Selection of Heterologous Protein-Producing Strains in Yarrowia lipolytica

2019 ◽  
pp. 153-168 ◽  
Author(s):  
Paul Soudier ◽  
Macarena Larroude ◽  
Ewelina Celińska ◽  
Tristan Rossignol ◽  
Jean-Marc Nicaud
Keyword(s):  
Yarrowia Lipolytica ◽  
Heterologous Protein ◽  
Selection Of

scholarly journals Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica

2020 ◽  
Author(s):  
Carmen Lopez ◽  
Mingfeng Cao ◽  
Zhanyi Yao ◽  
Zengyi Shao
Keyword(s):  
Yarrowia Lipolytica ◽  
Plasmid Stability ◽  
Critical Role ◽  
Fold Increase ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Expression Platform ◽  
Regulatory Dna ◽  
Selection Of

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 4.5-fold increase in gene expression and higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

scholarly journals Impact of overproduced heterologous protein characteristics on physiological response in Yarrowia lipolytica steady-state-maintained continuous cultures

2020 ◽  
Vol 104 (22) ◽  
pp. 9785-9800
Author(s):  
Paulina Korpys-Woźniak ◽  
Piotr Kubiak ◽  
Wojciech Białas ◽  
Ewelina Celińska
Keyword(s):  
Steady State ◽  
Yarrowia Lipolytica ◽  
Heterologous Protein ◽  
Target Protein ◽  
Host Cells ◽  
Synthesis Process ◽  
Secretory Proteins ◽  
Heterologous Proteins ◽  
Glycosylation Pattern ◽  
The Impact

Abstract Overproduction of recombinant secretory proteins triggers numerous physiological perturbations. Depending on a given heterologous protein characteristics, the producer cell is faced with different challenges which lead to varying responses in terms of its physiology and the target protein production rate. In the present study, we used steady-state-maintained Yarrowia lipolytica cells to investigate the impact of different heterologous proteins on the physiological behavior of the host cells. Such an approach allowed to uncouple the impact of the overproduction of a particular protein from the phenomena that result from growth phase or are caused by the heterogeneity of the analyzed populations. Altogether, eight variants of recombinant strains, individually overproducing heterologous proteins of varying molecular weight (27–65 kDa) and reporting activity (enzymatic and fluorescent) were subjected to chemostat cultivations. The steady-state-maintained cells were analyzed in terms of the substrate utilization, biomass and metabolites production, as well as the reporter protein synthesis. Simplified distribution of carbon and nitrogen between the respective products, as well as expression analysis of the heterologous genes were conducted. The here-obtained data suggest that using a more transcriptionally active promoter results in channeling more C flux towards the target protein, giving significantly higher specific amounts and production rates of the target polypeptide, at the cost of biomass accumulation, and with no significant impact on the polyols production. The extent of the reporter protein’s post-translational modifications, i.e., the number of disulfide bonds and glycosylation pattern, strongly impacts the synthesis process. Specific responses in terms of the protein formation kinetics, the gene expression levels, and transcript-to-protein linearity were observed. Key Points • Eight expression systems, producing different reporter proteins were analyzed. • The cells were maintained in steady-state by continuous chemostat culturing. • Protein- and promoter-specific effects were observed.

Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica

2005 ◽  
Vol 115 (4) ◽  
pp. 379-386 ◽  
Author(s):  
Patrick Fickers ◽  
Franck Fudalej ◽  
Jean-Marc Nicaud ◽  
Jacqueline Destain ◽  
Phillipe Thonart
Keyword(s):  
Yarrowia Lipolytica ◽  
Lipase Production ◽  
Extracellular Lipase ◽  
Yeast Yarrowia Lipolytica ◽  
Selection Of

Use of nystatin for selection of tributyrin non-utilising mutants in Yarrowia lipolytica

1988 ◽  
Vol 7 (1) ◽  
pp. 83-86 ◽  
Author(s):  
B.H. Nga ◽  
H. Heslot ◽  
C.M. Gaillardin ◽  
P. Fournier ◽  
K. Chan ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Selection Of

Feeding strategy impacts heterologous protein production in Yarrowia lipolytica fed‐batch cultures—Insight into the role of osmolarity

Yeast ◽  
2019 ◽  
Vol 36 (5) ◽  
pp. 305-318 ◽  
Author(s):  
Monika Kubiak ◽  
Monika Borkowska ◽  
Wojciech Białas ◽  
Paulina Korpys ◽  
Ewelina Celińska
Keyword(s):  
Yarrowia Lipolytica ◽  
Protein Production ◽  
Heterologous Protein ◽  
Feeding Strategy ◽  
Heterologous Protein Production ◽  
Fed Batch ◽  
Batch Cultures ◽  
Insight Into

So you want to express your protein in Escherichia coli?

2021 ◽  
Author(s):  
Aatir A. Tungekar ◽  
Angel Castillo-Corujo ◽  
Lloyd W. Ruddock
Keyword(s):  
Escherichia Coli ◽  
Protein Production ◽  
Heterologous Protein ◽  
Expression System ◽  
Heterologous Protein Expression ◽  
E Coli ◽  
Life Threatening ◽  
Economic Considerations ◽  
Selection Of ◽  
Suitable Expression

Abstract Recombinant proteins have been extensively employed as therapeutics for the treatment of various critical and life-threatening diseases and as industrial enzymes in high-value industrial processes. Advances in genetic engineering and synthetic biology have broadened the horizon of heterologous protein production using multiple expression platforms. Selection of a suitable expression system depends on a variety of factors ranging from the physicochemical properties of the target protein to economic considerations. For more than 40 years, Escherichia coli has been an established organism of choice for protein production. This review aims to provide a stepwise approach for any researcher embarking on the journey of recombinant protein production in E. coli. We present an overview of the challenges associated with heterologous protein expression, fundamental considerations connected to the protein of interest (POI) and designing expression constructs, as well as insights into recently developed technologies that have contributed to this ever-growing field.

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