mRNA Display Pipeline for Protein Biosensor Construction.

Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.

LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae.

Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. 'Turbo' mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37-33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages 'leaky' biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

EGFR Targeting of Liposomal Doxorubicin Improves Recognition and Suppression of Non-Small Cell Lung Cancer.

Introduction: Despite improvements in chemotherapy and molecularly targeted therapies, the life expectancy of patients with advanced non-small cell lung cancer (NSCLC) remains less than 1 year. There is thus a major global need to advance new treatment strategies that are more effective for NSCLC. Drug delivery using liposomal particles has shown success at improving the biodistribution and bioavailability of chemotherapy. Nevertheless, liposomal drugs lack selectivity for the cancer cells and have a limited ability to penetrate the tumor site, which severely limits their therapeutic potential. Epidermal growth factor receptor (EGFR) is overexpressed in NSCLC tumors in about 80% of patients, thus representing a promising NSCLC-specific target for redirecting liposome-embedded chemotherapy to the tumor site. Methods: Herein, we investigated the targeting of PEGylated liposomal doxorubicin (Caelyx), a powerful off-the-shelf antitumoral liposomal drug, to EGFR as a therapeutic strategy to improve the specific delivery and intratumoral accumulation of chemotherapy in NSCLC. EGFR-targeting of Caelyx was enabled through its complexing with a polyethylene glycol (PEG)/EGFR bispecific antibody fragment. Tumor targeting and therapeutic potency of our treatment approach were investigated in vitro using a panel of NSCLC cell lines and 3D tumoroid models, and in vivo in a cell line-derived tumor xenograft model. Results: Combining Caelyx with our bispecific antibody generated uniform EGFR-targeted particles with improved binding and cytotoxic efficacy toward NSCLC cells. Effects were exclusive to cancer cells expressing EGFR, and increments in efficacy positively correlated with EGFR density on the cancer cell surface. The approach demonstrated increased penetration within 3D spheroids and was effective at targeting and suppressing the growth of NSCLC tumors in vivo while reducing drug delivery to the heart. Conclusion: EGFR targeting represents a successful approach to enhance the selectivity and therapeutic potency of liposomal chemotherapy toward NSCLC.

Targeted delivery of polo-like kinase 1 siRNA nanoparticles using an EGFR-PEG bispecific antibody inhibits proliferation of high-risk neuroblastoma.

High-risk neuroblastoma has poor survival due to treatment failure and off-target side effects of therapy. Small molecule inhibitors have shown therapeutic efficacy at targeting oncogenic cell cycle dysregulators, such as polo-like kinase 1 (PLK1). However, their clinical success is limited by a lack of efficacy and specificity, causing off-target toxicity. Herein, we investigate a new treatment strategy whereby a bispecific antibody (BsAb) with dual recognition of methoxy polyethylene glycol (PEG) and a neuroblastoma cell-surface receptor, epidermal growth factor receptor (EGFR), is combined with a PEGylated small interfering RNA (siRNA) lipid nanoparticle, forming BsAb-nanoparticle RNA-interference complexes for targeted PLK1 inhibition against high-risk neuroblastoma. Therapeutic efficacy of this strategy was explored in neuroblastoma cell lines and a tumor xenograft model. Using ionizable lipid-based nanoparticles as a low-toxicity and clinically safe approach for siRNA delivery, we identified that their complexing with EGFR-PEG BsAb resulted in increases in cell targeting (1.2 to >4.5-fold) and PLK1 gene silencing (>2-fold) against EGFR+ high-risk neuroblastoma cells, and enhancements correlated with EGFR expression on the cells (r > 0.94). Through formulating nanoparticles with PEG-lipids ranging in diffusivity, we further identified a highly diffusible PEG-lipid which provided the most pronounced neuroblastoma cell binding, PLK1 silencing, and significantly reduced cancer growth in vitro in high-risk neuroblastoma cell cultures and in vivo in a tumor-xenograft mouse model of the disease. Together, this work provides an insight on the role of PEG-lipid diffusivity and EGFR targeting as potentially relevant variables influencing the therapeutic efficacy of siRNA nanoparticles in high-risk neuroblastoma.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

An immunomodulating peptide with potential to suppress tumour growth and autoimmunity.

Cancers and autoimmune diseases commonly co-exist and immune checkpoint inhibitor therapy (ICI) exacerbates autoimmune pathologies. We recently described a lipidic peptide, designated IK14004, that promotes expansion of immunosuppressive T regulatory (Treg) cells and uncouples interleukin-2 from interferon-gamma production while activating CD8+ T cells. Herein, we report IK14004-mediated inhibition of Lewis lung cancer (LLC) growth and re-invigoration of splenocyte-derived exhausted CD4+ T cells. In human immune cells from healthy donors, IK14004 modulates expression of the T cell receptor α/ß subunits, induces Type I IFN expression, stimulates natural killer (NK) cells to express NKG2D/NKp44 receptors and enhances K562 cytotoxicity. In both T and NK cells, IK14004 alters the IL-12 receptor ß1/ß2 chain ratio to favour IL-12p70 binding. Taken together, this novel peptide offers an opportunity to gain further insight into the complexity of ICI immunotherapy so that autoimmune responses may be minimised without promoting tumour evasion from the immune system.

An immunomodulating peptide to counteract solar radiation-induced immunosuppression and DNA damage.

Ultraviolet radiation (UVR) induces immunosuppression and DNA damage, both of which contribute to the rising global incidence of skin cancer including melanoma. Nucleotide excision repair, which is activated upon UVR-induced DNA damage, is linked to expression of interleukin-12 (IL-12) which serves to limit immunosuppression and augment the DNA repair process. Herein, we report an immunomodulating peptide, designated IK14800, that not only elicits secretion of IL-12, interleukin-2 (IL-2) and interferon-gamma (IFN-γ) but also reduces DNA damage in the skin following exposure to UVR. Combined with re-invigoration of exhausted CD4+ T cells, inhibition of UVR-induced MMP-1 release and suppression of B16F10 melanoma metastases, IK14800 offers an opportunity to gain further insight into mechanisms underlying the development and progression of skin cancers.

A universal reagent for detection of emerging diseases using bioengineered multifunctional yeast nanofragments.

Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.

Delivery of PEGylated liposomal doxorubicin by bispecific antibodies improves treatment in models of high-risk childhood leukemia.

High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia.

Pharmacokinetics and Biodistribution of 89Zr-Miltuximab and Its Antibody Fragments as Glypican-1 Targeting Immuno-PET Agents in Glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain cancer, accounting for about 85% of all primary central nervous system (CNS) tumors. With standard treatment strategies like surgery, radiation, and chemotherapy, the median survival time of patients with GBM is only 12-15 months from diagnosis. The poor prognosis of GBM is due to a very high tumor recurrence rate following initial treatment, indicating a dire need for improved diagnostic and therapeutic alternatives for this disease. Antibody-based immunotheranostics holds great promise in treating GBM, combining the theranostic applications of radioisotopes and target-specificity of antibodies. In this study, we developed and validated antibody-based positron emission tomography (PET) tracers targeting the heparan sulfate proteoglycan, glypican-1 (GPC-1), for noninvasive detection of disease using diagnostic molecular imaging. GPC-1 is overexpressed in multiple solid tumor types, including GBM, and is a promising biomarker for novel immunotheranostics. Here, we investigate zirconium-89 (89Zr)-conjugated Miltuximab (a clinical stage anti-GPC-1 monoclonal antibody developed by GlyTherix, Ltd.) and engineered fragments for their potential as immuno-PET tracers to detect GPC-1positive GBM tumors in preclinical models. We explore the effects of molecular size, avidity, and Fc-domain on the pharmacokinetics and biodistribution in vivo, by comparing in parallel the full-length antibody (Miltuximab), Fab'2, Fab, and single-chain variable fragment (scFv) formats. High radiolabeling efficiency (>95%) was demonstrated by all the formats and the stability post-radiolabeling was higher for larger constructs of Miltuximab and the Fab. Receptor-mediated internalization of all 89Zr-labeled formats was observed in a human GBM cell line in vitro, while full-length Miltuximab demonstrated the highest tumor retention (5.7 ± 0.94% ID/g, day-9 postinjection (p.i.)) and overall better tumor-to-background ratios than the smaller Fc-less formats. Results from in vivo PET image quantification and ex vivo scintillation counting were highly correlated. Altogether, 89Zr-DFO-Miltuximab appears to be an effective immuno-PET imaging agent for detecting GPC-1positive tumors such as GBM and the current results support utility of the Fc containing whole mAb format over smaller antibody fragments for this target.

Recent advances in the production of recombinant factor IX: bioprocessing and cell engineering.

Appropriate treatment of Hemophilia B is vital for patients' quality of life. Historically, the treatment used was the administration of coagulation Factor IX derived from human plasma. Advancements in recombinant technologies allowed Factor IX to be produced recombinantly. Successful recombinant production has triggered a gradual shift from the plasma derived origins of Factor IX, as it provides extended half-life and expanded production capacity. However, the complex post-translational modifications of Factor IX have made recombinant production at scale difficult. Considerable research has therefore been invested into understanding and optimizing the recombinant production of Factor IX. Here, we review the evolution of recombinant Factor IX production, focusing on recent developments in bioprocessing and cell engineering to control its post-translational modifications in its expression from Chinese Hamster Ovary (CHO) cells.

Detection of Dengue Virus 2 with Single Infected Mosquito Resolution Using Yeast Affinity Bionanofragments and Plasmonic SERS Nanoboxes.

Dengue disease is an emerging global threat triggered by dengue virus (DENV) transmission, primarily by the mosquito Aedes aegypti. The accurate surveillance and sensitive detection of DENV in mosquito populations are critical for the protection of human populations worldwide that are in the habitat of these mosquito species. There are four DENV serotypes with DENV2 reported to cause the most severe complications. There are limited ultrasensitive methods to early detect DENV2 mosquito infection and prevent human infection. Herein, we report an innovative nanobased immunoassay platform for early, specific, and ultrasensitive detection of DENV2-secreted nonstructural 1 (NS1) protein biomarker in single infected mosquitoes with the limit of detection of 500 fg of recombinant DENV2 NS1. The high sensitivity and DENV2 serotype specificity of the platform are the result of using nanomixing, plasmonic SERS nanoboxes, and yeast affinity bionanofragments displaying single-chain variable fragments (nanoyeast scFvs). Nanoyeast scFvs used for high affinity capture of DENV2 NS1 provided an innovative and cost-efficient alternative to monoclonal antibodies and differentiated DENV2 NS1 from other DENV serotypes and Zika virus NS1. The platform used electrohydrodynamically driven nanomixing to enhance NS1 capture by the nanoyeast scFvs while reducing nonspecific interactions. High sensitivity detection of captured DENV2 NS1 was achieved using NS1-specific surface-enhanced Raman scattering (SERS) nanotags. These nanotechnologies provide a significant innovation for early DENV2 detection in single infected mosquitoes, improving the accurate surveillance of mosquito habitats and preventing infection and severe disease arising from DENV2 transmission.

A novel lipidic peptide with potential to promote balanced effector-regulatory T cell responses.

T cell-dendritic cell (DC) interactions contribute to reciprocal stimulation leading to DC maturation that results in production of interleukin-12 (IL-12) and interferon-gamma (IFN-γ). Both cytokines have been implicated in autoimmune diseases while being necessary for effective immune responses against foreign antigens. We describe a lipidic peptide, designated IK14004, that modifies crosstalk between T cells and DCs resulting in suppression of IL-12p40/IFN-γ production. T cell production of interleukin-2 (IL-2) and IFN-γ is uncoupled and IL-12p70 production is enhanced. IK14004 induces expression of activating co-receptors in CD8+ T cells and increases the proportion of Foxp3-expressing CD4+ T regulatory cells. The potential for IK14004 to impact on signalling pathways required to achieve a balanced immune response upon stimulation of DCs and T cells is highlighted. This novel compound provides an opportunity to gain further insights into the complexity of T cell-DC interactions relevant to autoimmunity associated with malignancies and may have therapeutic benefit.

Confined microemulsion sono-polymerization of poly(ethylene glycol) nanoparticles for targeted delivery.

Confined sono-polymerization is developed to prepare poly(ethylene glycol) nanoparticles within water-in-oil microemulsion, followed by post-functionalization with a bispecific antibody (anti HER2 and anti PEG) for targeted delivery of photosensitizers (i.e., indocyanine green). The nanoparticles could specifically target to breast cancer cells (i.e., SKBR3) that overexpress HER2 receptors for the inhibition of cancer cell growth under 808 nm laser irradiation. This study highlights a facile and controllable method to fabricate therapeutic nanoparticles capable of targeted delivery.

An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae.

Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L-1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.

Antibody-Based Formats to Target Glioblastoma: Overcoming Barriers to Protein Drug Delivery.

Glioblastoma (GB) is recognized as the most aggressive form of primary brain cancer. Despite advances in treatment strategies that include surgery, radiation, and chemotherapy, the median survival time (∼15 months) of patients with GB has not significantly improved. The poor prognosis of GB is also associated with a very high chance of tumor recurrence (∼90%), and current treatment measures have failed to address the complications associated with this disease. However, targeted therapies enabled through antibody engineering have shown promise in countering GB when used in combination with conventional approaches. Here, we discuss the challenges in conventional as well as future GB therapeutics and highlight some of the known advantages of using targeted biologics to overcome these impediments. We also review a broad range of potential alternative routes that could be used clinically to administer anti-GB biologics to the brain through evasion of its natural barriers.

Next-Generation Molecular Discovery: From Bottom-Up In Vivo and In Vitro Approaches to In Silico Top-Down Approaches for Therapeutics Neogenesis.

Protein and drug engineering comprises a major part of the medical and research industries, and yet approaches to discovering and understanding therapeutic molecular interactions in biological systems rely on trial and error. The general approach to molecular discovery involves screening large libraries of compounds, proteins, or antibodies, or in vivo antibody generation, which could be considered "bottom-up" approaches to therapeutic discovery. In these bottom-up approaches, a minimal amount is known about the therapeutics at the start of the process, but through meticulous and exhaustive laboratory work, the molecule is characterised in detail. In contrast, the advent of "big data" and access to extensive online databases and machine learning technologies offers promising new avenues to understanding molecular interactions. Artificial intelligence (AI) now has the potential to predict protein structure at an unprecedented accuracy using only the genetic sequence. This predictive approach to characterising molecular structure-when accompanied by high-quality experimental data for model training-has the capacity to invert the process of molecular discovery and characterisation. The process has potential to be transformed into a top-down approach, where new molecules can be designed directly based on the structure of a target and the desired function, rather than performing screening of large libraries of molecular variants. This paper will provide a brief evaluation of bottom-up approaches to discovering and characterising biological molecules and will discuss recent advances towards developing top-down approaches and the prospects of this.

Understanding nanomedicine treatment in an aggressive spontaneous brain cancer model at the stage of early blood brain barrier disruption.

Personalised nanomedicine is an advancing field which has developed significant improvements for targeting therapeutics to aggressive cancer and with fewer side effects. The treatment of gliomas such as glioblastoma (or other brain tumours), with nanomedicine is complicated by a commonly poor accumulation of drugs in tumour tissue owing to the partially intact blood-brain barrier (BBB). Nonetheless, the BBB becomes compromised following surgical intervention, and gradually with disease progression. Increased vasculature permeability generated by a tumour, combined with decreased BBB integrity, offers a mechanism to enhance therapeutic outcomes. We monitored a spontaneous glioma tumour model in immunocompetent mice with ongoing T2-weighted and contrast-enhanced T1-weighted magnetic resonance imaging gradient echo and spin echo sequences to predict an optimal "leakiness" stage for nanomedicine injections. To ascertain the effectiveness of targeted nanomedicines in treating brain tumours, subsequent systemic administration of targeted hyperbranched polymers was then utislised, to deliver the therapeutic payload when both the tumour and brain vascularity had become sufficiently susceptible to allow drug accumulation. Treatment with either doxorubicin-loaded hyperbranched polymer, or the same nanomedicine targeted to an ephrin receptor (EphA2) using a bispecific antibody, resulted in uptake of chemotherapeutic doxorubicin in the tumour and in reduced tumour growth. Compared to vehicle and doxorubicin only, nanoparticle delivered doxorubicin resulted in increased tumour apoptosis, while averting cardiotoxicity. This suggests that polyethylene based (PEGylated)-nanoparticle delivered doxorubicin could provide a more efficient treatment in tumours with a disrupted BBB, and that treatment should commence immediately following detection of gadolinium permeability, with early detection and ongoing 'leakiness' monitoring in susceptible patients being a key factor.

Engineering eukaryote-like regulatory circuits to expand artificial control mechanisms for metabolic engineering in Saccharomyces cerevisiae.

Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast (Saccharomyces cerevisiae) suffers from several drawbacks; specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae. Trans- and cis- modules, including eukaryotic trans-activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L-1 in flask cultivation. These negative/positive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications.

Perfusion culture of Chinese Hamster Ovary cells for bioprocessing applications.

Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come.