Production of a sterol esterase fromOphiostomapiceae in batch and fed-batch bioprocesses using differentPichiapastorisphenotypes as cell factory

2014 ◽  
Vol 30 (5) ◽  
pp. 1012-1020 ◽  
Author(s):  
Víctor Barba Cedillo ◽  
María Jesús Martínez ◽  
Carolina Arnau ◽  
Francisco Valero
Keyword(s):  
Cell Factory ◽  
Fed Batch ◽  
Sterol Esterase

scholarly journals Automated Conditional Screening of Multiple Escherichia coli Strains in Parallel Adaptive Fed-Batch Cultivations

2020 ◽  
Vol 7 (4) ◽  
pp. 145
Author(s):  
Sebastian Hans ◽  
Benjamin Haby ◽  
Niels Krausch ◽  
Tilman Barz ◽  
Peter Neubauer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Developmental Stages ◽  
Added Value ◽  
Major Influence ◽  
Cell Factory ◽  
Sufficient Information ◽  
Limited Information ◽  
Fed Batch ◽  
Scale Down ◽  
Oscillating Glucose

In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance in larger reactors, has a major influence on the efficiency of the final process. To overcome this, scale-down approaches during screenings that show the real cell factory performance at industrial-like conditions are essential. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model-based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight different Escherichia coli strains in triplicate. The 24 fed-batch cultivations were run under the desired conditions, generating sufficient information to define the fastest-growing strain in an environment with oscillating glucose concentrations similar to industrial-scale bioreactors.

scholarly journals Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Pingping Wang ◽  
Wei Wei ◽  
Wei Ye ◽  
Xiaodong Li ◽  
Wenfang Zhao ◽  
...  
Keyword(s):  
Yeast Cell ◽  
Batch Fermentation ◽  
Cell Factory ◽  
Ginsenoside Rh2 ◽  
Fed Batch ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Fed Batch Fermentation ◽  
Shake Flasks ◽  
Produce Plant

AbstractSynthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3–OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones.

scholarly journals Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Javier Garrigós-Martínez ◽  
Kiira Vuoristo ◽  
Miguel Angel Nieto-Taype ◽  
Juha Tähtiharju ◽  
Jaana Uusitalo ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Cell Factory ◽  
The Novel ◽  
Expression Systems ◽  
Fed Batch ◽  
Model Protein

Abstract Background Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (PAOX1), and the constitutive GAP promoter (PGAP). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (PPDF, PUPP) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Results Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. Conclusions As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.

scholarly journals Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Chuanbo Zhang ◽  
Man Li ◽  
Guang-Rong Zhao ◽  
Wenyu Lu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mevalonate Pathway ◽  
Cell Factory ◽  
Fed Batch ◽  
Cell Factories ◽  
Fed Batch Fermentation ◽  
Pharmaceutical Industries ◽  
Engineered Saccharomyces Cerevisiae ◽  
Rate Limiting ◽  
Production Cell

Abstract Background Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction. Results α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L. Conclusions An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.

scholarly journals Automated Conditional Screening of Escherichia Coli Knockout Mutants in Parallel Adaptive Fed-Batch Cultivations

2020 ◽  
Author(s):  
Sebastian Hans ◽  
Benjamin Haby ◽  
Niels Krausch ◽  
Barz Tilman ◽  
Peter Neubauer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Developmental Stages ◽  
Added Value ◽  
Major Influence ◽  
Cell Factory ◽  
Sufficient Information ◽  
Limited Information ◽  
Fed Batch ◽  
Input Design ◽  
Scale Down

In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance of the selected cell factory in larger reactors, has a major influence on the performance of the final process. To overcome this, scaledown approaches are essential to run screenings that show the real cell factory performance at industrial like conditions. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight different Escherichia coli strains in triplicate. The 24 fed-batches ran under the desired conditions generating sufficient information to define the fastest growing strain in an environment with varying glucose concentrations similar to industrial scale bioreactors.

Simulation of UDMC on a Fed-Batch Bioreactor

1988 ◽  
Author(s):  
Hsin-Ying Lin ◽  
John C. Lewis ◽  
Richard H. Luecke
Keyword(s):  
Fed Batch ◽  
Batch Bioreactor
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