Yeast Screen Research Articles

Identification of inhibitors of human ChaC1, a cytoplasmic glutathione degrading enzyme through high throughput screens in yeast.

The cytosolic glutathione-degrading enzyme, ChaC1, is highly up-regulated in several cancers, with the up-regulation correlating to poor prognosis. The ability to inhibit ChaC1 is therefore important in different pathophysiological situations, but is challenging owing to the high substrate Km of the enzyme. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. Synthetic substrate analogs were also not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 µM. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds are the first inhibitor leads against the ChaC enzymes using a robust yeast screen.

Frozen dough steamed products: Deterioration mechanism, processing technology, and improvement strategies.

Fresh dough products lead to instability in product quality, high production costs, and more production time, which seriously affects the industrial production of the food industry. The frozen dough technology mitigates the problems of short shelf-life and easy deterioration of quality during storage and transportation. It has shown a series of advantages in large-scale industrialization, high-quality standardization, and chain operation. However, the further development of frozen dough is restricted by the deterioration of the main components (gluten, starch, and yeast) caused by freezing. This review summarizes the main production process of frozen steamed bread and buns, and the deterioration reasons for the main component of frozen dough. The improvement mechanisms of raw ingredients, processing technology, processing equipment, and additives on frozen dough quality were analyzed from the perspective of improving gluten network integrity and yeast freeze tolerance. From prefermented frozen raw to steamed products without thawing has become the preferred production process to improve production efficiency. Wheat flour mixed with other flour can maintain the gluten network continuity of frozen dough. The freeze tolerance of yeast was improved by treatment with yeast suspension, yeast cell encapsulation, screening hybridization, and genetic engineering. Process optimization and new technology-assisted fermentation and freezing effectively reduce freezing damage. Various additives improve the freeze resistance of the gluten-starch matrix by promoting protein cross-linking and inhibiting water migration. In addition, ice structural proteins and ice nucleating agents have been proven to change the growth morphology and formation temperature of ice crystals. More new technologies and additive synergies need to be further explored.

Rapid screening and identification strategy of lactic acid bacteria and yeasts based on Ramanomes technology and its application in fermented food

There is an urgent need for quick, sensitive, thorough, and low-cost preliminary screening and rapid identification method for probiotic resource development. Here, we constructed a culture-free, accurate and sensitive “separation-enrichment-detection” rapid evaluation and identification method of probiotics in fermented food based on Ramanomes technology, and established a Ramanome reference of lactic acid bacteria and yeasts by AgNPs nanostructure array (AgNPs NSA) chips with uniform “hot spots” and high signal enhancement ability as SERS substrates. Systematic cluster analysis could clearly distinguish among different genera of lactic acid bacteria and yeast, and could indicate the affinity and difference between lactic acid bacteria of the same genus and yeast of the same genus. The recognition accuracy of convolutive neural networks was 100 %, and the recognition sensitivity was less than 10 CFU/mL. We constructed a probiotic isolation and enrichment method by velocity gradient centrifugation and density gradient differential centrifugation, and the average recoveries of lactobacilli and yeasts were greater than 98 % in the practical application, and the accuracy of the verified classification was 100 %. In conclusion, this study has established a preliminary screening strategy of “screening before cultivating” for lactic acid bacteria and yeasts, which breaks through the principle limitation of the current traditional paradigm of “cultivating before screening”. It can greatly improve the preliminary screening rate of probiotics in fermented foods, and provide a good technical support for the mining of probiotic resources from environmental or complex matrix samples.

Screening and characterization of indigenous Saccharomyces cerevisiae and non-Saccharomyces yeasts isolated from Sicilian vineyards

The use of Saccharomyces cerevisiae as starter cultures in winemaking is widespread in all over the world. However, a growing interest in exploring native non-Saccharomyces yeast species as co-starters has emerged, driven by their potential to enhance wine complexity and regional identity. The aim of the present study was to investigate indigenous yeasts from Sicily, to select novel strains with promising technological traits. A comprehensive screening of indigenous S. cerevisiae and non-Saccharomyces yeasts was conducted across seven locations in Sicily, including the unique volcanic zone of Mount Etna. A total of 95 yeast isolates were obtained and genetically characterized by RFLP-PCR and sequencing of 5.8S-ITS rDNA. Isolates were screened for oenological traits and the most promising, belonging to Starmerella bacillaris, Hanseniaspora uvarum, and Metschnikowia pulcherrima species, were used for single and sequential fermentations in synthetic must. Results revealed a remarkable diversity of physiological traits within and among the species, regarding stress tolerance, enzymatic activity and growth at different temperatures. Notably, isolates of H. uvarum and St. bacillaris exhibited great ethanol tolerance, and the latter also strong glycerol production. Furthermore, M. pulcherrima exhibited a wide range of aroma-related enzymatic activities, particularly beta-glucosidase. The remarkable diversity, the phenotypic traits of the indigenous yeasts, represents a promise for the improvement of innovative fermentation strategies and for the development of wine indigenous starters related with the region's terroir.

Dynamic Changes in Yeast Populations and Aroma Composition of ‘Beimei’ (Vitis vinifera × Vitis amurensis) during Spontaneous Fermentation

‘Beimei’ is a hybrid cultivar of Vitis vinifera and wild Vitis amurensis with fungus- and cold-resistant characteristics that is widely grown in many regions of China. However, studies on the yeast diversity and aroma compound composition of this cultivar remain scarce. Here, indigenous yeast diversity, aroma compound composition, and their dynamic changes during the spontaneous fermentation of ‘Beimei’ were investigated and monitored using high-throughput sequencing and gas chromatography-mass spectrometry. The result indicated that non-Saccharomyces yeasts were predominant at the initial stages of ‘Beimei’ spontaneous fermentation and then sharply decreased. Cladosporium herbarum and Hanseniaspora uvarum were identified as the dominant species of filamentous fungi and yeast, respectively, at the beginning and on the first day of fermentation, whereas unclassified_f_Saccharomycetacease was dominant throughout fermentation, starting from the third day until the end of fermentation. The main volatile aroma chemicals were esters and alcohols, which accumulated abundantly at the late stage of fermentation. Taken together, this study represents the first step in exploring untapped yeast species and aroma compounds, which is helpful for the screening of native yeasts from ‘Beimei’ and producing distinctive aroma wines.

Nuclear proteasomes buffer cytoplasmic proteins during autophagy compromise

Autophagy is a conserved pathway where cytoplasmic contents are engulfed by autophagosomes, which then fuse with lysosomes enabling their degradation. Mutations in core autophagy genes cause neurological conditions, and autophagy defects are seen in neurodegenerative diseases such as Parkinson’s disease and Huntington’s disease. Thus, we have sought to understand the cellular pathway perturbations that autophagy-perturbed cells are vulnerable to by seeking negative genetic interactions such as synthetic lethality in autophagy-null human cells using available data from yeast screens. These revealed that loss of proteasome and nuclear pore complex components cause synergistic viability changes akin to synthetic fitness loss in autophagy-null cells. This can be attributed to the cytoplasm-to-nuclear transport of proteins during autophagy deficiency and subsequent degradation of these erstwhile cytoplasmic proteins by nuclear proteasomes. As both autophagy and cytoplasm-to-nuclear transport are defective in Huntington’s disease, such cells are more vulnerable to perturbations of proteostasis due to these synthetic interactions.

Characterization of human aquaporin ion channels in a yeast expression system as a tool for novel ion channel discovery.

Aquaporin (AQP) channels found in all domains of life are transmembrane proteins which mediate passive transport of water, glycerol, signaling molecules, metabolites, and charged solutes. Discovery of new classes of ion-conducting AQP channels has been slow, likely reflecting time- and labor-intensive methods required for traditional electrophysiology. Work here defines a sensitive mass-throughput system for detecting AQP ion channels, identified by rescue of cell growth in the K+-transport-defective yeast strain CY162 following genetic complementation with heterologously expressed cation-permeable channels, using the well characterized human AQP1 channel for proof of concept. Results showed AQP1 conferred transmembrane permeability to cations which rescued survival in CY162 yeast. Comprehensive testing showed that growth response properties fully recapitulated AQP1 pharmacological agonist and antagonist profiles for activation, inhibition, dose-dependence, and structure-function relationships, demonstrating validity of the yeast screening tool for AQP channel identification and drug discovery efforts. This method also provided new information on divalent cation blockers of AQP1, pH sensitivity of antagonists, and ion permeability of human AQP6. Site-directed mutagenesis of AQP1 channel regulatory domains confirmed that yeast growth rescue was mediated by the introduced channels. Optical monitoring with a lithium-sensitive photoswitchable probe in living cells independently demonstrated monovalent cation permeability of AQP1 channels in yeast plasma membrane. Ion channel properties of AQP1 expressed in yeast were consistent with those of AQP1 expressed in Xenopus laevis oocyte and K+-transport defective Escherichia coli. Outcomes here establish a powerful new approach for efficient screening of phylogenetically diverse AQPs for yet untested functions as cation channels.

An automated positive selection screen in yeast provides support for boron-containing compounds as inhibitors of SARS-CoV-2 main protease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to cause severe disease and deaths in many parts of the world, despite massive vaccination efforts. Antiviral drugs to curb an ongoing infection remain a priority. The virus-encoded 3C-like main protease (MPro; nsp5) is seen as a promising target. Here, with a positive selection genetic system engineered in Saccharomyces cerevisiae using cleavage and release of MazF toxin as an indicator, we screened in a robotized setup small molecule libraries comprising ~2,500 compounds for MPro inhibitors. We detected eight compounds as effective against MPro expressed in yeast, five of which are characterized proteasome inhibitors. Molecular docking indicates that most of these bind covalently to the MPro catalytically active cysteine. Compounds were confirmed as MPro inhibitors in an in vitro enzymatic assay. Among those were three previously only predicted in silico; the boron-containing proteasome inhibitors bortezomib, delanzomib, and ixazomib. Importantly, we establish reaction conditions in vitro preserving the MPro-inhibitory activity of the boron-containing drugs. These differ from the standard conditions, which may explain why boron compounds have gone undetected in screens based on enzymatic in vitro assays. Our screening system is robust and can find inhibitors of a specific protease that are biostable, able to penetrate a cell membrane, and are not generally toxic. As a cellular assay, it can detect inhibitors that fail in a screen based on an in vitro enzymatic assay using standardized conditions, and now give support for boron compounds as MPro inhibitors. This method can also be adapted for other viral proteases.IMPORTANCEThe coronavirus disease 2019 (COVID-19) pandemic triggered the realization that we need flexible approaches to find treatments for emerging viral threats. We implemented a genetically engineered platform in yeast to detect inhibitors of the virus's main protease (MPro), a promising target to curb severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Screening molecule libraries, we identified candidate inhibitors and verified them in a biochemical assay. Moreover, the system detected boron-containing molecules as MPro inhibitors. Those were previously predicted computationally but never shown effective in a biochemical assay. Here, we demonstrate that they require a non-standard reaction buffer to function as MPro inhibitors. Hence, our cell-based method detects protease inhibitors missed by other approaches and provides support for the boron-containing molecules. We have thus demonstrated that our platform can screen large numbers of chemicals to find potential inhibitors of a viral protease. Importantly, the platform can be modified to detect protease targets from other emerging viruses.

Research Progress on the Mechanism of Action and Screening of Acid-lowering Yeast in Wine

Research Progress on the Mechanism of Action and Screening of Acid-lowering Yeast in Wine

From yeast screening for suitability as single cell protein to fed-batch cultures.

Fed-batch cultures have rarely been used in single cell protein (SCP) research. This work evaluated multiple yeast species for suitability as SCP cultivated using glucose- and sucrose-based substrate and performed in-depth studies of fed-batch SCP cultivation kinetics for selected yeasts, including determination of specific crude nitrogen-to-protein conversion factors. SCP was cultivated using fully synthetic media in flask batch or bioreactor fed-batch cultures. Crude nitrogen and nucleic acid content were determined using the Dumas method and fluorescence assay kits, respectively. C. utilis compared favorably to other yeasts in flask batch cultures in terms of process yield (0.52 ± 0.01 gxgs-1) and crude nitrogen content (10.0 ± 0.5 and 9.9 ± 0.5%CDW for glucose and sucrose, respectively). This is the first time biomass composition data was reported for SCP cultivated in fed-batch mode. C. utilis crude nitrogen content was consistent across the tested conditions (protein content stabilized around 50%CDW in fed-batch), while that of the benchmark yeast S. cerevisiae was higher in batch cultures and at the beginning of fed-batch relative to the end (protein content decreased over time and stabilized around 43%CDW). Total nucleic acid content of the yeasts was similar (6.8%CDW and 6.3%CDW, for C. utilis and S. cerevisiae, respectively), with crude nitrogen-to-protein conversion factors of 4.97 and 5.80. This study demonstrated the suitability of C. utilis as SCP, notably the robustness of its crude nitrogen content (as an indicator of protein content) across batch and fed-batch conditions, compared to that of the benchmark yeast S. cerevisiae.

Yeast diversity and screening of biocontrol Yarrawia lipolitica against ochratoxin A biosynthesis by Aspergillus Niger in Panxian ham

Yeast diversity and screening of biocontrol <em>Yarrawia lipolitica</em> against ochratoxin A biosynthesis by <em>Aspergillus Niger</em> in Panxian ham

Isolation, Screening and Characterization of Xylose-Fermenting Yeasts Isolated from Saw Dust

Yeasts have been less frequently reported as xylanase producers compared to bacteria and filamentous fungi. Different cellulosic materials including sawdust are produced on a large scale and these can be used for the production of useful enzymes such as xylanases. Xylanases are a group of enzymes that hydrolyze plant fibers made of xylan hemicellulose. Xylose-fermenting yeasts isolated from soil at a wood processing factory were isolated and qualitatively and quantitatively screened for xylanase production using xylose supplemented medium and congo red as indicator. Xylanase enzyme was produced using different xylose concentrations (0.5%, 1.0%, 1.5% and 2.0%). Pichia chambardii isolate, which was later identified as Wickerhamomyces chambardii by molecular techniques, showed the highest xylanase activity of 199.31U mL-1. Maximum xylanase activity (275.83U mL-1) was achieved at 1.5 %w/v xylose. This study showed that yeasts have a high potential for the production of xylanase enzymes.

High-throughput screening of non-conventional yeasts for conversion of organic waste to microbial oils via carboxylate platform

Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse yeasts for microbial oil production by utilizing short-chain fatty acids (SCFAs) that can be produced from organic waste and focuses on identifying strains with the best SCFA utilisation, tolerance and lipid production. A collection of 1434 yeast strains was cultivated with SCFAs as the sole carbon source. Eleven strains emerged as candidates with promising growth rates and high lipid accumulation. Subsequent fermentation experiments in liquid SCFA-rich media, which focused on optimizing lipid accumulation by adjusting the carbon to nitrogen (C/N) ratio, showed an increase in lipid content at a C/N ratio of 200:1, but with a concurrent reduction in biomass. Two strains were characterized by their superior ability to produce lipids compared to the reference strain Yarrowia lipolytica CECT124: Y. lipolytica EXF-17398 and Pichia manshurica EXF-7849. Characterization of these two strains indicated that they exhibit a biotechnologically relevant balance between maximizing lipid yield and maintaining growth at high SCFA concentrations. These results emphasize the potential of using SCFAs as a sustainable feedstock for oleochemical production, offering a dual benefit of waste valorisation and microbial oil production.

Edeine B1 produced by Brevibacillus brevis reduces the virulence of a plant pathogenic fungus by inhibiting mitochondrial respiration.

Plant pathogenic fungi cause serious diseases, which result in the loss of crop yields and reduce the quality of crops worldwide. To counteract the escalating risks of chemical fungicides, interest in biological control agents to manage plant diseases has significantly increased. In this study, we comprehensively screened microbial culture filtrates using a yeast screening system to find microbes exhibiting respiratory inhibition activity. Consequently, we found a soil-borne microbe Brevibacillus brevis HK544 strain exhibiting a respiration inhibitory activity and identified edeine B1 (EB1) from the culture filtrate of HK544 as the active compound of the respiration inhibition activity. Furthermore, against a plant pathogenic fungus Fusarium graminearum, our results showed that EB1 has effects on multiple aspects of respiration with the downregulation of most of the mitochondrial-related genes based on transcriptome analysis, differential EB1-sensitivity from targeted mutagenesis, and the synergistic effects of EB1 with electron transport chain complex inhibitors. With the promising plant disease control efficacy of B. brevis HK544 producing EB1, our results suggest that B. brevis HK544 has potential as a biocontrol agent for Fusarium head blight.IMPORTANCEAs a necrotrophic fungus, Fusarium graminearum is a highly destructive pathogen causing severe diseases in cereal crops and mycotoxin contamination in grains. Although chemical control is considered the primary approach to control plant disease caused by F. graminearum, fungicide-resistant strains have been detected in the field after long-term continuous application of fungicides. Moreover, applying chemical fungicides that trigger mycotoxin biosynthesis is a great concern for many researchers. Biocontrol of Fusarium head blight (FHB) by biological control agents (BCAs) represents an alternative approach and could be used as part of the integrated management of FHB and mycotoxin production. The most extensive studies on bacterial BCAs-fungal communications in agroecosystems have focused on antibiosis. Although many BCAs in agricultural ecology have already been used for fungal disease control, the molecular mechanisms of antibiotics produced by BCAs remain to be elucidated. Here, we found a potential BCA (Brevibacillus brevis HK544) with a strong antifungal activity based on the respiration inhibition activity with its active compound edeine B1 (EB1). Furthermore, our results showed that EB1 secreted by HK544 suppresses the expression of the mitochondria-related genes of F. graminearum, subsequently suppressing fungal development and the virulence of F. graminearum. In addition, EB1 exhibited a synergism with complex I inhibitors such as rotenone and fenazaquin. Our work extends our understanding of how B. brevis HK544 exhibits antifungal activity and suggests that the B. brevis HK544 strain could be a valuable source for developing new crop protectants to control F. graminearum.

Isolation, Morphological Characterization and Screening of Yeast Isolates from Different Fruit Samples of Raichur District, Karnataka, India

Isolation, Morphological Characterization and Screening of Yeast Isolates from Different Fruit Samples of Raichur District, Karnataka, India

Isolation, screening, and characterization of the newly isolated osmotolerant yeast Wickerhamomyces anomalus BKK11-4 for the coproduction of glycerol and arabitol.

This study explored the isolation and screening of an osmotolerant yeast, Wickerhamomyces anomalus BKK11-4, which is proficient in utilizing renewable feedstocks for sugar alcohol production. In batch fermentation with high initial glucose concentrations, W. anomalus BKK11-4 exhibited notable production of glycerol and arabitol. The results of the medium optimization experiments revealed that trace elements, such as H3BO3, CuSO4, FeCl3, MnSO4, KI, H4MoNa2O4, and ZnSO4, did not increase glucose consumption or sugar alcohol production but substantially increased cell biomass. Osmotic stress, which was manipulated by varying initial glucose concentrations, influenced metabolic outcomes. Elevated glucose levels promoted glycerol and arabitol production while decreasing citric acid production. Agitation rates significantly impacted the kinetics, enhancing glucose utilization and metabolite production rates, particularly for glycerol, arabitol, and citric acid. The operational pH dictated the distribution of the end metabolites, with glycerol production slightly reduced at pH 6, while arabitol production remained unaffected. Citric acid production was observed at pH 6 and 7, and acetic acid production was observed at pH 7. Metabolomic analysis using GC/MS identified 29 metabolites, emphasizing the abundance of sugar/sugar alcohols. Heatmaps were generated to depict the variations in metabolite levels under different osmotic stress conditions, highlighting the intricate metabolic dynamics occurring post-glucose uptake, affecting pathways such as the pentose phosphate pathway and glycerolipid metabolism. These insights contribute to the optimization of W. anomalus BKK11-4 as a whole-cell factory for desirable products, demonstrating its potential applicability in sustainable sugar alcohol production from renewable feedstocks.

A strategy for successful dual-species protein expression of genes with non-optimal codon usage destined for bacterial and yeast cell factories.

Recombinant protein expression on an industrial scale traditionally utilizes one of two microbial workhorses: Escherichia coli or Saccharomyces cerevisiae. Additionally, random protein engineering of enzymes and proteins aimed for expression in S. cerevisiae are often mutagenized and pre-screened in E. coli before expression in yeast. This introduces artificial bottlenecks as the bacterial expression vector needs to be substituted for a yeast expression vector via sub-cloning, and the new library re-evaluated before a final screening in yeast. Here, we put forward a protein expression and engineering strategy that involves the use of a dual-host shuttle vector (pYB-Dual) designed with both a strong inducible yeast promoter (pGAL1), and a strong inducible bacterial promoter (pT7-RNAP), which allows for inducible protein expression in both species. Additionally, we demonstrate that by transforming the pYB-Dual vector into the E. coli strain Rosetta 2, which has elevated levels of 7 rare tRNAs, we can achieve high-level protein expression in both yeast and bacteria, even when using a mNeonGreen gene codon optimized for yeast. This dual expression vector is expected to remove bottlenecks during protein engineering of commercially important enzymes destined for high-titer expression in yeast.

Development of new steroid-based hydrazide and (thio)semicarbazone compounds with anticancer properties

Most breast and prostate cancers are caused by abnormal production or action of steroidal hormones. Hormonal drugs based on steroid scaffolds represent a significant class of chemotherapeutics that are routinely used in chemotherapy. In this study, the synthesis of new 17a-homo lactone and 17α-(pyridine-2-ylmethyl) androstane derivatives with hydrazide and semicarbazone motifs is presented. All compounds were screened for their effect on cell viability against a panel of five cancer cell lines and one healthy cell line. Two compounds showed significant cytotoxicity against cancer cells, with low toxicity against healthy cells. The relative binding affinities of compounds for the ligand-binding domains of estrogen receptor α, estrogen receptor β, androgen receptor and glucocorticoid receptor were tested using a fluorescence screen in yeast. Potential for inhibition of aldo-keto reductase 1C3 and 1C4 activity was measured in vitro. Experimental results are analyzed in the context of molecular docking simulations. Our results could help guide design of steroid compounds with improved anticancer properties against androgen- and estrogen-dependent cancers.

Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

The Dmc1 recombinase physically interacts with and promotes the meiotic crossover functions of the Mlh1-Mlh3 endonuclease.

The accurate segregation of homologous chromosomes during the Meiosis I reductional division in most sexually reproducing eukaryotes requires crossing over between homologs. In baker's yeast approximately 80% of meiotic crossovers result from Mlh1-Mlh3 and Exo1 acting to resolve double-Holliday junction intermediates in a biased manner. Little is known about how Mlh1-Mlh3 is recruited to recombination intermediates to perform its role in crossover resolution. We performed a gene dosage screen in baker's yeast to identify novel genetic interactors with Mlh1-Mlh3. Specifically, we looked for genes whose lowered dosage reduced meiotic crossing over using sensitized mlh3 alleles that disrupt the stability of the Mlh1-Mlh3 complex and confer defects in mismatch repair but do not disrupt meiotic crossing over. To our surprise we identified genetic interactions between MLH3 and DMC1, the recombinase responsible for recombination between homologous chromosomes during meiosis. We then showed that Mlh3 physically interacts with Dmc1 in vitro and in vivo. Partial complementation of Mlh3 crossover functions was observed when MLH3 was expressed under the control of the CLB1 promoter (NDT80 regulon), suggesting that Mlh3 function can be provided late in meiotic prophase at some functional cost. A model for how Dmc1 could facilitate Mlh1-Mlh3's role in crossover resolution is presented.