Compartmentalized biosynthesis of mycophenolic acid

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic to be isolated and crystallized, and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, some key biosynthetic mechanisms of such an old and important molecule have remained unclear. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and β-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB′ as the long-sought key enzyme responsible for the oxidative cleavage of the farnesyl side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC′ andO-methyltransferase MpaG′, the Golgi apparatus-associated prenyltransferase MpaA′, the endoplasmic reticulum-bound oxygenase MpaB′ and P450-hydrolase fusion enzyme MpaDE′, and the peroxisomal acyl-coenzyme A (CoA) hydrolase MpaH′. The whole pathway is elegantly comediated by these compartmentalized enzymes, together with the peroxisomal β-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic steps, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.

Genome-wide prediction of synthetic rescue mediators of resistance to targeted and immunotherapy

ABSTRACTMost patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Many of these events involvesynthetic rescue (SR) interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (therescuer). Here we perform a genome-wide prediction of SR rescuer genes by analyzing tumor transcriptomics and survival data of 10,000 TCGA cancer patients. Predicted SR interactions are validated in new experimental screens. We show that SR interactions can successfully predict cancer patients’ response and emerging resistance. Inhibiting predicted rescuer genes sensitizes resistant cancer cells to therapies synergistically, providing initial leads for developing combinatorial approaches to overcome resistance proactively. Finally, we show that the SR analysis of melanoma patients successfully identifies known mediators of resistance to immunotherapy and predicts novel rescuers.

Microhomology-Mediated High-Throughput Gene Inactivation Strategy for the Hyperthermophilic Crenarchaeon Sulfolobus islandicus

ABSTRACT Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the last archaeal eukaryote common ancestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD+ transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp, whereas argD marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of S. islandicus gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism. IMPORTANCE Current procedures for the construction of deletion mutants of S. islandicus are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in S. islandicus, providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the Sulfolobus genus that are recombinogenic.

Maternal expression of the novel centrosome assembly factor Wdr8 is required for vertebrate embryonic mitosis and development

The assembly of the first centrosome occurs upon fertilisation when the male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. While inaccuracy in the assembly of centrosomes leads to infertility and abnormal embryogenesis, the mechanism that ensures accurate assembly in vertebrate embryos remains obscure. Here we applied a CRISPR-Cas9-mediated gene knockout to show that Wdr8, a novel centrosomal protein, is maternally essential for PCM assembly during embryonic mitoses of medaka (Oryzias latipes). Maternal/zygotic Wdr8-null (Wdr8-/-) blastomeres exhibit severe defects in PCM assembly that cause them to divide asymmetrically and develop multipolar mitotic spindles and aneuploidy. We demonstrate that Wdr8 interacts via its WD40 domains with the centriolar satellite protein SSX2IP. Strikingly, exogenously provided Wdr8 fully rescues Wdr8-/-embryos to adulthood, except in variants with mutations in the WD40 domains. This combination of targeted gene inactivation andinvivoreconstitution of the maternally essential Wdr8-SSX2IP complex reveals an essential link between maternal PCM and the stability of the zygotic genome in the early vertebrate embryo.

Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation

ABSTRACTFunctional analysis ofBifidobacteriumgenes is essential for understanding host-Bifidobacteriuminteractions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover inBifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA, which lacks the plasmid replication generepA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harborsrepA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected inB. longum105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using theagagene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion ofaga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-basedaga-complemented strain. These functional analyses revealed thatagais the only gene encoding a functional α-galactosidase enzyme inB. longum105-A.

Isolation and Characterization of the Gene Cluster for Biosynthesis of the Thiopeptide Antibiotic TP-1161

ABSTRACT Recently, we isolated a new thiopeptide antibiotic, TP-1161, from the fermentation broth of a marine actinomycete typed as a member of the genus Nocardiopsis. Here we report the identification, isolation, and analysis of the TP-1161 biosynthetic gene cluster from this species. The gene cluster was identified by mining a draft genome sequence using the predicted structural peptide sequence of TP-1161. Functional assignment of a ∼16-kb genomic region revealed 13 open reading frames proposed to constitute the TP-1161 biosynthetic locus. While the typical core set of thiopeptide modification enzymes contains one cyclodehydratase/dehydrogenase pair, paralogous genes predicted to encode additional cyclodehydratases and dehydrogenases were identified. Although attempts at heterologous expression of the TP-1161 gene cluster in Streptomyces coelicolor failed, its identity was confirmed through the targeted gene inactivation in the original host.