High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast

Youngblood, Rachel A.; Keich, Uri; Dunham, Maitreya J.

doi:10.1101/gr.144659.112

Cited by 64 publications

(76 citation statements)

References 33 publications

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“…The template has a 100-bp sequence containing the ARS1 origin (Liachko et al., 2013) and an unbiased distribution of guanine and cytosine throughout. To the 5′ side of the origin, the top strand (leading-strand template) has an ∼7:1 adenine-to-thymine bias that is reversed to the 3′ side of the origin (Figure S1A).…”

Section: Resultsmentioning

confidence: 99%

How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

et al. 2017

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SummaryThe eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases α, δ, and ε and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min−1. We have now reconstituted this sophisticated machine with purified proteins, beginning with regulated CMG assembly and activation. We show that replisome-associated factors Mrc1 and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerase ε catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase δ can support leading-strand synthesis, but at slower rates. DNA polymerase δ is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase ε engagement. We propose that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress.

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Section: Resultsmentioning

confidence: 99%

How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

et al. 2017

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“…Furthermore, P. pastoris is readily amenable to genetic modification, and it is relatively easy to integrate multiple copies of an expression cassette, thereby further boosting yields (7)(8)(9). Expression cassettes containing the gene of interest are typically integrated into the genome, resulting in stable expression strains (despite recent advances [10], plasmids have been used infrequently). In contrast to the model organism Saccharomyces cerevisiae, however, in P. pastoris it is more difficult to specifically integrate cassettes at a desired locus in the genome.…”

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confidence: 99%

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

Vogl

Gebbie

Palfreyman

et al. 2018

Appl Environ Microbiol

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Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of Ͼ700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris. Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (Ͻ10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs).IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.KEYWORDS integration, Pichia pastoris, protein expression, genome analysis T he yeast Pichia pastoris (syn. Komagataella phaffii) is widely used for heterologous protein p...

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“…(a) Phylogenetic relationships and previously published ACS motifs are shown for S. cerevisiae (Broach et al 1983; Liachko et al 2013), L. waltii (Di Rienzi et al 2012), L. kluyveri (Liachko et al 2011), and K. lactis (Liachko et al 2010). (b) The sequences of the native (ARS) and optimized (OPT) panARS elements are shown.…”

Section: Supplementary Materialsmentioning

confidence: 99%

“…Yeast replication origins retain their initiation activity in a plasmid context allowing autonomous episomal plasmid maintenance (Stinchcomb et al 1980). This cis -acting a utonomously r eplicating s equence (ARS) function has been useful for both understanding the basic science of DNA replication (Nieduszynski et al 2007; Liachko et al 2013) and for industrial applications (Böer et al 2007). …”

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An autonomously replicating sequence for use in a wide range of budding yeasts

Liachko

Dunham

2013

FEMS Yeast Res

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The initiation of DNA replication at replication origins is essential for the duplication of genomes. In yeast, the autonomously replicating sequence (ARS) property of replication origins is necessary for the stable maintenance of episomal plasmids. However, because the sequence determinants of ARS function differ among yeast species, current ARS modules are limited for use to a subset of yeasts. Here we describe a short ARS sequence that functions in at least 10 diverse species of budding yeast. These include, but are not limited to members of the Saccharomyces, Lachancea, Kluyveromyces, and Pichia (Komagataella) genera spanning over 500 million years of evolution. In addition to its wide species range, this ARS and an optimized derivative confer improved plasmid stability relative to other currently used ARS modules.

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High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast

Cited by 64 publications

References 33 publications

How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

An autonomously replicating sequence for use in a wide range of budding yeasts

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