Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

BACKGROUND:Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS:The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. CONCLUSION:This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism

Evolutionary expansion and divergence in a large family of primate-specific zinc finger transcription factor genes

Although most genes are conserved as one-to-one orthologs in different mammalian orders, certain gene families have evolved to comprise different numbers and types of protein-coding genes through independent series of gene duplications, divergence and gene loss in each evolutionary lineage. One such family encodes KRAB-zinc finger (KRAB-ZNF) genes, which are likely to function as transcriptional repressors. One KRAB-ZNF subfamily, the ZNF91 clade, has expanded specifically in primates to comprise more than 110 loci in the human genome, yielding large gene clusters in human chromosomes 19 and 7 and smaller clusters or isolated copies at other chromosomal locations. Although phylogenetic analysis indicates that many of these genes arose before the split between old world monkeys and new world monkeys, the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. The paralogous loci are distinguished by sequence divergence within their zinc finger arrays indicating a selection for proteins with different DNA binding specificities. RT-PCR and in situ hybridization data show that some of these ZNF genes can have tissue-specific expression patterns, however many KRAB-ZNFs that are near-ubiquitous could also be playing very specific roles in halting target pathways in all tissues except for a few, where the target is released by the absence of its repressor. The number of variant KRAB-ZNF proteins is increased not only because of the large number of loci, but also because many loci can produce multiple splice variants, which because of the modular structure of these genes may have separate and perhaps even conflicting regulatory roles. The lineage-specific duplication and rapid divergence of this family of transcription factor genes suggests a role in determining species-specific biological differences and the evolution of novel primate traits

Prospects for utilizing microbial consortia for lignin conversion

Naturally occurring microbial communities are able to decompose lignocellulosic biomass through the concerted production of a myriad of enzymes that degrade its polymeric components and assimilate the resulting breakdown compounds by members of the community. This process includes the conversion of lignin, the most recalcitrant component of lignocellulosic biomass and historically the most difficult to valorize in the context of a biorefinery. Although several fundamental questions on microbial conversion of lignin remain unanswered, it is known that some fungi and bacteria produce enzymes to break, internalize, and assimilate lignin-derived molecules. The interest in developing efficient biological lignin conversion approaches has led to a better understanding of the types of enzymes and organisms that can act on different types of lignin structures, the depolymerized compounds that can be released, and the products that can be generated through microbial biosynthetic pathways. It has become clear that the discovery and implementation of native or engineered microbial consortia could be a powerful tool to facilitate conversion and valorization of this underutilized polymer. Here we review recent approaches that employ isolated or synthetic microbial communities for lignin conversion to bioproducts, including the development of methods for tracking and predicting the behavior of these consortia, the most significant challenges that have been identified, and the possibilities that remain to be explored in this field

Conceptualizing pathways linking women's empowerment and prematurity in developing countries.

BackgroundGlobally, prematurity is the leading cause of death in children under the age of 5. Many efforts have focused on clinical approaches to improve the survival of premature babies. There is a need, however, to explore psychosocial, sociocultural, economic, and other factors as potential mechanisms to reduce the burden of prematurity. Women's empowerment may be a catalyst for moving the needle in this direction. The goal of this paper is to examine links between women's empowerment and prematurity in developing settings. We propose a conceptual model that shows pathways by which women's empowerment can affect prematurity and review and summarize the literature supporting the relationships we posit. We also suggest future directions for research on women's empowerment and prematurity.MethodsThe key words we used for empowerment in the search were "empowerment," "women's status," "autonomy," and "decision-making," and for prematurity we used "preterm," "premature," and "prematurity." We did not use date, language, and regional restrictions. The search was done in PubMed, Population Information Online (POPLINE), and Web of Science. We selected intervening factors-factors that could potentially mediate the relationship between empowerment and prematurity-based on reviews of the risk factors and interventions to address prematurity and the determinants of those factors.ResultsThere is limited evidence supporting a direct link between women's empowerment and prematurity. However, there is evidence linking several dimensions of empowerment to factors known to be associated with prematurity and outcomes for premature babies. Our review of the literature shows that women's empowerment may reduce prematurity by (1) preventing early marriage and promoting family planning, which will delay age at first pregnancy and increase interpregnancy intervals; (2) improving women's nutritional status; (3) reducing domestic violence and other stressors to improve psychological health; and (4) improving access to and receipt of recommended health services during pregnancy and delivery to help prevent prematurity and improve survival of premature babies.ConclusionsWomen's empowerment is an important distal factor that affects prematurity through several intervening factors. Improving women's empowerment will help prevent prematurity and improve survival of preterm babies. Research to empirically show the links between women's empowerment and prematurity is however needed

The complete sequence of human chromosome 5

Chromosome 5 is one of the largest human chromosomes yet has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding and syntenic conservation with non-mammalian vertebrates, suggesting they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-encoding genes including the protocadherin and interleukin gene families and the first complete versions of each of the large chromosome 5 specific internal duplications. These duplications are very recent evolutionary events and play a likely mechanistic role, since deletions of these regions are the cause of debilitating disorders including spinal muscular atrophy (SMA)

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Evolutionary expansion and divergence in the ZNF91 subfamily of primate-specific zinc finger genes

Most genes are conserved in mammals, but certain gene families have acquired large numbers of lineage-specific loci through repeated rounds of gene duplication, divergence, and loss that have continued in each mammalian group. One such family encodes KRAB-zinc finger (KRAB-ZNF) proteins, which function as transcriptional repressors. One particular subfamily of KRAB-ZNF genes, including ZNF91, has expanded specifically in primates to comprise more than 110 loci in the human genome. Genes of the ZNF91 subfamily reside in large gene clusters near centromeric regions of human chromosomes 19 and 7 with smaller clusters or isolated copies in other locations. Phylogenetic analysis indicates that many of these genes arose before the split between the New and Old World monkeys, but the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. Paralogous loci are distinguished by divergence within their zinc finger arrays, indicating selection for proteins with different regulatory targets. In addition, many loci produce multiple alternatively spliced transcripts encoding proteins that may serve separate and perhaps even opposing regulatory roles because of the modular motif structure of KRAB-ZNF genes. The tissue-specific expression patterns and rapid structural divergence of ZNF91 subfamily genes suggest a role in determining gene expression differences between species and the evolution of novel primate traits

A comprehensive catalog of human KRAB-associated zinc finger genes: Insights into the evolutionary history of a large family of transcriptional repressors

Krüppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotes. KRAB-ZNF proteins, in which a potent repressor domain is attached to a tandem array of DNA-binding zinc-finger motifs, are specific to tetrapod vertebrates and represent the largest class of ZNF proteins in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the genome sequence for key motifs and then constructed and manually curated gene models incorporating those sequences. The resulting gene catalog contains 423 KRAB-ZNF protein-coding loci, yielding alternative transcripts that altogether predict at least 742 structurally distinct proteins. Active rounds of segmental duplication, involving single genes or larger regions and including both tandem and distributed duplication events, have driven the expansion of this mammalian gene family. Comparisons between the human genes and ZNF loci mined from the draft mouse, dog, and chimpanzee genomes not only identified 103 KRAB-ZNF genes that are conserved in mammals but also highlighted a substantial level of lineage-specific change; at least 136 KRAB-ZNF coding genes are primate specific, including many recent duplicates. KRAB-ZNF genes are widely expressed and clustered genes are typically not coregulated, indicating that paralogs have evolved to fill roles in many different biological processes. To facilitate further study, we have developed a Web-based public resource with access to gene models, sequences, and other data, including visualization tools to provide genomic context and interaction with other public data sets

Use of a Capture-Based Pathogen Transcript Enrichment Strategy for RNA-Seq Analysis of the <i>Francisella Tularensis</i> LVS Transcriptome during Infection of Murine Macrophages

<div><p><i>Francisella tularensis</i> is a zoonotic intracellular pathogen that is capable of causing potentially fatal human infections. Like all successful bacterial pathogens, <i>F. tularensis</i> rapidly responds to changes in its environment during infection of host cells, and upon encountering different microenvironments within those cells. This ability to appropriately respond to the challenges of infection requires rapid and global shifts in gene expression patterns. In this study, we use a novel pathogen transcript enrichment strategy and whole transcriptome sequencing (RNA-Seq) to perform a detailed characterization of the rapid and global shifts in <i>F. tularensis</i> LVS gene expression during infection of murine macrophages. We performed differential gene expression analysis on all bacterial genes at two key stages of infection: phagosomal escape, and cytosolic replication. By comparing the <i>F. tularensis</i> transcriptome at these two stages of infection to that of the bacteria grown in culture, we were able to identify sets of genes that are differentially expressed over the course of infection. This analysis revealed the temporally dynamic expression of a number of known and putative transcriptional regulators and virulence factors, providing insight into their role during infection. In addition, we identified several <i>F. tularensis</i> genes that are significantly up-regulated during infection but had not been previously identified as virulence factors. These unknown genes may make attractive therapeutic or vaccine targets.</p> </div

Comparison of the genes up- and down-regulated at each time point.

<p>The Venn diagrams depict the number of genes with significant changes in expression at both the 4 and 8-hour post-infection time points, with the number in the middle representing genes up- or down-regulated at both time points. A) Up-regulated genes. B) Down-regulated genes.</p