Wickerhamiella lachancei f.a. sp. nov., a novel ascomycetous yeast species isolated from flowers of Lantana camara in India.

Two isolates representing a novel species of the genus Wickerhamiella were obtained in India from nectar of flowers of Lantana camara, an ornamental exotic species native to Central and South America. Phylogenetic analyses of the D1/D2 domain of the 26S large subunit (LSU) rRNA gene, internal transcribed spacer (ITS) region, and physiological characteristics, supported the recognition of the novel species, that we designate Wickerhamiella lachancei sp. nov (MycoBank no. MB851709), with MCC 9929T as the holotype and PYCC 10003T as the isotype. Considering pairwise sequence similarity, the type strain of the novel species differs from the type strain of the most closely related species, Wickerhamiella drosophilae CBS 8459T, by 16 nucleotide substitutions and two gaps (3.9 % sequence variation) in the D1/D2 region (560 bp compared) and 28 nucleotide substitutions and five gaps (7.22 % sequence variation) in the ITS region (444 bp compared).

Cell growth and nutrient availability control the mitotic exit signaling network in budding yeast.

Cell growth is required for cell cycle progression. The amount of growth required for cell cycle progression is reduced in poor nutrients, which leads to a reduction in cell size. In budding yeast, nutrients can influence cell size by modulating the extent of bud growth, which occurs predominantly in mitosis. However, the mechanisms are unknown. Here, we used mass spectrometry to identify proteins that modulate bud growth in response to nutrient availability. This led to the discovery that nutrients regulate numerous components of the mitotic exit network (MEN), which controls exit from mitosis. A key component of the MEN undergoes gradual multisite phosphorylation during bud growth that is dependent upon bud growth and correlated with the extent of growth. Furthermore, activation of the MEN is sufficient to override a growth requirement for mitotic exit. The data suggest a model in which the MEN ensures that mitotic exit occurs only when an appropriate amount of bud growth has occurred.

Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production.

BACKGROUND: Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. RESULTS: This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. CONCLUSIONS: This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.

Hemolymph metabolism of black soldier fly (Diptera: Stratiomyidae), response to different supplemental fungi.

The black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae), is commonly used for organic waste recycling and animal feed production. However, the often inadequate nutrients in organic waste necessitate nutritional enhancement of black soldier fly larvae, e.g., by fungal supplementation of its diet. We investigated the amino acid composition of two fungi, Candida tropicalis (Castell.) Berkhout (Saccharomycetales: Saccharomycetaceae) and Pichia kudriavzevii Boidin, Pignal & Besson (Saccharomycetales: Pichiaceae), from the black soldier fly gut, and commercial baker's yeast, Saccharomyces cerevisiae Meyen ex E.C. Hansen (Saccharomycetales: Saccharomycetaceae), and their effects on larval growth and hemolymph metabolites in fifth-instar black soldier fly larvae. Liquid chromatography-mass spectrometry was used to study the effect of fungal metabolites on black soldier fly larval metabolism. Amino acid analysis revealed significant variation among the fungi. Fungal supplementation led to increased larval body mass and differential metabolite accumulation. The three fungal species caused distinct metabolic changes, with each over-accumulating and down-accumulating various metabolites. We identified significant alteration of histidine metabolism, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism in BSF larvae treated with C. tropicalis. Treatment with P. kudriavzevii affected histidine metabolism and citrate cycle metabolites, while both P. kudriavzevii and S. cerevisiae treatments impacted tyrosine metabolism. Treatment with S. cerevisiae resulted in down-accumulation of metabolites related to glycine, serine, and threonine metabolism. This study suggests that adding fungi to the larval diet significantly affects black soldier fly larval metabolomics. Further research is needed to understand how individual amino acids and their metabolites contributed by fungi affect black soldier fly larval physiology, growth, and development, to elucidate the interaction between fungal nutrients and black soldier fly physiology.

Kodamaea ohmeri: A rare yeast causing invasive infections in immunocompromised patients.

INTRODUCTION: Kodamaea ohmeri is a rare, recognized pathogen that has previously been isolated from environmental sources. The patients commonly affected by this yeast include immunocompromised as well as immunocompetent patients having several associated risk factors. METHODOLOGY: We report three cases in which K. ohmeri was isolated from blood using Bact T/ALERT. Identification was carried out by MALDI-TOF MS (Vitek-MS, BioMérieux, Marcy-l'Etoile, France) in addition to color characteristics on chromogenic media. The patients had diminished immune response on account of a multitude of comorbidities. RESULTS: K. ohmeri can be misidentified as Candida tropicalis, Candida albicans, or Candida hemolounii by conventional methods; correct and timely identification can be achieved by MALDI-TOF MS. Antifungal susceptibility breakpoints for K. ohmeri are currently not defined. An Echinocandin was added to the treatment regimen of all three of the cases. CONCLUSIONS: Identification of K. ohmeri using conventional methods is difficult and unusual yeasts should be carefully observed, especially upon prolonged incubation.

Simulation and experimental study of a cold atmospheric pressure plasma and comparison of efficiency in boosting recombinant Endoglucanase II production in Pichia pastoris.

Recombinant proteins are essential in various industries, and scientists employ genetic engineering and synthetic biology to enhance the host cell's protein production capacity. Stress response pathways have been found effective in augmenting protein secretion. Cold atmospheric pressure plasma (CAP) can induce oxidative stress and enhance protein production. Previous studies have confirmed the applicability of CAP jets on Phytase and green fluorescent protein (GFP) production in Pichia pastoris hosts. This study investigates the effect of CAP treatment on another valuable recombinant protein, Endoglucanase II (EgII), integrated into the Pichia pastoris genome. The results demonstrated that plasma induction via two different ignition modes: sinusoidal alternating current (AC) and pulsed direct current (DC) for 120, 180, and 240 s has boosted protein secretion without affecting cell growth and viability. The AC-driven jet exhibited a higher percentage increase in secretion, up to 45%. Simulation of plasma function using COMSOL software provided a pattern of electron temperature (Te) and density distribution, which determine the plasma cocktail's chemistry and reactive species production. Furthermore, electron density (ne) and temperature were estimated from the recorded optical spectrum. The difference in electron properties may explain the moderately different impressions on expression capability. However, cell engineering to improve secretion often remains a trial-and-error approach, and improvements are, at least partially, specific to the protein produced.

[Preparation of recombinant mussel mucin Mfp-3P and its promotion of wound healing].

To investigate the role of recombinant mussel mucin in wound healing, we aimed to prepare this mucin using Pichia pastoris as the host microbe. Our method involved constructing a genetically engineered strain of P. pastoris that expressed a fusion protein consisting of Mfp-3 and preCol-P peptide segments of mussel. After fermentation and purification, we obtained a pure recombinant mussel mucin product. We then conducted experiments to evaluate its effect at both the cellular and animal levels. At the cellular level, we examined its impact on the proliferation and migration of mouse fibroblast L929. At the animal level, we assessed its ability to promote wound healing after full-layer skin resection in rats. Our results showed that the recombinant mussel mucin protein has a content of 90.28% and a purity of 96.49%. The content of 3,4-dihydroxyphenylalanine (DOPA) was 0.73 wt%, and the endotoxin content was less than 0.5 EU/mg. Importantly, the recombinant mussel mucin protein significantly promoted both the migration and proliferation of mouse fibroblast, as well as the wound healing in rat skin. In conclusion, our findings demonstrate that recombinant mussel mucin has the potential to promote wound healing and can be considered a promising medical biomaterial.

Microbial preference for chlorate over perchlorate under simulated shallow subsurface Mars-like conditions.

The Martian surface and shallow subsurface lacks stable liquid water, yet hygroscopic salts in the regolith may enable the transient formation of liquid brines. This study investigated the combined impact of water scarcity, UV exposure, and regolith depth on microbial survival under Mars-like environmental conditions. Both vegetative cells of Debaryomyces hansenii and Planococcus halocryophilus, alongside with spores of Aspergillus niger, were exposed to an experimental chamber simulating Martian environmental conditions (constant temperatures of about - 11 °C, low pressure of approximately 6 mbar, a CO2 atmosphere, and 2 h of daily UV irradiation). We evaluated colony-forming units (CFU) and water content at three different regolith depths before and after exposure periods of 3 and 7 days, respectively. Each organism was tested under three conditions: one without the addition of salts to the regolith, one containing sodium chlorate, and one with sodium perchlorate. Our results reveal that the residual water content after the exposure experiments increased with regolith depth, along with the organism survival rates in chlorate-containing and salt-free samples. The survival rates of the three organisms in perchlorate-containing regolith were consistently lower for all organisms and depths compared to chlorate, with the most significant difference being observed at a depth of 10-12 cm, which corresponds to the depth with the highest residual water content. The postulated reason for this is an increase in the salt concentration at this depth due to the freezing of water, showing that for these organisms, perchlorate brines are more toxic than chlorate brines under the experimental conditions. This underscores the significance of chlorate salts when considering the habitability of Martian environments.

Recent progress on heterologous protein production in methylotrophic yeast systems.

Recombinant protein production technology is widely applied to the manufacture of biologics used as drug substances and industrial proteins such as recombinant enzymes and bioactive proteins. Various heterologous protein production systems have been developed using prokaryotic and eukaryotic hosts. Especially methylotrophic yeast in eukaryotic hosts is suggested to be particularly valuable because such systems have the following advantages: protein secretion into culture broth, eukaryotic quality control systems, a post-translational modification system, rapid growth, and established recombinant DNA tools and technologies such as strong promoters, effective selection markers, and gene knock-in and -out systems. Many methylotrophic yeasts such as the genera Candida, Ogataea, and Komagataella have been studied since methylotrophic yeast was first isolated in 1969. The methanol-consumption-related genes in methylotrophic yeast are strongly and strictly regulated under methanol-containing conditions. The well-regulated gene expression systems under the methanol-inducible gene promoter lead to the potential application of heterologous protein production in methylotrophic yeast. In this review, we describe the recent progress of heterologous protein production technology in methylotrophic yeast and introduce Ogataea minuta as an alternative production host as a substitute for K. phaffii and O. polymorpha.

Complete genome sequencing of Hortaea werneckii M-3 for identifying polyester polyurethane degrading enzymes.

Hortaea werneckii M-3, a black yeast isolated from the marine sediment of the West Pacific, can utilize polyester polyurethane (PU, Impranil DLN) as a sole carbon source. Here, we present the complete genome of Hortaea werneckii M-3 with the focus on PU degradation enzymes. The total genome size is 38,167,921 bp, consisting of 186 contigs with a N50 length of 651,266 bp and a GC content of 53.06%. Genome annotation analysis predicts a total of 13,462 coding genes, which include 99 tRNAs and 105 rRNAs. Some genes encoding PU degrading enzymes including cutinase and urease are identified in this genome. The genome analysis of Hortaea werneckii M-3 will be helpful for further understanding the degradation mechanism of polyester PU by marine yeasts.

Mrakia polaris sp. nov. and Mrakia amundsenii sp. nov. (Mrakiaceae, Cystofilobasidiales), two novel psychrophilic yeast species isolated from Arctic habitats.

Four yeast strains belonging to the basidiomycetous yeast genus Mrakia were isolated from diverse habitats in the Ny-Ålesund region (Svalbard, High Arctic): two from vascular plants, one from seawater and one from freshwater. Phylogenetic analysis, based on the ITS region and the D1/D2 domain of the 28S rRNA gene, identified these four strains as representing two novel species within the genus Mrakia. The names Mrakia polaris sp. nov. (MycoBank number: MB 852063) and Mrakia amundsenii sp. nov. (MycoBank number: MB 852064) are proposed. These two new species show distinct psychrophilic adaptations, as they exhibit optimal growth at temperatures between 10 and 15°C, while being unable to grow at 25°C. The holotype of M. polaris sp. nov. is CPCC 300345T, and the holotype of M. amundsenii sp. nov. is CPCC 300572T.

Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A.

Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5' untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline.

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here, we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.

Ceramide sorting into non-vesicular transport is independent of acyl chain length in budding yeast.

The transport of ceramide from the endoplasmic reticulum (ER) to the Golgi is a key step in the synthesis of complex sphingolipids, the main building blocks of the plasma membrane. In yeast, ceramide is transported to the Golgi either through ATP-dependent COPII vesicles of the secretory pathway or by ATP-independent non-vesicular transport that involves tethering proteins at ER-Golgi membrane contact sites. Studies in both mammalian and yeast cells reported that vesicular transport mainly carries ceramide containing very long chain fatty acids, while the main mammalian non-vesicular ceramide transport protein CERT only transports ceramides containing short chain fatty acids. However, if non-vesicular ceramide transport in yeast similarly favors short chain ceramides remained unanswered. Here we employed a yeast GhLag1 strain in which the endogenous ceramide synthase is replaced by the cotton-derived GhLag1 gene, resulting in the production of short chain C18 rather than C26 ceramides. We show that block of vesicular transport through ATP-depletion or the use of temperature-sensitive sec mutants caused a reduction in inositolphosphorylceramide (IPC) synthesis to similar extent in WT and GhLag1 backgrounds. Since the remaining IPC synthesis is a readout for non-vesicular ceramide transport, our results indicate that non-vesicular ceramide transport is neither blocked nor facilitated when only short chain ceramides are present. Therefore, we propose that the sorting of ceramide into non-vesicular transport is independent of acyl chain length in budding yeast.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Chromosome-level genome assembly of the yeast Lodderomyces beijingensis reveals the genetic nature of metabolic adaptations and identifies subtelomeres as hotspots for amplification of mating type loci.

Lodderomyces beijingensis is an ascosporic ascomycetous yeast. In contrast to related species Lodderomyces elongisporus, which is a recently emerging human pathogen, L. beijingensis is associated with insects. To provide an insight into its genetic makeup, we investigated the genome of its type strain, CBS 14171. We demonstrate that this yeast is diploid and describe the high contiguity nuclear genome assembly consisting of eight chromosome-sized contigs with a total size of about 15.1 Mbp. We find that the genome sequence contains multiple copies of the mating type loci and codes for essential components of the mating pheromone response pathway, however, the missing orthologs of several genes involved in the meiotic program raise questions about the mode of sexual reproduction. We also show that L. beijingensis genome codes for the 3-oxoadipate pathway enzymes, which allow the assimilation of protocatechuate. In contrast, the GAL gene cluster underwent a decay resulting in an inability of L. beijingensis to utilize galactose. Moreover, we find that the 56.5 kbp long mitochondrial DNA is structurally similar to known linear mitochondrial genomes terminating on both sides with covalently closed single-stranded hairpins. Finally, we discovered a new double-stranded RNA mycovirus from the Totiviridae family and characterized its genome sequence.

Xylitol fermentation characteristics with a newly isolated yeast Wickerhamomyces anomalus WA.

Xylitol is an increasingly popular functional food additive, and the newly isolated yeast Wickerhamomyces anomalus WA has shown extensive substrate utilization capability, with the ability to grow on hexose (d-galactose, d-glucose, d-mannose, l-fructose, and d-sorbose) and pentose (d-xylose and l-arabinose) substrates, as well as high tolerance to xylose at concentrations of up to 300 g/L. Optimal xylitol fermentation conditions were achieved at 32 °C, 140 rpm, pH 5.0, and initial cell concentration OD600 of 2.0, with YP (yeast extract 10 g/L, peptone 20 g/L) as the optimal nitrogen source. Xylitol yield increased from 0.61 g/g to 0.91 g/g with an increase in initial substrate concentration from 20 g/L to 180 g/L. Additionally, 20 g/L glycerol was found to be the optimal co-substrate for xylitol fermentation, resulting in an increase in xylitol yield from 0.82 g/g to 0.94 g/g at 140 rpm, enabling complete conversion of xylose to xylitol.

Genetic conflicts in budding yeast: The 2µ plasmid as a model selfish element.

Antagonistic coevolution, arising from genetic conflict, can drive rapid evolution and biological innovation. Conflict can arise both between organisms and within genomes. This review focuses on budding yeasts as a model system for exploring intra- and inter-genomic genetic conflict, highlighting in particular the 2-micron (2µ) plasmid as a model selfish element. The 2µ is found widely in laboratory strains and industrial isolates of Saccharomyces cerevisiae and has long been known to cause host fitness defects. Nevertheless, the plasmid is frequently ignored in the context of genetic, fitness, and evolution studies. Here, I make a case for further exploring the evolutionary impact of the 2µ plasmid as well as other selfish elements of budding yeasts, discuss recent advances, and, finally, future directions for the field.

Wickerhamomyces corioli f.a., sp. nov., a novel yeast species discovered in two mushroom species.

Two yeast strains, designated as 19-39-3 and 19-40-2, obtained from the fruiting bodies of Trametes versicolor and Marasmius siccus collected in Yunwu Mountain Forest Park, PR China, have been identified as representing a novel asexual ascomycetous yeast species. From the results of phylogenetic analyses of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA, small subunit (SSU) rRNA and translation elongation factor 1-α (TEF1) genes, it was determined that these strains represent a member of the genus Wickerhamomyces, with Wickerhamomyces alni and Candida ulmi as the closest relatives. The novel species exhibited 6.6 and 6.7% differences in the D1/D2 domains compared with W. alni and C. ulmi, respectively. Additionally, distinct biochemical and physiological differences were observed between the novel species and its related counterparts. No sexual reproduction was observed in these strains, leading to the proposal of the name Wickerhamomyces corioli f.a., sp. nov. for this newly discovered species.