Mixed-culture fermentation provides a means to recycle carbon from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, individual systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive product expression changes under differing conditions are currently poorly understood. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by CO2, acetate, and ethanol) to butyrate, valerate, and hexanoate. Lactate, a high-value biopolymer feedstock chemical, has been observed in transition states, but sustained production has not been described. In this work, a continuous stirred bioreactor was operated at low pH (5.5) with substrate concentration varied between limiting and non-limiting conditions. Using glucose as a model substrate, two sustained operational states were defined: butyrate production during substrate limitation, and lactate production in the non-limited state. Through SWATH-MS metaproteomics and 16S rDNA community profiling, the mechanism of change between butyrate and lactate was described primarily by redirected carbon flow through the methylglyoxal bypass by Megasphaera under substrate non-limiting concentrations. Crucially, butyrate production resumed upon return to substrate-limited conditions, demonstrating the reversibility of this transition.

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.

Incurable breast cancer bone metastasis causes widespread bone loss, resulting in fragility, pain, increased fracture risk, and ultimately increased patient mortality. Increased mechanical signals in the skeleton are anabolic and protect against bone loss, and they may also do so during osteolytic bone metastasis. Skeletal mechanical signals include interdependent tissue deformations and interstitial fluid flow, but how metastatic tumor cells respond to each of these individual signals remains under-investigated, a barrier to translation to the clinic. To delineate their respective roles, we report computed estimates of the internal mechanical field of a bone-mimetic scaffold undergoing combinations of high and low compression and perfusion using multiphysics simulations. Simulations were conducted in advance of multi-modal loading bioreactor experiments with bone metastatic breast cancer cells to ensure that mechanical stimuli occurring internally were physiological and anabolic. Our results show that mechanical stimuli throughout the scaffold were within the anabolic range of bone cells in all loading configurations, were homogenously distributed throughout, and that combined high magnitude compression and perfusion synergized to produce the largest wall shear stresses within the scaffold. These simulations, when combined with experiments, will shed light on how increased mechanical loading in the skeleton may confer anti-tumorigenic effects during metastasis.

We describe scalable and cost-efficient production of full length, His-tagged SARS-CoV-2 spike glycoprotein trimer by CHO cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both HEK and CHO cells mediated by PEI was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32ºC to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in ELISA format to detect IgG antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by PCR, including those who had displayed COVID-19 symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

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.

The development of highly productive, genetically stable manufacturing cell lines is on the critical path to IND filing for protein based biologic drugs. Here we describe Leap-In Transpoasase® platform, a novel transposon-based mammalian (e.g. CHO) cell line development system that produces high titer stable pools with productivity and product quality attributes that are highly comparable to clones that are subsequently derived therefrom. The productivity distributions of clones are strongly biased towards high producers and both genetic and expression stability is consistently high. By avoiding the poor integration rates, concatemer formation, detrimental transgene recombination, low average expression level, unpredictable product quality and inconsistent genetic stability characteristic of non-homologous recombination methods, Leap-In provides several opportunities to de-risk programs early and reduce timelines and resources.

The regulation of cell density is an important segment in microfluidic cell culture, particularly in the repeated assays. Traditionally, the consistent cell density is difficult to achieve owing to the inaccurate regulation of cell density with manual feedback. A novel microfluidic culture method with automatic feedback is proposed for real-time regulation of cell density in this paper. Here, an integrated microfluidic system combining cell culture, density detection and control of proliferation rate was developed. Interdigital electrode structures (IDES) are sputtered on the microchannel for automatically providing the real-time feedback information of impedance. The most sensitive frequency is studied to improve the detection resolution of the sensing chip. Cells were cultured on the chip surface and the cell density was detected by monitoring the alternation of the impedance. The feedback controller is established by least squares support vector machines (LS-SVM). Then, the cell proliferation rate was automatically controlled using the feedback controller to achieve the desired cell density in the repeated assays. The results show that the standard error of this method is 2.8% indicating that the method can keep consistency of cell density in the repeated assays. This study provides a basis for improving the accuracy and repeatability in the further assays of finding the optimal drug concentration.

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.

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 cell culture is the central piece of a biotechnological industrial process. It includes upstream (e.g. media preparation, fixed costs, etc.) and downstream steps (e.g. product purification, waste disposal, etc.). In the continuous mode of cell culture, a constant flow of fresh media replaces culture fluid until the system reaches a steady state. This steady state is the standard operation mode which, under very general conditions, is a function of the ratio between the cell density and the dilution rate and depends on the media supplied to the culture. To optimize the production process it is widely accepted that the concentration of the metabolites in this media should be careful tuned. A poor media may not provide enough nutrients to the culture, while a media too rich in nutrients may be a waste of resources because, either the cells do not use all of the available nutrients, or worse, they over-consume them producing toxic byproducts. In this work we show how an in-silico study of a genome scale metabolic network coupled to the dynamics of a chemostat could guide the strategy to optimize the media to be used in a continuous process. Given a known media we model the concentrations of the cells in a chemostat as a function of the dilution rate. Then, we cast the problem of optimizing the production process within a linear programming framework in which the goal is to minimize the cost of the media keeping fixed the cell concentration for a given dilution rate in the chemostat. We evaluate our results in two metabolic models: first a simplified model of mammalian cell metabolism, and then in a realistic genome-scale metabolic networks of mammalian cells, the Chinese Hamster Ovary (CHO) cell line. We explore the latter in more detail given specific meaning to the predictions of the concentrations of several metabolites.

Cell-to-cell variability in cell populations arises from a combination of intrinsic factors and extrinsic factors related to the milieu. However, the heterogeneity of high cell density suspension cultures for therapeutic protein production remains unknown. Here, we illustrate the increasing heterogeneity in the cellular transcriptome of serum-free adapted CHO K1 cells during high cell density suspension culture over time without concomitant changes in the genomic sequence. Cell cycle--dependent subpopulations and cell clusters, which typically appear in other single-cell transcriptome analyses of adherent CHO K1 cultures, were not found in these suspension cultures. Our results indicate that cell division changes the intracellular microenvironment and leads to cell cycle--dependent heterogeneity. Whole mitochondrial single-cell genome sequencing showed cell-to-cell mitochondrial genome variation and heteroplasmy within cells. Indeed, the mitochondrial genome sequencing method developed here enables the validation of cell clonality. The culture time-dependent increase in cellular heterogeneity observed in this study did not show any attenuation in this increasing heterogeneity. Future advances in bioengineering such as culture upscaling, prolonged culturing, and complex culture systems will be confronted with the need to assess and control cellular heterogeneity, and the method described here may prove useful for this purpose.

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.

Multiple impeller reactors are widely used due to their advanced gas utilization and an increased volumetric mass transfer coefficient. However, with the application of Rushton impellers, gas dispersion efficiency varies between the bottom and the upper impeller levels. The present study analyzes the individual flow regime, power input and gas hold-up in each compartment of a reactor equipped with four Rushton impellers. The results indicate that the pre-dispersion of the air introduced by the bottom impeller plays a key role in a better gas retention efficiency of the upper impellers. In contrast, a flooded bottom impeller adversely affects the gas dispersion of all impellers. A novel analysis of the bubble flow in the dispersed state via a two-phase CFD model reveals that a more homogenous distribution of air bubbles in the upper compartments leads to high compartment gas hold-up values, but fewer bubbles in the vicinity of the impellers. The measured and simulated data of this study indicate that the upper impellers' efficiency mostly depends on the flow regime of and the pre-dispersion by the bottom impeller rather than on the upper impellers' flow regimes. These results contribute to the understanding of essential mixing processes and scaling of aerated bioreactors.

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.

The use of electrochemical methods to study living systems, including cells, has been of interest to researchers for a long time. Thus, controlling the polarization of the electrode contacting living cells, one can influence, for example, their proliferation or the synthesis of specific proteins. Moreover, the electrochemical approach formed the basis of the biocompatibility improvement of the materials contacting with body tissues that use in carbon hemosorbents and implants development. It became possible to reach a fundamentally new level in the study of cell activity with the introduction of optically transparent electrodes in this area. The use of such materials allowed approaching to the study of the influence of the electrode potential on adhesion activity and morphology of the different cell types (HeLa cells, endothelial cell, etc.) more detailed. There are a negligible number of publications in this area despite the obvious advantages of the usage of optically transparent electrodes to study living cells. This mini review is devoted to some aspects of the interaction of living cells with conductive materials and current advances in the use of optically transparent electrodes for the study of living cells, as well as the prospects for their use in cellular technologies.

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.

This study explores the relation between biomass-specific succinic acid (SA) production rate and specific growth rate of an engineered industrial strain of Saccharomyces cerevisiae, with the aim to investigate the extent to which growth and product formation can be uncoupled. Ammonium-limited aerobic chemostat and retentostat cultures were grown at different specific growth rates under industrially relevant conditions, i.e., at a culture pH of 3 and with sparging of a 1:1 CO2-air mixture. Biomass-specific SA production rates decreased asymptotically with decreasing growth rate. At near-zero growth rates, the engineered strain maintained a stable biomass-specific SA production rate for over 500 h, with a SA yield on glucose of 0.61 mol.mol-1. These results demonstrate that uncoupling of growth and SA production could indeed be achieved. A linear relation between biomass-specific SA production rate and glucose consumption rate indicated a coupling of SA production rate and the flux through primary metabolism. The low culture pH resulted in an increased death rate, which was lowest at near-zero growth rates. Nevertheless, a significant amount of non-viable biomass accumulated in the retentostat cultures, thus underlining the importance of improving low-pH tolerance in further strain development for industrial SA production with S. cerevisiae.