Hypertension is a major risk factor for cardiovascular diseases, with high prevalence in low- and high-income countries. Among the various antihypertensive therapeutic strategies, synthetic Angiotensin I-converting enzyme inhibitors (ACEI) are one of the most used pharmacological agents. However, their use in hypertension therapy has been linked to various side effects. In recent years considerable research has been performed on the use of food-derived ACEI peptides (ACEIp) as antihypertensive agents. Although promising, the industrial production of these ACEIp through conventional methods, such as chemical synthesis and enzymatic hydrolysis of food proteins, has been proven troublesome and expensive. Limitations to the large-scale production of ACEIp for functional foods and supplements can be overcome by producing the precursors of these peptides in heterologous hosts. Bacterial hosts have been the privileged choice, particularly to test the success of the genetic engineering strategies, but new platforms based on plants and microalgae have also been emerging. This work provides an overview of the state of antihypertensive therapy, focusing on ACEI, illustrates the latest advances on ACEIp research, and describes current genetic engineer-based approaches for the heterologous production of ACEIp for antihypertensive therapy.

Technological advancements in the past few decades have made it possible to manufacture nanomaterials at large scale and ENPs are increasingly found in consumer products such as cosmetics, sports products and LED displays. A large amount of these ENPs are in wastewater and potentially impact the performance of wastewater treatment plants (WWTPs). One important function of the WWTP is nitrification, which is carried out by the actions of two groups of bacteria, ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). Since most ENPs are found to have or are designed to have antimicrobial activities, it is a legitimate concern that ENPs entering WWTPs may have negative impacts on nitrification. In this paper, the effects of ENPs on nitrification is discussed, focusing mainly on autotrophic nitrification by AOBs and NOBs. This review also covers ENPs effects on ANAMMOX. Generally, nitrifiers in pure and mixed culture can be inhibited by a variety of ENPs, but stress response mechanisms may attenuate toxicity. Long-term studies demonstrated that a wide range of NPs can cause severe deterioration of AOBs and/or NOBs when the influent concentration exceeded an inhibition threshold. Proposed mechanisms include the generation of reactive oxygen species, dissolved metals, physical disruption of cell membranes, bacterial engulfment, and intracellular accumulation of ENPs. Future research needs are also discussed.

Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5-kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with specific sites of DNA double strand breaks (DSBs) by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and 3 clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at small guide RNA (sgRNA) target sites. Our results indicated the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammal cells, but also inherently caused mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a non-encapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for a bacterial vaccine production. Batch, fed-batch and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least 4-fold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by Western blot and animal immunization assays, indicating that, so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole cell vaccine production, since it can be integrated to the cell separation process keeping the same vaccine quality.

Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing to manufacturers, demonstration of viral clearance for continuous processes is more complex. Regulators expect manufacturers to use an appropriate scale down model, based on solid scientific justification, to verify the viral reduction capacity of the manufacturing process. The output from chromatography columns operated in continuous processes fluctuates in concentration so that the load for the subsequent column is not homogenous. This must be considered when designing viral clearance studies. One way to approach clearance studies is to downscale the continuous process, using multi-column chromatography systems and in-line spiking of virus. The multi-column chromatography systems and experienced operators, however, may not be available at the CRO performing the study. Another approach is to evaluate each step in traditional batch mode, using existing chromatography systems, but to modify the spiking and loading conditions to mimic the variance introduced by the transition between the two connected process steps. Using a standard chromatography system, we have evaluated a flow through anion exchange chromatography step in a monoclonal antibody process using five different methods to introduce the virus to the column. We have shown that regardless of whether the virus spike is introduced in a well-mixed batch mode, introduced as a concentrated pulse of virus with homogeneous mAb or with a concentrated peak of mAb, the clearance of MMV was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process.

Mussel adhesive proteins (MAPs) have great potential as bioglues, in particular in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows production of bioengineered MAPs (bMAPs), the low production yield hinders the practical application of bMAPs. Such low production yield of Dopa-incorporated bMAPs (Dopa-bMAPs) was known to be caused by low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, in order to enhance the production yield of Dopa-bMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). In order to use the Dopa-specific Methanococcus jannascii TyrRS (MjTyrRS-Dopa), we altered the anti-codon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyrAUA) to recgonize a tyrosine codon (MjtRNATyrAUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyrAUA increased the production yield of Dopa-MAP by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of Dopa-incorporated bMAP. Even with coexpression of Dopa-recognizing TyrRSs, Dopa-bMAPs have a high Dopa incorporation yield (over 90%) compared to Dopa-bMAPs prepared without any coexpression of TyrRS.

Clinical use of pancreatic beta islets for regenerative medicine applications requires mass production of functional cells. Current technologies are insufficient for large-scale production in a cost-efficient manner. Here, we evaluate advantages of a porous cellulose scaffold and demonstrate scale-up to a wicking-matrix bioreactor as a platform for culture of human endocrine cells. Scaffold modifications were evaluated in a multi-well platform to find the optimum surface condition for pancreatic cell expansion followed by bioreactor culture to confirm suitability. Preceding scale-up, cell morphology, viability and proliferation of primary pancreatic cells were evaluated. Two optimal surface modifications were chosen and evaluated further for insulin secretion, cell morphology and viable cell density for human induced pluripotent stem cell-derived pancreatic cells at different stages of differentiation. Scale-up was accomplished with uncoated, amine-modified cellulose in a miniature bioreactor, and insulin secretion and cell metabolic profiles were determined for 13 days. We achieved 10-fold cell expansion in the bioreactor along with a significant increase in insulin secretion compared with cultures on tissue-culture plastic. Our findings define a new method for expansion of pancreatic cells on wicking-matrix cellulose platform to advance cell therapy biomanufacturing for diabetes.

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.