Endogenous 2μ Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae

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.

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Plasmid Copy Number and Plasmid Stability

New Trends and Developments in Biochemical Engineering - Advances in Biochemical Engineering/Biotechnology ◽

10.1007/b12440 ◽

2003 ◽

pp. 47-82 ◽

Cited By ~ 47

Author(s):

Karl Friehs

Keyword(s):

Copy Number ◽

Plasmid Stability ◽

Plasmid Copy Number ◽

Plasmid Copy

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Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation.

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1488 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1488-1497 ◽

Cited By ~ 53

Author(s):

K W Runge ◽

V A Zakian

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequences ◽

Copy Number ◽

Plasmid Copy Number ◽

High Copy Number ◽

Limiting Factor ◽

Telomeric Dna ◽

Base Pairs ◽

Plasmid Copy ◽

Telomere Elongation

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.

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Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system

Yeast ◽

10.1002/(sici)1097-0061(19960315)12:3<199::aid-yea895>3.0.co;2-u ◽

1996 ◽

Vol 12 (3) ◽

pp. 199-205 ◽

Cited By ~ 6

Author(s):

Concetta Compagno ◽

Danilo Porro ◽

Stefania Radice ◽

Enzo Martegani ◽

Bianca Maria Ranzi

Keyword(s):

Saccharomyces Cerevisiae ◽

Copy Number ◽

Plasmid Copy Number ◽

Yeast Cells ◽

Plasmid Copy ◽

Selection Of

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

10.1101/433417 ◽

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.

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Expression of plasmid R388-encoded type II dihydrofolate reductase as a dominant selective marker in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.4.3.407-414.1984 ◽

1984 ◽

Vol 4 (3) ◽

pp. 407-414

Author(s):

A Miyajima ◽

I Miyajima ◽

K Arai ◽

N Arai

Keyword(s):

Saccharomyces Cerevisiae ◽

Dihydrofolate Reductase ◽

Copy Number ◽

Enzyme Level ◽

Plasmid Copy Number ◽

Yeast Cells ◽

Type Ii ◽

Plasmid Copy ◽

Selective Media ◽

In Cells

The R388 plasmid-encoded drug-resistant type II dihydrofolate reductase gene (R . dhfr) was expressed in Saccharomyces cerevisiae by fusing the R . dhfr coding sequence to the yeast TRP5 promoter. Yeast cells harboring these recombinant plasmids grew in media with 10 micrograms of methotrexate per ml and 5 mg of sulfanilamide per ml, a condition which inhibits the growth of wild-type cells. Addition of a 390-base-pair fragment from the 3'-noncoding region of TRP5 downstream from R . dhfr increased expression. Presumably, the added segment promoted termination or polyadenylation or both of the R . dhfr transcript. The activity of the plasmid-encoded dihydrofolate reductase and the copy number of the R . dhfr plasmid in cells grown in drug-selective media were higher by one order of magnitude than those grown in nutrition-selective media. Plasmid copy number, as well as the plasmid-encoded enzyme level, decreased when cells were selected for prototrophy. In drug-selective media, the plasmid-encoded enzyme level and the content of R . dhfr transcripts were nearly constant in cells harboring R . dhfr plasmids containing different yeast promoters. In contrast, the plasmid copy number and beta-lactamase activity encoded in cis by plasmids were much higher when R . dhfr was associated with the weak TRP5 promoter than when it was fused to the strong ADC1 promoter. These results indicate that plasmid copy number, i.e., gene dosage of R . dhfr, correlates inversely with the strength of the promoter associated with R . dhfr, and cells with a higher plasmid copy number were enriched in drug-selective media. The transformation efficiency of R . dhfr fused to the ADC1 promoter was almost the same on drug-selective plates as on nutrition-selective plates, indicating that R . dhfr is suitable as a dominant selective transformation marker in S. cerevisiae.

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The 2μm Plasmid Causes Cell Death in Saccharomyces cerevisiae with a Mutation in Ulp1 Protease

Molecular and Cellular Biology ◽

10.1128/mcb.25.10.4299-4310.2005 ◽

2005 ◽

Vol 25 (10) ◽

pp. 4299-4310 ◽

Cited By ~ 31

Author(s):

Melanie J. Dobson ◽

Andrew J. Pickett ◽

Soundarapandian Velmurugan ◽

Jordan B. Pinder ◽

Lori A. Barrett ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Copy Number ◽

Plasmid Copy Number ◽

Partial Deletion ◽

Plasmid Copy ◽

Protein Substrates ◽

Mutant Cells ◽

Two Hybrid ◽

Complete Deletion

ABSTRACT The 2μm circle plasmid confers no phenotype in wild-type Saccharomyces cerevisiae but in a nib1 mutant, an elevated plasmid copy number is associated with cell death. Complementation was used to identify nib1 as a mutant allele of the ULP1 gene that encodes a protease required for removal of a ubiquitin-like protein, Smt3/SUMO, from protein substrates. The nib1 mutation replaces conserved tryptophan 490 with leucine in the protease domain of Ulp1. Complete deletion of ULP1 is lethal, even in a strain that lacks the 2μm circle. Partial deletion of ULP1, like the nib1 mutation, results in clonal variations in plasmid copy number. In addition, a subset of these mutant cells produces lineages in which all cells have reduced proliferative capacity, and this phenotype is dependent upon the presence of the 2μm circle. Segregation of the 2μm circle requires two plasmid-encoded proteins, Rep1 and Rep2, which were found to colocalize with Ulp1 protein in the nucleus and interact with Smt3 in a two-hybrid assay. These associations and the observation of missegregation of a fluorescently tagged 2μm circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification plays a role in both plasmid copy number control and segregation.

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