scholarly journals Cloning and functional characterization of Komagataella phaffii centromeres by a color-based plasmid stability assay

2018 ◽  
Author(s):  
Luiza Cesca Piva ◽  
Janice Lisboa De Marco ◽  
Lidia Maria Pepe de Moraes ◽  
Viviane Castelo Branco Reis ◽  
Fernando Araripe Gonçalves Torres
Keyword(s):  
Copy Number ◽  
Plasmid Stability ◽  
Functional Characterization ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Komagataella Phaffii ◽  
High Transformation ◽  
Colony Color ◽  
Stable Vectors

AbstractThe yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work we designed a color-based genetic assay to investigate genetic stability in K. phaffii. Plasmids bearing K. phaffii centromeres and the ADE3 marker were evaluated in terms of mitotic stability in an ade2/ade3 auxotrophic strain which allows plasmid screening through colony color. Plasmid copy number was verified through qPCR. Our results confirmed that the centromeric plasmids were maintained at low copy number as a result of typical chromosome-like segregation during cell division. These features, combined with high transformation efficiency and in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

scholarly journals Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation

2018 ◽  
Vol 65 (1) ◽  
pp. 179-192 ◽  
Author(s):  
Melissa S. Standley ◽  
Samuel Million-Weaver ◽  
David L. Alexander ◽  
Shuai Hu ◽  
Manel Camps
Keyword(s):  
Genetic Control ◽  
Copy Number ◽  
Plasmid Stability ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Cole1 Plasmid ◽  
Copy Number Regulation

scholarly journals Endogenous 2μ Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Bo-Xuan Zeng ◽  
Ming-Dong Yao ◽  
Wen-Hai Xiao ◽  
Yun-Zi Luo ◽  
Ying Wang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Plasmid Stability ◽  
Essential Gene ◽  
Expression System ◽  
Plasmid Copy Number ◽  
Pathway Engineering ◽  
Plasmid Copy ◽  
Control Plasmid ◽  
Cell Variation

In Saccharomyces cerevisiae, conventional 2μ-plasmid based plasmid (pC2μ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2μ plasmid (pE2μ) brings the issues concerning plasmid stability and copy number of pC2μ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2μ and built the pE2μ multi-copy system by insertion of the target DNA element and elimination of the original pE2μ plasmid. The resulting plasmid pE2μRAF1 and pE2μREP2 demonstrated higher copy number and slower loss rate than a pC2μ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2μRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2μ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2μ multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2μRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2μ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2μ due to the more stable pE2μ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.

scholarly journals Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability

2018 ◽  
Vol 36 (3) ◽  
pp. 472-486 ◽  
Author(s):  
Judith Ilhan ◽  
Anne Kupczok ◽  
Christian Woehle ◽  
Tanita Wein ◽  
Nils F Hülter ◽  
...  
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals Plasmid copy number noise in monoclonal populations of bacteria

2010 ◽  
Vol 81 (1) ◽  
Author(s):  
Jérôme Wong Ng ◽  
Didier Chatenay ◽  
Jérôme Robert ◽  
Michael Guy Poirier
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy
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