Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.

Yeast lacking the sterol C-5 desaturase Erg3 are tolerant to the anti-inflammatory triterpenoid saponin escin.

Escin is a mixture of over 30 glycosylated triterpenoid (saponin) structures, extracted from the dried fruit of horse chestnuts. Escin is currently used as an anti-inflammatory, and has potential applications in the treatment of arthritis and cancer. Engineered yeast would enable production of specific bioactive components of escin at industrial scale, however many saponins have been shown to be toxic to yeast. Here we report that a Saccharomyces cerevisiae strain specifically lacking the sterol C-5 desaturase gene ERG3, exhibits striking enhanced tolerance to escin treatment. Transcriptome analyses, as well as pre-mixing of escin with sterols, support the hypothesis that escin interacts directly with ergosterol, but not as strongly with the altered sterols present in erg3Δ. A diverse range of saponins are of commercial interest, and this research highlights the value of screening lipidome mutants to identify appropriate hosts for engineering the industrial production of saponins.

ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.

Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user's need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a ß-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

A systematic comparison of triterpenoid biosynthetic enzymes for the production of oleanolic acid in Saccharomyces cerevisiae.

Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a ß-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen ß-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of ß-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in ß-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives.

The wide-ranging phenotypes of ergosterol biosynthesis mutants, and implications for microbial cell factories.

Yeast strains have been used extensively as robust microbial cell factories for the production of bulk and fine chemicals, including biofuels (bioethanol), complex pharmaceuticals (antimalarial drug artemisinin and opioid pain killers), flavours, and fragrances (vanillin, nootkatone, and resveratrol). In many cases, it is of benefit to suppress or modify ergosterol biosynthesis during strain engineering, for example, to increase thermotolerance or to increase metabolic flux through an alternate pathway. However, the impact of modifying ergosterol biosynthesis on engineered strains is discussed sparsely in literature, and little attention has been paid to the implications of these modifications on the general health and well-being of yeast. Importantly, yeast with modified sterol content exhibit a wide range of phenotypes, including altered organization and dynamics of plasma membrane, altered susceptibility to chemical treatment, increased tolerance to high temperatures, and reduced tolerance to other stresses such as high ethanol, salt, and solute concentrations. Here, we review the wide-ranging phenotypes of viable Saccharomyces cerevisiae strains with altered sterol content and discuss the implications of these for yeast as microbial cell factories.

Monodehydroascorbate reductase mediates TNT toxicity in plants.

The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Due to the scale of affected areas, one of the most cost-effective and environmentally friendly means of removing explosives pollution could be the use of plants. However, mechanisms of TNT phytotoxicity have been elusive. Here, we reveal that phytotoxicity is caused by reduction of TNT in the mitochondria, forming a nitro radical that reacts with atmospheric oxygen, generating reactive superoxide. The reaction is catalyzed by monodehydroascorbate reductase 6 (MDHAR6), with Arabidopsis deficient in MDHAR6 displaying enhanced TNT tolerance. This discovery will contribute toward the remediation of contaminated sites. Moreover, in an environment of increasing herbicide resistance, with a shortage in new herbicide classes, our findings reveal MDHAR6 as a valuable plant-specific target.

Harnessing microbial gene pools to remediate persistent organic pollutants using genetically modified plants--a viable technology?

It has been 14 years since the international community came together to legislate the Stockholm Convention on Persistent Organic Pollutants (POPs), restricting the production and use of specific chemicals that were found to be environmentally stable, often bioaccumulating, with long-term toxic effects. Efforts are continuing to remove these pollutants from the environment. While incineration and chemical treatment can be successful, these methods require the removal of tonnes of soil, at high cost, and are damaging to soil structure and microbial communities. The engineering of plants for in situ POP remediation has had highly promising results, and could be a more environmentally-friendly alternative. This review discusses the characterization of POP-degrading bacterial pathways, and how the genes responsible have been harnessed using genetic modification (GM) to introduce these same abilities into plants. Recent advances in multi-gene cloning, genome editing technologies and expression in monocot species are accelerating progress with remediation-applicable species. Examples include plants developed to degrade 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), trichloroethylene (TCE), and polychlorinated biphenyls (PCBs). However, the costs and timescales needed to gain regulatory approval, along with continued public opposition, are considerable. The benefits and challenges in this rapidly developing and promising field are discussed.

Phytodetoxification of the environmental pollutant and explosive 2,4,6-trinitrotoluene.

Our recent study highlights the role of 2 glutathione transferases (GSTs) in the detoxification of the environmental pollutant, 2,4,6-trinitrotoluene (TNT) in Arabidopsis thaliana. TNT is toxic and highly resistant to biodegradation in the environment, raising both health and environmental concerns. Two GSTs, GST-U24 and GST-U25, are upregulated in response to TNT treatment, and expressed predominantly in the root tissues; the site of TNT location following uptake. Plants overexpressing GST-U24 and GST-U25 exhibited significantly enhanced ability to withstand and detoxify TNT, and remove TNT from contaminated soil. Analysis of the catalytic activities of these 2 enzymes revealed that they form 3 TNT-glutathionyl products. Of particular interest is 2-glutathionyl-4,6-dinitrotoluene as this represents a potentially favorable step toward subsequent degradation and mineralization of TNT. We demonstrate how GSTs fit into what is already known about pathways for TNT detoxification, and discuss the short and longer-term fate of TNT conjugates in planta.

Arabidopsis Glutathione Transferases U24 and U25 Exhibit a Range of Detoxification Activities with the Environmental Pollutant and Explosive, 2,4,6-Trinitrotoluene.

The explosive 2,4,6-trinitrotoluene (TNT) is a major worldwide military pollutant. The presence of this toxic and highly persistent pollutant, particularly at military sites and former manufacturing facilities, presents various health and environmental concerns. Due to the chemically resistant structure of TNT, it has proven to be highly recalcitrant to biodegradation in the environment. Here, we demonstrate the importance of two glutathione transferases (GSTs), GST-U24 and GST-U25, from Arabidopsis (Arabidopsis thaliana) that are specifically up-regulated in response to TNT exposure. To assess the role of GST-U24 and GST-U25, we purified and characterized recombinant forms of both enzymes and demonstrated the formation of three TNT glutathionyl products. Importantly, GST-U25 catalyzed the denitration of TNT to form 2-glutathionyl-4,6-dinitrotoluene, a product that is likely to be more amenable to subsequent biodegradation in the environment. Despite the presence of this biochemical detoxification pathway in plants, physiological concentrations of GST-U24 and GST-U25 result in only a limited innate ability to cope with the levels of TNT found at contaminated sites. We demonstrate that Arabidopsis plants overexpressing GST-U24 and GST-U25 exhibit significantly enhanced ability to withstand and detoxify TNT, properties that could be applied for in planta detoxification of TNT in the field. The overexpressing lines removed significantly more TNT from soil and exhibited a corresponding reduction in glutathione levels when compared with wild-type plants. However, in the absence of TNT, overexpression of these GSTs reduces root and shoot biomass, and although glutathione levels are not affected, this effect has implications for xenobiotic detoxification.