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Ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases is a frequent phenomenon that results in DNA structural change and genome instability. However, it is unclear whether the rNTP incorporation into DNA follows any specific sequence patterns. We analyzed multiple datasets of ribonucleoside monophosphates (rNMPs) embedded in DNA, generated from three rNMP-sequencing techniques. These rNMP libraries were obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late-firing autonomously replicating sequences (ARSs) of the yeast genome, where leading and lagging DNA synthesis starts bidirectionally. We found the preference of rNTP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNTP incorporation changes dramatically within the first 1,000 nucleotides from ARSs, highlighting the Pol δ - Pol ϵ handoff during early leading-strand synthesis. Furthermore, the pattern of rNTP incorporation is markedly distinct between the leading and lagging strands not only in mutant but also in wild-type polymerase cells. Such specific signatures of Pol δ and Pol ϵ provide a new approach to track the labor of these polymerases.

Eukaryotic DNA replication begins with multiple origins of replication (ORIs) and thus exploring and identifying ORIs is essential for further understanding of the DNA replication process. In budding yeast, certain autonomously replicating sequences (ARSs) initiate the replication as ORIs, maintaining the stability of chromosomes and plasmids during genome replication. Currently, ARS identification was further facilitated by the DNA microarray technology and bioinformatics, which is based on the previous experimental methods. The structural and functional properties of ARSs on yeast chromosomes are gradually explored in this field. In addition to the function of initiating replication, there is a growing interest for researchers in the effects of ARSs on gene silencing and expression of genes, particularly the relationship between ARSs and chromosome structure. In this review, we summarized the identification methods of ARSs, especially for the bioinformatics prediction methods over the past few years. The functions of ARSs are discussed in this research, moreover, ARS modification that combined with the high‐throughput sequencing was elaborated as well, shedding further light on the understanding of the roles of ARSs, and providing deep insights towards the optimization of ARSs.

Episomal plasmids are crucial expression tools for recombinant protein production and genome editing. In Saccharomyces cerevisiae, 2-μm artificial plasmids with a high copy number have been developed and used in metabolic engineering and synthetic biology. However, in unconventional yeasts such as Yarrowia lipolytica, episomal expression relies on a chromosome replication system; this system has the disadvantages of genetic instability and low copy numbers. In this study, we identified and characterized replication origins from the mitochondrial DNA (mtDNA) of Y. lipolytica. A 516-bp mtDNA sequence, mtORI, was confirmed to mediate the autonomous replication of circular plasmids with high protein expression levels and hereditary stability. However, the nonhomologous end-joining pathway could interfere with mtORI plasmid replication and engender genetic heterogeneity. In the Po 1f ΔKu70 strain, the homogeneity of the mtORI plasmid was significantly improved, and the highest copy number reached 5.0 per cell. Overall, mitochondrial-origin sequences can be used to establish highly stable and autonomously replicating plasmids, which can be a powerful supplement to the current synthetic biology tool library and promote the development of Y. lipolytica as a microbial cell factory.

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7kb-plasmid from seven DNA fragments ranging from 0.7kb to 1.7kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named “Rad53-dependent origins”. The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription.Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

Eukaryotic autonomously replicating sequences (ARSs) are composed of three domains, A, B, and C. Domain A is comprised of an ARS consensus sequence (ACS), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. In Saccharomyces cerevisiae and Kluyveromyces lactis ARS101, the ACS is commonly composed of 11 bp, 5ˊ-(A/T)AAA(C/T)ATAAA(A/T)-3ˊ. This core sequence is essential for S. cerevisiae and K. lactis ARS activity. In this study, we identified ARS-containing sequences from genomic libraries of the yeast Kluyveromyces marxianus DMKU3-1042 and validated their replication activities. The identified K. marxianus DMKU3-1042 ARSs (KmARSs) have very effective replication ability but their sequences are divergent and share no common consensus. We have carried out point mutations, deletions, and base pairs substitutions within the sequences of some of the KmARSs to identify the sequence(s) that influence the replication activity. Consensus sequences same as the 11 bp ACS of S. cerevisiae and K. lactis were not found in all minimum functional KmARSs reported here except KmARS7. Moreover, partial sequences from different KmARSs are interchangeable among each other to retain the ARS activity. We have also specifically identified the essential nucleotides, which are indispensable for replication, within some of the KmARSs. Our deletions analysis revealed that only 21 bp in KmARS18 could retain the ARS activity. The identified KmARSs in this study are unique compared to other yeasts’ ARSs, do not share common ACS, and are interchangeable., Highlights • Identification of minimal autonomously replicating sequences (ARSs) from the yeast Kluyveromyces marxianus DMKU3-1042. • The identities of the isolated ARSs are divergent and have no common consensus with the ARSs of other yeasts. • A short ARS sequence of twenty-one nucleotides functions as an effective replicator in K. marxianus DMKU3-1042. • Segments of ARSs from the yeast K. marxianus are interchangeable among each other. • Functional ARSs are found in both the intergenic and coding sequences of the strain DMKU3-1042.

Summary pMP7017 is a conjugative megaplasmid isolated from the gut commensal Bifidobacterium breve JCM7017 and was shown to encode two putative replicases, designated here as RepA and RepB. In the current work, RepB was identified as the pMP7017 replicative initiator, as the repB gene, and its surrounding region was shown to be sufficient to allow autonomous replication in two bifidobacterial species, B. breve and Bifidobacterium longum subsp. longum. RepB was shown to bind to repeat sequence downstream of its coding sequence and this region was determined to be essential for efficient replication. Based on our results, we hypothesize that pMP7017 is an iteron‐regulated plasmid (IRP) under strict auto‐regulatory control. Recombinantly produced and purified RepB was determined to exist as a dimer in solution, differing from replicases of other IRPs, which exist as a mix of dimers and monomers. Furthermore, a stable low‐copy Bifidobacterium‐E. coli shuttle vector, pRD1.3, was created which can be employed for cloning and expression of large genes, as was demonstrated by the cloning and heterologous expression of the 5.1 kb apuB gene encoding the extracellular amylopullulanase from B. breve UCC2003 into B. longum subsp. longum NCIMB8809., In the current work, RepB was identified as the replicative initiator of the 190 kb conjugative megaplasmid pMP7017 from gut commensal Bifidobacterium breve JCM7017. RepB was shown to bind to repeat sequences downstream of its coding sequence and this region was determined to be essential for efficient replication. In addition, a stable low‐copy Bifidobacterium‐E. coli shuttle vector, pRD1.3, was created which can be employed for cloning and expression of large genes.

DNA digital storage provides an alternative for information storage with high density and long-term stability. Here, we report the de novo design and synthesis of an artificial chromosome that encodes two pictures and a video clip. The encoding paradigm utilizing the superposition of sparsified error correction codewords and pseudo-random sequences tolerates base insertions/deletions and is well suited to error-prone nanopore sequencing for data retrieval. The entire 254 kb sequence was 95.27% occupied by encoded data. The Transformation-Associated Recombination method was used in the construction of this chromosome from DNA fragments and necessary autonomous replication sequences. The stability was demonstrated by transmitting the data-carrying chromosome to the 100th generation. This study demonstrates a data storage method using encoded artificial chromosomes via in vivo assembly for write-once and stable replication for multiple retrievals, similar to a compact disc, with potential in economically massive data distribution., A 254 kb artificial chromosome, storing two pictures and one video clip encoded with low-density parity-check codes and pseudo-random sequences, was manufactured and transmitted from generation to generation stably. The digital information residing in the chromosome was fast retrieved, tolerating base insertions and deletions in nanopore readout.

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.

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 4.5-fold increase in gene expression and 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.

Plasmids are autonomously replicating sequences that help cells adapt to diverse stresses. Theta plasmids are the most frequent plasmid class in enterobacteria. They co-opt two host replication mechanisms: replication at oriC , a DnaA-dependent pathway leading to replisome assembly (theta class A), and replication fork restart, a PriA-dependent pathway leading to primosome assembly through primer extension and D-loop formation (theta classes B, C, and D). To ensure autonomy from the host’s replication and to facilitate copy number regulation, theta plasmids have unique mechanisms of replication initiation at the plasmid origin of replication ( ori ). Tight plasmid copy number regulation is essential because of the major and direct impact plasmid gene dosage has on gene expression. The timing of plasmid replication and segregation are also critical for optimizing plasmid gene expression. Therefore, we propose that plasmid replication needs to be understood in its biological context, where complex origins of replication (redundant origins, mosaic and cointegrated replicons), plasmid segregation, and toxin-antitoxin systems are often present. Highlighting their tight functional integration with ori function, we show that both partition and toxin-antitoxin systems tend to be encoded in close physical proximity to the ori in a large collection of Escherichia coli plasmids. We also propose that adaptation of plasmids to their host optimizes their contribution to the host’s fitness while restricting access to broad genetic diversity, and we argue that this trade-off between adaptation to host and access to genetic diversity is likely a determinant factor shaping the distribution of replicons in populations of enterobacteria.

Initiation of DNA replication occurs at specialized regions along chromosomes called origins. The knowledge of replication origins is imperative to understand regulation of DNA replication. The properties of replication origins in the pathogenic budding yeastCandida albicansremain enigmatic in the absence of the knowledge of authentic chromosomal origins. Earlier, we identified centromere proximal (pCEN) chromosomal origins on chromosomes 5 and 7 ofC. albicans. Here, we identify another centromere-distal (dCEN) chromosomal origin by two-dimensional agarose gel electrophoresis corresponding to a previously reported autonomously replicating sequence (ARS), CARS2. We show that all the identified chromosomal origins are bound byC. albicanshomologs of conserved pre-replication complex proteins Orc2 and Mcm2. Previous reports from our lab and others have shown that there is a strong inter-relationship between centromere function and pericentric origin activity, suggesting that these origins are epigenetically regulated. However, we find that short intergenic regions corresponding to each of these origins functions as an ARS element in circular plasmids. Further, we use the novel strategy ofin vivogap repair to demonstrate that circular ARS plasmids can exist independentlyin vivoinC. albicans.Taken together, these results show that DNA sequence underlies the function of both centromere proximal and centromere distal chromosomal origins inC. albicans.

DNA replication is a fundamental process in all organisms; this event initiates at sites termed origins of replication. The characteristics of eukaryotic replication origins are best understood in Saccharomyces cerevisiae. For this species, origin prediction algorithms or web servers have been developed based on the sequence features of autonomously replicating sequences (ARSs). However, their performances are far from satisfactory. By utilizing the Z-curve methodology, we present a novel pipeline, Ori-Finder 3, for the computational prediction of replication origins in S. cerevisiae at the genome-wide level based solely on DNA sequences. The ARS exhibiting both an AT-rich stretch and ARS consensus sequence element can be predicted at the single-nucleotide level. For the identified ARSs in the S. cerevisiae reference genome, 83 and 60% of the top 100 and top 300 predictions matched the known ARS records, respectively. Based on Ori-Finder 3, we subsequently built a database of the predicted ARSs identified in more than a hundred S. cerevisiae genomes. Consequently, we developed a user-friendly web server including the ARS prediction pipeline and the predicted ARSs database, which can be freely accessed at http://tubic.tju.edu.cn/Ori-Finder3.

Ribonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARS’s) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARS’s, highlighting the Pol δ - Pol ε handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol δ and Pol ε.

Objective The objective of the research presented here was to determine whether autonomous replication sequences (ARS) discovered in the eccDNA replicon of glyphosate resistant Amaranthus palmeri enable self-replication in a yeast system. Results Sequence analysis of the eccDNA replicon revealed a region of sharp changes in A + T/G + C content with characteristic bending indicative of an autonomous replication sequence. Further sequence analysis revealed an extended autonomous replication sequence (EACS) in close proximity to multiple DNA unwinding element (DUE) sequences. This region of the eccDNA replicon enabled autonomous replication of an ARS-less yeast plasmid.

Mitochondrial genomes (mt-genomes) are characterized by a distinct codon usage and their autonomous replication. Mt-genomes encode highly conserved genes (mt-genes), like proteins involved in electron transport and oxidative phosphorylation but they also carry highly variable regions that are in part responsible for their high plasticity. The degree of conservation of their genes is such that they allow the establishment of phylogenetic relationships even across distantly related species. Here, we describe the mechanisms that generate changes along mt-genomes, which play key roles at enlarging the ability of fungi to adapt to changing environments. Within mt-genomes of fungal pathogens, there are dispensable as well as indispensable genes for survival, virulence and/or pathogenicity. We also describe the different complexes or mechanisms targeted by fungicides, thus addressing a relevant issue regarding disease management. Despite the controversial origin and evolution of fungal mt-genomes, the intrinsic mechanisms and molecular biology involved in their evolution will help to understand, at the molecular level, the strategies for fungal disease management., Centro de Investigaciones en Fitopatología, Instituto de Fisiología Vegetal, Facultad de Ciencias Agrarias y Forestales

Bacteriophages are the most abundant biological entities in the biosphere and are a source of uncharacterized biological mechanisms and genetic tools. Here, we identify segments of phage genomes that are used for stable extrachromosomal replication in the prophage state. Autonomous replication of some of these phages requires a RepA-like protein, although most lack repA and use RNA-based systems for replication initiation. We describe a suite of plasmids based on these prophage replication functions that vary in copy number, stability, host range, and compatibility. These plasmids expand the toolbox available for genetic manipulation of Mycobacterium and other Actinobacteria, including Gordonia terrae., Temperate bacteriophages are common and establish lysogens of their bacterial hosts in which the prophage is stably inherited. It is typical for such prophages to be integrated into the bacterial chromosome, but extrachromosomally replicating prophages have been described also, with the best characterized being the Escherichia coli phage P1 system. Among the large collection of sequenced mycobacteriophages, more than half are temperate or predicted to be temperate, most of which code for a tyrosine or serine integrase that promotes site-specific prophage integration. However, within the large group of 621 cluster A temperate phages, ∼20% lack an integration cassette, which is replaced with a parABS partitioning system. A subset of these phages carry genes coding for a RepA-like protein (RepA phages), which we show here is necessary and sufficient for autonomous extrachromosomal replication. The non-RepA phages appear to replicate using an RNA-based system, as a parABS-proximal region expressing a noncoding RNA is required for replication. Both RepA and non-RepA phage-based plasmids replicate at one or two copies per cell, transform both Mycobacterium smegmatis and Mycobacterium tuberculosis, and are compatible with pAL5000-derived oriM and integration-proficient plasmid vectors. Characterization of these phage-based plasmids offers insights into the variability of lysogenic maintenance systems and provides a large suite of plasmids for actinobacterial genetics that vary in stability, copy number, compatibility, and host range.

Trichoderma species play important roles in nature as plant growth promotors and antagonists of phytopathogenic fungi, and are used as models to study photomorphogenesis. Molecular tools have been implemented to manipulate and improve these fungi. However, instability of transformants or very low frequency of homologous recombination has been reported. Here, we report the fate of transforming DNA, demonstrating that it can follow two different fates. When a vector contains sequences also present in the Trichoderma atroviride genome, it mainly integrates by homologous recombination generating stable recombinant strains. In contrast, vectors with no sequence homology to the T. atroviride genome generate unstable transformants, losing the transforming DNA in the first generation of conidia produced without selection where, surprisingly, the vector behaves as autoreplicative. Integration by homologous recombination was demonstrated when transformants were generated with a truncated version of the blr2 gene, resulting in insertional mutants with phenotypes identical to those of knockout mutants. Our results indicate that T. atroviride is highly efficient in integrating DNA by homologous recombination and that plasmid vectors with no sequence homology to the genome are maintained for several generations in T. atroviride if kept under selective pressure even though they lacked fungal autonomous replication sequences.

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.

Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. Here, we describe an optimized conjugation protocol enabling the transfer of autonomously replicating plasmids from an E. coli donor strain into Roseburia inulinivorans and Eubacterium rectale. The modular nature of the plasmids and their ability to be maintained in the recipient bacterium by autonomous replication makes them ideal for investigating heterologous gene expression, and as a platform for other genetic tools including antisense RNA silencing or mobile group II interon gene disruption strategies.

Glutamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the formation of glucosamine-6-phosphate, and its gene is one of the genes essential for microbes. Using the GFAT-encoding gene can prevent the use of a drug-resistant gene as a selection marker in a bacterial system. Another unique property of the GFAT selection marker is that no particular compound is prohibited or required for creating a selective stress for a yeast. Filamentous fungi are major producers of industrial enzymes. However, there has been no report on the construction and application of the GFAT gene as a selection marker in filamentous fungi. To develop a new selection marker, the GFAT-encoding gene gfaA was deleted from the genome of the filamentous fungus Aspergillus nidulans, and the gfat gene of the straw mushroom Volvariella volvacea was used as the selection marker to mediate the transformation and overexpression of a thermostable bacterial laccase in A. nidulans. The GFAT-deficient strain A. nidulans ∆gfaA was not able to grow in the culture medium containing 0.5% yeast extract unless about 20 mM glucosamine was used to supplement to the medium. The gfat gene was amplified and inserted into the integration vector pAL5 and autonomous replication vector Prg3-AMA1-NotI for A. nidulans to generate the gfat vectors pALG and pAMAG, respectively. Using these gfat vectors, the laccase gene lcs from a hyperthermophilic bacterium was overexpressed intra- and extracellularly in A. nidulans ∆gfaA. Therefore, recombinant filamentous fungi can be constructed with gfat vectors, which can be maintained stably in host cells with the naturally occurred selective stress of a medium, forage, pulp, animal gut, wastewater, or soil.

With the aim of being able to co-express multiple genes, I searched for novel compatible plasmids and isolated two plasmid species, pRET1100 and pRET1200, from Rhodococcus erythropolis IAM1400. Sequencing analysis revealed that the pRET1100 plasmid is a double-stranded DNA molecule of 5444 bp with two possible open reading frames (ORFs), repT and div, and three minor ORFs. The cryptic replication protein, RepT, is not highly homologous to those from other plasmids that have been reported. The Rhodococcus-Escherichia coli shuttle vector pRET1102 was transformed into R. erythropolis JCM2895 harboring the pRE2895 plasmid. The recombinant R. erythropolis JCM2895 harbored two plasmid species. These results suggest that plasmid derivatives of pRET1100 and pRE2895 are fully compatible in R. erythropolis. I determined the minimum region of pRET1100 required for autonomous replication in R. erythropolis and constructed a high-copy plasmid, pRET1129, in R. erythropolis.

Autonomous replication sequences (ARS) are essential for the replication of Saccharomyces cerevisiae genome. The content and context of ARS sites are distinct from other segments of the genome and these factors influence the conformation and thermodynamic profile of DNA that favor binding of the origin recognition complex proteins. Identification of ARS sites in the genome is a challenging task because of their organizational complexity and degeneracy present across the intergenic regions. We considered a few properties of DNA segments and divided them into multiple subsets (views) for computational prediction of ARS sequences. Our approach utilized these views for learning classification models in an ensemble manner and accordingly predictions were made. This approach maximized the prediction accuracy over the traditional way where all features are selected at once. Our study also revealed that major groove width and major groove depth are the most prominent properties that distinguished ARS from other segments of the genome. Our investigation also provides clue about the most suitable classifier for a given feature set, and this strategy may be useful for finding ARS in other closely related species.

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum.

pHW126 belongs to a small group of rolling circle plasmids. So far, the region mediating autonomous replication has been identified and it was shown that the rep gene is required for replication. However, the regulation of rep expression remained elusive. Here evidence is presented that expression of the replication gene rep is auto-regulated. Sequence analysis revealed a conserved stretch in the rep promoter consisting of three imperfect direct repeats (DR2.1, DR2.2 and DR2.3). Assays for promoter activity showed that these direct repeats act as an enhancer of transcriptional activity. Interestingly, the activating effect was reduced in the presence of Rep protein. Electrophoretic mobility shift assays demonstrated that the Rep protein can directly bind to direct repeats DR2.1 and DR2.3 while DR2.2 is not bound but places DR2.1 and DR2.3 in an appropriate distance. These results show that the synthesis of Rep protein is auto-regulated. In the absence of Rep protein the promoter is, due to the presence of the direct repeats acting as a transcriptional enhancer, highly active. Binding of Rep to the direct repeats reduces the transcription rate significantly. Since this regulation mechanism is independent of a specialised regulator protein it is presumably a very economic strategy.

Microbial production of fuels and chemicals from renewable and readily available biomass is a sustainable and economically attractive alternative to petroleum-based production. Because of its unusual tolerance to highly acidic conditions, I. orientalis is a promising potential candidate for the manufacture of valued organic acids. Nevertheless, reliable and efficient genetic engineering tools in I. orientalis are limited. The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals., The nonconventional yeast Issatchenkia orientalis has emerged as a potential platform microorganism for production of organic acids due to its ability to grow robustly under highly acidic conditions. However, lack of efficient genetic tools remains a major bottleneck in metabolic engineering of this organism. Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1′-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. This genome editing tool can be used for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals. IMPORTANCE Microbial production of fuels and chemicals from renewable and readily available biomass is a sustainable and economically attractive alternative to petroleum-based production. Because of its unusual tolerance to highly acidic conditions, I. orientalis is a promising potential candidate for the manufacture of valued organic acids. Nevertheless, reliable and efficient genetic engineering tools in I. orientalis are limited. The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals.

The methylotrophic yeast Pichia pastoris 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 Kluyveromyces lactis 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.

Circular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, in Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of a C. albicans origin and an origin sequence from a distantly related yeast can both function as origins in C. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and baker’s yeast., The ability to generate autonomously replicating plasmids has been elusive in Candida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants (tiny, medium, and large, respectively). Candida albicans URA3 and a heterologous marker, ARG4, yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologous LEU2 marker was highly dependent upon the addition of a bona fide origin sequence. Several bona fide chromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin, panARS, from Kluyveromyces lactis, drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus, C. albicans maintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner. IMPORTANCE Circular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, in Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of a C. albicans origin and an origin sequence from a distantly related yeast can both function as origins in C. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and baker’s yeast.

The ability to generate autonomously replicating plasmids has been elusive inCandida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences.Furthermore, we could distinguish by colony size, transient, autonomously replicating and chromosomally integrated transformants (tiny, medium and large, respectively).Candida albicans URA3and a heterologous marker,ARG4,yielded many transient transformants indicative of weak origin activity; replication of plasmid carrying heterologousLEU2marker was highly dependent upon the addition of abona fideorigin sequence. Severalbona fidechromosomal origins, with an origin fragment of ~100 bp as well as a heterologous origin,panARS, fromKluyveromyces lactisdrove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus,C. albicansmaintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner.ImportanceCircular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, inCandida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate inC. albicans.We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that a ~100 bp fragment of aC. albicansorigin, as well as an origin sequence from a distantly related yeast, can both function as origins inC. albicans.Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ betweenC. albicansand baker’s yeast.

The white-fly borne begomoviruses (family Geminiviridae) have circular single-stranded (css) DNA genome, which is encapsidated as monopartite (DNA-A) or bipartite (DNA-A and DNA-B) in the twinned icosahedrons. During the course of their evolution and to escape host defense machinery, begomoviruses adopt small cssDNA satellites called alpha-, beta-, and deltasatellites. Alphasatellties are found to be associated with begomovirus–betasatellite complexes and encode their own replication-associated protein (Rep), thus capable of autonomous replication. These satellite-like molecules are not well known to serve any critical function for their helper begomovirus except for few reports about attenuation of helper-virus accumulation and/or occasionally suppression of the host defense. Most of the monopartite begomoviruses in the Old World (OW) are found to be associated with betasatellites; however, none of the New World (NW) begomoviruses are known to be associated with betasatellites. Begomoviruses replicate their genome through rolling circle replication (RCR), which requires the virus-encoded Rep to recognize and bind to the iterated sequences (iterons) in the origin of replication (ori) region. Betasatellites lack such iterated sequences; however, they can be transreplicated by a diverse range of begomoviruses, following a similar pattern for replication. Betasatellites play a significant role in viral pathogenesis by interacting with certain host factors, attenuation of disease symptoms, suppression of host defense, and sometimes inter- or intracellular shuttling of begomovirus genome. Likewise, the noncoding molecules deltasatellites depend upon their helper virus for their replication. However, their precise role in viral pathogenesis still needs to be explored.

Yellow fever virus, the prototype in the genus Flavivirus, was used to develop viruses in which the nonstructural protein NS1 is genetically fused to GFP in the context of viruses capable of autonomous replication. The GFP-tagging of NS1 at the amino-terminus appeared possible despite the presence of a small and functionally important domain at the NS1's amino-terminus which can be distorted by such fusing. GFP-tagged NS1 viruses were rescued from DNA-launched molecular clones. The initially produced GFP-tagged NS1 virus was capable of only poor replication. Sequential passages of the virus in cell cultures resulted in the appearance of mutations in GFP, NS4A, NS4B and NS5. The mutations which change amino acid sequences of GFP, NS4A and NS5 have the adaptive effect on the replication of GFP-tagged NS1 viruses. The pattern of GFP-fluorescence indicates that the GFP-NS1 fusion protein is produced into the endoplasmic reticulum. The intracellular GFP-NS1 fusion protein colocalizes with dsRNA. The discovered forms of extracellular GFP-NS1 possibly include tetramers and hexamers.

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

A lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A &beta, oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of &beta, oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.