Biology subjects

Rago, D.

Publications and source records attributed to Rago, D..

4 recordsLinked to original sources

bioRxiv2024

Metabolic engineering of yeast for de novo production of kratom monoterpene indole alkaloids

Monoterpene indole alkaloids (MIAs) from Mitragyna speciosa ("kratom"), such as mitragynine and speciogynine, are promising novel scaffolds for opioid receptor ligands for treatment of pain, addiction, and depression. While kratom leaves have been used for centuries in South-East Asia as stimulant and pain management substance, the biosynthetic pathway of these psychoactives have only recently been partially elucidated. Here, we demonstrate the de novo production of mitragynine and speciogynine in Saccharomyces cerevisiae through the reconstruction of a five-step synthetic pathway from common MIA precursor strictosidine comprising fungal tryptamine 4-monooxygenase to bypass an unknown kratom hydroxylase. Upon optimizing cultivation conditions, a titer of [~]290 {micro}g/L kratom MIAs from glucose was achieved. Untargeted metabolomics analysis of lead production strains led to the identification of numerous shunt products derived from the activity of strictosidine synthase (STR) and dihydrocorynantheine synthase (DCS), highlighting them as candidates for enzyme engineering to further improve kratom MIAs production in yeast. Finally, by feeding fluorinated tryptamine and expressing a human tailoring enzyme, we further demonstrate production of fluorinated and hydroxylated mitragynine derivatives with potential applications in drug discovery campaigns. Altogether, this study introduces a yeast cell factory platform for the biomanufacturing of complex natural and new-to-nature kratom MIAs derivatives with therapeutic potential.

synthetic biology↗

bioRxiv2023

High-yield enzymatic synthesis of mono- and trifluorinated alanine enantiomers

Fluorinated amino acids are a promising entry point for incorporating new-to-Nature chemistries in biological systems. Hence, novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers. To this end, the alanine dehydrogenase from Vibrio proteolyticus and the diaminopimelate dehydrogenase from Symbiobacterium thermophilum were applied to the in vitro production of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, an alanine racemase from Streptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in the {beta}-elimination of fluorine from the monosubstituted fluoroalanine. The in vitro enzymatic cascade based on the dehydrogenases of V. proteolyticus and S. thermophilum included a cofactor recycling system, whereby a formate dehydrogenase from Pseudomonas sp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. Moreover, the selected dehydrogenases were also able to catalyze the conversion of trifluoropyruvate into trifluorinated alanine, as a first-case example of biocatalysis with amino acids carrying a trifluoromethyl group.

synthetic biology↗

bioRxiv2023

Adaptive evolution of P. aeruginosa in human airways shows phenotypic convergence despite diverse patterns of genomic changes

Selective forces in the environment drive bacterial adaptation to novel niches, choosing the fitter variants in the population. However, in dynamic and changing environments, the evolutionary processes controlling bacterial adaptation are difficult to monitor. Here, we follow 9 cystic fibrosis patients chronically infected with Pseudomonas aeruginosa, as a proxy for bacterial adaptation. We identify and describe the bacterial changes and evolution occurring between 15 and 35 years of within host evolution. We combine whole genome sequencing, RNAseq and metabolomics, and compare the evolutionary trajectories directed by the adaptation of four different P. aeruginosa lineages to the lung. Our data suggest divergent evolution at the genomic level for most of the genes, with signs of convergent evolution with respect to acquisition of mutations in regulatory genes, which drive the transcriptional and metabolomic program at late time of evolution. Metabolomics further confirmed convergent adaptive phenotypic evolution as documented by reduction of the quorum sensing molecules acyl-homoserine lactone, phenazines and rhamnolipids (except for quinolones). The modulation of the quorum sensing repertoire suggests that similar selective forces characterize at late times of evolution independent of the patient. Collectively, our data suggest that similar environments and similar P. aeruginosa populations in the patients at prolonged time of infection are associated with an overall reduction of virulence-associated features and phenotypic convergence. SummarySelective forces in the human environment drive bacterial adaptation to novel niches, choosing the fitter variants in the population. We have investigated the evolutionary processes in 9 CF patients infected with Pseudomonas aeruginosa in the airways for several decades. To describe the within host evolution and trajectories of four different P. aeruginosa lineages to the lung environment we have combined whole genome sequencing, RNAseq and metabolomics. In this patient cohorte with persistent bacterial infections our data suggest that similar environments and similar P. aeruginosa populations in the patients at prolonged time of infection are associated with an overall reduction of virulence-associated features and phenotypic convergence

microbiology↗

bioRxiv2023

Pulmonary maternal immune activation does not extend through the placenta but leads to fetal metabolic adaptation

Maternal immune system activation (MIA) during pregnancy can disrupt the fetal environment, causing postnatal susceptibility to disorders. How the placenta and the fetus respond to acute MIA over time is unknown. Here, we characterized the response to acute maternal pulmonary inflammation across time in maternal and fetal organs using multi-omics. Unlike maternal organs which mounted strong innate immune responses, the placenta upregulated tissue-integrity genes, likely to prevent fetal exposure to infections, and downregulated growth-associated genes. Subsequently, the placenta upregulated biosynthesis and endoplasmic reticulum stress genes in order to return to homeostasis. These responses likely protected the fetus, since we observed no immune response in fetal liver. Instead, likely due to nutrient depletion, the fetal liver displayed metabolic adaptations, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. Our study shows, for the first time, the integrated temporal response to pulmonary MIA across maternal and fetal organs.

genomics↗