Biology subjects

Martins dos Santos, V.

Publications and source records attributed to Martins dos Santos, V..

7 recordsLinked to original sources

bioRxiv2026

Establishing de novo microbial biosynthesis of anisole

Anisole is a valuable aromatic ether and promising green solvent whose industrial manufacture relies exclusively on petroleum-derived phenol and hazardous alkali-mediated methylation. Here, we establish the first de novo microbial biosynthesis of anisole from glucose in Pseudomonas putida KT2440 by integrating retrosynthetic pathway design with growth-coupled directed evolution. Using a selection platform in an engineered double auxotroph, in which growth depends on S-adenosylmethionine-dependent methylation, we evolved a promiscuous plant chavicol O-methyltransferase to yield phenol O-methyltransferase 1 (POMT1; bearing a key D264N substitution), which improved anisole production by 2.3-fold over the wild-type enzyme. To overcome product volatility, we integrated an in situ two-phase extraction system using dodecane that achieved near-quantitative product recovery. Then, we compared two phenol routes from the shikimate pathway, selected the 4-hydroxybenzoate route, and raised precursor supply by deleting competing reactions and introducing feedback-resistant and reduced-flux variants in two shikimate pathway enzymes. The final strain produced up to 0.57 mM anisole (62 mg/L) from glucose.

bioengineering↗

bioRxiv2024

Integrative Omics reveals changes in the cellular landscape of yeast without peroxisomes

Peroxisomes are organelles that are crucial for cellular metabolism. However, these organelles play also important roles in non-metabolic processes, such as signalling. To uncover the consequences of peroxisome deficiency, we compared two extremes, namely Saccharomyces cerevisiae wild-type and pex3 cells, which lack functional peroxisomes, employing transcriptomics and quantitative proteomics technology. Cells were grown on acetate, a carbon source that involves peroxisomal enzymes of the glyoxylate cycle and does not repress peroxisomal proteins. Transcripts of peroxisomal {beta}-oxidation genes and the corresponding proteins were enhanced in pex3 cells. Peroxisome-deficiency also caused reduced levels of membrane bound peroxins, while the soluble receptors Pex5 and Pex7 were enhanced at the protein level. In addition, we observed alterations in non-peroxisomal transcripts and proteins, especially mitochondrial proteins involved in respiration or import processes. Our results not only reveal the impact of the absence of peroxisomes in yeast, but also represent a rich resource of candidate genes/proteins that are relevant in peroxisome biology. SummaryOmics comparison of wild-type and peroxisome-deficient (pex3) yeast cells uncovered processes that are affected by loss of peroxisomes. {beta}-oxidation enzymes were upregulated, whereas most peroxins were decreased. Also, several non-peroxisomal transcripts/proteins were significantly altered. Our data represent a rich source of candidate genes connected to peroxisome biology.

cell biology↗

bioRxiv2024

Model-guided metabolic engineering of curcuminoid Production in Pseudomonas putida

Production of value-added, plant-derived compounds in microbes increasingly attracts commercially interest in food and pharmaceutical industries. However, plant metabolic pathways are complex, require a robust balance of enzymes, cofactors, ATP and other metabolites, and often result in low production when transplanted to bacteria. This is exemplified by the biosynthesis of curcuminoids from the Curcuma longa plant. Here, we combine dynamic pathway modeling, systematic testing of isoenzymes, and the optimization of gene expression levels and substrate concentrations for the biosynthesis of curcuminoids in Pseudomonas putida, leading to unprecedented conversion rates of caffeic acid and tyrosine to curcumin. The development of kinetic ensemble models guided the design of production strains, emphasizing the necessity of high relative expression of c3h, curs2 and dcs and, the low relative expression of tal, comt, ccoaomt, and 4cl4. This optimization resulted in a strain that achieved a 10.8 {+/-}1.8% of the maximum theoretical yield of curcumin from tyrosine. This represents a 4.1-fold increase in production efficiency and the highest yield reported to date, demonstrating the potential of P. putida as a promising platform for curcuminoid production. Our findings highlight the effectiveness of our strategy not only in the advances in the production of curcuminoids but also in setting a framework for the biosynthesis of other complex compounds.

synthetic biology↗

bioRxiv2022

Tailoring and optimizing fatty acid production by oleaginous yeasts through the systematic exploration of their physiological fitness

BackgroundThe use of palm oil for our current needs is unsustainable. Replacing palm oil with oils produced by microbes through the conversion of sustainable feedstocks is a promising alternative. However, there are major technical challenges that must be overcome to enable this transition. Foremost among these challenges is the stark increase in lipid accumulation and production of higher content of specific fatty acids. Therefore, there is a need for more in-depth knowledge and systematic exploration of the oil productivity of the oleaginous yeasts. In this study, we cultivated Cutaneotrichosporon oleaginosus and Yarrowia lipolytica at various C/N ratios and temperatures in a defined medium with glycerol as carbon source and urea as nitrogen source. We ascertained the synergistic effect between various C/N ratios of a defined medium at different temperatures with Response Surface Methodology (RSM) and explored the variation in fatty acid composition through Principal Component Analysis. ResultsBy applying RSM, we determined a temperature of 30 {degrees}C and a C/N ratio of 175 g/g to enable maximal oil production by C. oleaginosus and a temperature of 21 {degrees}C and a C/N ratio of 140 g/g for Y. lipolytica. We increased production by 71 % and 66 % respectively for each yeast compared to the average lipid accumulation in all tested conditions. Modulating temperature enabled us to steer the fatty acid compositions. Accordingly, switching from higher temperature to lower cultivation temperature shifted the production of oils from more saturated to unsaturated by 14 % in C. oleaginosus and 31 % in Y. lipolytica. Higher cultivation temperatures resulted in production of even longer saturated fatty acids, 3 % in C. oleaginosus and 1.5 % in Y. lipolytica. ConclusionsIn this study, we provided the optimum C/N ratio and temperature for C. oleaginosus and Y. lipolytica by RSM. Additionally, we demonstrated that lipid accumulation of both oleaginous yeasts was significantly affected by the C/N ratio and temperature. Furthermore, we systematically analyzed the variation in fatty acids composition and proved that changing the C/N ratio and temperature steer the composition. We have further established these oleaginous yeasts as platforms for production of tailored fatty acids.

systems biology↗

bioRxiv2020

Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids

Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The cocultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcohols from syngas. To explore these possibilities, we report herein a genome-scale, constraint-based metabolic model to describe growth of a co-culture of Clostridium autoethanogenum and Clostridium kluyveri on syngas for the production of valuable compounds. Community flux balance analysis was used to gain insight into the metabolism of the two strains and their interactions, and to reveal potential strategies enabling production of butyrate and hexanoate. The model suggests that addition of succinate is one strategy to optimize the production of medium-chain fatty-acids from syngas with this co-culture. According to the predictions, addition of succinate increases the pool of crotonyl-CoA and the ethanol/acetate uptake ratio in C. kluyveri, resulting in the flux of up to 60% of electrons into hexanoate. Other potential way to optimize butyrate and hexanoate is to increase ethanol production by C. autoethanogenum. Deletion of either formate transport, acetaldehyde dehydrogenase or formate dehydrogenase (ferredoxin) from the metabolic model of C. autoethanogenum leads to a (potential) increase in ethanol production up to 150%, which is clearly very attractive.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2019

Identification of a novel therapeutic agent for Inflammatory Bowel Disease guided by systems medicine

ObjectiveInflammatory bowel diseases cause significant morbidity and mortality. Aberrant NF-{kappa}B signalling is strongly associated with these conditions, and several established drugs influence the NF-{kappa}B signalling network to exert their effect. This study aimed to identify drugs which alter NF-{kappa}B signalling and may be repositioned for use in inflammatory bowel disease. DesignThe SysmedIBD consortium established a novel drug-repurposing pipeline based on a combination of in-silico drug discovery and biological assays targeted at demonstrating an impact on NF-kappaB signalling, and a murine model of IBD. ResultsThe drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic Clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. Clarithromycins effects were validated in several experiments: it influenced NF-{kappa}B mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-{kappa}B protein shuttling in murine reporter enteroids; it suppressed NF-{kappa}B (p65) DNA binding in the small intestine of mice exposed to LPS, and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-{kappa}B (p65) nuclear translocation in human intestinal enteroids. ConclusionsThese findings demonstrate that in-silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of inflammatory bowel disease; and that further clinical assessment of clarithromycin in the management of inflammatory bowel disease is required.

systems biology↗

bioRxiv2018

Expected and observed genotype complexity in prokaryotes: correlation between 16S-rRNA phylogeny and protein domain content

BackgroundThe omnipresent 16S ribosomal RNA gene (16S-rRNA) is commonly used to identify and classify bacteria though it does not take into account the distinctive functional characteristics of taxa. We explored functional domain landscapes of over 5700 complete bacterial genomes, representing a wide coverage of the bacterial tree of life, and investigated to what extent the observed protein domain diversity correlates with the expected evolutionary diversity, using 16S-rRNA as metric for evolutionary distance.\n\nResultsAnalysis of protein domains showed that 83% of the bacterial genes code for at least one of the 9722 domain classes identified. By comparing clade specific and global persistence scores, candidate horizontal gene transfer and signifying domains could be identified. 16S-rRNA and functional domain content distances were used to evaluate and compare species divergence and overall a sigmoid curve is observed. Already at close 16S-rRNA evolutionary distances, high levels of functional diversity can be observed. At a larger 16S-rRNA distance, functional differences accumulate at a relatively lower pace.\n\nConclusionsAnalysis of 16S-rRNA sequences in the same taxa suggests that, in many cases, additional means of classification are required to obtain reliable phylogenetic relationships. Whole genome protein domain class phylogenies correlate with, and complement 16S-rRNA sequence-based phylogenies. Moreover, domain-based phylogenies can be constructed over large evolutionary distances and provide an in-depth insight of the functional diversity within and among species and enables large scale functional comparisons. The increased granularity obtained paves way for new applications to better predict the relationships between genotype, physiology and ecology.

genomics↗