Biology subjects

Coradini, A. L. V.

Publications and source records attributed to Coradini, A. L. V..

6 recordsLinked to original sources

bioRxiv2025

Synthetic collinearization of a chromosome segment enables genetic dissection of incompatibility between distant genera

Reproductive barriers and genomic structural differences hinder genetic analysis between highly divergent organisms. Here, we examined whether these challenges can be overcome by collinearization, the synthesis of chromosomes that retain the native gene content and organization of a host organism while incorporating the DNA sequences of another organism. We applied collinearization to Kluyveromyces marxianus, a yeast species that is both distantly related to and has a distinct genome structure from the model yeast Saccharomyces cerevisiae. We generated a [~]35-kb K. marxianus DNA segment that was collinear with one-sixth of a S. cerevisiae chromosome and contained 17 protein-coding genes. Although this synthetic chromosome segment successfully substituted for its native counterpart in an S. cerevisiae cell, it imposed a significant growth cost due to the incompatibility of two K. marxianus proteins with the host proteome. We predicted the disrupted protein-protein interactions using AlphaFold and alleviated their cost by supplementing orthologous protein partners from K. marxianus. Furthermore, we completely eliminated the growth cost by replacing the two incompatible K. marxianus genes with their S. cerevisiae orthologs. These findings demonstrate that collinearization can facilitate the creation of chimeric genomes to dissect the genetic basis of incompatibility and traits between organisms that cannot naturally hybridize. They also suggest that hundreds of genetic incompatibilities exist between S. cerevisiae and K. marxianus, reflecting disrupted protein-protein interactions that may be predictable in silico.

synthetic biology↗

bioRxiv2024

Diversified, miniaturized and ancestral parts for mammalian genome engineering and molecular recording

As the synthetic biology and genome engineering fields mature and converge, there is a clear need for a "parts list" of components that are diversified with respect to both functional activity (to facilitate design) and primary sequence (to facilitate assembly). Here we designed libraries composed of extant, ancestral, mutagenized or miniaturized variants of Pol III promoters or guide RNA (gRNA) scaffolds and quantified their ability to mediate precise edits to the mammalian genome via multiplex prime editing. We identified thousands of parts that reproducibly drive a range of editing activities in human and mouse stem cells and cancer cell lines, including hundreds exhibiting similar or greater activity than the sequences used in conventional genome engineering constructs. We further conducted saturation mutagenesis screens of canonical Pol III promoters (U6p, 7SKp, H1p) and the prime editing guide RNA (pegRNA) scaffold, which identified tolerated variants that can be superimposed on baseline parts to further enhance sequence diversity. While characterizing thousands of orthologous promoters from hundreds of extant or ancestral genomes, we incidentally mapped the functional landscape of mammalian Pol III promoter evolution. Finally, to showcase the usefulness of these parts, we designed a "ten key" molecular recording array that lacks repetitive subsequences in order to facilitate its one-step assembly in yeast. Upon delivering this 15.8 kb tandem array of promoters and guides to mammalian cells, individual pegRNAs exhibited balanced activities as predicted by the activity of component parts, despite their relocation to a single locus. Looking forward, we anticipate that the diversified parts and variant effect maps reported here can be leveraged for the design, assembly and deployment of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity, which will be useful for multiplex perturbation, advanced biological recorders and complex genetic circuits.

synthetic biology↗

bioRxiv2023

Building synthetic chromosomes from natural DNA

De novo chromosome synthesis is costly and time-consuming, limiting its use in research and biotechnology. Building synthetic chromosomes from natural components is an unexplored alternative with many potential applications. In this paper, we report CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), a method for constructing synthetic chromosomes from natural components in yeast. CReATiNG entails cloning segments of natural chromosomes and then programmably assembling them into synthetic chromosomes that can replace the native chromosomes in cells. We used CReATiNG to synthetically recombine chromosomes between strains and species, to modify chromosome structure, and to delete many linked, non-adjacent regions totaling 39% of a chromosome. The multiplex deletion experiment revealed that CReATiNG also enables recovery from flaws in synthetic chromosome design via recombination between a synthetic chromosome and its native counterpart. CReATiNG facilitates the application of chromosome synthesis to diverse biological problems.

synthetic biology↗

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↗

bioRxiv2018

Unraveling the layers of cryptic genetic variation in a yeast gene regulatory network

Cryptic genetic variation may be an important contributor to heritable traits, but its extent and regulation are not fully understood. Here, we investigate the cryptic variation underlying a Saccharomyces cerevisiae colony phenotype that is typically suppressed in a cross. Mapping of the traits genetic basis after induction by three different genetic perturbations identifies 21 total loci. The perturbations largely uncover distinct loci, and strongly influence additivity, epistasis, and genotype-environment interaction among these loci. Only a single locus-- the coding region of the cell surface gene FLO11--is common to all three perturbations. While the remaining loci mostly influence FLO11 transcription in cis or trans, we detect loci in different pathways and sub-pathways in the presence of each perturbation. Our work shows that complex gene regulatory networks can harbor multiple layers of cryptic variation, each of which may only be visible in highly specific genotypic and environmental contexts.\n\nArticle SummaryThe extent and regulation of cryptic genetic variation within populations is not fully understood. To address these questions, we comprehensively dissect the genetic basis of a budding yeast colony trait following its induction by three different genetic perturbations. In total, we identify 21 distinct loci, most of which show phenotypic effects that depend on particular perturbations, other segregating loci, and temperature. We find that nearly all involved loci participate in a common gene regulatory network (GRN). Thus, our work demonstrates the large amount of cryptic variation underlying a single trait and ties the visibility of this variation to GRN activity.

genetics↗