Algae are promising feedstock of biofuel. The screening of competent species and proper fertilizer supply are of the most important tasks. To accelerate this rather slow and laborious step, we developed an integrated high-throughput digital microfluidic (DMF) system that uses discrete droplet to serve as micro-bioreactor, encapsulating microalgal cells. Based on the fundamental understanding of various droplet hydrodynamics induced by the existence of different sorts of ions and biological species, an incorporation of capacitance-based position estimator, electrode-saving-based compensation and deterministic splitting approach was performed to optimize the DMF bioreactor. Thus, it enables all processes (e.g. nutrient gradient generation, algae culturing and analyzing of growth and lipid accumulation) occurring automatically on-chip especially in a high-fidelity way. The ability of the system to compare different micro algal strains on chip was investigated. Also, the Chlorella sp. were stressed by various conditions and then growth and oil accumulation were analyzed and compared, which demonstrated its potential as a powerful tool to investigate microalgal lipid accumulation at significantly lower laborites and reduced time.
Process analytical technology (PAT) has been defined by the Food and Drug Administration (FDA) as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality-by-design (QbD) principles, real-time or near-real-time data monitoring is essential for timely control of critical quality attributes (CQAs) to keep the process in a state of control. To facilitate next-generation continuous bioprocessing, deployment of PAT tools for real-time monitoring is integral for process understanding and control. Real-time monitoring and control of CQAs is essential to keep the process within the design space and align with the guiding principles of QbD. The contents of this manuscript are pertinent to the online/at-line monitoring of upstream titer and downstream product quality with timely process control. We demonstrated that a UPLC system interfaced with a process sample manager (UPLC-PSM) can be utilized to measure titer and CQAs directly from bioreactors and downstream unit operations, respectively. We established online titer measurements from fed-batch and perfusion-based alternating tangential flow (ATF) bioreactors as well as product quality assessments of downstream operations for real-time peak collection. This integrated, fully automated system for online data monitoring with feedback control is designed to achieve desired product quality.
E. coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, serious autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. The membrane structure and mRNA expression analysis showed that cells suffered programmed cell death (PCD) during autolysis period. However, blocking three known PCD pathway in BL21 (DE3) cannot alleviate autolysis completely. Furthermore, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in a host of BL21 (DE3-lac1G), which successfully withstand the PCD of the host. The GDH activity at 43h was greatly increased from 37.5 U/mL to 452.0 U/mL. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/mL/h at 43h, compared to the 3 U/mL/h of BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of other 10 enzymes. The engineered E. coli strain in the study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows potential industrial applications.
Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyse the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anti-cancer drug, paclitaxel (Taxol). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimisation of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimisation enhanced total oxygenated taxane titre to 39.0±5.7 mg/L and total taxane product titres were comparable at micro and mini-bioreactor scale at 95.4±18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titre to 19.2 mg/L, comparable to the 23.8±3.7 mg/L achieved at microscale. A combination of bioprocess optimisation and increased GC-MS resolution at 1L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titre of 3.7 mg/L. Total oxygenated taxane titres were improved 2.7-fold at this scale to 78 mg/L, the highest reported titre in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.
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.
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.
Affinity precipitation using stimulus-responsive biopolymers such as Elastin-like Polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a KD of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid phase modifiers such as NaCl, arginine or ethylene glycol was successful. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled > 80% product recovery. The overall process performance evaluated by SDS-PAGE and reversed-phase UPLC analyses, indicated the successful single-step purification of the biologic from an E. coli lysate resulting in ~90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.
Protein lipoylation is essential for the function of many key enzymes, but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate-protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (1) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate Lip-AMP; (2) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (3) both the lipoylation of H protein and its hydrolysis are enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles.
Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms due to the ability to produce complex biologics, use of simple chemically-defined, animal component-free media, robustness of host cells, and biosafety. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous techno-economic models or analyses have been published. Here we present techno-economic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes.
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.
Biofilms commonly develop in flowing aqueous environments, where the flow causes the biofilm to deform. Because biofilm deformation affects the flow regime, and because biofilms behave as complex heterogeneous viscoelastic materials, few models are able to predict biofilm deformation. In this study, a phase field continuum model coupled with the Oldroyd-B constitutive equation was developed and used to simulate biofilm deformation. The accuracy of the model was evaluated using two types of biofilms: a synthetic biofilm, made from alginate mixed with bacterial cells, and a Pseudomonas aeruginosa biofilm. Shear rheometry was used to experimentally determine the mechanical parameters for each biofilm, as inputs for the model. Biofilm deformation under fluid flow was monitored experimentally using optical coherence tomography. The fit between the experimental and modeling geometries after fluid-driven deformation was very good, with relative errors of 12.8% for synthetic biofilm and 22.2% for homogenized P. aeruginosa biofilm. This is the first demonstration of the effectiveness of a viscoelastic phase field biofilm model. This model provides an important tool for predicting biofilm viscoelastic deformation. It also can benefit the design and control of biofilms in engineering systems.
Human olfactory mucosa cells (hOMCs) have potential as a regenerative therapy for spinal cord injury. In our earlier work we derived the PA5 cells, a polyclonal population that retains functional attributes of primary OMCs. Microcarrier suspension culture is an alternative to planar 2D culture to produce cells in quantities that can meet the needs of clinical development. This study aimed to screen the effects of 10 microcarriers on PA5 hOMCs yield and phenotype. Studies performed in well plates led to a 2.9-fold higher cell yield on Plastic compared to Plastic Plus microcarriers with upregulation of neuronal markers β-III tubulin and nestin for both conditions. Microcarrier suspension culture resulted in concentrations of 1.4x105 cells/mL and 4.9x104 cells/mL for Plastic and Plastic Plus, respectively, after 7 days. p75NTR transcript was significantly upregulated for PA5 hOMCs grown on Plastic Plus compared to Plastic. Furthermore, co-culture of PA5 hOMCs grown on Plastic Plus with a neuronal cell line (NG108-15) led to increased neurite outgrowth. This study presents the successful expansion of PA5 cells using microcarrier suspension culture and it reveals competing effects of microcarriers on cell expansion versus functional attributes, showing that designing scalable bioprocesses should not only be driven by cell yields.
Abstract This study describes the response of Arthrospira platensis to a variety of temperature conditions as reflected in variations of photosynthetic parameters, pigmentation, and biomass productivity in indoor photobioreactor (PBR) cultivations. These experiments are designed to better understand the impact of temperature, seasonal variations, and acclimation effects on outdoor biomass production. The irradiance level and temperature range (20 – 39°C) are chosen to enable modeling of semi-continuous operation of large-scale outdoor PBR deployments. Overall, the cultivations were quite stable with some pigment-related instabilities after prolonged high temperature exposure. Changes in productivity with temperature, as reflected in measured photosynthetic parameters, are immediate and mainly attributable to the temperature dependence of the photosaturation parameter, a secondary factor being variation in pigment content on a longer time scale corresponding to turnover of the culture population. Though pigment changes have minimum impact on productivity, prolonged exposure at 35°C and above yields a clear degradation in performance. Productivities in a semi-continuous operation are quantitatively reproduced with a productivity model incorporating photosynthetic parameters measured herein. This study confirms the importance of temperature for biomass and pigment production in Arthrospira cultivations and provides a basis for risk assessments related to temperature mitigation for large-scale outdoor cultivations. Keywords: Arthrospira Platensis, photosynthetic parameters, pigment production, productivity modeling, photobioreactors
Vaccines provide effective protection against many infectious diseases as well as therapeutics for some serious diseases, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, that was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a higher doubling time in both 2D and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.
Previously, our lab developed high molecular weight (MW) tense (T) state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this work, we prepared PolybHb in the T- and relaxed (R) quaternary state with ultrahigh MW (> 500 kDa) with varying cross-link densities and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, autoxidation, and haptoglobin-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e. cross-linker:bovine Hb (bHb) molar ratio, gas/liquid exchange time, temperature during dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T-state and R-state PolybHbs. Our results showed that, the duration of the fast-step autoxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHb exhibited significantly higher biomolecular rate constants for binding to haptoglobin and unimoleular O2 offloading rate constants compared to R-state PolybHb. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHb. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.
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.
Recently, numerous synthetic small molecular peptidomimetics have been designed to overcome the shortcomings of antimicrobial peptides (AMPs), such as protease instability and high production cost. TZP4 is a triazine-based amphipathic polymer designed to mimic the amphipathic structure found in AMPs. Compared to melittin, TZP4 showed superior antimicrobial activity against antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. Results of membrane depolarization, SYTOX Green uptake, flow cytometry, and gel retardation assays suggested that the mechanism of antimicrobial action of TZP4 involved an intracellular target rather than the bacterial cell membrane. Furthermore, TZP4 suppressed the mRNA levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) and inhibited the release of NO and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BODIPY-TR-cadaverine displacement and dissociation of Fluorescein isothiocyanate (FITC) labelled lipopolysaccharides (LPS) assays revealed that TZP4 strongly bound to Escherichia coli-derived LPS and disaggregated the LPS oligomers. Additionally, flow cytometric analysis revealed that TZP4 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. These observations indicate that TZP4 may exert its anti-endotoxin activity by directly binding with LPS and inhibiting the interaction between LPS and CD14+ cells. Thus, we propose that TZP4 is a promising drug candidate for the treatment of endotoxic shock and sepsis caused by gram-negative bacterial infections.
Dibutyl phthalate (DBP) is an environmental pollutant that can threaten human health. The strain Arthrobacter sp. ZJUTW, isolated from the sludge of river of Hangzhou city, can efficiently degrade DBP. Its genomic and transcriptomic differences when cultivated with DBP and with glucose revealed specific DBP metabolic pathways in the ZJUTW strain. The degrading gene clusters distribute separately on a circular chromosome and a plasmid pQL1. Genes related to the initial steps of DBP degradation from DBP to phthalic acid (PA), the pehA gene and pht gene cluster, are located on the plasmid pQL1. While pca gene cluster related to the transforming of protocatechuic acid (PCA) to acetyl-CoA, is located on the chromosome. After homologous alignment analysis with the reported gene clusters, we found that there were a series of double copies of homologous genes in pht and pca gene clusters that contribute to the efficient degradation of DBP by ZJUTW. In addition, transcriptomic analysis showed a synergistic effect between pht and pca clusters, which also favor ZJUTW allowing it to efficiently degrade DBP. Combined genomic and transcriptomic analyses affords the complete DBP metabolic pathway in Arthrobacter sp. ZJUTW that is different from that of reported other Arthrobacter strains. After necessary modification based on its metabolic characteristics, Arthrobacter sp. ZJUTW or its mutants might represent promising candidates for use in the bioremediation of DBP pollution.