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Applied Biochemistry and Biotechnology Jun 2024Microbial proteins represent a promising solution to address the escalating global demand for protein, particularly in regions with limited arable land. Yeasts, such as...
Microbial proteins represent a promising solution to address the escalating global demand for protein, particularly in regions with limited arable land. Yeasts, such as Saccharomyces cerevisiae, are robust and safe protein-producing strains. However, the utilization of non-conventional yeast strains for microbial protein production has been hindered, partly due to a lack of comprehensive understanding of protein production traits. In this study, we conducted experimental analyses focusing on the growth, protein content, and amino acid composition of nine yeast strains, including one S. cerevisiae strain, three Yarrowia lipolytica strains, and five Pichia spp. strains. We identified that, though Y. lipolytica and Pichia spp. strains consumed glucose at a slower rate compared to S. cerevisiae, Pichia spp. strains showed a higher cellular protein content, and Y. lipolytica strains showed a higher glucose-to-biomass/protein yield and methionine content. We further applied computational approaches to explain that metabolism economy was the main underlying factor for the limited amount of scarce/carbon-inefficient amino acids (such as methionine) within yeast cell proteins. We additionally verified that the specialized metabolism was a key reason for the high methionine content in Y. lipolytica strains, and proposed Y. lipolytica strain as a potential producer of high-quality single-cell protein rich in scarce amino acids. Through experimental evaluation, we identified Pichia jadinii CICC 1258 as a potential strain for high-quality protein production under unfavorable pH/temperature conditions. Our work suggests a promising avenue for optimizing microbial protein production, identifying the factors influencing amino acid composition, and paving the way for the use of unconventional yeast strains to meet the growing protein demands.
PubMed: 38922492
DOI: 10.1007/s12010-024-04995-3 -
Metabolites May 2024Wild yeast suitable for kiwifruit wine fermentation was isolated and purified, and the fermentation process was optimized to increase the alcohol content of the...
Wild yeast suitable for kiwifruit wine fermentation was isolated and purified, and the fermentation process was optimized to increase the alcohol content of the kiwifruit wine. was isolated from kiwifruit pulp by lineation separating, screened by morphological characteristics in Wallerstein Laboratory Nutrient Agar (WL) medium and microscope observation, and further identified by 26S rDNA D1/D2 domain sequence analysis. Taking alcohol content and sensory evaluation as two indexes, the fermentation condition for kiwifruit wine was optimized by single factor and response surface experiment. The optimal fermentation conditions were optimized as follows: the fermentation temperature was at 24 °C, the initial pH was 3.8, the sugar dosage in second step was 8% (/), and the inoculating quantity of and was 0.15 g/L at equal proportion. Under these optimal conditions, the maximum estimated alcohol content was 15.6 vol%, and the kiwifruit wine was light green in color with strong kiwifruit aroma and mellow taste.
PubMed: 38921445
DOI: 10.3390/metabo14060310 -
Journal of Fungi (Basel, Switzerland) Jun 2024(formerly ) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained... (Review)
Review
(formerly ) 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 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.
PubMed: 38921397
DOI: 10.3390/jof10060411 -
Updates: Outbreak Evaluation through Molecular Assays and Antifungal Stewardship-A Narrative Review.Current Issues in Molecular Biology Jun 2024was reported by the WHO as second to , in the list of nineteen fungal priority pathogens, along with two species with a new nomenclature, () and (). This novel... (Review)
Review
was reported by the WHO as second to , in the list of nineteen fungal priority pathogens, along with two species with a new nomenclature, () and (). This novel classification was based on antifungal resistance, the number of deaths, evidence-based treatment, access to diagnostics, annual incidence, and complications and sequelae. We assessed which molecular assays have been used to diagnose outbreaks in the last five years. Using "; outbreak; molecular detection" as keywords, our search in PubMed revealed 32 results, from which we selected 23 original papers published in 2019-2024. The analyzed studies revealed that the detection methods were very different: from the VITEK® 2 System to MALDI TOF (Matrix-Assisted Laser Desorption Ionization-Time of Flight), NGS (Next-Generation Sequencing), WGS (Whole Genome Sequencing), and commercially available real-time PCR (Polymerase Chain Reaction) assays. Moreover, we identified studies that detected antifungal resistance genes (e.g., FKS for echinocandins and ERG11 for azoles). The analyzed outbreaks were from all continents, which confirms the capability of this yeast to spread between humans and to contaminate the environment. It is important that real-time PCR assays were developed for accurate and affordable detection by all laboratories, including the detection of antifungal resistance genes. This will allow the fast and efficient implementation of stewardship programs in hospitals.
PubMed: 38921033
DOI: 10.3390/cimb46060362 -
Journal of Agricultural and Food... Jun 2024Lipase from (ROL) exhibits remarkable -1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of...
Lipase from (ROL) exhibits remarkable -1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of 1,3-dioleoyl-2-palmitoyl glycerol (OPO, a high-quality substitute for human milk fat). In this work, a semirational method was proposed to engineer the thermostability and catalytic activity of 4M (ROL mutant in our previous study). First, a computer-aided design is performed using 4M as a template, and -glycosylation mutants are then recombinantly expressed and screened in , the optimal mutant N227 exhibited a half-life of 298.8 h at 45 °C, which is 7.23-folds longer than that of 4M. Its catalytic activity also reached 1043.80 ± 61.98 U/mg, representing a 29.2% increase compared to 4M (808.02 ± 47.02 U/mg). Molecular dynamics simulations of N227 suggested that the introduction of glycan enhanced the protein rigidity, while the strong hydrogen bonds formed between the glycan and the protein stabilized the lipase structure, thereby improving its thermostability. The acidolysis reaction between oleic acid (OA) and glycerol tripalmitate (PPP) was successfully carried out using immobilized N227, achieving a molar conversion rate of 90.2% for PPP. This engineering strategy guides the modification of lipases, while the glycomutants obtained in this study have potential applications in the biosynthesis of OPO.
PubMed: 38913033
DOI: 10.1021/acs.jafc.4c02623 -
Letters in Applied Microbiology Jun 2024Yeasts are unicellular eukaryotic microorganisms extensively employed in various applications, notably as an alternative source of protein in feeds, owing to their...
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.
Topics: Animals; Animal Feed; Cichlids; Pichia; Aquaculture; Diet; Liver; Intestines; Dietary Supplements; Philippines
PubMed: 38906842
DOI: 10.1093/lambio/ovae057 -
Bioprocess and Biosystems Engineering Jun 2024The trisaccharide 1-kestose, a major constituent of commercial fructooligosaccharide (FOS) formulations, shows a superior prebiotic effect compared to higher-chain FOS....
The trisaccharide 1-kestose, a major constituent of commercial fructooligosaccharide (FOS) formulations, shows a superior prebiotic effect compared to higher-chain FOS. The plant sucrose:sucrose 1-fructosyltransferases (1-SST) are extensively used for selective synthesis of lower chain FOS. In this study, enhanced recombinant (r) 1-SST production was achieved in Komagataella phaffii (formerly Pichia pastoris) containing three copies of a codon-optimized Festuca arundinacea 1-SST gene. R1-SST production reached 47 U/mL at the shake-flask level after a 96-h methanol induction phase. A chemostat-based strain characterization methodology was adopted to assess the influence of specific growth rate (µ) on cell-specific r1-SST productivity (Qp) and cell-specific oxygen uptake rate (Qo) under two different feeding strategies across dilution rates from 0.02 to 0.05 h. The methanol-sorbitol co-feeding strategy significantly reduced Qo by 46 ± 2.4% compared to methanol-only feeding without compromising r1-SST productivity. Based on the data, a dilution rate of 0.025 h was applied for continuous cultivation of recombinant cells to achieve a sustained r1-SST productivity of 5000 ± 64.4 U/L/h for 15 days.
PubMed: 38904715
DOI: 10.1007/s00449-024-03045-w -
Frontiers in Microbiology 2024Kefir beverage has beneficial microorganisms that have health-giving properties; therefore, they have a good potential to be probiotic. This study evaluated the...
INTRODUCTION
Kefir beverage has beneficial microorganisms that have health-giving properties; therefore, they have a good potential to be probiotic. This study evaluated the probiotic potential, technological, and safety characteristics of , , and isolated from traditional kefir beverages.
METHOD
First, isolates were evaluated in terms of resistance to acid, alkali, bile salts, trypsin, and pepsin of the gastrointestinal tract. The auto-aggregation and co-aggregation ability of isolates were measured using spectrophotometry. Antimicrobial activities were assayed against important food-borne pathogens using the agar well diffusion method. Moreover, gamma-aminobutyric acid (GABA) production was investigated by thin-layer chromatography (TLC).
RESULT
Among the isolates, had an 85% total survival rate, but its amount reached below 6 log CFU/ml which is considered non-resistant, and it showed the highest auto-aggregation (74.67%). Moreover, only showed antimicrobial activity and had the highest co-aggregation with PTCC 1338 (54.33%) and ATCC 7644 (78%). Finally, an evaluation of the technological and safety characteristics of the strains showed that the strains produced GABA and were safe.
DISCUSSION
Although the isolates were not resistant to the gastrointestinal tract, their supernatant contained valuable natural compounds, including antioxidants, GABA, and antimicrobials, which can be used to produce functional foods and medicines. In addition, other approaches, such as increasing the initial number of strains, using foods as carriers of isolates, and encapsulating the isolates, can effectively increase the survivability of isolates in the gastrointestinal tract.
PubMed: 38903778
DOI: 10.3389/fmicb.2024.1385301 -
Scientific Reports Jun 2024Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse...
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.
Topics: Fermentation; Fatty Acids, Volatile; Yeasts; Yarrowia; High-Throughput Screening Assays; Biomass; Biofuels; Carboxylic Acids; Pichia
PubMed: 38902520
DOI: 10.1038/s41598-024-65150-w -
Metabolic Engineering Jun 2024Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical...
Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs. The biosynthetic pathway of cis-trans nepetalactol, which serves as the universal iridoid scaffold for all MIAs, has been successfully identified and reconstituted. However, bottlenecks and challenges remain to construct a high-yielding platform strain for cis-trans nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of Pichia pastoris cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and cis-trans nepetalactol. By targeting the biosynthetic pathway from acetyl-CoA to geraniol in both peroxisomes and cytoplasm, we achieved comparable geraniol titers in both compartments. Through protein engineering, we found that either G8H or CPR truncation increased the production of 8-hydroxygeraniol, with a 47.8-fold and 14.0-fold increase in the peroxisomal and cytosolic pathway strain, respectively. Furthermore, through a combination of dynamical control of ERG20, precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of cis-trans nepetalactol, with a titer of 4429.4 mg/L using fed-batch fermentation in a 5-L bioreactor. We anticipate our systematic metabolic engineering strategies to facilitate the development of P. pastoris cell factories for sustainable production of MIAs and other plant natural products.
PubMed: 38897449
DOI: 10.1016/j.ymben.2024.06.007