Biology subjects

Vicente, C. M.

Publications and source records attributed to Vicente, C. M..

6 recordsLinked to original sources

bioRxiv2024

Role of a FAD-dependent monooxygenase in diazo group functionalisation of kinamycin in Streptomyces ambofaciens

Kinamycin biosynthesis is a complex process which has been extensively studied over the years, yet specific enzymatic steps continue to be unveiled. A diazo group present in the molecule is responsible for the promising antitumoral activity of kinamycins but its specific installation in Streptomyces ambofaciens is not fully understood. In this study, we explore the diazo functionalization of kinamycin in this strain. A new FAD-dependent monooxygenase is identified which is essential for kinamcyin biosynthesis. In its absence, stealthin C accumulates instead likely as a pathway shunt-product. Furthermore, as a result of the position of the gene encoding this monooxygenase, named alp2F, we also propose new boundaries of the kinamycin biosynthetic gene cluster resulting in a large cluster spanning over 72kb. This work paves the way for the continued understanding of the biosynthetic steps that are characteristic of diazo-containing natural products, and provides new biocatalysts for molecular engineering and accelerate bioactive compounds production.

microbiology↗

bioRxiv2024

Systems-level analysis provides insights on methanol-based production of L-glutamate and its decarboxylation product γ-aminobutyric acid by Bacillus methanolicus

BackgroundBacillus methanolicus is the next workhorse in biotechnology using methanol, an alternative and economical one-carbon feedstock that can be obtained directly from carbon dioxide, as both carbon and energy source for the production of various value-added chemicals. The wild-type strain B. methanolicus MGA3 naturally overproduces O_SCPLOWLC_SCPLOW-glutamate in methanol-based fed-batch fermentations. ResultsHere we generated, by induced mutagenesis, an evolved B. methanolicus strain exhibiting enhanced O_SCPLOWLC_SCPLOW-glutamate production capability (>150%). To showcase the potential of this evolved strain, further metabolic engineering enabled the production of {gamma}-aminobutyric acid (GABA) directly from O_SCPLOWLC_SCPLOW-glutamate, with a yield of 13.2 g/L from methanol during fed-batch fermentations. By using a systems-level analysis, encompassing whole-genome sequencing, RNA sequencing, fluxome analysis and genome-scale metabolic modelling, we were able to elucidate the metabolic and regulatory adaptations that sustain the biosynthesis of these products. The metabolism of the mutant strain evolved to prioritize energy conservation and efficient carbon utilization. Key metabolic shifts include the downregulation of energy-intensive processes such as flagellation and motility and the rerouting of carbon fluxes towards -ketoglutarate and its derivative, O_SCPLOWLC_SCPLOW-glutamate. Moreover, we observed that transformation of the evolved strain with a GABA biosynthesis plasmid had a positive effect on O_SCPLOWLC_SCPLOW-glutamate production, likely due to an upregulation of various transaminases involved in the O_SCPLOWLC_SCPLOW-glutamate biosynthesis from -ketoglutarate. ConclusionsThese results and insights provide a foundation for further rational metabolic engineering and bioprocess optimization, enhancing the industrial viability of B. methanolicus for sustainable production of O_SCPLOWLC_SCPLOW-glutamate and its derivatives.

systems biology↗

bioRxiv2024

SYNTERUPTOR: mining genomic islands for non-classical specialised metabolite gene clusters

Microbial specialised metabolite biosynthetic gene clusters (SMBGCs) are a formidable source of natural products of pharmaceutical interest. With the multiplication of genomic data available, very efficient bioinformatic tools for automatic SMBGC detection have been developed. Nevertheless, most of these tools identify SMBGCs based on sequence similarity with enzymes typically involved in specialised metabolism and thus may miss SMBGCs coding for under characterised enzymes. Here we present SYNTERUPTOR (https://bioi2.i2bc.paris-saclay.fr/synteruptor), a program that identifies genomic islands, known to be enriched in SMBGCs, in the genomes of closely related species. With this tool, we identified a SMBGC in the genome of Streptomyces ambofaciens ATCC23877, undetected by earlier versions of antiSMASH, and experimentally demonstrated that it directs the biosynthesis of two metabolites, one of which was identified as sphydrofuran. SYNTERUPTOR is also a valuable resource for the delineation of individual SMBGCs within antiSMASH regions that may encompass multiple clusters, and for refining the boundaries of these SMBGCs.

bioinformatics↗

bioRxiv2022

Modulation of multiple gene clusters expression by a single PAS-LuxR transcriptional regulator

PAS-LuxR transcriptional regulators are highly conserved enzymes governing polyene macrolide antifungal biosynthesis. PteF is one of such regulators, situated in the polyene macrolide filipin gene cluster from Streptomyces avermitilis. Its mutation leads to a drastic filipin production decline, but also to a severe loss of oligomycin production, an ATP-synthase inhibitor of macrolide structure, and a delay in sporulation, thus it has been considered as a transcriptional activator. Transcriptomic analyses were carried out in S. avermitilis {Delta}pteF and its parental strain S. avermitilis NRRL 8165 as control. Both strains were grown in YEME medium without sucrose, and samples were taken in the exponential and stationary growth phases. 257 genes showed altered expression in the PteF-deleted mutant, most of them in the exponential phase of growth. Surprisingly, despite PteF being an activator of filipin biosynthesis, a majority of the genes affected upon mutation showed overexpression thus suggesting a negative modulation of those genes. Genes affected were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation; among others, but particularly to secondary metabolite biosynthesis. Notably, ten secondary metabolite gene clusters out of 38 encoded by the genome, some of them encoding cryptic compounds, showed altered expression profiles in the mutant, suggesting a regulatory role for PteF wider than expected. Transcriptomic results were validated by quantitative reverse transcription polymerase chain reaction. These findings provide important clues to understand the intertwined regulatory machinery that modulates antibiotic biosynthesis in Streptomyces.

microbiology↗

bioRxiv2020

Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli

One-carbon (C1) compounds, such as methanol, have recently gained attention as alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable research efforts are thus currently focused on the generation of synthetic methylotrophs by transferring methanol assimilation pathways into established bacterial production hosts. In this study, we used an iterative combination of dry and wet approaches to design, implement and optimize this metabolic trait in the most common chassis, E. coli. Through in silico modeling, we designed a new route that "mixed and matched" two methylotrophic enzymes: a bacterial methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify the best combination of enzymes to introduce into E. coli, we built a library of 266 pathway variants containing different combinations of Mdh and Das homologues and screened it using high-throughput 13C-labeling experiments. The highest level of incorporation, 22% of labeled methanol carbon into the multi-carbon compound PEP, was obtained using a variant composed of a Mdh from A. gerneri and a codon-optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain did. Broadening the repertoire of methanol assimilation pathways is one step further toward synthetic methylotrophy in E. coli.

synthetic biology↗

bioRxiv2019

Charting the metabolic landscape of the facultative methylotroph Bacillus methanolicus

Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. This has motivated a number of omics studies of B. methanolicus at the genome, transcript, protein and metabolic levels. Intriguingly, the only substrates known to support B. methanolicus growth as sole source of carbon and energy are methanol, mannitol, and to a lesser extent glucose and arabitol. We hypothesized that comparing methylotrophic and non-methylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. 13C metabolic flux analysis (13C-MFA) is a powerful computational method to estimate carbon flows from substrate to biomass (i.e. the in vivo reaction rates of the central metabolic pathways) from experimental labeling data. In this study, we designed and performed a 13C-MFA of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol and arabitol to compare the associated metabolic states. The results obtained validate previous findings on the methylotrophy of B. methanolicus, allowed us to characterize the assimilation pathway of one of the studied carbon sources, and provide a better overall understanding of this strain. IMPORTANCEMethanol is cheap, easy to transport and can be produced both from renewable and fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as B. methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories, or to design methylotrophic capability in other strains already used by the industry.

systems biology↗