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 use 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 reveals 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.

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.

QTL mapping of a Brazilian bioethanol strain links the cell wall protein-encoding gene GAS1 to low pH tolerance in S. cerevisiae.

BACKGROUND: Saccharomyces cerevisiae is largely applied in many biotechnological processes, from traditional food and beverage industries to modern biofuel and biochemicals factories. During the fermentation process, yeast cells are usually challenged in different harsh conditions, which often impact productivity. Regarding bioethanol production, cell exposure to acidic environments is related to productivity loss on both first- and second-generation ethanol. In this scenario, indigenous strains traditionally used in fermentation stand out as a source of complex genetic architecture, mainly due to their highly robust background-including low pH tolerance. RESULTS: In this work, we pioneer the use of QTL mapping to uncover the genetic basis that confers to the industrial strain Pedra-2 (PE-2) acidic tolerance during growth at low pH. First, we developed a fluorescence-based high-throughput approach to collect a large number of haploid cells using flow cytometry. Then, we were able to apply a bulk segregant analysis to solve the genetic basis of low pH resistance in PE-2, which uncovered a region in chromosome X as the major QTL associated with the evaluated phenotype. A reciprocal hemizygosity analysis revealed the allele GAS1, encoding a ß-1,3-glucanosyltransferase, as the casual variant in this region. The GAS1 sequence alignment of distinct S. cerevisiae strains pointed out a non-synonymous mutation (A631G) prevalence in wild-type isolates, which is absent in laboratory strains. We further showcase that GAS1 allele swap between PE-2 and a low pH-susceptible strain can improve cell viability on the latter of up to 12% after a sulfuric acid wash process. CONCLUSION: This work revealed GAS1 as one of the main causative genes associated with tolerance to growth at low pH in PE-2. We also showcase how GAS1PE-2 can improve acid resistance of a susceptible strain, suggesting that these findings can be a powerful foundation for the development of more robust and acid-tolerant strains. Our results collectively show the importance of tailored industrial isolated strains in discovering the genetic architecture of relevant traits and its implications over productivity.

Building genomes to understand biology.

Genetic manipulation is one of the central strategies that biologists use to investigate the molecular underpinnings of life and its diversity. Thus, advances in genetic manipulation usually lead to a deeper understanding of biological systems. During the last decade, the construction of chromosomes, known as synthetic genomics, has emerged as a novel approach to genetic manipulation. By facilitating complex modifications to chromosome content and structure, synthetic genomics opens new opportunities for studying biology through genetic manipulation. Here, we discuss different classes of genetic manipulation that are enabled by synthetic genomics, as well as biological problems they each can help solve.

Layers of Cryptic Genetic Variation Underlie a Yeast Complex Trait.

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 genetic variation underlying a Saccharomyces cerevisiae colony phenotype that is typically suppressed in a cross of the laboratory strain BY4716 (BY) and a derivative of the clinical isolate 322134S (3S). To do this, we comprehensively dissect the trait's genetic basis in the BYx3S cross in the presence of three different genetic perturbations that enable its expression. This allows us to detect and compare the specific loci that interact with each perturbation to produce the trait. In total, we identify 21 loci, all but one of which interact with just a subset of the perturbations. Beyond impacting which loci contribute to the trait, the genetic perturbations also alter the extent of additivity, epistasis, and genotype-environment interaction among the detected loci. Additionally, we show that the single locus interacting with all three perturbations corresponds to the coding region of the cell surface gene FLO11 While nearly all of the other remaining loci influence FLO11 transcription in cis or trans, the perturbations tend to interact with loci in different pathways and subpathways. Our work shows how layers of cryptic genetic variation can influence complex traits. Here, these layers mainly represent different regulatory inputs into the transcription of a single key gene.