Acetaminophen production in the edible, filamentous cyanobacterium Arthrospira platensis.

Spirulina is the common name for the edible, nonheterocystous, filamentous cyanobacterium Arthrospira platensis that is grown industrially as a food supplement, animal feedstock, and pigment source. Although there are many applications for engineering this organism, until recently no genetic tools or reproducible transformation methods have been published. While recent work showed the production of a diversity of proteins in A.

Barcoded reciprocal hemizygosity analysis via sequencing illuminates the complex genetic basis of yeast thermotolerance.

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field cannot be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an interspecies hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research.

Chromosome assembled and annotated genome sequence of Aspergillus flavus NRRL 3357.

Aspergillus flavus is an opportunistic pathogen of crops, including peanuts and maize, and is the second leading cause of aspergillosis in immunocompromised patients. A. flavus is also a major producer of the mycotoxin, aflatoxin, a potent carcinogen, which results in significant crop losses annually.

Microevolution in the pansecondary metabolome of and its potential macroevolutionary implications for filamentous fungi.

Fungi produce a wealth of pharmacologically bioactive secondary metabolites (SMs) from biosynthetic gene clusters (BGCs). It is common practice for drug discovery efforts to treat species' secondary metabolomes as being well represented by a single or a small number of representative genomes. However, this approach misses the possibility that intraspecific population dynamics, such as adaptation to environmental conditions or local microbiomes, may harbor novel BGCs that contribute to the overall niche breadth of species.

Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass.

Background: Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates.

Multi-Omics Driven Metabolic Network Reconstruction and Analysis of Lignocellulosic Carbon Utilization in .

An oleaginous yeast is a promising host for converting lignocellulosic biomass to bioproducts and biofuels. In this work, we performed multi-omics analysis of lignocellulosic carbon utilization in and reconstructed the genome-scale metabolic network of . High-quality metabolic network models for model organisms and orthologous protein mapping were used to build a draft metabolic network reconstruction.

The Frequency of Sex: Population Genomics Reveals Differences in Recombination and Population Structure of the Aflatoxin-Producing Fungus Aspergillus flavus.

The apparent rarity of sex in many fungal species has raised questions about how much sex is needed to purge deleterious mutations and how differences in frequency of sex impact fungal evolution. We sought to determine how differences in the extent of recombination between populations of impact the evolution of genes associated with the synthesis of aflatoxin, a notoriously potent carcinogen. We sequenced the genomes of, and quantified aflatoxin production in, 94 isolates of sampled from seven states in eastern and central latitudinal transects of the United States.

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.

Genomewide and Enzymatic Analysis Reveals Efficient d-Galacturonic Acid Metabolism in the Basidiomycete Yeast Rhodosporidium toruloides.

Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as , are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast is a promising and robust host for lipid- and terpene-derived chemicals.

A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides.

Background: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R.

Multiplexed CRISPR-Cas9-Based Genome Editing of .

Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as and Significant advancements in the past few years have bolstered ' engineering capacity.

Monoterpene production by the carotenogenic yeast Rhodosporidium toruloides.

Background: Due to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks.

Transposon insertional mutagenesis in reveals -acting effects influencing species-dependent essential genes.

To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology.

Genetic dissection of interspecific differences in yeast thermotolerance.

Some of the most unique and compelling survival strategies in the natural world are fixed in isolated species. To date, molecular insight into these ancient adaptations has been limited, as classic experimental genetics has focused on interfertile individuals in populations. Here we use a new mapping approach, which screens mutants in a sterile interspecific hybrid, to identify eight housekeeping genes that underlie the growth advantage of Saccharomyces cerevisiae over its distant relative Saccharomyces paradoxus at high temperature.

Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.

Throughout history, the yeast has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically.

Functional genomics of lipid metabolism in the oleaginous yeast .

The basidiomycete yeast (also known as ) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded T-DNA insertions.

a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts.

Background: Economical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts.

Results: In this study, the carotenogenic yeast was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications.

Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S.

Quantitative Trait Loci (QTL)-Guided Metabolic Engineering of a Complex Trait.

Engineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness of Saccharomyces cerevisiae during fermentation of a Miscanthus x giganteus plant hydrolysate.

Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation.

Natural lipids can be used to make biodiesel and many other value-added compounds. In this work, we explored a number of different metabolic engineering strategies for increasing lipid production in the oleaginous yeast Rhodosporidium toruloides IFO0880. These included increasing the expression of enzymes involved in different aspects of lipid biosynthesis-malic enzyme (ME), pyruvate carboxylase (PYC1), glycerol-3-P dehydrogenase (GPD), and stearoyl-CoA desaturase (SCD)-and deleting the gene PEX10, required for peroxisome biogenesis.

Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S.

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

Unlabelled: Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars.

Complete Genome Sequences of Four Escherichia coli ST95 Isolates from Bloodstream Infections.

Finished genome sequences are presented for four Escherichia coli strains isolated from bloodstream infections at San Francisco General Hospital. These strains provide reference sequences for four major fimH-identified sublineages within the multilocus sequence type (MLST) ST95 group, and provide insights into pathogenicity and differential antimicrobial susceptibility within this group.

Engineering Rhodosporidium toruloides for increased lipid production.

Oleaginous yeast are promising organisms for the production of lipid-based chemicals and fuels from simple sugars. In this work, we explored Rhodosporidium toruloides for the production of lipid-based products. This oleaginous yeast natively produces lipids at high titers and can grow on glucose and xylose.