High-throughput microbioreactor provides a capable tool for early stage bioprocess development

Fink, Mathias; Cserjan‐Puschmann, Monika; Reinisch, Daniela; Striedner, Gerald

doi:10.1038/s41598-021-81633-6

Cited by 30 publications

(38 citation statements)

References 38 publications

(28 reference statements)

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“…Efficient, standardized methods for the detailed characterization of candidate strains under process relevant conditions are critical to alleviating key bottlenecks and accelerating bioprocess development. [ 6–8 ] Quality by design is a strategic approach, which aims to maximize process insight and understanding from the outset to minimize the risks associated with bioprocess scale‐up. Statistical design of experiments (DoE), a key tool in the implementation of quality by design, provide a structured approach to exploring relationships between factors of interest and the measured response.…”

Section: Introductionmentioning

confidence: 99%

Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories

Walls

Martínez

Chanona

et al. 2021

Biotechnology Journal

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Background Recent technological advancements in synthetic and systems biology have enabled the construction of microbial cell factories expressing diverse heterologous pathways in unprecedentedly short time scales. However, the translation of such laboratory scale breakthroughs to industrial bioprocesses remains a major bottleneck. Methods and Major Results In this study, an accelerated bioprocess development approach was employed to optimize the biosynthetic pathway of the blockbuster chemotherapy drug, Taxol. Statistical design of experiments approaches were coupled with an industrially relevant high‐throughput microbioreactor system to optimize production of key Taxol intermediates, Taxadien‐5α‐ol and Taxadien‐5α‐yl‐acetate, in engineered yeast cell factories. The optimal factor combination was determined via data driven statistical modelling and validated in 1 L bioreactors leading to a 2.1‐fold improvement in taxane production compared to a typical defined media. Elucidation and mitigation of nutrient limitation enhanced product titers a further two‐fold and titers of the critical Taxol precursors, Taxadien‐5α‐ol and Taxadien‐5α‐yl‐acetate were improved to 34 and 11 mg L‐1, representing a three‐fold improvement compared to the highest literature titers in S. cerevisiae. Comparable titers were obtained when the process was scaled up a further five‐fold using 5 L bioreactors. Conclusions The results of this study highlight the benefits of a holistic design of experiments guided approach to expedite early stage bioprocess development.

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Section: Introductionmentioning

confidence: 99%

Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories

Walls

Martínez

Chanona

et al. 2021

Biotechnology Journal

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“…DO was set to 30% and maintained by adjusting the stirrer speed, aeration rate, and in-gas composition. The batch was inoculated from precultures according to ref ( 94 ). Cells were grown to 6 g in the batch phase after which the exponential carbon-limited feed was started.…”

Section: Methodsmentioning

confidence: 99%

“…Compositions of batch and fed-batch media are described elsewhere. 94 To prevent the formation of foam, PPG2000 antifoam (BASF, Germany) was added on demand. Cultivations used for RNA-seq were conducted in triplicate.…”

Section: Methodsmentioning

confidence: 99%

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in Escherichia coli Affects Product Yield

et al. 2022

Self Cite

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Antibody fragments such as Fab’s require the formation of disulfide bonds to achieve a proper folding state. During their recombinant, periplasmic expression in Escherichia coli , oxidative folding is mediated by the DsbA/DsbB system in concert with ubiquinone. Thereby, overexpression of Fab’s is linked to the respiratory chain, which is not only immensely important for the cell’s energy household but also known as a major source of reactive oxygen species. However, the effects of an increased oxidative folding demand and the consequently required electron flux via ubiquinone on the host cell have not been characterized so far. Here, we show that Fab expression in E. coli BL21(DE3) interfered with the intracellular redox balance, thereby negatively impacting host cell performance. Production of four different model Fab’s in lab-scale fed-batch cultivations led to increased oxygen consumption rates and strong cell lysis. An RNA sequencing analysis revealed transcription activation of the oxidative stress-responsive soxS gene in the Fab-producing strains. We attributed this to the accumulation of intracellular superoxide, which was measured using flow cytometry. An exogenously supplemented ubiquinone analogue improved Fab yields up to 82%, indicating that partitioning of the quinone pool between aerobic respiration and oxidative folding limited ubiquinone availability and hence disulfide bond formation capacity. Combined, our results provide a more in-depth understanding of the profound effects that periplasmic Fab expression and in particular disulfide bond formation has on the host cell. Thereby, we show new possibilities to elaborate cell engineering and process strategies for improved host cell fitness and process outcome.

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“…Calculate the volume needed for the target normalized OD in a total 150 𝜇L volume. 13 5. The 20X diluted sample, or further diluted samples, can also be used directly for final analyses requiring suspended cells, such as relative fluorescence of GFP.…”

Section: Day 3 (Harvest)mentioning

confidence: 99%

The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations

Moreb

et al. 2022

Preprint

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Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of optimal protocols used for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, the development approach described is adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.

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High-throughput microbioreactor provides a capable tool for early stage bioprocess development

Cited by 30 publications

References 38 publications

Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories

Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in Escherichia coli Affects Product Yield

The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations

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