Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1.

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

Synchronization of Saccharomyces cerevisiae Cells for Analysis of Progression Through the Cell Cycle.

The cell division cycle is a fundamental process required for proliferation of all living organisms. The eukaryotic cell cycle follows a basic template with an ordered series of events beginning with G1 (Gap1) phase, followed successively by S (Synthesis) phase, G2 (Gap 2) phase, and M-phase (Mitosis). The process is tightly regulated in response to signals from both the internal and external milieu. The budding yeast S. cerevisiae is an outstanding model for the study of the cell cycle and its regulatory process. The basic events and regulatory processes of the S. cerevisiae cell cycle are highly conserved with other eukaryotes. The organism grows rapidly in simple medium, has a sequenced annotated genome, well-established genetics, and is amenable to analysis by proteomics and microscopy. Additionally, a range of tools and techniques are available to generate cultures of S. cerevisiae that are homogenously arrested or captured at specific phases of the cell cycle and upon release from that arrest these can be used to monitor cell cycle events as the cells synchronously proceed through a division cycle. In this chapter, we describe a series of commonly used techniques that are used to generate synchronized populations of S. cerevisiae and provide an overview of methods that can be used to monitor the progression of the cells through the cell division cycle.

Bioengineered Probiotics: Synthetic Biology Can Provide Live Cell Therapeutics for the Treatment of Foodborne Diseases.

The rising prevalence of antibiotic resistant microbial pathogens presents an ominous health and economic challenge to modern society. The discovery and large-scale development of antibiotic drugs in previous decades was transformational, providing cheap, effective treatment for what would previously have been a lethal infection. As microbial strains resistant to many or even all antibiotic drug treatments have evolved, there is an urgent need for new drugs or antimicrobial treatments to control these pathogens. The ability to sequence and mine the genomes of an increasing number of microbial strains from previously unexplored environments has the potential to identify new natural product antibiotic biosynthesis pathways. This coupled with the power of synthetic biology to generate new production chassis, biosensors and "weaponized" live cell therapeutics may provide new means to combat the rapidly evolving threat of drug resistant microbial pathogens. This review focuses on the application of synthetic biology to construct probiotic strains that have been endowed with functionalities allowing them to identify, compete with and in some cases kill microbial pathogens as well as stimulate host immunity. Weaponized probiotics may have the greatest potential for use against pathogens that infect the gastrointestinal tract: Vibrio cholerae, Staphylococcus aureus, Clostridium perfringens and Clostridioides difficile. The potential benefits of engineered probiotics are highlighted along with the challenges that must still be met before these intriguing and exciting new therapeutic tools can be widely deployed.

Ume6 Acts as a Stable Platform To Coordinate Repression and Activation of Early Meiosis-Specific Genes in Saccharomyces cerevisiae.

In response to nutrient starvation, the budding yeast Saccharomyces cerevisiae abandons mitotic proliferation and embarks on a differentiation process that leads through meiosis to the formation of haploid spores. This process is driven by cascading waves of meiosis-specific-gene expression. The early meiosis-specific genes are repressed during mitotic proliferation by the DNA-binding protein Ume6 in combination with repressors Rpd3 and Sin3. The expression of meiosis-specific transcription factor Ime1 leads to activation of the early meiosis-specific genes. We investigated the stability and promoter occupancy of Ume6 in sporulating cells and determined that it remains bound to early meiosis-specific gene promoters when those genes are activated. Furthermore, we find that the repressor Rpd3 remains associated with Ume6 after the transactivator Ime1 has joined the complex and that the Gcn5 and Tra1 components of the SAGA complex bind to the promoter of IME2 in an Ime1-dependent fashion to induce transcription of the early meiosis-specific genes. Our investigation supports a model whereby Ume6 provides a platform allowing recruitment of both activating and repressing factors to coordinate the expression of the early meiosis-specific genes in Saccharomyces cerevisiae.

Biosynthesis of Fatty Alcohols in Engineered Microbial Cell Factories: Advances and Limitations.

Concerns about climate change and environmental destruction have led to interest in technologies that can replace fossil fuels and petrochemicals with compounds derived from sustainable sources that have lower environmental impact. Fatty alcohols produced by chemical synthesis from ethylene or by chemical conversion of plant oils have a large range of industrial applications. These chemicals can be synthesized through biological routes but their free forms are produced in trace amounts naturally. This review focuses on how genetic engineering of endogenous fatty acid metabolism and heterologous expression of fatty alcohol producing enzymes have come together resulting in the current state of the field for production of fatty alcohols by microbial cell factories. We provide an overview of endogenous fatty acid synthesis, enzymatic methods of conversion to fatty alcohols and review the research to date on microbial fatty alcohol production. The primary focus is on work performed in the model microorganisms, Escherichia coli and Saccharomyces cerevisiae but advances made with cyanobacteria and oleaginous yeasts are also considered. The limitations to production of fatty alcohols by microbial cell factories are detailed along with consideration to potential research directions that may aid in achieving viable commercial scale production of fatty alcohols from renewable feedstock.

Lipomyces starkeyi: an emerging cell factory for production of lipids, oleochemicals and biotechnology applications.

Oils and oleochemicals produced by microbial cells offer an attractive alternative to petroleum and food-crop derived oils for the production of transport fuel and oleochemicals. An emerging candidate for industrial single cell oil production is the oleaginous yeast Lipomyces starkeyi. This yeast is capable of accumulating storage lipids to concentrations greater than 60% of the dry cell weight. From the perspective of industrial biotechnology L. starkeyi is an excellent chassis for single-cell oil and oleochemical production as it can use a wide variety of carbon and nitrogen sources as feedstock. The strain has been used to produce lipids from hexose and pentose sugars derived from cellulosic hydrolysates as well as crude glycerol and even sewage sludge. L. starkeyi also produces glucanhydrolases that have a variety of industrial applications and displays potential to be employed for bioremediation. Despite its excellent properties for biotechnology applications, adoption of L. starkeyi as an industrial chassis has been hindered by the difficulty of genetically manipulating the strain. This review will highlight the industrial potential of L. starkeyi as a chassis for the production of lipids, oleochemicals and other biochemicals. Additionally, we consider progress and challenges in engineering this organism for industrial applications.

Optimization of C16 and C18 fatty alcohol production by an engineered strain of Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi was engineered for the production of long-chain fatty alcohols by expressing a fatty acyl-CoA reductase, mFAR1, from Mus musculus. The optimal conditions for production of fatty alcohols by this strain were investigated. Increased carbon-to-nitrogen ratios led to efficient C16 and C18 fatty alcohol production from glucose, xylose and glycerol. Batch cultivation resulted in a titer of 1.7 g/L fatty alcohol from glucose which represents a yield of 28 mg of fatty alcohols per gram of glucose. This relatively high level of production with minimal genetic modification indicates that L. starkeyi may be an excellent host for the bioconversion of carbon-rich waste streams, particularly lignocellulosic waste, to C16 and C18 fatty alcohols.

Genetic Approaches to Study Meiosis and Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae has a long history as a model organism for studies of meiosis and the cell cycle. The popularity of this yeast as a model is in large part due to the variety of genetic and cytological approaches that can be effectively performed with the cells. Cultures of the cells can be induced to synchronously progress through meiosis and sporulation allowing large-scale gene expression and biochemical studies to be performed. Additionally, the spore tetrads resulting from meiosis make it possible to characterize the haploid products of meiosis allowing investigation of meiotic recombination and chromosome segregation. Here we describe genetic methods for analysis progression of S. cerevisiae through meiosis and sporulation with an emphasis on strategies for the genetic analysis of regulators of meiosis-specific genes.

Selection of G1 Phase Yeast Cells for Synchronous Meiosis and Sporulation.

Centrifugal elutriation is a procedure that allows the fractionation of cell populations based upon their size and shape. This allows cells in distinct cell cycle stages can be captured from an asynchronous population. The technique is particularly helpful when performing an experiment to monitor the progression of cells through the cell cycle or meiosis. Yeast sporulation like gametogenesis in other eukaryotes initiates from the G1 phase of the cell cycle. Conveniently, S. cerevisiae arrest in G1 phase when starved for nutrients and so withdrawal of nitrogen and glucose allows cells to abandon vegetative growth in G1 phase before initiating the sporulation program. This simple starvation protocol yields a partial synchronization that has been used extensively in studies of progression through meiosis and sporulation. By using centrifugal elutriation it is possible to isolate a homogeneous population of G1 phase cells and induce them to sporulate synchronously, which is beneficial for investigating progression through meiosis and sporulation. An additionally benefit of this protocol is that cell populations can be isolated based upon size and both large and small cell populations can be tested for progression through meiosis and sporulation. Here we present a protocol for purification of G1 phase diploid cells for examining synchronous progression through meiosis and sporulation.

Dual phosphorylation of cdk1 coordinates cell proliferation with key developmental processes in Drosophila.

Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Here, we expressed wild-type and phospho-acceptor mutant Cdk1 proteins to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2-phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the premitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1-Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2-phase cells are ready for mitosis.

Among B-type cyclins only CLB5 and CLB6 promote premeiotic S phase in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae cyclin Clb5 is required for premeiotic S phase, meiotic recombination, and successful progression through meiosis. Clb5 is not essential for mitotic proliferation because Clb1-Clb4 can support DNA replication in clb5 clb6 mutants. Clb1, Clb3, and Clb4 accumulate in clb5 clb6 cells during meiotic differentiation yet fail to promote premeiotic DNA replication. When expressed under the regulation of the CLB5 promoter, Clb1 and Clb3 accumulate and are active in the early stages of meiotic differentiation but cannot induce premeiotic DNA replication, suggesting that they do not target Cdk1 to the necessary substrates. The Clb5 hydrophobic patch (HP) residues are important for Clb5 function but this motif alone does not provide the specificity required for Clb5 to induce premeiotic S phase. Domain exchange experiments demonstrated that the amino terminus of Clb5 when fused to Clb3 confers upon Clb3 the ability to induce premeiotic S phase. Chimeric cyclins containing smaller regions of the Clb5 amino terminus displayed reduced ability to activate premeiotic DNA replication despite being more abundant and having greater associated histone H1 kinase activity than endogenous Clb5. These observations suggest that Clb5 has a unique ability to trigger premeiotic S phase and that the amino-terminal region of Clb5 contributes to its specificity and regulates the functions performed by the cyclin-Cdk complex.

A comparison of fluorescent DNA binding dyes for flow cytometric analysis of sporulating Saccharomyces cerevisiae.

Flow cytometry is important tool for investigating DNA replication in sporulating Saccharomyces cerevisiae. However, flow cytometry data from maturing spores is often difficult to interpret due to extensive broadening of the fluorescence peaks. This problem is markedly improved by treatment of the spores with potassium hydroxide prior to staining.

Structural elucidation of a PRP8 core domain from the heart of the spliceosome.

The spliceosome is a complex ribonucleoprotein (RNP) particle containing five RNAs and more than 100 associated proteins. One of these proteins, PRP8, has been shown to interact directly with the splice sites and branch region of precursor-mRNAs (pre-mRNAs) and spliceosomal RNAs associated with catalysis of the two steps of splicing. The 1.85-A X-ray structure of the core of PRP8 domain IV, implicated in key spliceosomal interactions, reveals a bipartite structure that includes the presence of an RNase H fold linked to a five-helix assembly. Analysis of mutant yeast alleles and cross-linking results in the context of this structure, coupled with RNA binding studies, suggests that domain IV forms a surface that interacts directly with the RNA structures at the catalytic core of the spliceosome.

The Saccharomyces cerevisiae CLB5 promoter contains two middle sporulation elements (MSEs) that are differentially regulated during sporulation.

The B-type cyclins Clb5 and Clb6 are essential activators of DNA replication during sporulation in Saccharomyces cerevisiae. The expression of CLB5 is maximally induced during the middle phase of sporulation by the transcription factor Ndt80. We have performed an analysis of the CLB5 promoter and have identified two middle sporulation elements (MSEs) that act as binding sites for Ndt80. Although both MSE sequences bind Ndt80 in vitro, they display differential effectiveness in their ability to function as cis-acting regulatory sequences in vivo. Mutation of both MSE sequences in the CLB5 promoter profoundly reduces the induction of CLB5 transcription during the middle phase of sporulation but results in no obvious defect in progression through meiosis and sporulation, implying that the Ndt80-dependent induction of CLB5 is not required for effective DNA replication or chromosome division.

Analyses of phosphorylation events in the rubella virus capsid protein: role in early replication events.

The Rubella virus capsid protein is phosphorylated prior to virus assembly. Our previous data are consistent with a model in which dynamic phosphorylation of the capsid regulates its RNA binding activity and, in turn, nucleocapsid assembly. In the present study, the process of capsid phosphorylation was examined in further detail. We show that phosphorylation of serine 46 in the RNA binding region of the capsid is required to trigger phosphorylation of additional amino acid residues that include threonine 47. This residue likely plays a direct role in regulating the binding of genomic RNA to the capsid. We also provide evidence which suggests that the capsid is dephosphorylated prior to or during virus budding. Finally, whereas the phosphorylation state of the capsid does not directly influence the rate of synthesis of viral RNA and proteins or the assembly and secretion of virions, the presence of phosphate on the capsid is critical for early events in virus replication, most likely the uncoating of virions and/or disassembly of nucleocapsids.

Meiosis-specific regulation of the Saccharomyces cerevisiae S-phase cyclin CLB5 is dependent on MluI cell cycle box (MCB) elements in its promoter but is independent of MCB-binding factor activity.

In proliferating S. cerevisiae, genes whose products function in DNA replication are regulated by the MBF transcription factor composed of Mbp1 and Swi6 that binds to consensus MCB sequences in target promoters. We find that during meiotic development a subset of DNA replication genes exemplified by TMP1 and RNR1 are regulated by Mbp1. Deletion of Mbp1 deregulated TMP1 and RNR1 but did not interfere with premeiotic S-phase, meiotic recombination, or spore formation. Surprisingly, deletion of MBP1 had no effect on the expression of CLB5, which is purportedly controlled by MBF. Extensive analysis of the CLB5 promoter revealed that the gene is largely regulated by elements within a 100-bp fragment containing a cluster of MCB sequences. Surprisingly, induction of the CLB5 promoter requires MCB sequences, but not Mbp1, implying that another MCB-binding factor may exist in cells undergoing meiosis. In addition, full activation of CLB5 during meiosis requires Clb5 activity, suggesting that CLB5 may be regulated by a positive feedback mechanism. We further demonstrate that during meiosis MCBs function as effective transcriptional activators independent of MBP1.

Purification and characterization of the DNA binding domain of Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80.

Ndt80 is a Saccharomyces cerevisiae meiosis-specific transcription factor responsible for promoting the stage-specific expression of a family of genes referred to as middle sporulation genes. Many members of this gene family are essential for the completion of meiotic chromosome segregation. Thus, Ndt80 is essential for the completion of meiosis. Ndt80 is highly regulated both transcriptionally and post-translationally. To facilitate biochemical analysis of Ndt80, we have expressed the DNA binding domain in Escherichia coli and purified the recombinant protein with an affinity chromatography procedure. In addition we have dissected the amino-terminus of Ndt80 to delimit the functional DNA binding domain. This analysis shows that the amino-terminal 40 amino-acids of Ndt80, although not essential for its DNA binding activity, do have an effect on its ability to bind specifically to its target DNA sequence. In addition, we show that the Ndt80 DNA binding domain can be phosphorylated by the meiosis-specific protein kinase Ime2 in vitro, but contrary to our initial hypothesis this phosphorylation does not significantly affect the affinity of Ndt80 for its target DNA sequence.

Purification and some properties of Saccharomyces cerevisiae meiosis-specific protein kinase Ime2.

Ime2 is the founding member of a family of protein kinases that are required for effective progression through meiotic development. Ime2 is essential for the induction of meiosis-specific genes and for the activation of meiotic DNA replication in the budding yeast Saccharomyces cerevisiae. Aside from the fact that Ime2 is a protein kinase and shares several amino acid motifs with cyclin dependent kinases, virtually nothing is known about its enzymatic properties or substrates. Biochemical characterization of Ime2 has been hindered by its low abundance and short half-life. We have created baculovirus expression vectors to produce recombinant Ime2 in insect cells. In this report, we describe the overproduction of Ime2 and its purification using affinity chromatography. Using this procedure, we have been able to purify up to 2mg Ime2 from 1L of infected insect cells. The Ime2 isolated by this method displays properties similar to those of the native enzyme that has been immunoprecipitated from yeast. The high level expression of Ime2 in this system and its ease of purification will be beneficial for more extensive biochemical analysis of Ime2 and related meiosis-specific kinases.