A Library of Aspergillus niger Chassis Strains for Morphology Engineering Connects Strain Fitness and Filamentous Growth With Submerged Macromorphology

Submerged fermentation using filamentous fungal cell factories is used to produce a diverse portfolio of useful molecules, including food, medicines, enzymes, and platform chemicals. Depending on strain background and abiotic culture conditions, different macromorphologies are formed during fermentation, ranging from dispersed hyphal fragments to approximately spherical pellets several millimetres in diameter. These macromorphologies are known to have a critical impact on product titres and rheological performance of the bioreactor. Pilot productivity screens in different macromorphological contexts is technically challenging, time consuming, and thus a significant limitation to achieving maximum product titres. To address this bottleneck, we developed a library of conditional expression mutants in the organic, protein, and secondary metabolite cell factory Aspergillus niger. Thirteen morphology-associated genes transcribed during fermentation were placed via CRISPR-Cas9 under control of a synthetic Tet-on gene switch. Quantitative analysis of submerged growth reveals that these strains have distinct and titratable macromorphologies for use as chassis during strain engineering programs. We also used this library as a tool to quantify how pellet formation is connected with strain fitness and filamentous growth. Using multiple linear regression modelling, we predict that pellet formation is dependent largely on strain fitness, whereas pellet Euclidian parameters depend on fitness and hyphal branching. Finally, we have shown that conditional expression of the putative kinase encoding gene pkh2 can decouple fitness, dry weight, pellet macromorphology, and culture heterogeneity. We hypothesize that further analysis of this gene product and the cell wall integrity pathway in which it is embedded will enable more precise engineering of A. niger macromorphology in future.

Enhancing Saccharomyces cerevisiae Taxane Biosynthesis and Overcoming Nutritional Stress-Induced Pseudohyphal Growth

The recent technological advancements in synthetic biology have demonstrated the extensive potential socio-economic benefits at laboratory scale. However, translations of such technologies to industrial scale fermentations remains a major bottleneck. The existence and lack of understanding of the major discrepancies in cultivation conditions between scales often leads to the selection of suboptimal bioprocessing conditions, crippling industrial scale productivity. In this study, strategic design of experiments approaches were coupled with state-of-the-art bioreactor tools to characterize and overcome nutritional stress for the enhanced production of precursors to the blockbuster chemotherapy drug, Taxol, in S. cerevisiae cell factories. The batch-to-batch variation in yeast extract composition was found to trigger nutritional stress at a mini-bioreactor scale, resulting in profound changes in cellular morphology and the inhibition of taxane production. The cells shifted from the typical budding morphology into striking pseudohyphal cells. Doubling initial yeast extract and peptone concentrations (2×YP) delayed filamentous growth, and taxane accumulation improved to 108 mg/L. Through coupling a statistical definitive screening design approach with the state-of-the-art high-throughput micro-bioreactors, the total taxane titers were improved a further two-fold, compared to the 2×YP culture, to 229 mg/L. Filamentous growth was absent in nutrient-limited microscale cultures, underlining the complex and multifactorial nature of yeast stress responses. Validation of the optimal microscale conditions in 1L bioreactors successfully alleviated nutritional stress and improved the titers to 387 mg/L. Production of the key Taxol precursor, T5αAc, was improved two-fold to 22 mg/L compared to previous maxima. The present study highlights the importance of following an interdisciplinary approach combining synthetic biology and bioprocessing technologies for effective process optimization and scale-up.

Plasma membrane phosphatidylinositol-4-phosphate is not necessary for Candida albicans viability, yet is key for cell wall integrity and systemic infection

Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi is required for the budding to filamentous growth transition in the human pathogenic fungus Candida albicans, however the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e. Efr3, Ypp1 and Stt4. The amount of plasma membrane PI(4)P in the efr3∆/∆ and ypp1∆/∆ mutant was ~60% and ~40% of the wild-type strain, respectively, whereas it was nearly undetectable in the stt4∆/∆ mutant. All three mutants had reduced plasma membrane phosphatidylserine (PS). Although these mutants had normal yeast phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity and had an increased exposure of cell wall β(1,3)-glucan, yet they induced a range of hyphal specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the efr3∆/∆ had wild-type virulence, the ypp1∆/∆ mutant had attenuated virulence and the stt4∆/∆ mutant caused no lethality. In the mouse model of orpharyngeal candidiasis, only the ypp1∆/∆ mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity and virulence in C. albicans.

Phenotypic Switching and Filamentation in Candida haemulonii, an Emerging Opportunistic Pathogen of Humans

The capacity to switch between distinct cell types, known as phenotypic switching, is a common strategy adopted by Candida species to adapt to diverse environments. Despite considerable studies on phenotypic plasticity of various Candida species, Candida haemulonii is considered to be incapable of phenotypic switching or filamentous growth.

Clarifying intercellular signalling in yeast: Saccharomyces cerevisiae does not undergo a quorum sensing-dependent switch to filamentous growth

Saccharomyces cerevisiae can alter its morphology to a filamentous form associated with unipolar budding in response to environmental stressors. Induction of filamentous growth is suggested under nitrogen deficiency in response to alcoholic signalling molecules through a quorum sensing mechanism. To investigate this claim, we analysed the budding pattern of S. cerevisiae cells over time under low nitrogen while concurrently measuring cell density and extracellular metabolite concentration. We found that the proportion of cells displaying unipolar budding increased between local cell densities of 4.8x106 and 5.3x107 cells/ml within 10 to 20 hours of growth. However, the observed increase in unipolar budding could not be reproduced when cells were prepared at the critical cell density and in conditioned media. Removing the nutrient restriction by growth in high nitrogen conditions also resulted in an increase in unipolar budding between local cell densities of 5.2x106 and 8.2x107 cells/ml within 10 to 20 hours of growth, but there were differences in metabolite concentration compared to the low nitrogen conditions. This suggests that neither cell density, metabolite concentration, nor nitrogen deficiency were necessary or sufficient to increase the proportion of unipolar budding cells. It is therefore unlikely that quorum sensing is the mechanism controlling the switch to filamentous growth in S. cerevisiae. Only a high concentration of the putative signalling molecule, 2-phenylethanol resulted in an increase in unipolar budding, but this concentration was not physiologically relevant. We suggest that the compound 2-phenylethanol acts through a toxicity mechanism, rather than quorum sensing, to induce filamentous growth.

Mitochondrial perturbation reduces susceptibility to xenobiotics through altered efflux in Candida albicans

Candida albicans is a leading human fungal pathogen, which can cause superficial infections or life-threatening systemic disease in immunocompromised individuals. The ability to transition between yeast and filamentous forms is a major virulence trait of C. albicans, and a key regulator of this morphogenetic transition is the molecular chaperone Hsp90. To explore the mechanisms governing C. albicans morphogenesis in response to Hsp90 inhibition, we performed a functional genomic screen using the gene replacement and conditional expression (GRACE) collection to identify mutants that are defective in filamentation in response to the Hsp90 inhibitor, geldanamycin. We found that transcriptional repression of genes involved in mitochondrial function blocked filamentous growth in response to the concentration of Hsp90 inhibitor used in the screen, and this was attributable to increased resistance to the compound. Further exploration revealed that perturbation of mitochondrial function reduced susceptibility to two structurally distinct Hsp90 inhibitors, geldanamycin and radicicol, such that filamentous growth was restored in the mitochondrial mutants by increasing the compound concentration. Deletion of two representative mitochondrial genes, MSU1 and SHY1, enhanced cellular efflux and reduced susceptibility to diverse intracellularly acting compounds. Additionally, screening a C. albicans efflux pump gene deletion library implicated Yor1 in efflux of geldanamycin and Cdr1, in efflux of radicicol. Deletion of these transporter genes restored sensitivity to Hsp90 inhibitors in MSU1 and SHY1 homozygous deletion mutants, thereby enabling filamentation. Taken together, our findings suggest that mitochondrial dysregulation elevates cellular efflux and consequently reduces susceptibility to xenobiotics in C. albicans.

A Case of Alopecia and Its Homoeopathic Approach

Hair is an ornament & beauty of every person life. It is an outward filamentous growth made up of keratin protein. It has major cosmetic role along with other sensory & homeostatic function. Homoeopathy has wonderful role to reduce hair fall & re-growth of new hair. This article is an attempt to overview of hair cycle, why hair is fall, homoeopathic approach of hair fall or alopecia with different repertorial rubric analysis & a very interesting case history. Keywords Hair fall; Homoeopathy; Constitutional medicine; Miasm

Actin Cytoskeleton Regulation by the Yeast NADPH Oxidase Yno1p Impacts Processes Controlled by MAPK Pathways

Reactive oxygen species (ROS) that exceed the antioxidative capacity of the cell can be harmful and are termed oxidative stress. Increasing evidence suggests that ROS are not exclusively detrimental, but can fulfill important signaling functions. Recently, we have been able to demonstrate that a NADPH oxidase-like enzyme (termed Yno1p) exists in the single-celled organism Saccharomyces cerevisiae. This enzyme resides in the peripheral and perinuclear endoplasmic reticulum and functions in close proximity to the plasma membrane. Its product, hydrogen peroxide, which is also produced by the action of the superoxide dismutase, Sod1p, influences signaling of key regulatory proteins Ras2p and Yck1p/2p. In the present work, we demonstrate that Yno1p-derived H2O2 regulates outputs controlled by three MAP kinase pathways that can share components: the filamentous growth (filamentous growth MAPK (fMAPK)), pheromone response, and osmotic stress response (hyperosmolarity glycerol response, HOG) pathways. A key structural component and regulator in this process is the actin cytoskeleton. The nucleation and stabilization of actin are regulated by Yno1p. Cells lacking YNO1 showed reduced invasive growth, which could be reversed by stimulation of actin nucleation. Additionally, under osmotic stress, the vacuoles of a ∆yno1 strain show an enhanced fragmentation. During pheromone response induced by the addition of alpha-factor, Yno1p is responsible for a burst of ROS. Collectively, these results broaden the roles of ROS to encompass microbial differentiation responses and stress responses controlled by MAPK pathways.

Translational control as a novel regulator of gradient sensing and chemotropism in yeast

The yeast mating pathway regulates haploid cell fusion to form diploids in response to pheromone signaling. Study of this pathway has led to important insights into the structure and function of mitogen-activated protein kinase (MAPK) cascades, yet our understanding of how external signals are converted into specific changes in gene expression and cell morphology is incomplete. For example, the regulators of directional growth (chemotropism) remain poorly defined. Upon pheromone exposure, yeast grow asymmetrically towards a nearby mating partner (chemotropic morphogenesis) and form a mating projection (shmoo). Using non-biased genome-wide screening, we identify >20 novel positive and negative regulators of pheromone gradient sensing, shmoo development, and mating. In addition to known regulators of exocytic and endocytic pathways, several are directly involved in translational control downstream of the G-protein-regulated pheromone and filamentous growth MAPK pathways. These include the Scp160 RNA-binding protein and ribosomal proteins Asc1, Rpl12b and Rpl19b. Importantly, pheromone treatment and Gα (Gpa1) activation both stimulate Scp160 binding to, and inhibition of, Asc1, which acts downstream of glucose-activated Gα (Gpa2) on the filamentous growth pathway. We also identify Rpl12b and Rpl19b as paralog-specific positive regulators of translation of specific mating pathway components, including Scp160. Thus, the different MAPK pathways converge at the level of translational control to regulate signaling.