scholarly journals Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Sophia A. H. Heyde ◽  
Morten H. H. Nørholm
Keyword(s):  
Gene Expression ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Rna Stability ◽  
Solution Space ◽  
Cell Factory ◽  
Rnase E ◽  
Biological Phenomenon ◽  
Dominant Mutation ◽  
Major Bottleneck

AbstractGene expression toxicity is an important biological phenomenon and a major bottleneck in biotechnology. Escherichia coli BL21(DE3) is the most popular choice for recombinant protein production, and various derivatives have been evolved or engineered to facilitate improved yield and tolerance to toxic genes. However, previous efforts to evolve BL21, such as the Walker strains C41 and C43, resulted only in decreased expression strength of the T7 system. This reveals little about the mechanisms at play and constitutes only marginal progress towards a generally higher producing cell factory. Here, we restrict the solution space for BL21(DE3) to evolve tolerance and isolate a mutant strain Evo21(DE3) with a truncation in the essential RNase E. This suggests that RNA stability plays a central role in gene expression toxicity. The evolved rne truncation is similar to a mutation previously engineered into the commercially available BL21Star(DE3), which challenges the existing assumption that this strain is unsuitable for expressing toxic proteins. We isolated another dominant mutation in a presumed substrate binding site of RNase E that improves protein production further when provided as an auxiliary plasmid. This makes it easy to improve other BL21 variants and points to RNases as prime targets for cell factory optimisation.

Transient gene expression: Recombinant protein production with suspension-adapted HEK293-EBNA cells

2001 ◽  
Vol 75 (2) ◽  
pp. 197-203 ◽  
Author(s):  
Petra Meissner ◽  
Horst Pick ◽  
Alexandra Kulangara ◽  
Philippe Chatellard ◽  
Kirstin Friedrich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Transient Gene Expression

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 Optogenetic lac operon to control chemical and protein production in Escherichia coli with light

2019 ◽  
Author(s):  
Makoto A. Lalwani ◽  
Samantha S. Ip ◽  
Cesar Carrasco-Lopez ◽  
Evan M. Zhao ◽  
Hinako Kawabe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Engineering ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Lac Operon ◽  
Iptg Induction ◽  
Spatial Control ◽  
Chemical Inducers ◽  
Cell Functions

AbstractControl of the lac operon with IPTG has been used for decades to regulate gene expression in E. coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities such as easy tunability, reversibility, dynamic induction strength, and spatial control that are difficult to obtain with chemical inducers. We developed an optogenetic lac operon in a series of circuits we call OptoLAC. With these circuits, we control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least 2L bioreactors. Furthermore, OptoLAC circuits enable light control of recombinant protein production, reaching yields comparable to IPTG induction, but with enhanced tunability of expression and spatial control. OptoLAC circuits are potentially useful to confer light controls over other cell functions originally engineered to be IPTG-inducible.

Recent Advances in Recombinant Protein Production by Bacillus subtilis

2020 ◽  
Vol 11 (1) ◽  
pp. 295-318 ◽  
Author(s):  
Kang Zhang ◽  
Lingqia Su ◽  
Jing Wu
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Genetic Manipulation ◽  
Microbial Cell ◽  
Secretory Proteins ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Recent Advances

Bacillus subtilis has become a widely used microbial cell factory for the production of recombinant proteins, especially those associated with foods and food processing. Recent advances in genetic manipulation and proteomic analysis have been used to greatly improve protein production in B. subtilis. This review begins with a discussion of genome-editing technologies and application of the CRISPR–Cas9 system to B. subtilis. A summary of the characteristics of crucial legacy strains is followed by suggestions regarding the choice of origin strain for genetic manipulation. Finally, the review analyzes the genes and operons of B. subtilis that are important for the production of secretory proteins and provides suggestions and examples of how they can be altered to improve protein production. This review is intended to promote the engineering of this valuable microbial cell factory for better recombinant protein production.

Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement

2015 ◽  
Vol 112 (5) ◽  
pp. 934-946 ◽  
Author(s):  
Laura Cervera ◽  
Sonia Gutiérrez-Granados ◽  
Nicholas Simon Berrow ◽  
Maria Mercedes Segura ◽  
Francesc Gòdia
Keyword(s):  
Gene Expression ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Transient Transfection ◽  
Medium Exchange

scholarly journals Development of a constitutive and an auto-inducible high-yield expression system for recombinant protein production in the microalga Nannochloropsis oceanica

2020 ◽  
Vol 104 (20) ◽  
pp. 8747-8760
Author(s):  
Imke de Grahl ◽  
Sweta Suman Rout ◽  
Jodi Maple-Grødem ◽  
Sigrun Reumann
Keyword(s):  
Gene Expression ◽  
Nitrate Reductase ◽  
Recombinant Protein ◽  
Reporter Gene ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Reporter Gene Expression ◽  
Total Soluble Protein ◽  
High Yield ◽  
Nannochloropsis Oceanica

Abstract Photoautotrophic microalgae offer a great potential as novel hosts for efficient recombinant protein production. Nannochloropsis oceanica produces an extraordinarily high content of polyunsaturated fatty acids, and its robust growth characteristics, published genome sequence and efficient nuclear transformation make N. oceanica a promising candidate for biotechnological applications. To establish a robust and flexible system for recombinant protein production, we cloned six endogenous, potentially constitutive or inducible promoters from N. oceanica strain CCMP1779 and investigated their strength using monomeric Venus as reporter gene. Microscopic pre-screening of individual transformants revealed that the promoters of elongation factor (EF), tubulin (TUB) and nitrate reductase (NR) enabled high reporter gene expression. Comparative quantitative analyses of transformant populations by flow cytometry and qRT-PCR demonstrated the highest Venus expression from the EF promoter and the NR promoter if extended by an N-terminal 14-amino acid leader sequence. The kinetics of reporter gene expression were analysed during photobioreactor cultivation, achieving Venus yields of 0.3% (for EF) and 4.9% (for NR::LS) of total soluble protein. Since inducible expression systems enable the production of toxic proteins, we developed an auto-induction medium for the NR promoter transformants. By switching the N source from ammonium to nitrate in the presence of low ammonium concentrations, the starting point of Venus induction could be fine-tuned and shifted towards exponential growth phase while maintaining high recombinant protein yields. Taken together, we demonstrate that a model recombinant protein can be produced robustly and at very high levels in N. oceanica not only under constitutive but also under auto-inducible cultivation conditions. Key points • Nannochloropsis oceanica might serve as host for recombinant protein production. • Comparative promoter strength analyses were conducted for twelve different constructs. • Robust high-yield recombinant protein production was achieved under constitutive conditions. • The nitrate reductase promoter enabled protein production under auto-induction conditions.

scholarly journals Bioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast Yarrowia lipolytica

2019 ◽  
Vol 7 (2) ◽  
pp. 40 ◽  
Author(s):  
Marie Vandermies ◽  
Patrick Fickers
Keyword(s):  
Recombinant Protein ◽  
Yarrowia Lipolytica ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Genetic Manipulation ◽  
Research Field ◽  
Cell Factory ◽  
Cell Factories ◽  
A Cell ◽  
High Level

Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as a cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and the possibility of post-translational modifications. Yarrowia lipolytica is considered as one of the most attractive hosts due to its ability to metabolize raw substrate, to express genes at a high level, and to secrete protein in large amounts. In recent years, several reviews have been dedicated to genetic tools developed for this purpose. Though the construction of efficient cell factories for recombinant protein synthesis is important, the development of an efficient process for recombinant protein production in a bioreactor constitutes an equally vital aspect. Indeed, a sports car cannot drive fast on a gravel road. The aim of this review is to provide a comprehensive snapshot of process tools to consider for recombinant protein production in bioreactor using Y. lipolytica as a cell factory, in order to facilitate the decision-making for future strain and process engineering.

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