conversion was observed. This reaction was scaled to a reactor having dimensions of 14 inches long by 0.75 inches outer diameter and 0.652 inches inner diameter (Example 2.13 below). 98% conversion to product (19.6 mM glyceric acid) was observed in 66 hours under the same conditions as above.

[0141] (2.2 Production of pyruvate from glycerate using immobilized dihydroxyacid dehydratase (DHAD) (Figure 15): A 13.5 inch long, 0.25 inch OD and 0.180 inch ID reactor containing immobilized DHAD enzyme (360 mg enzyme on 3.70 g resin) was heated to 55°C and equilibration buffer (250 mM HEPES, pH 7.4, 2.5 mM MgCl2·6H2O) was passed through the reactor for 1 hour to equilibrate. After allowing to settle for 1 hour, substrate solution (40 mL, 250 mM HEPES, pH 7.4, 2.5 mM MgCl2·6H2O, 20 mM glycerate) was run through the reactor at a flow rate of 10 μL / min (72 min residence time, Figure 15). After this solution had passed through the reactor, it was collected in the same beaker as the starting solution to allow this solution to be recycled through a separate reactor. The reaction was allowed to proceed for 72 hours, with sampling every 24 hours. For sampling, 100 μl of the reaction fluid was tested on an HPLC system to check the amount of glyceric acid and pyruvate. The HPLC method was the same as above, with pyruvate eluting at 15.8 minutes and glyceric acid eluting at 17.2 minutes. Analysis gave the following data; 24 hour reactivity = 17% pyruvate and 48% glyceric acid, 48 hour reactivity = 23% pyruvate and 47% glyceric acid. At 48 hours, the yield of the reaction converting glyceric acid to pyruvate was 23% (4.51 mM, 0.4 g / L). The reaction was scaled to a reactor with dimensions of 14 inches long x 0.75 inches outer diameter and 0.652 inches inner diameter (Example 2.13 below). 20% conversion to product (2 mM pyruvate) was observed at 16 hours under the same conditions as above.

[0142] (2.3 Production of acetyl phosphate from pyruvate using immobilized pyruvate oxidase (PyOx) (Figure 16): A 13.5 inch long, 0.25 inch OD and 0.180 inch ID reactor containing immobilized PyOX enzyme (140 mg on 3.5 g resin) was heated to 37°C and equilibration buffer (10 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4, pH 6.5, 5.0 mM MgCl2, and 100 mM NaCl) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (10 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4, pH 6.5, 5.0 mM MgCl2, 100 mM NaCl) was passed through the reactor for 1 hour to equilibrate. A mixture of 5 mM NaCl, 5 mM pyruvate, 5 mM thiamine pyrophosphate (TPP) was flowed through the reactor at a flow rate of 10 μL / min (72 min residence time, FIG. 16). After the solution passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through the separate reactors. The reaction was allowed to proceed for 16 hours. The HPLC method was as follows: Agilent 1200 HPLC with 30 cm Aminex An HPX-87H column and Microguard Cation H refill cartridge were installed. 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 an isocratic mobile phase consisting of 100% sulfuric acid (10 mM). The run time was 25 min total with acetyl phosphate eluting at 23.6 min and pyruvate eluting at 16.0 min. A refractive index detector (Agilent) was used for analysis after a 2-h equilibration period to generate a stable baseline. Over the 16-h reaction, the reaction yield of pyruvate converted to acetyl phosphate was 10% (0.5 mM or 92 mg / L).

[0143] (2.4 Production of Acetyl-Coenzyme A from Acetyl Phosphate Using Immobilized Acetyl Phosphate Transferase (PTA) (FIG. 17): A 13.5 inch long, 0.25 inch OD and 0.180 inch ID reactor containing immobilized PTA enzyme (112 mg enzyme on 3.50 g resin) was heated to 55° C. and equilibration buffer (10 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4, pH 8.0, 5.0 mM MgCl2, 100 mM NaCl) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (10 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4, pH 8.0, 5.0 mM MgCl2, 100 mM NaCl, 3.2 mM acetyl phosphate, and 3.2 mM acetyl coenzyme A) was added. Acetyl phosphate and acetyl-CoA) were run through the reactor at a flow rate of 10 μL / min (72 min residence time, FIG. 17). After the solution passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through a separate reactor. The reaction was allowed to proceed for 8 hours. For sampling, the reaction fluid was tested on an HPLC system to check the amount of acetyl phosphate and acetyl-CoA. The HPLC method was as follows: an Agilent 1200 HPLC was fitted 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 used a 5 μl sample injection volume and the mobile phase consisted of 75 mM CH3COONa (sodium acetate) and 100 mM The column consisted of a mixture of NaH2PO4 (sodium dihydrogen phosphate) and acetonitrile (volume ratio 94:6). 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 target molecules at 259 nm. After 8 h of reaction, the reaction yield for the conversion of acetyl phosphate to acetyl-CoA was 12% (0.384 mM or 337 mg / L).

[0144] (2.5 Production of acetoacetyl-Coenzyme A from acetyl-Coenzyme A using immobilized acetyl-CoA acetyltransferase (PhaA) (Figure 18): A 5.5 inch long, 0.25 inch OD and 0.218 inch ID reactor containing immobilized PhaA enzyme (41 mg on 1.5 g resin) was heated to 32°C and equilibration buffer (10 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4, pH 8.0, 5.0 mM MgCl2, 100 mM NaCl) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (10 mM Tris, 50 mM KH2PO4, 50 mM K2HPO4, pH 8.0, 5.0 mM MgCl2, 100 mM NaCl, 2.5 mM NaCl) was added to the reactor. AcCoA) was run through the reactor at a flow rate of 10 μL / min (43 min residence time, FIG. 18). After the solution passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through a separate reactor. The reaction was allowed to proceed for 8 hours. For sampling, the reaction fluid was tested on an HPLC system to determine the amount of AcCoA and CoA. The HPLC method was as follows: Agilent 1200 HPLC 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 sample injection volume of 5 μl, with 75 mM CH3COONa and 100 mM CoA mixed in a 94:6 ratio with acetonitrile (ACN). An isocratic gradient consisting of NaH2PO4 was used as the mobile phase. 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 to detect the molecules of interest at 259 nm. Over an 8-h reaction, the reaction yield for the conversion of acetyl phosphate to acetoacetyl-coenzyme A was 21% (0.94 mM or 930 mg / L).

[0145] (2.6 Production of β-hydroxyβ-methylglutaryl-coenzyme A (HMG-CoA) from acetoacetyl-coenzyme A using immobilized HMG-CoA synthase A110G (HMGS A110G) (FIG. 19): A 5.5 inch long, 0.25 inch OD and 0.218 inch ID reactor containing immobilized HMGS A110G enzyme (34.5 mg on 1.5 g resin) was heated to 32° C. and equilibration buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 7.0) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 7.0, 5 mM acetoacetyl-coenzyme A) was added. Acetoacetyl-CoA) was flowed through the reactor at a flow rate of 10 μL / min (43 min residence time, FIG. 19). After the solution passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through a separate reactor. The reaction was allowed to proceed for 2 hours. After 2 hours, the reaction solution was incubated with 20.7 μM HMGR and 5 mM nicotinamide adenine dinucleotide phosphate (NADPH). HMGR converts acetoacetyl-CoA to HMG-CoA using the cofactor NADPH. The activity of HMGR was measured by monitoring the loss of NADPH at 340 nm using a spectrophotometer. Over the 2 hour reaction, the reaction yield of converting acetoacetyl-CoA to HMG-CoA was 13% (0.65 mM or 621 mg / L).

[0146] (2.7 Production of mevalonate from HMG-CoA using immobilized HMG-CoA reductase (HMGR) (Figure 20): A 5.5 inch long, 0.25 inch OD and 0.218 inch ID reactor containing immobilized HMGS A110G enzyme (31 mg on 1.5 g resin) was heated to 37°C and equilibration buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 7.0) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 7.0, 5 mM NADPH) was flowed through the reactor at a flow rate of 10 μL / min (residence time 43 min, FIG. 20). After the solution passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through the separate reactors. HMGR activity was measured by monitoring the loss of NADPH at 340 nm using a spectrophotometer. Over a 2-hour reaction, the reaction yield for converting HMG-COA to mevalonate was 98.3% (2.45 mM or 378 mg / L).

[0147] (2.8 Production of mevalonate-5-phosphate from mevalonate using immobilized mercaptokinase (MVK) (Figure 21): A 5.5 inch long, 0.25 inch outer diameter and 0.218 inch inner diameter reactor containing immobilized MVK enzyme (60 mg on 1.5 g resin) was heated to 37°C and equilibration buffer (50 mM Tris, 5 mM MgCl, pH 8) was passed through the reactor for 1 hour. After settling for 1 hour, substrate solution (50 mM Tris, 5 mM MgCl, pH 8) was passed through the reactor for 1 hour. MgCl2, pH 8, 4 mM adenosine triphosphate (ATP), 4 mM mevalonate) was run through the reactor at a flow rate of 10 μL / min (43 min residence time, FIG. 21). After the solution passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through the separate reactors. The reaction was allowed to proceed for 16 hours. For sampling, the reaction fluid was tested on an HPLC system to determine the amount of ATP and ADP (adenosine diphosphate) present in the final reaction solution. The HPLC method was as follows: Agilent 1200 HPLC with a HYPERSIL ODS COLUMN equipped with a BetaSil C18 20 mm×2.1 mm guard column. A 150 mm x 3 mm column was fitted. 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 (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. Over a 16-h reaction, the reaction yield for the conversion of mevalonate to mevalonate-5-phosphate was 0.3 mM (68 mg / L).

[0148] (2.9 Production of mevalonate-5-diphosphate from mevalonate-5-phosphate using immobilized phosphomevalonate kinase (PMVK) (Figure 22): A 5.5 inch long, 0.25 inch outer diameter and 0.218 inch inner diameter reactor containing immobilized PMVK enzyme (54 mg on 1.5 g resin) was heated to 37°C and equilibration buffer (50 mM Tris, 5 mM MgCl2, pH 8) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (50 mM Tris, 5 mM MgCl2, pH 8, 4 mM ATP, 4 mM ATP) was added. Mevalonate-5-phosphate) was run through the reactor at a flow rate of 10 μL / min (43 min residence time, FIG. 22). After the solution had passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through a separate reactor. The reaction was allowed to proceed for 32 hours. For sampling, the reaction fluid was tested on an HPLC system to determine the amount of ATP and ADP present in the reaction mixture. The HPLC method was as follows: an 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 used a 5 μl sample injection volume and 100 mM KH2PO4, 8 mM TBAHS, pH 6.0, 20% methanol. The mobile phase consisted of an isocratic mixture of 1000 mM tetrahydrofuran (TFA) and 1000 mM tetrahydrofuran (ADP) (v / v). The run time was 10 min in total, with ATP eluting at 5.7 min and ADP eluting at 4.6 min. Target molecules were detected at 254 nm using a diode array detector (Agilent). Over the 32-h reaction, the reaction converted mevalonate to mevalonate-5-pyrophosphate (2.1 mM, 697 mg / L).

[0149] (2.10 Production of isopentenyl pyrophosphate from mevalonate-5-diphosphate using immobilized diphosphomevalonate kinase (MDC) (Figure 23): A 5.5 inch long, 0.25 inch outer diameter and 0.218 inch inner diameter reactor containing immobilized MDC enzyme (30 mg on 1.5 g resin) was heated to 37°C and equilibration buffer (50 mM Tris, 5 mM MgCl2, pH 8.0) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (50 mM Tris, 5 mM MgCl2, pH 8, 4 mM ATP, 4 mM ATP) was added. Mevalonate-5-pyrophosphate) was run through the reactor at a flow rate of 10 μL / min (43 min residence time, FIG. 23). After the solution had passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to be recycled through the separate reactors. The reaction was allowed to proceed for 16 hours. For sampling, the reaction was checked on an HPLC system to check the amount of ATP and ADP. The HPLC method was as follows: Agilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150mm×3mm with a BetaSil C18 20mm×2.1mm 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, 100 mM KH2PO4, 8 mM An isocratic gradient consisting of TBAHS, pH 6.0, 20% methanol (vol / vol) was used as the mobile phase. 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 target molecules at 254 nm. Over the 16-h reaction, mevalonate-5-pyrophosphate was converted to isopentenyl pyrophosphate at 0.9 mM (237 mg / L).

[0150] (2.11) Geranyl pyrophosphate (GPP) production from dimethylallyl pyrophosphate using immobilized farnesyl-PP synthase (FPPS) and a reporter prenyltransferase for CBGA production (Figure 24): A 5.5 inch long, 0.25 inch OD and 0.218 inch ID reactor containing immobilized FPPS enzyme (68 mg on 1.5 g resin) was heated to 25°C and equilibration buffer (50 mM Tris pH 8.0, 5 mM MgCl2, 10 mM NaCl) was passed through the reactor for 1 hour to equilibrate. After settling for 1 hour, substrate solution (50 mM Tris, pH 8, 5 mM MgCl2, 10 mM NaCl) was added. NaCl, 3.5 mM isopentenyl pyrophosphate, 3.5 mM dimethylallyl pyrophosphate) were flowed through the reactor at a flow rate of 10 μL / min with intermittent stops of 50 seconds after each 10 seconds of pumping to meet a 4 hour residence time (Figure 24). Unlike the other examples above, the reaction solution was not recycled in this experiment. The reaction solution collected after the completion of the designated time was incubated with 3.5 mM olivetolic acid (OA) and 120 μM prenyltransferase (NphB) at 25°C for 2 hours. 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 (cannabigerolic acid). This conjugation reaction was capable of converting 25% of the starting material to the product. The HPLC method was as follows: Agilent 1200 HPLC was equipped with a 250 mm x 4.6 mm 5 μm liChrospher 1000 HPLC column with a guard column. An RP8 column was attached. 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). Analysis of the coupled reaction showed that the reaction yielded 0.9 mM or 285 mg / L of the final product CBGA.

[0151] (2.12 Removal of oxygen from the reaction stream before each individual reactor: In some cases, the partial pressure of gaseous oxygen in the reaction mixture must be reduced to avoid enzyme deactivation. Therefore, before pumping the reaction solution through the reactor, the solution must be pumped through a deoxygenation and / or degassing device to reduce the oxygen partial pressure in the reaction solution prior to the next reaction. Coupling a deoxygenation and / or degassing device to the system allowed removal of up to 3.68 ppm oxygen, a reduction of almost 50% (Figure 17). For testing, deionized water was passed through the deoxygenation / degassing device at a flow rate of 0.25 mL / min. This process takes approximately 20 min to complete. In addition to the deoxygenation / degassing components, nitrogen gas can also be introduced into the solution to further remove oxygen. Nitrogen gas input, controlled by a mass flow controller, is added to the solution before pumping through the reactor (Figures 25A-25B).

[0152] (2.13 Multi-step enzymatic reaction for the conversion of glycerol to pyruvate (Figure 26): A 14 inch long, 0.75 inch od and 0.652 inch id reactor containing immobilized Aldo enzyme (2.7 g enzyme on 56 g resin) was heated to 37°C and equilibrated with equilibration buffer (50 mM tris, pH 8.5) for 6 hours by pumping the equilibration buffer through the reactor. After this time, substrate solution (40 mL, 50 mM tris, pH 8.5) was added. A solution of 10 mM Tris, pH 8.5, 20 mM glycerol) was flowed through the reactor at a flow rate of 10 μL / min. Before entering the reactor, the substrate solution was mixed with an equal flow rate of compressed air (0.01 sccm) resulting in a total flow rate of 20 μL / min (8 h residence time) through the reactor. After the solution has passed through the reactor, the fluid flow, now containing only glyceric acid (98% conversion from the previous reactor), is pH adjusted in-line. For this purpose, a pH buffer (50 mM Tris, pH 12.0) was added to the reaction flow using a T-mixer at a flow rate of 10 μL / min of pH buffer solution. The resulting fluid, now pH adjusted, is immediately held in a stirred tank reactor (CSTR) that is degassed by using bubbling nitrogen flow. Finally, another pump draws fluid from the CSTR into a second reactor, 14 inches long, 0.75 inch OD and 0.652 inch ID, containing immobilized DHAD enzyme (1.8 g enzyme on 56 g resin) heated at 45°C. The total flow rate of the reaction mixture through this reactor was 10 μL / min (16.25 hours residence time). The solution was collected in a glass beaker at the end of the second reactor. The reaction mixture was then analyzed every 24 hours. For sampling, 100 μl of the reaction fluid was tested in an HPLC system to determine the amount of glycerol, glyceric acid and pyruvate. The HPLC method was as follows: Agilent 1200 The HPLC was equipped with a 30 cm Aminex HPX-87H column and 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 mobile phase consisting of 100% sulfuric acid (10 mM) was used.The total sample run time was 45 min, with glyceric acid eluting at 17.2 min, glycerol eluting at 21.0 min, and pyruvate eluting at 15.8 min. For detection, a RID detector (Agilent) was used after a 2-h equilibration period to generate a stable baseline. It was found that the first reactor converted 98% of the glycerol to glyceric acid, and the second reactor converted the glyceric acid to a final concentration of 2 mM pyruvate. We found that the ALDO reactor was capable of achieving a conversion level of 98% in 3 days without loss of productivity, and this enzymatic conversion remained viable during 10 days of continuous processing.

[0153] (Embodiment) The following embodiments are intended to be illustrative only and not limiting in any way.

[0154] Embodiment 1: A cell-free manufacturing platform for chemical production, the platform comprising: one or more individual reactors, each of the one or more individual reactors comprising a cylindrical tube with a first end and a second end, both the first end of the cylindrical tube and the second end of the cylindrical tube comprising fittings, the cylindrical tube interior of the individual reactors comprising a resin and an enzyme; and a pumping system adapted to flow solutions through the one or more individual reactors, each of the one or more individual reactors comprising input tubing connected to the first end of the cylindrical tube and output tubing connected to the second end of the cylindrical tube to create a closed system.

[0155] Embodiment 2: The platform of embodiment 1, wherein the fitting is a stainless steel fitting.

[0156] Embodiment 3: The platform of embodiment 1, wherein the inside of the cylindrical tube of the individual reactor further comprises one or more sensors.

[0157] Embodiment 4: The platform of embodiment 3, wherein the one or more sensors include a temperature sensor, a pH sensor, a pressure sensor, a flow sensor, a dissolved oxygen (DO) sensor, a spectrometer, or a combination thereof.

[0158] Embodiment 5: A platform described in any one of embodiments 1 to 4, wherein each of the one or more individual reactors comprises an individual reactor housing.

[0159] Embodiment 6: The platform of embodiment 5, wherein the individual reactor housings surround and are secured to the individual reactors.

[0160] Embodiment 7: A platform described in any one of embodiments 1 to 6, further comprising a temperature changing element attached to the individual reactor housing or the individual reactor.

[0161] Embodiment 8: The platform of embodiment 7, wherein the temperature changing element is a thermoelectric cooler (TEC).

[0162] Embodiment 9: A platform described in any one of embodiments 1 to 8, further comprising a spectrometer mounted in series with one or more individual reactors.

[0163] Embodiment 10: A platform described in any one of embodiments 1 to 9, further comprising a degassing module adapted to remove gas from the solution.

[0164] Embodiment 11: The platform described in embodiment 10, wherein the degassing module is a deoxygenation module.

[0165] Embodiment 12: The platform of embodiment 11, wherein the deoxygenation module is adapted to remove oxygen from the solution.

[0166] Embodiment 13: The platform of any one of embodiments 1 to 12, further comprising a gas addition module adapted to introduce a gas into the solution.

[0167] Embodiment 14: A platform described in any one of embodiments 1 to 13, further comprising a pH module adapted to introduce an acid or a base into the solution.

[0168] Embodiment 15: A platform described in any one of embodiments 1 to 14, further comprising a graphical user interface (GUI) adapted to control the automation software and hardware.

[0169] Embodiment 16: A platform described in any one of embodiments 1 to 15, wherein the cell-free manufacturing platform is capable of automatically altering each of the conditions of the one or more reactors based on input from a sensor.

[0170] Embodiment 17: The platform according to any one of embodiments 1 to 16, wherein the enzyme comprises MBP-Aldo (Aldo), dihydroxy acid dehydratase (TvDHAD), 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), farnesyl-PP synthase S82F (FPPS), prenyltransferase (NphB), or a combination thereof.

[0171] Embodiment 18: A platform according to any one of embodiments 1 to 17, wherein an enzyme is immobilized.

[0172] Embodiment 19: A platform according to any one of embodiments 1 to 17, wherein the enzyme is not immobilized.

[0173] As used herein, the term "about" refers to the referenced number ±10%.

[0174] 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 cell-free manufacturing platform for the production of chemicals, the platform comprising: one or more individual reactors, each of the one or more individual reactors comprising a cylindrical tube having a first end and a second end, both the first end of the cylindrical tube and the second end of the cylindrical tube comprising fittings, an input tubing connected to the first end of the cylindrical tube and an output tubing connected to the second end of the cylindrical tube, the interior of the cylindrical tube of the individual reactor comprising: Resin; and enzyme; one or more individual reactors, a pumping system adapted to flow a solution through the one or more individual reactors; A platform that provides:

2. 10. The platform of claim 1, wherein the inside of the cylindrical tube of the individual reactor further comprises one or more sensors, and optionally, the fittings are stainless steel fittings.

3. The platform of claim 2 , wherein the one or more sensors include a temperature sensor, a pH sensor, a pressure sensor, a flow sensor, a dissolved oxygen (DO) sensor, a spectrometer, or a combination thereof.

4. 4. The platform of claim 1, wherein each of the one or more individual reactors comprises an individual reactor housing, optionally surrounding the individual reactor and secured to the individual reactor.

5. The platform of any one of claims 1 to 4, further comprising a temperature changing element attached to said individual reactor housing or said individual reactor, optionally said temperature changing element being a thermoelectric cooler (TEC).

6. The platform of any one of claims 1 to 5, further comprising a spectrometer mounted in series with the one or more individual reactors.

7. The platform of any one of claims 1 to 6, further comprising a degassing module adapted to remove gas from the solution.

8. 8. The platform of claim 7, wherein the degassing module is a deoxygenation module, optionally adapted to remove oxygen from the solution.

9. The platform of any one of claims 1 to 8, further comprising a gas addition module adapted to introduce a gas into the solution.

10. The platform of any one of claims 1 to 9, further comprising a pH module adapted to introduce an acid or a base into the solution.

11. The platform of any one of claims 1 to 10, further comprising a Graphical User Interface (GUI) adapted to control the automation software and hardware.

12. The platform of any one of claims 1 to 11, wherein the cell-free manufacturing platform is capable of automatically altering each of the conditions of the one or more reactors based on input from the sensors.

13. 13. The platform of any one of claims 1 to 12, wherein the enzymes comprise MBP-Aldo (Aldo), dihydroxyacid dehydratase (TvDHAD), 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), farnesyl-PP synthase S82F (FPPS), prenyltransferase (NphB), or a combination thereof.

14. The platform according to any one of claims 1 to 13, wherein the enzyme is immobilized.

15. The platform of any one of claims 1 to 13, wherein the enzyme is not immobilized.