Engineered yeast tolerance enables efficient production from toxified lignocellulosic feedstocks.

Lignocellulosic biomass remains unharnessed for the production of renewable fuels and chemicals due to challenges in deconstruction and the toxicity its hydrolysates pose to fermentation microorganisms. Here, we show in that engineered aldehyde reduction and elevated extracellular potassium and pH are sufficient to enable near-parity production between inhibitor-laden and inhibitor-free feedstocks. By specifically targeting the universal hydrolysate inhibitors, a single strain is enhanced to tolerate a broad diversity of highly toxified genuine feedstocks and consistently achieve industrial-scale titers (cellulosic ethanol of >100 grams per liter when toxified).

A Morgan Legacy.

The Thomas Hunt Morgan Medal recognizes lifetime contributions to the field of genetics. The 2020 recipient is Gerald R. Fink of Massachusetts Institute of Technology and the Whitehead Institute for Biomedical Research, recognizing the discovery of principles central to genome organization and regulation in eukaryotic cells.

Xrn1p acts at multiple steps in the budding-yeast RNAi pathway to enhance the efficiency of silencing.

RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection.

Critical Roles of the Pentose Phosphate Pathway and GLN3 in Isobutanol-Specific Tolerance in Yeast.

Branched-chain alcohols are attractive advanced biofuels; however, their cellular toxicity is an obstacle to engineering microbes to produce them at high titers. We performed genome-wide screens on the Saccharomyces cerevisiae gene deletion library to identify cell systems involved in isobutanol-specific tolerance. Deletion of pentose phosphate pathway genes GND1 or ZWF1 causes hypersensitivity to isobutanol but not to ethanol.

mA modification of a 3' UTR site reduces RME1 mRNA levels to promote meiosis.

Despite the vast number of modification sites mapped within mRNAs, known examples of consequential mRNA modifications remain rare. Here, we provide multiple lines of evidence to show that Ime4p, an N6-methyladenosine (mA) methyltransferase required for meiosis in yeast, acts by methylating a site in the 3' UTR of the mRNA encoding Rme1p, a transcriptional repressor of meiosis. Consistent with this mechanism, genetic analyses reveal that IME4 functions upstream of RME1.

Complex modifier landscape underlying genetic background effects.

The phenotypic consequence of a given mutation can be influenced by the genetic background. For example, conditional gene essentiality occurs when the loss of function of a gene causes lethality in one genetic background but not another. Between two individual strains, S288c and Σ1278b, ∼1% of yeast genes were previously identified as "conditional essential.

Excised linear introns regulate growth in yeast.

Spliceosomal introns are ubiquitous non-coding RNAs that are typically destined for rapid debranching and degradation. Here we describe 34 excised introns in Saccharomyces cerevisiae that-despite being rapidly degraded in log-phase growth-accumulate as linear RNAs under either saturated-growth conditions or other stresses that cause prolonged inhibition of TORC1, which is a key integrator of growth signalling. Introns that become stabilized remain associated with components of the spliceosome and differ from other spliceosomal introns in having a short distance between their lariat branch point and 3' splice site, which is necessary and sufficient for their stabilization.

Galectin-3 Regulates γ-Herpesvirus Specific CD8 T Cell Immunity.

To gain insights into the molecular mechanisms and pathways involved in the activation of γ-herpesvirus (MHV68)-specific T cell receptor transnuclear (TN) CD8 T cells, we performed a comprehensive transcriptomic analysis. Upon viral infection, we observed differential expression of several thousand transcripts encompassing various networks and pathways in activated TN cells compared with their naive counterparts. Activated cells highly upregulated galectin-3.

Biosynthesis of monoethylene glycol in Saccharomyces cerevisiae utilizing native glycolytic enzymes.

Monoethylene glycol (MEG) is an important commodity chemical with applications in numerous industrial processes, primarily in the manufacture of polyethylene terephthalate (PET) polyester used in packaging applications. In the drive towards a sustainable chemical industry, bio-based production of MEG from renewable biomass has attracted growing interest. Recent attempts for bio-based MEG production have investigated metabolic network modifications in Escherichia coli, specifically rewiring the xylose assimilation pathways for the synthesis of MEG.

New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi.

We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in , , , and These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both and rely heavily on homology-directed repair, whereas and use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs.

Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell.

Current techniques to characterize leukocyte subgroups in blood require long sample preparation times and sizable sample volumes. A simplified method for leukocyte characterization using smaller blood volumes would thus be useful in diagnostic settings. Here we describe a flow system comprised of two functionalized graphene oxide (GO) surfaces that allow the capture of distinct leukocyte populations from small volumes blood using camelid single-domain antibodyfragments (VHHs) as capture agents.

A CRISPR system permits genetic engineering of essential genes and gene families.

is a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families.

Biofuels. Engineering alcohol tolerance in yeast.

Ethanol toxicity in the yeast Saccharomyces cerevisiae limits titer and productivity in the industrial production of transportation bioethanol. We show that strengthening the opposing potassium and proton electrochemical membrane gradients is a mechanism that enhances general resistance to multiple alcohols. The elevation of extracellular potassium and pH physically bolsters these gradients, increasing tolerance to higher alcohols and ethanol fermentation in commercial and laboratory strains (including a xylose-fermenting strain) under industrial-like conditions.

Interactions between chromosomal and nonchromosomal elements reveal missing heritability.

The measurement of any nonchromosomal genetic contribution to the heritability of a trait is often confounded by the inability to control both the chromosomal and nonchromosomal information in a population. We have designed a unique system in yeast where we can control both sources of information so that the phenotype of a single chromosomal polymorphism can be measured in the presence of different cytoplasmic elements. With this system, we have shown that both the source of the mitochondrial genome and the presence or absence of a dsRNA virus influence the phenotype of chromosomal variants that affect the growth of yeast.

Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption.

Phenotyping single cells based on the products they secrete or consume is a key bottleneck in many biotechnology applications, such as combinatorial metabolic engineering for the overproduction of secreted metabolites. Here we present a flexible high-throughput approach that uses microfluidics to compartmentalize individual cells for growth and analysis in monodisperse nanoliter aqueous droplets surrounded by an immiscible fluorinated oil phase. We use this system to identify xylose-overconsuming Saccharomyces cerevisiae cells from a population containing one such cell per 10(4) cells and to screen a genomic library to identify multiple copies of the xylose isomerase gene as a genomic change contributing to high xylose consumption, a trait important for lignocellulosic feedstock utilization.

High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis.

N(6)-methyladenosine (m(6)A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m(6)A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated eight out of eight methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence.

Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.

Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1.

Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols.

Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeting metabolic pathways to mitochondria can increase production compared with overexpression of the enzymes involved in the same pathways in the cytoplasm. Compartmentalization of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overproducing enzymes involved in only the first three steps of the biosynthetic pathway.

Genetic variation in Saccharomyces cerevisiae: circuit diversification in a signal transduction network.

The connection between genotype and phenotype was assessed by determining the adhesion phenotype for the same mutation in two closely related yeast strains, S288c and Sigma, using two identical deletion libraries. Previous studies, all in Sigma, had shown that the adhesion phenotype was controlled by the filamentation mitogen-activated kinase (fMAPK) pathway, which activates a set of transcription factors required for the transcription of the structural gene FLO11. Unexpectedly, the fMAPK pathway is not required for FLO11 transcription in S288c despite the fact that the fMAPK genes are present and active in other pathways.

Genes come and go: the evolutionarily plastic path of budding yeast RNase III enzymes.

Our recent finding that the Candida albicans RNase III enzyme CaDcr1 is an unusual, multifunctional RNase III coupled with data on the RNase III enzymes from other fungal species prompted us to seek a model that explained the evolution of RNase III's in modern budding yeast species. CaDcr1 has both dicer function (generates small RNA molecules from dsRNA precursors) and Rnt1 function, (catalyzes the maturation of 35S rRNA and U4 snRNA). Some budding yeast species have two distinct genes that encode these functions, a Dicer and RNT1, whereas others have only an RNT1 and no Dicer.

Global gene deletion analysis exploring yeast filamentous growth.

The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae strain, Σ1278b.

Ruler arrays reveal haploid genomic structural variation.

Despite the known relevance of genomic structural variants to pathogen behavior, cancer, development, and evolution, certain repeat based structural variants may evade detection by existing high-throughput techniques. Here, we present ruler arrays, a technique to detect genomic structural variants including insertions and deletions (indels), duplications, and translocations. A ruler array exploits DNA polymerase's processivity to detect physical distances between defined genomic sequences regardless of the intervening sequence.

Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae.

Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S.

RNA methylation by the MIS complex regulates a cell fate decision in yeast.

For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program.