A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase-mediated plasmid supercoiling. SGS from 3 different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57-SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total % supercoiled monomer was maintained to 85-90%. A two-fold increase in plasmid yield was also observed for pUC57-SGS in comparison to pUC57. pUC57-SGS displayed greater segregational stability than pUC57-cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. This article is protected by copyright. All rights reserved

Pichia pastoris (Komagataella phaffii) as a Cost-Effective Tool for Vaccine Production for Low- and Middle-Income Countries (LMICs)

Vaccination is of paramount importance to global health. With the advent of the more recent pandemics, the urgency to expand the range has become even more evident. However, the potential limited availability and affordability of vaccines to resource low‐ and middle‐income countries has created a need for solutions that will ensure cost‐effective vaccine production methods for these countries. Pichia pastoris (P. pastoris) (also known as Komagataella phaffii) is one of the most promising candidates for expression of heterologous proteins in vaccines development. It combines the speed and ease of highly efficient prokaryotic platforms with some key capabilities of mammalian systems, potentially reducing manufacturing costs. This review will examine the latest developments in P. pastoris from cell engineering and design to industrial production systems with focus on vaccine development and with reference to specific key case studies

Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring

Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50 pg (4.91 × 103 copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300 pg gDNA (2.95 × 104 copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600 = 80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600 = 80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard

Improving Fab’ fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA

OBJECTIVES: To reduce unwanted Fab’ leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab’ fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. RESULTS: We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab’ grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab’ coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab’ leakage relative to the original autonucleolytic Fab’ strain with OmpA-fused staphylococcal nuclease. CONCLUSIONS: We successfully rescued Fab’ leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab’ fragment to the surrounding growth media

Application of Magnetic Field for Improvement of Microbial Productivity

Continued attempt by the industry and research sectors to improve productivity of commercially viable microbial products fall into three general approaches including microbial-based (e.g. isolation, selection, and manipulation of microbes as higher producers), environmental-based (e.g. media development), and bioreactor/bioprocessbased studies. Application of electromagnetic field to microbial cultures is a recent bioprocess-based technique. Current literature shows some effects on characteristics of microbial species (fungi and bacteria). These include enhancement of ethanol production capacity of Saccharomyces cerevisiae, citric acid and cellulase production by Aspergillus niger species and insulase production by Geotrichum candidum after the cultures were exposed to electromagnetic field. In this paper we report the application of electromagnetic field to cultures of Bacillus licheniformis to enhance productivity of bacitracin, a water-soluble branched polypeptide used as an antimicrobial agent against grampositive and some gram-negative bacteria. Electromagnetic field was applied on cultures of B. licheniformis in stirred tank reactors (STRs) with working volume of 1.5 litres circulating into an in-house designed and constructed magnetic field generator with low magnetic field intensity. The experiments were carried out both with and without pH control of the culture. Samples were assayed for bacitracin concentration to confirm the effects of electromagnetic field. The microbial growth and pH profiles were also monitored. The results showed that circulation of culture at flow rate of 10 mL.min-1 into magnetic field with 10 millitesla intensity leads to an increase in bacitracin concentration. The increase was higher when the pH of the culture was controlled compared to non-controlled culture. The highest percentage increase in bacitracin concentration was 36 % after 35 hours without pH control, while the highest bacitracin percentage increase obtained from the controlled culture under pH 7 exposed to electromagnetic field, was almost 89 % after 43 hours

Effect of the oxygen transfer rate on oxygen-limited production of plasmid DNA by Escherichia coli

Oxygen limitation can increase the pDNA yield in cultures of Escherichia coli. Nevertheless, such effect has not been studied systematically. Namely, only cultures at low DOT have been performed, excluding important factors like the oxygen transfer rate (OTR). Moreover, to the best of our knowledge, there is no information regarding the impact of oxygen availability on the topology of the plasmid. The supercoiling of DNA requires energy and it is hypothesized that oxygen availability will affect the produced isoforms. In the present study, we performed fully aerobic and oxygen-limited cultures of E. coli bearing a high copy number plasmid. Cultures at OTRmax values of 10, 14, 30, 45 (for oxygen-limited cultures) and 110 mmol L-1 h-1 (for aerobic cultures) were performed in microtiter plates with DOT, pH, biomass (measured as scattered light) and NADH fluorescence online monitoring. To further investigate the impact of oxygen limitation on pDNA topology, an E. coli strain constitutively expressing the Vitreoscilla hemoglobin (VHb) was used. VHb is known to improve aerobic respiration and consequently ATP generation at low oxygen availability. Our results show that the pDNA yields on biomass (YpDNA/X) were inversely proportional to the OTRmax for both strains, and increased more than two-fold in cultures at the lowest OTRmax, compared to aerobic cultures. Expression of VHb resulted in lower YpDNA/X, compared to cultures of the parent strain. The strain expressing the VHb displayed higher specific growth rates at OTRmax of 10, 14 and 30 mmol L-1 h-1, compared to the parent strain. However, at OTRmax of 45 and 110 mmol L-1 h-1, the growth rate of the parent strain was higher. In general, the specific NADH fluorescence was lower in cultures of the engineered strain, which can be associated to a more oxidized intracellular state, in agreement with the proposed effect of VHb on the cellular metabolism. The pDNA supercoiled fraction (SCF) was maximum in cultures at OTRmax of 30 mmol L-1 h-1, reaching 92.9 % for the wild type strain and 98.7 % for the strain expressing VHb, while no linearized pDNA was detected. This condition was replicated in a 1 L stirred tank bioreactor (STB) for W3110 recA-, due to the higher productivity of this strain. The performance of cultures in the STB was very similar to that of cultures in the MTP concerning accumulated fermentative by-products, cell growth and pDNA production and SCF. Altogether, these results show the existence of an optimal OTRmax for oxygen-limited production of plasmid DNA. Furthermore, we demonstrate that studies in microtiter plates are excellent to predict culture performance of STB and to scale-up plasmid DNA production cultures

High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017

Importin-7 mediates nuclear trafficking of DNA in mammalian cells

Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy-dependent way. The imp7-dependent pathway was specifically competed by excess DNA but not by excess of maltose-binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly-l-lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems