Recent research has demonstrated that synthetic methanotroph-photoautotroph cocultures offer a highly promising route to convert biogas into value-added products. However, there is a lack of techniques for fast and accurate characterization of cocultures, such as determining the individual biomass concentration of each organism in real-time. To address this unsolved challenge, we propose an experimental-computational protocol for fast, easy and accurate quantitative characterization of the methanotroph-photoautotroph cocultures. Besides determining the individual biomass concentration of each organism in the coculture, the protocol can also obtain the individual consumption and production rates of O2 and CO2 for the methanotroph and photoautotroph, respectively. The accuracy and effectiveness of the proposed protocol was demonstrated using two model coculture pairs, Methylomicrobium alcaliphilum 20ZR - Synechococcus sp. PCC7002 that prefers high pH high salt condition, and Methylococcus capsulatus - Chlorella sorokiniana that prefers low salt and neutral pH medium. The performance of the proposed protocol was compared with a flow cytometry based cell counting approach. The experimental results show that the proposed protocol is much easier to carry out and delivers faster and more accurate results in measuring individual biomass concentration than the cell counting approach without requiring any special equipment.
In this study, the binding of multimodal chromatographic ligands to the IgG1 FC domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated 15N-labeled FC domain indicated that while single mode ion exchange ligands interacted very weakly throughout the FC surface, multimodal ligands interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the Fc. The multimodal ligand binding sites on the FC were concentrated in the hinge region and near the interface of the CH2 and CH3 domains. Further, the multimodal binding sites were primarily composed of positively charged, polar and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on FC corroborated molecular level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand-FC binding in these preferred regions was shown to be electrostatic interactions and pi-pi stacking of surface exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular level understanding of multimodal ligand-FC interactions and sets the stage for future analyses of even more complex biotherapeutics.
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.
ABSTRACT: AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase. AcCHMOM6 is a mutant of AcCHMO we obtained previously that could oxidase the omeprazole sulfide to chiral sulfoxide drug esomeprazole. Based on the structural characteristics of AcCHMO, focused mutagenesis strategy was adopted at the intersections of FAD binding domain, NADPH binding domain and α-helical domain. By the focused mutagenesis and subsequent global evolution, two key residues (55-Leu and 497-Pro) at the intersection of subdomains were identified, of which the L55Y mutagenesis accelerated the H- transfer from NADPH to FAD, while the P497S mutagenesis widened the bottleneck radius of the substrate tunnel and alleviated the substrate inhibition remarkably. By combination of the two mutagenesis, AcCHMOM7 (L55Y/P497S) increased its specific activity from 18.5 U/g to 108 U/g, and its Ki of the substrate sulfide was increased from 34 μM to 265 μM. These results indicated that the catalytic performance can be elevated by modification of the sensitive sites in the intersection of subdomains of AcCHMO, which also provided some insights for the engineering of other type I BVMOs or other multi-subdomain proteins.
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.
Yeast has been engineered for cost-effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single-cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes and phenotypes. The results showed that the average growth of single yeast cells in microdroplets was identical to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell-to-cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value-added bioproducts.
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.
We recently demonstrated that HepaRG cells encapsulated into 1.5% alginate beads are capable of self-assembling into spheroids. They adequately differentiate into hepatocyte-like cells, with hepatic features observed at day 14 post-encapsulation required for external bioartificial liver applications. Preliminary investigations performed within a bioreactor under shear stress conditions and using a culture medium mimicking acute liver failure (ALF) highlighted the need to reinforce beads with a polymer coating. We demonstrated in a first step that a Poly-L-Lysine coating improved the mechanical stability, without altering the metabolic activities necessary for bioartificial liver applications (such as ammonia and lactate elimination). In a second step, we tested the optimized biomass in a newly-designed perfused dynamic bioreactor (PDB), in the presence of the medium model for pathological plasma for 6 hours. Performances of the biomass were enhanced as compared to the steady configuration, demonstrating its efficacy in decreasing the typical toxins of ALF. This type of bioreactor is easy to scale up as it relies on the number of micro-encapsulated cells, and could provide an adequate hepatic biomass for liver supply. Its design allows it to be integrated into a hybrid artificial/bioartificial liver setup for further clinical studies regarding its impact on ALF animal models.
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.
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.
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.