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16 pages, 3834 KiB

Open AccessArticle

Construction of a Yeast Cell-Based Assay System to Analyze SNAP25-Targeting Botulinum Neurotoxins

by Shilin Chen, Feng Li, Guoyu Liu, Yuqing Li, Zijie Li, Yishi Liu and Hideki Nakanishi

Microorganisms 2023, 11(5), 1125; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms11051125 - 26 Apr 2023

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Herein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins, including synaptosomal-associated protein 25 (SNAP25). BoNT-LCs [...] Read more.

Herein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins, including synaptosomal-associated protein 25 (SNAP25). BoNT-LCs are metalloproteases, and each BoNT-LC recognizes and cleaves conserved domains in SNAREs termed the SNARE domain. In the budding yeast Saccharomyces cerevisiae, the SNAP25 ortholog Spo20 is required for production of the spore plasma membrane; thus, defects in Spo20 cause sporulation deficiencies. We found that chimeric SNAREs in which SNARE domains in Spo20 are replaced with those of SNAP25 are functional in yeast cells. The Spo20/SNAP25 chimeras, but not Spo20, are sensitive to digestion by BoNT-LCs. We demonstrate that spo20∆ yeasts harboring the chimeras exhibit sporulation defects when various SNAP25-targeting BoNT-LCs are expressed. Thus, the activities of BoNT-LCs can be assessed by colorimetric measurement of sporulation efficiencies. Although BoNTs are notorious toxins, they are also used as therapeutic and cosmetic agents. Our assay system will be useful for analyzing novel BoNTs and BoNT-like genes, as well as their manipulation. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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11 pages, 1753 KiB

Open AccessArticle

An Integrated Process for the Xylitol and Ethanol Production from Oil Palm Empty Fruit Bunch (OPEFB) Using Debaryomyces hansenii and Saccharomyces cerevisiae

by Efri Mardawati, Emilda Ayu Febrianti, Hana Nur Fitriana, Tri Yuliana, Norisca Aliza Putriana, Sri Suhartini and Kasbawati

Microorganisms 2022, 10(10), 2036; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10102036 - 14 Oct 2022

Cited by 7 | Viewed by 1839

Abstract

Oil palm empty fruit bunch (OPEFB) is the largest biomass waste from the palm oil industry. The OPEFB has a lignocellulose content of 34.77% cellulose, 22.55% hemicellulose, and 10.58% lignin. Therefore, this material’s hemicellulose and cellulose content have a high potential for xylitol [...] Read more.

Oil palm empty fruit bunch (OPEFB) is the largest biomass waste from the palm oil industry. The OPEFB has a lignocellulose content of 34.77% cellulose, 22.55% hemicellulose, and 10.58% lignin. Therefore, this material’s hemicellulose and cellulose content have a high potential for xylitol and ethanol production, respectively. This study investigated the integrated microaerobic xylitol production by Debaryomyces hansenii and anaerobic ethanol semi simultaneous saccharification and fermentation (semi-SSF) by Saccharomyces cerevisiae using the same OPEFB material. A maximum xylitol concentration of 2.86 g/L was obtained with a yield of 0.297 g/g_xylose. After 96 h of anaerobic fermentation, the maximum ethanol concentration was 6.48 g/L, corresponding to 71.38% of the theoretical ethanol yield. Significant morphological changes occurred in the OPEFB after hydrolysis and xylitol and ethanol fermentation were shown from SEM analysis. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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15 pages, 1780 KiB

Open AccessArticle

Towards the Development of Microbial Ecotoxicology Testing Using Chlorpyrifos Contaminated Sediments and Marine Yeast Isolates as a Model

by Gustavo Echeverri-Jaramillo, Beatriz Jaramillo-Colorado, Howard Junca and Claudia Consuegra-Mayor

Microorganisms 2022, 10(10), 2019; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10102019 - 13 Oct 2022

Viewed by 1454

Abstract

Chlorpyrifos (CP), a widely used pesticide, and its metabolite 3,5,6-trichloro-2-pyridinol (3,5,6-TCP), are xenobiotic compounds detected in many biomes, notably in marine sediments, all over the world. These compounds are posing a serious environmental and health problem given their toxicity to wildlife and possible [...] Read more.

Chlorpyrifos (CP), a widely used pesticide, and its metabolite 3,5,6-trichloro-2-pyridinol (3,5,6-TCP), are xenobiotic compounds detected in many biomes, notably in marine sediments, all over the world. These compounds are posing a serious environmental and health problem given their toxicity to wildlife and possible exposure effects to human neurodevelopment. Microorganisms at CP-impacted environments could harbor metabolic capabilities that can be used as indicators of the biological effects of the contaminant and could encode selected functions reactive against contaminants. Those features could be used for microbial ecotoxicology applications by collectively using analytical, enzymatic, microbiological and toxicological techniques in order to assess the biological effects of pollutants and other environmental/climatic stressors in ecosystems. The objective of this study was to assess the variability in the metabolic responses of yeast isolates from CP-contaminated marine sediments as potential biological indicators for microbial ecotoxicology testing. Sediment samples from a South Caribbean tropical shore (Cartagena Bay, Colombia) were collected, and deoxyribonucleic acid (DNA) was recovered from lyophilized aliquots. The DGGE (Denaturing Gradient Gel Electrophoresis) technique targeting fungal Internal Transcribed Spacer (ITS) showed the great diversity of fungal types. Simultaneously, yeast strains were isolated from the freshly collected sediment samples. Physiological characterization including API 20C and antibiosis tests, growth patterns at salt concentrations (2/4/10/25%), temperatures (4/25/37/45 °C), esterase activity assay and resistance tests to CP/TCP toxicity resulted in 10 isolated yeast strains, identified as Candida spp. (6), Cryptococcus spp. (3). and Rhodotorula spp. (1), showing promising characteristics to be used as a test for yeast-based ecotoxicity indicators. The patterns of carbohydrate assimilation, low antibiosis, presence of esterases/lipases, growth in a wide range of temperatures and salt concentrations, and tolerance to minimal inhibitory concentrations of CP and TCP are factors useful for testing environmental samples. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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20 pages, 1694 KiB

Open AccessArticle

The Effect of Different Substrates on the Morphological Features and Polyols Production of Endomyces magnusii Yeast during Long-Lasting Cultivation

by Anastasia S. Kokoreva, Elena P. Isakova, Vera M. Tereshina, Olga I. Klein, Natalya N. Gessler and Yulia I. Deryabina

Microorganisms 2022, 10(9), 1709; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10091709 - 25 Aug 2022

Cited by 3 | Viewed by 1801

Abstract

The study on the influence of different glucose concentrations (2%, 0.5%, and 0.2%) and glycerol (1%) on the morphological and physiological features, as well as the composition of soluble carbohydrates, was performed using Endomyces magnusii yeast. Two-factor analysis of variance with repetitions to [...] Read more.

The study on the influence of different glucose concentrations (2%, 0.5%, and 0.2%) and glycerol (1%) on the morphological and physiological features, as well as the composition of soluble carbohydrates, was performed using Endomyces magnusii yeast. Two-factor analysis of variance with repetitions to process the data of the cell size changes showed that the substrate type affected cell size the most. The cells with 2% glucose were 30–35% larger than those growing on glycerol. The decrease in the initial glucose concentration up to 0.5–0.2% slightly changed the cell length. However, even in the logarithmic growth phase pseudo-mycelium of two to four cells appeared in the cultures when using low glucose, unlike those using glycerol. Throughout the whole experiment, more than 90% of the populations remained viable on all of the substrates tested. The ability for colony formation decreased during aging. Nevertheless, at the three-week stage, upon substrate restriction (0.2% glucose), it was twice higher than those under the other conditions. The respiration rate also decreased and exceeded not more than 10% of that in the logarithmic phase. By the end of the experiment, the cyanide-sensitive respiration share decreased up to 40% for all types of substrates. The study of soluble cytosol carbohydrates showed that the cultures using 2% glucose and 1% glycerol contained mainly arabitol and mannitol, while at low glucose concentrations they were substituted for inositol. The formation of inositol is supposed to be related to pseudo-mycelium formation. The role of calorie restriction in the regulation of carbohydrate synthesis and the composition in the yeast and its biotechnological application is under consideration. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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15 pages, 2385 KiB

Open AccessArticle

Establishment of a Cre-loxP System Based on a Leaky LAC4 Promoter and an Unstable panARS Element in Kluyveromyces marxianus

by Haiyan Ren, Anqi Yin, Pingping Wu, Huanyu Zhou, Jungang Zhou, Yao Yu and Hong Lu

Microorganisms 2022, 10(6), 1240; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10061240 - 17 Jun 2022

Cited by 1 | Viewed by 2361

Abstract

The Cre-loxP system produces structural variations, such as deletion, duplication, inversion and translocation, at specific loci and induces chromosomal rearrangements in the genome. To achieve chromosomal rearrangements in Kluyveromyces marxianus, the positions and sequences of centromeres were identified in this species for [...] Read more.

The Cre-loxP system produces structural variations, such as deletion, duplication, inversion and translocation, at specific loci and induces chromosomal rearrangements in the genome. To achieve chromosomal rearrangements in Kluyveromyces marxianus, the positions and sequences of centromeres were identified in this species for the first time. Next, a Cre-loxP system was established in K. marxianus. In this system, the Cre recombinase was expressed from a leaky LAC4 promoter in a plasmid to alleviate the cytotoxicity of Cre, and the unstable plasmid contained a panARS element to facilitate the clearance of the plasmid from the cells. By using LAC4 as a reporter gene, the recombination frequencies between loxP sites or loxPsym sites were 99% and 73%, respectively. A K. marxianus strain containing 16 loxPsym sites in the genome was constructed. The recombination frequency of large-scale chromosomal rearrangements between 16 loxPsym sites was up to 38.9%. Our study provides valuable information and tools for studying chromosomal structures and functions in K. marxianus. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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15 pages, 5742 KiB

Open AccessArticle

Municipal Landfill Leachate Treatment and Sustainable Ethanol Production: A Biogreen Technology Approach

by Mahmod Sidati Ali Abobaker, Husnul Azan Tajarudin, Abdul Latif Ahmad, Wan Maznah Wan Omar and Charles Ng Wai Chun

Microorganisms 2022, 10(5), 880; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10050880 - 22 Apr 2022

Cited by 3 | Viewed by 1629

Abstract

Sustainable material sources are an important agenda to protect the environment and to meet human needs. In this study, Scenedesmus sp. was used to treat municipal landfill leachate via batch and continuous cultivation modes to protect the environment and explore sufficient biomass production [...] Read more.

Sustainable material sources are an important agenda to protect the environment and to meet human needs. In this study, Scenedesmus sp. was used to treat municipal landfill leachate via batch and continuous cultivation modes to protect the environment and explore sufficient biomass production for bioethanol production using Saccharomyces cerevisiae. Physicochemical characteristics of leachate were determined for the phases before, during, and after the process. Batch and continuous cultivation were used to treat raw leachate to determine optimum conditions for treatment. Then, the biomass of Scenedesmus sp. with and without sonication was used as a substrate for ethanol production. Sonication was carried out for biomass cell disruption for 20 min at a frequency of 40 kHz. Through batch cultivation mode, it was found that pH 7 was the optimum condition for leachate treatment. Continuous cultivation mode had the highest removal values for COD, phosphorus, and carbohydrate, namely 82.81%, 79.70%, and 84.35%, respectively, among other modes. As for ethanol production, biomass without sonication with 9.026 mg·L⁻¹ ethanol, a biomass concentration of 3.300 µg·L⁻¹, and pH 5 were higher than biomass with sonication with 5.562 mg·L⁻¹ ethanol, a biomass concentration of 0.110 µg·L⁻¹, and pH 5. Therefore, it is evident that the leachate has the potential to be treated by Scenedesmus sp. and converted to bioethanol in line with the concept of sustainable materials. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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Review

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14 pages, 887 KiB

Open AccessReview

The Production of Pyruvate in Biological Technology: A Critical Review

by Wei Yuan, Yongbao Du, Kechen Yu, Shiyi Xu, Mengzhu Liu, Songmao Wang, Yuanyuan Yang, Yinjun Zhang and Jie Sun

Microorganisms 2022, 10(12), 2454; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10122454 - 12 Dec 2022

Cited by 7 | Viewed by 3391

Abstract

Pyruvic acid has numerous applications in the food, chemical, and pharmaceutical industries. The high costs of chemical synthesis have prevented the extensive use of pyruvate for many applications. Metabolic engineering and traditional strategies for mutation and selection have been applied to microorganisms to [...] Read more.

Pyruvic acid has numerous applications in the food, chemical, and pharmaceutical industries. The high costs of chemical synthesis have prevented the extensive use of pyruvate for many applications. Metabolic engineering and traditional strategies for mutation and selection have been applied to microorganisms to enhance their ability to produce pyruvate. In the past decades, different microbial strains were generated to enhance their pyruvate production capability. In addition to the development of genetic engineering and metabolic engineering in recent years, the metabolic transformation of wild-type yeast, E. coli, and so on to produce high-yielding pyruvate strains has become a hot spot. The strategy and the understanding of the central metabolism directly related to pyruvate production could provide valuable information for improvements in fermentation products. One of the goals of this review was to collect information regarding metabolically engineered strains and the microbial fermentation processes used to produce pyruvate in high yield and productivity. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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19 pages, 717 KiB

Open AccessReview

Accumulation and Enrichment of Trace Elements by Yeast Cells and Their Applications: A Critical Review

by Jie Sun, Shiyi Xu, Yongbao Du, Kechen Yu, Yi Jiang, Hao Weng and Wei Yuan

Microorganisms 2022, 10(9), 1746; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10091746 - 30 Aug 2022

Cited by 6 | Viewed by 3085

Abstract

Maintaining the homeostasis balance of trace elements is crucial for the health of organisms. Human health is threatened by diseases caused by a lack of trace elements. Saccharomyces cerevisiae has a wide and close relationship with human daily life and industrial applications. It [...] Read more.

Maintaining the homeostasis balance of trace elements is crucial for the health of organisms. Human health is threatened by diseases caused by a lack of trace elements. Saccharomyces cerevisiae has a wide and close relationship with human daily life and industrial applications. It can not only be used as fermentation products and single-cell proteins, but also as a trace elements supplement that is widely used in food, feed, and medicine. Trace-element-enriched yeast, viz., chromium-, iron-, zinc-, and selenium-enriched yeast, as an impactful microelements supplement, is more efficient, more environmentally friendly, and safer than its inorganic and organic counterparts. Over the last few decades, genetic engineering has been developing large-scaled genetic re-design and reconstruction in yeast. It is hoped that engineered yeast will include a higher concentration of trace elements. In this review, we compare the common supplement forms of several key trace elements. The mechanisms of detoxification and transport of trace elements in yeast are also reviewed thoroughly. Moreover, genes involved in the transport and detoxification of trace elements are summarized. A feasible way of metabolic engineering transformation of S. cerevisiae to produce trace-element-enriched yeast is examined. In addition, the economy, safety, and environmental protection of the engineered yeast are explored, and the future research direction of yeast enriched in trace elements is discussed. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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26 pages, 1678 KiB

Open AccessReview

Yeasts Inhabiting Extreme Environments and Their Biotechnological Applications

by Claudia Segal-Kischinevzky, Lucero Romero-Aguilar, Luis D. Alcaraz, Geovani López-Ortiz, Blanca Martínez-Castillo, Nayeli Torres-Ramírez, Georgina Sandoval and James González

Microorganisms 2022, 10(4), 794; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10040794 - 09 Apr 2022

Cited by 33 | Viewed by 6366

Abstract

Yeasts are microscopic fungi inhabiting all Earth environments, including those inhospitable for most life forms, considered extreme environments. According to their habitats, yeasts could be extremotolerant or extremophiles. Some are polyextremophiles, depending on their growth capacity, tolerance, and survival in the face of [...] Read more.

Yeasts are microscopic fungi inhabiting all Earth environments, including those inhospitable for most life forms, considered extreme environments. According to their habitats, yeasts could be extremotolerant or extremophiles. Some are polyextremophiles, depending on their growth capacity, tolerance, and survival in the face of their habitat’s physical and chemical constitution. The extreme yeasts are relevant for the industrial production of value-added compounds, such as biofuels, lipids, carotenoids, recombinant proteins, enzymes, among others. This review calls attention to the importance of yeasts inhabiting extreme environments, including metabolic and adaptive aspects to tolerate conditions of cold, heat, water availability, pH, salinity, osmolarity, UV radiation, and metal toxicity, which are relevant for biotechnological applications. We explore the habitats of extreme yeasts, highlighting key species, physiology, adaptations, and molecular identification. Finally, we summarize several findings related to the industrially-important extremophilic yeasts and describe current trends in biotechnological applications that will impact the bioeconomy. Full article

(This article belongs to the Special Issue Biotechnological Applications of Yeasts)

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