Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree.

The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.

A booster for vaccines from plants.

Reconstituting a plant biosynthetic pathway enables a sustainable supply of vaccine adjuvants.

Significance of Catecholamine Biosynthetic/Metabolic Pathway in SARS-CoV-2 Infection and COVID-19 Severity.

The SARS-CoV-2 infection was previously associated with the expression of the dopamine biosynthetic enzyme L-Dopa decarboxylase (DDC). Specifically, a negative correlation was detected between DDC mRNA and SARS-CoV-2 RNA levels in in vitro infected epithelial cells and the nasopharyngeal tissue of COVID-19 patients with mild/no symptoms. However, DDC, among other genes related to both DDC expression and SARS-CoV-2-infection (ACE2, dACE2, EPO), was upregulated in these patients, possibly attributed to an orchestrated host antiviral response. Herein, by comparing DDC expression in the nasopharyngeal swab samples of severe/critical to mild COVID-19 cases, we showed a 20 mean-fold reduction, highlighting the importance of the expression of this gene as a potential marker of COVID-19 severity. Moreover, we identified an association of SARS-CoV-2 infection with the expression of key catecholamine biosynthesis/metabolism-related genes, in whole blood samples from hospitalized patients and in cultured cells. Specifically, viral infection downregulated the biosynthetic part of the dopamine pathway (reduction in DDC expression up to 7.5 mean-fold), while enhanced the catabolizing part (increase in monoamine oxidases A and B expression up to 15 and 10 mean-fold, respectively) in vivo, irrespectively of the presence of comorbidities. In accordance, dopamine levels in the sera of severe cases were reduced (up to 3.8 mean-fold). Additionally, a moderate positive correlation between DDC and MAOA mRNA levels (r = 0.527, p < 00001) in the blood was identified upon SARS-CoV-2-infection. These observations were consistent to the gene expression data from SARS-CoV-2-infected Vero E6 and A549 epithelial cells. Furthermore, L-Dopa or dopamine treatment of infected cells attenuated the virus-derived cytopathic effect by 55% and 59%, respectively. The SARS-CoV-2 mediated suppression of dopamine biosynthesis in cell culture was, at least in part, attributed to hypoxia-like conditions triggered by viral infection. These findings suggest that L-Dopa/dopamine intake may have a preventive or therapeutic value for COVID-19 patients.

Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda).

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

Sesterterpenoids: chemistry, biology, and biosynthesis.

Covering: July 2012 to December 2019Over the last seven years, expanding research efforts focused on sesterterpenoids has led to the isolation, identification, and characterization of numerous structurally novel and biologically active sesterterpenoids. These newly reported sesterterpenoids provide diverse structures that often incorporate unprecedented ring systems and new carbon skeletons, as well as unusual functional group arrays. Biological activities of potential biomedical importance including suppression of cancer cell growth, inhibition of enzymatic activity, and modulation of receptor signaling, as well as ecologically important functions such as antimicrobial effects and deterrence of herbivorous insects have been associated with a variety of sesterterpenoids. There has also been a rapid growth in our knowledge of the genomics, enzymology, and specific pathways associated with sesterterpene biosynthesis. This has opened up new opportunities for future sesterterpene discovery and diversification through the expression of new cryptic metabolites and the engineered manipulation of associated biosynthetic machinery and processes. In this paper we reviewed 498 new sesterterpenoids, including their structures, source organisms, country of origin, relevant bioactivities, and biosynthesis.

Caffeoylquinic acids: chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity.

Caffeoylquinic acids (CQAs) are specialized plant metabolites we encounter in our daily life. Humans consume CQAs in mg-to-gram quantities through dietary consumption of plant products. CQAs are considered beneficial for human health, mainly due to their anti-inflammatory and antioxidant properties. Recently, new biosynthetic pathways via a peroxidase-type p-coumaric acid 3-hydroxylase enzyme were discovered. More recently, a new GDSL lipase-like enzyme able to transform monoCQAs into diCQA was identified in Ipomoea batatas. CQAs were recently linked to memory improvement; they seem to be strong indirect antioxidants via Nrf2 activation. However, there is a prevalent confusion in the designation and nomenclature of different CQA isomers. Such inconsistencies are critical and complicate bioactivity assessment since different isomers differ in bioactivity and potency. A detailed explanation regarding the origin of such confusion is provided, and a recommendation to unify nomenclature is suggested. Furthermore, for studies on CQA bioactivity, plant-based laboratory animal diets contain CQAs, which makes it difficult to include proper control groups for comparison. Therefore, a synthetic diet free of CQAs is advised to avoid interferences since some CQAs may produce bioactivity even at nanomolar levels. Biotransformation of CQAs by gut microbiota, the discovery of new enzymatic biosynthetic and metabolic pathways, dietary assessment, and assessment of biological properties with potential for drug development are areas of active, ongoing research. This review is focused on the chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity recently reported for mono-, di-, tri-, and tetraCQAs.

Antiviral strategies should focus on stimulating the biosynthesis of heparan sulfates, not their inhibition.

Antiviral strategies for viruses that utilize proteoglycan core proteins (syndecans and glypicans) as receptors should focus on heparan sulfate (HS) biosynthesis rather than on inhibition of these sugar chains. Here, we show that heparin and certain xylosides, which exhibit in vitro viral entry inhibitory properties against HSV-1, HSV-2, HPV-16, HPV-31, HVB, HVC, HIV-1, HTLV-1, SARS-CoV-2, HCMV, DENV-1, and DENV-2, stimulated HS biosynthesis at the cell surface 2- to 3-fold for heparin and up to 10-fold for such xylosides. This is consistent with the hypothesis from a previous study that for core protein attachment, viruses are glycosylated at HS attachment sites (i.e., serine residues intended to receive the D-xylose molecule for initiating HS chains). Heparanase overexpression, endocytic entry, and syndecan shedding enhancement, all of which are observed during viral infection, lead to glycocalyx deregulation and appear to be direct consequences of this hypothesis. In addition to the appearance of type 2 diabetes and the degradation of HS observed during viral infection, we linked this hypothesis to that proposed in a previous publication.

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses.

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

Deciphering the Molecular Details of the Lipoxin Formation Mechanism in the 5(S),15(S)-DiHpETE Biosynthetic Pathway Catalyzed by Reticulocyte 15-Lipoxygenase-1.

Chronic inflammation is now widely recognized to play important roles in many commonly occurring diseases, including COVID-19. The resolution response to this chronic inflammation is an active process governed by specialized pro-resolving mediators (SPMs) like the lipid mediators known as lipoxins. The biosynthesis of lipoxins is catalyzed by several lipoxygenases (LOXs) from arachidonic acid. However, the molecular details of the mechanisms involved are not well known yet. In this paper, we have combined molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations to analyze how reticulocyte 15-LOX-1 catalyzes the production of lipoxins from 5(S),15(S)-diHpETE. Our results indicate that the dehydration mechanism from 5(S),15(S)-diHpETE, via the formation of an epoxide, presents huge energy barriers even though it was one of the two a priori synthetic proposals. This result is compatible with the fact that no epoxide has been directly detected as an intermediate in the catalytic formation of lipoxins from 5(S),15(S)-diHpETE. Conversely, the oxygenation of 5(S),15(S)-diHpETE at C14 is feasible because there is an open channel connecting the protein surface with this carbon atom, and the energy barrier for oxygen addition through this channel is small. The analysis of the following steps of this mechanism, leading to the corresponding hydroperoxide at the 15-LOX-1 active site, indicates that the oxygenation mechanism will lead to the formation of lipoxinB4 after the final action of a reductase. In contrast, our calculations are in agreement with experiments that lipoxinA4 cannot derive from 5(S),15(S)-diHpETE by either of the two proposed mechanisms and that 5(S),15(S)-diHETE is not an intermediate of lipoxin biosynthesis catalyzed by 15-LOX-1.

Melatonin: Roles in influenza, Covid-19, and other viral infections.

There is a growing appreciation that the regulation of the melatonergic pathways, both pineal and systemic, may be an important aspect in how viruses drive the cellular changes that underpin their control of cellular function. We review the melatonergic pathway role in viral infections, emphasizing influenza and covid-19 infections. Viral, or preexistent, suppression of pineal melatonin disinhibits neutrophil attraction, thereby contributing to an initial "cytokine storm", as well as the regulation of other immune cells. Melatonin induces the circadian gene, Bmal1, which disinhibits the pyruvate dehydrogenase complex (PDC), countering viral inhibition of Bmal1/PDC. PDC drives mitochondrial conversion of pyruvate to acetyl-coenzyme A (acetyl-CoA), thereby increasing the tricarboxylic acid cycle, oxidative phosphorylation, and ATP production. Pineal melatonin suppression attenuates this, preventing the circadian "resetting" of mitochondrial metabolism. This is especially relevant in immune cells, where shifting metabolism from glycolytic to oxidative phosphorylation, switches cells from reactive to quiescent phenotypes. Acetyl-CoA is a necessary cosubstrate for arylalkylamine N-acetyltransferase, providing an acetyl group to serotonin, and thereby initiating the melatonergic pathway. Consequently, pineal melatonin regulates mitochondrial melatonin and immune cell phenotype. Virus- and cytokine-storm-driven control of the pineal and mitochondrial melatonergic pathway therefore regulates immune responses. Virus-and cytokine storm-driven changes also increase gut permeability and dysbiosis, thereby suppressing levels of the short-chain fatty acid, butyrate, and increasing circulating lipopolysaccharide (LPS). The alterations in butyrate and LPS can promote viral replication and host symptom severity via impacts on the melatonergic pathway. Focussing on immune regulators has treatment implications for covid-19 and other viral infections.