Biology subjects

de Mello, F. d. S. B.

Publications and source records attributed to de Mello, F. d. S. B..

3 recordsLinked to original sources

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

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↗

bioRxiv2021

Genetic mapping of a bioethanol yeast strain reveals new targets for aldehyde- and thermotolerance

Current technology that enables bioethanol production from agricultural biomass imposes harsh conditions for Saccharomyces cerevisiaes metabolism. In this work, the genetic architecture of industrial bioethanol yeast strain SA-1 was evaluated. SA-1 segregant FMY097 was previously described as highly aldehyde resistant and here also as thermotolerant: two important traits for the second-generation industry. A Quantitative Trait Loci (QTL) mapping of 5-hydroxymethylfurfural (HMF) -resistant segregants of hybrid FMY097/BY4742 disclosed a region in chromosome II bearing alleles with uncommon non-synonymous (NS) single nucleotide polymorphisms (SNPs) in FMY097: MIX23, PKC1, SEA4, and SRO77. Allele swap to susceptible laboratory strain BY4742 revealed that SEA4FMY097 enhances robustness towards HMF, but the industrial fitness could not be fully recovered. The genetic network arising from the causative genes in the QTL window suggests that intracellular signaling TOR (Target of Rapamycin) and CWI (Cell Wall Integrity) pathways are regulators of this phenotype in FMY097. Because the QTL mapping did not result in one major allelic contribution to the evaluated trait, a background effect in FMY097s HMF resistance is expected. Quantification of NADPH - cofactor implied in endogenous aldehyde detoxification reactions - supports the former hypothesis, given its high availability in FMY097. Regarding thermotolerance, SEA4FMY097 grants BY4742 ability to grow in temperatures as high as 38 {degrees}C in liquid, while allele PKC1FMY097 allows growth up to 40 {degrees}C in solid medium. Both SEA4FMY097 and PKC1FMY097 encode rare NS SNPs, not found in other >1,013 S. cerevisiae. Altogether, these findings point towards crucial membrane and stress mediators for yeast robustness. KEY POINTSO_LIQTL mapping of the HMF-resistant strain FMY097 reveals a region enriched with SNPs in Chr II C_LIO_LISEA4FMY097 has rare non-synonymous mutations and improves cell growth at 10 mM HMF and 38{degrees}C C_LIO_LIPKC1FMY097 has rare non-synonymous mutations and improves cell growth at 40 {degrees}C in solid media C_LI

genetics↗