Harnessing dual applications of a novel ascomycetes yeast, Starmerella cerana sp. nov., as a biocatalyst for stereoselective ketone reduction and biosurfactant production.

Introduction: New bioresources for catalytic application and fine chemical synthesis are the need of the hour. In an effort to find out new biocatalyst for oxidation-reduction reaction, leading to the synthesis of chiral intermediates, novel yeast were isolated from unique niche and employed for the synthesis of value added compounds. Methods: To determine the genetic relatedness of the isolated strain, HSB-15T, sequence analysis of the internal transcribed spacer (ITS) and D1/D2 domains of the 26S rRNA gene sequence was carried out. The distinctive features of the strain HSB-15T were also identified by phenotypic characterization. The isolated strain HSB-15T was employed for the reduction of selected naphthyl ketones to their corresponding alcohols and a biosurfactant was isolated from its culture broth. Results: The analysis of the ITS and D1/D2 domains of the 26S rRNA gene revealed that strain HSB-15T is closely related to the type strain of Starmerella vitae (CBS 15147T) with 96.3% and 97.7% sequence similarity, respectively. However, concatenated sequences of the ITS gene and D1/D2 domain showed 94.6% sequence similarity. Phenotypic characterization indicated significant differences between strain HSB-15T and its closely related species and consequently, it was identified as a novel species, leading to the proposal of the name Starmerella cerana sp. nov. The strain was able to reduce selected naphthyl ketones to their corresponding alcohols with remarkable efficiency, within a 12-hours. The strain HSB-15T also produced a surfactant in its culture broth, identified as sophorolipid upon analysis. Discussion: The study explored the potential of the novel strain, HSB-15T, as a whole-cell biocatalyst for the reduction of naphthyl ketones to their corresponding alcohols and also reports its capability to produce sophorolipid, a biosurfactant, in its culture broth. This dual functionality of HSB-15T both as biocatalyst and biosurfactant producer enhances its applicability in biotechnology and environmental science.

Unraveling the mechanistic insights of sophorolipid-capped gold nanoparticle-induced cell death in Vibrio cholerae.

IMPORTANCE: Vibrio cholerae, a Gram-negative bacterium, is the causative agent of a fatal disease, "cholera." Prevention of cholera outbreak is possible by eliminating the bacteria from the environment. However, antimicrobial resistance developed in microorganisms has posed a threat and challenges to its treatment. Application of nanoparticles is a useful and effective option for the elimination of such microorganisms. Metal-based nanopaticles exhibit microbial toxicity through non-specific mechanisms. To prevent resistance development and increase antibacterial efficiency, rational designing of nanoparticles is required. Thus, knowledge on the exact mechanism of action of nanoparticles is highly essential. In this study, we explore the possible mechanisms of antibacterial activity of AuNPs-SL against V. cholerae. We show that the interaction of AuNPs-SL with V. cholerae enhances ROS production and membrane depolarization, change in permeability, and leakage of intracellular content. This action leads to the depletion of cellular ATP level, DNA damage, and subsequent cell death.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Deciphering the Antibacterial Role of Peptide From Bacillus subtilis subsp. spizizenii Ba49 Against Staphylococcus aureus.

An increase in antibiotic resistance has led to escalating the need for the development of alternate therapy. Antimicrobial peptides (AMPs) are at the forefront of replacing conventional antibiotics, showing slower development of drug resistance, antibiofilm activity, and the ability to modulate the host immune response. The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that jeopardize most conventional antibiotics are known to be involved in severe respiratory tract, bloodstream, urinary tract, soft tissue, and skin infections. Among them, S. aureus is an insidious microbe and developed resistance against conventional antibiotics. In the present study, an AMP (named as peptide-Ba49) isolated from Bacillus subtilis subsp. spizizenii strain from Allium cepa (the common onion) exhibited strong antibacterial efficacy against S. aureus ATCC 25923. The mode of action of this peptide-Ba49 on S. aureus was deciphered through various sensitive probes, i.e., DiSC3 (5) and H2DCFDA, suggesting the peptide-Ba49 to be acting upon through change in membrane potential and by triggering the production of reactive oxygen species (ROS). This induced disruption of the cell membrane was further supported by morphological studies using scanning electron microscopy (SEM). Investigations on a possible post-antibiotic effect (PAE) of peptide-Ba49 showed prolonged PAE against S. aureus. Furthermore, the peptide-Ba49 prevented the formation of S. aureus biofilm at low concentration and showed its potential to degrade the mature biofilm of S. aureus. The peptide-Ba49 also exhibited intracellular killing potential against S. aureus ATCC 25923 in the macrophage cells, and moreover, peptide-Ba49 was found to bolster the fibroblast cell migration in the scratch assay at low concentration, exhibiting a wound healing efficacy of this peptide. These studies demonstrated that peptide-Ba49 isolated from the strain B. subtilis subsp. spizizenii could be a therapeutic candidate to combat the pathogenic S. aureus infections.

Bacteriocin isolated from the natural inhabitant of Allium cepa against Staphylococcus aureus.

Extensive usage of antibiotics has led to the emergence of drug-resistant strains of pathogens and hence, there is an urgent need for alternative antimicrobial agents. Antimicrobial Peptides (AMPs) of bacterial origin have shown the potential to replace some conventional antibiotics. In the present study, an AMP was isolated from Bacillus subtilis subsp. spizizenii strain Ba49 present on the Allium cepa, the common onion and named as peptide-Ba49. The isolated AMP was purified and characterized. The purified peptide-Ba49, having a molecular weight of ~ 3.3 kDa as determined using mass spectroscopy, was stable up to 121 °C and in the pH range of 5-10. Its interaction with protein degrading enzymes confirmed the peptide nature of the molecule. The peptide exhibited low minimum inhibitory concentration (MIC) against Staphylococcus aureus and its (Methicillin-resistant Staphylococcus aureus) MRSA strains (MIC, 2-16 µM/mL). Further, time kill kinetic assay was performed and analysis of the results of membrane depolarization and permeabilization assays (TEM, DiBAC4 (3) and PI) suggested peptide-Ba49 to be acting through the change in membrane potential leading to disruption of S. aureus membrane. Additionally, cytotoxicity studies of peptide-Ba49, carried out using three mammalian cell lines viz. HEK 293T, RAW 264.7, and L929, showed limited cytotoxicity on these cell lines at a concentration much higher than its MIC values. All these studies suggested that the AMP isolated from strain Ba49 (peptide-Ba49) has the potential to be an alternative to antibiotics in terms of eradicating the pathogenic as well as drug-resistant microorganisms.

Facile One Pot Greener Synthesis of Sophorolipid Capped Gold Nanoparticles and its Antimicrobial Activity having Special Efficacy Against Gram Negative Vibrio cholerae.

Microbes develop several strategies to survive in the adverse condition such as biofilm formation, attaining non-dividing state, altering drug target or drug, thereby increases the burden of drug dosage. To combat these issues, nanoparticles have shown an alternative approach for new treatment strategy but synthesis via chemical synthetic route limits their application in biomedical field. Here, green method for the synthesis of gold nanoparticles using sophorolipid (SL) is discussed that is characterized by various techniques. Initially, the antimicrobial activity was checked against metabolically active state of microbes; Gram-positive Staphylococcus aureus and Gram-negative Vibrio cholerae using XTT assay and growth kinetics assay. Results suggested higher efficacy of nanoparticles for Gram-negative, therefore further analyzed against Escherichia coli that confirmed its potency for the same. AuNPs-SL also signifies its efficiency at least metabolically active state; non dividing cells and biofilm of these microbes. Induced morphological changes were studied by SEM that revealed AuNPs-SL led to disruption of cell membrane and leakage of intracellular fluid to the surroundings. Inhibition of respiratory enzymes activity also plays a crucial role in bactericidal action as indicated by LDH assay. Synergy of AuNPs-SL with different antibiotics was also analyzed using checkerboard assay. These results suggested the possible use of AuNPs-SL as an antimicrobial therapy in the field of nanomedicine.

Sophorolipid exhibits antifungal activity by ROS mediated endoplasmic reticulum stress and mitochondrial dysfunction pathways in Candida albicans.

In the present study, we investigated the mechanism of cell death in C. albicans due to treatment with sophorolipid (SL). SL is an extracellular glycolipid biosurfactant produced by various species of non-pathogenic yeasts and is known to inhibit the growth and biofilm formation of C. albicans. This study revealed that treatment of C. albicans cells with SL increases the ROS production and expression of oxidative stress-related genes significantly (SOD1, CAT1). Increased ROS level within the cells causes ER stress and release of Ca2+ in the cytoplasm and alteration of the mitochondrial membrane potential (MMP). Quantitative real time-polymerase chain reaction (qRT-PCR) data showed that SL also upregulates the Endoplasmic Reticulum (ER) stress marker HAC1. Flow cytometric analysis (AnnexinV/PI) indicated that the cell death may have occurred due to necrosis which was further confirmed by LDH release assay and transmission electron microscopy (TEM). Further experiments with the null mutant Δ hog1 strain of C. albicans SC5314 indicated the activation of the osmotic stress response pathway (HOG-MAPK) and SAP9. This study gave an insight into the mechanism of cell death initiation by glycolipids and indicated that further modification of these molecules can lead to the development of new therapeutic agent against C. albicans.

Inhibitory Effect of Sophorolipid on Candida albicans Biofilm Formation and Hyphal Growth.

Candida albicans causes superficial and life-threatening systemic infections. These are difficult to treat often due to drug resistance, particularly because C. albicans biofilms are inherently resistant to most antifungals. Sophorolipid (SL), a glycolipid biosurfactant, has been shown to have antimicrobial and anticancer properties. In this study, we investigated the effect of SL on C. albicans biofilm formation and preformed biofilms. SL was found to inhibit C. albicans biofilm formation as well as reduce the viability of preformed biofilms. Moreover, SL, when used along with amphotericin B (AmB) or fluconazole (FLZ), was found to act synergistically against biofilm formation and preformed biofilms. Effect of SL on C. albicans biofilm formation was further visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed absence of hyphae, typical biofilm architecture and alteration in the morphology of biofilm cells. We also found that SL downregulates the expression of hypha specific genes HWP1, ALS1, ALS3, ECE1 and SAP4, which possibly explains the inhibitory effect of SL on hyphae and biofilm formation.

Exploitation of marine bacteria for production of gold nanoparticles.

BACKGROUND: Gold nanoparticles (AuNPs) have found wide range of applications in electronics, biomedical engineering, and chemistry owing to their exceptional opto-electrical properties. Biological synthesis of gold nanoparticles by using plant extracts and microbes have received profound interest in recent times owing to their potential to produce nanoparticles with varied shape, size and morphology. Marine microorganisms are unique to tolerate high salt concentration and can evade toxicity of different metal ions. However, these marine microbes are not sufficiently explored for their capability of metal nanoparticle synthesis. Although, marine water is one of the richest sources of gold in the nature, however, there is no significant publication regarding utilization of marine micro-organisms to produce gold nanoparticles. Therefore, there might be a possibility of exploring marine bacteria as nanofactories for AuNP biosynthesis. RESULTS: In the present study, marine bacteria are exploited towards their capability of gold nanoparticles (AuNPs) production. Stable, monodisperse AuNP formation with around 10 nm dimension occur upon exposure of HAuCl(4) solution to whole cells of a novel strain of Marinobacter pelagius, as characterized by polyphasic taxonomy. Nanoparticles synthesized are characterized by Transmission electron microscopy, Dynamic light scattering and UV-visible spectroscopy. CONCLUSION: The potential of marine organisms in biosynthesis of AuNPs are still relatively unexplored. Although, there are few reports of gold nanoparticles production using marine sponges and sea weeds however, there is no report on the production of gold nanoparticles using marine bacteria. The present work highlighted the possibility of using the marine bacterial strain of Marinobacter pelagius to achieve a fast rate of nanoparticles synthesis which may be of high interest for future process development of AuNPs. This is the first report of AuNP synthesis by marine bacteria.

Asymmetric reduction of a ketone by wet and lyophilized cells of Geotrichum candidum in organic solvents.

Sixteen organic co-solvents were screened for stereoselective reduction of 1-acetonapthone in aqueous media by whole cells of Geotrichum candidum. Benzyl alcohol was found to be a good co-solvent as it afforded a high coversion and reduced deactivation of the cells. Half-lives of the wet and lyophilized whole cell biocatalysts in pure benzyl alcohol were 23.07 and 11.21 hours, respectively. The initial reaction rates at 30°C were 13.1 and 11.0µmol/min, respectively, for the wet and lyophilized cells. With optimized conditions in a reaction medium containing phosphate buffer and benzyl alcohol (1:1 by vol) with 230mM 1-acetonapthone, more than 98% and 81% conversion (ee >99%) was achieved in 5 hours with the wet and lyophilized cells, respectively. Both the cell preparations showed maximum conversion at 30°C. A thermodynamic characterization revealed that the wet cells were more thermostable than the lyophilized cells. The calculated half-life of the wet cells at pH 7 was 93 hours, whereas that of the lyophilized cells was 71 hours at the same condition.

Immobilization of intracellular carbonyl reductase from Geotrichum candidum for the stereoselective reduction of 1-naphthyl ketone.

Different cell disintegration methods were used for the liberation of intracellular carbonyl reductase from Geotrichum candidum, in its active form. Solid shear (bead milling) was proved to be the best method for the extraction of the enzyme. Various solid supports were checked for the immobilization of the purified enzyme. Carbonyl reductase was immobilized on silica with an optimized protein loading of 4 mg/g support. Cross-linking with glutaraldehyde rendered the preparation more stable and suitable for use in consecutive batches. Carbonyl reductase of G. candidum immobilized on silica support and cross-linked by glutaraldehyde was found to be highly efficient biocatalyst formulation for the production of S(-)-1-(1'-naphthyl) ethanol.

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor.

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77 x 10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.

Improvement of carbonyl reductase production of Geotrichum candidum for the transformation of 1-acetonaphthone to S(-)-1-(1'-napthyl) ethanol.

The aim of this work was to study the influence of media components and physico-chemical parameters on the growth and carbonyl reductase production by Geotrichum candidum. Under optimized conditions, the conversion of the substrate increased to >93%, while the specific growth rate and enzyme activity were increased by 200% and 29%, respectively. The rate of conversion of the substrate was also very high in the cells grown in optimized medium. The volumetric productivity of the biotransformation process was much higher (0.27g/lh) with the cells grown in the optimized medium compared to that of grown in un-optimized medium (0.16g/lh). The cells were also highly stable in the operational condition, indicating the feasibility of their use in multiple batches of reaction.