The rare ginsenosides are recognized as the functionalized molecules after oral administration of Panax ginseng and its products. The sources of rare ginsenosides are extremely limited because of low ginsenoside contents in wild plants, hindering their application in functional foods and drugs. We developed an effective combinatorial biotechnology approach including tissue culture, immobilization, and hydrolyzation methods. Rh2 and nine other rare ginsenosides were produced by MeJA-induced culture of adventitious roots in a 10 L bioreactor associated with enzymatic hydrolysis using six β-glycosidases and their combination with yields ranging from 5.54-32.66 mg L-1. The yield of Rh2 was furthermore increased 7% by using immobilized BglPm and Bgp1 in optimized pH and temperature condition, with the highest yield reaching 51.17 mg L-1 (17.06% of PPD-type ginsenosides mixture). Our combinatorial biotechnology method provides a highly efficient approach to acquiring diverse rare ginsenosides, replacing direct extraction from Panax plants, and can also be used to supplement yeast cell factories.
Site-specific integration has emerged as a promising strategy for precise Chinese hamster ovary (CHO) cell line engineering and predictable cell line development. CRISPR/Cas9 with homology-directed repair (HDR) pathway enables precise integration of transgenes into target genomic sites. However, inherent recalcitrance to HDR-mediated targeted integration (TI) of transgenes results in low targeting efficiency, thus requires selection process to acquire targeted integrant in CHO cells. Here we explored several parameters that influence the targeting efficiency using the promoter-trap based single or double knock-in (KI) monitoring system. A simple change in the donor template design by adding sgRNA recognition sequences strongly increased KI efficiency by 2.9–36 fold depending on integration sites and culture mode, compared with conventional circular donor plasmids. Furthermore, sequential and simultaneous KI strategies enabled the generation of double KI populations about 1–4% without the need of additional enrichment processes. This simple optimized strategy not only allowed efficient CRISPR/Cas9-mediated TI in CHO cells but also paved the way for the applicability of multiplexed KIs in one experimental step without the requirement of sequential and independent CHO cell line development procedures.
Endotoxins are considered as the major contributors to the pyrogenic response observed with contaminated pharmaceutical products. Recombinant biopharmaceutical products are manufactured using living organisms, including gram-negative bacteria. Upon the death of a gram-negative bacteria, endotoxins (also known as lipopolysaccharides; LPS) in the outer cell membrane are released into the lysate where it can interact with and form bonds with biomolecules, including target therapeutic compounds. Endotoxin contamination of biologic products may also occur through water, raw materials such as excipients, media, additives, sera, equipment, containers closure systems, and expression systems used in manufacturing. The manufacturing process is therefore in critical need to reduce and remove endotoxins by monitoring raw materials and in-process intermediates at critical steps, in addition to final drug product release testing. In this review, a discussion regarding the progression of endotoxin detection techniques, from crude to refined are presented. We provide a brief overview of the upstream processed used to manufacture therapeutic products and then discuss various downstream purification techniques widely used to purify the products off endotoxins. Finally, we investigate the effectiveness of endotoxin purification processes, both from a perspective of precision as well as cost-effectiveness.
Lactic acid producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology-based process design and can aid the development of sustainable and energy-efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.
3D cell culture has developed rapidly over the past 5-10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability and distribution in real-time, non-destructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration and outcomes of a range of impedance spectroscopy studies and multi-parametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.