Biology subjects

Perea, S.

Publications and source records attributed to Perea, S..

7 recordsLinked to original sources

bioRxiv2026

Expanding the scope of redox-balance growth coupling techniques with a carbon cofeeding strategy

Metabolic engineering to produce molecules not naturally synthesized by the host often requires directed evolution to improve pathway enzyme performance. Growth-coupled selection can dramatically increase directed-evolution throughput, and manipulation of redox balance has proven effective for tying reductase fitness to microbial growth. However, most redox-balance selections require feeding the reductase substrate because of stoichiometric constraints. This is impractical for many biosynthetic pathways either due to practical limitations on cost or complexity of bulk substrate synthesis, or the lack of an ability to transport substrate into cells, for example intracellular acyl-CoA/ACP intermediates. Here we define stoichiometric constraints that make substrate feeding necessary for many acetyl-CoA-derived reduction pathways in NADPH-imbalanced hosts. We overcome these constraints with a dual-feedstock strategy in which glucose provides reducing power while acetate supplies additional acetyl-CoA without directly perturbing redox balance. In an engineered E. coli selection strain, acetate co-feeding enabled growth coupling of acetaldehyde, 3-hydroxybutyrate, and mevalonate production and produced a linear correlation between product formation and growth. We then used this selection to evolve a class II HMG-CoA reductase (HMGR) from Delftia acidovorans toward NADPH utilization, enriching variants with improved NADPH-dependent activity. Finally, propionate co-feeding enabled growth coupling of propionyl-CoA reduction, supporting the generality of carbon co-feeding for selecting enzymes in pathways involving acyl-chain elongation and reduction. HighlightsO_LIStoichiometric limits of redox-balance growth coupling are defined C_LIO_LIAcetate co-feeding supplies acetyl-CoA without perturbing redox balance C_LIO_LICo-feeding enables growth coupling of acetaldehyde, 3-HB, and mevalonate C_LIO_LIGrowth coupling enables evolution of HMGR toward NADPH specificity C_LIO_LIPropionate co-feeding extends growth coupling to additional acyl-CoA substrates C_LI

bioengineering↗

bioRxiv2025

Activation-Free Upgrading of Carboxylic Acids to Aldehydes and Alcohols

Advances in organic and gas waste valorization have enabled high-yield production of carboxylic acids, positioning them as promising feedstocks for the bioeconomy. However, carboxylic acids must be activated before downstream use, typically requiring ATP, CoA, or reduced ferredoxin to overcome unfavorable thermodynamics. These activators are costly to generate and divert carboxylic acids into CO2-releasing pathways, reducing carbon efficiency. Here, we demonstrate that aldehyde dehydrogenases (ALDHs) can directly reduce carboxylic acids to aldehydes without prior activation, a process previously thought to be biologically inaccessible. Screening 133 ALDHs revealed that this activity is remarkably widespread within the protein family, enabling production of aliphatic aldehydes and alcohols, diols, and aromatic alcohols, at titers >1 g/L, in some cases, after optimization of thermodynamic driving forces. Additionally, we applied this system to upgrade waste-derived carboxylic acid effluent streams from wastewater sludge, food waste, and waste gas (CO2). This activation-free process, termed "reverse aldehyde oxidation" (rAOX), establishes a broadly applicable, energy-efficient platform for carboxylic acid valorization at 100% carbon yield. Analogous to the reverse tricarboxylic acid cycle (rTCA) and reverse {beta}-oxidation (rBOX), rAOX exemplifies that metabolic reactions classically defined as unidirectional may have unexpected plasticity to operate in reverse and open new avenues in biomanufacturing.

synthetic biology↗

bioRxiv2025

Dating and quantifying past gene flow due to hybridization events in Iberian chubs

Hybridization - the successful reproductive cross between individuals from different species - is increasingly recognized as a relatively common phenomenon in nature, mostly due to the increasing availability of genomic data for many organisms and the development of sophisticated methods to detect past gene flow. As a result, evidence of past gene flow has been detected in the genomes of many species, allowing us to investigate the evolutionary forces that act upon hybrid genomes. Biological systems where multiple instances of hybridization have been detected at different time scales are especially useful for this, as they allow investigating the effects of selection and recombination at different time scales post-hybridization. The Iberian chubs (Squalius spp.) present an ideal study system for this. In this group of primary fish inhabiting the rivers of the Iberian Peninsula, multiple instances of past hybridization at different geographical and time scales were reported between different species. However, before we investigate the effects of selection and recombination, it is crucial that we date the hybridization events and quantify the contribution from each parental species. In this study, we infer the demographic history of two Iberian chub hybrid lineages, one resulting from a proposed ancient hybridization event (S. pyrenaicus) and another potentially much more recent (Sao Martinho). We generated whole genome resequencing data for 52 individuals from the two hybrid lineages and their putative parental populations. Our demographic modelling results confirm the hypothesis of ancient hybridization between S. carolitertii and S. tartessicus around 600,000-800,000 years ago, giving rise to the S. pyrenaicus lineage. We also confirm that S. carolitertii is the major parental, with S. tartessicus only contributing <10% to the S. pyrenaicus genome. Furthermore, we confirm the more recent hybridization between S. caetobrigus and S. pyrenaicus (around 4500 years ago) in the Sao Martinho population. We observe similar contributions from both parental species (around 50%) to the genome of the individuals of this population. Overall, our results give us a deeper understanding of how and when these past gene flow events happened and the contributions of each parental species to the genomes of the individuals of the hybrid lineages.

evolutionary biology↗

bioRxiv2023

Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor

Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate life-essential redox reaction directions. However, in whole cells the reliance on these canonical cofactors limits flexible control of redox reaction direction as these reactions are permanently tied to catabolism or anabolism. In cell-free systems, NADP+ is too expensive in large scale. We have previously reported the use of nicotinamide mononucleotide, (NMN+) as a low-cost, noncanonical redox cofactor capable of specific electron delivery to diverse chemistries. Here, we present Nox Ortho, an NMNH-specific water-forming oxidase, that completes the toolkit to modulate NMNH/NMN+ ratio. This work uncovers an enzyme design principle that succeeds in parallel engineering of six butanediol dehydrogenases as NMN(H)-orthogonal biocatalysts consistently with a 103 - 106 -fold cofactor specificity switch from NAD(P)+ to NMN+. We combine these to produce chiral-pure 2,3-butanediol (Bdo) isomers without interference from NAD(H) or NADP(H) in vitro and in E. coli cells. We establish that NMN(H) can be held at a distinct redox ratio on demand, decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo.

synthetic biology↗

bioRxiv2022

CIGB-300 peptide targets the CK2 phospho-acceptor domain on Human Papillomavirus E7 and disrupts the Retinoblastoma (RB) complex in cervical cancer cells

CIGB-300 is a clinical-grade anti- Protein Kinase CK2 peptide, binding both its substrates phospho-acceptor site and the CK2 catalytic subunit. The cyclic p15 inhibitory domain of CIGB-300 was initially selected in a phage display library screen for its ability to bind the CK2 phospho-acceptor domain of HPV-16 E7. However, the actual role of this targeting in CIGB-300s antitumoral mechanism remains unexplored. Here, we investigated the physical interaction of CIGB-300 with HPV-E7 and its impact on CK2-mediated phosphorylation. Hence, we studied the relevance of targeting E7 phosphorylation for the cytotoxic effect induced by CIGB-300. Finally, co-immunoprecipitation experiments followed by western blot were performed to study the impact of the peptide on the E7-pRB interaction. Interestingly, we found a clear binding of CIGB-300 to the N terminal region of E7 proteins from HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduces the CK2-mediated phosphorylation of E7, as well as its binding to the tumour suppressor pRB. However, the targeting of E7 phosphorylation by CIGB-300 seemed to be dispensable for the induction of cell death in HPV-18 cervical cancer-derived C4-1 cells. These findings unveil novel molecular clues to the means by which the CIGB-300 triggers cell death in cervical cancer cells.

cancer biology↗

bioRxiv2020

Influence of historical and human factors on genetic structure and diversity patterns in peripheral populations: implications for the conservation of Moroccan trout

O_LIThe brown trout s.l. has been the focus of numerous phylogeographic and conservation studies due to its socioeconomic importance, its marked genetic and phenotypic differentiation and its broad distribution range. Especially interesting evolutionary patterns are observed for populations occupying peripheral areas of a distribution range, such as in the case of the highly isolated trout populations in Morocco. C_LIO_LIContinuous stocking programs may conceal natural genetic patterns, making it challenging to discern evolutionary patterns. In Morocco, trout stocking programs have been implemented to increase the genetic diversity of native populations by pooling fish of different origins in the Ras el Ma hatchery (Azrou region) and then stocking them in the different basins. In this study, phylogenetic and phylogeographic patterns, as well as genetic structure and diversity, of Moroccan trout populations were analyzed to evaluate the impact of continuous fish stocking on evolutionary processes in order to better distinguish between natural and human-mediated patterns. C_LIO_LITwo mitochondrial and nine microsatellite markers were analyzed for all populations along the entire distribution range of brown trout in Morocco. Phylogenetic and phylogeographic analyses rendered two highly divergent evolutionary lineages, one comprising populations in the Draa Basin and a second grouping the remaining Moroccan populations. Divergence of the Draa lineage occurred during the Upper Pliocene, whilst differentiation within the second lineage coincided with the onset of the Pleistocene. C_LIO_LIGenetic structuring among populations was evident. Nevertheless, populations exhibiting higher levels of genetic diversity were those affected by human-mediated processes, making it difficult to associate this diversity with natural processes. In fact, highly geographically isolated, not stocked populations showed the lowest values of genetic diversity. Although stocking management may increase the genetic diversity of these populations, it could also lead to the loss of local adaptive genotypes. Hence, current trout conservation programs should be revised. C_LI

evolutionary biology↗

bioRxiv2017

Whole-exome sequencing of plasma cell-free DNA portrays the somatic mutation landscape of refractory metastatic colorectal cancer and enables the discovery of mutated KDR/VEGFR2 receptors as modulators of anti-angiogenic therapies.

The non-invasive detection of cancer mutations is a breakthrough in oncology. Here, we applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens including VEGFR inhibitors. Using WES-cfDNA, we could detect 73% (54/74) of the somatic mutations uncovered by WES-tumor including a variety of mutation types: frameshift (indels), missense, noncoding (splicing), and nonsense mutations. Additionally, WES-cfDNA discovered 14 high-confidence somatic mutations not identified by WES-tumor. Importantly, in the absence of the tumor specimen, WES-cfDNA could identify 68 of the 88 (77.3%) total mutations that could be identified by both techniques. Of tumor biology relevance, we identified the novel KDR/VEGFR2 L840F somatic mutation, which we showed was a clonal mutation event in this tumor. Comprehensive in vitro and in vivo functional assays confirmed that L840F causes strong resistance to anti-angiogenic drugs, whereas the KDR/VEGFR2 hot-spot mutant R1032Q confers sensitivity to cabozantinib. Moreover, we found a 1-3% of recurrent KDR somatic mutations across large and non-overlapping cancer sequencing projects, and the majority of these mutations were located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK, suggesting a functional role.\n\nIn summary, the current study highlights the capability of exomic sequencing of cfDNA from plasma of cancer patients as a powerful platform for somatic landscape analysis and discovery of resistance-associated cancer mutations. Because of its advantage to generate results highly concordant to those of tumor sequencing without the hurdle of conventional tumor biopsies, we anticipate that WES-cfDNA will become frequently used in oncology. Moreover, our study identified for the first-time KDR/VEGFR2 somatic mutations as potential genetic biomarkers of response to anti-angiogenic cancer therapies and will serve as reference for further studies on the topic.

cancer biology↗