A scalable platform to identify fungal secondary metabolites and their gene clusters.

The genomes of filamentous fungi contain up to 90 biosynthetic gene clusters (BGCs) encoding diverse secondary metabolites-an enormous reservoir of untapped chemical potential. However, the recalcitrant genetics, cryptic expression, and unculturability of these fungi prevent scientists from systematically exploiting these gene clusters and harvesting their products. As heterologous expression of fungal BGCs is largely limited to the expression of single or partial clusters, we established a scalable process for the expression of large numbers of full-length gene clusters, called FAC-MS. Using fungal artificial chromosomes (FACs) and metabolomic scoring (MS), we screened 56 secondary metabolite BGCs from diverse fungal species for expression in Aspergillus nidulans. We discovered 15 new metabolites and assigned them with confidence to their BGCs. Using the FAC-MS platform, we extensively characterized a new macrolactone, valactamide A, and its hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS). The ability to regularize access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity.

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics.

A functional metagenomic approach identified novel and diverse soil-derived DNAs encoding inhibitors to methicillin-resistant Staphylococcus aureus (MRSA). A metagenomic DNA soil library containing 19 200 recombinant Escherichia coli BAC clones with 100 Kb average insert size was screened for antibiotic activity. Twenty-seven clones inhibited MRSA, seven of which were found by LC-MS to possess modified chloramphenicol ( Cm) derivatives, including three new compounds whose structures were established as 1-acetyl-3-propanoylchloramphenicol, 1-acetyl-3-butanoylchloramphenicol, and 3-butanoyl-1-propanoylchloramphenicol. Cm was used as the selectable antibiotic for cloning, suggesting that heterologously expressed enzymes resulted in derivatization of Cm into new chemical entities with biological activity. An esterase was found to be responsible for the enzymatic regeneration of Cm, and the gene trfA responsible for plasmid copy induction was found to be responsible for inducing antibacterial activity in some clones. Six additional acylchloramphenicols were synthesized for structure and antibacterial activity relationship studies, with 1- p-nitrobenzoylchloramphenicol the most active against Mycobacterium intracellulare and Mycobacterium tuberculosis, with MICs of 12.5 and 50.0 µg/mL, respectively.

Fungal artificial chromosomes for mining of the fungal secondary metabolome.

BACKGROUND: With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80 kb in size, breakthrough techniques are needed to characterize this SM wealth. RESULTS: Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery. CONCLUSION: The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.

Efficient de novo assembly of large and complex genomes by massively parallel sequencing of Fosmid pools.

BACKGROUND: Sampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality. RESULTS: In order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with ~40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS. CONCLUSIONS: By exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process.We have made public the input data (FASTQ format) for the set of pools used in this study:ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/.

DNA is structured as a linear "jigsaw puzzle" in the genomes of Arabidopsis, rice, and budding yeast.

Knowledge of how a genome is structured and organized from its constituent elements is crucial to understanding its biology and evolution. Here, we report the genome structuring and organization pattern as revealed by systems analysis of the sequences of three model species, Arabidopsis, rice and yeast, at the whole-genome and chromosome levels. We found that all fundamental function elements (FFE) constituting the genomes, including genes (GEN), DNA transposable elements (DTE), retrotransposable elements (RTE), simple sequence repeats (SSR), and (or) low complexity repeats (LCR), are structured in a nonrandom and correlative manner, thus leading to a hypothesis that the DNA of the species is structured as a linear "jigsaw puzzle". Furthermore, we showed that different FFE differ in their importance in the formation and evolution of the DNA jigsaw puzzle structure between species. DTE and RTE play more important roles than GEN, LCR, and SSR in Arabidopsis, whereas GEN and RTE play more important roles than LCR, SSR, and DTE in rice. The genes having multiple recognized functions play more important roles than those having single functions. These results provide useful knowledge necessary for better understanding genome biology and evolution of the species and for effective molecular breeding of rice.

The Identification and Heterologous Expression of the Biosynthetic Gene Cluster Encoding the Antibiotic and Anticancer Agent Marinomycin.

With the rise in antimicrobial resistance, there is an urgent need for new classes of antibiotic with which to treat infectious disease. Marinomycin, a polyene antibiotic from a marine microbe, has been shown capable of killing methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF), as well as having promising activity against melanoma. An attractive solution to the photoprotection of this antibiotic has been demonstrated. Here, we report the identification and analysis of the marinomycin biosynthetic gene cluster (BGC), and the biosynthetic assembly of the macrolide. The marinomycin BGC presents a challenge in heterologous expression due to its large size and high GC content, rendering the cluster prone to rearrangement. We demonstrate the transformation of Streptomyces lividans using a construct containing the cluster, and the heterologous expression of the encoded biosynthetic machinery and production of marinomycin B.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites.

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, Penicillium fuscum and P. camembertii/clavigerum, which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form "biosynthetic gene clusters" (BGCs), which might remain silent (inactive) under various fermentation conditions. Fungi may also harbor cryptic BGCs that are not associated with previously characterized molecules. We turned to the tools of Fungal Artificial Chromosomes (FAC)-Next-Gen-Sequencing (NGS) to understand how co-culture activated cryptic biosynthesis of BPLA and several related berkeleylactones and to further investigate the true biosynthetic potential of these two fungi. FAC-NGS enables the capture of BGCs as individual FACs for heterologous expression in a modified strain of Aspergillus nidulans (heterologous host, FAC-AnHH). With this methodology, we created ten BGC-FACs that yielded fourteen different SMs, including strobilurin, which was previously isolated exclusively from basidiomycetes. Eleven of these compounds were not detected in the extracts of the FAC-AnHH. Of this discrete set, only the novel compound citreohybriddional had been isolated from either Penicillium sp. before and only at very low yield. We propose that through heterologous expression, FACs activated these silent BGCs, resulting in the synthesis of new natural products (NPs) with yields as high as 50%-60% of the crude organic extracts.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize due to cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a new twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wildtype, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1-2, 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wildtype chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli.

Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed 'pJAZZ' vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT-rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries.

Numbers of genes in the NBS and RLK families vary by more than four-fold within a plant species and are regulated by multiple factors.

Many genes exist in the form of families; however, little is known about their size variation, evolution and biology. Here, we present the size variation and evolution of the nucleotide-binding site (NBS)-encoding gene family and receptor-like kinase (RLK) gene family in Oryza, Glycine and Gossypium. The sizes of both families vary by numeral fold, not only among species, surprisingly, also within a species. The size variations of the gene families are shown to correlate with each other, indicating their interactions, and driven by natural selection, artificial selection and genome size variation, but likely not by polyploidization. The numbers of genes in the families in a polyploid species are similar to those of one of its diploid donors, suggesting that polyploidization plays little roles in the expansion of the gene families and that organisms tend not to maintain their 'surplus' genes in the course of evolution. Furthermore, it is found that the size variations of both gene families are associated with organisms' phylogeny, suggesting their roles in speciation and evolution. Since both selection and speciation act on organism's morphological, physiological and biological variation, our results indicate that the variation of gene family size provides a source of genetic variation and evolution.

BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.).

BACKGROUND: Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. RESULT: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. CONCLUSION: BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.

Artificial chromosome formation in maize (Zea mays L.).

We report on the construction of maize minichromosomes using shuttle vectors harboring native centromeric segments, origins of replication, selectable marker genes, and telomeric repeats. These vectors were introduced into scutellar cells of maize immature embryos by microprojectile bombardment. Several independent transformation events were identified containing minichromosomes in addition to the normal diploid complement of 20 maize chromosomes. Immunostaining indicated that the minichromosomes recruited centromeric protein C, which is a specific component of the centromere/kinetochore complex. Minichromosomes were estimated to be 15-30 Mb in size based on cytological measurements. Fluorescent in situ hybridization (FISH) showed that minichromosomes contain the centromeric, telomeric, and exogenous unique marker sequences interspersed with maize retrotransposons. Minichromosomes were detected for at least a year in actively dividing callus cultures, providing evidence for their stability through numerous cell cycles. Plants were regenerated and minichromosomes were detected in root tips, providing confirmation of their normal replication and transmission during mitosis and through organogenesis. Assembly of maize artificial chromosomes may provide a tool to study centromere function and a foundation for developing new high capacity vectors for plant functional genomics and breeding.

Heterologous Expression of the Unusual Terreazepine Biosynthetic Gene Cluster Reveals a Promising Approach for Identifying New Chemical Scaffolds.

Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates that FAC-based approaches targeting unusual biosynthetic machinery provide a promising avenue forward for targeted discovery of novel scaffolds and their biosynthetic enzymes, and it also represents another example of a biosynthetic gene cluster "repurposing" a primary metabolic enzyme to diversify its secondary metabolite arsenal.IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the numerous strategies fungi employ for diversifying and protecting themselves against secondary metabolites. The observations in this study set the stage for further in-depth studies into the function of duplicated IDOs present in fungal biosynthetic gene clusters and presents a strategy for accessing the biosynthetic potential of gene clusters containing duplicated primary metabolic genes.

Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species.

BACKGROUND: Manduca sexta, Heliothis virescens, and Heliconius erato represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date. RESULTS: We report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152-175 kb. We estimated that the genome coverage of each library ranged from 6-9 x, with the two combined libraries of each species being equivalent to 13.0-16.3 x haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6 approximately 8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of approximately 200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the H. erato genome is somewhat more abundant in repeat elements and simple repeats than those of M. sexta and H. virescens. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of H. virescens being the most conserved as a typical lepidopteran, whereas both genomes of H. erato and M. sexta appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences. CONCLUSION: The high-quality and large-insert BAC libraries of the insects, together with the identified BACs containing genes of interest, provide valuable information, resources and tools for comprehensive understanding and studies of the insect genomes and for addressing many fundamental questions in Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and evolution of the insect genomes.

The Soybean Genome Database (SoyGD): a browser for display of duplicated, polyploid, regions and sequence tagged sites on the integrated physical and genetic maps of Glycine max.

Genomes that have been highly conserved following increases in ploidy (by duplication or hybridization) like Glycine max (soybean) present challenges during genome analysis. At http://soybeangenome.siu.edu the Soybean Genome Database (SoyGD) genome browser has, since 2002, integrated and served the publicly available soybean physical map, bacterial artificial chromosome (BAC) fingerprint database and genetic map associated genomic data. The browser shows both build 3 and build 4 contiguous sets of clones (contigs) of the soybean physical map. Build 4 consisted of 2854 contigs that encompassed 1.05 Gb and 404 high-quality DNA markers that anchored 742 contigs. Many DNA markers anchored sets of 2-8 different contigs. Each contig in the set represented a homologous region of related sequences. GBrowse was adapted to show sets of homologous contigs at all potential anchor points, spread laterally and prevented from overlapping. About 8064 minimum tiling path (MTP2) clones provided 13,473 BAC end sequences (BES) to decorate the physical map. Analyses of BES placed 2111 gene models, 40 marker anchors and 1053 new microsatellite markers on the map. Estimated sequence tag probes from 201 low-copy gene families located 613 paralogs. The genome browser portal showed each data type as a separate track. Tetraploid, octoploid, diploid and homologous regions are shown clearly in relation to an integrated genetic and physical map.

Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology.

Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated CR) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives.

Structural diversity and differential transcription of the patatin multicopy gene family during potato tuber development.

The patatin multicopy gene family encodes the major storage protein in potato tubers and is organized as a single cluster in the potato genome. We sequenced a 154-kb bacterial artificial chromosome (BAC) clone containing a portion of the patatin gene cluster. Two putatively functional patatin genes were found in this BAC. These two genes are embedded within arrays of patatin pseudogenes. Using a chromatin immunoprecipitation method we demonstrate that the dramatic increase of patatin gene expression during the transition from stolons to tubers coincides with an increase of histone H4 lysine acetylation. We used 3' rapid amplification of cDNA ends to profile expression of different patatin genes during tuber development. The profiling results revealed differential expression patterns of specific patatin gene groups throughout six different stages of tuber development. One group of patatin gene transcripts, designated patatin gene group A, was found to be the most abundant group during all stages of tuber development. Other patatin gene groups, with a 48-bp insertion in the 3'-untranslated region, are not expressed in stolons but display a gradual increase in expression level following the onset of tuberization. These results demonstrate that the patatin genes exhibit alterations in chromatin state and differential transcriptional regulation during the developmental transition from stolons into tubers, in which there is an increased demand for protein storage.

Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum.

Physical mapping with large-insert clones is becoming an active area of genomics research, and capillary electrophoresis (CE) promises to revolutionize the physical mapping technology. Here, we demonstrate the utility of the CE technology for genome physical mapping with large-insert clones by constructing a robust, binary bacterial artificial chromosome (BIBAC)-based physical map of Penicillium chrysogenum. We fingerprinted 23.1x coverage BIBAC clones with five restriction enzymes and the SNaPshot kit containing four fluorescent-ddNTPs using the CE technology, and explored various strategies to construct quality physical maps. It was shown that the fingerprints labeled with one or two colors, resulting in 40-70 bands per clone, were assembled into much better quality maps than those labeled with three or four colors. The selection of fingerprinting enzymes was crucial to quality map construction. From the dataset labeled with ddTTP-dROX, we assembled a physical map for P.chrysogenum, with 2-3 contigs per chromosome and anchored the map to its chromosomes. This map represents the first physical map constructed using the CE technology, thus providing not only a platform for genomic studies of the penicillin-producing species, but also strategies for efficient use of the CE technology for genome physical mapping of plants, animals and microbes.