Bioprocess systems and / or methods and optogenetic latching switches

Abstract

Bioprocess systems and methods are provided for industrial control of microbial metabolism using optogenetic photoswitches. A microbial culture is maintained in at least one culture vessel and circulated through an in-line illumination module defining a high surface-area-to-volume flow path that improves light penetration in turbid, high-cell-density broths. The illumination module may be a shell-and-tube device with parallel transparent conduits, a configuration in which culture flowing through the conduits can be illuminated, or a wetted-wall thin-film column. One or more light sources, including LEDs and / or scanned laser illumination, deliver one or more wavelengths to actuate optogenetic switches, including latching switches that maintain state after illumination. A recirculating pump sets exposure frequency and residence time. Optional sensors and a controller provide feedback control of illumination parameters, flow rate, feedstock addition, and product extraction, including solvent-based recovery of hydrophobic products.

Description

Docket: 0412-0005W01BIOPROCESS SYSTEMS AND / OR METHODS AND OPTOGENETIC LATCHING SWITCHESRELATED APPLICATIONS[oooi] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 749,894, filed on January 27, 2025, which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] Industrial-scale biomanufacturing commonly employs bioreactors and fermenters to grow microorganisms that convert feedstocks into products of interest. In many commercial processes, pathway expression and / or pathway flux is managed using process conditions (e.g., nutrient limitation, oxygen availability, temperature, pH) and / or inducible genetic regulation. Conventional inducible regulation frequently relies on chemical inducers or media additives. At production scale, however, chemical induction can introduce added raw-material cost, mixing and mass-transfer limitations, downstream purification burdens, and operational complexity, particularly where repeated switching between metabolic states is desired during a run.

[0003] Continuous and semi-continuous bioprocess architectures have been developed to improve productivity and reduce downtime, including systems that recirculate biomass and media through different unit operations. These architectures can be particularly useful for products that benefit from in situ removal (e.g., to reduce product toxicity, relieve feedback inhibition, or enable continuous product capture). Such systems often incorporate pumps, conduits, sensors, and control logic to maintain stable operation over extended runtimes.

[0004] For hydrophobic products, traditional fermentation frequently involves growing biomass, harvesting cells, and then extracting product in a downstream operation. To reduce the need for separate harvest-and-extraction steps, solvent-based extraction approaches have been explored in which product partitions into an immiscible organic phase. Implementing solvent extraction in a bioprocess environment, however, can present challenges including oxygen transfer limitations, multiphase flow management, emulsification / foaming control, solvent containment, and reliable separation of solvent from product and / or aqueous media.

[0005] A prior circulating bioreactor approach addressing certain hydrophobic-product challenges is described in previous patents numbers: US11987782B2 andDocket: 0412-0005W01WO2024036256A1, both of which are incorporated herein in their entirety by this reference. In those systems, biofilm biomass is provided as suspended biofilm particles, including biofilm on solid supports (e.g., beads) and / or self-aggregated biofilm, and is circulated between a synthesis (growth) vessel (or multiple synthesis vessels) and an extraction vessel containing an immiscible organic solvent. The architecture enables continuous or periodic extraction of hydrophobic products into the solvent phase while recirculating the aqueous phase and biomass back to the synthesis vessel(s). The prior systems further contemplate multiple synthesis vessels arranged in parallel and / or series, optional bubble trapping for gasliquid management, and separation arrangements (including membrane-based separation) to isolate product and recycle solvent.

[0006] Separately, optogenetic systems have emerged as a powerful class of genetic control technologies for microbial bioprocessing. Optogenetic regulation can use light-responsive proteins and / or domains to modulate gene expression or protein activity in response to illumination of one or more wavelengths. In microbial systems, optogenetic control is often implemented by coupling light-responsive elements to transcription factors and promoters to activate or repress transcription, and / or by controlling post-transcriptional or post-translational mechanisms. Such approaches can, in principle, provide rapid switching, tunable dose-response behavior, and multiplexed control using multiple wavelengths, while reducing or eliminating the need for chemical inducers.

[0007] Despite these advantages, applying optogenetic control at production scale presents significant engineering constraints. Industrial bioprocesses often operate at high cell densities and / or in turbid broths that strongly attenuate light through absorption and scattering, causing steep spatial gradients in photon flux. In more detail, for many optogenetic implementations, maintaining a desired regulatory state can require continuous illumination or a high illumination duty cycle. At industrial scale, attempting to illuminate substantially all cells continuously is often ineffective because photon flux is rapidly attenuated by absorption and scattering in high-density cultures, resulting in strong spatial gradients and subpopulations that receive insufficient doses. Continuous, large-area illumination, when required to maintain an induced state, can also increase thermal loads and energy consumption, may complicate reactor design (e.g., internal light sources, sterile optical interfaces, and cleaning / sterilization constraints), and may raise concerns regarding phototoxicity or undesirable side reactions.Docket: 0412-0005W01

[0008] In many cases, large-volume reactors can exhibit non-uniform induction, poorly defined “on / off ’ states, and batch-to-batch variability when illumination is attempted through external light sources.

[0009] Photobioreactor designs for photosynthetic organisms (e.g., algae) address light delivery using specialized geometries, internal illumination structures, and / or thin optical path lengths. However, many industrial microbial processes using non-photosynthetic hosts (e.g., bacteria or yeast) are not otherwise designed around light delivery, and thus may not readily accommodate optogenetic control without imposing substantial changes to reactor hardware, sterilization strategies, and process economics.

[0010] In addition, many optogenetic circuits used in laboratory settings are designed such that the induced state relaxes when illumination ceases. This often necessitate continuous or frequent illumination to maintain gene expression. While bistable and / or latching regulatory designs can reduce illumination duty cycle by maintaining a regulatory state after a triggering stimulus, reliably implementing such control in industrial bioprocesses remains a challenge.SUMMARY OF THE INVENTION

[0011] Considerations noted above motivate architectures that enable reliable optogenetic control using intermittent illumination delivered to flowing culture in a dedicated illumination module.

[0012] In light of existing approaches, there remains a need for improved bioprocess systems and methods that enable practical optogenetic control — preferably including intermittent illumination compatible with latching or bistable responses — under industrially relevant conditions such as high cell density and / or high turbidity. There also remains a need for architectures that can integrate optogenetic control with circulating process configurations (including, in some cases, recirculating extraction and other unit operations), while maintaining controllability, scalability, and economical operation. Also desirable are practical and scalable means for exposing cells to defined light doses (potentially at multiple wavelengths) with adequate uniformity, while maintaining overall process performance (e.g., mixing, gas transfer, feed delivery, and — where applicable — product extraction).

[0013] In intermittent illumination systems, microbial cells are briefly exposed to light to induce the desired response, allowing use of such systems with high cell density cultures, often reducing process costs associated with illumination.Docket: 0412-0005W01

[0014] The present invention encompasses an apparatus suitable for controlling an optogenetic bioprocess at industrial-scale with intermittent illumination. The apparatus includes a continuously or intermittently flowing system with several components, one of which is an illumination module. A second component is the main culture vessel that often holds the majority of cells and culture medium. Additional apparatus components may be installed including other vessels or pumps used for extraction of products or addition of process feedstocks. Apparatus configurations and additional components can include those described in previous patents numbers: US11987782B2 and WO2024036256A1, both of which are incorporated herein in their entirety by this reference.

[0015] In embodiments of the present invention, the cells and medium are recirculated through the main culture vessel, illumination module, and other apparatus components. The illumination module allows for flow of medium and cells through a space (region) having a high surface area to volume ratio. This enables the exposure of cells to the desired light wavelengths. In typical approaches, the surface area to volume ratio is high enough to achieve good light penetration into often turbid high density cell cultures. Continuous or intermittent flow of culture medium and cells allows for periodic illumination of cells to activate or deactivate photocontrolled systems (including optogenetic elements).

[0016] In a preferred embodiment, the apparatus is designed to work with latching photoswitches that enable use of transient illumination. In response to illumination, these latching photoswitches maintain state, such as activation of a pathway in the cells or deactivation of a pathway. Then the flowrate of the system, being controlled by a pump, for example, and can be adjusted to fit the specific requirements of each photocontrollable system where the period of illumination may need to be shorter or longer, depending on individual photosystems and the current photoswitch(es) being latched.

[0017] In some embodiments, the system employs optogenetic latching photoswitches that stay activated until a secondary signal (i.e. light, temperature, chemical) deactivates the genetic circuit. Latching photosystems can involve one or more mechanisms to accomplish the desired genetic regulatory effect. Regulatory effects can be imposed at the transcriptional or post-transcriptional level.

[0018] Optogenetic photo-active mechanisms often involve use of UV receptors, BLUF domains, opsins, light-oxygen-voltage (LOV) domains, cryptochromes, fluorescent proteins, cobalamin-binding domains, and / or phytochromes. These photo-active switches induce theDocket: 0412-0005W01desired genetic circuit either directly, or more commonly through one or more indirect mechanisms that enable transcriptional or post-transcriptional responses.

[0019] In some embodiments, the illumination model includes one or more optically transparent tubes that cells and culture medium flow through and are illuminated by one or more wavelengths of light. A shell-and-tube design, for instance, might include an outer opaque shell with internal illumination, and one or more transparent or translucent tubes that run through the internal compartment where the cell culture flows. This design increases surface area to volume ratio for improved light penetration and transmission into cell cultures. The shell and tube design can be of various geometries including cylindrical or rectangular tubes. A parallel-plate type design, with a high aspect ratio, gives rise to a short path-length for illumination. In some embodiments of the shell-and-tube illumination model, a mirrored surface is used on the inside surface of the opaque shell and / or between tubes to increase reflection of light within the device to aid in illumination uniformity of the cell culture.

[0020] In still other embodiments of the shell-and-tube illumination model design, the inner tubes are or house the illumination source (such as fiber optic cables or other tube-like light sources) and the cells and culture medium flow around between the outside (shell side) of the inner tubes and the inside (inner) surface of the opaque shell.

[0021] In some embodiments, the illumination model employs a thin-film flowing fluid illuminated by one or more wavelengths. The thin-film may be produced in a falling-film (or wetted-wall) column that is equipped to illuminate the thin-film that is generated. In this approach, the thin-film column has an opaque shell with internal illumination directed at the wetted-wall that supports the flow of the thin film of cell culture. This design increases the surface area to volume ratio using the thin-film mechanism to enable improved light penetration into the cell culture. In some implementations of the thin-film illumination module, the illumination source is located on the wetted wall, across the wetted-wall, and / or employs mirrors inside the opaque shell inner surface to reflect light inside the illumination module.

[0022] In other embodiments the apparatus may have a component for separation of gas and liquid phases, known as a bubble trap. The thin-film illumination model can be used as a modification to such a bubble trap system, where the bubble trap is made opaque or outfitted with mirrors and at least one illumination source. The bubble trap has an inlet flow of cellsDocket: 0412-0005W01and culture media that fall down the side of the bubble trap, creating the thin-film type wetted-wall. This allows separation of the gas phase which leaves via a vent in the bubble trap, illumination of the thin-film, and at least one liquid outlet in or near the bottom of the bubble trap illumination module.

[0023] In some embodiments, the apparatus may be used to induce optogenetic elements in a latching switch structure to activate or deactivate transcription of feedstock (i.e. carbon, nitrogen, sulfur, and / or phosphate containing feed materials) catabolism genes and / or genes related to production of a desired bioproduct.

[0024] In some embodiments the cell culture is an engineered fungal culture, including yeast. Yeast can include strains from the genera Yarrowia (e.g., Yarrowia lipolytica), Saccharomyces (i.e. Saccharomyces cerevisiae), Komagataella (i.e. Komagataella pastoris), Pichia, Candida, Schizosaccharomyces (i.e. Schizosaccharomyces pombe), Rhodotorula (i.e. Rhodotorula glutinis and Rhodotorula mucHaginosa , and Lipomyces (i.e. Lipomyces starkeyi). Other fungal strains can include those from genera Aspergillus (i.e. Aspergillus niger, Aspergillus wentii, Aspergillus oryzae, and Aspergillus terreus), Penicillium (i.e. Penicillium chrysogenum and Penicillium rubens), Trichoderma (i.e. Trichoderma reesei), avAMucor (i.e. Mucor indicus).

[0025] In some embodiments the cell culture is an engineered bacterial culture. Bacteria can include strains from the genera Escherichia (i.e. Escherichia coll), Marinobacter (i.e. Marinobacter atlanticus), Pseudomonas, Vibrio, Bacillus (i.e. Bacillus subtilis), and Cyanobacteria (i.e Synechocystis, Anabaena, Arthrospira, and Cyanothece).

[0026] In some embodiments the cell culture is an algae, such as strains from the genus Scenedesmus, Chlorella (i.e. Chlorella protothecoides and Chlorella vulgaris), Desmodesmus, Dunaliella, Nitzschia, Phormidium, Pseudokirchneriella, Euglena, and Haematococcuspluvialis .

[0027] In some embodiments, the apparatus is fitted with process sensors, such as Raman spectrophotometers or electrochemical sensors, that monitor process conditions. The outputs of these sensors are used to control which latching photoswitches are activated and deactivated by modulating wavelengths of light in the illumination module.

[0028] In some embodiments, the engineered microorganisms comprise at least one biosensor circuit that produces a measurable output indicative of a process state, such as product concentration, intermediate concentration, pathway flux, stress response, orDocket: 0412-0005W01contamination. The biosensor circuit may be placed under control of an optogenetic photoswitch so that biosensor expression and / or biosensor activation occurs only during periodic illumination in the illumination module, thereby reducing cellular burden during non-illuminated periods. A process sensor may measure the biosensor output (e.g., fluorescence, luminescence, absorbance, electrochemical signal, or secreted marker concentration) and provide the output to a controller for closed-loop adjustment of feedstock addition, recirculation rate, illumination parameters, and / or extraction conditions.

[0029] The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as a limitation of the invention. The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Of the drawings:

[0031] Fig. 1 is a schematic view of a photocontrolled / photoswitching circulating bioreactor system according to aspects of the present invention;

[0032] Fig. 2 shows one embodiment of the illumination module, i.e., a shell-and-tube illumination module;

[0033] Fig. 3 shows another embodiment of the illumination module, i.e., thin-film illumination module;

[0034] Fig. 4 shows another embodiment of the photocontrolled / photoswitching circulating bioreactor, i.e., a bioreactor photocontrolled bioproduction system for chemicals from various feedstocks;

[0035] Fig. 5 is a schematic diagram showing a photoswitching genetic circuit that could be employed in the instant photocontrolled circulating bioreactor;

[0036] Fig. 6 is a schematic diagram illustrating a dual-wavelength optogenetic latching switch that could be employed in the disclosed photocontrolled circulating bioreactor;Docket: 0412-0005W01

[0037] Fig. 7 is a schematic diagram showing a dual-wavelength optogenetic latching with a bidirectional promotor that is used with the disclosed the photocontrolled circulating bioreactor in some examples;

[0038] Fig. 8 is a schematic diagram showing a single-wavelength optogenetic latching switch with chemical reset that is used with the disclosed photocontrolled circulating bioreactor in some examples; and

[0039] Figs. 9A, 9B, and 9C are schematic top cross section views of different embodiments of the illumination module.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0041] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Also, all conjunctions used are to be understood in the most inclusive sense possible. Thus, the word "or" should be understood as having the definition of a logical "or" rather than that of a logical "exclusive or" unless the context clearly necessitates otherwise. Further, the singular forms and the articles "a", "an" and "the" are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and / or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groupsthereof. Further, it will be understood that when an element, including component or subsystem, is referred to and / or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

[0042] It will be understood that although terms such as “first” and “second” are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, an elementDocket: 0412-0005W01discussed below could be termed a second element, and similarly, a second element may be termed a first element without departing from the teachings of the present invention.

[0043] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0044] Fig. 1 is a schematic view of a photocontrolled / photoswitching circulating bioreactor system 100 according to embodiments of the present invention. The system 100 generally provides (i) a primary cultivation environment in which an engineered microorganism is grown at industrially relevant scale and density, and (ii) a recirculation loop that periodically exposes at least a portion of the circulating culture to a defined light dose (including one or more wavelengths) to activate and / or deactivate one or more photocontrolled genetic circuits, including optogenetic latching switches. In preferred implementations, the primary cultivation environment is maintained with minimal or no illumination, while the desired optical stimulus is delivered in a dedicated illumination module that is optimized for light penetration in turbid, high-cell-density broths.

[0045] In the illustrated embodiment, the system 100 includes a main culture or synthesis vessel 12, an illumination module 112, a recirculating pump 24, and additional apparatus components 116, which can include a product extractor 14. These components are fluidically coupled by process piping 118 to form a closed or semi -closed circulation loop, with flow direction indicated by arrows. In operation, culture broth (including cells and culture medium) is circulated from the main culture vessel 12, through the illumination module 112 for intermittent light exposure, then through the pump 24 and one or more additional components 116 (optionally including a product extractor 14) and returned to the main culture vessel 12. The circulation can be continuous or intermittent, and the illumination can be continuous, pulsed, or otherwise time-modulated to implement a desired optogenetic control strategy.

[0046] The main culture or synthesis vessel 12 is a primary bioprocess vessel configured to hold a majority of the working volume of the system 100 and to provide the principal environment for cell growth and / or bioproduction. Physically, the vessel 12 may beDocket: 0412-0005W01implemented as a stirred tank (e.g., a stainless steel sanitary fermenter), an airlift vessel, a bubble column, or another suitable industrial bioreactor geometry, and may include conventional features such as a headplate with sanitary ports, one or more inlets for media, inoculum, antifoam, and / or gases, one or more outlets, and a bottom drain. The vessel 12 may further include agitation (e.g., one or more impellers), sparging / aeration hardware (e.g., spargers, diffusers), a thermal jacket and / or internal heat-exchange coils, and / or cleaning / sterilization features (e.g., CIP / SIP (clean-in place / sterilization-in-place) hardware). Functionally, the vessel 12 maintains the culture under conditions (e.g., temperature, pH, dissolved oxygen, nutrient levels) suitable for growth and production, while the optogenetic “state changes” are effected primarily by controlled illumination that occurs when circulating culture passes through the illumination module 112, rather than by attempting to illuminate the full bulk volume of vessel 12.

[0047] Process piping 118 fluidically couples the main culture vessel 12, illumination module 112, recirculating pump 24, and additional apparatus components 116 into a circulation loop. Physically, the process piping 118 may comprise sanitary tubing (e.g., stainless steel tubing, single-use polymer tubing, hoses, and / or rigid manifolds) and may include fittings (e.g., tri-clamp sanitary fittings), aseptic connectors, sample ports, sterile filters for gas lines (where applicable), and optional inline instrumentation (not shown) such as pressure sensors, temperature probes, turbidity / optical density sensors, flow meters, and / or spectroscopic probes. Functionally, process piping 118 defines the flow path and residence time between modules, enables recirculation of cells and medium, and provides locations for optional control elements (not shown) such as valves, bypass lines, drain lines, and isolation points to support operation, cleaning, and / or maintenance.

[0048] Inlet tube 118-1 is the upstream segment of process piping 118 that delivers circulating culture (cells and culture medium) into the illumination module 112. Physically, inlet tube 118-1 may be sized and routed to provide a desired flow regime through the illumination module 112 (e.g., laminar, transitional, or turbulent flow depending on the illumination geometry and shear tolerance of the cells). In some embodiments, inlet tube 118-1 includes (not shown) an upstream distribution element, splitter manifold, or flow conditioner to ensure that the culture is evenly distributed across multiple flow paths within the illumination module 112 (for example, across an array of parallel tubes or channels), thereby improving uniformity of light exposure across the circulating biomass.Docket: 0412-0005W01

[0049] The illumination module 112 is a dedicated illumination device constructed to expose flowing culture to one or more wavelengths of light while maintaining sterility and compatibility with industrial bioprocess operation. Physically, module 112 is preferably constructed as a closed, cleanable / sterilizable flow-through unit that provides a high surface-area-to-volume ratio and a short optical path length through the culture, thereby enabling effective light penetration even in high optical density, turbid broths. In some embodiments, module 112 is implemented as a shell-and-tube design (e.g., multiple transparent or translucent tubes carrying culture within an opaque housing that contains one or more light sources); as a thin-film (wetted-wall / falling-film) illumination column; or another high-aspect-ratio flow geometry, as further illustrated elsewhere in the application. Functionally, module 112 delivers a controlled light “dose” (wavelength(s), intensity, pulse timing, exposure duration), as specified by controller 20, to activate and / or deactivate optogenetic elements — including latching switches — so that desired metabolic pathways (e.g., feedstock utilization pathways and / or product synthesis pathways) can be turned on, turned off, or toggled between states, without requiring bulk illumination of the main vessel 12.

[0050] Outlet tube 118-2 is the downstream segment of process piping 118 that receives culture exiting the illumination module 112 and conveys the illuminated culture onward through the circulation loop. Physically, outlet tube 118-2 may include (not shown) optional inline features such as a pressure relief element, a temperature sensor (to monitor any heat input from illumination), a back-pressure regulator, and / or a static mixer to recombine streams where the illumination module 112 employs parallel channels. Functionally, outlet tube 118-2 transports culture that has been optically “switched” (or partially switched) back into the bulk process so that the optogenetically induced state can be expressed in the main culture vessel 12 and / or acted upon in downstream unit operations (e.g., extraction, feeding, conditioning).

[0051] The recirculating pump 24 provides the motive force that drives circulation of culture through the loop comprising vessel 12, illumination module 112, and additional components 116. Physically, pump 24 may be implemented as a sanitary centrifugal pump, a peristaltic pump, a diaphragm pump, a progressive cavity pump, or another pump suitable for sterile bioprocess use, with materials selected for chemical compatibility with the culture medium and any products / solvents that may be encountered in the loop. In preferred embodiments, pump 24 is controlled (e.g., variable speed) by controller 20 to adjust flow rate and thus control (i) the frequency with which cells pass through the illumination module 112,Docket: 0412-0005W01(ii) the residence time within the illumination module 112, and (iii) the overall circulation time constant of the system 100. This enables matching the optical stimulation schedule to the kinetics of the optogenetic system (including latching behavior) and also enables operational adjustments in response to process conditions (e.g., cell density, oxygen demand, product accumulation).

[0052] In some embodiments, the illumination module 112 is additionally configured to perform microbial inactivation for sanitization and / or waste treatment. For example, the illumination source 158 (described below) may be operable to emit an antimicrobial wavelength (e.g., UV and / or visible wavelengths suitable for photoinactivation) to inactivate contaminating organisms in a purge stream, harvest stream, waste stream, or recirculation loop segment. In some embodiments, microbial inactivation is enhanced by inclusion of a photosensitizer in a treated stream to enable photodynamic inactivation, with treated material directed to a disposal or waste treatment subsystem. Such sanitization cycles may be executed intermittently (e.g., between production phases, during downtime, or upon detection of contamination) while maintaining sterility and protecting the primary culture when desired.

[0053] Additional apparatus components 116 represent one or more optional unit operations and / or auxiliary process modules that are installed in-line (as shown) or otherwise integrated with system 100 and controlled by controller 20. Physically, components 116 can include, by way of non-limiting examples, one or more of: feedstock addition modules (e.g., metering pumps and injection tees), nutrient and pH adjustment modules, heat exchangers, bubble traps or gas-liquid separators, inline sensors (e.g., spectroscopic probes such as Raman, electrochemical probes, optical density probes), sample ports, and / or associated actuators operated by the controller 20 for feedback control of flow rate, illumination wavelength selection, and / or illumination duty cycle. In some embodiments, components 116 include process equipment for product extraction and handling, including solvent management and / or phase separation equipment. For example, in recirculating architectures, integrating components such as (i) synthesis / growth vessel(s), (ii) extraction vessels containing an immiscible organic solvent for hydrophobic product capture, (iii) optional bubble traps to reduce emulsification and manage gas carryover, and (iv) optional solvent / product separation loops (including membrane-based separation) facilitate the isolation of product and recycling of solvent.

[0054] Product extractor 14 is one example of an additional apparatus in component 116; it can be configured to remove, recover, and / or concentrate one or more products ofDocket: 0412-0005W01interest from the circulating culture. Physically, extractor 14 may be implemented in various forms depending on the product and host organism, including (without limitation) a two-phase liquid-liquid extraction vessel, a membrane contactor, an adsorption column, a filtration module, a centrifugation module, or combinations thereof. In embodiments directed to hydrophobic products, extractor 14 may be implemented as a solvent extraction unit in which an immiscible organic solvent contacts the culture to partition hydrophobic product into the organic solvent phase, while an aqueous / cell -containing phase is recirculated back toward the main culture vessel 12. In such embodiments, extractor 14 can incorporate features including an extraction vessel geometry supporting stable phase separation (e.g., organic solvent phase above an aqueous phase), optional headspace handling and solvent vapor management (e.g., condensation and / or solvent capture elements), optional inerting to reduce oxidation for oxygen-sensitive products, and optional downstream separation equipment (e.g., membrane-based separation) to separate product from solvent and recycle solvent back to the extractor. Functionally, extractor 14 enables in situ or continuous product removal so that the optogen etically controlled bioprocess in vessel 12 can proceed with reduced product inhibition and / or reduced product toxicity, so that downstream processing load can be reduced by concentrating product during the run.

[0055] This system 100 is often configured to allow for controlled growth of organisms, such as, for instance, Yarrow ia lipolytica or Marinobacter allanlicus. where the organisms are engineered with latching optogenetic switches that regulate feedstock utilization enzymatic pathways and / or product formation enzymatic pathways.

[0056] Fig. 2 shows one embodiment of the illumination module 112, implemented as a shell-and-tube illumination module for use in the photocontrolled / photoswitching circulating bioreactor system 100. In this embodiment, cell culture (cells plus culture medium) is flowed through a plurality of optically transparent or translucent tubes 154 contained within an opaque shell or housing 150, while one or more illumination sources 158 deliver one or more selected wavelengths to the tubes to provide a controlled optical stimulus to the flowing culture (e.g., Yarrowia lipolytica cultures of 600 nm optical densities of >20). This geometry is configured to provide a high surface-area-to-volume ratio and a short optical path length, which can improve light penetration and dose uniformity in turbid, high-cell-density cultures.

[0057] The illumination module 112 is preferably a closed, sanitary, flow -through unit of generally cylindrical or other suitable geometry designed for installation in-line with process piping 118 of the circulating system 100. Physically, the module 112 can beDocket: 0412-0005W01constructed as a cartridge-like assembly (e.g., with removable end caps or headers) that houses the splitter 152, tube bundle 154, and mixer / collector 156 within the shell 150, and that provides external interfaces for inlet and outlet tubing 118-1, 118-2. Functionally, module 112 is the location where optical dosing is concentrated: rather than attempting to illuminate the full working volume of the main culture vessel, a fraction (or all) of the circulating broth is repeatedly passed through module 112 so that cells receive intermittent and / or periodic illumination appropriate to activate and / or deactivate one or more optogenetic circuits (including latching switches).

[0058] The shell or housing 150 is an outer enclosure that surrounds the tube bundle 154 and defines an internal illumination chamber. The shell 150 is preferably opaque to confine illumination within the module and reduce unintended light exposure elsewhere in the process (e.g., in the main vessel or upstream / downstream piping). In various embodiments, shell 150 is cylindrical, rectangular, or another geometry selected for manufacturability and / or for arranging light sources relative to the tube bundle. The shell 150 can be fabricated from stainless steel or other sanitary metals, or from polymeric materials compatible with cleaning and sterilization protocols. In some embodiments, an inner surface of shell 150 includes a reflective and / or mirrored region to increase reflection and improve illumination uniformity across the tube bundle. Functionally, shell 150 provides mechanical support, environmental isolation, and (when reflective) optical efficiency, enabling the illumination module 112 to operate as a dedicated “light dosing chamber” within a recirculating industrial bioprocess.

[0059] Inlet tube 118-1 is the upstream fluid connection that introduces the circulating cell culture into the illumination module 112. Physically, inlet tube 118-1 can be implemented as sanitary tubing or piping connected to the module using aseptic fittings, clamps, gaskets, and / or sterile connectors, and it may pass through or terminate at an inlet port on shell 150. In some embodiments, inlet tube 118-1 includes (or is adjacent to) optional instrumentation (not shown) such as a flow sensor, pressure sensor, temperature sensor, or optical density sensor to support control of residence time and verification of operating conditions. Functionally, inlet tube 118-1 delivers a defined flow of culture into the module so that cells can be exposed to light in a controlled, repeatable manner as part of the circulation loop driven by the recirculating pump (e.g., pump 24 in Fig. 1).

[0060] The splitter 152 is a flow-distribution structure that receives the incoming stream from inlet tube 118-1 and divides it into a plurality of substantially parallel flow pathsDocket: 0412-0005W01through tubes 154. Physically, splitter 152 can be formed as a header, a manifold block, a distribution plate, or a molded / printed plenum with multiple outlets corresponding to the individual tubes. In some embodiments, splitter 152 includes flow -balancing features (e.g., restrictors, equal -length channels, orifices, or tuned outlet geometries) so that each tube 154 receives approximately similar flow rate and residence time. These measures can improve dose uniformity across the circulating biomass. Functionally, splitter 152 enables division and parallelization of the flow to increase illuminated surface area and reduce optical path length for each sub-stream, thereby improving illumination effectiveness in high turbidity broths.

[0061] Tubes 154 are the primary illuminated flow conduits within the shell-and-tube illumination module 112. Physically, tubes 154 are preferably fabricated from optically transmissive materials such as glass, quartz, or suitable transparent / translucent polymers (e.g., fluoropolymers), and are sized to provide a short optical path length through the flowing culture. In one configuration, the light traversing each tube is orthogonal to the flow. The tubes 154 can be arranged as a bundle in parallel, as depicted, and can be straight, curved, or otherwise shaped to increase illuminated area within a given housing footprint. In some embodiments, the tubes are removable and / or replaceable to support cleaning, maintenance, and scalability (e.g., increasing the number of tubes accommodates higher flow rates). Functionally, tubes 154 provide high surface-area-to-volume ratio flow paths so that cells passing through the tubes experience light exposure with reduced shading relative to bulk-vessel illumination, enabling intermittent illumination strategies suited for optogenetic switching (including latching systems).

[0062] In some embodiments, each tube 154 has an internal diameter selected to balance light penetration, flow capacity, and fouling resistance. By way of non-limiting example, the tubes 154 may have an internal diameter in a range of about 0.125 inches (about 3.2 mm) to about 1 inch (about 25.4 mm), preferably about 0.25 inches (about 6.4 mm) to about 1 inch (about 25.4 mm). Tube length may be selected to provide a target illumination residence time and may be in a range of about 1 foot (about 0.30 m) to about 10 feet (about 3.05 m); the length can be optionally increased by routing a tube 154 in a coiled or multi-pass configuration within the housing 150. For larger working volumes and / or higher recirculation flow rates, the illumination module 112 may be scaled analogously to a shell-and-tube heat exchanger by increasing the number of tubes, the tube length, and / or the illuminated footprint so that a desired optical dose is delivered to a desired fraction of cells for a desired durationDocket: 0412-0005W01without requiring an excessive optical path length through the culture. In many cases, the tube lengths and flow rates represent the primary tunable variables.

[0063] The illumination source 158 generates emissions at one or more wavelengths selected to activate and / or deactivate optogenetic elements in the circulating culture.Physically, illumination source 158 can include one or more LED arrays, laser sources, diode laser arrays, fiber-coupled light engines, or other illumination devices positioned on or within shell 150 to direct light toward the tubes 154. In certain embodiments, the illumination source 158 includes multiple independently addressable wavelength channels to support multiplexed control of multiple photoswitches, including two- wavelength activation / deactivation schemes. In operation, illumination source 158 can be controlled to deliver continuous, pulsed, or time-varying illumination (intensity and / or duty cycle) by the controller 20 so that exposure is matched to the switching kinetics of the optogenetic circuit and to the circulation rate through module 112. In some embodiments, illumination source 158 (and / or associated optics) is implemented to increase illumination uniformity, including use of reflective inner surfaces of shell 150 and / or placement of light emitters around the tube bundle.

[0064] Mixer 156 is a flow-collection structure that receives the individual sub-streams exiting the parallel tubes 154 and recombines them into a single outlet stream. Physically, mixer 156 can be a header, plenum, or manifold block, and may optionally include passive mixing features (e.g., baffles, static mixing elements, or angled channels) to reduce tube-to-tube concentration differences and to homogenize the illuminated fraction before it returns to the remainder of the process. Functionally, mixer 156 (i) consolidates flow for return to the process, (ii) promotes uniformity of the illuminated culture leaving the module, and (iii) can reduce the risk that only a subpopulation of the culture receives effective light dosing due to maldistribution or channeling.

[0065] Outlet tube 118-2 is the downstream fluid connection that conveys illuminated culture out of the illumination module 112 and back into the circulation loop of system 100 (e.g., toward the main culture vessel and / or additional apparatus components).

[0066] Although Fig. 2 depicts the illumination module 112 as a stand-alone unit, it is preferably integrated within a broader circulating process architecture (as in Fig. 1), an architecture that can include pumps, sensors, and additional unit operations (e.g., extraction equipment). The illumination module 112 can be deployed as an in-line specialty module that adds a controllable optical “unit operation” to the circulation loop. In certain embodiments,Docket: 0412-0005W01sensor data (including, in some cases, data from sensors that circulate with process flow) can be used to adjust flow rate and / or illumination parameters (wavelength selection, intensity, pulse timing), thereby coordinating optogenetic switching with the broader process state and downstream unit operations.

[0067] Fig. 3 shows another embodiment of the illumination module 112, implemented as a thin-film (wetted-wall / falling-film) illumination module for use in the photocontrolled / photoswitching circulating bioreactor system 100. In this embodiment, a circulating cell culture (cells plus culture medium) enters the module 112 via inlet tube 118-1, is distributed to form a thin fluid film 160 that flows along an interior surface of the module, and is illuminated by one or more illumination sources 158 while in the thin-film state. The illuminated culture is then collected at an outlet collector 156 and discharged via outlet tube 118-2 back into the recirculating process. This thin-film geometry can provide a high surface-area-to-volume ratio and short optical path length, thereby improving light penetration and dose uniformity in turbid, high-cell-density cultures.

[0068] The illumination module 112 is preferably a closed, sanitary, flow -through device configured for in-line installation in process piping (e.g., piping 118 in Fig. 1).Physically, the module 112 may be configured as a generally vertical column, cartridge, or chamber having an upper region that receives the incoming stream, a central region in which the thin fluid film 160 is formed and illuminated, and a lower region that funnels and recombines the flowing thin film into a collected outlet stream at outlet collector 156.Functionally, module 112 provides a controlled optical dosing environment that enables intermittent illumination strategies suitable for optogenetic switching (including latching photoswitches) without requiring bulk illumination of the main culture vessel. The module 112 can be operated continuously or intermittently, and the illumination source 158 can be controlled to deliver continuous, pulsed, or time-modulated light of one or more wavelengths selected to activate and / or deactivate optogenetic circuits.

[0069] The shell or housing 150 forms the outer body of the thin-film illumination module 112 and defines an internal chamber in which the thin fluid film 160 is generated and illuminated. Physically, shell 150 is preferably constructed as an opaque enclosure to confine light within the illumination module and reduce unintended illumination of other process components. In various embodiments, shell 150 may be fabricated from stainless steel or other sanitary metals, or from polymeric materials compatible with cleaning and sterilization protocols (e.g., CIP / SIP). The internal geometry of shell 150 can be selected to encourageDocket: 0412-0005W01formation of a stable thin film, including smooth walls, textured walls, wettability-enhancing coatings, and / or internal structures that promote uniform wetting. In some embodiments, one or more internal surfaces of shell 150 include reflective and / or mirrored regions to improve light utilization and illumination uniformity by reflecting photons back toward the thin film region. Functionally, shell 150 provides mechanical support, sterility containment, optical isolation, and (where reflective) improved optical efficiency for illumination of the thin fluid film 160.

[0070] In some embodiments, the thin-film illumination module 112 is implemented as a vertical column having dimensions selected to provide stable thin-film formation and sufficient illuminated residence time. By way of non-limiting example, the column may have a height in a range of about 3 feet (about 0.91 m) to about 20 feet (about 6.10 m) and an internal diameter in a range of about 0.5 feet (about 0.15 m) to about 2 feet (about 0.61 m). Multiple thin-film columns may be installed in parallel to accommodate higher volumetric circulation rates while maintaining a target thin-film residence time and thin-film thickness. As with the tube-based embodiment, the thin-film module 112 may be scaled to larger process volumes by increasing column height and / or diameter and / or by deploying multiple modules in parallel so that adequate optical dosing is achieved without bulk illumination of the main culture vessel 12.

[0071] Inlet tube 118-1 delivers the circulating cell culture into the thin-film illumination module 112. Physically, inlet tube 118-1 is preferably a sanitary connection (e.g., tubing, piping, or a manifold connection) that couples to an inlet port of shell 150 and is sized to provide a desired flow rate and inlet velocity. In some embodiments, inlet tube 118-1 terminates in (or is fluidically coupled to) an integrated distribution feature such as a slot, weir, spray nozzle, distribution ring, perforated header, or flow spreader that directs incoming culture to the interior surface of shell 150 to initiate formation of the thin fluid film 160. Functionally, inlet tube 118-1 supplies culture to the module in a manner that supports repeatable thin-film formation and predictable residence time in the illuminated region, thereby enabling consistent optical stimulation of cells passing through the module.

[0072] The thin fluid film 160 is a flowing, wetted-wall layer of culture medium and cells formed on an interior surface of the module 112. Physically, film 160 is created as culture introduced at the upper region of the module spreads over and flows downward along the interior surface under gravity (and / or under the influence of pressure-driven flow), forming a thin layer with a short optical path length. Film thickness may be influenced byDocket: 0412-0005W01flow rate, viscosity, surface tension, wettability of the wall surface, and the internal geometry of shell 150. In preferred embodiments, the film thickness is selected to be less than (or comparable to) the effective light penetration depth in the culture at the relevant wavelength(s), such that a substantial fraction of cells in the thin film receive sufficient photon dose for optogenetic activation and / or deactivation. In some embodiments, the film 160 exhibits surface ripples or waviness (as schematically depicted), which can enhance mixing within the film, reduce boundary layer limitations, and improve uniformity of optical exposure and / or mass transfer for cells moving within the film. Functionally, the thin-film state provides an illumination-efficient regime for high-density and / or highly scattering cultures by reducing self-shading and enabling intermittent illumination compatible with latching photoswitch operation.

[0073] The illumination source 158 generates light emissions at one or more wavelengths selected to control optogenetic elements in the circulating culture. Physically, illumination source 158 can include one or more LED strips or arrays, laser sources, diode laser arrays, fiber-coupled illuminators, or other light engines mounted within the shell 150 and arranged to illuminate the thin fluid film 160 over a desired axial length of the module. In some implementations this could take the form of a commercial display, such as an activematrix light emitting diode (AMOLED display). In some embodiments, illumination source 158 provides multiple independently controllable wavelength channels (e.g., red and blue channels for a dual -wavelength latching switch), and / or is segmented along the height of the module to deliver spatially controlled illumination profiles. In some embodiments, illumination source 158 includes optical elements (not shown) such as diffusers, lenses, reflectors, or light guides to improve uniformity of illumination and to reduce hotspots that could cause localized heating or phototoxic effects. Functionally, illumination source 158 delivers controlled photon doses to cells in the thin fluid film 160, enabling rapid “trigger” illumination events and / or periodic reinforcement illumination events consistent with photocontrolled and photoswitching bioprocess operation under the control of the controller 20.

[0074] Outlet collector 156 receives the descending thin fluid film 160 and consolidates it into a collected flow for discharge through outlet tube 118-2. Physically, outlet collector 156 may be formed as a funnel, sump, header region, or shaped lower section of shell 150 that directs liquid toward the outlet while minimizing pooling and dead zones. In some embodiments, outlet collector 156 includes features (not shown) such as baffles or calmingDocket: 0412-0005W01regions to reduce entrained bubbles and to stabilize flow into outlet tube 118-2. Functionally, outlet collector 156 recombines the illuminated thin film into a bulk outlet stream, thereby delivering optically stimulated culture back to the circulation loop for continued growth and / or production in the main culture vessel and / or for processing in other apparatus components. Outlet tube 118-2 conveys the illuminated, recombined culture from the outlet collector 156 to downstream portions of the circulating system.

[0075] In some embodiments, the thin-film illumination module 112 may additionally provide a gas-management function analogous to a bubble trap or gas-liquid separator, particularly where the circulating culture contains entrained gas from aeration, agitation, or upstream unit operations. For example, in recirculating bioprocess architectures that include downstream solvent extraction of hydrophobic products, removing entrained gas prior to an extraction vessel can reduce foaming and emulsification and improve phase stability. In such embodiments, the thin-film module 112 can be configured with an optional vent or headspace outlet to allow separated gas to exit while simultaneously providing thin-film illumination of the culture, thereby combining optical dosing and degassing in a single in-line module when desirable.

[0076] Fig. 4 shows another embodiment of the photocontrolled / photoswitching circulating bioreactor system 100, i.e., a bioreactor photocontrolled bioproduction system for producing one or more chemicals from one or more feedstocks. In this embodiment, process flow (cells and culture medium) is recirculated from the main culture or synthesis vessel 12 through a product extractor 14, then through a recirculating pump 24, then through an illumination module 112, and back to the main culture vessel 12. In this arrangement, the extractor 14 is located upstream of the pump 24, and the illumination module 112 is located downstream of the pump 24, which can be advantageous for: (i) maintaining stable extraction conditions in extractor 14 and / or (ii) providing a predictable, controllable flow rate and pressure through the illumination module 112 for repeatable optical dosing.

[0077] The main culture or synthesis vessel 12 is the principal cultivation vessel that retains the majority of the working volume of the bioprocess, including cells and aqueous culture medium. Physically, vessel 12 can be implemented as an industrial stirred tank (e.g., stainless steel fermenter) or other suitable bioreactor format, and may include (not shown) agitation hardware, spargers / diffusers for aeration, temperature control hardware (jackets / coils), and sanitary ports for inoculation, sampling, and addition of nutrients, such as acid / base, antifoam, and / or other reagents. Functionally, vessel 12 provides a stableDocket: 0412-0005W01environment for growth and / or product formation while the optogenetic control action is accomplished by periodically sending culture through the illumination module 112 rather than attempting to illuminate the full bulk volume of vessel 12.

[0078] Process piping 118 fluidically couples the components of system 100 into a recirculation loop. In the illustrated embodiment of Fig. 4, line 118-1 is the vessel-to-extractor transfer conduit carrying culture from vessel 12 to the product extractor 14, with flow direction indicated by arrows. Physically, piping 118 may be implemented as sanitary tubing / piping (e.g., stainless steel or single-use polymer) with aseptic fittings, and may include (not shown) isolation valves, check valves, sample ports, pressure relief, and inline instrumentation such as flow / pressure / temperature sensors. Functionally, line 118-1 carries a portion (or all) of the circulating culture to the extractor 14 so that product can be removed continuously or semi-continuously, while maintaining the bulk cultivation environment in vessel 12.

[0079] The product extractor 14 is an operation operation configured to remove and / or recover product from the circulating culture before the culture is returned to vessel 12. The extractor 14 can be implemented in various forms depending on the product and / or organism, including (without limitation) a liquid-liquid extraction column, a membrane contactor, an adsorption module, a filtration / centrifugation module, or combinations thereof. In embodiments directed to hydrophobic products (e.g., free fatty acids or other hydrophobic metabolites), extractor 14 can be configured as an extraction vessel that contacts the culture with an immiscible organic solvent so that product partitions into the organic solvent phase while an aqueous / cell-containing phase is returned to the process. In such embodiments, extractor 14 may be configured to promote stable phase separation and to manage multiphase issues such as foaming and emulsification — e.g., by providing a headspace region, an immiscible solvent region, and an aqueous region, and / or by employing internal distributors and / or calming sections as described in circulating extraction-based systems.

[0080] The extractor 14 includes at least one outlet stream 14B that withdraws a product-containing stream from the recirculation loop for collection, downstream purification, storage, and / or disposal. Physically, the subsystem 15 may include a product hold tank, solvent / product decanter, collection vessel, or other downstream process equipment sized for the desired duty cycle (continuous draw, periodic draw, or batch draw). A continuous or semi-continuous product removal at outlet 14B can improve productivity by reducing product inhibition and / or toxicity in the main culture. It can also simplifyDocket: 0412-0005W01downstream processing by concentrating product during the run. In solvent extraction embodiments, outlet 14B may carry a solvent-rich product stream, which can be sent to downstream separation (e.g., distillation, membrane separation, adsorption, or other purification techniques). Solvent may optionally be recycled back to extractor 14 as part of a closed-loop extraction configuration.

[0081] The feedstock inj ector 120 is a feed introduction module configured to add one or more feedstocks and / or nutrients to the circulating process stream. Physically, injector 120 can include one or more feed reservoirs, metering pumps, and an injection interface (e.g., sterile injection tee, nozzle, or mixing junction) that introduces feed into line 118-2 as shown. In some embodiments, injector 120 is configured for multiple feeds (e.g., two or more carbon sources and / or nutrient solutions) that can be delivered independently or as a blended stream. Functionally, injector 120 enables continuous or dynamic feeding strategies (e.g., fed -batchlike operation in a recirculating loop), and, in optogenetic implementations, it enables coordinated “feed + illumination” control, where the genetic state of the organism is adjusted to match the feedstock(s) being introduced.

[0082] The process sensor 122 is a sensing module configured to measure at least one process variable and to provide a signal usable for automated control of one or more system components by the controller 20. Physically, sensor 122 may be implemented as an inline probe, a flow-cell-based analyzer, and / or a slipstream sampler returning to the process. In one preferred class of embodiments, sensor 122 is a spectroscopic sensor (e.g., Raman, NIR / IR, UV-Vis absorbance, fluorescence) capable of estimating feedstock composition and / or concentrations of key metabolites. In other embodiments, sensor 122 includes (or is supplemented by) electrochemical sensors (pH, dissolved oxygen, oxidation-reduction potential (ORP), conductivity), gas sensors (e.g., CO2 / O2), biomass sensors (e.g., optical density / turbidity or capacitance), and / or product sensors (e.g., solvent-phase absorbance for hydrophobic product concentration). Multiple sensors 122 may be deployed in combination — e.g., a Raman sensor for feedstock identification plus a DO / pH sensor suite for physiological state plus an optical density sensor for biomass — so that the controller 20 can distinguish whether changes in process performance are driven by feed composition, culture physiology, extraction performance, or illumination duty cycle.

[0083] In some embodiments, the at least one process sensor 122 includes one or more sensors for dissolved chemical species and nutrient ions, such as sensors configured to measure ammonium (NHf), nitrate, phosphate, sulfate, dissolved carbon species, and / orDocket: 0412-0005W01conductivity as a proxy for ionic strength. Such sensors can be implemented as ion-selective electrodes, wet-chemistry analyzers on a slipstream, microfluidic analyzers, or spectroscopic estimators. Outputs from these dissolved-species sensors may be used by a controller, e.g., controller 20 or a separate controller, to adjust feedstock injection rate via the feedstock injector 120, to select illumination wavelength(s) and duty cycle in the illumination module 112 (e.g., to activate nutrient assimilation pathways when a nutrient is present), and / or to adjust recirculation rate using the pump 24 to maintain stable process operation.

[0084] In some embodiments, process sensor 122 is configured to detect a biosensor output generated by the microbial culture, including optical readouts (e.g., fluorescence or luminescence measured in an inline flow cell) and / or chemical readouts (e.g., secreted reporter molecules measured by spectroscopy or electrochemical sensing). The controller may execute a control policy in which biosensor measurement is temporally coordinated with illumination events (e.g., illuminating a reporter induction pulse, waiting a programmed expression / maturation interval, then sampling the reporter signal), thereby enabling periodic “measurement windows” without requiring continuous illumination or continuous reporter expression.

[0085] The recirculating pump 24 provides the motive force that drives culture around the loop. In this Fig. 4 embodiment, pump 24 is positioned downstream of extractor 14 and upstream of illumination module 112. Physically, pump 24 may be a sanitary centrifugal pump, peristaltic pump, diaphragm pump, or other bioprocess-compatible pump selected for sterility and compatibility with the circulating fluid(s). Functionally, pump 24 sets the circulation flow rate and therefore directly influences (i) the frequency with which cells pass through illumination module 112, (ii) the residence time and hydraulic regime within module 112, and (iii) the overall turnover time of vessel 12. In preferred implementations, pump 24 is a variable-speed pump, controlled in closed loop by controller 20, thus allowing dynamic adjustment of optical dosing frequency to match the kinetics of the optogenetic switch (including latching behavior) and to respond to process conditions measured by sensor(s) 122 and / or other sensors.

[0086] The illumination module 112 is an in-line optical dosing device configured to expose flowing culture to one or more selected wavelengths of light with sufficient dose and uniformity to activate and / or deactivate optogenetic elements in high-density, turbid cultures. In theembodiment of Fig. 4, module 112 is positioned after pump 24, an arrangement that can provide a stable and controllable flow rate through module 112 and can support illuminationDocket: 0412-0005W01geometries that benefit from defined pressure / flow conditions (e.g., parallel channel flow in shell-and-tube modules or stable thin-film formation in wetted-wall modules). Physically, module 112 can be constructed in accordance with the shell-and-tube configuration and / or thin-film configuration described elsewhere herein, and may include one or more independently controllable light sources that can emit at multiple wavelengths for multiplexed control of latching switches. The module 112 provides intermittent illumination that can “set” or “reset” latching optogenetic switches while keeping the main culture volume in vessel 12 substantially unilluminated, thereby reducing illumination energy cost and improving scalability.

[0087] Fig. 4 further schematically depicts a control relationship (dashed control line; “PID”) between process sensor 122 and one or more controlled elements, including the illumination module 112, optionally pump 24 and feedstock injector 120. In practice, controller 20 may execute a proportional-integral-derivative (PID) control algorithm, rulebased logic, model-predictive control, and / or hybrid approaches to maintain one or more target setpoints or operating objectives. For example, sensor 122 can identify feedstock composition at injector 120 (e.g., determining which carbon source is presently being dosed, and at what concentration), and the controller 20 can responsively (i) adjust injector 120 feed rate and / or blend ratio, (ii) select illumination wavelength(s) and duty cycle in illumination module 112 to activate catabolic pathways corresponding to the detected feedstock(s), and / or (iii) adjust pump 24 speed to change the frequency and / or duration of optical exposure events per cell as culture recirculates.

[0088] In some embodiments, a multi-sensor arrangement is employed in which process sensor(s) 122 include (or are supplemented by additional sensors positioned at vessel 12, extractor 14, and / or in piping 118) to enable coordinated, closed-loop control of the overall system 100. By way of non-limiting examples:

[0089] (a) Vessel-state sensors (vessel 12): pH, dissolved oxygen, temperature, ORP (oxidation reduction potential), conductivity, turbidity / OD (optical density ) / capacitance (biomass) can be used to control aeration / agitation (if present), acid / base dosing (if present), temperature control, and / or feed rate via injector 120.

[0090] (b) Extraction sensors (extractor 14): interface / level sensors (solvent / aqueous boundary), foam sensors, solvent vapor sensors, and optical / UV-Vis sensors in the solvent phase (product concentration and / or product quality) can be used to control extractionDocket: 0412-0005W01intensity, solvent recycle (if present), product draw via outlet 14B, and / or to trigger adjustments in vessel 12 conditions to reduce unwanted byproducts.

[0091] (c) Flow / pressure sensors (pump 24 / lines 118): flow meters and pressure sensors can be used to control pump 24 speed for a target residence time through illumination module 112, and / or to detect fouling / clogging or process deviations (e.g., increased pressure drop suggesting channel blockage in module 112).

[0092] (d) Illumination verification sensors (module 112): internal light sensors (photodiodes), temperature sensors, and / or downstream reporter measurements (e.g., fluorescence reporters measured in-line) can be used to verify delivered photon dose and / or to adjust LED intensity, duty cycle, wavelength selection, and / or pump speed to ensure reliable switching of optogenetic circuits.

[0093] (e) Feedstock composition sensors (injector 120 / sensor 122): Raman or NIR can quantify the concentration of specific feedstocks (e.g., sugars, glycerol, organic acids) and can be used to select which latching switch is activated (illumination wavelength selection) and / or to avoid unnecessary expression burden by turning off unneeded catabolic pathways.

[0094] In some embodiments, the controller 20 uses sensor 122 (and optionally additional sensors) to coordinate optogenetic switching with extraction performance. For instance, when a product concentration measurement in extractor 14 (or in outlet 14B) indicates that product is accumulating too rapidly in the culture (risking toxicity) or is not being removed efficiently, the controller 20 may (i) increase pump 24 speed to increase extraction turnover frequency, (ii) adjust extractor operating conditions (e.g., phase levels and / or solvent recycle, if present), and / or (iii) alter illumination patterns in module 112 to shift metabolic flux (e.g., temporarily reduce production and increase growth or feedstock utilization) until extraction recovers. Conversely, when sensor data indicates that the feedstock composition has changed (e.g., a different carbon source is being dosed), the controller 20 can change illumination wavelength(s) and / or timing to toggle the corresponding latching switch(es) so the organism expresses the appropriate catabolic machinery for the new feed, improving overall yield and reducing metabolic burden.

[0095] Fig. 5 shows a photoswitching genetic circuit that could be employed in the disclosed photocontrolled circulating bioreactor. Shown are feedstocks 1 through 16, illumination wavelengths 1-n, and associated cellular metabolism pathways to make a desiredDocket: 0412-0005W01product 205. Optogenetic switches 210, linked to genetic circuits, control specific feedstock utilization pathways.

[0096] The latching photoswitches are turned on or off by wavelength-specific illumination generated in the circulating bioreactor system 100. Once on, the switches remain on until a secondary stimulus is sequentially applied in the circulating bioreactor system, which could be another wavelength of light or a chemical switch. Photoswitches are latchable due to continuous self-activation of regulatory pathways which remain active until the secondary stimulus represses the self-activation. Several of these latching optogenetic switches can be multiplexed together to allow control over multiple pathways. One example of an optogenetic switch that can be used is the EL222 system, from Erythrobacter litoralis , which has been demonstrated as a functional switch in bacteria, yeast, and fish, where blue light induces dimerization of the protein. EL222 can be combined with nuclear localization signals, DNA binding domains, and transcription activation domains to enable precise localization and control of gene transcription.

[0097] In some embodiments, the secondary stimulus used to deactivate or reset a latching optogenetic system comprises a physical stimulus in addition to, or instead of, a second wavelength of light and / or a chemical signal. Non-limiting physical stimuli include a controlled change in temperature (e.g., by heating or cooling the main culture vessel 12 or using a recirculation loop heat exchanger) and / or a controlled change in pH (e.g., by addition of acid / base into the main culture vessel 12 or into process piping 118). Physical stimuli may be selected to alter activity, localization, stability, or binding interactions of one or more regulatory proteins in the latching circuit so as to terminate self-activation and return the circuit to a baseline state.

[0098] In some embodiments, a reset stimulus comprises a programmed temperature shift and / or pH shift that is applied for a defined duration and magnitude sufficient to reset the optogenetic latching switch, after which the temperature and / or pH is returned to a production setpoint. Such reset protocols can be executed under controller control and coordinated with flow rate (pump 24) and illumination duty cycle (illumination module 112) to ensure that a desired fraction of cells experiences the reset stimulus during circulation.

[0099] Latching optogenetic switches can be combined to create tight regulatory control over desired pathways. For example, Yarrowia lipolytica engineered with diverse feedstock utilization pathways under control of optogenetic switches would have advantages over aDocket: 0412-0005W01strain with chemically inducible or constitutively expressed feedstock utilization pathways. This advantage is realized by reduction in burden on cells that occurs when feedstock utilization proteins are transcribed without their respective feedstocks present (leaky or constitutive expression). Here, optogenetic switches can be utilized to enable less leaky expression of desired pathways. Several photocontrolled pathways can be multiplexed when their respective activation wavelengths are sufficiently independent to prevent crossactivation.

[0100] The photoswitching bioreactor system 100 allows for transient illumination of cell cultures enabled by illumination modules. Illumination module design enables illumination of high optical density cultures by creating a high surface area to volume ratio. This ensures that the majority of cells present are exposed to the wavelengths needed to activate the desired optogenetic switches.

[0101] Fig. 6 shows a dual -wavelength optogenetic latching switch that could be employed in the disclosed photocontrolled circulating bioreactor 100.

[0102] Optogenetic regulation system 212 is shown in the absence of light (initial state prior to any illumination). Here E1222 (a LOV domain optogenetic switch, blue light sensitive) and PhyB (a phytochrome B type optogenetic switch, red light sensitive) are linked via a spacer sequence to a repressor (REP) or an activator (ACT) respectively. Optogenetic DNA binding sequences for an inducible promoter are upstream of the genes of interest and of a self-activation sequence that codes for an activator with a binding domain that recognizes the inducible promoter (represented in Fig. 6 by ACT with a triangle).

[0103] State of the optogenetic latching switch 214 is shown when the disclosed photocontrolled circulating bioreactor exposes the cell culture to a red light. PhyB-activator activates transcript! on / translati on of both the gene of interest and the self-activation sequence.

[0104] State of the optogenetic latching switch 216 is shown in the dark after exposure to red light has been terminated. Self-activation continues and maintains expression of the gene of interest in the circulating bioreactor.

[0105] State of optogenetic latching switch 218 is shown when the photocontrolled circulating bioreactor exposes the cell culture to blue light with or without red light present to repress both the gene of interest and the self-activation system due to dimerization and binding of the EL222 repressor that enables binding of its DNA binding region upstream of the promoters.Docket: 0412-0005W01

[0106] Fig. 7 shows a dual -wavelength optogenetic latching with a bidirectional promotor that is used with the disclosed photocontrolled circulating bioreactor in some examples.

[0107] This is a variant of the optogenetic latching system shown in Fig. 8. Instead of two instances of the promoter sequences needed for the gene of interest and the selfactivation, here a bidirectional promoter system allows a single promoter with optogenetic binding regions to control both the gene of interest and self-activation transcripts. The table summarizes the state of the optogenetic latching switch in the presence of either wavelength and the activator.

[0108] Fig. 8 shows a single-wavelength optogenetic latching switch with chemical reset that can be used with the disclosed photocontrolled circulating bioreactor in some examples.

[0109] An optogenetic regulation system 220 functions similarly to that of Fig. 6, except it utilizes a single activation wavelength and a chemical molecule sensitive reset / deactivation.

[0110] Panel 220 is the initial no-light system in the absence of chemical repressor (pentagon labeled Rep in Fig. 8).

[0111] Panel 222 shows the red light activation of the system in the absence of chemical repressor.

[0112] Panel 224 shows the continuous self-activation in the absence of light after exposure to red light is finished.

[0113] Panel 226 shows the deactivation by exposure to a chemical repressor that stops transcription of the gene of interest and the self-activator.

[0114] Figs. 9 A, 9B, and 9C are schematic top cross-sectional views looking down into the illumination module 112 along the axis of flow of the circulating cell culture.Accordingly, the transparent tubes 154 (through which the culture flows) are shown in crosssection as circular lumens arranged in an array. These embodiments are generally similar to the shell -and-tube illumination module of Fig. 2 (which is a side cross-sectional view), but Figs. 9A-9C emphasize alternative optical architectures for delivering one or more illumination wavelengths to the tube array 154 with improved spatial uniformity and / or controllable dosing across high-density, turbid cultures.

[0115] In the embodiment of Fig. 9A, the illumination module 112 includes an array of transparent tubes 154 positioned within an optically enclosed housing (e.g., an opaque shellDocket: 0412-0005W01as described with reference to Fig. 2). The culture flows through the tubes 154 substantially parallel to the viewing axis (i.e., into / out of the page), such that the optical design shown in Fig. 9A is configured to illuminate the culture in each tube 154 from one or more directions around the tube bundle.

[0116] The tubes 154 are optically transmissive conduits (e.g., glass, quartz, or transmissive polymer) arranged as a bundle / array to increase illuminated surface area and reduce optical path length through the culture. In operation, each tube 154 carries a portion of the circulating culture stream so that cells are repeatedly exposed to controlled illumination as they pass through the illumination module 112, thereby enabling intermittent illumination strategies and / or multi-wavelength switching for optogenetic circuits.

[0117] The illumination source 158A in Fig. 9A is a supercontinuum laser, other white light laser source or other white light source that provides a high-brightness optical output suitable for delivering short-duration, high-intensity optical stimuli (and, in some embodiments, for selecting one or more wavelength bands from a broader emission spectrum using optical filtering and / or wavelength selection hardware, not shown). Functionally, the laser-based approach can be advantageous where the optogenetic system benefits from precise, repeatable optical dose delivery and / or where multiple distinct wavelengths are used to activate and deactivate latching photoswitches.

[0118] In some embodiments, the light wavelength is made tunable by adding a spatially variable bandpass tunable filter 190, mounted, for instance, on a linear stage 192. By moving the tunable filter 190 on the stage 192, the light from the illumination source 158A is bandpass filtered to thereby control the wavelength of the light illuminating the culture.

[0119] The illumination module 112 includes a rotationally movable or scanning optic, shown as element 176, which, in preferred embodiments, is implemented as a mirror mounted on a galvanometer (or another beam-steering actuator). Another option is a spatial light modulator such as a Digital Micromirror Device (DMD). DMDs are a specific type of Micro-Electro-Mechanical System (MEMS), also technically referred to as MEMS micromirror array.

[0120] The optic element 176 receives light from illumination source 158A and dynamically directs the beam along different trajectories to sequentially illuminate different portions of the tube array 154. This scanning approach can distribute optical doses across the tube bundle without requiring the light source to physically surround the module and can beDocket: 0412-0005W01used to create time-varying illumination patterns (e.g., to equalize doses across tubes or to implement programmed pulse trains).

[0121] A track 180 is added in some examples; it carries a moving optic 182 (e.g., a mirror or lens) positioned near the tube bundle 154. The moving optic 182 can be translated along the track 180 to intercept and redirect a scanned beam (or portions thereof) toward areas of the tube array that might otherwise receive lower illumination. In this manner, the combination of beam steering by optic 176 and secondary redistribution by moving optic 182 improves spatial uniformity of illumination across the array, compensate for geometric shadowing, and / or provide a controllable optical “sweep” over the tube bundle for consistent dosing of the flowing culture.

[0122] The arrows 170 schematically indicate representative light paths from the illumination (e.g., laser) source 158A, reflected by the scanning optic 176, optionally redirected by the moving optic 182, and delivered toward the tube array 154. In operation, the optical trajectories can be adjusted by controlling the angular position of optic 176 and / or the position of optic 182 along track 180, thereby tuning dose distribution, dwell time per region, and total exposure per pass through the illumination module 112.

[0123] Fig. 9B illustrates another tube-array illumination module 112 in the same general shell-and-tube format, but using a predominantly fixed-optics architecture to spread the output of a supercontinuum laser across the tube array 154. As in Fig. 9A, the tubes 154 are shown in cross-section because the view is taken along the axis of culture flow through the tubes.

[0124] In this approach, a reflective surface 178 such as a curved reflective wall segment is included within the illumination module 112. The reflective surface 178 may be implemented as a polished / metalized internal wall, a mirror insert, or another reflective element positioned to redirect incident light back through the tube array 154. Functionally, reflector 178 can increase optical efficiency, reduce wasted light, and improve dose uniformity by providing reflected illumination to portions of the tube array opposite the primary illumination direction.

[0125] The optical element 174 is a fixed lens and / or mirror positioned to receive light from the illumination (e.g., laser) source 158A and direct it toward the expansion optics 172 and / or toward the tube array 154. The element 174 may include one or more lenses,Docket: 0412-0005W01reflectors, prisms, or beam-conditioning elements, and may be selected to collimate, focus, and / or redirect the beam to achieve a desired illumination geometry within the module.

[0126] The light expander 172 expands the beam from the source 158A to illuminate a larger area of the tube array 154. In the illustrated schematic, the expander 172 creates a widened, fan-like distribution (as indicated by multiple rays) so that multiple tubes receive light concurrently. Functionally, this approach can reduce mechanical complexity relative to scanning architectures while still providing controllable dose delivery (e.g., by controlling laser power, exposure duration, and / or employing wavelength selection components not shown).

[0127] Fig. 9C illustrates a further embodiment of the illumination module 112 employing an area illumination source to illuminate the tube array 154. As in Figs. 9A and9B, this is a top cross-sectional view looking down the axis of flow through the tubes 154, which are therefore shown in cross-section.

[0128] The illumination source is an LED array panel 158B positioned adjacent the tube bundle 154. The LED array panel 158B may be implemented as an array of discrete LEDs, an LED backlight assembly, or a display-like light engine (including, in some embodiments, an addressable panel such as a commercial display, for instance, an active-matrix light emitting diode (AMOLED) display capable of emitting one or more wavelengths used for optogenetic switching. Functionally, panel 158B can illuminate a broad field of view across the tube array concurrently, which can simplify dose delivery and can be advantageous for high-throughput illumination where a large number of tubes 154 must receive substantially similar exposure during each pass through the module.

[0129] A reflective surface 178 is positioned on the side of the tube array opposite the primary light source. In this configuration, reflector 178 reflects incident light back through the tube array 154, increasing the effective optical path utilization and improving illumination uniformity across the bundle by providing a reflected light component that reaches tubes that are farther from the panel 158B.

[0130] In some embodiments, the illumination module 112 is used to control a transition between a growth mode and a production mode by actuating optogenetic switches controlling expression of one or more enzymes, transporters, regulatory proteins, or pathway branches. For example, during a growth phase the controller may maintain low-duty-cycle illumination (or no illumination) to minimize burden, and upon reaching a target biomass the controllerDocket: 0412-0005W01may apply an illumination protocol that activates one or more production pathways, optionally using latching logic to maintain expression after the illumination event. The controller may further coordinate production-mode activation with extraction performance (extractor 14) and nutrient feeding (injector 120) to maintain target productivity and product quality.

[0131] Example 1 : Bioproduction of free fatty acids by engineered Yarrow ia lipolytica from two or more feedstocks.

[0132] The apparatus can be configured as seen in Fig. 4 to enable continuous feedstock addition and product separation. One application of this design is to remove hydrophobic products, such as free fatty acids (FFAs), from microbial culture with a solvent extraction. Here, if a oleaginous, solvent-tolerant microbe, like Yarrowia lipolytica, is engineered to overproduce FFAs, the FFAs can be removed with a solvent extraction continuously, and the yeast cells and aqueous culture medium can be recirculated back to the culture vessel by a pump. Production of FFAs from various carbon feedstocks is desirable. To accomplish this, several feedstock catabolism pathways are engineered into the yeast. Constitutive expression for catabolic pathways in the absence of their respective feedstocks would increase protein burden, reduce product yields, and potentially reduce cell fitness. As such, the catabolic pathways can be placed under the control of latching optogenetic switches, allowing them to be activated or deactivated when needed. Here, a FFA producing Yarrowia lipolytica strain engineered with 2 or more latching photoswitch controlled feedstock catabolic pathways is suitable for use in the apparatus depicted in Fig. 4, where the illumination module transiently illuminates cell culture in a shell-and-tube configuration with specific wavelengths to activate the feedstock utilization pathways required for current feeds to the apparatus. Activation of feedstock catabolism genes can be automated by employing a Raman spectrophotometer to monitor incoming feedstocks to the process, which then, through a control scheme, determine which wavelengths the illumination module uses.

[0133] Optogenetic latching switches can take several structural forms. Dual -wavelength systems can be implemented (Figs. 6 and 7) where a specific wavelength activates the switch, self-activation latches the switch to continue its active state after light induced activation, then a secondary, independent wavelength deactivates the switch and represses the selfactivation. The DNA regions coding for the expression of the gene of interest and the selfactivation can be discrete and are independent of their proximity to each other (Fig. 7).Alternatively, a bidirectional promoter can be employed to control both the gene of interestDocket: 0412-0005W01and the self-activator; in this case only a single instance of the optogenetic sensitive regions is needed, and the gene of interest and self activator are localized together on either side of the bidirectional promoter. Single-wavelength systems can also be used where deactivation is controlled by chemical responsive repressors (Fig. 8).

[0134] Optogenetic latching systems such as those illustrated in Figs. 6, 7, and 8 are suitable for multiplexing given that that wavelengths and activators are sufficiently independent of one another. In the context of the Yarrowia lipolytica example, the genes of interest could be feedstock utilization genes or genes involved with product formation (free fatty acids). Here, the use of hybrid optogenetic and chemical switches (Fig. 8) could be particularly beneficial to feedstock switching, where unneeded feedstock catabolism pathways can be repressed when other feedstocks are present; one example would be to repress glucose metabolism when glycerol is present. Such strains can be utilized in bioreactors like the one depicted in Fig 4.

[0135] In other embodiments, the apparatus is a more traditional cell culture vessel, such as a continuously stirred tank reactor (CSTR) with a recirculation loop installed for the purpose of intermittent illumination. Here, the recirculation loop sends media out of the main vessel, through the illumination module, then back to the main culture vessel. The speed and residence time of the recirculation can be adjusted as needed for optimal photocontrol performance of the optogenetic switches.

[0136] Example 2: Periodic product monitoring using an optogenetically actuated biosensor.

[0137] Engineered microorganisms in the main culture vessel 12 can comprise an optogenetic photoswitch controlling expression of a biosensor reporter correlated with concentration of a target product or pathway intermediate. Culture is circulated through the illumination module 112, where a brief illumination pulse induces biosensor expression and / or activation. After a defined interval, an inline process sensor 122 measures the reporter output in a recirculating stream (e.g., in a flow cell), and the controller adjusts at least one of (i) illumination duty cycle and wavelength, (ii) recirculation rate via pump 24, (iii) feedstock injection rate via injector 120, and / or (iv) extraction intensity via extractor 14 to maintain the process within target operating bounds.

[0138] Example 3 : Sanitization or waste treatment using illumination module photoinacti vati on .Docket: 0412-0005W01

[0139] The illumination module 112 can be operated in a sanitization mode to reduce viability of contaminating microorganisms in a purge or waste stream. The controller actuates the illumination source 158 to deliver an antimicrobial optical dose within the illumination module 112 as material flows through the module. Treated material is then routed to a waste treatment or disposal subsystem. In some implementations, a photosensitizer is added to the treated stream to enhance photodynamic inactivation, and the sanitization mode is executed based on a schedule or in response to sensor-indicated contamination.

[0140] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. Docket: 0412-0005W01CLAIMSWhat is claimed is:

1. A bioprocess system, comprising:at least one culture vessel configured to contain a microbial culture comprising engineered microorganisms having at least one optogenetic photoswitch that is actuatable by light;an illumination module fluidically coupled to the at least one culture vessel and defining a flow path for the microbial culture, the illumination module comprising at least one illumination source configured to emit light at one or more wavelengths to actuate the at least one optogenetic photoswitch while the microbial culture flows through the illumination module; and a recirculating pump configured to circulate at least a portion of the microbial culture from the at least one culture vessel through the illumination module and back to the at least one culture vessel.

2. A system as claimed in claim 1, further comprising a controller operatively coupled to the illumination source and the recirculating pump, the controller being configured to intermittently actuate the illumination source and to control a flow rate through the illumination module by control of the recirculating pump such that microorganisms in the microbial culture are periodically exposed to the light to control a metabolic pathway of the microorganisms.

3. The system of any of claims 1-2, further comprising a product extractor fluidically coupled to the main culture vessel and configured to contact at least a portion of the microbial culture with an immiscible organic solvent to extract a hydrophobic bioproduct, the product extractor having (i) a recirculation outlet configured to return a culture stream comprising cells and culture medium to the at least one culture vessel and (ii) a product outlet configured to withdraw a solvent stream comprising the hydrophobic bioproduct.

4. The system of any of claims 1-3, wherein the illumination module comprises: an opaque housing; an inlet; an outlet; a splitter coupled to the inlet; a plurality of optically transmissive tubes fluidically coupled in parallel between the splitter and a collector coupled to the outlet, the plurality of optically transmissive tubes beingDocket: 0412-0005W01configured to convey the microbial culture; and the at least one illumination source arranged to illuminate the plurality of optically transmissive tubes.

5. The system of claim 4, wherein the at least one illumination source comprises a white light laser and optics configured to distribute light across the plurality of optically transmissive tubes.

6. The system of claim 4, wherein the at least one illumination source comprises an array panel configured to illuminate the plurality of optically transmissive tubes across a field of view of the illumination module, and wherein the illumination module optionally comprises a reflective surface positioned opposite the array panel to reflect light back through the plurality of optically transmissive tubes.

7. The system of any of claims 1-3, wherein the illumination module comprises a thin-film illumination module defining a wetted-wall thin film flow path of the microbial culture, and wherein the at least one illumination source is configured to illuminate the microbial culture while the microbial culture flows as a thin film.

8. The system of any of claims 1-7, wherein the at least one optogenetic photoswitch comprises an optogenetic latching switch configured to maintain a regulatory state after cessation of illumination, and wherein the optogenetic latching switch is reset by a reset stimulus comprising at least one of (i) illumination at a second wavelength different from a first wavelength used to activate the optogenetic latching switch (ii) exposure to a chemical signal and (iii) a physical stimulus comprising a change in temperature and / or pH.

9. The system of any of claims 1-8, further comprising at least one process sensor configured to generate a sensor output indicative of at least one of ammonium, feedstock composition, biomass, pH, dissolved oxygen, oxidation-reduction potential, temperature, pressure, product concentration, and optical density, wherein the controller is configured to select at least one illumination parameter comprising at least one of wavelength, intensity, pulse duration, and duty cycle based on the sensor output.

10. The system of any of claims 1-9, further comprising a feedstock injector configured to introduce one or more feedstocks into the microbial culture, wherein aDocket: 0412-0005W01process sensor is configured to measure a composition of the one or more feedstocks, and wherein the controller is configured to select the one or more wavelengths to actuate the at least one optogenetic photoswitch based on the measured composition.

11. The system of any of claims 1-10, wherein the microorganisms include Yarrowia lipolytic and / or Marinobacter atlanticus.

12. An illumination module for use in an optogenetically controlled bioprocess, comprising:a housing;a fluid inlet and a fluid outlet;a culture-conveyance structure disposed in a flow path between the fluid inlet and the fluid outlet and configured to convey a microbial culture comprising engineered microorganisms having at least one optogenetic photoswitch that is actuatable by light;at least one illumination source arranged to emit light at one or more wavelengths toward the microbial culture while the microbial culture is conveyed through the flow path to actuate the at least one optogenetic photoswitch;wherein the culture-conveyance structure comprises at least one selected from the group consisting ofone or more optically transmissive conduits configured to convey the microbial culture; anda wetted-wall surface configured to form the microbial culture into a flowing thin film within the housing for illumination by the at least one illumination source.

13. A method of operating a bioprocess system having an illumination module, comprising:introducing one or more feedstocks into a microbial culture using a feedstock injector;measuring, using at least one process sensor, a parameter indicative of at least one of (i) a composition of the one or more feedstocks and (ii) a state of the microbial culture;Docket: 0412-0005W01determining, using a controller, an illumination protocol comprising at least one of a wavelength selection, an intensity, and a duty cycle based on an output of the at least one process sensor; andilluminating, within the illumination module, a flowing portion of the microbial culture according to the illumination protocol to actuate an optogenetic latching switch that controls expression of a pathway associated with utilization of the one or more feedstocks and / or production of a bioproduct.

14. The method of claim 13 executed with the system of any of claims 1-11.

15. A bioprocess system, comprising:at least one culture vessel configured to contain a microbial culture comprising engineered microorganisms, the engineered microorganisms comprising: (i) at least one optogenetic photoswitch actuatable by light and (ii) a biosensor circuit that produces a measurable reporter signal indicative of at least one process parameter, wherein expression and / or activation of the biosensor circuit is controlled by the at least one optogenetic photoswitch;an illumination module fluidically coupled to the at least one culture vessel and defining a flow path for the microbial culture, the illumination module comprising at least one illumination source configured to emit light at one or more wavelengths to actuate the at least one optogenetic photoswitch while the microbial culture flows through the illumination module;a recirculating pump configured to circulate at least a portion of the microbial culture from the at least one culture vessel through the illumination module and back to the at least one culture vessel;at least one process sensor configured to detect the measurable reporter signal and to provide a sensor output corresponding to the process parameter; and a controller operatively coupled to the at least one illumination source, the recirculating pump, and the at least one process sensor, the controller being configured to:(i) intermittently actuate the at least one illumination source to cause the biosensor circuit to produce the measurable reporter signal;(ii) receive the sensor output from the at least one process sensor; andDocket: 0412-0005W01(iii) adjust at least one operating parameter of the bioprocess system based on the sensor output, the at least one operating parameter comprising at least one of: a flow rate through the illumination module, an illumination parameter comprising at least one of wavelength, intensity, pulse duration, and duty cycle, a feedstock addition rate, a product extraction rate, a pH setpoint, a temperature setpoint, an aeration rate, and an agitation rate.

Images