A chemical genetic approach using genetically encoded reporters to detect and assess the toxicity of plant secondary metabolites against bacterial pathogens.

Plant secondary metabolites are emerging as attractive alternatives in the development of therapeutics against infectious and chronic diseases. Due to the present pandemic, therapeutics showing toxicity against bacterial pathogens and viruses are gaining interest. Plant metabolites of terpenoid and phenylpropanoid categories have known antibacterial and antiviral properties. These metabolites have also been associated with toxicity to eukaryotic cells in terms of carcinogenicity, hepatotoxicity, and neurotoxicity. Sensing methods that can report the exact antibacterial dosage, formation, and accumulation of these antibacterial compounds are needed. The whole-cell reporters for such antibacterial metabolites are cost-effective and easy to maintain. In the present study, battery of toxicity sensors containing fluorescent transcriptional bioreporters was constructed, followed by fine-tuning the response using gene-debilitated E. coli mutants. This study shows that by combining regulatory switches with chemical genetics strategy, it may be possible to detect and elucidate the mode of action of effective antibacterial plant secondary metabolites - thymol, cinnamaldehyde, eugenol, and carvacrol in both pure and complex formats. Apart from the detection of adulteration of pure compounds present in complex mixture of essential oils, this approach will be useful to detect authenticity of essential oils and thus reduce unintended harmful effects on human and animal health.

Draft Genome Sequence of Limosilactobacillus fermentum Strain NKN-51, Isolated from Fermented Yak Milk in the Western Himalayas of India.

Here, we report the draft genome sequence of Limosilactobacillus fermentum strain NKN-51, which was isolated from naturally processed yak cheese from the western Himalayas of India. The genome was assembled in 101 contigs with a total length of 1,879,705 bp and a GC content of 53.5%. Genome annotation predicted 1,730 protein-coding genes and 50 tRNA genes.

Mechanistic Insights Into Probiotic Properties of Lactic Acid Bacteria Associated With Ethnic Fermented Dairy Products.

Gut microbes and their metabolites maintain the health and homeostasis of the host by communicating with the host via various biochemical and physical factors. Changing lifestyle, chronic intake of foods rich in refined carbohydrates and fats have caused intestinal dysbiosis and other lifestyle-based diseases. Thus, supplementation with probiotics has gained popularity as biotherapies for improving gut health and treating disorders. Research shows that probiotic organisms enhance gastrointestinal health, immunomodulation, generation of essential micronutrients, and prevention of cancer. Ethnically fermented milk and dairy products are hotspots for novel probiotic organisms and bioactive compounds. These ethnic fermented foods have been traditionally prepared by indigenous populations, and have preserved unique microflora for ages. To apply these unique microflora for amelioration of human health, it is important that probiotic properties of the bacterial species are well studied. Majority of the published research and reviews focus on the probiotic organisms and their properties, fermented food products, isolation techniques, and animal studies with their health pathologies. As a consequence, there is a dearth of information about the underlying molecular mechanism behind probiotics associated with ethnically prepared dairy foods. This review is targeted at stimulating research on understanding these mechanisms of bacterial species and beneficial attributes of ethnically fermented dairy products.

Physiological growth and galactose utilization by dairy yeast Kluyveromyces marxianus in mixed sugars and whey during fermentation.

The dairy yeast Kluyveromyces marxianus represents a promising industrial strain useful for the production of bioethanol from cheese whey. Physiology of the five K. marxianus strains on galactose was examined during batch cultivation under controlled aerobic conditions on minimal media and one of the strains designated K. marxianus strain 6C17 which presented the highest specific galactose consumption rate. A maximum specific growth rate of 0.34 and 0.37 h-1, respectively, was achieved using batch cultivation in a minimal medium and a complex medium amended with galactose (50 g/L) at 37 °C. The sugar was metabolized for the production of ethanol as the chief metabolite with a maximum ethanol yield of 0.39 g/g of galactose. Different growth behaviors were observed when galactose was used with other sugar such as glucose, lactose and fructose. The growth rates on hydrolyzed cheese whey were also measured, and a maximum specific growth rate of 0.39 and 0.32 h-1 was observed with glucose and galactose, respectively, with the maximum flux diverted toward ethanol production. This approach of studying the physiology of thermotolerant K. marxianus on hydrolyzed whey during fermentation would be helpful in achieving higher yields of ethanol.

Comparative Analysis of Oxidative Stress During Aging of Kluyveromyces marxianus in Synthetic and Whey Media.

During the aging of yeast culture, Kluyveromyces marxianus undergoes a number of changes in physiology and these changes play a significant role during fermentation. Aged stationary phase cells were found to contain more reactive oxygen species. Additionally, the level of oxidant is counteracted by the antioxidant defense system of the cells. Comparison of 3-day-old culture of K. marxianus with 45-day stationary phase culture represents an increased level of ROS inside the cells. Moreover, a decrease in glutathione content was observed over the set of the incubation period. The increased level of superoxide dismutase (SOD) and catalase also revealed that there is oxidative stress during the long period incubation of the stationary phase cells of K. marxianus. The actual phenomenon of aging in dairy yeast K. marxianus is a complex process, but the present study signifies that role of antioxidant defense system during aging in stationary phase cells of K. marxianus.

Utilization of Cheese Whey Using Synergistic Immobilization of ß-Galactosidase and Saccharomyces cerevisiae Cells in Dual Matrices.

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the ß-galactosidase using whey. The maximum ß-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, ß-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.