Transcriptional Downregulation of Methanol Metabolism Key Genes During Yeast Death in Engineered Pichia pastoris.

Pichia pastoris possesses the unique ability to utilize methanol as its sole carbon source, which makes it a proper host for producing various high-value-added products via metabolic engineering. Nevertheless, cell death has been observed during the fermentation of modified P. pastoris, with limited literature elucidating the underlying causes and mechanisms. Understanding the death mechanisms during methanol-based fermentation is crucial for optimizing fermentation strategies, enhancing the accumulation of target products, and reducing production costs. Here, we first sought to eliminate the potential causes of cell death during fermentation, such as inadequate inorganic salts and toxic by-product accumulation. The elimination of these potential causes was achieved efficiently utilizing the high-throughput fermentation equipment. Subsequently, we established a correlation between yeast cell death and the duration of the methanol metabolism period by monitoring the growth of the yeast at different fermentation stages. A critical revelation from this work came from analyzing the yeast's transcriptomic data at various stages of methanol metabolism. It was observed that a significant characteristic of yeast cell death during fermentation was the marked down-regulation of transcript levels of key enzymes involved in the methanol assimilation pathway and genes related to their biosynthesis process. The findings of this work are crucial for better understanding the causes and mechanisms of cell death for engineered P. pastoris during methanol-utilized fermentation.

Isolation, identification, and technological characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid.

Pyracantha fortuneana (P. fortuneana), as a medicinal and edible plant resource, is rich in nutrients. In order to screen the high quality yeast used in Firebone fruit wine, 12 strains of yeast were isolated and purified from P. fortuneana fermentation broth by traditional pure culture method. They were identified by molecular biology as Pichia kudriavzevii (P. kudriavzevii) and Saccharomyces cerevisiae (S. cerevisiae), respectively. Strain HJ-2 could grow normally at 30℃, alcohol content 15%, SO2 mass concentration 360 mg/L, pH 3.2, sucrose mass concentration 400 g/L and glucose mass concentration 250 g/L. Strain HJ-6 could grow normally at 30℃, alcohol content 3%, SO2 concentration 360 mg/L, pH 3.2, sucrose concentration 250 g/L, glucose concentration 300 g/L. Based on the technological characteristics of fruit wine, S. cerevisiae HJ-2 has the potential of brewing P. fortuneana fruit wine. P. kudriavzevii HJ-6 has a low tolerance to ethanol and is suitable for the production of fermented beverages such as low-alcohol wine or beer.

Efficient expression and purification of rat CRP in Pichia pastoris.

C-reactive protein (CRP) plays a crucial role in the diagnosis and monitoring of the non-specific acute phase response in humans. In contrast, rat CRP (rCRP) is an atypical acute-phase protein that possesses unique features, such as a possible incapacity to trigger the complement system and markedly elevated baseline plasma concentrations. To facilitate in vitro studies on these unique characteristics, obtaining high-quality pure rCRP is essential. Here we explored various strategies for rCRP purification, including direct isolation from rat plasma and recombinant expression in both prokaryotic and eukaryotic systems. Our study optimized the recombinant expression system to enhance the secretion and purification efficiency of rCRP. Compared to traditional purification methods, we present a streamlined and effective approach for the expression and purification of rCRP in the Pichia pastoris system. This refined methodology offers significant improvements in the efficiency and effectiveness of rCRP purification, thereby facilitating further structural and functional studies on rCRP.

Brazilian mangrove sediments as a source of biosurfactant-producing yeast Pichia pseudolambica for bioremediation.

This work highlights the biosurfactant production potential of yeasts from mangroves in northeastern Brazil. The biosurfactants were evaluated by their emulsifying capacity (EI24), with 6 isolates showing values between 50% and 62%. Surfactant properties from crude extract were measured using drop collapse, oil displacement, Parafilm® M, surface tension and critical micellar concentration tests. The effects of temperature, salinity, pH, and the ability to emulsify different hydrocarbons were analyzed, showing a promising potential of the yeast species investigated to tolerance to high temperatures and acidic pH, in addition to emulsifying different sources of hydrocarbons with environmental impact. It is important to note that the Pichia pseudolambica isolates showed a remarkable ability to reduce the surface tension of water, from 70.82 mN/m to 36.47 mN/m. In addition, the critical micellar concentration (CMC) values ranged from 7 to 16 mg/mL, highlighting the promising surfactant activity of these isolates for future applications. It was identified that the biosurfactant adhered to the yeast cell wall, and FTIR and 1H NMR spectroscopy analysis was carried out on the yeast biomass and its post-sonication supernatant. The results indicate the presence of characteristic functional groups and peaks found in biosurfactants of a glycolipid nature. Taking together the results reveals the promising potential of biosurfactant biosynthesis of P. pseudolambica yeast, a trait not reported in the literature so far for this species. P. pseudolambica presents a relevant metabolic potential for alternative substrate use and resilience to adverse conditions that could enable it to produce biosurfactants for the biotechnological remediation of areas contaminated by oil derivatives. The metabolic properties herein investigated, together with their presence in Brazilian mangroves, make P. pseudolambica an emerging candidate for developing industrial processes and sustainable strategies for the recovery of ecosystems impacted by oil spills, being positioned as a sustainable alternative to conventional surfactants.

Expression in Pichia pastoris of Thermostable Endo-1,4-ß-xylanase from the Actinobacterium Nocardiopsis halotolerans: Properties and Use for Saccharification of Xylan-Containing Products.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-ß-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-ß-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-ß-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

Physiological responses contributing to multiple stress tolerance in Pichia kudriavzevii with potential enhancement for ethanol fermentation.

Economically feasible ethanol production requires efficient hydrolysis of lignocellulosic biomass and high-temperature processing to enable simultaneous saccharification and fermentation. During the lignocellulolysic hydrolysate, the yeast must encounter with a multiple of inhibitors such as heat and furfural. To solve this problem, a potential fermentative yeast strain that tolerated simultaneous multistress and enhance ethanol concentration was investigated. Twenty yeast isolates were classified into two major yeast species, namely Pichia kudriavzevii (twelve isolates) and Candida tropicalis (eight isolates). All P. kudriavzevii isolates were able to grow at high temperature (45 °C) and exhibited stress tolerance toward furfural. Among P. kudriavzevii isolates, NUCG-S3 presented the highest specific growth rate under each stress condition of heat and furfural, and multistress. Morphological changes in P. kudriavzevii isolates (NUCG-S2, NUCG-S3, NUKL-P1, NUKL-P3, and NUOR-J1) showed alteration in mean cell length and width compared to the non-stress condition. Ethanol production by glucose was also determined. The yeast strain, NUCG-S3, gave the highest ethanol concentrations at 99.46 ± 0.82, 62.23 ± 0.96, and 65.80 ± 0.62 g/l (P < 0.05) under temperature of 30 °C, 40 °C, and 42 °C, respectively. The tolerant isolated yeast NUCG-S3 achieved ethanol production of 53.58 ± 3.36 and 48.06 ± 3.31 g/l (P < 0.05) in the presence of 15 mM furfural and multistress (42 °C with 15 mM furfural), respectively. Based on the results of the present study, the novel thermos and furfural-tolerant yeast strain P. kudriavzevii NUCG-S3 showed promise as a highly proficient yeast for high-temperature ethanol fermentation.

Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris.

Protective vaccines are crucial for preventing and controlling coronavirus disease 2019 (COVID-19). Updated vaccines are needed to confront the continuously evolving and circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These vaccines should be safe, effective, amenable to easily scalable production, and affordable. Previously, we developed receptor binding domain (RBD) dimer-based protein subunit vaccines (ZF2001 and updated vaccines) in mammalian cells. In this study, we explored a strategy for producing RBD-dimer immunogens in Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 gene (encoding histidine dehydrogenase) to increase histidine synthesis for better growth. In addition, RBD-dimer protein was truncated to remove the R328/F329 cleavage sites in P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris expression system. We further resolved the cryo-EM structure of prototype-Beta RBD-dimer complexed with the neutralizing antibody CB6 to reveal the completely exposed immune epitopes of the RBDs. In a murine model, we demonstrated that the yeast-produced RBD-dimer induces robust and protective antibody responses, which is suitable for boosting immunization. This study developed the yeast system for producing SARS-CoV-2 RBD-dimer immunogens, providing a promising platform and pipeline for the future continuous updating and production of SARS-CoV-2 vaccines.

D-Limonene Inhibits Pichia kluyveri Y-11519 in Sichuan Pickles by Disrupting Metabolism.

The Pichia kluyveri, a proliferation commonly found in Sichuan pickles (SCPs), can accelerate the growth and reproduction of spoilage bacteria, causing off-odor development and decay. Although D-limonene, a common natural preservative, effectively restricts P. kluyveri, its inhibitory mechanism remains unclear. This study aimed to elucidate this molecular mechanism by investigating the impact on basic P. kluyveri metabolism. The findings revealed that D-limonene inhibited P. kluyveri growth and disrupted the transcription of the genes responsible for encoding the enzymes involved in cell wall and membrane synthesis, oxidative phosphorylation, glycolysis, and the tricarboxylic acid (TCA) cycle pathway. The results indicated that these events disrupted crucial metabolism such as cell wall and membrane integrity, adenosine triphosphate (ATP) synthesis, and reactive oxygen species (ROS) balance. These insights provided a comprehensive understanding of the inhibitory effect of D-limonene on the growth and reproduction of P. kluyveri while highlighting its potential application in the SCP industry.

Biosynthesis of a Functional Fragment of Human Collagen II in Pichia pastoris.

Collagen II (COL2) is the major component of cartilage tissue and is widely applied in pharmaceuticals, food, and cosmetics. In this study, COL fragments were extracted from human COL2 for secretory expression in Pichia pastoris. Three variants were successfully secreted by shake flask cultivation with a yield of 73.3-100.7 mg/L. The three COL2 variants were shown to self-assemble into triple-helix at 4 °C and capable of forming higher order assembly of nanofiber and hydrogel. The bioactivities of the COL2 variants were validated, showing that sample 205 exhibited the best performance for inducing fibroblast differentiation and cell migration. Meanwhile, sample 205 and 209 exhibited higher capacity for inducing in vitro blood clotting than commercial mouse COL1. To overexpress sample 205, the expression cassettes were constructed with different promoters and signal peptides, and the fermentation condition was optimized, obtaining a yield of 172 mg/L for sample 205. Fed-batch fermentation was carried out using a 5 L bioreactor, and the secretory protease Pep4 was knocked out to avoid sample degradation, finally obtaining a yield of 3.04 g/L. Here, a bioactive COL2 fragment was successfully identified and can be overexpressed in P. pastoris; the variant may become a potential biomaterial for skin care.

Promoters in Pichia pastoris: A Toolbox for Fine-Tuned Gene Expression.

This chapter reviews the different promoters used to control gene expression in the yeast Pichia pastoris, mainly for recombinant protein production. It covers natural inducible, derepressed, and constitutive promoters, as well as engineered synthetic/hybrid promoters, orthologous promoters from related yeasts, and emerging bidirectional promoters. Key examples, characteristics, and regulatory mechanisms are discussed for each promoter class. Recent efforts in promoter engineering through rational design, mutagenesis, and computational approaches are also highlighted. Looking ahead, we anticipate further developments that will enhance promoter design for Pichia pastoris. Overall, this comprehensive overview underscores the importance of promoter choice and engineering for fully harnessing Pichia pastoris biotechnological potential.

Efficient Secretory Expression and Purification on Three Insoluble Amidohydrolases for Ochratoxin A Hydrolysis by Pichia pastoris.

As a highly toxic mycotoxin, ochratoxin A (OTA) is widely contaminating agricultural products and has various toxicological effects. Bioenzymes for OTA degradation have shown promising potential for detoxification. Other than the efficient amidohydrolase ADH3 previously, two novel amidohydrolases ADH1 and AMD3 were obtained in this study. During Escherichia coli expression, the expressed protein solubility was very low and will limit future industrial application. Here, high copy number integrations were screened, and the amidohydrolases were efficiently secretory expressed by Pichia pastoris GS115. The protein yields from 1.0 L of fermentation supernatant were 53.5 mg for ADH1, 89.15 mg for ADH3, and 79.5 mg for AMD3. The catalytic efficiency (Kcat/Km) of secretory proteins was 124.95 s-1 mM-1 for ADH3, 123.21 s-1 mM-1 for ADH1, and 371.99 s-1 mM-1 for AMD3. In comparison to E. coli expression, the active protein yields substantially increased 15.78-51.53 times. Meanwhile, two novel amidohydrolases (ADH1 and AMD3) showed much higher activity than ADH3 that produced by secretory expression.

Novel transcriptional regulation of the GAP promoter in Pichia pastoris towards high expression of heterologous proteins.

BACKGROUND: Pichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries. RESULTS: To improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris. CONCLUSION: These findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.

Pichia kudriavzevii as feed additive in Nile tilapia (Oreochromis niloticus) diet.

Yeasts are unicellular eukaryotic microorganisms extensively employed in various applications, notably as an alternative source of protein in feeds, owing to their nutritional benefits. Despite their potential, marine and mangrove yeast species used in the aquaculture industry have received little attention in the Philippines. Pichia kudriavzevii (A2B R1 ISO 3), sourced from bark samples, was selected and mass-produced due to its high protein content and amino acid profile. The dried biomass of P. kudriavzevii was incorporated into the diets of Nile tilapia (Oreochromis niloticus) juveniles at varying inclusion levels (0, 1, 2, and 4 g/kg diet) and its effect on their growth performance, body composition, and liver and intestinal morphology was assessed after 40 days of feeding. The groups that received P. kudriavzevii at a concentration of 2 g/kg diet exhibited higher final body weight, percent weight gain, and specific growth rate in comparison to the other treatment groups. Whole body proximate composition did not vary among the dietary groups. Intestinal and liver histopathology also indicated no abnormalities. These findings suggest the potential of ascomycetous P. kudriavzevii as a beneficial feed additive in Nile tilapia diets, warranting further investigation into its long-term effects and broader applications in fish culture.

Komagataella phaffii (Pichia pastoris) as a Powerful Yeast Expression System for Biologics Production.

Komagataella phaffii (K. phaffii) (Pichia pastoris), also called biotech yeast, is a yeast species with many applications in the biotechnology and pharmaceutical industries. This methylotrophic yeast has garnered significant interest as a platform for the production of recombinant proteins. Numerous benefits include effective secretory expression that facilitates the easy purification of heterologous proteins, high cell density with rapid growth, post-translational changes, and stable gene expression with integration into the genome. In the last thirty years, K. phaffii has also been refined as an adaptable cell factory that can produce hundreds of biomolecules in a laboratory setting and on an industrial scale. Indeed, over 5000 recombinant proteins have been generated so far using the K. phaffii expression method, which makes up 30% of the total cell protein or 80% of the total released protein. K. phaffii has been used to manufacture more than 70 commercial products in addition to over 300 industrial processes that have been granted licenses. Among these are useful enzymes for industrial biotechnology, including xylanase, mannanase, lipase, and phytase. The others are biopharmaceuticals, which include human serum albumin, insulin, hepatitis B surface antigen, and epidermal growth factor. Compared to other expression systems, this yeast is also considered a special host for synthesizing subunit vaccines, which have recently been supplanted by alternative vaccination types, such as inactivated/killed and live attenuated vaccines. Moreover, efficient production of recombinant proteins is achieved through multi-level optimization methods, such as codon bias, gene dosage, promoters, signal peptides, and environmental factors. Therefore, although K. phaffii expression systems are efficient and simple with clearly established process procedures, it is still necessary to determine the ideal conditions since these vary depending on the target protein to ensure the highest recombinant protein generation. This review addresses the K. phaffii expression system, its importance in industrial and biopharmaceutical protein production, and some bioprocessing and genetic modification strategies for efficient protein production. K. phaffii will eventually continue contributing as a potent expression system in research areas and industrial applications.

Expression and characterization of scFv-6009FV in Pichia pastoris with improved ability to neutralize the neurotoxin Cn2 from Centruroides noxius.

Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % ß-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.

Comprehensive reutilization of Glycyrrhiza uralensis residue by extrusion-biological pretreatment for coproduction of flavonoids, cellulase, and ethanol.

A continuous chemical-free green approach was investigated for the comprehensive reutilization of all components in herbal extraction residues (HERs), taking Glycyrrhiza uralensis residue (GUR) as an example. The GUR structural changes induced by mechanical extrusion which improve the specific surface area and enzyme accessibility of GUR. With 3 % pretreated GUR loading of high-tolerance Penicillium oxalicum G2. The reducing sugar yield of 11.45 g/L was achieved, along with an 81.06 % in situ enzymatic hydrolysis. Finally, 8.23 g/L bioethanol (0.40 g/g total sugar) was produced from GUR hydrolysates after 24 h fermentation of Pichia stipitis G32. The amount of functional medicinal ingredients extracted from GUR after hydrolysis (39.63 mg/g) was 37.69 % greater than that of un-pretreated GUR. In total, 1.49 g flavonoids, 294.36 U cellulase, and 14.13 g ethanol could be produced from 100 g GUR using this process, illustrating that this green and efficient process has the potential for industrial production.

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.

Effects of pickle brine and glycine addition on biogenic amine production in pickle fermentation.

This study investigated the effects of different pickle brines and glycine additions on biogenic amine formation in pickle fermentation. The results showed that the brines with higher biogenic amine content led to the production of more biogenic amines in the simulated pickle fermentation system. This was related to the abundance of biogenic amine-producing microorganisms in the microbial communities of the brines. Metagenome analysis of the brines and metatranscriptome analysis of the fermentation systems showed that putrescine was primarily from Lactobacillus, Oenococcus, and Pichia, while histamine and tyramine were primarily from Lactobacillus and Tetragenococcus. Addition of glycine significantly reduced the accumulation of biogenic amines in the simulated pickle fermentation system by as much as 70 %. The addition of glycine had no inhibitory effect on the amine-producing microorganisms, but it down-regulated the transcription levels of the genes for enzymes related to putrescine synthesis in Pichia, Lactobacillus, and Oenococcus, as well as the histidine decarboxylase genes in Lactobacillus and Tetragenococcus. Catalytic reaction assay using crude solutions of amino acid decarboxylase extracted from Lactobacillus brevis showed that the addition of glycine inhibited 45 %-55 % of ornithine decarboxylase and tyrosine decarboxylase activities. This study may provide a reference for the study and control of the mechanism of biogenic amine formation in pickle fermentation.

The Hansenula polymorpha mitochondrial carrier family protein Mir1 is dually localized at peroxisomes and mitochondria.

Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full-length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full-length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information. Data are available via ProteomeXchange with identifier PXD050324.

Decoding Polysaccharides from Two Pichia Yeasts and Their Molecular Interaction with Wine Fruity Esters.

This study extensively characterized yeast polysaccharides (YPs) from Pichia fermentans (PF) and Pichia kluyveri (PK), with a specific focus on their structural attributes and their interaction with wine fruity esters in a model wine system. By finely tuning enzymatic reactions based on temperature, pH, and enzyme dosage, an optimal YP yield of 77.37% was achieved, with a specific mass ratio of cellulase, pectinase, and protease set at 3:5:2. There were four YP fractions (YPPF-W, YPPF-N, YPPK-W, and YPPK-N) isolated from the two yeasts. YPPF-N and YPPK-N were identified as glucans based on monosaccharide analysis and Fourier-transform infrared spectroscopy analysis. "Specific degradation-methylation-nuclear magnetic" elucidated YPPF-W's backbone structure as 1,3-linked α-l-Man and 1,6-linked α-d-Glc residues, while YPPK-W displayed a backbone structure of 1,3-linked α-Man residues, indicative of a mannoprotein nature. Isothermal titration calorimetry revealed spontaneous interactions between YPPK-W/YPPF-W and fruity esters across temperatures (25-45 °C), with the strongest interaction observed at 30 °C. However, distinct esters exhibited varying interactions with YPPK-W and YPPF-W, attributed to differences in molecular weights and hydrophobic characteristics. While shedding light on these intricate interactions, further experimental data is essential for a comprehensive understanding of yeast polysaccharides' or mannoproteins' impact on fruity esters. This research significantly contributes to advancing our knowledge of yeast polysaccharides' role in shaping the nuanced sensory attributes of wine.