Biology subjects

Pereira, G. A. G.

Publications and source records attributed to Pereira, G. A. G..

10 recordsLinked to original sources

bioRxiv2025

A rational approach for multicopy delta integration in Saccharomyces cerevisiae targeting a conserved Ty1 LTR sequence

Genomic integration is a foundation in yeast-based cell factories, ensuring phenotypic stability and adequate gene dosage to sustain desired metabolic fluxes. Retrotransposon-associated sequences are attractive genomic integration targets for metabolic engineering, as they are present in multiple copies throughout the genome and their disruption should not jeopardize the cell. In S. cerevisiae, multiple strategies have been proposed to target Ty elements, but little attention has been given to the homology sequence itself. Through analysis of all long terminal repeats in S. cerevisiae, we propose a novel consensus {delta} sequence for efficient multicopy gene integration. Reducing homology length to favor a more conserved sequence greatly increases the number of potential genomic integration sites - reaching 85 targets in this work. The designed integration sequence was validated by introducing a non-native xylose fermentation pathway into this yeast, achieving a single-step simultaneous integration of 15 xylose isomerase copies, enabling efficient ethanol production in the engineered strain.

synthetic biology↗

bioRxiv2025

Epistatic mutations in ISC metabolism synergize with cell cycle regulation and the PPP to enhance xylose fermentation and acetic acid tolerance in industrial yeast

Efficient utilization of complex biomass-derived sugars and tolerance to inhibitors are key requirements for the viability of lignocellulosic-based biorefineries. In this study, a two-stage evolution of an industrial yeast strain engineered with a xylose isomerase pathway yielded strain AceY.14, which exhibited improved fermentative performance and increased tolerance to acetic acid. Whole-genome sequencing of the evolved strain identified SNPs in ZWF1, a component of the pentose phosphate pathway (PPP), and in the G1 cyclin gene CLN3, both of which were functionally validated through CRISPR and reverse engineering. The zwf1E191D mutation reduced xylitol accumulation, alleviating inhibition of xylose isomerase and enhancing flux through the non-oxidative branch of the PPP, while the frameshift cln3T556fsmutation unexpectedly improved acetic acid tolerance and xylose consumption in the evolved strain, also affecting cell size and growth. Genome sequencing of AceY.14 also revealed a significant reduction in the xylA gene copy number, likely decreasing the metabolic burden associated with high xylose isomerase expression. A synergistic effect was observed in the isu1{Delta}/zwf1{Delta} double mutant, further boosting xylose consumption rates. A diploid derivative (AceY-2n) demonstrated high productivity and robustness in fermentations using hydrolysates from various lignocellulosic feedstocks, highlighting the strains potential for industrial-scale applications. These findings reveal novel metabolic targets for strain optimization and offer valuable insights for the rational engineering of yeast platforms for sustainable biofuel and bioproduct production.

synthetic biology↗

bioRxiv2025

A Transcriptomic Atlas of Macauba Palm Reveals Organ-Specific Gene Expression and Stress-Related Pathways

The macauba palm (Acrocomia aculeata) is an emerging oilseed species with promising applications in biodiesel production, as well as in food and cosmetic industries. Native to the Neotropical, it is incipiently domesticated and distributed across diverse environments and edaphoclimatic conditions. However, genomic studies of macauba are limited due to the scarcity of publicly available sequence data, as it is considered a non-model plant. In this study, we present an exploratory analysis of a transcriptome dataset comprising seven organs (roots, bulbs, male and female flowers, leaves, leaf sheath, and fruits). A total of 22,703 transcripts were assembled into a single reference dataset. Of these, 9,729 transcripts (42.85%) were annotated using KEGG orthology. Gene expression profiling revealed 306, 32, 41, 159, 158 and 916 organ-specific transcripts in leaves, leaf sheaves, bulbs, flowers, fruit and root, respectively. Comparative analysis with African oil palm (Elaeis guineensis) and date palm (Phoenix dactylifera) revealed 55 gene families exclusive to macauba palm. In addition, 221 transcripts related to drought stress were identified, grouped into 112 families. Root libraries revealed 7,091 fungal transcripts - approximately 3.9% of all reads - mainly derived from arbuscular mycorrhizal fungi (AMF) Rhizophagus spp. These findings highlight the central role of signal transduction pathways in response to environmental stresses in macauba palm. The transcriptome dataset generated in this study provides a valuable genomic resource for future genotype-phenotype investigations in macauba palm. Furthermore, the presence of AMF-associated transcripts suggests a potentially important role for these symbiotic fungi in macauba palm growth and development.

plant biology↗

bioRxiv2024

Unveiling novel mechanisms of strobilurin resistance in the cacao pathogen Moniliophthora perniciosa

Witches broom disease (WBD) is a major constraint for cacao production in the Americas. The severe socioeconomic impact of WBD encouraged the evaluation of different control strategies, including the use of strobilurin fungicides. These molecules inhibit mitochondrial respiration, thus impairing ATP generation and leading to oxidative stress. These chemicals, however, have proven ineffective against the WBD pathogen Moniliophthora perniciosa. Here, we demonstrate that M. perniciosa tolerates high concentrations of strobilurins under in vitro conditions and highlight a set of molecular alterations that correlate with strobilurin tolerance in this fungus. Short-term exposure of M. perniciosa to the commercial strobilurin azoxystrobin led to the up-regulation of genes encoding enzymes of the glyoxylate cycle, gluconeogenesis, and fatty acid and amino acid catabolism, indicating that the fungal metabolism is remodeled to compensate for reduced ATP production. Furthermore, cell division, ribosome biogenesis, and sterol metabolism were repressed, which agrees with the impaired mycelial growth on azoxystrobin. Genes associated with cellular detoxification and response to oxidative stress (e.g., cytochrome P450s, membrane transporters and glutathione s-transferases) were strongly induced by the drug and represent potential strategies used by the pathogen to mitigate the toxic effects of the fungicide. Remarkably, exposure of M. perniciosa to azoxystrobin resulted in the spontaneous generation of a mutant with increased resistance to strobilurin. Comparative genomics and transcriptomics revealed alterations that may explain the resistance phenotype, including a large deletion in a putative transcriptional regulator and significant changes in the mutant transcriptome. Overall, this work provides important advances towards a comprehensive understanding of the molecular basis of strobilurin resistance in a tropical fungal pathogen. This is a fundamental step to efficiently employ these fungicides in agriculture and to prevent the emergence of strobilurin resistance.

microbiology↗

bioRxiv2024

Unveiling genetic anchors in Saccharomyces cerevisiae: QTL mapping identifies IRA2 as a key player in ethanol tolerance and beyond

Ethanol stress in Saccharomyces cerevisiae is a well-studied phenomenon, but pinpointing specific genes or polymorphisms governing ethanol tolerance remains a subject of ongoing debate. Naturally found in sugar-rich environments, this yeast has evolved to withstand high ethanol concentrations, primarily produced during fermentation in the presence of suitable oxygen or sugar levels. Originally a defense mechanism against competing microorganisms, yeast-produced ethanol is now a cornerstone of brewing and bioethanol industries, where customized yeasts require high ethanol resistance for economic viability. However, yeast strains exhibit varying degrees of ethanol tolerance, ranging from 8% to 20%, making the genetic architecture of this trait complex and challenging to decipher. In this study, we introduce a novel QTL mapping pipeline to investigate the genetic markers underlying ethanol tolerance in an industrial bioethanol S. cerevisiae strain. By calculating missense mutation frequency in a prominent QTL region within a population of 1011 S. cerevisiae strains, we uncovered rare occurrences in gene IRA2. Following molecular validation, we confirmed the significant contribution of this gene to ethanol tolerance, particularly in concentrations exceeding 12% of ethanol. IRA2 pivotal role in stress tolerance due to its participation in the Ras-cAMP pathway was further supported by its involvement in other tolerance responses, including thermotolerance, low pH tolerance, and resistance to acetic acid. Understanding the genetic basis of ethanol stress in S. cerevisiae holds promise for developing robust yeast strains tailored for industrial applications.

genetics↗

bioRxiv2023

Comparative Genomics of Firmicutes reveals probable adaptations for xylose fermentation in Thermoanaerobacterium saccharolyticum

Second-generation (2G) ethanol is one potential biofuel that could be used to achieve the goal of reducing greenhouse gas emissions. Many challenges still need to be overcome for the feasibility of this technology, most of them related to consumption of xylose, a pentose sugar not easily metabolized by industrial microorganisms. Thus, exploring genes, pathways and other organisms that can ferment xylose is a strategy implemented to solve industrial bottlenecks. Thermoanaerobacterium saccharolyticum (T. sac) is an organism from the firmicutes phylum, capable of naturally fermenting compounds of industrial interest, such as xylan and xylose. Understanding evolutionary adaptations may help not only to solidify this bacterium as a potential substitute to the yeast Saccharomyces cerevisiae in industry, but also bring novel genes and information that can be used for yeast, enhance its fermenting capabilities, and increase production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve T. sac as a new microbial cell factory, which may help to solve current problems found in the biofuels industry.

evolutionary biology↗

bioRxiv2022

Strategies for improved xylitol production in batch fermentation of sugarcane hydrolysate using Saccharomyces cerevisiae

A plethora of studies have focused on improvements of xylitol production. The challenges of establishing a biotechnological route for the industrial production of this sugar have been explored using different microorganisms and renewable feedstock. Nevertheless, sugarcane biomass has been neglected as the pentose source for xylitol production using Saccharomyces cerevisiae. Therefore, here we investigate the use of an industrial S. cerevisiae strain for xylitol production in batch fermentation of non-detoxified sugarcane straw hydrolysate, envisioning the diversification of the current infrastructure used for second-generation bioethanol production from the same lignocellulosic material. In order to optimize the xylose conversion in a non-fed cultivation system, guidelines in cell inoculum and medium supplementation are suggested, as well as the first attempt to use electro-fermentation for this purpose. Accordingly, our results show that the increase in initial cell density and hydrolysate supplementation allows a xylitol production of 19.24 {+/-} 0.68 g/L, representing 0,132 g/L.h productivity.

microbiology↗

bioRxiv2021

Adaptive evolution of Moniliophthora PR-1 proteins towards its pathogenic lifestyle

Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that harbor a large number of Pathogenesis-Related 1 genes, many of which are induced in the biotrophic interaction with Theobroma cacao. Here, we provide evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Phylogenetic analysis revealed conserved PR-1 genes, shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora, as well as in recent specialization cases within both species. PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arm race. Furthermore, we show that the highly diversified MpPR-1 genes are not induced by two phytoalexins, suggesting detoxification might not be their main function as proposed before.

evolutionary biology↗

bioRxiv2020

Transcriptome analysis of three Agave fiber-producing cultivars suitable for biochemicals and biofuels production in semiarid regions

O_LIAgaves, which have been grown commercially for fiber or alcoholic beverages, are emerging as a candidate crop for biochemicals and biofuels production in semiarid regions because of their high productivity in low rainfall areas, drought tolerance, and low lignin content. C_LIO_LIIn this work, we present the transcriptomic atlas of Agave sisalana, Agave fourcroydes, and agave hybrid 11648 (A. amaniensis x A. angustifolia) under prolonged drought in field conditions. Leaf, stem, and root tissues were sequenced, and gene expression profiles were correlated with biomass composition, enzymatic hydrolysis of cell wall carbohydrates, histochemical analysis, and non-structural carbohydrates content. C_LIO_LIDifferences in biomass accessibility were attributed to either lignin content or lignin composition, possibly through modification of s/g ratio promoted by changes in Caffeic Acid 3-O-Methyltransferase (COMT) transcript abundance. Unlike most plants, the most highly expressed transcripts do not encode photosynthetic proteins, but rather involved in stress response. Although the three cultivars presented quantitative differences in global gene expression, they activated a highly overlapping set of genes. The main molecular strategies employed by agave to cope with high-temperature and drought seem to consist in overexpressing HSP and LEA, as well as promoting raffinose accumulation as an osmolyte. C_LIO_LIIn conclusion, our data provide vital new genetic information for the study of Agave species and provide new insights into cell wall architecture, recalcitrance, and resistance to abiotic stresses for these species. C_LI

plant biology↗

bioRxiv2017

Mitotic systemic genomic instability in yeast

Conventional models of genome evolution generally include the assumption that mutations accumulate gradually and independently over time. We characterized the occurrence of sudden spikes in the accumulation of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, suggesting the existence of a mitotic systemic genomic instability process (mitSGI). We characterized the emergence of a rough colony morphology phenotype resulting from an LOH event spanning a specific locus (ACE2/ace2-A7). Surprisingly, half of the clones analyzed also carried unselected secondary LOH tracts elsewhere in their genomes. The number of secondary LOH tracts detected was 20-fold higher than expected assuming independence between mutational events. Secondary LOH tracts were not detected in control clones without a primary selected LOH event. We then measured the rates of single and double LOH at different chromosome pairs and found that coincident LOH accumulated at rates 30-100 fold higher than expected if the two underlying single LOH events occurred independently. These results were consistent between two different strain backgrounds, and in mutant strains incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population experience systemic genomic instability episodes, resulting in multiple chromosomal rearrangements over one or few generations. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in the cancer and genomic disorder contexts, all of which challenge the idea of gradual accumulation of structural genomic variation. Our experimental approach provides a model to further dissect the fundamental mechanisms responsible for mitSGI.\n\nSIGNIFICANCE STATEMENTPoint mutations and alterations in chromosome structure are generally thought to accumulate gradually and independently over many generations. Here, we combined complementary genetic approaches in budding yeast to track the appearance of chromosomal changes resulting in loss-of-heterozygosity (LOH). Contrary to expectations, our results provided evidence for the occurrence of non-independent accumulation of multiple LOH events over one or a few cell generations. These results are analogous to recent reports of bursts of chromosomal instability in humans. Our experimental approach provides a framework to further dissect the fundamental mechanisms underlying systemic chromosomal instability processes, including in the human cancer and genomic disorder contexts.

genetics↗