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Foods (Basel, Switzerland) Jun 2024There is a growing market for craft beverages with unique flavors. This study aimed to obtain a palate-pleasing mead derived from 4A as a monoculture. Different culture...
There is a growing market for craft beverages with unique flavors. This study aimed to obtain a palate-pleasing mead derived from 4A as a monoculture. Different culture media were evaluated to compare the fermentation kinetics and final products. The crucial factors in the medium were ~200 mg L of yeast assimilable nitrogen and a pH of 3.5-5.0. A panel of judges favored the mead derived from 4A (fermented in a medium with honey initially at 23 °Bx) over a commercial sample produced from , considering its appearance, fruity and floral flavors (provided by esters, aldehydes, and higher alcohols), and balance between sweetness (given by the 82.91 g L of residual sugars) and alcohol. The present mead had an 8.57% / ethanol concentration, was elaborated in 28 days, and reached a maximum biomass growth (2.40 g L) on the same fermentation day (6) that the minimum level of pH was reached. The biomass growth yield peaked at 24 and 48 h (~0.049 g g), while the ethanol yield peaked at 24 h (1.525 ± 0.332 g g), in both cases declining thereafter. The Gompertz model adequately describes the kinetics of sugar consumption and the generation of yeast biomass and ethanol. Pathogenic microorganisms, methanol, lead, and arsenic were absent in the mead. Thus, 4A produced a safe and quality mead with probable consumer acceptance.
PubMed: 38928890
DOI: 10.3390/foods13121948 -
Biotechnology Letters Feb 2024D-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have...
D-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of D-glucose to D-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of D-glucose to biomass, and to increase D-glucaric acid yield, the four step D-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High D-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled C-glucose confirmed conversion of D-glucose to D-glucaric acid. In batch bioreactor cultures with pulsed D-fructose and ethanol provision 1.3 g D-glucaric acid L was produced. The D-glucaric acid titer (0.71 g D-glucaric acid L) was lower in nitrogen limited conditions, but the yield, 0.23 g D-glucaric acid [g D-glucose consumed], was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield D-glucaric acid yeast strains.
Topics: Saccharomyces cerevisiae; Glucose-6-Phosphate Isomerase; Glucaric Acid; Escherichia coli; Inositol; Glucose
PubMed: 38064042
DOI: 10.1007/s10529-023-03443-2 -
Journal of Microbiology and... May 2024Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important...
Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important enzyme from an environmental perspective because it not only promotes the growth of livestocks but also prevents phosphorus contamination released into the environment. Here we present a novel phytase derived from , TmPhy, which has distinctive structure and properties compared to other previously known phytases. TmPhy gene expressed in the system was confirmed to be 41 kDa in size and was used in purified form to evaluate optimal conditions for maximum activity. TmPhy has a dual optimum pH at pH3 and pH6.8 and exhibited the highest activity at 70°C. However, the heat tolerance of the wildtype was not satisfactory for feed application. Therefore, random mutation, disulfide bond introduction, and N-terminal mutation were performed to improve the thermostability of the TmPhy. Random mutation resulted in TmPhyM with about 45% improvement in stability at 60°C. Through further improvements, a total of three mutants were screened and their heat tolerance was evaluated. As a result, we obtained TmPhyMD1 with 46.5% residual activity, TmPhyMD2 with 74.1%, and TmPhyMD3 with 66.8% at 80°C heat treatment without significant loss of or with increased activity.
Topics: 6-Phytase; Enzyme Stability; Hydrogen-Ion Concentration; Hot Temperature; Mutation; Pichia; Temperature; Animal Feed; Kinetics; Recombinant Proteins
PubMed: 38563103
DOI: 10.4014/jmb.2311.11044 -
Regulatory Toxicology and Pharmacology... Feb 2024Serendipity berry plant (Dioscoreophyllum cumminsii (Stapf) Diels) is the source of a naturally sweet protein referred to as monellin. The safety of serendipity berry...
Serendipity berry plant (Dioscoreophyllum cumminsii (Stapf) Diels) is the source of a naturally sweet protein referred to as monellin. The safety of serendipity berry sweet protein (SBSP) containing single polypeptide monellin (MON) expressed in Komagataella phaffii (formerly Pichia pastoris) and produced via precision fermentation was examined comprehensively through assessments of in vitro and in silico protein digestion, in silico allergenicity, in vitro genotoxicity (reverse mutation and mammalian micronucleus assays), and 14-day and 90-day oral (dietary) toxicity studies in rats. There was no indication of allergenicity for SBSP in the in silico analyses. Results from both in vitro and in silico protein digestibility assessments indicated that SBSP is digested upon ingestion and would therefore be unlikely to pose a toxigenic or allergenic risk to consumers. SBSP was non-genotoxic in in vitro assays and showed no adverse effects in the 14-day or 90-day toxicity studies up to the highest dose tested. The 90-day toxicity study supports a NOAEL for SBSP of 1954 mg/kg bw/day, which corresponds to a NOAEL for MON of 408 mg/kg bw/day.
Topics: Rats; Animals; Fruit; Plants; Plant Proteins; Mammals; Saccharomycetales
PubMed: 38190935
DOI: 10.1016/j.yrtph.2024.105562 -
Frontiers in Bioengineering and... 2024Xylitol is a pentose-polyol widely applied in the food and pharmaceutical industry. It can be produced from lignocellulosic biomass, valorizing second-generation...
Xylitol is a pentose-polyol widely applied in the food and pharmaceutical industry. It can be produced from lignocellulosic biomass, valorizing second-generation feedstocks. Biotechnological production of xylitol requires scalable solutions suitable for industrial scale processes. Immobilized-cells systems offer numerous advantages. Although fungal pellet carriers have gained attention, their application in xylitol production remains unexplored. In this study, the yeast strain WC 1507 was employed for xylitol production. The optimal conditions were observed with free-cell cultures at pH above 3.5, low oxygenation, and medium containing (NH)SO and yeast extract as nitrogen sources (xylitol titer 79.4 g/L, Y 66.3%, and volumetric productivity 1.3 g/L/h). Yeast cells were immobilized using inactive pellet mycelial carrier (MC) and alginate beads (AB) and were tested in flasks over three consecutive production runs. Additionally, the effect of a 0.2% w/v alginate layer, coating the outer surface of the carriers (cMC and cAB, respectively), was examined. While Y values observed with both immobilized and free cells were similar, the immobilized cells exhibited lower final xylitol titer and volumetric productivity, likely due to mass transfer limitations. AB and cAB outperformed MC and cMC. The uncoated AB carriers were tested in a laboratory-scale airlift bioreactor, which demonstrated a progressive increase in xylitol production in a repeated batch process: in the third run, a xylitol titer of 63.0 g/L, Y of 61.5%, and volumetric productivity of 0.52 g/L/h were achieved. This study confirmed WC 1507 as a promising strain for xylitol production in both free- and entrapped-cells systems. Considering the performance of the wild strain, a metabolic engineering intervention aiming at further improving the efficiency of xylitol production could be justified. MC and AB proved to be viable supports for cell immobilization, but additional process development is necessary to identify the optimal bioreactor configuration and fermentation conditions.
PubMed: 38303913
DOI: 10.3389/fbioe.2024.1339093 -
Mycopathologia Dec 2023We performed a retrospective survey of non-Candida albicans candidemia in patients with cancer, including those with solid tumors and those with hematological...
We performed a retrospective survey of non-Candida albicans candidemia in patients with cancer, including those with solid tumors and those with hematological malignancies as well as transplants patients both, solid-organ transplant recipients and hematopoietic stem cell transplant recipients. The study was performed at two healthcare centers in New York City and covered the years 2018-2022. A total of 292 patients (318 isolates) were included in the study. In order of frequency, C. glabrata (38%) was the most common species recovered, followed by C. parapsilosis (19.2%), C. tropicalis (12.6%), C. krusei (10.7%), C. lusitaniae (5.7%), and C. guilliermondii (4.4%). Micafungin was the most common antifungal treatment and 18.5% of patients were on antifungal prophylaxis. The 30-day crude mortality was 40%. 4.5% of patients had more than one non-albicans species detected. In conclusion, this study represents one of the largest surveys of non-albicans species in cancer and transplant patients and provides data on the current epidemiology of these Candida species in this patient population.
Topics: Humans; Antifungal Agents; Transplant Recipients; Retrospective Studies; Microbial Sensitivity Tests; Candida; Candidemia; Candida glabrata; Candida parapsilosis; Candida tropicalis; Neoplasms
PubMed: 37365379
DOI: 10.1007/s11046-023-00765-7 -
Microbial Cell Factories Sep 2023Methanol, synthesized from CO, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell...
BACKGROUND
Methanol, synthesized from CO, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production.
RESULTS
In the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris, associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine).
CONCLUSIONS
This study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits.
Topics: Animals; Methanol; Pichia; Carbon; Recombinant Proteins
PubMed: 37770920
DOI: 10.1186/s12934-023-02198-9 -
Microbial Cell Factories Apr 2024The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like...
BACKGROUND
The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein.
RESULTS
The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct.
CONCLUSIONS
The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.
Topics: Animals; Green Fluorescent Proteins; Weevils; Flow Cytometry; Saccharomycetales
PubMed: 38609906
DOI: 10.1186/s12934-024-02382-5 -
Food Chemistry: X Oct 2023Microbial activity during spontaneous fermentation in alcoholic beverages have driven in developing the chemical and aromatic characteristic of products but not clear in...
Microbial activity during spontaneous fermentation in alcoholic beverages have driven in developing the chemical and aromatic characteristic of products but not clear in apricot wines. We have characterised the composition of fungal communities and volatile metabolites in apricot wine spontaneous fermentation among two Shaanxi regions. Results showed that , , and , were the dominant fungi in apricot wine fermentation. A total of 80 volatiles including esters, alcohols, acids and terpenes were detected from two apricot wines. Their correlations suggested that apricot wine aroma was mainly affected by , rather than we commonly considered. Furthermore, reinforced inoculation of LQD20 has exhibited the commendable potential in enhancing sensory qualities. The results of this study provide fundamental information of the indigenous microbiota in microbial dynamic during apricot wine fermentation, which would be helpful in exploiting the strains with potential for industrial use as starter cultures.
PubMed: 37780311
DOI: 10.1016/j.fochx.2023.100862 -
The Protein Journal Aug 2023COVID-19 is a disease that have affected the entire world, and it continues to spread with new variants. A patient's innate immune system plays a critical role in the...
COVID-19 is a disease that have affected the entire world, and it continues to spread with new variants. A patient's innate immune system plays a critical role in the mild and severe transition of COVID-19. Antimicrobial peptides (AMPs), which are important components of the innate immune system, are potential molecules to fight pathogenic bacteria, fungi, and viruses. Human β-defensin 2 (hBD-2), a 41-amino-acid antimicrobial peptide, is one of the defensins inducibly expressed in the skin, lungs, and trachea in humans. In this study, it was aimed to investigate the interaction of hBD-2 produced recombinantly in Pichia pastoris with the human angiotensin-converting enzyme 2 (ACE-2) under in vitro conditions. First, hBD-2 was cloned in P. pastoris X-33 via the pPICZαA vector, a yeast expression platform, and its expression was confirmed by SDS-PAGE, western blotting, and qRT-PCR. Then, the interaction between recombinant hBD-2 and ACE-2 proteins was revealed by a pull-down assay. In light of these preliminary experiments, we suggest that the recombinantly produced hBD-2 may be protective against SARS-CoV-2 and be used as a supplement in treatment. However, current findings need to be supported by cell culture studies, toxicity analyses, and in vivo experiments.
Topics: Humans; beta-Defensins; Angiotensin-Converting Enzyme 2; COVID-19; SARS-CoV-2; Pichia
PubMed: 37291459
DOI: 10.1007/s10930-023-10130-8