Biology subjects

Vargas, B. d. O.

Publications and source records attributed to Vargas, B. d. O..

3 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↗

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↗

bioRxiv2021

Rational engineering of industrial S. cerevisiae: towards xylitol production from sugarcane bagasse

BACKGROUNDSugarcane hemicellulosic material is a compelling source of usually neglected xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as xylitol. In this context, Saccharomyces cerevisiae strains typically used in the Brazilian bioethanol industry are a robust chassis for genetic engineering, given their robustness towards harsh operational conditions and outstanding fermentation performance. Nevertheless, there are no reports on the use of these strains for xylitol production using sugarcane hydrolysate. RESULTSPotential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5-NGG 20 nt sequences, and guidelines for the fast and cost-effective CRISPR-editing of such strains. After genomic integration of a NADPH-preferring xylose reductase (XR), FMYX (SA-1 ho{Delta}::xyl1) and CENPKX (CEN.PK-122 ho{Delta}::xyl1) were tested in varying cultivation conditions for xylitol productivity to infer influence of the genetic background. Near-theoretical yields were achieved for all strains, however the industrial consistently outperformed the laboratory strain. Batch fermentation of raw sugarcane bagasse hydrolysate with remaining solid particles represented a challenge for xylose metabolization and 3.65 {+/-} 0.16 g/L xylitol titre was achieved by FMYX. Finally, quantification of NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX. CONCLUSIONSAlthough widely used in several S. cerevisiae strains, this is the first report of CRISPR-Cas9 editing major yeast of the Brazilian bioethanol industry. Fermentative assays of xylose consumption revealed that NADPH availability is closely related to mutant strains performance. We also pioneer the use of sugarcane bagasse as a substrate for xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation industry, given its inhibitor tolerance and better redox environment that may favor production of reduced sugars.

molecular biology↗