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Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR–Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d. In this extension to their original protocol applying TAR cloning to mammalian genomes, the authors apply the technique to microbes and environmental DNA samples, by adding ARS-like elements not commonly found in microbial genomes to the TAR cloning vector.

Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomyces cerevisiae , whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichia pastoris ( Komagataella phaffii ). Establishment of surface display in P. pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments (DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P. pastoris formaldehyde dehydrogenase ( FLD1 ) promoter, S. cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyces lactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S. cerevisiae , similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure. Key points • First report of antibody display in P. pastoris using episomal plasmids. • Identification of genetic elements conferring highest levels of antibody display. • Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.

The methylotrophic yeast Pichiapastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P.pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyceslactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P.pastoris.

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 2.2-fold increase in gene expression and 1.7-fold 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.Key points• In Y. lipolytica, an ARS comprises an origin of replication, a spacer, and a centromere.• The unannotated spacer present in the wild-type ARS stabilizes episomal expression.• Plasmid stability and intramolecular rearrangements exhibit a clear cargo dependency.

ABSTRACT Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA–protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements. The centromeres of R. toruloidesIFO0880 were identified by using CUT&RUN to identify the chromosomal regions bound by the centromeric histone Cse4.

Background Komagataella phaffii (Pichia pastoris ) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers. Results Here, we describe a linear “nano”chromosome (of 15–25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70 -knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii -derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii -like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI , a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of P AOX1 and demonstrated that the secreted protein was active as a regulator of the complement system. Conclusions We have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain.

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.

Routine molecular manipulation of any organism is inefficient and difficult without the existence of a plasmid. Although transformation is possible in C. auris, no plasmids are available that can serve as cloning or shuttle vectors. C. auris centromeres have been well characterized but have not been explored further as molecular tools. We tested C. auris centromeric sequences to identify which, if any, could be used to create a plasmid that was stably maintained after transformation. We cloned all seven C. auris centromeric sequences and tested them for transformation frequency and stability. Transformation frequency varied significantly; however, one was found to transform at a very high frequency. A 1.7 Kb subclone of this sequence was used to construct a shuttle vector. The vector was stable with selection and maintained at ~1 copy per cell but could be easily lost when selection was removed, which suggested that the properties of the centromeric sequence were more Autonomously Replicating Sequence (ARS)-like than centromere-like when part of a plasmid. Rescue of this plasmid from transformed C. auris cells into E. coli revealed that it remained intact after the initial C. auris transformation, even when carrying large inserts. The plasmid was found to be able to transform all four clades of C. auris, with varying frequencies. This plasmid is an important new reagent in the C. auris molecular toolbox, which will enhance the investigation of this human fungal pathogen.

Autonomously replicating sequences (ARSs) are important accessories in episomal vectors that allow them to be replicated and stably maintained within transformants. Despite their importance, no information on ARSs in diatoms has been reported. Therefore, we attempted to identify ARS candidates in the model diatom, Phaeodactylum tricornutum , via chromatin immunoprecipitation sequencing. In this study, subunits of the origin recognition complex (ORC), ORC2 and ORC4, were used to screen for ARS candidates. ORC2 and ORC4 bound to 355 sites on the P. tricornutum genome, of which 69 were constantly screened after multiple attempts. The screened ARS candidates had an AT-richness of approximately 50% (44.39–52.92%) and did not have conserved sequences. In addition, ARS candidates were distributed randomly but had a dense distribution pattern at several sites. Their positions tended to overlap with those of the genetic region (73.91%). Compared to the ARSs of several other eukaryotic organisms, the characteristics of the screened ARS candidates are complex. Thus, our findings suggest that the diatom has a distinct and unique native ARSs.

Manipulating gene expression using different types of plasmids (centromeric, 2‐micron, and autonomously replicating sequence) can expand expression levels and enable a quick characterization of combinations, both of which are highly desired for metabolic engineering applications. However, for the powerful industrial workhorse Yarrowia lipolytica, only a low‐copy CEN plasmid is available. In this study, we engineered the CEN plasmid from Y. lipolytica by fusing different promoters upstream of the centromeric region to regulate its function and expand its range. By doing this, we successfully improved the copy number and gene expression at plasmid level by 80% and enabled a dynamic range of nearly 2.7‐fold. This improvement was seen to be independent from both the promoter and gene used in the expression cassette. These results present a better starting point for more potent plasmids in Y. lipolytica.