An atlas of rational genetic engineering strategies for improved xylose metabolism in Saccharomyces cerevisiae.

Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.

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

Current technology that enables bioethanol production from agricultural biomass imposes harsh conditions for Saccharomyces cerevisiae's 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 FMY097's 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 ºC in liquid, while allele PKC1FMY097 allows growth up to 40 ºC in solid medium. Both SEA4FMY097 and PKC1FMY097 encode rare NS SNPs, not found in other > 1013S. cerevisiae. Altogether, these findings point towards crucial membrane and stress mediators for yeast robustness.

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

BACKGROUND: Sugarcane 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. RESULTS: Potential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5'-NGG 20 nucleotide 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 hoΔ::xyl1) and CENPKX (CEN.PK-122 hoΔ::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 straw hydrolysate with remaining solid particles represented a challenge for xylose metabolization, and 3.65 ± 0.16 g/L xylitol titer was achieved by FMYX. Finally, quantification of NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX. CONCLUSIONS: Although 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 straw 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.

Exploring G protein-coupled receptors and yeast surface display strategies for viral detection in baker's yeast: SARS-CoV-2 as a case study.

Viral infections pose intense burdens to healthcare systems and global economies. The correct diagnosis of viral diseases represents a crucial step towards effective treatments and control. Biosensors have been successfully implemented as accessible and accurate detection tests for some of the most important viruses. While most biosensors are based on physical or chemical interactions of cell-free components, the complexity of living microorganisms holds a poorly explored potential for viral detection in the face of the advances of synthetic biology. Indeed, cell-based biosensors have been praised for their versatility and economic attractiveness, however, yeast platforms for viral disease diagnostics are still limited to indirect antibody recognition. Here we propose a novel strategy for viral detection in Saccharomyces cerevisiae, which combines the transductive properties of G Protein-Coupled Receptors (GPCRs) with the Yeast Surface Display (YSD) of specific enzymes enrolled in the viral recognition process. The GPCR/YSD complex might allow for active virus detection through a modulated signal activated by a GPCR agonist, whose concentration correlates to the viral titer. Additionally, we explore this methodology in a case study for the detection of highly pathogenic coronaviruses that share the same cell receptor upon infection (i.e. the Angiotensin-Converting Enzyme 2, ACE2), as a conceptual example of the potential of the GPCR/YSD strategy for the diagnosis of COVID-19.

Genome Assembly of a Highly Aldehyde-Resistant Saccharomyces cerevisiae SA1-Derived Industrial Strain.

Here, we report the genome assembly of a Saccharomyces cerevisiae SA1-derived haploid (FMY097) indigenous strain isolated from a Brazilian ethanol distillery. FMY097 was recently reported to be a highly aldehyde-resistant strain capable of producing bioethanol in the presence of up to 40 mM furfural and 80 mM 5-hydroxymethylfurfural.