An Origin of Replication and a Centromere Are Both Needed To Establish a Replicative Plasmid in the YeastYarrowia lipolytica

Abstract

. We have used the integration properties of centromeric sequences to show that all Y. lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere. The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere. A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid. The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome. Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not. Although the nature of higher eukaryotic replication originsis not yet clear (12), that of origins in the yeast Saccharomyces cerevisiae is relatively well understood. It has been possible to clone S. cerevisiae genomic sequences that confer on a plasmid the ability to replicate extrachromosomally in this yeast. These sequences are called ARS (autonomously replicating sequence) elements. Most ARS elements are active as origins in their chromosomal context, as shown by two-dimensional (2D) gel replicon mapping studies (8,44,62). ARS elements are usually smaller than 150 bp, and they contain an essential motif that matches the 11-bp ARS consensus sequence (ACS; WTT TAYRTTTW) in at least 9 of 11 positions (61, 72). The ACS is essential for initiation of replication, both on plasmids and in the chromosome (17). Also essential is domain B, located at the 3Ј end of the ACS (54,55,76). Depending on the ARS element, domain B can be subdivided into two or three subdomains with variable sequences but conserved roles (42, 66). Finally, a DNA unwinding element is frequently present in ARS elements and is important for replication of plasmids (60) and chromosomes (41). Initiation at ARS elements is under strict cell cycle control (reviewed in reference 18).In contrast to the defined origin sequences of S. cerevisiae, the degree to which specific sequences are employed as origins in the differentiated cells of higher organisms is not yet clear. Most studies employing labeling techniques suggest that initiation takes place at specific sites or in very small (a few kilobases or less) initiation zones (15,19,35). In contrast, all studies employing 2D gel replicon mapping techniques-and some studies using labeling techniques (31, 71)-suggest that initiation can take place at any of numerous locations within large initiation zones of 50 kb or more (reviewed in reference 36). Furthermore, in contrast to the discrete ARS elements of S. cerevisiae, most human DNA pieces larger than ϳ10 kb permit extrachromosomal plasmid replication (38,51). In this case, initiation does not start at a preferred locus, a result which is also observed in plasmid transformation in Xenopus eggs (46,53). Demonstrations that the choice of initiation sites ...

Genomic Exploration of the Hemiascomycetous Yeasts: 17. Yarrowia lipolytica

“…lipolytica chromosomal origins of replication are not able to sustain plasmid replication without a centromeric sequence [9]. No sequence homology was found neither between centromeres or origins of replication within Y. lipolytica nor with those from other organisms.…”

Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals

“…To facilitate gene cloning and assembly, a self-replicative and assembly-friendly Yarrowia vector was constructed to accommodate centromere and replication origin (38,39) as well as the ePathBrick gene assembly feature (40). Specifically, a gene fragment containing centromere 1.1 (CEN 1.1), TEF promoter, exon, incomplete intron, multiple cloning sites, XPR2 terminator, and replication origin ORI1001 was synthesized by Invitrogen (the first gene fragment in SI Appendix, Table S4).…”

Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach

“…lipolytica is a unique host for biochemical production and heterologous protein excretion on account of its abilities to accumulate high levels of lipids (4)(5)(6), utilize hydrophobic and waste carbon sources (2,3,15), and secrete native and heterologous proteins at high levels (12,27,32). The availability of Y. lipolytica's genome sequence (13,42) along with basic genetic tools such as transformation methods (8,10,17), gene knockouts (16), and both episomal (17,29,45,46) and integrative (21,24,44) expression cassettes enable metabolic engineering approaches. However, many of the methods using this organism rely on ill-defined genetic elements (3), especially in the area of promoters.…”