Enhancing the yield and quality of supercoiled plasmid through Plasmid Engineering

There has been a rise in the interest of plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. To be effective as therapeutics, high yield and high level of supercoiling are required from the bioprocessing point of view. We have approached meeting these requirements through plasmid engineering. Potentially, the level of supercoiling can have an impact on ease of downstream processing. A 7.2kb plasmid was developed by cloning of Bacteriophage-Mu Strong gyrase-binding sequence (Mu-SGS) into 6.8kb pSVβ-Gal. Four E. coli strains were transformed with both the modified pSVβ-Gal398 plasmid and pSVβ-Gal. Small scale fermentations and analysis were carried out in triplicate cultures to screen for best performing strains. Two of the four strains selected amplified the plasmids efficiently. There was over 20% increase in the total plasmid yield with pSVβ-Gal398 in both strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The two strains were investigated further in shake flasks. Increases in supercoiling and plasmid yield were also observed. The extent of supercoiling was examined by superhelical density quantification, with pSVβ-Gal398 maintaining a supercoil density of -0.022 and pSVβ-Gal -0.019 in both strains. The compactness of the plasmid DNA was also quantified by hydrodynamic diameter measurement using the Nanoparticle Tracking Analysis (NTA) and it was observed that pSVβ-Gal398 was more compact with a Dh of 40-59nm compared to pSVβ-Gal with Dh of 70-90nm for both strains examined. The report of this study has shown that plasmid engineered to contain the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing. References: Hassan, S., Keshavarz‐Moore, E., & Ward, J. (2016). A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy. Biotechnology and bioengineering, 113(9), 2064-2071. Yau, S. Y., Keshavarz‐Moore, E., & Ward, J. (2008). Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large‐scale processes. Biotechnology and bioengineering, 101(3), 529-544

A New Approach to Plasmid Upstream Processing for Vaccine and Gene Therapy Applications

There has been a rise in the interest of plasmid DNA as therapeutics. The rise is evident in the number of ongoing clinical trials involving the use of plasmid DNA. To be useful as therapeutics, the DNA needs to be of high yield and high level of supercoiling. From the bioprocessing point of view, the level of supercoiling can potentially have an impact on the ease of downstream processing. We have approached meeting these requirements through plasmid engineering and developing an optimized fermentation strategy. Two different plasmids (small and large size) were developed. A 7.2kb plasmid was developed by insertion of Bacteriophage-Mu Strong gyrase-binding sequence (Mu-SGS) to 6.8kb pSVβ-Gal. Four E. coli strains were transformed with both the modified pSVβ-Gal398 plasmid and pSVβ-Gal. Small scale fermentation and analysis were carried out in triplicates cultures to screen for best-performing strains. There was over 20% increase in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The two strains were investigated further, and an increase in supercoiling and plasmid yield was also observed. The extent of supercoiling was examined by superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of -0.022 and pSVβ-Gal -0.019 in both strains. The compactness of the plasmid DNA was also quantified by hydrodynamic diameter (Dh) measurement using the Nanoparticle Tracking Analysis (NTA), and it was observed that pSVβ-Gal398 was more compact with a Dh of 40-59 nm compared to pSVβ-Gal with Dh of 70-90 nm for both strains examined. In order to investigate this approach can be scaled on plasmid, a 27 kb plasmid pSTFλ398 was constructed with its respective control plasmid, 26.6 kb pSTFλ. There was an almost 2-fold increase in the plasmid yield for the SGS containing plasmid pSTFλ398 and a supercoiled content of 93%. In addition, SGS containing plasmid was maintained integrity when subjected to shear. An approach was also developed to increase the plasmid yield by developing a batch fermentation for high cell density which involves supplementation with minimal media with amino acids. The media was supplemented with histidine and glycine which have been reported to be implicated in increasing plasmid yield, and methionine for suppressing acetate inhibition. The supplementation allowed the use of initial glycerol concentration as high as 100 g/L with a volumetric yield and plasmid yield of twice as much as using 100 g/L glycerol in the media without supplementation. The report of this study has shown that plasmid modification with the Mu-phage SGS sequence and optimizing fermentation strategies have beneficial effects on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing

Enhancing the productivity of supercoiled plasmid upstream bioprocessing through plasmid engineering

This study was set out to develop an approach for producing highly supercoiled plasmid DNA. Potentially, the level of supercoiling can have an impact on ease of downstream processing. A 7.2kb plasmid was developed by cloning of Bacteriophage-Mu Strong gyrase-binding sequence (Mu-SGS) into 6.8kb pSVβ-Gal. Four E. coli strains were transformed with both the modified pSVβ-Gal398 plasmid and pSVβ-Gal. Small scale fermentations and analysis were carried out in triplicate cultures to screen for best performing strains. Two of the four strains selected amplified the plasmids efficiently. There was over 20% increase in the total plasmid yield with pSVβ-Gal398 in both strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The two strains were investigated further in shake flasks. Increases in supercoiling and plasmid yield were also observed. The extent of supercoiling was examined by superhelical density quantification, with pSVβ-Gal398 maintaining a supercoil density of -0.022 and pSVβ-Gal -0.019 in both strains. The compactness of the plasmid DNA was also quantified by hydrodynamic diameter measurement using the Nanoparticle Tracking Analysis (NTA) and it was observed that pSVβ-Gal398 was more compact with a Dh of 40-59nm compared to pSVβ-Gal with Dh of 70-90nm for both strains examined. The report of this study has shown that plasmid engineered to contain the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing. References: Hassan, S., Keshavarz‐Moore, E., & Ward, J. (2016). A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy. Biotechnology and bioengineering, 113(9), 2064-2071. Yau, S. Y., Keshavarz‐Moore, E., & Ward, J. (2008). Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large‐scale processes. Biotechnology and bioengineering, 101(3), 529-544

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

Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing

Genetic variability of spiralling whitefly Aleurodicus disperses Russell on Citrus aurantifolia Christm and Ocimum gratissimum L.

The spiralling whitefly, Aleurodicus dispersus Russell (Hemiptera: Aleyrodidae), has a wide range of anatomy, physiology, behaviour, and ecology depending on the host plant. This research looked at the genetic variations between A. dispersus populations on two separate host plants (Citrus aurantifolia and Ocimum gratissimum). The existence of host-related genetic variation in A. dispersus populations was determined using Rapid Amplification of Polymorphic DNA (RAPD) markers. Our findings revealed that the A. dispersus populations on the two host plants had a considerable amount of genetic divergence. The spiralling whiteflies on the adaxial part of Citrus aurantifolia were genetically distinct from those on the abaxial part of the same plants. Various population genetic parameters such as heterozygosity, Nei's genetic gap, and fixation indices (FST) revealed that spiralling whitefly populations vary genetically, which may be attributable to spiralling whitefly populations originating from multiple sources. These findings also have consequences for the invasive pest's quarantine safety strategy

Rapid assessments of the impact of COVID-19 on the availability of quality seed to farmers : Advocating immediate practical, remedial and preventative action

Rapid assessments of the impacts of the COVID-19 crisis on the seed sector were conducted by a coalition of partners in Ethiopia, Myanmar, Nigeria, and Uganda in May and June 2020. The method was rapid, iterative, inclusive and valuable in revealing threats to the availability and timely access of farmers to quality seed and to food, nutrition and income security, and in advocating for remedial and preventative action. Via mobile application and web survey, and focus group discussions on virtual conferencing platforms a panel of 36 or more local experts operating particularly in formal seed systems in each country identified potential disruptions to activities in the seed sector and recommended immediate practical action to ensure continuity in performance. Recommendations, and the stakeholders best positioned to propel their action, were proposed to and approved by senior leadership in the sector. The entire process from survey to publication of a seed alert in each iteration was completed within two weeks. Due to the highly seasonal nature of agriculture, and recognition that activities are time-bound, quick turnaround on assessments was essential. Dashboards indicated where impact was felt the hardest, also showing how dynamic the situation was. Countries were at different stages in their agricultural seasons, which made the data highly contextual, but also interesting for getting a glimpse into the future. Lessons were offered from one country to another. Reduced mobility was the root cause of many disruptions in supplying seed to farmers. Disruptions caused seed and related industry to operate at reduced capacity. The cost of transactions and doing business during these times may have increased the scarcity and price of inputs beyond what farmers can recover. Sales of quality seed in formal markets were perceived to decline due to delays in distribution, weakened promotion efforts and fewer farmers present. Farmers are less likely to benefit from investments in crop improvement for more seasons to come due to delays in the development and release of new varieties. Social distancing prevents stakeholders from meeting to exchange goods, services and information, but the sector is gradually getting up to speed with information technology. For all concerns, practical options were offered and often implemented. The pandemic has exacerbated structural weaknesses in the organization of the seed sector, for which reforms are not only justified, but overdue.</p

Diversity of plant-based food products involving alkaline fermentation

One of the hallmarks of alkaline fermentation is the rich diversity of products. e broad range of alkaline-fermented foods (AFFs) attributes to the variety of substrates used and the metabolic activities of the microorganisms involved in various fermentation processes. e knowledge of microbial diversity in AFFs will help in establishing their genetic resources and preparing knowledge databases.</p