Biology subjects

Barreto, J. A.

Publications and source records attributed to Barreto, J. A..

4 recordsLinked to original sources

bioRxiv2025

QTL mapping, breeding, and debugging Saccharomyces cerevisiae strains through Reiterated Mass Selection and backcrosSing (ReMaSSing)

BackgroundProducing second-generation ethanol from lignocellulosic hydrolysates (LCHs) poses significant challenges for Saccharomyces cerevisiae due to the presence of fermentation inhibitors. Quantitative trait loci (QTL) mapping of stress-tolerant S. cerevisiae strains is important for identifying adaptive alleles that can enhance yeast fermentation of LCHs. However, the QTL mapping process is labor-intensive, requiring the screening of numerous recombinants and repeated crossings to improve mapping resolution. ResultsWe developed Reiterated Mass Selection and backcrosSing (ReMaSSing) to facilitate the identification of adaptive alleles through QTL mapping and to enhance LCH tolerance in yeast strains. ReMaSSing was applied to populations obtained by crossing the stress-resistant yeast PE-2_H4 with the laboratory strain S288C. Using alternative protocols, we selected haploid or diploid populations with dominant markers, enriching millions of segregants carrying adaptive alleles by propagating them in standard or LCH-supplemented media. The enriched pools were then bulk backcrossed with S288C, and germination of millions of spores generated new recombinant populations for subsequent selection cycles. After five rounds of ReMaSSing, whole-genome sequencing and QTL mapping identified key alleles associated with LCH tolerance, linked to VPS70, CAT5, GCY1, UBP2, MKT1/SAL1, HAP1, and PHO84, which influence growth and mitochondrial function in S288C. Mutations in IRA1 and HTA1, unique to our S288C strain, were also mapped, highlighting ReMaSSings ability to detect and correct deleterious alleles ("bugs"). Allele swapping and competition assays confirmed that the identified QTL improved LCH tolerance and growth, with strains combining adaptive alleles performing over 20% better than the parental S288C. Finally, applying ReMaSSing to breed an LCH-tolerant yeast with a xylose-consuming strain produced recombinants with improved fermentation of xylose-enriched LCH. ConclusionReMaSSing offers a practical protocol for generating QTL mapping populations to identify adaptive alleles in tolerant strains and correct genetic defects in inferior ones. Notably, recombinant populations and clones derived from ReMaSSing outperformed both parental strains in LCH tolerance and growth. Furthermore, we applied ReMaSSing to breed strains with enhanced LCH tolerance, efficient xylose catabolism, and robust ethanol production. Together, these results demonstrate that ReMaSSing is a powerful tool for engineering industrial yeast strains that integrate desirable traits from multiple parental backgrounds.

genomics↗

bioRxiv2024

Engineering Adaptive Alleles for Escherichia coli Growth on Sucrose Using the EasyGuide CRISPR System

Adaptive Laboratory Evolution (ALE) is a powerful approach for mining genetic data to engineer industrial microorganisms. This evolution-informed design requires robust genetic tools to incorporate the discovered alleles into target strains. Here, we introduce the EasyGuide CRISPR, a five-plasmid platform that exploits E. colis natural recombination system to assemble gRNA plasmids from overlapping PCR fragments. The production of gRNAs and donor DNA is further facilitated by using recombination cassettes generated through PCR with 40 to 60-mer oligos. With the new CRISPR toolkit, we constructed 22 gene edits in E. coli DH5, most of which corresponded to alleles mapped in E. coli DH5 and E2348/69 ALE populations selected for sucrose propagation. For DH5 ALE, sucrose consumption was supported by the cscBKA operon expression from a high-copy plasmid. During ALE, plasmid integration into the chromosome, or its copy number reduction due to the pcnB deletion, conferred a 30-35% fitness gain, as demonstrated by CRISPR-engineered strains. A [~]5% advantage was also associated with a [~]40.4 kb deletion involving fli operons for flagella assembly. In E2348/69 ALE, inactivation of the hfl system suggested selection pressures for maintaining {lambda}-prophage dormancy (lysogeny). We further enhanced our CRISPR toolkit using yeast for in vivo assembly of donors and expression cassettes, enabling the establishment of polyhydroxybutyrate synthesis from sucrose. Overall, our study highlights the importance of combining ALE with streamlined CRISPR-mediated allele editing to advance microbial production using cost-effective carbon sources.

genetics↗

bioRxiv2024

Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks

The design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. We conducted an adaptive laboratory evolution by challenging four populations (P1-P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68-82 cycles of 2-h ethanol shocks (19%-30% v/v) and outgrowths. Colonies isolated from the final populations (P1c-P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c-P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1{Delta} conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1{Delta} strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production. These results demonstrate that some alleles selected for acute stress survival may further confer stress tolerance and optimal performance under industrial conditions.

evolutionary biology↗

bioRxiv2022

EasyGuide plasmids support in vivo assembly of gRNAs for CRISPR/Cas9 applications in Saccharomyces cerevisiae

Most CRISPR/Cas9 applications in yeast rely on a plasmid-based expression of Cas9 and its guide RNA (gRNA) containing a 20-nucleotides (nts) spacer tailored to each genomic target. The lengthy assembly of this customized gRNA requires at least 3-5 days for its pre-cloning in Escherichia coli, purification, validation, and co-transformation with Cas9 into a yeast strain. Here, we constructed a series of 12 EasyGuide plasmids to simplify CRISPR/Cas9 applications in Saccharomyces cerevisiae. The new vectors provide templates for generating PCR fragments that can assemble up to six functional gRNAs directly into yeasts via homologous recombination between the 20-nts spacers. By dispensing pre-cloning in E. coli, yeast in vivo gRNA assembly significantly reduces the CRISPR/Cas9 experimental workload. A highly efficient yeast genome editing procedure, involving PCR amplification of gRNAs and donors, followed by their transformation into a Cas9-expressing strain, can be easily accomplished in a single day.

synthetic biology↗