Adenosine: a partially discovered medicinal agent

Background A plethora of chemicals exists in human body which can alter physiology in one way or other. Scientists have always been astounded by such abilities of chemicals but as the technology advances, even the chemical which was once expected to be well known changes its status to not really well known. Adenosine is one of the chemicals which is in consonance with the aforementioned statements, although previous articles have covered vast information on role of adenosine in cardiovascular physiology, bacterial pathophysiology and inflammatory diseases. In this review we have discussed adenosine and its congeners as potential promising agents in the treatment of Huntington’s disease, post-traumatic stress disorder, erectile dysfunction, viral infections (SARS-CoV) and anxiety. Main text Adenosine is a unique metabolite of ATP; which serves in signalling as well. It is made up of adenine (a nitrogenous base) and ribo-furanose (pentose) sugar linked by β-N9-glycosidic bond. Adenosine on two successive phosphorylation forms ATP (Adenosine Triphosphate) which is involved in several active processes of cell. It is also one of the building blocks (nucleotides) involved in DNA (Deoxy-ribonucleic Acid) and RNA (Ribonucleic Acid) synthesis. It is also a component of an enzyme called S-adenosyl-L-methionine (SAM) and cyano-cobalamin (vitamin B-12). Adenosine acts by binding to G protein-coupled receptor (GPCR: A1, A2A, A2B and A3) carries out various responses some of which are anti-platelet function, hyperaemic response, bone remodelling, involvement in penile erection and suppression of inflammation. On the other hand, certain microorganisms belonging to genus Candida, Staphylococcus and Bacillus utilize adenosine in order to escape host immune response (phagocytic clearance). These microbes evade host immune response by synthesizing and releasing adenosine (with the help of an enzyme: adenosine synthase-A), at the site of infection. Conclusion With the recent advancement in attribution of adenosine in physiology and pathological states, adenosine and its congeners are being looked forward to bringing a revolution in treatment of inflammation, viral infections, psychiatric and neurodegenerative disorders.

Overexpression of an oxidoreductase YghA confers tolerance against furfural in ethanologenic Escherichia coli strain SSK42

Furfural is a common furan inhibitor formed due to dehydration of pentose sugar like xylose and acts as an inhibitor of microbial metabolism. Overexpression of NADH specific FucO and deletion of NADPH specific YqhD had been a successful strategy in the past in conferring tolerance against furfural in E. coli , which highlight the importance of oxidoreductases in conferring tolerance against furfural. In a screen consisting of various oxidoreductases, dehydrogenases, and reductases, we identified yghA gene as an overexpression target to confer tolerance against furfural. YghA preferably used NADH as a cofactor and had apparent K m value of 0.03 mM against furfural. In presence of 1 g L −1 furfural and 10% xylose (wt/vol), yghA overexpression in an ethanologenic E. coli strain SSK42 resulted in a 5.3-fold increase in ethanol titers as compared to the control strain with an efficiency of ∼97%. YghA also exhibited activity against the lesser toxic inhibitor 5-hydroxymethyl furfural that is formed due to dehydration of hexose sugars and thus is a formidable target for overexpression in ethanologenic strain for fermentation of sugars in biomass hydrolysate. IMPORTANCE Lignocellulosic biomass represents an inexhaustible source of carbon for second-generation biofuels. Thermo-acidic pretreatment of biomass is performed to loosen the lignocellulosic fibers and make the carbon bioavailable for microbial metabolism. The pretreatment process also results in the formation of inhibitors that inhibit microbial metabolism and increase production costs. Furfural is a potent furan inhibitor that increases the toxicity of other inhibitors present in the hydrolysate. Thus it is desirable to engineer furfural tolerance in E. coli for efficient fermentation of hydrolysate sugars.

Escherichia coli AraJ boosts utilization of arabinose in metabolically engineered cyanobacterium Synechocystis sp. PCC 6803

Lignocellulosic biomass can serve as an inexpensive and renewable source of carbon for the biosynthesis of commercially important compounds. L-arabinose is the second most abundant pentose sugar present in the plant materials. Model cyanobacterium Synechocystis sp. PCC 6803 is incapable of catabolism of L-arabinose as a source of carbon and energy. In this study, all the heterologous genes expressed in Synechocystis were derived from Escherichia coli K-12. Initially we constructed four Synechocystis strains that expressed AraBAD enzymes involved in L-arabinose catabolism, either in combination with or without one of the three arabinose transporters, AraE, AraFGH or AraJ. Among the recombinants, the strain possessing AraJ transporter was observed to be the most efficient in terms of dry biomass production and L-arabinose consumption. Later, an additional strain was generated by the expression of AraJ in the AraE-possessing strain. The resultant strain was shown to be advantageous over its parent. This study demonstrates that AraJ, a protein with hitherto unknown function plays a role in the uptake of L-arabinose to boost its catabolism in the transgenic Synechocystis strains. The work also contributes to the current knowledge regarding metabolic engineering of cyanobacteria for the utilization of pentose sugars.

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

Transcriptomic changes induced by de-activation of lower glycolysis and its advantage on pentose sugar metabolism in Saccharomyces cerevisiae

As a microbial host for cellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway. However, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (pho13) has been reported to be a crucial genetic perturbation for improving xylose fermentation. A confirmed mechanism of the pho13-positive effect on xylose fermentation is that the deletion of PHO13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the present study, we reported that a pho13-positive effect was not observed from a couple of engineered strains, among the many others we have examined. To extend our knowledge of pho13-mediated metabolic regulation, we performed genome sequencing of pho13-negative strains. We identified a loss-of-function mutation in GCR2 responsible for the pho13-negative phenotype. Gcr2 is a transcriptional activator of the lower glycolytic pathway. Thus, the deletion of GCR2 (gcr2) led to deactivation of lower glycolysis as confirmed by RNA-seq. Also, gcr2 resulted in the up-regulation of PPP genes, which explains the improved xylose fermentation of gcr2 mutants. As pho13 and gcr2 cause similar transcriptional changes with PPP genes, there was no synergistic effect between pho13 and gcr2 for improving xylose fermentation. The present study identified GCR2 as a new knockout target to improve xylose fermentation and cellulosic biofuel production. Now published in Frontiers in Bioengineering and Biotechnology doi: 10.3389/fbioe.2021.654177

A novel d-xylose isomerase from the gut of the wood feeding beetle Odontotaenius disjunctus efficiently expressed in Saccharomyces cerevisiae

Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar d-xylose is often present in significant amounts along with hexoses. Saccharomyces cerevisiae can acquire the ability to metabolize d-xylose through expression of heterologous d-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen XIs have been reported to be active so far. We cloned a new d-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetle Odontotaenius disjunctus. Although somewhat homologous to the XI from Piromyces sp. E2, the new gene was identified as bacterial in origin and the host as a Parabacteroides sp. Expression of the new XI in S. cerevisiae resulted in faster aerobic growth than the XI from Piromyces on d-xylose media. The d-xylose isomerization rate conferred by the new XI was also 72% higher, while absolute xylitol production was identical in both strains. Interestingly, increasing concentrations of xylitol (up to 8 g L−1) appeared not to inhibit d-xylose consumption. The newly described XI displayed 2.6 times higher specific activity, 37% lower KM for d-xylose, and exhibited higher activity over a broader temperature range, retaining 51% of maximal activity at 30 °C compared with only 29% activity for the Piromyces XI.

Evaluating the Effect of Lignocellulose-Derived Microbial Inhibitors on the Growth and Lactic Acid Production by Bacillus coagulans Azu-10

Effective lactic acid (LA) production from lignocellulosic biomass materials is challenged by several limitations related to pentose sugar utilization, inhibitory compounds, and/or fermentation conditions. In this study, a newly isolated Bacillus coagulans strain Azu-10 was obtained and showed homofermentative LA production from xylose with optimal fermentation conditions at 50 °C and pH 7.0. Growth of strain Azu-10 and LA-fermentation efficiency were evaluated in the presence of various lignocellulose-derived inhibitors (furans, carboxylic acids, and phenols) at different concentrations. Furanic lignocellulosic-derived inhibitors were completely detoxified. The strain has exhibited high biomass, complete xylose consumption, and high LA production in the presence of 1.0–4.0 g/L furfural and 1.0–5.0 g/L of hydroxymethyl furfural, separately. Moreover, strain Azu-10 exhibited high LA production in the presence of 5.0–15.0 g/L acetic acid, 5.0 g/L of formic acid, and up to 7.0 g/L of levulinic acid, separately. Besides, for phenolic compounds, p-coumaric acid was most toxic at 1.0 g/L, while syringaldehyde or p-hydroxybenzaldehyde, and vanillin at 1.0 g/L did not inhibit LA fermentation. The present study provides an interesting potential candidate for the thermophilic LA fermentation from lignocellulose-derived substrates at the industrial biorefinery level.

Predicting experimental designs leading to rewiring of transcription program and evolution of anticipatory regulation in E. coli

Environmental cues in an ecological niche are temporal. In response to these temporal cues, bacteria have been known to exhibit learning or conditioning, whereby they trigger response to a yet to appear cue, anticipating its actual arrival in the near future. Such an anticipatory response in known to enhance Darwinian fitness, and hence, is likely an important feature in the regulatory networks in microorganisms. However, the conditions under which an anticipatory response optimizes cellular fitness are not known. Nor has evolution of anticipatory regulation in laboratory conditions been experimentally demonstrated. In this work, we develop a quantitative model to study response of a population to two temporal environmental cues, and present the key variables in cellular physiology associated with response to the cues whose modulation is likely to lead to evolution of anticipatory regulatory response. We predict experimental conditions, which are likely to lead to demonstration of rewiring of regulation, and evolution of anticipatory response in bacterial populations. Using inputs from the modeling results, we evolve E. coli in alternating environments of the pentose sugar rhamnose and paraquat, which induces oxidative stress. We demonstrate that growth in this cyclical environment leads to evolution of anticipatory regulation. Thus, we argue that in niches where environmental stimuli have a cyclical nature, conditioning evolves in a population as an adaptive response.

Metal Catalyzed Synthesis of Pentose-Sugar-Based Chiral 2-Substituted-1H-Benzimidazoles

The authors have withdrawn this preprint due to author disagreement.