Search Results (96)

Background: Tay–Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the HEXA gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast Pichia pastoris, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. Methods: Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, GM2AP and HEXB expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. Results: Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the HEXB gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. Conclusions: Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells. Full article

Cellulosic biomass is considered an important and sustainable source of renewable energy, which needs a complex mixture of different enzymes for its degradation. After amylase, cellulases are the second most important enzymes, gain more importance due to their broad range of applications at the industrial level, and are considered more economical and environmentally friendly; researchers have focused more on the production of cellulase with its higher expression rate and low cost. Pichia pastoris, a methylotrophic yeast strain, has a more effective and well-established system for the production of heterologous proteins, particularly for industrial enzymes. Moreover, its readily achievable high-density fermentation, high capacity for protein secretion, tractable genetic modifications, typical post-transcriptional modifications, and strong regulated promoters makes it superior to other expression systems. In this review, we address the P. pastoris expression system including protein expression platforms, plasmids, and cellular metabolism quantification as a potential candidate for heterologous protein production, particularly for cellulase enzymes. Full article

The methylotrophic yeast Komagataella kurtzmanii belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typically contain a mixture of sexually dimorphic cells that are prone to self-diploidisation and spore formation accompanied by genetic rearrangements. These characteristics pose a significant challenge to the development of genetically stable producers for industrial use. In the present study, we constructed heterothallic strains of K. kurtzmanii, ensuring a constant mating type by unifying the genetic sequences in the active and silent MAT loci. To obtain such strains, we performed site-directed inactivation of one of the two yeast MAT loci, replacing its sequence with a selective HIS4 gene surrounded by I-SceI meganuclease recognition sites. We then used transient expression of the SCE1 gene, encoding a recombinant I-SceI meganuclease, to induce site-specific cleavage of HIS4, followed by damage repair by homologous recombination in mutant cells. As a result, heterothallic strains designated ‘Y-727-2(alpha)’ and ‘Y-727-9(a)’, which correspond to the α and a mating type, respectively, were obtained. The strains demonstrated a loss of the ability to self-diploidize. The results of PCR and whole genome analysis confirmed the identity of the contents of the MAT loci. Analysis of the genomes of the final strains, however, revealed a fusion of chromosome 3 and chromosome 4 in strain Y-727-2(alpha)-1. This finding was subsequently confirmed by pulsed-field gel electrophoresis of yeast chromosomes. However, the ability of the Y-727-2(alpha)-derived producers to efficiently secrete recombinant β-galactosidase was unaffected by this genomic rearrangement. Full article

Phosphorus is a key nutrient for all organisms. The study of phosphate metabolism and its regulation is important for understanding the evolutionary processes of regulatory systems in eukaryotic cells. The methylotrophic yeast Komagataella phaffii is an efficient producer organism, and it is actively used in biotechnological production. The high practical importance of K. phaffii has stimulated active research to find new tools to work with this yeast and optimize its cultivation conditions. In this work, we observed the effect of phosphate starvation on gene expression in K. phaffii at the transcriptome level. Phosphate starvation had a significant effect on general cell metabolism. K. phaffii cells demonstrated a response to this macronutrient deficiency through an altered gene expression of carbon and amino acid metabolism. We observed the activation of phosphate and polyphosphate metabolism gene expression. In this case, there was a suppression of ribosome biogenesis genes and genes involved in fatty acid beta-oxidation and translation processes. Full article

Oxidation of methane by soil microorganisms is an important mechanism controlling the content of this potent greenhouse gas in the atmosphere. Agricultural soils operate under stressful conditions, and ammonium (N-fertilization) and drying (global warming) may have a significant impact on methane oxidation. In order to investigate how soil methanotrophs respond to drying–rewetting (DW), ammonium addition (100 mg/g) (A), and their combined action (MS), agricultural soil microcosms were incubated over the three months and methane oxidation was measured before and after perturbations, while community composition was monitoring using 16S rRNA gene sequencing. A significant decline in the methane-oxidation activity after perturbations was found, with subsequent restoration, and the combined treatment was more effective than the sum of individual treatments, indicating a synergistic effect. After rewetting, the structure of the bacterial community returned to pre-dry-down levels, but the application of ammonia and combined action lead to irreversible changes in the structure of soil methanotrophic communities. Methanotroph Methylomicrobium were significantly reduced under disturbances, while there was a significant increase in the representation of Methylobacter accompanied by the facultative methylotroph Methylovorus. We concluded that methanotrophic communities in agricultural soil demonstrated flexibility, and even when the abundance of dominant populations drops, ecosystem functions can recover. Full article

Background/Objectives: The current epidemic of drug-resistance bacterial strains is one of the most urgent threats to human health. Antimicrobial peptides (AMPs) are known for their good activity against multidrug resistance bacteria. Specifically targeted AMPs (STAMPs) are a fraction of AMPs that target specific bacteria and maintain the balance of the healthy microbiota of a host. We reported a STAMP Peptide K (former name: peptide 13) for E. coli. The aim of this study was to effectively produce peptide K using methylotrophic yeast Pichia pastoris. Methods: Three inserts (sequence of peptide K (K), two copies of peptide K fused with 2A sequence (KTK), and two copies of peptide K fused with 2A and an extra α mating factor (KTAK)) were designed to investigate the effect of the number of repeats and the trafficking of peptide on the yield. Results: The yield from KTK was the highest—more than two-fold higher compared with K—implying the role of the 2A sequence in heterologous peptide expression apart from the co-translation. Then, the fermentation condition for KTK was optimized. The optimized yield of KTK was 6.67 mg/mL, suggesting the efficiency of the expression system. Selectivity, antibacterial activity, biocompatibility, and the stability of the fermentation product were equivalent to the chemically synthesized peptide. The actional mechanism of the fermentation product included membrane permeabilization and ROS induction. Conclusions: Together, our work provided a new perspective to augment the yield of the antimicrobial peptide in the microbial system, building a technological foundation for their large-scale production and expanding the market application of AMPs. Full article

Methylotrophic yeast Ogataea polymorpha BKM Y-2559 was immobilized in organosilicon sol–gel matrices using precursors isobutyltriethoxysilane (iBTES) and tetraethoxysilane (TEOS) to create an effective biocatalyst. The analytical and metrological performance of the biosensor permitted the determination of the optimum ratio of iBTES and TEOS, which was found to be 20/80 vol.%. The results of the scanning electron microscopy method demonstrated the formation of organosilicon material around microorganisms, as well as the ease with which metabolic products of yeast cells and substrates could diffuse through the obtained pores. A laboratory model of the biofilter was developed, exhibiting an oxidative capacity that varied from 0.14 to 1.25 gO₂/(m³ × cycle) in accordance with the initial level of water pollution and the degree of purification of moderately polluted water. The latter was found to be 20%, which aligns with the norm for drip biofilters operating in cyclic mode. Full article

Alkaline coal mine drainage represents one of the most critical issues in the coal industry, driven by complex hydro-biogeochemical processes. However, the interplay of hydrogeochemical and biogeochemical interactions in alkaline coal mine drainage is still poorly understood. To this end, water samples were systematically collected from alkaline coal mine drainage sites from five coal mining areas in Chongqing coal mining district, located in southwestern China. Hydrogeochemical analyses showed that the main water type of the coal mine drainage sample was HCO₃-SO₄~K-Na, which primarily originated from local meteoric water. The microbial community compositions in the studied alkaline coal drainage were critically associated with sulfate, bicarbonate, DOC, nitrate, and pH, and linked to three putative keystone genera via network analysis (Thiothrix, Methylophilaceae_MM1, and an unclassified genus from Comamonadaceae family). Functional predictions from FAPROTAX suggested a high abundance of metabolic pathways involving the oxidation of sulfide and sulfur compounds, potentially underscoring their importance in controlling sulfate enrichment in alkaline coal mine drainage. Interestingly, members of the Methylomonadaceae family (methanotrophs) and the Methylotenera genus (methylotrophs) had positive Spearman correlations with both ammonium and sulfate, potentially inferring that the enhanced activities of methanotrophs might help capture methane in the alkaline coal mine drainage. This study further enhances our comprehension of the intricate interplay between hydrogeochemical and biogeochemical interactions in alkaline coal mine drainage, contributing to the carbon budget. Full article

Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts. Full article

The PHO91 and PHO87 are parts of yeast’s phosphate metabolism system. We investigated the influence of PHO91 deletion on the methanol utilization as a sole carbon source in Ogataea parapolymorpha through comparison of wet biomass, protein content, methanol oxidase (MOX) activity and acid-soluble and acid-insoluble inorganic polyphosphate (polyP) content, while growing on methanol and without any carbon source other than trace amounts from the yeast extract. The Δpho91 strain has a lack of wet biomass, protein content and MOX activity while grown on methanol (as a ΔMOX strain) and has the same numbers as a WT strain in media without methanol. We found two ways of recovering methanol utilizing capabilities for Δpho91 mutant—either adding MOX on plasmid under constitutive promoter control, or adding a Δpho87 mutation. Both Δpho91 + MOX and Δpho91Δpho87 strains showed all the parameters as a WT strain on methanol as a sole carbon source and without methanol addition. Further, we compare the polyP content of the strains in methanol media, media without a sole carbon source and media with glucose. All of the WT, Δpho91 + MOX and Δpho91Δpho87 strains, capable of utilizing methanol, have the P_i, acid-soluble and acid-insoluble polyP at the same levels with some variations. The ΔMOX and Δpho91 strains, despite both not being able to utilize methanol, showed very different levels of P_i and polyP’s, which can be interpreted as different mechanisms of the inner cell, leading to a loss of capability utilizing methanol in both of these strains. Full article

Polyhydroxyalkanoates (PHAs) are intracellular biopolymers that microorganisms use for energy and carbon storage. They are mechanically similar to petrochemical plastics when chemically extracted, but are completely biodegradable. While they have potential as a replacement for petrochemical plastics, their high production cost using traditional carbon sources remains a significant challenge. One potential solution is to modify heterotrophic PHA-producing strains to utilize alternative carbon sources. An alternative approach is to utilize methylotrophic or autotrophic strains. This article provides an overview of bacterial strains employed for PHA production, with a particular focus on those exhibiting the highest PHA content in dry cell mass. The strains are organized according to their carbon source utilization, encompassing autotrophy (utilizing CO₂, CO) and methylotrophy (utilizing reduced single-carbon substrates) to heterotrophy (utilizing more traditional and alternative substrates). Full article

An ever-increasing amount of research is being performed on the stability and recovery of soil methane-oxidizing bacteria since this is one of the fundamental processes controlling the amount of methane in the atmosphere. Mineral fertilizers may alter the methane oxidation processes in agricultural soils when they are introduced. Although ammonium (NH₄⁺) is believed to have a significant impact on aerobic methane oxidation activity in soils, there is still little data on how it reacts with lanthanum (La). The recent identification of a novel class of lanthanum-containing enzymes in methanotrophic bacteria may be the foundation for controlling the function of the soil “methane filter” and related microbiota. In the current study, microcosms with agricultural sod-podzolic soils were created and incubated in air or 20% CH₄ in the gas phase with the addition of NH₄⁺ (100 µg/g) and La (5 µg/g) to the soil. Using GC analysis and high-performance 16S rRNA sequencing, the methane oxidation potential and composition of soil bacterial communities were studied over the month of incubation. A negative impact of NH₄⁺ on the oxidation of methane was observed, whereas La had a somewhat beneficial effect. Ammonium had an impact on the composition of methanotrophs, and a significant shift was observed upon La addition. Proteobacteria made up a larger share of the soil microbial community, and Gammaproteobacteria dominated the methanotrophic populations. Methylobacter, a methanotroph, and Methylotenera, an obligatory methylotroph, were the two absolute dominants in the La-amended variants. These findings could help evaluate how lanthanum regulates methanotrophic communities in agricultural soils and lead to the creation of new strategies for controlling the “methane filter” in soil. Full article

This study examined the impact of sodium propionate concentration (0–40 g/L) on the methanogenic archaea in an inoculum which was cultured in basal nutrient medium, exploring its mechanisms and nonlinear stress intensity. The results indicated that at low concentrations, propionate-maintained homeostasis of the anaerobic digestion (AD) system and enriched Methanosaeta. However, when the concentration exceeded 16 g/L, the stability of the AD system was disrupted. The methanogenic pathway shifted towards a predominantly hydrogenotrophic pathway, resulting in a significant increase in methane yield. Below concentrations of 28 g/L, the AD system gradually enhanced its ability to utilize propionate in an orderly manner. At concentrations of 24–28 g/L, genera (e.g., Advenella and Methanosarcina) were enriched to adapt to the high-VFA environment. This was accompanied by a significant upregulation of genes related to the methylotrophic and hydrogenotrophic pathways, effectively mitigating propionate inhibition and enhancing methanogenesis. Conversely, excess concentrations (>30 g/L) suppressed methanogenesis-related genes and led to methane production arrest despite activating specialized propionate-metabolizing bacteria such as genus Pelotomaculum schinkii. As such, an increase in the stress intensity of propionate promotes a change in the metabolic pathways of methanogens and increases methane production; however, excessive sodium propionate was not conducive to maintaining the steady state of the system. Full article

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast Komagataella phaffii, a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in K. phaffii. In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%). Full article

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules. Full article