Yeast Protein Research Articles

Metabolic Engineering of Komagataella phaffii for Xylose Utilization from Cellulosic Biomass

Cellulosic biomass hydrolysates are rich in glucose and xylose, but most microorganisms, including Komagataella phaffii, are unable to utilize xylose effectively. To address this limitation, we engineered a K. phaffii strain optimized for xylose metabolism through the xylose oxidoreductase pathway and promoter optimization. A promoter library with varying strengths was used to fine-tune the expression levels of the XYL1, XYL2, and XYL3 genes, resulting in a strain with a strong promoter for XYL2 and weaker promoters for XYL1 and XYL3. This engineered strain exhibited superior growth, achieving 14 g cells/L and a maximal growth rate of 0.4 g cells/L-h in kenaf hydrolysate, outperforming a native strain by 17%. This study is the first to report the introduction of the xylose oxidoreductase pathway into K. phaffii, demonstrating its potential as an industrial platform for producing yeast protein and other products from cellulosic biomass.

Proteome-wide forced interactions reveal a functional map of cell-cycle phospho-regulation in S. cerevisiae

ABSTRACT Dynamic protein phosphorylation and dephosphorylation play an essential role in cell cycle progression. Kinases and phosphatases are generally highly conserved across eukaryotes, underlining their importance for post-translational regulation of substrate proteins. In recent years, advances in phospho-proteomics have shed light on protein phosphorylation dynamics throughout the cell cycle, and ongoing progress in bioinformatics has significantly improved annotation of specific phosphorylation events to a given kinase. However, the functional impact of individual phosphorylation events on cell cycle progression is often unclear. To address this question, we used the Synthetic Physical Interactions (SPI) method, which enables the systematic recruitment of phospho-regulators to most yeast proteins. Using this method, we identified several putative novel targets involved in chromosome segregation and cytokinesis. The SPI method monitors cell growth and, therefore, serves as a tool to determine the impact of protein phosphorylation on cell cycle progression.

ReCRAC: A Stringent Method for Precise Mapping of Protein-RNA Interactions in Yeast.

Intricate interactions between RNA-binding proteins (RBPs) and RNA play pivotal roles in cellular homeostasis, impacting a spectrum of biological processes vital for survival. UV crosslinking methods to study protein-RNA interactions have been instrumental in elucidating their interactions but can be limited by degradation of target proteins during the process, low signal-to-noise ratios, and nonspecific interactions. Addressing these limitations, we describe reCRAC (reverse CRAC), a novel adaptation of the CRAC (crosslinking and analysis of cDNA) technique, optimized for yeast Saccharomyces cerevisiae. Like CRAC, reCRAC applies tandem affinity purification to yield highly enriched protein preparations. However, reCRAC is redesigned to work with unstable proteins. This is achieved by lysing the cells directly into highly denaturing buffer conditions, followed by stringent purification steps. The reCRAC method was successfully applied to the easily degraded yeast protein Pin4, allowing identification of precise binding sites at base-pair resolution with greatly reduced target protein degradation and improved signal-to-noise ratios.

Bimolecular Fluorescence Complementation as a Tool to Study Specific Dynamic Interactions Between Proteins in Fission Yeast.

Bimolecular fluorescence complementation (BiFC) is a technique that enables real-time observation within living cells of the interaction between two proteins forming a complex, determining the location where such interaction occurs within the cell, and even the association and dissociation cycles in response to physiological cues. Here, we describe in detail the use of bimolecular fluorescence complementation to visualize the assembly and disassembly of cohesin over the fission yeast cell cycle.

Subtleties in Clathrin heavy chain binding boxes provide selectivity among adaptor proteins of budding yeast

Clathrin forms a triskelion, or three-legged, network that regulates cellular processes by facilitating cargo internalization and trafficking in eukaryotes. Its N-terminal domain is crucial for interacting with adaptor proteins, which link clathrin to the membrane and engage with specific cargo. The N-terminal domain contains up to four adaptor-binding sites, though their role in preferential occupancy by adaptor proteins remains unclear. In this study, we examine the binding hierarchy of adaptors for clathrin, using integrative biophysical and structural approaches, along with in vivo functional experiments. We find that yeast epsin Ent5 has the highest affinity for clathrin, highlighting its key role in cellular trafficking. Epsins Ent1 and Ent2, crucial for endocytosis but thought to have redundant functions, show distinct binding patterns. Ent1 exhibits stronger interactions with clathrin than Ent2, suggesting a functional divergence toward actin binding. These results offer molecular insights into adaptor protein selectivity, suggesting they competitively bind clathrin while also targeting three different clathrin sites.

A Semi-supervised Protein Complex Identification Algorithm Based on Sparseness Constraint

Abstract Recognising protein complexes in protein interaction networks is crucial, but poses a major challenge due to the frequency of noisy interactions. These networks typically involve numerous protein complexes, with each protein generally only participating in a few complexes. Current recognition models often ignore this aspect. To address this problem, we present a semi-supervised protein complex identification algorithm that extends non-negative matrix factorization (NMF) with sparsity constraints. In contrast to conventional approaches that apply a global sparsity constraint to the entire matrix, our method imposes individual sparsity constraints on protein membership indicator vectors. This targeted strategy controls the algorithm more effectively. Our experimental results with yeast and human protein interaction networks show that our algorithm achieves higher accuracy in identifying protein complexes than leading contemporary methods.

Substituting imported soybean meal with locally produced novel yeast protein in concentrates for Norwegian Red dairy cows: implications for rumen microbiota and fatty acid composition.

This research paper addresses the hypothesis that substituting soybean meal with locally produced yeast protein from Cyberlindnera jadinii in barley-based concentrates for Norwegian Red (NR) dairy cows does not have adverse effects on milk fatty acid (FA) composition, rumen microbiota and sensory quality of milk. As soybeans also represent valuable protein sources for human consumption, alternative protein sources need to be investigated for animal feed. A total of 48 NR dairy cows were allocated into three feeding treatments, with the same basal diet of grass silage, but different concentrates. The concentrates were all based on barley, but 7% of the barley in the barley-concentrate (BAR; negative control) was replaced by either soybean meal (SBM; conventional control) or yeast microbial protein (YEA). The experiment lasted for a total of 10 weeks, including 2 weeks of adaptation with the soybean meal concentrate. Analysis of the feed revealed some differences in the FA composition of the YEA concentrate compared to the SBM and BAR concentrates. In milk, only two FAs (C17:1n-8cis9 and an unidentified isomer of C18:3) were significantly different between the YEA- and SBM-group, while six FAs differed between the BAR- and SBM-group. However, the amount of these FAs was low compared to the entire FA profile (<0.7 g/100 g). The experimental diets did not affect rumen microbiota nor the milk sensory quality. This study shows that C. jadinii can replace soybean meal as a protein source in concentrates (7% inclusion) for NR dairy cows fed a diet composed of grass silage and concentrates without any effects on rumen microbiota, and without compromising the FA composition or sensory quality of milk.

Regulation of tapioca starch 3D printability by yeast protein: Rheological, textural evaluation, and machine learning prediction

This article investigated the effects of yeast protein (YP) on gel rheology, texture, and 3D printability of tapioca starch and the feasibility of Principal component analysis (PCA) and support vector machine (SVM) algorithms for classification and prediction of printability. The results indicated that increasing YP content enhanced the viscosity, storage and loss moduli, and hardness, thereby improving extrudability and supportability of 3D printing. The addition of 15% YP exhibited the best 3D printing performance, but excessively high YP addition hindered ink extrusion. PCA analysis based on rheological and texture indices categorized the ink's 3D printing performance into four classes: poor support and low printing accuracy; good support but low printing accuracy; good support and high printing accuracy; and non-smooth extrusion. Furthermore, SVM algorithm used texture data to predict printability classification, with the highest prediction accuracy (91.67%) achieved at polynomial kernel among four different kernel functions. These results confirm that YP can serve as a potential ink for 3D printing and underscore SVM's efficacy in predicting ink's 3D printing performance.

Yeast protein‐derived γ‐glutamyl peptides prepared by transpeptidation reaction exhibit a pronounced taste‐enhancing effect

AbstractA high‐salt diet can induce hypertension, so salt reduction can prevent hypertension. γ‐Glutamyl peptides (GGPs) have obvious taste‐enhancing effects, while their contents in natural foods are relatively low. Yeast protein rich in Glu/Gln is a good precursor for the preparation of GGPs. In this study, yeast protein‐derived GGPs were prepared through hydrolysis and transpeptidation reactions, followed by sensory evaluation and E‐tongue analysis. Peptide sequences were identified by LC−MS/MS and screened for molecular docking. The optimal reaction conditions were hydrolysis for 4 h, enzyme concentration of 16 U/g, and transpeptidation for 4 h. GGPs could increase salt and umami intensity by 60.78% and 40.93% based on sensory evaluation, 22.52%, and 16.40% according to E‐tongue analysis. Fifteen γ‐glutamyl peptides with different peptide lengths were selected for molecular docking. Molecular docking confirmed their binding to calcium‐sensing receptors (CaSr) through hydrogen bonds and hydrophobic interaction, while interaction between CaSR receptor and γ‐glutamyl di‐, tri‐, and oligo‐peptides varied in binding energy. The stimulation received by CaSR lasted a longer time and varied in intensity. It was further proved that the flavor of mixed peptides has a layered sense and can give people a rich taste experience. Overall, yeast protein‐derived GGPs can enhance salt and umami taste, which can reduce salt usage without compromising taste.

Evaluation of the nutritional quality of yeast protein in comparison to animal and plant proteins using growing rats and INFOGEST model

Yeast, identified as a microorganism, boasts a considerable protein content, positioning yeast protein as a highly promising alternative in the quest for sustainable protein sources. The primary aim of this study is to evaluate the protein quality of yeast protein and compare it with animal proteins (whey concentrate/isolate proteins) and plant proteins (soy, wheat, pea proteins). Notably, yeast protein exhibits the highest ratio of indispensable/dispensable amino acids (IAAs/DAAs, 0.91). However, in both in vivo and in vitro digestion experiments, yeast protein demonstrated lower true protein digestibility (TPD) and true ileal digestibility (TID) compared to other proteins. Despite this, the yeast protein's amino acid score (AAS, 1.37 for >3 years), protein digestibility-corrected amino acid score (PDCAAS, 100 % for >3 years), and digestibility-corrected amino acid score (DIAAS, 82.42 % for >3 years) of yeast protein surpassed those of plant proteins, yet remained lower than animal proteins primarily due to its lower digestibility.

Overexpression of CBK1 or deletion of SSD1 confers fludioxonil resistance in yeast by suppressing Hog1 activation

Group III hybrid histidine kinases (HHK3) are known molecular targets of the widely used fungicidal agent fludioxonil which indirectly converts these kinases to a phosphatase form that causes constitutive activation of Hog1 MAPK. To better understand the fungicidal effect of fludioxonil we have screened S. cerevisiae haploid deletion collection for fludioxonil resistant mutant and identified Ssd1 as a critical factor for this. Deletion of SSD1 not only promoted resistance to fludioxonil but also abrogated Hog1 activation and other cellular damages caused by fludioxonil. Our results showed that fludioxonil perturbed the localization of Cbk1 kinase, an essential protein in yeast, at the bud neck triggering the accumulation of Ssd1 in P-bodies. As a result, localized synthesis of Ssd1 bound mRNA encoding cell wall proteins at the polarized growth site was impaired which created a sustained cell wall stress causing constitutive activation of Hog1. Our data, for the first time, clearly indicated the role of Cbk1 upstream of Hog1 and provided a novel paradigm in the mechanism of action of fludioxonil.

Yeast Proteins: Proteomics, Extraction, Modification, Functional Characterization, and Structure: A Review.

Proteins are essential for human tissues and organs, and they require adequate intake for normal physiological functions. With a growing global population, protein demand rises annually. Traditional animal and plant protein sources rely heavily on land and water, making it difficult to meet the increasing demand. The high protein content of yeast and the complete range of amino acids in yeast proteins make it a high-quality source of supplemental protein. Screening of high-protein yeast strains using proteomics is essential to increase the value of yeast protein resources and to promote the yeast protein industry. However, current yeast extraction methods are mainly alkaline solubilization and acid precipitation; therefore, it is necessary to develop more efficient and environmentally friendly techniques. In addition, the functional properties of yeast proteins limit their application in the food industry. To improve these properties, methods must be selected to modify the secondary and tertiary structures of yeast proteins. This paper explores how proteomic analysis can be used to identify nutrient-rich yeast strains, compares the process of preparing yeast proteins, and investigates how modification methods affect the function and structure of yeast proteins. It provides a theoretical basis for solving the problem of inadequate protein intake in China and explores future prospects.

Identification of Novel Umami Peptides from Yeast Protein through Enzymatic, Sensory, and In Silico Approaches.

This study aimed to rapidly develop novel umami peptides using yeast protein as an alternative protein source. Yeast protein hydrolysates exhibiting pronounced umami intensity were produced using flavorzyme under optimum conditions determined via a sensory-guided response surface methodology. Six out of 2138 peptides predicted to possess umami taste by composite machine learning and assessed as nontoxic, nonallergenic, water-soluble, and stable using integrated bioinformatics were screened as potential umami peptides. Sensory evaluation results revealed these peptides exhibited multiple taste attributes (detection threshold: 0.37 ± 0.10-1.1 ± 0.30 mmol/L), including umami. In light of the molecular docking outcomes, it is inferred that hydrogen bond, hydrophobic, and electrostatic interactions enhanced the theoretically stable binding of peptides to T1R1/T1R3, with their contributions gradually diminishing. Hydrophilic amino acids within T1R1/T1R3, especially Ser, may play a particularly pivotal role in binding with umami peptides. Future research will involve establishing heterologous cell models expressing T1R1 and T1R3 to delve into the cellular physiology of umami peptides. Peptide sequences (FADL, LPDP, and LDIGGDF) also had synergistic saltiness-enhancing effects; to overcome the limitation of not investigating the saltiness enhancement mechanism, comprehensive experiments at the molecular and cellular levels will also be conducted. This study offers a rapid umami peptide development framework and lays the groundwork for exploring yeast protein taste compounds.

Protein Arginine Methylation of the Translation Initiation Factor eIF1A Increases Usage of a Near-cognate Start Codon.

Protein arginine methylation has emerged as a key post-translational modification responsible for many facets of eukaryotic gene expression. To better understand the extent of this modification in cellular pathways, we carried out bioorthogonal methylation profiling in Saccharomyces cerevisiae to comprehensively identify the in vivo substrates of the major yeast protein arginine methyltransferase Hmt1. Gene ontology analysis of candidate substrates revealed an enrichment of proteins involved in the process of translation. We verified one such factor, eIF1A, by in vitro methylation. Three sites on eIF1A were found to be responsible for its methylation: R13, R14, and R62, with varied capacity by which each site contributed to the overall methylation capacity in vitro. To determine the role of methylation in eIF1A function, we used a battery of arginine-to-alanine substitution mutants to evaluate translation fidelity in these mutants. Our data show that substitution mutants at R13 and R14 in the N-terminal tail improved the fidelity of start codon recognition in an initiation fidelity assay. Overall, our data suggest that Hmt1-mediated methylation of eIF1A fine-tunes the fidelity of start codon recognition for proper translation initiation.

Assessment of the potential risks in SD rats gavaged with genetically modified yeast containing the cp4-epsps gene.

Despite the absence of definitive evidence indicating that the cp4-epsps gene and its resultant recombinant proteins have significant harmful effects on either human or animal health, the safety assessment of genetically modified (GM) crops expressing the CP4-EPSPS proteins has been controversial. This study endeavor was aimed at evaluating the potential risks posed by the CP4-EPSPS protein in transgenic crops, thereby contributing to the advancement of risk assessment methodologies in the context of genetically engineered crops. To ascertain the appropriate daily dosages for oral gavage administration, the expression levels of the CP4-EPSPS protein in a recombinant yeast were quantified. Subsequently, physiological and biochemical analysis, metabolomics, and metagenomic analysis were conducted based on a 90-day Sprague-Dawley (SD) rats feeding experiment, respectively, thereby enhancing the depth and precision of our risk assessment framework. The results from the physiological and biochemical analysis, organ pathological, blood metabolism, gut microbiota, and correlation analysis of metabolites and gut microbiota revealed several biomarkers for further risk assessment. These biomarkers include clinical biochemical indexes such as total bilirubin (TBIL), direct bilirubin (DBIL), creatine kinase (CK), and lactate dehydrogenase (LDH); metabolites like Methionine, 2-Oxovaleric acid, and LysoPC (16:0); and gut microbiota including Blautia wexlerae, Holdemanella biformis, Dorea sp. CAG 317, Coriobacteriaceae and Erysipelotrichaceae. In conclusion, the risk can be significantly reduced by directly consuming inactivated recombinant CP4-EPSPS. Therefore, in everyday life, the risk associated with consuming GM foods containing recombinant CP4-EPSPS is substantially reduced after heat treatment.

A mechanistic investigation into combined influences of NaCl and extrusion temperature on fibrous structures of high-moisture textured yeast protein

NaCl and extrusion temperature have an important influence on the qualities of high-moisture textured proteins, but the influence mechanism is still unclear. Therefore, this study prepared high-moisture textured yeast protein (HMTYP) with different NaCl contents (0%–4%) under different extrusion temperatures (170 °C, 180 °C) and characterized their physicochemical properties. The results showed that the HMTYP containing 1% and 2% NaCl prepared at 180 °C contained a strong fibrous structure. The possible mechanism was as follows: YP could not be sufficiently melted at 170 °C after adding NaCl, causing a decrease in the structural strength; however, at 180 °C, YP still reached a fully molten state even though 1%–2% NaCl was added. After YP sufficiently melted, NaCl enhanced the cross-linking and aggregation of proteins during cooling, which improved the textural properties of HMTYP. Accordingly, NaCl and extrusion temperature could combine to adjust the fibrous structure and texture of HMTYP.

A biallelic variant of the RNA exosome gene, EXOSC4, associated with neurodevelopmental defects impairs RNA exosome function and translation

The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Pathogenic variants in EXOSC genes, which encode structural subunits of this complex, are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene that causes a collection of clinical features in two affected siblings. This missense variant (NM_019037.3: exon3:c.560T>C) changes a leucine residue within a conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals show prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified by exome sequencing with Sanger sequencing to confirm segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show growth defects. Steady-state levels of both Rrp41-L187P and EXOSC4-L187P are decreased compared to controls, and EXOSC4-L187P shows decreased copurification with other RNA exosome subunits. RNA exosome target transcripts accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a decrease in actively translating ribosomes in rrp41-L187P cells as compared to control cells with the incorporation of 7S pre-rRNA into polysomes. This work adds EXOSC4 to the structural subunits of the RNA exosome that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse phenotypes caused by EXOSC pathogenic variants.

Carp growth, body composition, lipidomics, and flesh quality in response to replacing dietary fish meal with a complex protein source

Alternatives to fish meal (FM) in fish feed are urgently required in aquaculture. We therefore examined the effect of replacing FM with a complex protein source (CPS) comprising soybean meal, peanut meal, corn protein meal, feather meal, and yeast protein, on carp (Cyprinus carpio) growth, body composition, lipidomics, and flesh quality. Lipidomics analysis was conducted to reveal the impact of dietary changes on fish lipid composition and metabolism, which is closely related to flesh quality and nutritional value. Over an eight-week growth trial, fish meal was replaced with CPS at 0 % (FM only), 20 %, 40 %, 60 %, 80 %, or 100 %. Weight gain and other growth parameters did not differ significantly among the treatments. Lipid deposition (LD) was positively correlated, and protein deposition negatively correlated, with CPS levels. LD and fillet ether extract were significantly elevated at 100 % CPS, whereas superoxidase dismutase expression, claudin-1 expression, and lipase activity were significantly reduced. The n-3 PUFA level and n-3/n-6 PUFA ratio were significantly lower following CPS feeding, reducing flesh quality. These findings indicate that CPS successfully replaced 60–80 % of dietary FM in carp diets, while the 100 % replacement group provided insights into the comprehensive effects of total substitution. Future studies will further optimize formulations based on these results to resemble practical production more closely. CPS with nutrient supplementation may therefore present a viable and safe partial replacement for dietary FM.

Advanced Protocol for Molecular Characterization of Viral Genome in Fission Yeast (Schizosaccharomyces pombe).

Fission yeast, a single-cell eukaryotic organism, shares many fundamental cellular processes with higher eukaryotes, including gene transcription and regulation, cell cycle regulation, vesicular transport and membrane trafficking, and cell death resulting from the cellular stress response. As a result, fission yeast has proven to be a versatile model organism for studying human physiology and diseases such as cell cycle dysregulation and cancer, as well as autophagy and neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's diseases. Given that viruses are obligate intracellular parasites that rely on host cellular machinery to replicate and produce, fission yeast could serve as a surrogate to identify viral proteins that affect host cellular processes. This approach could facilitate the study of virus-host interactions and help identify potential viral targets for antiviral therapy. Using fission yeast for functional characterization of viral genomes offers several advantages, including a well-characterized and haploid genome, robustness, cost-effectiveness, ease of maintenance, and rapid doubling time. Therefore, fission yeast emerges as a valuable surrogate system for rapid and comprehensive functional characterization of viral proteins, aiding in the identification of therapeutic antiviral targets or viral proteins that impact highly conserved host cellular functions with significant virologic implications. Importantly, this approach has a proven track record of success in studying various human and plant viruses. In this protocol, we present a streamlined and scalable molecular cloning strategy tailored for genome-wide and comprehensive functional characterization of viral proteins in fission yeast.

Benchmarking and Automating the Biotinylation Proteomics Workflow.

Protein biotinylation has been widely used in biotechnology with various labeling and enrichment strategies. However, different enrichment strategies have not been systematically evaluated due to the lack of a benchmarking model for fair comparison. Most biotinylation proteomics workflows suffer from lengthy experimental steps, non-specific bindings, limited throughput, and experimental variability. To address these challenges, we designed a two-proteome model, where biotinylated yeast proteins were spiked in unlabeled human proteins, allowing us to distinguish true enrichment from non-specific bindings. Using this benchmarking model, we compared common biotinylation proteomics methods and provided practical selection guidelines. We significantly optimized and shortened sample preparation from 3 days to 9 hours, enabling fully-automated 96-well plate sample processing. Next, we applied this optimized and automated workflow for proximity labeling to investigate the intricate interplay between mitochondria and lysosomes in living cells under both healthy state and mitochondrial damage. Our results suggested a time-dependent proteome remodeling and dynamic translocation within mitochondria and between mitochondria and lysosomes upon mitochondrial damage. This newly established benchmarking model and the fully-automated 9-hour workflow can be readily applied to the broad fields of protein biotinylation to study protein interaction and organelle dynamics.