Frontiers in Bioengineering and Biotechnology

Galvanic Vestibular Stimulation(GVS) induce the sensation of movement in subjects in flight simulators and in cosmonauts, creating a cognitive simulation of movement. The system consists of a control unit, a function generator, and a power amplifier. GVS is capable of activating the neurons of the vestibular system and inducing the sensation of movement. When applied in coordination with a flight simulation program GVS modifies the eye movement control responses, electrically activating the vestibular-ocular, vestibule-colic, and vestibule-spinal reflexes. The ultimate goal of this type of stimulation is to generate augmented reality in the pilots during training or potentially also during a flight in microgravity.

Fungal peroxygenases are promising biocatalysts for hydroxylation steps in various industry-relevant synthesis pathways. In this application note we describe a bioprocess for the production of unspecific peroxygenase (UPO) in Pichia pastoris. The process was divided in four phases, with different carbon requirements. Precise timing of culture feeding was crucial for optimal cell growth and protein expression. We demonstrate how the automation of culture feeding reduced manual work as well as the risk of process failure due to operator error.

Gene drive systems could be a viable strategy to prevent pathogen transmission or suppress vector populations by propagating drive alleles with super-Mendelian inheritance. CRISPR-based homing gene drives, perhaps the most powerful gene drive strategy, convert wild type alleles into drive alleles in heterozygotes with the help of Cas9 and gRNA. However, achieving successful outcomes with these drives often requires high performance. Specifically, it is desirable to identify Cas9 promoters that yield high drive conversion rates, minimize the formation rate of resistance alleles in both the germline and the early embryo, and limit somatic Cas9 expression. Thus far, high-performance promoters have only been discovered in Anopheles species. In Drosophila, the nanos promoter avoids leaky somatic expression, but at the cost of high embryo resistance from maternally deposited Cas9. To improve drive efficiency, we tested eleven Drosophila melanogaster germline promoters in several configurations. Some of the new promoters achieved higher drive conversion efficiency with minimal embryo resistance, but none could completely avoid somatic expression like nanos. However, such somatic expression often did not carry detectable fitness costs when the promoter-Cas9 elements supported a rescue homing drive targeting a haplolethal gene, suggesting somatic drive conversion. Based on our findings, we selected two Cas9 promoter lines for cage experiments with a 4-gRNA suppression drive. While one promoter exhibited substantial somatic effects, leading to a low drive equilibrium frequency, the other outperformed nanos, resulting in the successful suppression of the cage population. Overall, these novel Cas9 promoters hold potential advantages for homing drives in Drosophila species and may also possess valuable homologs in other organisms.

Advanced robotic systems that replicate musculoskeletal structure and function have significant potential for a wide range of applications. Although they are proposed to be better platforms for biomedical applications, little is known about how well current musculoskeletal humanoid systems mimic the motion and force profiles of humans. This is particularly relevant to the field of tendon tissue engineering, where engineered grafts require advanced bioreactor systems that accurately replicate the kinetic and kinematic profiles experienced by the humans in vivo. A motion study was conducted comparing the kinetic and kinematic profiles produced by a musculoskeletal humanoid robot shoulder to a group of human participants completing abduction/adduction tasks. Results from the study indicate that the humanoid arm can be programed to either replicate the kinematic profile or the kinetic profile of human participants during task completion, but not both simultaneously. This study supports the use of humanoid robots for applications such as tissue engineering and highlights suggestions to further enhance the physiologic relevance of musculoskeletal humanoid robotic platforms.

Gene drive is a novel approach for controlling vector borne disease via either population modification or suppression. Even with high efficiency, though, overall drive performance can be reduced by somatic Cas9 expression and by maternal deposition of Cas9, leading to resistance allele formation. The nanos promoter for Cas9 shows very little leaky somatic expression, but it causes high rates of embryo resistance allele formation in Drosophila melanogaster. By truncating the promoter, we reduced rates of embryo resistance to undetectable levels, but germline cutting in females decreased by over half. Germline cutting and successful drive conversion was eventually lost when only the 5' UTR was present, though males still retained moderate germline drive efficiency. Several additional methods were tested to improve performance, including additional suppressor elements to the 3' UTR and introns to increase expression level. The most successful of these was the addition of a second nuclear localization signal, which substantially increased activity when coupled with a full-length or truncated nanos promoter. Overall, these experiments show the potential to modulate Cas9 regulatory elements to achieve desired expression for gene drive applications, while also showcasing the difficulty of obtaining an optimal activity profile.

BackgroundCommercially-available microprocessor-controlled prosthetic knees are unable to fully replicate the biomechanical function of the missing biological limb. While powered prostheses have the capacity to restore joint level kinetics, current systems rely on intrinsic control schemes that do not allow the user to volitionally modulate movement under neural commands. This limitation may compromise functional performance and hinder prosthetic embodiment, the sense that the device is part of the users body. In a case study on one test participant, we evaluate the functional and perceptual benefits of a bone-anchored, neurally-controlled knee prosthesis by comparing it to the participants microprocessor-controlled prosthesis. MethodsWe conducted a within-subject study on an individual with a transfemoral amputation, with an osseointegrated implant and surgically reconstructed agonist-antagonist muscle pairs. We tested a neurally-controlled powered knee and conventional microprocessor knee across a set of activities, including seated volitional control tasks, sit-to-stand transitions, squatting, level-ground walking, stair ascent, and uninstructed standing. Performance metrics included knee kinematics, prosthesis-generated mechanical power, and functional outcomes such as gait speed, stair ascent time, and weight-bearing symmetry derived from ground reaction forces. Functional mobility and control were complemented by self-reported embodiment, assessed through a questionnaire targeting agency, ownership, and body representation. ResultsThe neurally-controlled prosthesis enabled intuitive and responsive control. Compared to the subjects prescribed prosthesis, the prosthesis yielded improved temporal gait symmetry during walking (symmetry index: 0.93 vs. 0.59, with 1 indicating perfect stance time symmetry), increased prosthetic-side weight-bearing during sit-to-stand and squatting, and successful execution of a step-over-step stair ascent strategy--an outcome not achievable with the subjects prescribed device. Embodiment scores were consistently higher with the neurally-controlled prosthesis compared to the prescribed device across multiple domains, including agency, ownership and body representation. ConclusionsThis study is the first to directly compare a prescribed microprocessor knee with a bone-anchored, neurally-controlled powered prosthesis. By combining osseointegration, surgically reconstructed agonist-antagonist muscle pairs, and powered actuation, the system improved gait symmetry, greater prosthetic-side loading, and step-over-step stair ascent. These results demonstrate the novelty and promise of integrating surgical and mechatronic innovations to restore both functional mobility and embodied control after transfemoral amputation. Trial registrationThis study was approved by the Institutional Review Board at MIT (Protocol No. 2503001589).

Process systems engineering methods and tools have been difficult to apply in bioprocess engineering, mainly due to the high complexity of biological systems and the low reproducibility of the experiments. High throughput robotic cultivation platforms in combination with computational tools for experimental design, resource scheduling, and operation, are rapidly gaining popularity. One important contribution being the generation of data in high throughput needed to overcome this lack of data with high information content and the worrying reproducibility crisis in life sciences. In this work, directed acyclic graphs are used to represent, manage and track all experimental workflows in a robotic platform. They support data provenance and enable traceability and reproducibility of workflows in robotic facilities. The experimental workflows are automated using Apache Airflow enabling to manage all necessary steps for fed-batch cultivations, including sampling, sample transport by a mobile robot, feed additions, data collection, storage in a SQL database and model fitting. The added value of this system is demonstrated in scale-down experiments, where E. coli BL21 (DE3), producing elastin like proteins, exhibits robustness towards glucose oscillations that mimic industrial cultivation conditions.

Although numerous cell and gene therapies have received regulatory approval, their adoption has been hampered by high cost and challenges in scaling out manufacturing. Many autologous cell therapies are individually manufactured for each patient using traditional manual methods in high-cost environments. Therefore, robotics and automation offer a potential solution to meet the growing demand for such therapies. To ensure identical biological outcomes, automation must replicate validated manual workflows, a requirement that poses significant engineering challenges, especially for aseptic manipulation and compatibility with manual-centric instruments and consumables. Here we describe the design and performance of a modular robotic cluster consisting of specialized modules containing widely adopted equipment. The robotic arm uses custom end-effectors to handle standard consumables and instruments such as syringes, vials, bags, cell counters, bioreactors, incubators, and closed centrifuges. We compared the performance of skilled human operators against the robotic cluster across multiple tasks: transferring cells between sterile bags, cell counting, drawing volume from a vial to a syringe, and resuspension and sampling from both a bag and G-Rex 100M-CS bioreactor. The robotic system also executed high-complexity operations with industry-standard instruments: cell selection using a CytoSinct 1000 and wash/buffer exchange with a CTS Rotea Counterflow centrifugation system. Experimental results for each unit operation show that the robotic clusters performance is equivalent to manual operations on the selected key metrics. These data demonstrate that the robotic system can efficiently and robustly perform specific unit operations, which can be combined in any order for end-to-end cell therapy manufacturing processes. One Sentence summaryA modular robotic system can perform key unit operations in cell therapy manufacturing with accuracy comparable to human operators while ensuring throughput and flexibility.

Challenges in normothermic machine perfusion (NMP) remain, particularly concerning the duration for which individual organs can be safely preserved. We hypothesize that optimal preservation can be achieved by perfusing organs together in a multivisceral block. Therefore, our aim was to establish a platform for ex vivo multivisceral organ perfusion. Multivisceral grafts containing the liver, kidneys, pancreas, spleen and intestine were obtained from Yorkshire pigs. Three generation (gen) setups were tested during the iterative design process, and minor changes were made throughout. Gen1 (n=4) used a custom-designed single perfusion circuit. Gen2 (n=3) employed a dual perfusion circuit. Gen3 (n=4) featured a single perfusion circuit with an optimized basin and reservoir. Grafts underwent NMP using an autologous blood-based perfusate, while hemostatic parameters and function were assessed. With each iteration, aortic flow improved, resistance decreased, urine output increased, oxygen consumption rose, perfusate lactate levels dropped, and pH stability improved. Cellular injury trended lower in Gen3. Histological evaluation demonstrated minimal differences in Gen2 and 3. We demonstrate the feasibility of abdominal multivisceral NMP for up to 8 hours. Adequate arterial flow, stable perfusate pH, and high oxygen consumption in setup 3 indicate organ viability. Multivisceral perfusion may serve as a platform for long-term NMP.

Total knee arthroplasty aims to relieve pain and restore function in the affected joint through artificial implants. Despite advances in design and surgical techniques, there are complications associated with surgery, especially concerning the patella. The use of musculoskeletal models in orthopedic surgery allows for objective prediction of postoperative function and optimization of results for each patient. To ensure that simulations are trustworthy and can be used for predictive purposes, comparing simulation results with experimental data is crucial. Although progress has been made in obtaining 3D bone geometry and estimating contact forces, validation of these predictions has been limited due to the lack of direct in vivo measurements and the economic and ethical constraints associated with available alternatives. In this study, an existing commercial surgical training station was transformed into a sensorized test bench. The use of 3D-printed models and sensors allowed low-cost and reproducible experimental validation of computer simulation (patellar movement and forces) while avoiding ethical issues.

Bionic reconstruction techniques that employ surgical neuroprosthetic interfaces, biomimetic control systems, and powered mechatronics have enabled versatile and biomimetic legged gait without reliance on intrinsic gait controllers. However, relative emphasis has been placed on the emulation of sagittal plane biomechanics while neglecting to provide control over frontal plane mechanics critical for terrain adaptation. Here, we present a 2-degree-of-freedom (DOF) bionic reconstruction at the transtibial amputation level that enables continuous neural control of both sagittal and frontal ankle and subtalar joint mechanics. To demonstrate its capabilities in a case study design, we integrated a 2-DOF robotic ankle-foot device via surface electromyographic electrodes to an individual provisioned with a surgical neuroprosthetic interface that augments residual muscle afferents. The subject was able to neurally control both degrees of freedom to regain nominal gait mechanics during both level-ground walking and continuous cross-slope navigation. Furthermore, the subject strategically traversed an obstacle course by dynamically hopping between a series of discrete cross-slope blocks, adapting to the slopes, and responding to rapid foot slips. These preliminary findings suggest that bionic reconstruction techniques can restore continuous neural control over multi-DOF prostheses to achieve agile locomotion over complex terrain. One-Sentence SummaryA multi-DOF ankle-foot prosthesis under continuous neural control enables agile locomotion over complex terrain.

The Lokomat provides task-oriented therapy for patients with gait disorders. This robotic technology drives the lower limbs in the sagittal plane. However, unconstrained gait involves motions also in the coronal and transverse planes. This study aimed to compare the Lokomat with Treadmill gait through 3D-joint kinematics and inter-joint coordination. Lower limb kinematics was recorded in 18 healthy participants who walked at 3 km/h on a Treadmill or in a Lokomat with nine combinations of Guidance (30, 50, 70%) and body-weight-support (30, 50,70%). Compared to Treadmill, the Lokomat altered pelvis rotation, decreased pelvis obliquity and hip adduction, and increased ankle rotation. Moreover, the Lokomat resulted in a significantly slower velocity at the hip, knee, and ankle flexion compared to the treadmill condition. Moderate to strong correlations were observed between the Treadmill and Lokomat conditions in terms of inter-joint coordination between hip-knee (r=0.67-0.91), hip-ankle (r=0.66-0.85), and knee-ankle (r=0.90-0.95). In conclusion, this study showed that some gait determinants such as pelvis obliquity and rotation, and hip adduction are altered when walking with Lokomat in comparison to Treadmill. Kinematic deviations induced by the Lokomat were most prominent at high levels of body-weight-support. Interestingly, different levels of Guidance did not affect gait kinematics.

Although regenerative actuators can extend the operating durations of robotic lower-limb exoskeletons and prostheses, these energy-efficient powertrains have been exclusively designed and evaluated for continuous level-ground walking. ObjectiveHere we analyzed the lower-limb joint mechanical power during stand-to-sit movements using inverse dynamic simulations to estimate the biomechanical energy available for electrical regeneration. MethodsNine subjects performed 20 sitting and standing movements while lower-limb kinematics and ground reaction forces were measured. Subject-specific body segment parameters were estimated using parameter identification, whereby differences in ground reaction forces and moments between the experimental measurements and inverse dynamic simulations were minimized. Joint mechanical power was calculated from net joint torques and rotational velocities and numerically integrated over time to determine joint biomechanical energy. ResultsThe hip produced the largest peak negative mechanical power (1.8 {+/-} 0.5 W/kg), followed by the knee (0.8 {+/-} 0.3 W/kg) and ankle (0.2 {+/-} 0.1 W/kg). Negative mechanical work from the hip, knee, and ankle joints per stand-to-sit movement were 0.35 {+/-} 0.06 J/kg, 0.15 {+/-} 0.08 J/kg, and 0.02 {+/-} 0.01 J/kg, respectively. Conclusion and SignificanceAssuming an 80-kg person and previously published regenerative actuator efficiencies (i.e., maximum 63%), robotic lower-limb exoskeletons and prostheses could theoretically regenerate ~26 Joules of total electrical energy while sitting down, compared to ~19 Joules per walking stride. Given that these regeneration performance calculations are based on healthy young adults, future research should include seniors and/or rehabilitation patients to better estimate the biomechanical energy available for electrical regeneration among individuals with mobility impairments.

Widespread treatment of human diseases with gene therapies necessitates the development of gene transfer vectors that integrate genetic information effectively, safely and economically. Accordingly, significant efforts have been devoted to engineer novel tools that i) achieve high-level stable gene transfer at low toxicity to the host cell; ii) induce low levels of genotoxicity and possess a safe integration profile with a high proportion of integrations into safe genomic locations; and iii) are associated with acceptable cost per treatment and scalable/exportable vector production to serve large numbers of patients. The Sleeping Beauty (SB) transposon has been transformed into a vector system that is fulfilling these requirements.\n\nIn the CARAMBA project, we use SB transposition to genetically modify T cells with a chimeric antigen receptor (CAR) specific for the SLAMF7 antigen, that is uniformly and highly expressed on malignant plasma cells in multiple myeloma. We have demonstrated that SLAMF7 CAR-T cells confer specific and very potent anti-myeloma reactivity in pre-clinical models, and are therefore preparing a Phase I/IIa clinical trial of adoptive immunotherapy with autologous, patient-derived SLAMF7-CAR T cells in multiple myeloma (EudraCT Nr. 2019-001264-30/CARAMBA-1).\n\nHere we report on the characterization of genomic safety attributes in SLAMF7 CAR-T cells that we prepared in three clinical-grade manufacturing campaigns under good manufacturing practice (GMP), using T cells that we obtained from three healthy donor volunteers. In the SLAMF7 CAR-T cell product, we determined the average transposon copy number, the genomic insertion profile, and presence of residual SB100X transposase. The data show that the SLAMF7 CAR transposon had been inserted into the T cell genome with the close-to-random distribution pattern that is typical for SB, and with an average transposon copy number ranging between 6 and 12 per T cell. No residual SB100X transposase could be detected by Western blotting in the infusion products. With these attributes, the SLAMF7 CAR-T products satisfy criteria set forth by competent regulatory authorities in order to justify administration of SLAMF7 CAR-T cells to humans in the context of a clinical trial. These data set the stage for the CARAMBA clinical trial, that will be the first in the European Union to use virus-free SB transposition for CAR-T engineering.\n\nDisclosuresThis project is receiving funding from the European Unions Horizon 2020 research and innovation programme under grant agreement No 754658 (CARAMBA).

When striving for maximal throughput at minimal volumes while cultivating close to industrial conditions, simple and robust feeding strategies offer important advantages. Enzyme-mediated glucose cleavage from dextrin is an easy way of imitating continuous fed-batch in the small scale, with no complex equipment required. While the release rate - and thus the feed rate - can be controlled by adapting the enzyme concentration, it strongly depends on the concentration of the involved substances and the environmental conditions. Thus, it is a challenge to use the technology for controlling the specific growth rate, as it is commonly done with feed pumps. For solving this problem, we present here a mathematical model that extends simple Michaelis-Menten kinetics by considering different substrate fractions and can be applied to control the glucose release rate even in high throughput experiments. The fitted model was used during automated microbial cultivations to control the growth rate in quasi-continuous fed-batch processes and to realize different exponential growth rates by intermittent additions of enzyme and dextrin by a liquid handling robot system. We thus present an approach for defined biocatalytically controlled glucose supply of small-scale systems, where - if at all - continuous feeding was only possible with low accuracy or high technical efforts until now.

Bioprocess development is commonly characterized by long development times, especially in the early screening phase. After promising candidates have been pre-selected in screening campaigns, an optimal operating strategy has to be found and verified under conditions similar to production. Cultivating cells with pulse-based feeding and thus exposing them to oscillating feast and famine phases has shown to be a powerful approach to study microorganisms closer to industrial bioreactor conditions. In view of the large number of strains and the process conditions to be tested, high-throughput cultivation systems provide an essential tool to sample the large design space in short time. We have recently presented a comprehensive platform, consisting of two liquid handling stations coupled with a model-based experimental design and operation framework to increase the efficiency in High Throughput bioprocess development. Using calibrated macro-kinetic growth models, the platform has been successfully used for the development of scale-down fed-batch cultivations in parallel mini-bioreactor systems. However, it has also been shown that parametric uncertainties in the models can significantly affect the prediction accuracy and thus the reliability of optimized cultivation strategies. To tackle this issue, we implemented a multi-stage Model Predictive Control (MPC) strategy to fulfill the experimental objectives under tight constraints despite the uncertainty in the parameters and the measurements. Dealing with uncertainties in the parameters is of major importance, since constraint violation would easily occur otherwise, which in turn could have adverse effects on the quality of the heterologous protein produced. Multi-stage approaches build up scenario tree, based on the uncertainty that can be encountered and computing optimal inputs that satisfy the constrains despite of such uncertainties. Using the feedback information gained through the evolution along the tree, the control approach is significantly more robust than standard MPC approaches without being overly conservative. We show in this study that the application of multi-stage MPC can increase the number of successful experiments, by applying this methodology to a mini-bioreactor cultivation operated in parallel.

Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. Photorhabdus luminescens toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe like nano-machine for delivery of protein toxins from different species. Additionally, we loaded the highly active copepod luciferase Metridia longa M-Luc7 for accurate quantification of injected molecules. We suggest that besides the size also the charge of the cargo defines the efficiency of packing and transport into mammalian cells. Our data show that the Photorhabdus luminescens toxin complex constitutes a powerful system to inject recombinant proteins, peptides and potentially other molecules like aptamers into mammalian cells. In contrast to other protein transporters based on pore formation, the cargo is protected from degradation. The system opens new perspectives for cell research and pharmacology.

Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease, tick-borne encephalitis, and anaplasmosis. The study of gene function in I. scapularis and other ticks has been hampered by the lack of genetic tools, such as an inducible promoter to permit temporal control over transgenes encoding protein or double-stranded RNA expression. Studies of vector-pathogen relationships would also benefit from the capability to activate anti-pathogen genes at different times during pathogen infection and dissemination. We have characterized an intergenic sequence upstream of the heat shock protein 70 (HSP70) gene that can drive Renilla luciferase expression and mCherry fluorescence in the I. scapularis cell line ISE6. In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter in the synthetic 3xP3 promoter with a minimal portion of the I. scapularis HSP70 promoter and generated an I. scapularis specific 3xP3 (Is3xP3) promoter. Both promoter constructs, IsHSP70 and Is3xP3, allow for heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent positive cells upon exposure to a 2 h heat shock. These promoters described here will be valuable tools for gene function studies and temporal control of gene expression, including anti-pathogen genes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/569248v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@b5ae25org.highwire.dtl.DTLVardef@1bc267borg.highwire.dtl.DTLVardef@1826baborg.highwire.dtl.DTLVardef@16acf4e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Engineering animal models with self-sustained luminescence could enable non-invasive longitudinal monitoring of molecular events in living animals. To create animal models that report physiology with autoluminescence, both luciferin biosynthesis enzymes and the luciferase need to be optimised. Previous work on engineering the autoluminescence pathway from fungi resulted in the development of nnLuz_v3, a version of Neonothopanus nambi luciferase with enhanced thermal stability. Here, we generated an nnLuz_v3 reporter of endogenous Cyp1a1 expression as a measure of aryl hydrocarbon receptor (AHR) activation, assessing the performance of nnLuz_v3 in vivo at physiologically relevant expression levels. As AHR dynamically responds to metabolic, environmental and dietary changes it provides a validated platform to assess novel luminescence approaches. In Cyp1a1-nnLuz mice bioluminescence signal was stable, allowing the generation of well-resolved luminescence images both on standard in vivo imaging equipment and consumer-grade cameras. Using mice and nematode models, we demonstrated limited oral availability of the fungal luciferin, potentially compatible with delivering the substrate via food or the microbiome. Our results are an encouraging first step in the generation of an autoluminescent mammalian model of a molecular event and encourage optimisation of other enzymes of the fungal luciferase pathway.

Coordination of multi-gene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features of the 5UTR in combination with the start of the gene, referred to as the "gene ramp", such as rare codons and mRNA secondary structures. These features strongly influence translation yield and protein quality by regulating ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5UTRs in combination with different target genes leads to a wide variety of gene ramp compositions with irregular translation rates leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramps (SIGER) system for controlling protein expression. The SIGER system makes use of inteins to decouple the translation initiation features from the gene of a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways. As a proof of concept of the potential of the system, we also used a SIGER system to express two difficult-to-produce proteins, GumM and CBM73. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/471673v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1c759acorg.highwire.dtl.DTLVardef@d03c87org.highwire.dtl.DTLVardef@135d6f9org.highwire.dtl.DTLVardef@1b48eac_HPS_FORMAT_FIGEXP M_FIG C_FIG