Dataset Information

Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution.

ABSTRACT: The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (?Ph = 0.2-0.3), and cells were maintained at each pH value for 2-3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

SUBMITTER: Correia R

PROVIDER: S-EPMC7711658 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution.

Correia Ricardo R Fernandes Bárbara B Alves Paula M PM Carrondo Manuel J T MJT Roldão António A

Vaccines 20201007 4

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying ...[more]

PMID: 33036359

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Project description:Protein production processes based on stable insect cell lines require intensification to be competitive with the insect cell-baculovirus expression vector system (IC-BEVS). High cell density (HCD) cultures operate continuously, capable of maintaining specific production rates for extended periods of time which may lead to significant improvements in production yields. However, setting up such processes is challenging (e.g., selection of cell retention device and optimization of dilution rate), often demanding the manipulation of large volumes of culture medium with associated high cost. In this study, we developed a process for continuous production of Gag virus–like particles (VLP) pseudotyped with a model membrane protein (influenza hemagglutinin, HA) at HCD using stable insect cells adapted to low culture temperature. The impact of the cell retention device (ATF vs. TFF) and cell-specific perfusion rate (CSPR) on cell growth and protein expression kinetics was evaluated. Continuous production of Gag-HA VLPs was possible using both retention devices and CSPR of 0.04 nL/cell.d; TFF induces higher cell lysis when compared to ATF at later stages of the process (kD = 0.009 vs. 0.005 h−1, for TFF and ATF, respectively). Reducing CSPR to 0.01–0.02 nL/cell.d using ATF had a negligible impact on specific production rates (rHA = 72–68 titer/109 cell.h and rp24 = 12–11 pg/106 cell.h in all CSPR) and on particle morphology (round-shaped structures displaying HA spikes on their surface) and size distribution profile (peaks at approximately 100 nm). Notably, at these CSPRs, the amount of p24 or HA formed per volume of culture medium consumed per unit of process time increases by up to 3-fold when compared to batch and perfusion operation modes. Overall, this work demonstrates the potential of manipulating CSPRs to intensify the continuous production of Gag-HA VLPs at HCD using stable insect cells to make them an attractive alternative platform to IC-BEVS.

Project description:BackgroundThe demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important.ResultsIn this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the L-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive laboratory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h-1 by the parental strain to 0.17 h-1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L-1 h-1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of L-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L-1, a yield of 0.23 g g-1 and a volumetric productivity of 0.35 g L-1 h-1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream processing results.ConclusionFlux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies.

Project description:Among naturally produced polymers, bacterial cellulose is receiving enormous attention due to remarkable properties, making it suitable for a wide range of industrial applications. However, the low yield, the instability of microbial strains and the limited knowledge of the mechanisms regulating the metabolism of producer strains, limit the large-scale production of bacterial cellulose. In this study, Komagataeibacter xylinus K2G30 was adapted in mannitol based medium, a carbon source that is also available in agri-food wastes. K. xylinus K2G30 was continuously cultured by replacing glucose with mannitol (2% w/v) for 210 days. After a starting lag-phase, in which no changes were observed in the utilization of mannitol and in bacterial cellulose production (cycles 1-25), a constant improvement of the phenotypic performances was observed from cycle 26 to cycle 30, accompanied by an increase in mannitol consumption. At cycle 30, the end-point of the experiment, bacterial cellulose yield increased by 38% in comparision compared to cycle 1. Furthermore, considering the mannitol metabolic pathway, D-fructose is an intermediate in the bioconversion of mannitol to glucose. Based on this consideration, K. xylinus K2G30 was tested in fructose-based medium, obtaining the same trend of bacterial cellulose production observed in mannitol medium. The adaptive laboratory evolution approach used in this study was suitable for the phenotypic improvement of K. xylinus K2G30 in bacterial cellulose production. Metabolic versatility of the strain was confirmed by the increase in bacterial cellulose production from D-fructose-based medium. Moreover, the adaptation on mannitol did not occur at the expense of glucose, confirming the versatility of K2G30 in producing bacterial cellulose from different carbon sources. Results of this study contribute to the knowledge for designing new strategies, as an alternative to the genetic engineering approach, for bacterial cellulose production.

Project description:Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution. Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions. Conclusion: C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes.

Dataset Information

Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution.

Publications

Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution.

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