A rapid method for the determination of microbial biomass by dry weight using a moisture analyser with an infrared heating source and an analytical balance

Aims: Microbial biomass is an important biotechnological parameter. The traditional method for its determination involves an oven‐drying step and equilibration to room temperature before weighing, and it is tedious and time consuming. This work studied the utilisation of a moisture analyser consisting of an efficient infrared‐heating module and an analytical balance for the determination of microbial biomass by dry weight. Methods and Results: The method duration depended on the sample volume and was between 7 and 40 min for sample volumes of 1–10 ml. The method precision depended on the total dry weight analysed – 10 mg of total dry weight being sufficient to achieve coefficients of variation of 5% or less. Comparison with the conventional oven method provided a correlation coefficient r² of 0·99. The recovery of an internal standard ranged between 94·2 and 106·4% with a precision of 1·39–4·53%CV. Conclusions: Validation revealed sufficient method accuracy, precision and robustness and was successfully applied to the study of yeast and bacterial growth kinetics. Techniques are discussed that allow for increased method precision at low biomass concentrations, and equations are provided to estimate required drying time and method precision based on sample volume and total sample dry weight, respectively. Significance and Impact of the Study: This work presents a rapid method for the determination of microbial biomass, allowing for the timely implementation of biomass‐based information in biotechnological and laboratory protocols.     

A genome‐wide screen identifies genes that suppress the accumulation of spontaneous mutations in young and aged yeast cells

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age‐dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear whether the spontaneous mutation rate changes during aging and whether specific pathways are important for genome maintenance in old cells. We developed a high‐throughput replica‐pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age‐specific mutation suppression gene. While wild‐type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes may be important to preserve genome integrity specifically in old cells.     

An ex-situ and in vitro approach towards the bioremediation of carcinogenic hexavalent chromium

Abstract

Chromium, ranking the second most among toxic heavy metal pollutants in the world, causing respiratory, cardiovascular and renal problems in human beings is under study herein. We examined the biological remediation of the carcinogenic Cr (VI) polluted soils by indigenous yeast isolates. The total element analysis of the treated sample was determined by Energy Dispersion X-ray Micro Analysis (EDXMA). The sample under study was observed to have a high concentration of 458.29 mgKg^?1 Cr (VI), determined by Atomic Absorption Spectroscopy (AAS) and DPC analysis. The most tolerant isolate designated as CSR was used for in vitro and ex-situ bioremediation studies of Cr (VI). The isolate achieved significant bioremediation of 86% in vitro and 75.12% in ex-situ method. The optimal conditions for in vitro bioremediation were found to be 28?°C and a pH of 6. The ITS1, 5.8S rRNA and D1, D2 domain of LSU rRNA gene characterization of the isolate CSR illustrated that it belongs to Ustilago genera. The isolate was deposited in NCBI GenBank as Ustilago sp. CSR (KY284846). Although, Ustilago is generally a pathogenic fungus, our study opens up the scope of using Ustilago spp. for bioremediation of the carcinogenic heavy metal Chromium.     

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata

Riboflavin (vitamin B₂) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.     

Integrative structure and function of the yeast exocyst complex

Exocyst is an evolutionarily conserved hetero‐octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative‐stain electron microscopy (EM) at ~16 Å resolution, 434 disuccinimidyl suberate and 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride cross‐links from chemical‐crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo‐EM structure, cross‐links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo‐EM structure, except for Sec5. While not observed in the cryo‐EM map, the integrative model localizes the N‐terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low‐resolution structural data can inform biologically relevant hypotheses, even in the absence of high‐resolution data.     

In-vivo Single-Molecule Imaging in Yeast: Applications and Challenges

Single-molecule imaging has gained momentum to quantify the dynamics of biomolecules in live cells, as it provides direct real-time measurements of various cellular activities under their physiological environment. Yeast, a simple and widely used eukaryote, serves as a good model system to quantify single-molecule dynamics of various cellular processes because of its low genomic and cellular complexities, as well as its facile ability to be genetically manipulated. In the past decade, significant developments have been made regarding the intracellular labeling of biomolecules (proteins, mRNA, fatty acids), the microscopy setups to visualize single-molecules and capture their fast dynamics, and the data analysis pipelines to interpret such dynamics. In this review, we summarize the current state of knowledge for the single-molecule imaging in live yeast cells to provide a ready reference for beginners. We provide a comprehensive table to demonstrate how various labs tailored the imaging regimes and data analysis pipelines to estimate various biophysical parameters for a variety of biological processes. Lastly, we present current challenges and future directions for developing better tools and resources for single-molecule imaging in live yeast cells.     

The Degree and Length of O-Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, P_GAP and P_AOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with P_GAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with P_AOX1. The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system.     

Forest Saccharomyces paradoxus are robust to seasonal biotic and abiotic changes

Microorganisms are famous for adapting quickly to new environments. However, most evidence for rapid microbial adaptation comes from laboratory experiments or domesticated environments, and it is unclear how rates of adaptation scale from human‐influenced environments to the great diversity of wild microorganisms. We examined potential monthly‐scale selective pressures in the model forest yeast Saccharomyces paradoxus. Contrary to expectations of seasonal adaptation, the S. paradoxus population was stable over four seasons in the face of abiotic and biotic environmental changes. While the S. paradoxus population was diverse, including 41 unique genotypes among 192 sampled isolates, there was no correlation between S. paradoxus genotypes and seasonal environments. Consistent with observations from other S. paradoxus populations, the forest population was highly clonal and inbred. This lack of recombination, paired with population stability, implies that selection is not acting on the forest S. paradoxus population on a seasonal timescale. Saccharomyces paradoxus may instead have evolved generalism or phenotypic plasticity with regard to seasonal environmental changes long ago. Similarly, while the forest population included diversity among phenotypes related to intraspecific interference competition, there was no evidence for active coevolution among these phenotypes. At least ten percent of the forest S. paradoxus individuals produced “killer toxins,” which kill sensitive Saccharomyces cells, but the presence of a toxin‐producing isolate did not predict resistance to the toxin among nearby isolates. How forest yeasts acclimate to changing environments remains an open question, and future studies should investigate the physiological responses that allow microbial cells to cope with environmental fluctuations in their native habitats.     

优良菌种发酵葡萄酒优化条件的探讨

以诱变耐低温果酒酵母菌种YU2.28和产香酵母S15.3为发酵菌株,进行了葡萄酒发酵条件优化的试验研究.探讨了菌种生长温度、通氧量等因素,通过对菌种的生长情况和发酵醪液中总酯含量的变化分析,确定了自选酵母酿制葡萄酒的最佳技术参数,并对优化条件下发酵得到的葡萄酒进行GC/MS分析.结果显示：YU2.28和S15.3以1：3比例的混合发酵,接种量3%,调节醪液pH值为4.0,SO2添加量40 mg/L,发酵温度20℃,主发酵6 d内控制以230r/min的摇床转速进行摇瓶发酵,并进行9 h（每天1.5 h）供氧处理,后发酵30 d,酿造出的葡萄酒品质较佳,具有酒体丰盈,酒液澄清透亮,香气醇和的特征.成品酒香气成分共检测出醇类9种,酯类8种,酸类6种和少量的醛类、酮类等成分.