Maureen E. Hillenmeyer

The Chemical Genomic Portrait of Yeast: Uncovering a Phenotype for All Genes

Genetics aims to understand the relation between genotype and phenotype. However, because complete deletion of most yeast genes (∼80%) has no obvious phenotypic consequence in rich medium, it is difficult to study their functions. To uncover phenotypes for this nonessential fraction of the genome, we performed 1144 chemical genomic assays on the yeast whole-genome heterozygous and homozygous deletion collections and quantified the growth fitness of each deletion strain in the presence of chemical or environmental stress conditions. We found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.

Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action

We systematically analyzed the relationships between gene fitness profiles (co-fitness) and drug inhibition profiles (co-inhibition) from several hundred chemogenomic screens in yeast. Co-fitness predicted gene functions distinct from those derived from other assays and identified conditionally dependent protein complexes. Co-inhibitory compounds were weakly correlated by structure and therapeutic class. We developed an algorithm predicting protein targets of chemical compounds and verified its accuracy with experimental testing. Fitness data provide a novel, systems-level perspective on the cell.

Introns Regulate RNA and Protein Abundance in Yeast

The purpose of introns in the architecturally simple genome of Saccharomyces cerevisiae is not well understood. To assay the functional relevance of introns, a series of computational analyses and several detailed deletion studies were completed on the intronic genes of S. cerevisiae. Mining existing data from genomewide studies on yeast revealed that intron-containing genes produce more RNA and more protein and are more likely to be haplo-insufficient than nonintronic genes. These observations for all intronic genes held true for distinct subsets of genes including ribosomal, nonribosomal, duplicated, and nonduplicated. Corroborating the result of computational analyses, deletion of introns from three essential genes decreased cellular RNA levels and caused measurable growth defects. These data provide evidence that introns improve transcriptional and translational yield and are required for competitive growth of yeast.

Update of the Anopheles gambiae PEST genome assembly

BackgroundThe genome of Anopheles gambiae, the major vector of malaria, was sequenced and assembled in 2002. This initial genome assembly and analysis made available to the scientific community was complicated by the presence of assembly issues, such as scaffolds with no chromosomal location, no sequence data for the Y chromosome, haplotype polymorphisms resulting in two different genome assemblies in limited regions and contaminating bacterial DNA.ResultsPolytene chromosome in situ hybridization with cDNA clones was used to place 15 unmapped scaffolds (sizes totaling 5.34 Mbp) in the pericentromeric regions of the chromosomes and oriented a further 9 scaffolds. Additional analysis by in situ hybridization of bacterial artificial chromosome (BAC) clones placed 1.32 Mbp (5 scaffolds) in the physical gaps between scaffolds on euchromatic parts of the chromosomes. The Y chromosome sequence information (0.18 Mbp) remains highly incomplete and fragmented among 55 short scaffolds. Analysis of BAC end sequences showed that 22 inter-scaffold gaps were spanned by BAC clones. Unmapped scaffolds were also aligned to the chromosome assemblies in silico, identifying regions totaling 8.18 Mbp (144 scaffolds) that are probably represented in the genome project by two alternative assemblies. An additional 3.53 Mbp of alternative assembly was identified within mapped scaffolds. Scaffolds comprising 1.97 Mbp (679 small scaffolds) were identified as probably derived from contaminating bacterial DNA. In total, about 33% of previously unmapped sequences were placed on the chromosomes.ConclusionThis study has used new approaches to improve the physical map and assembly of the A. gambiae genome.

Differential gene expression in abdomens of the malaria vector mosquito, Anopheles gambiae, after sugar feeding, blood feeding and Plasmodium berghei infection

BackgroundLarge scale sequencing of cDNA libraries can provide profiles of genes expressed in an organism under defined biological and environmental circumstances. We have analyzed sequences of 4541 Expressed Sequence Tags (ESTs) from 3 different cDNA libraries created from abdomens from Plasmodium infection-susceptible adult female Anopheles gambiae. These libraries were made from sugar fed (S), rat blood fed (RB), and P. berghei-infected (IRB) mosquitoes at 30 hours after the blood meal, when most parasites would be transforming ookinetes or very early oocysts.ResultsThe S, RB and IRB libraries contained 1727, 1145 and 1669 high quality ESTs, respectively, averaging 455 nucleotides (nt) in length. They assembled into 1975 consensus sequences – 567 contigs and 1408 singletons. Functional annotation was performed to annotate probable molecular functions of the gene products and the biological processes in which they function. Genes represented at high frequency in one or more of the libraries were subjected to digital Northern analysis and results on expression of 5 verified by qRT-PCR.Conclusion13% of the 1965 ESTs showing identity to the A. gambiae genome sequence represent novel genes. These, together with untranslated regions (UTR) present on many of the ESTs, will inform further genome annotation. We have identified 23 genes encoding products likely to be involved in regulating the cellular oxidative environment and 25 insect immunity genes. We also identified 25 genes as being up or down regulated following blood feeding and/or feeding with P. berghei infected blood relative to their expression levels in sugar fed females.

Computational identification and analysis of orphan assembly-line polyketide synthases.

The increasing availability of DNA sequence data offers an opportunity for identifying new assembly-line polyketide synthases (PKSs) that produce biologically active natural products. We developed an automated method to extract and consolidate all multimodular PKS sequences (including hybrid PKS/non-ribosomal peptide synthetases) in the National Center for Biotechnology Information (NCBI) database, generating a non-redundant catalog of 885 distinct assembly-line PKSs, the majority of which were orphans associated with no known polyketide product. Two in silico experiments highlight the value of this search method and resulting catalog. First, we identified an orphan that could be engineered to produce an analog of albocycline, an interesting antibiotic whose gene cluster has not yet been sequenced. Second, we identified and analyzed a hitherto overlooked family of metazoan multimodular PKSs, including one from Caenorhabditis elegans. We also developed a comparative analysis method that identified sequence relationships among known and orphan PKSs. As expected, PKS sequences clustered according to structural similarities between their polyketide products. The utility of this method was illustrated by highlighting an interesting orphan from the genus Burkholderia that has no close relatives. Our search method and catalog provide a community resource for the discovery of new families of assembly-line PKSs and their antibiotic products.

Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast

The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathways is therefore important toward understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered. Here, using Saccharomyces cerevisiae as a heterologous host and a phylogenetically guided enzyme mining approach, we combinatorially assembled IDT biosynthetic pathways using IDTCs homologues identified from different fungal hosts. We identified the genetically standalone IDTCs involved in the cyclization of aflavinine and anominine and produced new IDTs not previously isolated. The cyclization mechanisms of the new IDTCs were proposed based on the yeast reconstitution results. Our studies demonstrate heterologous pathway assembly is a useful tool in the reconstitution of unclustered biosynthetic pathways.

Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters

Significance Type II polyketide natural products are powerful antimicrobial agents that are biosynthesized within bacteria by enzyme-encoding clusters of genes. We present a method to elucidate the evolution of these gene clusters as a whole, illuminating how natural selection has led to the chemical diversity of type II polyketides. Our approach can be applied to understand how other natural product gene clusters evolve. This understanding may aid efforts to access novel natural products and to design rational enzyme assemblies that produce chemicals of desired structures and activities. Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties.

HEx: A heterologous expression platform for the discovery of fungal natural products

We develop a novel synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and encoded metabolites. For decades, fungi have been a source of U.S. Food and Drug Administration–approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.

Heterologous expression of diverse propionyl-CoA carboxylases affects polyketide production in Escherichia coli

Heterologous expression of diverse propionyl-CoA carboxylases affects polyketide production in Escherichia coli