Combined 6-benzylaminopurine and H2O2 stimulate the astaxanthin biosynthesis in Xanthophyllomyces dendrorhous.

Astaxanthin is one of the most attractive carotenoids due to its high antioxidant activity and beneficial biological properties, while Xanthophyllomyces dendrorhous is one of its main microbial sources. Since astaxanthin is synthesized as a response to oxidative stress, several oxidative agents have been evaluated to increase X. dendrorhous astaxanthin yields. However, the extent of the stimulation is determined by the cellular damage caused by the applied oxidative agent. Phytohormones have also been reported as stimulants of astaxanthin biosynthesis acting directly on its metabolic pathway and indirectly promoting cellular resistance to reactive oxygen species. We reasoned that both oxidative agents and phytohormones lead to increased astaxanthin synthesis, but the latter could mitigate the drawbacks of the former. Thus, here, the stimulation on astaxanthin biosynthesis, as well as the cellular and transcriptional responses of wild type X. dendrorhous to phytohormones (6-benzylaminopurine, 6-BAP; abscisic acid, ABA; and indole-3-acetic acid, IAA), and oxidative agents (glutamate, menadione, H2O2, and/or Fe2+) were evaluated as a single or combined treatments. ABA and 6-BAP were the best individual stimulants leading to 2.24- and 2.60-fold astaxanthin biosynthesis increase, respectively. Nevertheless, the effect of combined 6-BAP and H2O2 led to a 3.69-fold astaxanthin synthesis increase (0.127 ± 0.018 mg astaxanthin/g biomass). Moreover, cell viability (> 82.75%) and mitochondrial activity (> 82.2%) remained almost intact in the combined treatment (6-BAP + H2O2) compared to control (< 52.17% cell viability; < 85.3% mitochondrial activity). On the other hand, mRNA levels of hmgR, idi, crtYB, crtR, and crtS, genes of the astaxanthin biosynthetic pathway, increased transiently along X. dendrorhous fermentation due to stimulations assayed in this study. KEY POINTS: • Combined 6-BAP and H2O2 is the best treatment to increase astaxanthin yields in X. dendrorhous. • 6-BAP preserves cell integrity under oxidative H2O2 stress conditions. • 6-BAP and H2O2 increase transcriptional responses of hmgR, idi, and crt family genes transiently.

Yeast diversity in open agave fermentations across Mexico.

Yeasts are a diverse group of fungal microorganisms that are widely used to produce fermented foods and beverages. In Mexico, open fermentations are used to obtain spirits from agave plants. Despite the prevalence of this traditional practice throughout the country, yeasts have only been isolated and studied from a limited number of distilleries. To systematically describe the diversity of yeast species from open agave fermentations, here we generate the YMX-1.0 culture collection by isolating 4524 strains from 68 sites with diverse climatic, geographical, and biological contexts. We used MALDI-TOF mass spectrometry for taxonomic classification and validated a subset of the strains by ITS and D1/D2 sequencing, which also revealed two potential novel species of Saccharomycetales. Overall, the composition of yeast communities was weakly associated with local variables and types of climate, yet a core set of six species was consistently isolated from most producing regions. To explore the intraspecific variation of the yeasts from agave fermentations, we sequenced the genomes of four isolates of the nonconventional yeast Kazachstania humilis. The genomes of these four strains were substantially distinct from a European isolate of the same species, suggesting that they may belong to different populations. Our work contributes to the understanding and conservation of an open fermentation system of great cultural and economic importance, providing a valuable resource to study the biology and genetic diversity of microorganisms living at the interface of natural and human-associated environments.

Sustainable production of single-cell oil and protein from wastepaper hydrolysate: identification and optimization of a Rhodotorula mucilaginosa strain as a promising yeast.

This study investigated the potential of wastepaper hydrolysate as a sustainable and low-cost carbon source for single-cell oil and protein production, attending to the growing need for alternative feedstocks and waste management strategies. Wastepaper, characterized by its high carbohydrate content, was subjected to enzymatic and chemo-enzymatic treatments for carbohydrate release. The chemo-enzymatic treatment performed better, yielding 65.3 g l-1 of fermentable sugars. A total of 62 yeast strains were screened for single-cell oil accumulation, identifying Rhodotorula mucilaginosa M1K4 as the most advantageous oleaginous yeast. M1K4 lipid production was optimized in liquid culture, and its fatty acid profile was analyzed, showing a high content of industrially valuable fatty acids, particularly palmitic (28%) and oleic (51%). Batch-culture of M1K4 in a 3-l reactor demonstrated the strain's ability to utilize wastepaper hydrolysate as a carbon source, with dry cell weight, total lipid and protein production of 17.7 g l-1, 4.5 g l-1, and 2.1 g l-1, respectively. Wastepaper as a substrate provides a sustainable solution for waste management and bioproduction. This research highlights the potential of R. mucilaginosa for lipid and protein production from wastepaper hydrolysate.

Draft Genome Sequence of a Kazachstania humilis Strain Isolated from Agave Fermentation.

The ascomycetous yeast Kazachstania humilis is an active species in backslopped sourdough and in the spontaneous fermentation of several traditional foods and beverages. Here, we report the draft genome sequence of a K. humilis strain isolated from agave must from a traditional distillery in Mexico.

Metabolic engineering for high yield synthesis of astaxanthin in Xanthophyllomyces dendrorhous.

Astaxanthin is a carotenoid with a number of assets useful for the food, cosmetic and pharmaceutical industries. Nowadays, it is mainly produced by chemical synthesis. However, the process leads to an enantiomeric mixture where the biologically assimilable forms (3R, 3'R or 3S, 3'S) are a minority. Microbial production of (3R, 3'R) astaxanthin by Xanthophyllomyces dendrorhous is an appealing alternative due to its fast growth rate and easy large-scale production. In order to increase X. dendrorhous astaxanthin yields, random mutant strains able to produce from 6 to 10 mg/g dry mass have been generated; nevertheless, they often are unstable. On the other hand, site-directed mutant strains have also been obtained, but they increase only the yield of non-astaxanthin carotenoids. In this review, we insightfully analyze the metabolic carbon flow converging in astaxanthin biosynthesis and, by integrating the biological features of X. dendrorhous with available metabolic, genomic, transcriptomic, and proteomic data, as well as the knowledge gained with random and site-directed mutants that lead to increased carotenoids yield, we propose new metabolic engineering targets to increase astaxanthin biosynthesis.

The intestinal mycobiota and its relationship with overweight, obesity and nutritional aspects.

BACKGROUND: The fungal community of the gastrointestinal tract has recently become of interest, and knowledge of its relationship with the development of obesity is scarce. The present study aimed to evaluate the cultivable fungal fraction from the microbiota and to analyze its relationship with obesity. METHODS: Samples were taken from 99 participants with normal weight, overweight and obesity (n = 31, 34 and 34, respectively) and were cultivated in selective medium, and the cultivable yeasts were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anthropometric and biochemical measures were also evaluated. RESULTS: Eutrophic, overweight and obese groups presented concentrations of 1.6, 2.16 and 2.19 log10  colony-forming units g-1 yeast, respectively. Ascomycota and Basidiomycota were the two identified phyla. At the genus level, Candida spp. showed a relatively high prevalence, and 10 different species were detected: Candida glabrata, Candida orthopsilosis, Candida lambica, Candida kefyr, Candida albicans, Candida krusei, Candida valida, Candida parapsilosis, Candida utilis and Candida humilis (with relative abundances of 71.72%, 5.05%, 21.21%, 6.06%, 29.29%, 27.27%, 8.08%, 16.16%, 1.01% and 2.02%, respectively). CONCLUSIONS: The obese group presented a higher prevalence of Candida albicans. Furthermore, Candida albicans, Candida kefyr and Rhodotorula mucilaginosa showed a high positive correlation with obesity, weight gain and fat mass and showed a negative correlation with high-density lipoprotein and lean mass, parameters related to weight loss.

Screening of Gastrointestinal Lipase Inhibitors Produced by Microorganisms Isolated from Soil and Lake Sediments.

Gastrointestinal lipase inhibitors are molecules of pharmaceutical interest due to their use as anti-obesity drugs. In this study, forty strains isolated from soil and sediments were identified with the ability to produce inhibition of gastrointestinal lipase activity. The biomass extract of these strains showed at least 50% inhibition in the hydrolysis of tributyrin by recombinant human pancreatic lipase (rHPL) or rabbit gastric lipase (RGL) by in vitro assays. Based on gene sequencing, the isolates were identified mainly as Streptomycetes. Moreover, none of the identified strains has been reported to be lipase inhibitor producers, so they can be viewed as potential sources for obtaining new drugs. IC50 values of the three best inhibitor extracts showed that AC104-10 was the most promising strain for production of gastrointestinal lipase inhibitors. AC104-10 shows 99% homology (16S rRNA gene fragment) to Streptomyces cinereoruber strain NBRC 12756. An inhibitory study over trypsin activity revealed that AC104-10 extract, as well as THL, had no significant effect on the activity of this protease, showing its specificity for lipases. In addition, analyzes by MALDI-TOF mass spectrometry of the enzyme-inhibitor complex revealed that there is a covalent interaction of the AC104-10 inhibitor with the catalytic serine of the pancreatic lipase, and that the molecular weight of the inhibitor is approximately 686.19 Da.

Yeast Species, Strains, and Growth Media Mediate Attraction of Drosophila suzukii (Diptera: Drosophilidae).

Specific ecological interactions between insects and microbes have potential in the development of targeted pest monitoring or control techniques for the spotted wing drosophilid, Drosophila suzukii (Matsumura), an exotic invasive pest of soft fruit. To evaluate D. suzukii attraction to yeast species from preferred types of fruit, three yeasts were isolated from blackberry fruit and two yeasts from raspberry fruit and used to bait simple plastic bottle traps. Saccharomyces cerevisiae and Hanseniaspora uvarum were identified from blackberries, whereas a different H. uvarum strain was identified from raspberry. Yeast identification was based on sequence analysis of the D1/D2 domain of the large subunit 26S rRNA gene. Commercial baker's yeast (S. cerevisiae) was similar or more effective for the capture of D. suzukii males and females than yeasts isolated from blackberry or raspberry when grown in sucrose. However, when grown in corn syrup, a strain of S. cerevisiae from blackberry captured the highest number of females and a strain of H. uvarum isolated from raspberry captured high numbers of males and females. Species of Candida, Hanseniaspora, and Pichia from a laboratory yeast collection did not outperform baker's yeast in pairwise tests when grown in sucrose solution or yeast-peptone-dextrose medium. The raspberry strain of H. uvarum grown in corn syrup outperformed S. cerevisiae grown in sucrose, in terms of captures in baited traps under laboratory conditions. We conclude that yeast species, strain, and growth medium can have a marked influence on D. suzukii attraction to baited traps, a finding that could assist in the development of yeast-related monitoring or control techniques targeted at this pest.

Biotechnological potential of bacteria isolated from cattle environments of desert soils in Sonora Mexico.

The aim of this research was to study the hydrolytic potential of bacteria isolated from cattle environments of two desert soils in one of the driest and hottest zones in America. A total of 26 points were sampled, 144 strains were isolated, and 50 strains were selected for the characterization of esterase, lipase, protease, and amylase activities and for 16S rRNA identification. Strains of the Bacillus, Pseudomonas, Acinetobacter, Enterobacter, Providencia, Escherichia, and Pantoea genera were identified. Comparisons of the proteolytic activity of the secretome from 14 strains (Bacillus n = 7, Escherichia n = 2; Providencia, Pseudomonas, Enterobacter, Pantoea and Acinetobacter n = 1) were performed. Four strains of Bacillus showed the highest proteolytic activity. These strains were characterized through a comparative analysis of pH and temperature as well as the effects of salt concentration on protease activity. Maximum proteolytic activity occurred in the range of pH 7-9 and temperatures between 50 and 70 °C for B. subtilis WD01, B. tequilensis WS11, B. tequilensis WS13, and B. tequilensis WS14. At a 20% NaCl concentration, the proteolytic activity retained was 71.4%, 65%, and 79.8% for WD01, WS11, and WS13, respectively; the activity of strain WS14 increased with 45% NaCl. Protease production by B. tequilensis WS14 with wheat, fish, and bone flours as low-cost substrates showed no differences between bone and fish flours and showed a decrease in protease production with wheat flour. The proteolytic activity in flour extracts with 20% NaCl was 82%, 75.61% and 38.04% for fish, bone and wheat flours, respectively. Data obtained in this work allow us to propose that strains isolated from environments with extreme conditions have a biotechnological potential.

Correction: Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

[This corrects the article DOI: 10.7717/peerj.3524.].

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.