Biology subjects

Rabelo, S. C.

Publications and source records attributed to Rabelo, S. C..

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↗

bioRxiv2023

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate

AO_SCPLOWBSTRACTC_SCPLOWSimultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers an advance towards more cost-effective second-generation (2G) ethanol production. As xylan is one of the most abundant polysaccharides present in lignocellulosic residues, the transport and breakdown of XOS in an intracellular environment might bring a competitive advantage for recombinant strains in competition with contaminating microbes, which are always present in fermentation tanks; furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing NADPH-linked xylose reductase (XR), NAD+-linked xylitol dehydrogenase (XDH) (for xylose assimilation), as well as NADH-linked acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) (for an NADH-dependent acetate reduction pathway), was used as the host for expressing of two {beta}-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered strain SR8A6S3-CDT2-GH432/7. Both {beta}-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, which catalyse steps in xylitol production. Xylitol accumulation during xylose fermentation is a problem for 2G ethanol production since it reduces final ethanol yield. The engineered strain, SR8A6S3-CDT2-GH432/7, produced ethanol through simultaneous co-utilization of XOS, xylose, and acetate. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan compared with the parental strain. The consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production. HighlightsO_LIIntegration of XOS pathway in an acetate-xylose-consuming S. cerevisiae strain; C_LIO_LIIntracellular fermentation of XOS, acetate and xylose improved ethanol production; C_LIO_LIDeletion of both sor1{Delta} and gre3{Delta} reduced xylitol production. C_LI

bioengineering↗

bioRxiv2021

Biochemical Methane Potential (BMP) from sugarcane biorefinery residues: maximizing their use by co-digestion

This is a pioneer study evaluating the methane (CH4) production potential from residues of integrated 1st (vinasse and filter cake) and 2nd (deacetylation pretreatment liquor from straw) generation (1G2G) sugarcane biorefinery, providing a fully chemical characterization of them and their relation with the anaerobic digestion (AD) process. Small-scale assays provided fundamentals for basing the co-digestion optimization by assessing the optimal co-substrates synergistic conditions. Biochemical Methane Potential (BMP) tests showed co-digestion enhanced CH4 yield of isolated substrates, reaching up to 605 NmLCH4 gVS-1. The association of vinasse and deacetylation liquor as co-substrates increased the BMP by ~38% mostly by nutritionally benefiting the methanogenic activity. The kinetic analysis confirmed that the deacetylation liquor was the co-substrate responsible for improving the CH4 production in the co-digestion systems due to the highest CH4 conversion rate. The alkaline characteristic of the liquor (pH~12) also prevented alkalizing from being added to the co-digestion, an input that normally makes the process economically unfeasible to implement on an industrial scale due to the large quantities required for buffering the reactor. The filter cake had the lowest BMP (262 NmLCH4 gVS-1) and digestibility ([≤] 40%), further limited by the required stirring to improve the mass transfer of biochemical reactions. The present study drives towards more sustainable use of vinasse, the most voluminous waste from the sugarcane industry, and lignin-rich residues derived from pre-treatment alkaline methods, aiming at an energy-efficient utilization, by at least 16% when compared to the traditional vinasse AD. The experimental and modeling elements from this work indicated the lignin-rich liquor is the main responsible for putting the co-digestion as a disruptive technological arrangement within the 1G2G sugarcane biorefineries, reinforcing the biogas production as the hub of the bioeconomy in the agroindustrial sector.

bioengineering↗

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↗