US20220193655A1

US20220193655A1 - Versatile continuous manufacturing platform for cell-free chemical production

Info

Publication number: US20220193655A1
Application number: US17/554,426
Authority: US
Inventors: Joshua Britton; Matthew Hutcheson; Nicholas Brideau
Original assignee: Debut Biotechnology Inc
Current assignee: Debut Biotechnology Inc
Priority date: 2020-12-18
Filing date: 2021-12-17
Publication date: 2022-06-23
Also published as: EP4263843A1; EP4263792A1; WO2022133223A9; EP4263843A4; WO2022133223A1; US12006528B2; JP2024501517A; WO2022133213A1; JP2024500848A; US20220195469A1

Abstract

The present invention features a versatile continuous manufacturing platform for cell-free chemical production.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a non-provisional and claims benefit of U.S. Provisional Application No. 63/127,758 filed Dec. 18, 2020, and U.S. Provisional Application No. 63/127,836 filed Dec. 18, 2020, the specifications of which are incorporated herein in their entirety by reference

FIELD OF THE INVENTION

The present invention relates to devices and methods for the production of chemicals in a cell-free continuous manufacturing platform.

BACKGROUND OF THE INVENTION

A natural product is defined as being a molecule found in Nature created from a natural process. These broad classes of molecules find use as therapeutics, agrochemicals, or industrial starting materials. The natural processes that form these materials are typically multi-step enzyme pathways. Such enzyme pathways convert simple starting materials such as glycerol and glucose into complex materials through multi-step enzyme reactions. Currently, the majority of natural products are cultivated and extracted from plants, synthesized via complex chemical synthesis, or biomanufactured through cell-based factories also known as biofoundries. The present invention details the workings of a scalable continuous system to house immobilized enzymes that mimic how Nature creates diverse ranges of natural products.
Manufacturing natural products via cultivation, chemical synthesis, or the use of modified cells suffers from many problems that limit commercial viability of bio-based specialty chemical industrialization. First, cultivation requires vast amounts of land/energy/water, and the plant is only capable of producing the high value material in low amounts. Next, chemical synthesis requires extensive, elaborate, expensive, toxic, and inefficient multi-step chemical reactions to produce natural products that often are too complex to make in the laboratory. Finally, the cell suffers from product toxicity, carbon flux redirection, diffusion problems through cell walls, and toxic byproduct generation. To overcome these problems, the use of enzymes in the present system provides a viable alternative. As the same enzymes are used as the cell, but without the limitations of the cell, this research has been dubbed ‘cell-free’.
However, for cell-free manufacturing to be competitive new technology must be developed to allow each enzyme to experience its optimal reaction conditions to drive higher titers.

BRIEF SUMMARY OF THE INVENTION

It is an objective of the present invention to provide devices and methods that allow for the production of chemicals in a cell-free manner, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.
In a provisional patent application entitled “CELL-FREE PRODUCTION OF GERANYL PYROPHOSPHATE FROM GLYCEROL IN A CELL-FREE MANUFACTURING SYSTEM”, the inventors demonstrated glycerol conversion 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 into a reactor and mixed for five days to afford 49 mg/L of GPP (FIG. 1). However, using these different immobilized enzymes in a single batch operation confines the chemistry opportunity. Because only one reaction temperature, time, and pH could be accommodated in a single reactor, the reaction efficiency would be hindered. Additionally, to draw on the benefits of continuous manufacturing with this number of enzymes would require creating and optimizing a system from first principles.
The individual requirements of the 12 immobilized enzymes involved in the biochemical pathway to convert glycerol into geranyl pyrophosphate were explored first (Table 1); each enzyme has a unique set of conditions needed for optimal reactivity. Furthermore, deviation away from optimal conditions would lead to loss of reactivity through protein precipitation as well as product and substrate degradation; factors that would leave this process unscalable and commercially unviable. Thus, a system was needed that could provide these individual enzyme requirements, could overcome protein precipitation, could provide multiple temperatures, multiple pH's, multiple reaction times, and different reaction solution oxygenation levels at any given time. In effect, it was impossible to satisfy all the individual enzyme requirements in the current manufacturing equipment of today. To overcome this limitation, the 12 immobilized enzymes were placed into continuous manufacturing reactors that are strictly controlled by an array of external sensors and algorithmic feedback loops to create optimal reaction conditions and for these enzymes and others in the future.

TABLE 1

The optimal reaction conditions for each enzyme involved in
the biochemical pathways converting glycerol into GPP and
then through to CBGA through use of a reporter enzyme system
(NphB enzyme). In these reactions, between 10-100 mg of enzyme-
resin complex was used, and the reaction was monitored through
use of high-performance liquid chromatography (HPLC) coupled
with a refractive index detector (RID).

	Reaction Conditions
	Screened

	Temp		Time	Yield
Enzyme Name	(° C.)	pH	(h)	(%)

MBP-Aldo (Aldo)	37	9.0	21	98
Dihydroxy Acid Dehydratase	45	8.0	16	32
(TvDHAD)
Pyruvate Oxidase (PyOx)	37	6.5	16	96
Acetyl-phosphate transferase	32	8.0	8	60
(PTA)
Acetyl-CoA acetyltransferase	32	8.0	8	44
(PhaA)
HMG-CoA Synthase A110G	32	7.5	2	54
(HMGS)
HMG-CoA Reductase (HMGR)	37	7.0	2	55
Melvonate Kinase (MVK)	37	8.0	—	87
Phosphomevalonate Kinase	37	8.0	32	96
(PMVK)
Diphosphomevalonate Kinase	37	8.0	16	94
(MDC)
Isopentyl-PP Isomerase (IDI)	22	8.0	2	28
Farnesyl-PP synthase S82F	25	8.3	4	81
(FPPS)
Prenyl transferase (NphB)	50	8.0	6	16

The inventors have created a versatile continuous manufacturing platform that allows cell-free biomanufacturing to be scaled while providing the necessary conditions for the enzyme reactions to work. This patent application describes the manufacturing system and its use in an important biomanufacturing approach.
As described herein, a cell-free system and the key reactor drivers (see Table 1) are used in a cell-free chemical reaction (i.e., without the cell being present). The required enzymes are first created in vivo (typically through protein overexpression), isolated via chromatography, and then added into a bioreactor with a low-cost substrate. The enzymes transform the low-cost substrate into product via the exact same way that occurs in plants, animals, and bacteria but without the complexity of the organism. In this way, natural pathways can be harnessed to create natural molecules.
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 comprising 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, a cylindrical tube interior of the individual reactor comprises a resin and an enzyme. In some embodiments, each of the one or more individual reactors has an input tubing connected at the first end of the cylindrical tube and an output tubing connected at the second end of the cylindrical tube to create a closed system. In some embodiments, the cylindrical tube interior of the individual reactor further comprises one or more sensors.
One of the unique and inventive technical features of the present invention is the use of cell-free manufacturing. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for higher reaction concentrations, no cell-wall to battle for product and substrate diffusion, no battling the cell for carbon flux and byproduct formation, no cell death due to the formation of toxic compounds as there is no cell, and cell-free offers a platform solution to create a large number of compounds; cells have to be re-programmed and this invention simply allows a reactor column to be switched out for another one containing a different immobilized enzyme. None of the presently known prior references or work has the unique inventive technical feature of the present invention.
Furthermore, the prior references teach away from the present invention. For example, the current use of immobilized enzymes typically use a single reactor (batch or continuous) that only allows for one set of reactor conditions (time, pH, temperature, etc.) The present invention allows for the use of different reactor conditions between each individual reactor (or reactors in sequence) without intermediate isolation. Moreover, the addition of gases via a controlled module allows for enzymes requiring oxygen (or a lack of) to be used in a continuous reactor, which previous devices have not been able to do. Additionally, the deoxygenation module allows oxygen “phobic” and oxygen “philic” enzymes to be used in sequence, again, without intermediate purification.
Furthermore, the inventive technical features of the present invention contributed to a surprising result. For example, the device of the present invention can fit the same amount of enzymes in a 79 mL continuous reactor as compared to a 1,000 L traditional fermenter, allowing for a higher reaction concentration. When a 45 L reactor is achieved, this will be the equivalent of a 567,000 L traditional fermenter.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

FIG. 1 shows the biochemical pathway to create GPP from glycerol that has been previously shown. Here glycerol is transformed into GPP using 12 individual enzymes, as described herein.

FIG. 2 shows the optimal reaction temperature for DHAD. Here, 45° C. is the optimal temperature with significant drop off in yield observed when moving away from 45° C.

FIG. 3 shows a reactor contained within a housing with heating/cooling Peltier elements attached and supporting electronics circuits.

FIG. 4 shows the Grafana Data dashboard showing temperature and electrical output data of four reactors over time along with each reactor setpoint.

FIG. 5A shows the pressure over time (24 hrs) of the first reactor in the system, one sample per second. Mean=0.092 PSI.

FIG. 5B shows the pressure over time (24hrs) as seen by the pH adjustment pump between the first two reactors in the system. Mean=0.789 PSI.

FIG. 6 shows a screen capture of the graphical user interface (GUI).

FIG. 7A shows a standard voltage divider circuit. As R2 changes, V_outchanges.

FIG. 7B shows the standard equation to calculate output voltage (V_out) based on input voltage (V_in) and resistor values (R1, R2)

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

FIGS. 9A-9B show a Circuitry for Temperature Control, Standard (FIG. 9A) or if both heating and cooling is required without reorienting the Peltier elements (FIG. 9B).

FIG. 10A shows a thermal image of four reactors mounted and being heated/cooled.

FIG. 10B shows a thermal image of a reactor being cooled to 12° C.

FIG. 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, then was placed into treated deoxygenated/degassed water and was continuously stirred. Dissolved oxygen went from 7.75 ppm to 4.07 ppm, a reduction of almost 50%.

FIGS. 12A-12D show ¼″ and ¾″ Outer Diameter Reactor Housings—¼″ Outer Diameter Reactor Housing Tapped Side (FIG. 12A) or Screw Side (FIG. 12B) and a ¾″ Outer Diameter Reactor Housing—Tapped Side (FIG. 12C), or Screw Side (FIG. 12D).

FIG. 13 shows a data flow from capture to display.

FIG. 14 shows a reaction set-up for the reaction in example 2.1.

FIG. 15 shows a reaction set-up for the reaction in example 2.2.

FIG. 16 shows a reaction set-up for the reaction in example 2.3.

FIG. 17 shows a reaction set-up for the reaction in example 2.4.

FIG. 18 shows a reaction set-up for the reaction in example 2.5.

FIG. 19 shows a reaction set-up for the reaction in example 2.6.

FIG. 20 shows a reaction set-up for the reaction in example 2.7.

FIG. 21 shows a reaction set-up for the reaction in example 2.8.

FIG. 22 shows a reaction set-up for the reaction in example 2.9.

FIG. 23 shows a reaction set-up for the reaction in example 2.10.

FIG. 24 shows a reaction set-up for the reaction in example 2.11.

FIG. 25A shows the reaction set-up for removal of oxygen from a reaction. A pump pushes liquid through all four channels of a deoxygenation/degassing machine prior to entering a reactor.

FIG. 25B shows a diagram illustrating the addition of nitrogen gas prior to being pumped through the deoxygenation and/or degassing module and a reactor.

FIG. 26 shows a reaction set-up for the reaction in example 2.13.

FIG. 27 shows a testing set-up for achieving equal throughput of two parallel reactors connected to a single pump.

FIG. 28 shows commercial software to visualize the wavelength intensity across the spectrum for a volume flowing through the flow cell that is connected to the spectrometer.

FIG. 29 shows the output of software written to capture and/or display the absorbance data across the spectrum for a volume flowing through the flow cell that is connected to the spectrometer.

FIG. 30 shows several views of an individual reactor as described herein.

FIG. 31A and 31B show 2D diagram of a cell-free manufacturing platform as described herein. FIG. 31A shows a cell-free manufacturing platform described herein comprising a pH module. FIG. 31B shows a cell-free manufacturing platform described herein comprising a pressure sensor and a gas addition module or degassing module.

FIG. 32 shows one embodiment of a cell-free manufacturing platform as described herein. FIG. 32 shows a 3D cell-free manufacturing platform diagram comprising 4 individual reactors (i.e., four reactions) with a pH control module attached.

FIG. 33 shows a circuit schematic that can be used to heat/cool thermoelectric coolers or heat electric silicon heaters.

FIG. 34A and 34B show certain embodiments of the present invention as described herein. FIG. 34A shows a 2D diagram of the individual reactor of the cell-free manufacturing platform as described herein and FIG. 34B shows a 2D diagram of the cell-free manufacturing platform as described herein.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compounds, compositions, and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods or to specific compositions, as such 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.
Additionally, although embodiments of the disclosure have been described in detail, certain variations and modifications will be apparent to those skilled in the art, including embodiments that do not provide all the features and benefits described herein. 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 obvious modifications and equivalents thereof. Moreover, while a number of variations have been shown and described in varying detail, other modifications, which are within the scope of the present disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the present disclosure. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the present disclosure. Thus, it is intended that the scope of the present disclosure herein disclosed should not be limited by the particular disclosed embodiments described herein.
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 that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”
As used herein, a “reactor” or an “individual reactor” may refer to a continuous reactor containing an enzyme-resin complex. An individual reactor means one reactor with an entry and exit as defined by the fluid entering or leaving the reactor, respectfully. Additionally, two or more individual reactors may be linked together to form a reactor system.
As used herein, a “control system” may refer to software and/or hardware that is implemented to receive conditions parameters and respond by performing calculations and presenting data to the user and/or changing hardware state or configuration in response to the data to reach a required state.
As used herein, a “pumping system” may refer to an isocratic metering pump or syringe pump or equivalent thereof used to pump various fluids in the continuous manufacturing system.
As used herein, a “pumping buffer” may refer to a water-based solution containing salts that are used to perform enzyme-based reactions. Examples include, but are not limited to, protein buffer solution (PBS) or sodium acetate buffer.
The size of an individual reactor may vary in length, outside diameter and internal diameter depending on the desired throughput. An illustrative embodiment may be an individual reactor having a length between 1.5 inches and 14.5 inches or greater, an outside diameter (OD) between 0.125 inches and 0.75 inches or greater, or an internal diameter (ID) between 0.055 inches and 0.652 inches or greater, or some combination thereof. Other embodiments are contemplated based upon the desired throughput of the reactor. Other sizes of individual reactors may be used in accordance with the platforms described herein.
The reactor housing may be manufactured from any material having suitable properties, such as durability, strength, inertness toward reactor contents, etc. In some embodiments, the housing of the individual reactor may be made from 6061 aluminum or similar material.
The reactor may be manufactured from any material having suitable properties, such as durability, strength, inertness toward reactor contents, etc. In some embodiments, the individual reactor may made of 304 stainless steel cylindrical tubing or a similar material.
As used herein, “reactor conditions” may refer to conditions that ensure the enzyme-resin complex(es) remain active to convert substrate to product and/or conditions that ensure the substrate and the product are stable and/or conditions that are optimal for enzyme reactions. Non-limiting examples of conditions the reactor may control include, but are not limited to, temperature, pressure, throughput volume, solvent(s), pH, oxygen level, other gas level(s), or combinations thereof.
As used herein, a “graphical user interface (GUI)” may refer to a visual method of interacting with and/or controlling the continuous manufacturing system from a computer including but not limited to text boxes and buttons within a software application.
As used herein, “reaction medium” may refer to a solution that is flowed through the reactor that contains the chemicals required to perform the enzyme-controlled reaction. This typically includes, but is not limited to, a substrate (i.e., starting material), cofactor, gas, buffer salts, and other solvents. The chemical reaction takes place in the reaction medium.
As used herein, “equilibrium buffer” may refer to a buffer that has the same composition as the “reactor buffer” or “substrate solution,” but devoid (or nearly devoid) of the substrate.
As used herein, “substrate solution” may refer to a solution that is flowed through the reactor that contains the chemicals required to perform the enzyme-controlled reaction. This may include one or more of the following: one or more substrates (starting material), one or more cofactors, one or more gases, one or more buffer salts, and one or more solvent(s).
In some embodiments, the time for the conversion of the substrate to product may be varied to optimize the throughput and yield of the reaction. In some embodiments, the conversion of the substrate to product may proceed between 0.01 hours and 10 hours. In some embodiments, the conversion of the substrate to product may proceed between 0.01 hours and 100 hours, e.g., between about 10 hours and 100 hours. In some embodiments, the conversion of the substrate to product may proceed between about 0.01 hours and 1,000 hours, or between about 10 hours and 1,000 hours or between about 100 hours and 1,000 hours. In some embodiments, the conversion of the substrate to product may proceed more than 1,000 hours.
As used herein, “flow rate” may refer to the rate at which a fluid is passing through a reactor and can be measured by a flowmeter inserted prior and/or after an individual reactor.
In some embodiments, the flow rate of an individual reactor may be varied to optimize the throughput and yield of the reaction. In some embodiments, the flow rate may range from about 0.1 μL/min to 1000 μL/min, or about 0.1 mL/min to 100 mL/min, or about 0.1 μL/min to 10 μL/min, or about 0.1 μL/min to 1 μL/min, or about 1 μL/min to 1000 μL/min, or about 1 mL/min to 100 mL/min, or about 1 μL/min to 10 μL/min, or about 10 μL/min to 1000 μL/min, or about 10 mL/min to 100 mL/min, or about 100 μL/min to 1000 μL/min. In some embodiments, the flow rate may range from about 10 μL/min to 100 μL/min. In other embodiments, the flow rate 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 about 1 mL/min to 100 mL/min, or about 1 mL/min to 10 mL/min, or 10 mL/min to 1000 mL/min, or about 10 mL/min to 100 mL/min, or 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 may range from about 10 L/min to 100 L/min. In other embodiments the flow rate may range from about 1 L/min to 1000 L/min, or about 1 L/min to 100 L/min, or about 1 L/min to 10 L/min, or about 10 L/min to 1000 L/min, or about 10 L/min to 100 L/min, or about 100 L/min to 1000 L/min. In further embodiments, the flow rate may be greater than 100 L/min.
As used herein, “residence time” may refer to the length of time a unit of fluid is inside a reactor.
In other embodiments, the residence time may be varied to optimize the throughput and yield of a reaction. In some embodiments, the residence time may range from about 0.1 minutes to 1 minutes, about 0.1 minutes to 10 minutes, or about 0.1 minutes to 100 minutes, or about 1.0 minutes to 100 minutes. In other embodiments, the residence time may range from about 0.5 minutes to 10 minutes, or about 0.5 minutes to 100 minutes, or about 0.5 minutes to 1000 minutes. In some embodiments, the residence time may range from about 1 minute to 10 minutes, or about 1 minutes to 100 minutes, or about 1 minute to 1000 minutes. In other embodiments, the residence time may range from about 10 minutes to 100 minutes, or about 10 minutes to 1000 minutes. In some embodiments, the residence time may range from about 100 minutes to 1000 minutes. In some embodiments, the residence time may be greater than 100 minutes.
In some embodiments, the cell free manufacturing platform comprises a cylindrical tube interior of the individual reactor comprising a resin. In some embodiments, the amount of resin packed inside (i.e., in the cylindrical tube interior) an individual reactor may vary. In some embodiments, an individual reactor may have about 0.01 g and 1.0 g of resin packed inside. In some embodiments, an individual reactor may have about 0.1 g and 1.0 g of resin packed inside. In some embodiments, an individual reactor may have about 1.0 g and 10 g of resin packed inside. In some embodiments, an individual reactor may have about 10 g and 100 g of resin packed inside. In some embodiments, an individual reactor may have about 100 g and 1.0 kg of resin packed inside. In some embodiments, an individual reactor may have about 1.0 kg and 10 kg of resin packed inside. In some embodiments, an individual reactor may have about 10 kg and 100 kg of resin packed inside. In some embodiments, an individual reactor may have more than 100 kg of resin packed inside.
In some embodiments, the amount of resin packed inside an individual reactor may 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 packed inside an individual reactor may 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.
In some embodiments, the cell free manufacturing platform comprises a cylindrical tube interior of the individual reactor comprising an enzyme. In some embodiments, the amount of enzyme inside (i.e., in the cylindrical tube interior) an individual reactor may vary. In some embodiments, an individual reactor may have about 0.01 mg to 1000 mg of enzyme inside, or about 0.01 mg to 100 mg of enzyme inside, about 0.01 mg to 10 mg of enzyme inside, about 0.01 mg to 1 mg of enzyme inside, about 0.01 mg to 0.1 mg of enzyme inside. In other embodiments, individual reactor may have about 0.1 mg to 1000 mg of enzyme inside, or about 0.1 mg to 100 mg of enzyme inside, about 0.1 mg to 10 mg of enzyme inside, about 0.1 mg to 1 mg of enzyme inside. In further embodiments, individual reactor may have about 1 mg to 1000 mg of enzyme inside, or about 1 mg to 100 mg of enzyme inside, about 1 mg to 10 mg of enzyme inside. In some embodiments, an individual reactor may have about 10 mg to 1000 mg of enzyme inside, or about 10 mg to 100 mg of enzyme inside. In other embodiments, an individual reactor may have about 100 mg to 1 g of enzyme inside. In some embodiments, an individual reactor may have about 1 g to 1000 g of enzyme inside, or about 1 g to 100 g of enzyme inside, or about 1 g to 10 g of enzyme inside. In other embodiments, an individual reactor may have about 10 g to 1000 g of enzyme inside, or about 10 g to 100 g of enzyme inside. In some embodiments, an individual reactor may have about 100 g to 1 kg of enzyme inside. In some embodiments, an individual reactor may have about 1 kg to 1000 kg of enzyme inside, or about 1 kg to 100 kg of enzyme inside, or about 1 kg to 10 kg of enzyme inside. In other embodiments, an individual reactor may have about 10 kg to 1000 kg of enzyme inside, or about 10 kg to 100 kg of enzyme inside. In some embodiments, an individual reactor may have about 100 kg to 1 Mg of enzyme inside. In some embodiments, an individual reactor may have about 1 Mg to 1000 Mg of enzyme inside, or about 1 Mg to 100 Mg of enzyme inside, or about 1 Mg to 10 Mg or enzyme inside. In other embodiments, an individual reactor may have about 10 Mg to 1000 Mg of enzyme inside, or about 10 Mg to 100 Mg of enzyme inside. In some embodiments, an individual reactor may have greater than 100 Mg of enzyme inside.
In some embodiments, the temperature of an individual reactor may be varied to optimize the throughput and yield of the reaction. In some embodiments, the temperature of an individual reactor is varied with a temperature altering element. In some embodiments, the temperature altering element is attached to an individual reactor housing or an individual reactor. In other embodiments, the temperature altering element is attaching to a cylindrical tube exterior. In some embodiments, the temperature of an individual reactor 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 an individual reactor 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 an individual reactor 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 an individual reactor may be about 40° C. to 70° C., or about 40° C. to 60° C., or about 40° C. to 50° C. In some embodiments, the temperature of an individual reactor may be about 50° C. to 70° C., or about 50° C. to 60° C. In some embodiments, the temperature of an individual reactor may be between 60° C. and 70° C. In some embodiments, the temperature of an individual reactor may be greater than 70° C.
The temperature within an individual reactor may be measured at various intervals. In some embodiments, the temperature within an individual reactor may be measured by a temperature sensor. In other embodiments, the temperature within an individual reactor may be measured by a temperature sensor within the cylindrical tube interior. For example, the temperature of an individual reactor may be measured once per second. In some embodiments, the temperature of an individual reactor may be measured less than once per second. In other embodiments, the temperature of an individual reactor is measured more than once per second.
In some embodiments, the pH of an individual reactor (or of a solution therein) may be varied to optimize the throughput and yield of the reaction. In some embodiments, the pH of an individual reactor (or of a solution therein) may be varied using a pH measurement module adapted to introduce an acid or a base into the solution within the individual reactor. In some embodiments, the pH of an individual reactor may be about 4.0 to 10.0, or about 4.0 to 9.0, or about 4.0 to 8.0, or about 4.0 to 7.0, or about 4.0 to 6.0, or about 4.0 to 5.0. In some embodiments, the pH of an individual reactor may be about 5.0 to 10.0, or about 5.0 to 9.0, or 5.0 to 8.0, or about 5.0 to 7.0, or about 5.0 to 6.0. In some embodiments, the pH of an individual reactor may be about 6.0 to 10, or about 6.0 to 9.0, or about 6.0 to 8.0, or about 6.0 to 7.0. In some embodiments, the pH of an individual reactor may be about 7.0 to 10.0, or about 7.0 to 9.0, or about 7.0 to 8.0. In some embodiments, the pH of an individual reactor may be about 8.0 to 10.0 or about 8.0 to 9.0. In some embodiments, the pH of an individual reactor may be about 9.0 to 10.0. In some embodiments, the pH may be less than 4.0. In some embodiments, the pH may be greater than 10.0
The pH in an individual reactor (or of a solution therein) may be measured at various intervals. In some embodiments, the pH within an individual reactor (or within a solution therein) may be measured by a pH sensor. In other embodiments, the pH within an individual reactor (or within a solution therein) may be measured by pH sensor within the cylindrical tube interior. In preferred embodiments, the pH of an individual reactor (or of a solution therein) may be measured once per second. In some embodiments, the pH of an individual reactor (or of a solution therein) may be measured less than once per second. In other embodiments, the pH of an individual reactor (or of a solution therein) may be measured more than once per second. In some embodiments, the pH of an individual reactor may be changed through the addition of acidic or basic solutions.
In some embodiments, the pressure of an individual reactor may be varied to optimize the throughput and yield of the reaction. In other embodiments, the pressure variation of an individual reactor may be a byproduct of introducing gases into the individual reactor. In further embodiments, the pressure variation of an individual reactor may be a byproduct of removing gases from the individual reactor. In some embodiments, the pressure may be about 0 psig (pound-force per square inch) to 500 psig, or about 0 psig to 250 psig, or about 0 psig to 100 psig, or about 0 psig to 50 psig, or about 0 psig to 10 psig, or about 0 psig to 1 psig, or about 0 psig to 0.1 psig, or about 0 psig to 0.01 psig. In other embodiments, the pressure may be about 0.01 psig to 500 psig, or about 0.01 psig to 250 psig, or about 0.01 psig to 100 psig, or about 0.01 psig to 50 psig, or about 0.01 psig to 10 psig, or about 0.01 psig to 1 psig, or about 0.01 psig to 0.1 psig. In some embodiments, the pressure may be about 0.1 psig to 500 psig, or about 0.1 psig to 250 psig, or about 0.1 psig to 100 psig, or about 0.1 psig to 50 psig, or about 0.1 psig to 10 psig, or about 0.1 psig to 1 psig. In other embodiments, the pressure may be about 10 psig to 500 psig, or about 10 psig to 250 psig, or about 10 psig to 100 psig, or about 10 psig to 50 psig. In some embodiments, the pressure may be about 50 psig to 500 psig, or about 50 psig to 250 psig, or about 50 psig to 100 psig. In some embodiments, the pressure may be about 100 psig to 250 psig, or about 100 psig to 500 psig. In other embodiments, the pressure may be about 250 psig to 500 psig. In further embodiments, the pressure may be greater than 500 psig.
The pressure within an individual reactor may be measured at various intervals. In some embodiments, the pressure within an individual reactor may be measured by a pressure sensor. In other embodiments, the pressure within an individual reactor may be measured by pressure sensor within the cylindrical tube interior. In some embodiments, the pressure of an individual reactor may be measured once per second. In some embodiments, the pressure of an individual reactor may be measured less than once per second. In other embodiments, the pressure of an individual reactor may be measured more than once per second.
In some embodiments, the amount of dissolved oxygen in an individual reactor may vary. In other embodiments, the amount of dissolved oxygen in a solution within an individual reactor may vary. In some embodiments, the amount of dissolved oxygen may be varied using a gas addition module adapted to introduce gas into the solution within the individual reactor. In other embodiments, the amount of dissolved oxygen may be varied using a gas addition module adapted to introduce gas into the cylindrical tubing interior of the individual reactor. In other embodiments, the amount of dissolved oxygen may be varied using a degassing module adapted to remove gasses from the solution. In further embodiments, the amount of dissolved oxygen may be varied using a degassing module adapted to remove gasses from the cylindrical tubing interior of the individual reactor. In some embodiments, the amount of dissolved oxygen of an individual reactor may be about 0.0 ppm (parts per million) to 10 ppm, or about 0.0 ppm to 9.0 ppm, or about 0.0 ppm or about 8.0 ppm, or about 0.0 ppm to 7.0 ppm, or about 0.0 ppm to 6.0 ppm, or about 0.0 ppm to 5.0 ppm, or about 0.0 ppm to 4.0 ppm, or about 0.0 ppm to 3.0 ppm, or about 0.0 ppm to 2.0 ppm, or about 0.0 ppm to 1.0 ppm. In some embodiments, the amount of dissolved oxygen of an individual reactor may be about 1.0 ppm 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 of an individual reactor may be about 2.0 ppm to 10 ppm, or about 2.0 ppm to 9.0 ppm, or about 2.0 ppm or about 8.0 ppm, or about 2.0 ppm to 7.0 ppm, or about 2.0 ppm to 6.0 ppm, or about 2.0 ppm to 5.0 ppm, or about 2.0 ppm to 4.0 ppm, or about 2.0 ppm to 3.0 ppm. In some embodiments, the amount of dissolved oxygen of an individual reactor may 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 of an individual reactor may 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 of an individual reactor may be about 5.0 ppm 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 of an individual reactor 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 of an individual reactor 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 of an individual reactor may be between 8.0 ppm to 10 ppm, or about 8.0 ppm to 9.0 ppm. In some embodiments, the amount of dissolved oxygen of an individual reactor may be between 9.0 ppm to 10.0 ppm. In further embodiments, the amount of dissolved oxygen in an individual reactor is greater than 10.0 ppm.
The amount of dissolved oxygen within an individual reactor (or within a solution therein) may be measured at various intervals. In some embodiments, the amount of dissolved oxygen within an individual reactor (or within a solution therein) may be measured by a dissolved oxygen (DO) sensor. In other embodiments, the amount of dissolved oxygen within an individual reactor (or within a solution therein) may be measured by a dissolved oxygen (DO) sensor within the cylindrical tube interior. In some embodiments, the amount of dissolved oxygen of an individual reactor (or of a solution therein) may be measured once per second. In some embodiments, the amount of dissolved oxygen of an individual reactor (or of a solution therein) may be measured less than once per second. In other embodiments, the amount of dissolved oxygen of an individual reactor (or of a solution therein) may be measured more than once per second.
In some embodiments, dissolved oxygen may be removed via a deoxygenation/degassing machine (i.e., deoxygenation/degassing module). As used herein, a “deoxygenation machine” or “degassing machine” or “deoxygenation module” or “degassing module” may refer to a device which removes the amount of dissolved oxygen and/or other gasses (e.g., nitrogen) in a fluid (i.e., a solution) when the fluid is flowed through the device. In other embodiments, nitrogen gas is introduced to an individual reactor to reduce the levels of oxygen.
As used herein, a “gassing machine” or “gassing module” may refer to a device which adds an amount of dissolved oxygen and/or other gasses (e.g., nitrogen) into a fluid (i.e., a solution) when the fluid is flowed through the device. In some embodiments, the cell manufacturing platform comprises a gas addition module adapted to introduce gas into the solution. In some embodiments, oxygen, nitrogen or a combination thereof may be added to an individual reactor. Other gases may be added or removed from an individual reactor in accordance with the platform described herein.
As used herein, a “chemical stream” may refer to a solution that contains a substrate, product, intermediate, cofactor or another chemical.
Referring now to FIGS. 1-34B, the present invention features a cell free manufacturing platform for continuous chemical production.
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 comprising 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, a cylindrical tube interior of the individual reactor comprises a resin and an enzyme. In some embodiments, each of the one or more individual reactors has an input tubing connected at the first end of the cylindrical tube and an output tubing connected at the second end of the cylindrical tube. In some embodiments, the cell-free manufacturing platform is able to automatically change each of the one or more reactors conditions based on input from the sensors.
In some embodiments, the cell-free manufacturing platform described herein is a closed system. In other embodiments, the cell-free manufacturing platform described herein is an open system.
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 fastened to the individual reactor. In embodiments, the cell-free manufacturing platform further comprises a temperature altering element attached to the individual reactor housing or the individual reactor. In some embodiments, the temperature altering element is a thermoelectric cooler (TEC). In some embodiments, the temperature altering element is a flexible heating element.
In some embodiments, the cell-free manufacturing platform further comprises a spectrometer attached in series with the one or more individual reactors. In other embodiments, the cell-free manufacturing platform further comprises a degassing module adapted to remove gasses 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 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 automation software and hardware.
In some embodiments, present 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 at 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 fastened to the individual reactor. In some embodiments, the platform comprises a temperature altering 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 gasses 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 with the reactor(s). In some embodiments, the platform comprises a graphical user interface (GUI). In some embodiments, the platform comprises an automation software and hardware. In some embodiments, the GUI is adapted to control the automation software and hardware. In some embodiments, the individual reactor has input, and output tubing connected at each end of the cylindrical tube to create a closed system. In some embodiments, the platform is able to automatically change each of the one or more reactors conditions based on input from the sensors.
In other embodiments, the present 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 a reactor. In some embodiments, the platform comprises a plurality of reactors connected in series. In other embodiments, the platform comprises a plurality of reactors in parallel. In some embodiments, the reactor comprises resin, enzymes, and sensors. In some embodiments, the platform comprises an individual or multiple reactor housing, wherein the housing surrounds and is fastened to the individual reactor(s). In some embodiments, the platform comprises a combination of thermoelectric coolers (TEC) or other temperature altering element(s) attached to the housing(s), a pumping system, a deoxygenation and/or degassing module, a gas addition module, a graphical user interface (GUI), and an automation software and hardware the device is ran on. In some embodiments, the individual reactor is sealed at each end of the cylindrical tube to create a closed system. In some embodiments, the platform is able to automatically change the reactor conditions based on input from the sensors. In some embodiments, the platform comprises a spectrometer to measure wavelength and absorption of volume entering and exiting. In other embodiments, the platform comprises a spectrometer to measure output color wavelength and/or absorption. In further embodiments, the platform is able to automatically change the reactor conditions based on input from the sensors.
In some embodiments, the 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, the one or more individual reactors connected in parallel are connected to a single pump. In some embodiments, the parallel reactors are connected to a single pump. In some embodiments, the flow through the parallel reactors is controlled via software and control valves.
In some embodiments, the enzymes are chosen from a group consisting of: MBP-Aldo (Aldo), Dihydroxy Acid Dehydratase (TvDHAD), Pyruvate Oxidase (PyOx), Acetyl-phosphate transferase (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 Prenyl transferase (NphB). In some embodiments, the enzymes comprise MBP-Aldo (Aldo), Dihydroxy Acid Dehydratase (TvDHAD), Pyruvate Oxidase (PyOx), Acetyl-phosphate transferase (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), Prenyl transferase (NphB), or a combination thereof.
In some embodiments, the enzymes are immobilized. In other embodiments, the enzymes are non-immobilized.
In some embodiments, the plurality of sensors comprises a temperature sensor. In some embodiments, the plurality of sensors comprises a pH sensor. In some embodiments, the plurality of sensors comprises a pressure sensor. In some embodiments, the plurality of sensors comprises a flow rate sensor. In some embodiments, the plurality of sensors comprises a dissolved oxygen (DO) sensor. In some embodiments, the plurality of sensors comprises a spectrometer. In some embodiments, the cylindrical tube interior of the individual reactor further comprises one or more sensors. In some embodiments, the one or more sensors comprises a temperature sensor, a pH sensor, a pressure sensor, a flow rate sensor, a dissolved oxygen (DO) sensor, a spectrometer, or a combination thereof.
In some embodiments, an individual reactor has a percent yield of about 10-100%. In some embodiments, an individual reactor has a percent yield of about 10%. In some embodiments, an individual reactor has a percent yield of about 20%. In some embodiments, an individual reactor has a percent yield of about 30%. In some embodiments, an individual reactor has a percent yield of about 40%. In some embodiments, an individual reactor has a percent yield of about 50%. In some embodiments, an individual reactor has a percent yield of about 60%. In some embodiments, an individual reactor has a percent yield of about 70%. In some embodiments, an individual reactor has a percent yield of about 80%. In some embodiments, an individual reactor has a percent yield of about 90%. In some embodiments, an individual reactor has a percent yield of about 95%. In some embodiments, an individual reactor has a percent yield of about 98%. In some embodiments, an individual reactor has a percent yield of about 99%. In some embodiments, an individual reactor has a percent yield of about 100%.
In some embodiments, each individual reactor allows an enzyme-resin complex to be contained within the reactor. In some embodiments, the reactor contains a single enzyme-resin complex. In other embodiments, the reactor contains multiple enzyme-resin complexes.
In some embodiments, each individual reactor contains a set amount of enzyme-resin complex. In other embodiments, a set amount of enzyme resin complex may allow for a tunable concentration of enzyme can be achieved in each individual reactor. For example, if more enzyme is desired, the individual reactor may be packed with more enzyme-resin complex, and vice versa.
In some embodiments, each individual reactor provides the necessary reactor conditions to ensure that the enzyme-resin complex(es) remain active to convert substrate to product. In some embodiments, the reactor conditions are finely controlled to ensure the lifetime of the enzyme. Non-limiting examples of conditions the reactor may control include but are not limited to temperature or pH or oxygen level.
In some embodiments, each individual reactor has customizable temperature control to ensure each enzyme obtains its optimal reaction temperature. In some embodiments, each individual reactor has the ability to control reactor temperature within +/−0.1° C. by using the graphical user interface (GUI). There is no manual readjustment to the system, only algorithmic feedback, and control after user temperature setpoint to a computer.
Without wishing to limit the present invention to any theory or mechanism it is believed that temperature is highly important for any enzymes during a reaction. For example, Aldo (that converts glycerol into glyceric acid) requires an optimal temperature of 37° C. (19.6 mM, 98% yield) however, the next enzyme in the pathway (DHAD) requires 45° C. to convert glyceric acid into pyruvic acid (6.4 mM, 32% yield, Table 1). If these reaction temperatures are not adhered to, reaction yield and rate suffer. FIG. 2 shows that 45° C. is optimal for DHAD, if the reaction temperature drops to 32° C. or increases to 55° C. then a significant move from optimal conversion is observed.
In some embodiments, each individual reactor has reactor fluid pH adjustment and control to afford the unique pH requirements for each enzyme (Table 1). In some embodiments, the pH sensor and automated feedback loops to ensure that the pH of the reaction medium can be changed between individual continuous reactors. In some embodiments, to change the pH of the reaction medium a certain volume of a certain pH solution is injected into the reactor system to change and maintain a specific pH. This allows each reactor to have optimal pH to avoid protein precipitation, loss of reactivity, or other degrading influences.
In some embodiments, the reactor system has the ability to add gases to individual reactors for enzymes that require a gas. Non-limiting examples may include oxygen or nitrogen. As described herein, oxygen and nitrogen addition to the reactors have been demonstrated to ensure effective enzyme transformations. This was crucial, as without oxygen addition, immobilized ALDO afforded 0% product. However, after introduction of oxygen into the reactor, 98% yield was obtained (19.6 mM).
In some embodiments, the reactor system can reduce the oxygen present in the reaction medium when required. In some embodiments, oxygen is removed through the use of sonication and a vacuum. In some embodiments, oxygen is removed through the addition of nitrogen.
As described herein, the DHAD enzyme evaluated required oxygen removal to increase reaction efficiency. A module for this system was introduced that removed the oxygen present in the fluid down to 4.07 ppm through the use of sonication and a vacuum. This device allows removal of pO₂from 7.50 ppm to 4.07 ppm in 10 minutes at room temperature. In addition to the module, nitrogen gas can be introduced to further reduce the amount of oxygen in the medium.
In some embodiments, each individual reactor allows a starting material (substrate solution) to be pumped through the reactor using a standard laboratory pump. The substrate solution is injected into the reactor, the solution moves through the reactor to interact with the enzyme and cause a chemical reaction. Once the chemical reaction is complete, the reacted fluid moves out of the reactor to a collection flask.
In some embodiments, the reactor can operate in a continuous mode, wherein the pump injecting the fluid into the individual reactor would always be pumping fluid. In other embodiments, the reactor can operate in a semi-continuous mode, wherein the pump injects the fluid into the individual reactor for a certain amount of time and then stops for a certain amount of time to hold the fluid in the reactor. In some embodiments, after the set period of time, the pump restarts pushing the reacted fluid out of the reactor system. Without wish to limit the present invention to any theories or mechanisms, it is thought that a semi-continuous mode allows longer enzyme reaction times to be accommodated.
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, a computer-controlled pump allows chemical solutions to be injected into the continuous manufacturing system when instructed. Non-limited 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 at pH 12.0 to adjust process flow pH from pH 7.7 to pH 8.5, 100 mM Tris K₂HPO₄: KH₂PO₄(1:1) at pH 6.33 in order to adjust process flow from pH 7.7 to pH 6.5, and 50 mM Tris at pH 12.85 in order to adjust process flow from pH 6.47 to pH 8.0.
In some embodiments, the platform contains a wide range of sensors allowing full system automation. These sensors include, but are not limited to temperature sensors/thermistors, pressure sensors, flowmeters, pH sensors/probes, dissolved oxygen sensors/probes, and spectrometers. Deviation away from a programmed optimal value in the GUI or code (temperature, flow rate, pH, pressure, etc.) triggers system adjustment through an algorithmic feedback loop(s) and corresponding changes in hardware state(s) to allow for system conditions to reach a required state.
In some embodiments, the individual reactor is scalable. In some embodiments, the length, diameter, or number of individual reactors may be increased to achieve a higher throughput. There is no theoretical limit on the size of these reactors or the number. In some embodiments, the individual reactors can be different sizes, shapes, and configurations.
In some embodiments, the cylindrical tubing of an individual reactor may be virtually any diameter tubing and of any length the user wants to allow for more or less resin/enzyme, or shorter/longer reaction times. In other embodiments, the fittings (e.g., stainless steel fittings) at the ends of the cylindrical tubing of an individual reactor may be customized as well to allow for various sizes of input/output tubing to be used. In some embodiments, the material of the cylindrical tubing the individual reactor is made out of can be changed to allow for more optimal thermal conductivity or other parameters if the reaction permits coming into contact with the material.
In some embodiments, the reactor system has a customizable user interface for users to control the reactor with a mouse click, commonly known as a GUI (graphical user interface).

EXAMPLE

The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.
1.1 Individual Reactor Design: The individual reactors are made of 304 stainless steel cylindrical tubing. The individual reactor housings are made of 6061 aluminum. The individual reactor housings are used as heat transfer vehicles to change the temperature of the reactors held within by attaching thermoelectric coolers (TEC) elements to the housings (FIG. 3). The temperature(s) of the reactor(s) can also be altered through attaching flexible heating elements to the exterior of the reactor(s). The individual reactors can be heated and cooled from 12° C. and 55° C. Compared to a conventional batch reactor, these reactors can reach the desired temperature in 11 minutes when tested at a set temperature of 45° C. (FIG. 4). Each individual reactor is fitted with Swagelok fittings at each end of the 304 stainless steel cylindrical tubing to seal the tubing to create a reactor. Additionally, the Swagelok fittings allow inlet and outlet tubing to be attached to the reactor to introduce and remove fluids from the individual reactor. To note, the volume, shape, size and material of the reactor can be changed, and different materials such as plastics and different volumes including 1 g to 330 g have been tested.
To ensure the individual reactors would comply with the pumping system, the pressure of the reactors was tested. Internal pressure of the individual reactors was monitored in real time over 24 hours by pumping a buffer (50 mM Tris, 20 mM glyceric Acid, pH 7.7) through the first two reactors in the pathway. Buffer was pumped through the individual reactor containing resin at a temperature of 21° C. (i.e., room temperature) with a flow rate of 10 mL/min. After the first reactor, there is a joint where a buffer (50 mM Tris, pH 12.0) was pumped into the process flow along with the Tris solution in order to modify its pH level. To monitor the pressure of the individual reactor in real time, a python program was written to retrieve the pressure level from both the process pumps once per second. In this testing system, the pumps are directly connected to the reactors through PFA tubing and thus the pressure calculated by the pumps will be a direct sampling of the internal pressure of the individual reactors.
The calculated pressure inside the individual reactors ranged from 0-70 psig. The average internal pressure of the first reactor in the system was <0.01 psig when tested over a 24-hour window (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 window. This low pressure is required for scale up of the reactor and also indicates that the system does not induce resin swelling through uptake of fluid. Finally, the pressure monitoring used in this system also allows the user to set pressure warning limits in the GUI for safety and control aspects. This feeds into the automated control of the continuous manufacturing system as a whole.
The amount of enzyme that a standard individual reactor can hold was also calculated. The individual reactors tested here can hold 57 g of enzyme-resin complex (<5.5 g of isolated enzyme). This means that one reactor with a size of 14″ length (L)×0.652″ inner diameter (ID) (79 mL total volume, FIG. 6), can hold as much enzyme as a 1000 L fermenter or greater when the enzyme expression level in a batch reactor is 5.5 mg/L or greater. This is just one example and the comparison changes when enzymes are expressed at different levels. However, this dramatic improvement means that a 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 a batch reactor is 5.5 mg/L.
1.2 Temperature Control for the Individual Reactors: Individual reactor temperature control is achieved through a proportional-integral-derivative (PID) algorithm and supporting code contained within an Arduino Mega 2560 microcontroller or STM32L476RG microcontroller or similar. The temperature sensors used are NTC thermistors that operate by a change in electrical resistance as their temperatures change. This change in resistance is relayed to the microcontroller as a voltage through use of a voltage divider circuit (FIGS. 7A-7B). The thermistors are placed within indentions in the reactor housings and are held in place once the housings are fastened close. The thermistors contact both the reactor housing and the reactor itself. In the case of a reactor being heated with a flexible silicon heating element or similar, no reactor housing is used. In this case the thermistor is inserted between the exterior of the reactor and the heating element. The difference in temperature between where the thermistors are placed and inside the reactor has been measured and is accounted for in the software. Within the PID algorithm software, the thermistors' resistance values are converted to an accurate temperature reading by converting the incoming voltage reading back to an electrical resistance, and then is further calculated into degrees Kelvin by using a simplified version of the Steinhart-Hart equation (FIG. 8). This value is then converted from Kelvin to Celsius. The temperature of the individual reactor(s) is read once per second. The temperature reading is fed into a PID loop that responds to the current temperature and modifies the pulse width modulation (PWM) duty cycle of the electrical output that is powering the heating elements to change the temperature of the individual reactor to the correct level. The circuitry can be seen in FIGS. 9A-9B. The PID software framework is licensed under the MIT permissive license. This software foundation has been edited and expanded to control the system described herein. The PID loop has been fine tuned to minimize the amount of temperature overshooting and equilibration time (FIG. 4). The TECs causing the temperature change are adhered to the individual reactor housings using a thermal conductive glue. The glue keeps the TECs securely fastened to the housings while allowing for heat transfer to continue uninterrupted (FIG. 3). The flexible silicon heating elements or similar have adhesive on one side to allow for attaching to various objects.
The temperature control system can also cool the individual reactors to 12° C. (FIGS. 10A-10B), no change was observed in the performance. Additionally, the amount of power that was required to cool to this temperature was 0.027 kW; this has a staggering benefit compared to large batch reactors that require large amounts of electricity to cool. Cooling can be achieved by flipping the TECs to their other side, as the other side of the TEC is the “cold” side. Alternatively, if there is an application where a reactor needs to be both heated and cooled, an alternate circuit can be implemented that allows for heating/cooling to be switched by inserting a separate wire into the corresponding microcontroller digital pulse width modulation (PWM) pin, instead of the wire used to activate heating (FIG. 9B). If cooling is required, it is necessary to attach a heatsink apparatus onto the Peltier elements. The elements work on a temperature differential between each side of the plate, so if the cold side is being used to cool down the reactor the other side will heat up. If no heatsink is used, the hot side will overpower the cold side until an equilibrium temperature above ambient is reached. The heat sink keeps the “hot” side cooler, which allows for the cold side to maintain a cooler temperature. A thermal image of this occurring can be seen in FIG. 10B.
1.3 The Individual Reactors have strict pH control: Between each reactor will be an injection point where acidic or basic solution can be continuously injected into the process flow to provide a change in pH that matches the pH requirement for the next reactor in line. The correct flow rate and pH level of the required injections have been determined through experimentation. Additionally, a pH sensor was implemented that can measure the pH of a flowing solution in real-time to provide accurate pH monitoring. The pH sensor is connected in-line and sends pH values to the control system serially once per second. This provides the user(s) additional data on the accuracy of the pH adjustment as well as automatically changes the flowrate of the pH injection in order to maintain an accurate pH output.
1.4 Gases can be added to the Individual Reactors: The system has additional injection points for gas injection into the fluid flow before an individual reactor. The required gas tank is connected to the system via a mass flow controller. Here, the user can set a flow rate of gas to enter into the individual reactors in a continuous manner. The mass flow controller(s) are controlled via voltage signals. These signals can be adjusted both in an analog manner using a voltage divider circuit with a potentiometer (manual turning of a dial), or digitally via sending digital signals through a digital to analog converter (DAC). Three gases have been tested thus far: compressed air, oxygen, and nitrogen. The compressed air was used for the Aldo enzyme that requires supplemental oxygen to react properly, and compressed nitrogen can be used to reduce oxygen concentrations of the fluid. Gas tanks are connected to the mass flow controller(s) via tubing and stainless-steel fittings. Mass flow controllers are connected to the individual reactors via tubing and Swagelok fittings. Mass flow controllers are programmed to allow a specific volume of gas per minute to pass through the mass flow controller into the individual reactor. The correct ratio of gas to fluid has been experimentally tested and optimized and is roughly a 1:1 volumetric ratio.
1.5 A module to decrease oxygen content in the fluid entering the individual reactors: In addition to the nitrogen gas being added to reduce oxygen, the system utilizes a deoxygenation and/or degassing machine to reduce the amount of oxygen within the process flow. The deoxygenation/degassing module will be placed in series with the flow and the fluid exiting the machine will have up to 3.68 ppm of oxygen removed. A reduction of dissolved oxygen from 7.75 ppm to 4.07 ppm, a reduction of almost 50% was shown (FIG. 11).
1.6 Injection of additional chemical streams into the individual reactors: In addition to the injection of solutions to adjust pH, there will be injection points to add chemical streams to the process flow. These chemical streams are specific to the reactors they are associated with and will alter the chemistry required to yield a product as intended.
1.7 System control and automation on the Individual Reactors: The system contains various sensors to monitor and/or adapt reaction conditions when told to by the user or automatically. Sensors include, but are not limited to, temperature sensors/thermistors, flowmeters, pressure sensors, pH probes, dissolved oxygen probes, mass flow controllers, and spectrometers. The temperature sensors for each individual reactor feed data to the microcontroller which then autonomously adjusts the power output to the heating elements, which alters the temperature of said reactor. The flow meter monitors the rate at which the fluid within the system is flowing through the reactor and streams that data to the microcontroller. If the process flow has slowed to a rate considered not optimal, the microcontroller sends commands to incrementally increase or decrease the flow rate of the pumps until an acceptable flow rate is achieved. The pH probe(s) monitor the pH of the volume passing across it and adjusts the amount of acidic or basic solution being injected into the process flow to achieve the desired pH level. The dissolved oxygen probe measures the amount of oxygen in the solution and the microcontroller can adjust the rate at which oxygen or other gases are being added to the process flow via mass flow controller. The pressure sensors measure the pressure within the process flow. The spectrometer measures the wavelength of light of the volume exiting the reactor and reads the intensity across the spectrum of 340.6 nm to 1010 nm and the absorbance levels across the same spectrum of light.
1.8 Usage of Parallel Reactors with equal flow: The system can utilize reactors in a parallel fashion. Two individual reactors can be connected to a single pump in parallel. The two reactors have achieved equal flow through each by implementing control software with accompanying hardware. The accompanying hardware in this configuration is a flowmeter(s) and control valves. The control valves are placed upstream from the reactors and the flowmeter(s) are placed downstream from the reactor in series. The software is connected to the flowmeter(s) and calculates the flow rate of the output of each reactor connected in parallel. The software keeps a total of the amount flowed through each reactor. The software then makes determinations based on the flow rates and amounts flowed to temporarily stop or continue flow in individual reactors via solenoid valves to keep the total amount of solution flowing through each to be even (FIG. 27)
1.9 System output color and absorption collection: The system contains a spectrometer in which system output wavelength and absorption can be measured. A flow cell is connected in series with system output through which the output flows. A light source is connected to the flow cell via fiber optic cabling. The flow cell is connected to the spectrometer via fiber optic cabling. The spectrometer readings can be retrieved in two ways. One way is by using a commercial software package included with the spectrometer in tandem with the spectrometer unit to read the wavelength of the color of the output or the absorption of the output in real time (FIG. 28). The second way is through software that was written to communicate with the spectrometer via RS232 to retrieve the data. The software can retrieve the sample's wavelength intensity over the spectrum from 340.6 nm to 1010 nm. The software also performs calculations to convert the intensity levels of the sample to an absorbance value. The software has the capability to plot its findings on a graph and/or save them to a log file or spreadsheet document or similar (FIG. 29). The software was written in Python. The data from the sample's color and absorbance can be further analyzed to make predictions of the output's concentration based on the color/spectral data.
1.10 Reactors can be different sizes, shapes, and configurations: Individual reactors can be different diameters and lengths to allow for different residence times for the fluid being flowed through the reactor. Currently, three different sizes of reactors have been tried:

- 1. The first size reactor is 13.5″ in length with a 0.25″ outside diameter (OD) and a 0.180″ inner diameter (ID) resulting in a volume of 5.63 mL (Table 2)
- 2. The second size reactor is 14.5″ in length with a 0.75″ OD and a 0.652″ (ID), resulting in a volume of 79.33 mL (Table 2).
- 3. The third size reactor is 5.5″ in length with a 0.25″ OD and a 0.218″ ID, resulting in a volume of 1.64 mL (Table 2).

TABLE 2

Dimensions of the three sizes of reactors
along with volume and residence time data.

	Reactor #1	Reactor #2	Reactor #3

Outside diameter (in)	0.25	0.25	0.75
Inside Diameter (in)	0.180	0.218	0.652
Height (in)	13.5	5.5	14
PSI max @ 72 F.	6125	2000	2190
Reactor Volume (in³)	0.344	0.205	4.674
Reactor volume (cm³)	5.629	3.364	76.598
Resin volume (cm³)	4.912	2.935	66.831
Volume for media (cm³)	0.718	0.429	9.766
Flow rate (mL/min)	0.01	0.01	0.01
Residence Time	71.776	42.892	976.619
(minutes)
Residence Time (hours)	1.196	0.715	16.277

There were two different sizes of reactor housings made, one for the ¼″ OD reactor and one for the ¾″ OD reactor. The complete dimensions can be seen on their drawings in FIGS. 12A-12D. To accommodate the shorter length of the third reactor, a reactor housing meant for the ¼ OD reactor was cut to length. The reactor sizes and their calculated residence times can be seen in Table 2.
1.11 The reactor system has a user interface to control the reactor: The system has an optional user interface to allow users to specify what temperature each reactor should be programmed to, start and stop the pumps within the system, manually check the pressures each pump is under, and set upper pressure limits on the pumps (FIGS. 9A-9B). The GUI was written in Python using the Kivy framework.
1.12 The system has a graphical dashboard to monitor the variables for each reactor in real time: In addition to the control the user interface will provide, there is a monitoring dashboard that displays each reactor's temperature, the flowrate(s) as provided by the flowmeter(s), the pressure as seen by the pumps, and the PWM duty cycle being applied to each Peltier element. The data will be displayed using Grafana and stored in a database using InfluxDB, in conjunction with NodeRED. This environment will be powered and hosted by a raspberry pi. The data flow can be seen in FIG. 13.
1.13 Individual Reactors can be linked into a sequence to afford multi-step enzyme reactors: Reactors may be connected in series via ¼″ OD tubing to allow for multi-step enzyme reactions. The pathway requires 13 different enzymes which translates to 13 different reactors. These reactors will be connected in series along with injection points for pH control and gas addition.

Example 2.0

Experimental Examples

2.1 Production of glyceric acid from glycerol using immobilized alditol oxidase (Aldo) (FIG. 14): A reactor of 13.5″ in length with a 0.25″ outside diameter and a 0.180″ internal diameter containing immobilized Aldo enzyme (240 mg enzyme on 4.00 g of resin) was heated to 37° C. and equilibrated for one hour with equilibration buffer (50 mM tris, pH 8.5) being passed through the reactor. After one hour had subsided, the 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)) to yield a total flow rate through the reactor of 40 μL/min (18-minute residence time). After the solution had passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to recycle through the individual reactor. The reaction was allowed to proceed for 72 hours with sampling performed every 24 hours. For sampling, 100 μl of the reaction fluid was examined on a high-performance liquid chromatography (HPLC) system to examine 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 micro-guard 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 comprised of 100% sulfuric acid (10 mM) was used. The sample run time was a total of 45 minutes with glyceric acid eluting at 17.2 mins and glycerol eluting at 21.0 minutes. For detection, a RID detector (Agilent) was used after a 2 h equilibration period produced 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. Upon 72 h the yield of the reaction converting glycerol to glyceric acid was 99% (19.6 mM, 2.06 g/L). To note, the same reaction was carried out without addition of compressed air and 0% conversion was observed. This reaction was scaled to a reactor with dimensions 14″ length×0.75″ outside diameter and 0.652″ inner diameter (Example 2.13 below). 98% conversion to product was observed (19.6 mM glyceric acid) in 66 hours under the same conditions as described above.
2.2 Production of pyruvic acid from glyceric acid using immobilized dihydroxy acid dehydratase (DHAD) (FIG. 15): A reactor of 13.5″ in length with a 0.25″ outside diameter and a 0.180″ internal diameter containing immobilized DHAD enzyme (360 mg on 3.70 g of resin) was heated to 55° C. and allowed to equilibrate for one hour while passing equilibration buffer (250 mM HEPES, pH 7.4, 2.5 mM MgCl₂.6H₂O) through the reactor. After one hour had subsided, the substrate solution (40 mL, 250 mM HEPES, pH 7.4, 2.5 mM MgCl₂.6H₂O, 20 mM glyceric acid) was flowed through the reactor at a flow rate of 10 μL/min (72 min residence time, FIG. 15). After the solution had passed through the reactor it was collected in the same beaker as the starting solution to allow the solution to recycle through the individual reactor. The reaction was allowed to proceed for 48 hours with sampling performed every 24 hours. For sampling, 100 μl of the reaction fluid was examined on a HPLC system to examine the amount of glyceric acid and pyruvic acid. The HPLC method was the same as above with pyruvic acid eluting at 15.8 mins and glyceric acid eluting at 17.2 minutes. Upon analysis, the following data was obtained; 24 hours reactivity=17% pyruvic acid and 48% glyceric acid, 48 hours reactivity=23% pyruvic acid and 47% glyceric acid. Upon 48 h the yield of the reaction converting glyceric acid to pyruvic acid was 23% (4.51 mM, 0.4 g/L). This reaction was scaled to a reactor with dimensions 14″ length×0.75″ outside diameter and 0.652″ inner diameter (Example 2.13 below). 20% conversion to product was observed (2 mM pyruvic acid) in 16 hours under the same conditions as described above
2.3 Production of acetyl phosphate from pyruvic acid using immobilized pyruvate oxidase (PvOx) (FIG. 16): A reactor of 13.5″ in length with a 0.25″ outside diameter and a 0.180″ internal diameter containing immobilized PyOX enzyme (140 mg on 3.5 g of resin) was heated to 37° C. and allowed to equilibrate for 1 hour while passing equilibration buffer (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 6.5, 5.0 mM MgCl2, and 100 mM NaCl) through the reactor. After one hour had subsided, the substrate solution (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 6.5, 5.0 mM MgCl₂, 100 mM NaCl, 5 mM pyruvic acid, 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 had passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to recycle through the individual reactor. The reaction was allowed to proceed for 16 hours. The HPLC method was as follows: An Agilent 1200 HPLC was equipped with a 30 cm Aminex HPX-87H column and a micro-guard cation H refill cartridge. The column was heated to 55° C. with the sample block being maintained at 25° C. The HPLC method consisted of 5 μl sample injection volume and an isocratic mobile phase comprised of 100% sulfuric acid (10 mM). The run time was a total of 25 minutes with acetyl phosphate eluting at 23.6 mins and pyruvate eluting at 16.0 minutes. A refractive index detector (Agilent) was used for analysis after a two-hour equilibration period to produce a stable baseline. Upon 16 h of reactivity, the reaction yield converting pyruvic acid into acetyl phosphate was 10% (0.5 mM or 92 mg/L). 00
2.4 Production of acetyl coenzyme A from acetyl phosphate using immobilized Acetyl-phosphate transferase (PTA) (FIG. 17): A reactor of 13.5″ in length with a 0.25″ outside diameter and a 0.180″ internal diameter containing immobilized PTA enzyme (112 mg on 3.50 g of resin) was heated to 55° C. and allowed to equilibrate for one hour while passing equilibration buffer (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 8.0, 5.0 mM MgCl₂, 100 mM NaCl) through the reactor. After 1 h had subsided, the 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 CoA) was flowed through the reactor at a flow rate of 10 μL/min (72 min residence time, FIG. 17). After the solution had passed through the reactor, it was collected in the same beaker as the starting solution to allow the solution to recycle through the individual reactor. The reaction was allowed to proceed for 8 h. For sampling, the reaction fluid was examined on a HPLC to examine 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 equipped with a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to 25° C. with the sample block being maintained at 4° C. The HPLC method used a 5 μl sample injection volume and a mobile phase comprised of 75 mM CH₃COONa (sodium acetate) and 100 mM NaH₂PO4 (sodium dihydrogen phosphate) mixed with acetonitrile (94:6 volumetric ratio). The run time was a total of 12 minutes with acetyl-coA eluting at 8.5 mins and coenzyme A eluting at 3.9 minutes. A diode array detector (Agilent) was used for the detection of the molecule of interest at 259 nm. Upon 8 h of reactivity, the reaction yield converting acetyl phosphate to acetyl coenzyme A was 12% (0.384 mM or 337 mg/L).
2.5 Production of acetoacetyl coenzyme A from acetyl co-enzyme A using immobilized Acetyl-CoA acetyltransferase (PhaA) (FIG. 18): A reactor of 5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diameter containing immobilized PhaA enzyme (41 mg on 1.5 g of resin) was heated to 32° C. and allowed to equilibrate for 1 h while passing equilibration buffer (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 8.0, 5.0 mM MgCl₂, 100 mM NaCl) through the reactor. After 1 h had subsided, the substrate solution (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 8.0, 5.0 mM MgCl₂, 100 mM NaCl, 2.5 mM acetyl CoA) was flowed through the reactor at a flow rate of 10 μL/min (43 min residence time, FIG. 18). After the solution had passed through the reactor it was collected in the same beaker as the starting solution to allow the solution to recycle through the individual reactor. The reaction was allowed to proceed for 8 h. For sampling, the reaction fluid was examined on the HPLC system to examine the amount of AcCoA and CoA. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm×3 mm equipped with a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to 25° C. with the sample block being maintained at 4° C. HPLC method consisted of 5 μl sample injection volume and an isocratic gradient comprised of 75 mM CH₃COONa and 100 mM NaH₂PO₄mixed with acetonitrile (ACN) in a ratio 94:6 was used as the mobile phase. The run time was a total of 12 minutes with acetyl-coA eluting at 8.5 mins and coenzyme A eluting at 3.9 minutes. A diode array detector (Agilent) was used for the detection of the molecule of interest at 259 nm. Upon 8 h of reactivity, the reaction yield converting acetyl phosphate to acetoacetyl coenzyme A was 21% (0.94 mM or 930 mg/L).
2.6 Production of β-Hydroxy β-methylglutaryl-Coenzyme A (HMG-CoA) from acetoacetyl coenzyme A using immobilized HMG-CoA Svnthase A110G (HMGS A110G) (FIG. 19): A reactor of 5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diameter containing immobilized HMGS A110G enzyme (34.5 mg on 1.5 g of resin) was heated to 32° C. and allowed to equilibrate for 1 h while passing equilibration buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 7.0) through the reactor. After 1 h had subsided, the substrate solution (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 7.0, 5 mM acetoacetyl CoA) was flowed through the reactor at a flow rate of 10 μL/min (43 min residence time, FIG. 19). After the solution had passed through the reactor it was collected in the same beaker as the starting solution to allow the solution to recycle through the individual reactor. The reaction was allowed to proceed for 2 h. After 2 h, the reaction solution was incubated with 20.7 μM HMGR and 5 mM nicotinamide adenine dinucleotide phosphate (NADPH). HMGR converts acetoacetyl CoA into HMG-CoA using the cofactor NADPH. The activity of HMGR was measured by monitoring loss of NADPH at 340 nm using a spectrophotometer. Upon 2 h of reactivity, the reaction yield converting acetoacetyl CoA to HMG-CoA was 13% (0.65 mM or 621 mg/L).
2.7 Production of mevalonate from HMG-CoA using immobilized HMG-CoA Reductase (HMGR) (FIG. 20): A reactor of 5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diameter containing immobilized HMGS A110G enzyme (31 mg on 1.5 g of resin) was heated to 37° C. and allowed to equilibrate for 1 h while passing equilibration buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 7.0) through the reactor. After 1 h had subsided, the 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 (43 min residence time, FIG. 20). After the solution had passed through the reactor it was collected in the same beaker as the starting solution to allow the solution to recycle through the individual reactor. The activity of HMGR was measured by monitoring the loss of NADPH at 340 nm using a spectrophotometer. Upon 2 h of reactivity, the reaction yield converting HMG-COA to mevalonate was 98.3% (2.45 mM or 378 mg/L).
2.8 Production of Mevalonate-5-Phosphate from mevalonate using immobilized Melvonate Kinase (MVK) (FIG. 21): A reactor of 5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diameter containing immobilized MVK enzyme (60 mg on 1.5 g of resin) was heated to 37° C. and allowed to equilibrate for 1 h while passing equilibration buffer (50 mM Tris, 5 mM MgCl₂, pH 8) through the reactor. After 1 h had subsided, the substrate solution (50 mM Tris, 5 mM MgCl₂, pH 8, 4 mM adenosine triphosphate (ATP), 4 mM mevalonic acid) was flowed through the reactor at a flow rate of 10 μL/min (43 min residence time, FIG. 21). After the solution had passed through the reactor it was collected in the same beaker as the starting solution to allow the solution to recycle through the individual reactor. The reaction was allowed to proceed for 16 hours. For sampling, the reaction fluid was examined on a HPLC system to examine the amount of ATP and ADP (adenosine diphosphate) present in the final reaction solution. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm×3 mm equipped with a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to 25° C. with the sample block being maintained at 4° C. HPLC method consisted of 5 μl sample injection volume and an isocratic mobile phase comprised of 100 mM KH₂PO₄(potassium dihydrogen phosphate), 8 mM TBAHS (tetrabutylammonium hydrogen sulfate), pH 6.0, 20% methanol (v/v). The run time was a total of 10 mins with ATP eluting at 5.7 mins and ADP eluting at 4.6 minutes. A diode array detector (Agilent) was used for the detection of the molecule of interest at 254 nm. Upon 16 h of reactivity, the reaction yields converting mevalonic acid to mevalonic acid-5-phosphate 0.3 mM (68 mg/L).
2.9 Production of Mevalonate-5-Disphosphate from Mevalonate-5-Phosphate using Immobilized Phosphomevalonate Kinase (PMVK) (FIG. 22): A reactor of 5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diameter containing immobilized PMVK enzyme (54 mg on 1.5 g of resin) was heated to 37° C. and allowed to equilibrate for 1 h while passing equilibration buffer (50 mM Tris, 5 mM MgCl₂, pH 8) through the reactor. After 1 h had subsided, the substrate solution (50 mM Tris, 5 mM MgCl₂, pH 8, 4 mM ATP, 4 mM mevalonic acid-5-phosphate) was flowed 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 recycle through the individual reactor. The reaction was allowed to proceed for 32 hours. For sampling, the reaction fluid was examined on a HPLC system to examine the amount of ATP and ADP present in the reaction mixture. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm×3 mm equipped with a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to 25° C. with the sample block being maintained at 4° C. HPLC method consisted of 5 μl sample injection volume and an isocratic mobile phase comprised of 100 mM KH₂PO₄, 8 mM TBAHS, pH 6.0, 20% methanol (v/v). The run time was a total of 10 mins with ATP eluting at 5.7 mins and ADP eluting at 4.6 minutes. A diode array detector (Agilent) was used for the detection of the molecule of interest at 254 nm. Upon 32 h of reactivity, the reaction converted mevalonic acid to mevalonic acid-5-pyrophosphate (2.1 mM, 697 mg/L).
2.10 Production of Isopentenyl Pyrophosphate from Mevalonate-5-Diphosphate using Immobilized Diphosphomevalonate Kinase (MDC) (FIG. 23): A reactor of 5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diameter containing immobilized MDC enzyme (30 mg on 1.5 g of resin) was heated to 37° C. and allowed to equilibrate for one hour while passing equilibration buffer (50 mM Tris, 5 mM MgCl₂, pH 8.0) through the reactor. After 1 h had subsided, the substrate solution (50 mM Tris, 5 mM MgCl₂, pH 8, 4 mM ATP, 4 mM mevalonic acid-5-pyrophosphate) was flowed 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 recycle through the individual reactor. The reaction was allowed to proceed for 16 hours. For sampling, the reaction fluid was examined on the HPLC system to examine the amount of ATP and ADP. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm×3 mm equipped with a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to 25° C. with the sample block being maintained at 4° C. HPLC method consisted of 5 μl sample injection volume and an isocratic gradient comprised of 100 mM KH₂PO₄, 8 mM TBAHS, pH 6.0, 20% methanol (v/v) was used as the mobile phase. The run time was a total of 10 mins with ATP eluting at 5.7 mins and ADP eluting at 4.6 minutes. A diode array detector (Agilent) was used for the detection of the molecule of interest at 254 nm. Upon 16 h of reactivity, mevalonic acid-5-pyrophosphate was converted into isopentenyl pyrophosphate at 0.9 mM (237 mg/L).
2.11 Production of Geranyl Pyrophosphate (GPP) from Dimethylallyl pyrophosphate using immobilized Farnesvl-PP svnthase (FPPS) and a reporter prenyl transferase for CBGA production (FIG. 24): A reactor of 5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diameter containing immobilized FPPS enzyme (68 mg on 1.5 g of resin) was heated to 25° C. and allowed to equilibrate for 1 h while passing equilibration buffer (50 mM Tris pH 8.0, 5 mM MgCl₂, 10 mM NaCl) through the reactor. After 1 h had subsided, the substrate solution (50 mM Tris, pH 8, 5 mM MgCl₂, 10 mM NaCl, 3.5 mM isopentenyl pyrophosphate, 3.5 mM dimethylallyl pyrophosphate) was flowed through the reactor at a flow rate of 10 μL/min with an intermittent pausing of 50 sec after each 10 seconds of pumping to meet the residence time of 4 hours (FIG. 24). Unlike other examples above, the reaction solution was not recycled in the experiment. The reaction solution collected after the completion of designated time was incubated with 3.5 mM olivetolic acid (OA) and 120 μM prenyl transferase (NphB) for 2 hours at 25° C. Completed reactions were extracted 3× with ethyl acetate, evaporated, and resuspended in methanol for analysis on a HPLC system to examine the amount of CBGA (cannabigerolic acid). The coupled reaction was able to convert 25% of the starting material to the product. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a 250 mm×4.6 mm, 5 μm liChrospher RP8 column equipped with a guard column. The column was heated to 30° C. with the sample block being maintained at 25° C. HPLC method consisted of 5 μl sample injection volume and an isocratic mobile phase comprised of 25% buffer A (water, 0.1% formic acid, 5 mM ammonia formate) and 75% buffer B (acetonitrile, 0.1% formic acid, 5 mM ammonia formate) was used as the mobile phase. CBGA produced in the reaction was measured using DAD at 228 nm. The run time was a total of 10 minutes with CBGA eluting at 3.68 mins. Upon the analysis of the coupled reactions, the reaction yielded 0.9 mM or 285 mg/L of the final product, CBGA.
2.12 Removal of oxygen from a reaction stream prior to an individual reactor: In some instances, the partial pressure of gaseous oxygen in the reaction mixture must be decreased to avoid enzyme deactivation. Thus, prior to a reaction solution being pumped through a reactor, the solution must be pumped through a deoxygenation and/or degassing device to decrease the partial pressure of oxygen in the reaction solution prior to the next reaction. The use of a deoxygenation and/or degassing machine when coupled with the system allowed the removal of up to 3.68 ppm of oxygen, a reduction of almost 50% (FIG. 17). For testing, deionized water passed through the deoxygenation/degassing device at a flow rate of 0.25 mL/min. This process takes approximately 20 mins to complete. In addition to the deoxygenation/degassing component, nitrogen gas may be introduced into a solution to further remove oxygen. Nitrogen gas input controlled by a mass flow controller is added to a solution prior to being pumped through a reactor (FIGS. 25A-25B).
2.13 Multi-step enzyme reaction to convert glycerol to pyruvic acid (FIG. 26): A reactor of 14″ in length with a 0.75″ outside diameter and a 0.652″ internal diameter containing immobilized Aldo enzyme (2.7 g enzyme on 56 g of resin) was heated to 37° C. and equilibrated for six hours with equilibration buffer (50 mM tris, pH 8.5) by pumping it through the reactor. After this time, the substrate solution (40 mL, 50 mM Tris, pH 8.5 20 mM glycerol) was flown through the reactor at a flow rate of 10 μL/min. Prior to entering the reactor, the substrate solution was mixed with an equal flow rate of compressed air (0.01 sccm) to yield a total flow rate through the reactor of 20 μL/min (8 hour residence time). 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, pH buffer (50 mM Tris, pH 12.0) was added into the reaction flow using a T-piece mixer with a pH buffer solution flow rate of 10 μL/min. The resulting fluid now pH adjusted is held momentarily in a stirred tank reactor (CSTR) where it was degassed by using a bubbling nitrogen flow. Finally, another pump draws the fluid from the CSTR into the second reactor of 14″ in length with a 0.75″ outside diameter and a 0.652″ internal diameter containing immobilized DHAD enzyme (1.8 g enzyme on 56 g of resin) heated at 45° C. The total flow rate of reaction mixture through the reactor was 10 μL/min (16.25-hour residence time). The solution was collected at the end of the second reactor into a glass beaker. The reaction mixture was then analyzed every 24 hours. For sampling, 100 μl of the reaction fluid was examined on a HPLC system to examine the amount of glycerol, glyceric acid, and pyruvic acid. The HPLC method was as follows: An Agilent 1200 HPLC was equipped with a 30 cm Aminex HPX-87H column and a micro-guard 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 comprised of 100% sulfuric acid (10 mM) was used. The sample run time was a total of 45 minutes with glyceric acid eluting at 17.2 mins, glycerol eluting at 21.0 minutes, and pyruvic acid eluting at 15.8 mins. For detection, a RID detector (Agilent) was used after a 2 h equilibration period produced 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 pyruvic acid at a final concentration of 2 mM. We found that the ALDO reactor was able to achieve 98% conversion levels for 3 days with no loss in productivity and remained able to do the enzyme conversion for 10 days of continual processing.

EMBODIMENTS

The following embodiments are intended to be illustrative only and not to be limiting in any way.
Embodiment 1: A cell-free manufacturing platform for chemical production, the platform comprising: one or more individual reactors, wherein each of the one or more individual reactors comprises: a cylindrical tube comprising a first end and a second end, wherein both the first end of the cylindrical tube and the second end of the cylindrical tube comprise fittings, wherein a cylindrical tube interior of the individual reactor comprises: a resin and an enzyme and, a pumping system adapted to flow a solution through the one or more individual reactors, wherein each of the one or more individual reactors has an input tubing connected at the first end of the cylindrical tube and an output tubing connected at the second end of the cylindrical tube to create a closed system.
Embodiment 2: The platform of Embodiment 1, wherein the fittings are stainless steel fittings.
Embodiment 3: The platform of Embodiment 1, wherein the cylindrical tube interior of the individual reactor further comprises one or more sensors.
Embodiment 4: The platform of Embodiment 3, wherein the one or more sensors comprises a temperature sensor, a pH sensor, a pressure sensor, a flow rate sensor, a dissolved oxygen (DO) sensor, a spectrometer, or a combination thereof.
Embodiment 5: The platform of any one of Embodiments 1-4, wherein each of the one or more individual reactors comprises an individual reactor housing.
Embodiment 6: The platform of Embodiment 5, wherein the individual reactor housing surrounds and is fastened to the individual reactor.
Embodiment 7: The platform of any one of Embodiments 1-6, further comprising a temperature altering element attached to the individual reactor housing or the individual reactor.
Embodiment 8: The platform of Embodiment 7, wherein the temperature altering element is a thermoelectric cooler (TEC).
Embodiment 9: The platform of any one of Embodiments 1-8, further comprising a spectrometer attached in series with the one or more individual reactors.
Embodiment 10: The platform of any one of Embodiments 1-9, further comprising a degassing module adapted to remove gasses from the solution.
Embodiment 11: The platform of Embodiment 10, wherein the degassing module is a deoxygenation module.
Embodiment 12: The platform of Embodiment 11, wherein the deoxygenation module is adapted to remove oxygen from the solution.
Embodiment 13: The platform of any one of Embodiments 1-12, further comprising a gas addition module adapted to introduce gas into the solution.
Embodiment 14: The platform of any one of Embodiments 1-13, further comprising a pH module adapted to introduce an acid or base into the solution
Embodiment 15: The platform of any one of Embodiments 1-14, further comprising a graphical user interface (GUI) adapted to control automation software and hardware
Embodiment 16: The platform of any one of Embodiments 1-15, wherein the cell-free manufacturing platform is able to automatically change each of the one or more reactors conditions based on input from the sensors.
Embodiment 17: The platform of any one of Embodiments 1-16, wherein the enzyme comprises: MBP-Aldo (Aldo), Dihydroxy Acid Dehydratase (TvDHAD), Pyruvate Oxidase (PyOx), Acetyl-phosphate transferase (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), Prenyl transferase (NphB) or a combination thereof.
Embodiment 18: The platform of any one of Embodiments 1-17, wherein the enzymes are immobilized.
Embodiment 19: The platform of any one of Embodiments 1-17, wherein the enzymes are non-immobilized
As used herein, the term “about” refers to plus or minus 10% of the referenced number.
Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of” or “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of” or “consisting of” is met.

Claims

What is claimed is:

1. A cell-free manufacturing platform for chemical production, the platform comprising:

a) one or more individual reactors, wherein each of the one or more individual reactors comprises: a cylindrical tube comprising a first end and a second end, wherein both the first end of the cylindrical tube and the second end of the cylindrical tube comprise fittings, wherein an input tubing is connected at the first end of the cylindrical tube and an output tubing is connected at the second end of the cylindrical tube, wherein a cylindrical tube interior of the individual reactor comprises:

i) a resin; and

ii) an enzyme; and

b) a pumping system adapted to flow a solution through the one or more individual reactors.

2. The platform of claim 1, wherein the fittings are stainless steel fittings.

3. The platform of claim 1, wherein the cylindrical tube interior of the individual reactor further comprises one or more sensors.

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

5. The platform of claim 1, wherein each of the one or more individual reactors comprises an individual reactor housing.

6. The platform of claim 5, wherein the individual reactor housing surrounds and is fastened to the individual reactor.

7. The platform of claim 1, further comprising a temperature altering element attached to the individual reactor housing or the individual reactor.

8. The platform of claim 7, wherein the temperature altering element is a thermoelectric cooler (TEC).

9. The platform of claim 1, further comprising a spectrometer attached in series with the one or more individual reactors.

10. The platform of claim 1, further comprising a degassing module adapted to remove gasses from the solution.

11. The platform of claim 10, wherein the degassing module is a deoxygenation module.

12. The platform of claim 11, wherein the deoxygenation module is adapted to remove oxygen from the solution.

13. The platform of claim 1, further comprising a gas addition module adapted to introduce gas into the solution.

14. The platform of claim 1, further comprising a pH module adapted to introduce an acid or base into the solution.

15. The platform of claim 1, further comprising a graphical user interface (GUI) adapted to control automation software and hardware.

16. The platform of claim 1, wherein the cell-free manufacturing platform is able to automatically change each of the one or more reactors conditions based on input from the sensors.

17. The platform of claim 1, wherein the enzyme comprises: MBP-Aldo (Aldo), Dihydroxy Acid Dehydratase (TvDHAD), Pyruvate Oxidase (PyOx), Acetyl-phosphate transferase (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), Prenyl transferase (NphB) or a combination thereof.

18. The platform of claim 1, wherein the enzymes are immobilized.

19. The platform of claim 1, wherein the enzymes are non-immobilized.