Real-Time Detection of Yeast Growth on Solid Medium through Passive Microresonator Biosensor.

This study presents a biosensor fabricated based on integrated passive device (IPD) technology to measure microbial growth on solid media in real-time. Yeast (Pichia pastoris, strain GS115) is used as a model organism to demonstrate biosensor performance. The biosensor comprises an interdigital capacitor in the center with a helical inductive structure surrounding it. Additionally, 12 air bridges are added to the capacitor to increase the strength of the electric field radiated by the biosensor at the same height. Feasibility is verified by using a capacitive biosensor, and the change in capacitance values during the capacitance detection process with the growth of yeast indicates that the growth of yeast can induce changes in electrical parameters. The proposed IPD-based biosensor is used to measure yeast drop-added on a 3 mm medium for 100 h at an operating frequency of 1.84 GHz. The resonant amplitude of the biosensor varies continuously from 24 to 72 h due to the change in colony height during vertical growth of the yeast, with a maximum change of 0.21 dB. The overall measurement results also fit well with the Gompertz curve. The change in resonant amplitude between 24 and 72 h is then analyzed and reveals a linear relationship with time with a coefficient of determination of 0.9844, indicating that the biosensor is suitable for monitoring yeast growth. Thus, the proposed biosensor is proved to have potential in the field of microbial proliferation detection.

Physiological and morphological plasticity in response to nitrogen availability of a yeast widely distributed in the open ocean.

Yeasts are prevalent in the open ocean, yet we have limited understanding of their ecophysiological adaptations, including their response to nitrogen availability, which can have a major role in determining the ecological potential of other planktonic microbes. In this study, we characterized the nitrogen uptake capabilities and growth responses of marine-occurring yeasts. Yeast isolates from the North Atlantic Ocean were screened for growth on diverse nitrogen substrates, and across a concentration gradient of three environmentally relevant nitrogen substrates: nitrate, ammonium, and urea. Three strains grew with enriched nitrate while two did not, demonstrating that nitrate utilization is present but not universal in marine yeasts, consistent with existing knowledge of nonmarine yeast strains. Naganishia diffluens MBA_F0213 modified the key functional trait of cell size in response to nitrogen concentration, suggesting yeast cell morphology changes along chemical gradients in the marine environment. Meta-analysis of the reference DNA barcode in public databases revealed that the genus Naganishia has a global ocean distribution, strengthening the environmental applicability of the culture-based observations. This study provides novel quantitative understanding of the ecophysiological and morphological responses of marine-derived yeasts to variable nitrogen availability in vitro, providing insight into the functional ecology of yeasts within pelagic open ocean environments.

Physiological evaluation of yeast strains under anaerobic conditions using glucose, fructose, or sucrose as the carbon source.

The aim of this study was to evaluate the physiology of 13 yeast strains by assessing their kinetic parameters under anaerobic conditions. They included Saccharomyces cerevisiae CAT-1 and 12 isolated yeasts from different regions in Brazil. The study aimed to enhance understanding of the metabolism of these strains for more effective applications. Measurements included quantification of sugars, ethanol, glycerol, and organic acids. Various kinetic parameters were analyzed, such as specific substrate utilization rate (qS), maximum specific growth rate (µmax), doubling time, biomass yield, product yield, maximum cell concentration, ethanol productivity (PEth), biomass productivity, and CO2 concentration. S. cerevisiae CAT-1 exhibited the highest values in glucose for µmax (0.35 h-1), qS (3.06 h-1), and PEth (0.69 gEth L-1 h-1). Candida parapsilosis Recol 37 did not fully consume the substrate. In fructose, S. cerevisiae CAT-1 stood out with higher values for µmax (0.25 h-1), qS (2.24 h-1), and PEth (0.60 gEth L-1 h-1). Meyerozyma guilliermondii Recol 09 and C. parapsilosis Recol 37 had prolonged fermentation times and residual substrate. In sucrose, only S. cerevisiae CAT-1, S. cerevisiae BB9, and Pichia kudriavzevii Recol 39 consumed all the substrate, displaying higher PEth (0.72, 0.51, and 0.44 gEth L-1 h-1, respectively) compared to other carbon sources.

PicoShells: Hollow Hydrogel Microparticles for High-Throughput Screening of Clonal Libraries.

Microfluidics enables the creation of monodisperse, micron-scale aqueous droplets, or other compartments. These droplets serve as picolitre-volume reaction chambers which can be utilized for various chemical assays or reactions. Here we describe the use of a microfluidic droplet generator to encapsulate single cells within hollow hydrogel microparticles called PicoShells. The PicoShell fabrication utilizes a mild pH-based crosslinking modality of an aqueous two-phase prepolymer system, avoiding the cell death and unwanted genomic modifications that accompany more typical, ultraviolet light crosslinking techniques. The cells are grown inside of these PicoShells into monoclonal colonies in any number of environments, including scaled production environments using commercially relevant incubation methods. Colonies can be phenotypically analyzed and/or sorted using standard, high-throughput laboratory techniques, namely, fluorescence-activated cell sorting (FACS). Cell viability is maintained throughout particle fabrication and analysis, and cells exhibiting a desired phenotype can be selected and released for re-culturing and downstream analysis. Large-scale cytometry runs are of particular use when measuring the protein expression of heterogeneous cells in response to environmental stimuli, notably to identify targets early in the drug discovery process. The sorted cells can also be encapsulated multiple times to direct the evolution of a cell line to a desired phenotype.

Whole-cell modeling in yeast predicts compartment-specific proteome constraints that drive metabolic strategies.

When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here, we present a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions. The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data suggests that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation-known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimisation, providing a framework to understand metabolic adaptation in eukaryal cells.

Tyrosols retrieved from traditionally brewed yeasts assist in tolerance against heavy metals and promote the growth of cells.

Heavy metals act as cofactors for several microbial enzymes and are required in low concentrations for the proper biological functioning of yeasts. Because concentrations beyond the permitted threshold can damage a cell's functionality and viability, metal tolerance in yeasts towards such heavy metals is therefore desirable during fermentation. Tyrosol, a quorum-sensing molecule in yeasts, protects yeasts from oxidative stress induced by various factors, but the performance of the molecule under heavy metal-induced stress is not known. In this investigation, the metal tolerance of four species of endemic yeasts from northeast India, Wickerhamomyces anomalus, Candida tropicalis, Saccharomyces cerevisiae and Candida glabrata, isolated from traditional starter culture cakes, was tested towards zinc (Zn+2), manganese (Mn+2), cobalt (Co+2) and copper (Cu+2) in the presence and absence of tyrosols retrieved from these isolates. The decreasing order of the tolerance of isolates was found to be Mn+2 > Zn+2 > Co+2 > Cu+2. Under the influence of tyrosols, isolates showed enhanced growth at their upper metal tolerance limit. Candida tropicalis showed enhanced growth (2-48-fold, P < 0.0001) in all the tested metal consisting medium (2 mM Zn+2, 5 mM Mn+2, 2 mM Co+2 and 1 mM Cu+2), while W. anomalus, C. glabrata and S. cerevisiae showed increased growth (3-17-fold, P < 0.0001) in Zn+2 (2 mM), Mn+2 (5 mM) and Cu+2 (1 mM) augmented medium. The overall result suggests that tyrosol exerts a protective effect under heavy metal-induced stress, which could be useful in enhancing the quality of fermented products.

The Discovery of Actinospene, a New Polyene Macrolide with Broad Activity against Plant Fungal Pathogens and Pathogenic Yeasts.

Phytopathogenic fungi infect crops, presenting a worldwide threat to agriculture. Polyene macrolides are one of the most effective antifungal agents applied in human therapy and crop protection. In this study, we found a cryptic polyene biosynthetic gene cluster in Actinokineospora spheciospongiae by genome mining. Then, this gene cluster was activated via varying fermentation conditions, leading to the discovery of new polyene actinospene (1), which was subsequently isolated and its structure determined through spectroscopic techniques including UV, HR-MS, and NMR. The absolute configuration was confirmed by comparing the calculated and experimental electronic circular dichroism (ECD) spectra. Unlike known polyene macrolides, actinospene (1) demonstrated more versatile post-assembling decorations including two epoxide groups and an unusual isobutenyl side chain. In bioassays, actinospene (1) showed a broad spectrum of antifungal activity against several plant fungal pathogens as well as pathogenic yeasts with minimum inhibitory concentrations ranging between 2 and 10 µg/mL.

Wine yeast species show strong inter- and intra-specific variability in their sensitivity to ultraviolet radiation.

While the trend in winemaking is toward reducing the inputs and especially sulphites utilization, emerging technologies for the preservation of wine is a relevant topic for the industry. Amongst yeast spoilage in wine, Brettanomyces bruxellensis is undoubtedly the most feared. In this study, UV-C treatment is investigated. This non-thermal technique is widely used for food preservation. A first approach was conducted using a drop-platted system to compare the sensitivity of various strains to UV-C surface treatment. 147 strains distributed amongst fourteen yeast species related to wine environment were assessed for six UV-C doses. An important variability in UV-C response was observed at the interspecific level. Interestingly, cellar resident species, which are mainly associated with wine spoilage, shows higher sensitivity to UV-C than vineyard-resident species. A focus on B. bruxellensis species with 104 screened strains highlighted an important effect of the UV-C, with intra-specific variation. This intra-specific variation was confirmed on 6 strains in liquid red wine by using a home-made pilot. 6624 J.L-1 was enough for a reduction of 5 log10 of magnitude for 5 upon 6 strains. These results highlight the potential of UV-C utilization against wine yeast spoiler at cellar scale.

Customized yeast cell factories for biopharmaceuticals: from cell engineering to process scale up.

The manufacture of recombinant therapeutics is a fastest-developing section of therapeutic pharmaceuticals and presently plays a significant role in disease management. Yeasts are established eukaryotic host for heterologous protein production and offer distinctive benefits in synthesising pharmaceutical recombinants. Yeasts are proficient of vigorous growth on inexpensive media, easy for gene manipulations, and are capable of adding post translational changes of eukaryotes. Saccharomyces cerevisiae is model yeast that has been applied as a main host for the manufacture of pharmaceuticals and is the major tool box for genetic studies; nevertheless, numerous other yeasts comprising Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Yarrowia lipolytica have attained huge attention as non-conventional partners intended for the industrial manufacture of heterologous proteins. Here we review the advances in yeast gene manipulation tools and techniques for heterologous pharmaceutical protein synthesis. Application of secretory pathway engineering, glycosylation engineering strategies and fermentation scale-up strategies in customizing yeast cells for the synthesis of therapeutic proteins has been meticulously described.

Structure and antimicrobial comparison between N-(benzyl) chitosan derivatives and N-(benzyl) chitosan tripolyphosphate nanoparticles against bacteria, fungi, and yeast.

Chitosan (Ch) was reacted with seven benzaldehyde analogs separately through reductive amination in which the corresponding imines were formed and followed by reduction to produce N-(benzyl) chitosan (NBCh) derivatives. 1H NMR spectroscopy was used to characterize the products. The nanoparticles (NPs) of Ch and NBCh derivatives were prepared according to the ionotropic gelation mechanism between Ch products and sodium tripolyphosphate, followed by high-energy ultrasonication. Scanning electron microscopy, particle size, polydispersity index, and zeta potential were applied for the NPs examination. The particle size was ranged from 235.17 to 686.90 nm and narrow size distribution (PDI <1). The zeta potential of NPs was varied between -1.26 and -27.50 mV. The antimicrobial activity was evaluated against bacteria (Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. carotovora, and Ralstonia solanacearum), fungi (Aspergillus flavus and Aspergillus niger), and yeast (Candida albicans). The action of NBCh derivatives was significantly higher than Ch. The NPs had considerably higher than the Ch and NBCh derivatives. The activity was directly proportional to the chemical derivatization of Ch and the zeta potential of the NPs. The antimicrobial efficacy of these derivatives formulated in a greener approach could become an alternative to using traditional antimicrobial applications in an environmentally friendly manner.

Non-conventional yeasts from fermented honey by-products: Focus on Hanseniaspora uvarum strains for craft beer production.

The increasing interest in novel beer productions focused on non-Saccharomyces yeasts in order to pursue their potential in generating groundbreaking sensory profiles. Traditional fermented beverages represent an important source of yeast strains which could express interesting features during brewing. A total of 404 yeasts were isolated from fermented honey by-products and identified as Saccharomyces cerevisiae, Wickerhamomyces anomalus, Zygosaccharomyces bailii, Zygosaccharomyces rouxii and Hanseniaspora uvarum. Five H. uvarum strains were screened for their brewing capability. Interestingly, Hanseniaspora uvarum strains showed growth in presence of ethanol and hop and a more rapid growth than the control strain S. cerevisiae US-05. Even though all strains showed a very low fermentation power, their concentrations ranged between 7 and 8 Log cycles during fermentation. The statistical analyses showed significant differences among the strains and underlined the ability of YGA2 and YGA34 to grow rapidly in presence of ethanol and hop. The strain YGA34 showed the best technological properties and was selected for beer production. Its presence in mixed- and sequential-culture fermentations with US-05 did not influence attenuation and ethanol concentration but had a significant impact on glycerol and acetic acid concentrations, with a higher sensory complexity and intensity, representing promising co-starters during craft beer production.

The antagonistic Metschnikowia andauensis produces extracellular enzymes and pulcherrimin, whose production can be promoted by the culture factors.

Biological control against microbial infections has a great potential as an alternative approach instead of fungicidal chemicals, which can cause environmental pollution. The pigment producer Metschnikowia andauensis belongs to the antagonistic yeasts, but details of the mechanism by which it inhibits growth of other microbes are less known. Our results confirmed its antagonistic capacity on other yeast species isolated from fruits or flowers and demonstrated that the antagonistic capacity was well correlated with the size of the red pigmented zone. We have isolated and characterized its red pigment, which proved to be the iron chelating pulcherrimin. Its production was possible even in the presence of 0.05 mg/ml copper sulphate, which is widely used in organic vineyards because of its antimicrobial properties. Production and localisation of the pulcherrimin strongly depended on composition of the media and other culture factors. Glucose, galactose, disaccharides and the presence of pectin or certain amino acids clearly promoted pigment production. Higher temperatures and iron concentration decreased the diameter of red pigmented zones. The effect of pH on pigment production varied depending of whether it was tested in liquid or solid media. In addition, our results suggest that other mechanisms besides the iron depletion of the culture media may contribute to the antagonistic capacity of M. andauensis.

Co-cultivation of sake yeast and Kocuria isolates from the sake brewing process.

Kocuria isolates collected from the sake brewing process have inhabited the Narimasa Sake Brewery in Toyama, Japan. To investigate the effect of these actinobacterial isolates on the growth and metabolism of sake yeast, co-cultivation of sake yeast and Kocuria isolates was performed in a medium containing tryptone, glucose and yeast extract (TGY), and a solution containing koji (steamed rice covered with Aspergillus oryzae) and glucose. In the TGY medium, the ethanol concentration and the number of living cells of each microorganism were measured. In the koji solution, the concentrations of ethanol and organic acids (citric acid, lactic acid and succinic acid) were measured. The results showed that in TGY media, the growth of each Kocuria isolate in the co-culture of the two Kocuria isolates was similar to that in each monoculture. However, the growth of both Kocuria isolates was inhibited in the co-cultures of sake yeast and Kocuria isolates. On the other hand, the growth and ethanol productivity of sake yeast did not differ between its monoculture and co-cultures with Kocuria isolates. In the koji solution, Kocuria isolates TGY1120_3 and TGY1127_2 affected the concentrations of ethanol and lactic acid, respectively. Thus, Kocuria isolates affected the microbial metabolism, but the effects were not identical between the two isolates. This strongly suggests that bacteria inhabiting a sake brewery may influence the flavor and taste of sake products of the brewery.

Novel use of Kombucha consortium to reduce Escherichia coli in dairy shed effluent.

BACKGROUND: Faecal contamination from dairy farm effluent is a major risk to water quality in New Zealand. In this experiment we have tested the efficacy of Kombucha SCOBY (symbiotic culture of bacteria and yeast), to reduce the concentration of Escherichia coli in dairy shed effluent (DSE). RESULTS: Kombucha SCOBY was highly effective in lowering the number of E. coli colony forming units (CFUs) to levels that were undetectable. The decrease in CFUs occurred rapidly within 48 h of Kombucha SCOBY being inoculated to the effluent matrix and was accompanied by a corresponding decline in pH. CONCLUSION: We conclude that Kombucha SCOBY was effective in reducing the abundance of E. coli in DSE due to its effect on solution pH. Further work is required to assess the practicality of treating DSE with Kombucha SCOBY within a farm environment where effluent management and climatic complexities are important. © 2021 Society of Chemical Industry.

A buffered media system for yeast batch culture growth.

BACKGROUND: Fungi are premier hosts for the high-yield secretion of proteins for biomedical and industrial applications. The stability and activity of these secreted proteins is often dependent on the culture pH. As yeast acidifies the commonly used synthetic complete drop-out (SD) media that contains ammonium sulfate, the pH of the media needs to be buffered in order to maintain a desired extracellular pH during biomass production. At the same time, many buffering agents affect growth at the concentrations needed to support a stable pH. Although the standard for biotechnological research and development is shaken batch cultures or microtiter plate cultures that cannot be easily automatically pH-adjusted during growth, there is no comparative study that evaluates the buffering capacity and growth effects of different media types across pH-values in order to develop a pH-stable batch culture system. RESULTS: We systematically test the buffering capacity and growth effects of a citrate-phosphate buffer (CPB) from acidic to neutral pH across different media types. These media types differ in their nitrogen source (ammonium sulfate, urea or both). We find that the widely used synthetic drop-out media that uses ammonium sulfate as nitrogen source can only be effectively buffered at buffer concentrations that also affect growth. At lower concentrations, yeast biomass production still acidifies the media. When replacing the ammonium sulfate with urea, the media alkalizes. We then develop a medium combining ammonium sulfate and urea which can be buffered at low CPB concentrations that do not affect growth. In addition, we show that a buffer based on Tris/HCl is not effective in maintaining any of our media types at neutral pH even at relatively high concentrations. CONCLUSION: Here we show that the buffering of yeast batch cultures is not straight-forward and addition of a buffering agent to set a desired starting pH does not guarantee pH-maintenance during growth. In response, we present a buffered media system based on an ammonium sulfate/urea medium that enables relatively stable pH-maintenance across a wide pH-range without affecting growth. This buffering system is useful for protein-secretion-screenings, antifungal activity assays, as well as for other pH-dependent basic biology or biotechnology projects.

Co-culture fermentation of Pediococcus acidilactici XZ31 and yeast for enhanced degradation of wheat allergens.

Previous researchers have shown the potential of sourdough and isolated lactic acid bacteria in reducing wheat allergens. As the interactions of lactic acid bacteria with yeast is a key event in sourdough fermentation, we wished to investigate how yeast affects metabolism of lactic acid bacteria, thereby affecting protein degradation and antigenic response. In this study, three strains isolated from sourdough were selected for dough fermentation, namely Pediococcus acidilactici XZ31, Saccharomyces cerevisiae JM1 and Torulaspora delbrueckii JM4. The changes in dough protein during the fermentation process were studied. Protein degradation and antigenic response in dough inoculated with Pediococcus acidilactici XZ31 monoculture and co-culture with yeast were mainly evaluated by SDS-PAGE, immunoblotting, ELISA and Liquid chromatography-tandem mass spectrometry assay. The whole-genome transcriptomic changes in Pediococcus acidilactici XZ31 were also investigated by RNA sequencing. The results showed that water/salt soluble protein and Tri a 28/19 allergens content significantly decreased after 24 h fermentation. Co-culture fermentation accelerated the degradation of protein, and reduced the allergen content to a greater extent. RNA-sequencing analysis further demonstrated that the presence of yeast could promote protein metabolism in Pediococcus acidilactici XZ31 for a certain period of time. These results revealed a synergistic effect between Pediococcus acidilactici XZ31 and yeast degrading wheat allergens, and suggested the potential use of the multi-strain leavening agent for producing hypoallergenic wheat products.

Variable and dose-dependent response of Saccharomyces and non-Saccharomyces yeasts toward lignocellulosic hydrolysate inhibitors.

Lignocellulosic hydrolysates will also contain compounds that inhibit microbial metabolism, such as organic acids, furaldehydes, and phenolic compounds. Understanding the response of yeasts toward such inhibitors is important to the development of different bioprocesses. In this work, the growth capacity of 7 industrial Saccharomyces cerevisiae and 7 non-Saccharomyces yeasts was compared in the presence of 3 different concentrations of furaldehydes (furfural and 5-hydroxymetil-furfural), organic acids (acetic and formic acids), and phenolic compounds (vanillin, syringaldehyde, ferulic, and coumaric acids). Then, Candida tropicalis JA2, Meyerozyma caribbica JA9, Wickerhamomyces anomalus 740, S. cerevisiae JP1, B1.1, and G06 were selected for fermentation in presence of acetic acid, HMF, and vanillin because they proved to be most tolerant to the tested compounds, while Spathaspora sp. JA1 because its xylose consumption rate. The results obtained showed a dose-dependent response of the yeasts toward the eight different inhibitors. Among the compared yeasts, S. cerevisiae strains presented higher tolerance than non-Saccharomyces, 3 of them with the highest tolerance among all. Regarding the non-Saccharomyces yeasts, C. tropicalis JA2 and W. anomalus 740 appeared as the most tolerant, whereas Spathaspora strains appeared very sensitive to the different compounds.

Production of antimicrobial metabolites against pathogenic bacteria and yeasts by Fusarium oxysporum in submerged culture processes.

The antimicrobial activity of the metabolites produced by Fusarium oxysporum PR-33 in submerged culture was evaluated against Gram-positive and Gram-negative bacteria and yeasts. Metabolites were determined by HPLC-DAD-MS/MS. An extract was obtained following the removal of mycelium by centrifugation and lyophilisation of the supernatant. The compounds in this extract demonstrated broad-spectrum antimicrobial action, with rates of inhibition between 60 and 80%, depending on the species and extract tested. The major compounds of the extracts were identified as fusarinolic acid and its isomer [56.9% flask extract (FE)] and 59.2% bioreactor extract (BE), dehydrofusaric acid (35.7% FE and 31.6% BE), and fusaric acid (6.5% FE and 1.1% BE). Fusaric acid has been shown to be responsible for antimicrobial activity. The cytotoxicity of the extracts was evaluated in culture of HEK-293 and SH-SY5Y animal cells and toxicity of these extracts was verified even in the lowest tested concentrations. Therefore, our results indicate that the compounds identified exhibit potential as antimicrobial agents.

Influence of ultrasound on selected microorganisms, chemical and structural changes in fresh tomato juice.

The paper presents the possibility of applying ultrasonic technology for inactivation of mesophilic aerobic microorganisms, lactic acid bacteria, coliform bacteria, and yeast with the maintenance of the chemical and structural properties of tomato juice. The research was conducted on fresh tomato juice obtained from the Apis F1 variety. Pressed juice was exposed to high power ultrasound and frequency 20 kHz with three operational parameters: ultrasound intensity (28 and 40 W cm-2), treatment time (2, 5, and 10 min), and product storage time (1, 4, 7 and 10 days). The temperature of the juice during the sonication ranged from 37 to 52 °C depending on the intensity of ultrasound and time of treatment. Effectiveness of the tested microorganisms eradication in the juice depended on the amplitude and duration of the ultrasound treatment. It was shown that the juice exposed to an ultrasonic field with an intensity of 40 W cm-2 for 10 min was microbiologically pure and free from spoilage microorganism even after 10 storage days. No statistically significant differences in pH were found between the untreated juice and the sonicated samples. The ultrasonic treatment was found to change the content of lycopene in small degree (both an increase and a decrease, depending on the processing time) and to induce a small decrease in the vitamin C content. The study suggests that the ultrasonic treatment can be successfully implemented on an industrial scale for the production of not-from-concentrate (NFC) tomato juice.

Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands.

We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.75 µM for YAP and 0.68 µM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.