The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate with the induction of apoptosis. Recent progress in mammalian synthetic biology provides unique opportunities to endow cells with programmable, user-defined behaviors, thereby addressing some of the challenges of current methods. In this review, we will discuss advances in synthetic biology to design efficient strategies for biomanufacturing.

The liver is one of the vital organs in the body, and the gold standard of treatment for liver function impairment is liver transplantation, which poses many challenges. The specific 3D structure of liver, which significantly impacts the growth and function of its cells, has made biofabrication with the 3D printing of scaffolds suitable for this approach. In this study, to investigate the effect of scaffold geometry on the performance of HepG2 cells, Poly-Lactic acid (PLA) polymer was used as the input of the Fused Deposition Modeling (FDM) 3D-printing machine. Samples with simple square and bioinspired hexagonal cross-section designs were printed. 1% and 2% of gelatin-coating were applied to the 3D printed PLA to improve the wettability and surface properties of the scaffold. SEM pictures were used to analyze the structural properties of PLA-Gel hybrid scaffolds, EDS to investigate the presence of gelatin, water contact angle measurement for wettability, and weight loss for degradation. In vitro tests were performed by culturing HepG2 cells on the scaffold to evaluate the cell adhesion, viability, cytotoxicity, and specific liver functions. Then, high-precision scaffolds were printed and the presence of gelatin was detected. Also, the effect of geometry on cell function was confirmed in viability, adhesion, and functional tests. The albumin and urea production of the Hexagonal PLA scaffold was about 1.22 ­±0.02 fold higher than the square design in 3 days. This study will hopefully advance our understanding of liver tissue engineering toward a promising perspective for liver regeneration.

The mechanical properties of biofilms can be used to predict biofilm deformation, for example under fluid flow. We used magnetic tweezers to spatially map the compliance of Pseudomonas aeruginosa biofilms at the micron scale, then used modeling to assess its effects on biofilm deformation. Biofilms were grown in capillary flow cells with Reynolds numbers (Re) ranging 0.28 to 13.9, bulk dissolved oxygen (DO) concentrations from 1 mg/L to 8 mg/L, and bulk calcium ion (Ca2+) concentrations of 0 and 100 mg CaCl2/L. Higher Re numbers resulted in more uniform biofilm morphologies. The biofilm was stiffer at the center of the flow cell than near the walls. Lower bulk DO led to more stratified biofilms. Higher Ca2+ led to increased stiffness and more uniform mechanical properties. Using the experimental mechanical properties, fluid-structure interaction models predicted up to 64% greater deformations for heterogeneous biofilms, compared to a homogeneous biofilms with the same average properties. However, the error depended on the biofilm morphology and flow regime. Our results show significant spatial mechanical variability exists at the micron scale, and that this variability can potentially affect biofilm deformation. The average mechanical properties, provided in many studies, should be used with caution when predicting biofilm deformation.

Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells to enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to sulfur- and cell-free control experiments. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to further 85% increase in the corrosion of the steel. Thus, the modification of the culturing conditions and cell line, led to a more than 3-fold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This work demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target.

Since 2014, an Asian lineage of Zika virus has caused outbreaks, and it has been associated with neurological disorders in adults and congenital defects in newborns. The resulting threat of the Zika virus to human health has prompted the development of new vaccines, which have yet to be approved for human use. Vaccines based on the attenuated or chemically inactivated virus will require large-scale production of the intact virus to meet potential global demands. Intact viruses are produced by infecting cultures of susceptible cells, a dynamic process that spans from hours to days and has yet to be optimized. Here, we infected Vero cells adhesively cultured in well-plates with two Zika virus strains: a recently isolated strain from the Asian lineage, and a cell-culture-adapted strain from the African lineage. At different time points post-infection, virus particles in the supernatant were quantified; further, microscopy images were used to quantify cell density and the proportion of cells expressing viral protein. These measurements were performed across multiple replicate samples of one-step infections every four hours over 60 hours and for multi-step infections every four to 24 hours over 144 hours, generating a rich dataset. For each set of data, mathematical models were developed to estimate parameters associated with cell infection and virus production. The African-lineage strain was found to produce a 14-fold higher yield than the Asian-lineage strain in one-step growth and a 7-fold higher titer in multi-step growth, suggesting a benefit of cell-culture adaptation for developing a vaccine strain. We found that image-based measurements were critical for discriminating among different models, and different parameters for the two strains could account for the experimentally observed differences. An exponential-distributed delay model performed best in accounting for multi-step infection of the Asian strain, and it highlighted the significant sensitivity of virus titer to the rate of viral degradation, with implications for optimization of vaccine production. More broadly, this work highlights how image-based measurements can contribute to discrimination of virus-culture models for the optimal production of inactivated and attenuated whole-virus vaccines.

We evaluated filtration behavior and virus removal capability for a mAb and plasma IgG under constant flow rate directly following flow-through column chromatography in an integrated process. For mAb solution with quantified host cell protein (HCP) content processed in flow-through mode on in-series mixed-mode AEX and modified CEX columns connected to the Planova BioEX filter (pool-less), HCP logarithmic reduction value (LRV) of 2.3 and 93.9% protein recovery were achieved for the process. Filtration behavior for 5 to 15 mg/mL plasma IgG run at flux of 10 to 100 LMH to 100 L/m2 throughput on Planova BioEX filters showed similar behavior across the protein concentrations tested although filtration pressure increased with throughput at 50 LMH and above, indicating the suitability of lower flux processes for continuous processing. Comparing both plasma IgG and mAb filtration behavior to four clogging models showed little difference in fit among the models, but with slightly better fit to the cake filtration model. Viral clearance tested by in-line spiking X-MuLV or MVM into purified plasma IgG showed robust removal at low flux. Integrating the Planova BioEX filter into continuous processes with column chromatography can achieve efficient downstream processing with reduced footprint and process time.

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ) the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a 3-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to analyze changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.

Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α-D-glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. Using G1P at a concentration excluding the substrate-bound E-Glc, the product ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group transfer reactions by hydrolytic enzymes.

The pathophysiological response following spinal cord injury (SCI) is characterized by a complex cellular cascade that limits regeneration. Biomaterial and stem cell combination therapies have shown synergistic effects, compared to their interventions independent of each other, and represent a promising approach towards regaining function after injury. In this study, we combine our polyethylene glycol (PEG) cell delivery platform with lentiviral-mediated overexpression of the anti-inflammatory cytokine interleukin (IL)-10 to improve embryonic day 14 (E14) spinal progenitor transplant survival. PEG tubes loaded with lentivirus encoding for IL-10 were implanted immediately following injury into a mouse SCI hemisection model. Two weeks after tube implantation, mouse E14 spinal progenitors were injected directly into the integrated tubes, which served as a soft substrate for cell transplantation. Together, the tubes with the IL-10 encoding lentivirus improved E14 spinal progenitor survival, assessed at two weeks post-transplantation (four weeks post-injury). Mice receiving IL-10 lentivirus-laden tubes had on average 8.1% of E14 spinal progenitors survive compared to 0.7% in mice receiving transplants without tubes, an 11.5-fold difference. Surviving E14 spinal progenitors gave rise to neurons when injected into tubes. Additionally, axon elongation and remyelination was observed, in addition to a faster rate of functional recovery in mice receiving anti-inflammatory tubes with E14 spinal progenitor delivery. This system affords increased control over the transplantation microenvironment, offering the potential to improve stem cell-mediated tissue regeneration.

Nonwoven membranes are highly engineered fibrous materials that can be manufactured on a large scale from a wide range of different polymers, and their surfaces can be modified using a large variety of different chemistries and ligands. The fiber diameters, surface areas, pore sizes, total porosities, and thicknesses of the nonwoven mats can be carefully controlled, providing many opportunities for creative approaches for the development of novel membranes with unique properties to meet the needs of the future of downstream processing. Fibrous membranes are already finding use in ultrafiltration, microfiltration, depth filtration, and, more recently, in membrane chromatography for product capture and impurity removal. This article summarizes the various methods of manufacturing nonwoven fabrics, and the many methods available for the modification of the fiber surfaces. It also reviews recent studies focused on the use of nonwoven fabric devices in membrane chromatography and provides some perspectives on the challenges that need to be overcome to increase binding capacities, decrease residence times, and reduce pressure drops so that eventually they can replace resin column chromatography in downstream process operations.

Growth decoupling can be used to optimize production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference (CRISPRi) to increase production of a single domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways, and found that repression of 11 pathway genes led to increased GFP production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.

Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor-stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three-dimensional, in vitro, vascularized, IBC platform to quantify the spatial and temporal dynamics of tumor-vasculature and tumor-ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non-IBC cells, MDA-MB-231 (triple negative). An acellular collagen platform with an endothelial blood vessel served as control. SUM149 and MDA-MB-231 platforms exhibited a significantly (p<0.05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher levels of VEGF (p<0.05) and increased collagen ECM porosity compared to non-IBC MDA-MB-231 (p<0.05) and control (p<0.01) platforms. Additionally, unique to the MDA-IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA-IBC3 cells replicating a feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well-described in vivo and clinical IBC phenotypes and provide an adaptable, high throughout tool for systematically and quantitatively investigating tumor-stromal mechanisms and dynamics of tumor progression.

Generally, high bioelectroactivity of anodophilic biofilm favors high power generation of microbial fuel cell (MFC), however, it is not clear whether it can promote denitrification of MFC synchronously. In this study, the impact of anodophilic biofilms bioelectroactivity on denitrification behavior of single-chamber air-cathode MFC (SAMFC) in steady state was studied for the first time. Anodophilic biofilms of various bioelectroactivity were acclimated at conditions of open circuit (OC), Rext of 1000Ω and 20Ω (denoted as SAMFC-OC, SAMFC-1000Ω and SAMFC-20Ω, respectively) and run for 100 days in the presence of nitrate. Electrochemical tests and microbial analysis results showed that the anode of the SAMFC-20Ω delivered higher oxidation and denitrification current response and had a higher abundance of electroactive bacteria, like Geobacter, Pseudomonas and Comamonas, which possessed bidirectional electron transfer function, demonstrating a higher bioelectroactivity of the anodophilic biofilm. Moreover, these electroactive bacteria favored the accumulation of denitrifers, like Thauera and Alicycliphilus, probably by consuming trace oxygen through catalyzing oxygen reduction. The SAMFC-20Ω not only delivered a 61.7% higher power than the SAMFC-1000Ω, but also achieved a stable and high denitrification rate constant (kDN) of 1.9, which was 50% and 40% higher than that of the SAMFC-OC and SAMFC-1000Ω, respectively. It could be concluded that the high bioelectroactivity of the anodophilic biofilms not only favored high power generation of the SAMFC, but also promote the growth of denitrifers at the anodes and strengthened denitrification. This study provided an effective method and important theoretical basis for enhancing power generation and denitrification performance of the SAMFC synchronously.

Fatty acid metabolism has been widely studied in various organisms. However, fatty acid transport has received less attention even though it plays vital roles for the cells, such as export of toxic free fatty acids or uptake of exogenous fatty acids. Hence, there are important knowledge gaps in how fatty acids cross biological membranes and many mechanisms and proteins involved in these processes still need to be determined. The lack of information is more predominant in microorganisms, even though the identification of fatty acids transporters in these cells could lead to new drug targets or improvements in microbial cell factories. This review provides a thorough analysis of the current information on fatty acid transporters in microorganisms, including bacteria, yeasts and microalgae species. Most information available relates to the model organisms Escherichia coli and Saccharomyces cerevisiae, but transport systems of other species are also discussed. Intracellular trafficking of fatty acids and their transport through organelle membranes in eukaryotic organisms is described as well. Finally, applied studies and engineering efforts using fatty acids transporters are presented to show the applied potential of these transporters and to stress the need for further identification of new transporters and their engineering.

The actinomycete Lentzea aerocolonigenes produces the antitumor antibiotic rebeccamycin. In previous studies the rebeccamycin production was significantly increased by the addition of glass beads during cultivation in different diameters between 0.5 – 2 mm and the induced mechanical stress by the glass beads was proposed to be responsible for the increased production. Thus, this study was conducted to be a systematic investigation of different parameters for macroparticle addition, such as bead diameter, concentration and density (glass and ceramic) as well as shaking frequency, for a better understanding of the particle induced stress on L. aerocolonigenes. The induced stress for optimal rebeccamycin production can be estimated by a combination of stress energy and stress frequency. In addition, the macroparticle-enhanced cultivation of L. aerocolonigenes was combined with soy lecithin addition to further increase the rebeccamycin concentration. With 100 g L-1 glass beads in a diameter of 969 µm and 5 g L-1 soy lecithin a concentration of 388 mg L 1 rebeccamycin was reached after 10 days of cultivation, which corresponds to the highest rebeccamycin concentrations achieved in shake flask cultivations of L. aerocolonigenes stated in literature so far.

As a precursor of graphene, graphene oxide (GO) exhibits excellent mechanical, thermal, and electrical properties, besides appreciable biocompatibility in tissue engineering applications. However, the current GO-3D fabrication technology is still in need of optimization and simplification in order to ensure fine architecture and reasonable mechanical property, which would further promote the performance of GO as bio-scaffolds in cell or microorganism attachment and in material transformation. To address this issue, we proposed a GO ink, with appreciable rheological properties and excellent printing performance via high-speed centrifugation and ferric ion-assisted cross-linking. A woodpile structure with controllable micro-pores was produced by micro-extrusion-based 3D printing technology followed by an optimized freeze-drying process. Cellular adhesion and viability were verified by inoculation and culture of HepaRG cells using the fabricated GO 3D structure, thus suggesting ferric ion-assisted cross-linking and controllable pore distribution to improve the performance of GO construct as a bio-scaffold for in-vitro liver tissue models.

Mucociliary clearance is a crucial event that supports the elimination of inhaled particles, bacteria, pollution and hazardous agents from the human airways, and it also limits the diffusion of aerosolized drugs into the airway epithelium. In spite of its relevance, few in vitro models sufficiently address the cumulative effect of the steric and interactive barrier function of mucus on the one hand, and the dynamic mucus transport imposed by ciliary mucus propulsion on the other hand. Here, ad hoc mucus models of physiological and pathological mucus are combined with magnetic artificial cilia to model mucociliary transport in both physiological and pathological states. The Lego®-like concept adopted, in this study, enables the development of mucociliary clearance models with high versatility, since these can be easily modified to reproduce phenomena characteristic of healthy and diseased human airways, while allowing to determine the effect of each parameter and/or structure separately on the overall mucociliary transport. These Lego®-like airway models can be available off-the-shelf because they are exclusively made of readily available materials, thus ensuring reproducibility across different laboratories.

Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this work, a rapid, highly efficient and easily manipulated base editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base editing window could be readily and efficiently achieved by the pCBEso system without requiring double strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5. With this tool, the roles of the key genes involving in N-acetyl-glucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild type when glucose or GlcNAc was used as the sole carbon source. Such a base editing system could be readily applied to other EEB. This work not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1, but also accelerates the robust construction of engineered strains for environmental bioremediation.