The production of ethanol by yeast fermentation represents the largest of all global biotechnologies. Consequently, the yeast Saccharomyces cerevisiae is the world's premier industrial microorganism, ...which is responsible not only for the production of alcoholic beverages, including beer, wine, and distilled spirits, but also for the billions of liters of bioethanol produced annually for use as a renewable transportation fuel. Although humankind has exploited the fermentative activities of yeasts for millennia, many aspects of alcohol fermentation remain poorly understood. This chapter will review some of the key considerations in optimizing industrial alcohol fermentations with a particular emphasis on enhancement opportunities involving cell physiology and strain engineering of the major microbial ethanologen, the yeast S. cerevisiae.
Engineered yeast are an important production platform for the biosynthesis of high-value compounds with medical applications. Recent years have witnessed several new developments in this area, ...largely spurred by advances in the field of synthetic biology and the elucidation of natural metabolic pathways. This minireview presents an overview of synthetic biology applications for the heterologous biosynthesis of biopharmaceuticals in yeast and demonstrates the power and potential of yeast cell factories by highlighting several recent examples. In addition, an outline of emerging trends in this rapidly-developing area is discussed, hinting upon the potential state-of-the-art in the years ahead.
Many yeast strains secrete proteins that are lethal to other yeast and filamentous fungi. These toxins constitute a highly diverse system of targeted cell toxicity with different modes of ...action.Technological developments in synthetic biology offer an opportunity to unlock the potential of these systems and overcome their inherent limitations.These include engineering protein toxins towards user-defined physicochemical properties, systematically refactoring producer yeasts to achieve user-defined targeted killer functions, and bioprospecting for completely novel variants with either narrow or broad target ranges.Potential applications of protein toxins are diverse, but they likely represent a crucial new tool to address the looming challenges of food security, agricultural sustainability, and human health.The double-stranded (ds) RNA genomes of these systems may also represent a novel means of producing designer RNA.
Killer yeasts secrete protein toxins that are selectively lethal to other yeast and filamentous fungi. These exhibit exceptional genetic and functional diversity, and have several biotechnological applications. However, despite decades of research, several limitations hinder their widespread adoption. In this perspective we contend that technical advances in synthetic biology present an unprecedented opportunity to unlock the full potential of yeast killer systems across a spectrum of applications. By leveraging these new technologies, engineered killer toxins may emerge as a pivotal new tool to address antifungal resistance and food security. Finally, we speculate on the biotechnological potential of re-engineering host double-stranded (ds) RNA mycoviruses, from which many toxins derive, as a safe and noninfectious system to produce designer RNA.
Killer yeasts secrete protein toxins that are selectively lethal to other yeast and filamentous fungi. These exhibit exceptional genetic and functional diversity, and have several biotechnological applications. However, despite decades of research, several limitations hinder their widespread adoption. In this perspective we contend that technical advances in synthetic biology present an unprecedented opportunity to unlock the full potential of yeast killer systems across a spectrum of applications. By leveraging these new technologies, engineered killer toxins may emerge as a pivotal new tool to address antifungal resistance and food security. Finally, we speculate on the biotechnological potential of re-engineering host double-stranded (ds) RNA mycoviruses, from which many toxins derive, as a safe and noninfectious system to produce designer RNA.
Abstract
Yeast research is entering into a new period of scholarship, with new scientific tools, new questions to ask and new issues to consider. The politics of emerging and critical technology can ...no longer be separated from the pursuit of basic science in fields, such as synthetic biology and engineering biology. Given the intensifying race for technological leadership, yeast research is likely to attract significant investment from government, and that it offers huge opportunities to the curious minded from a basic research standpoint. This article provides an overview of new directions in yeast research with a focus on
Saccharomyces cerevisiae
, and places these trends in their geopolitical context. At the highest level, yeast research is situated within the ongoing convergence of the life sciences with the information sciences. This convergent effect is most strongly pronounced in areas of AI‐enabled tools for the life sciences, and the creation of synthetic genomes, minimal genomes, pan‐genomes, neochromosomes and metagenomes using computer‐assisted design tools and methodologies. Synthetic yeast futures encompass basic and applied science questions that will be of intense interest to government and nongovernment funding sources. It is essential for the yeast research community to map and understand the context of their research to ensure their collaborations turn global challenges into research opportunities.
Take‐away
The opportunities for basic and applied yeast research are likely to accelerate in the current global geopolitical environment.
Synthetic yeast research interfaces with many critical technologies and the yeast research community must now navigate a funding environment adapting to global technology competition.
Key opportunities for synthetic yeast research are found in the areas of minimal genomics, neochromosome design, multicellular community and consortia interactions, biosensing, and extraterrestrial applications.
The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of ...multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or “CRISPR D-BUGS,” to map phenotypic variants caused by specific designer modifications, known as “bugs.” We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.
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•Consolidation of synthetic chromosomes using advanced endoreduplication intercrossing•CRISPR D-BUGS to map “bugs” of designer modifications•Combinatorial interaction links transcription, inositol metabolism, and tRNA abundance•Next-generation chromosome consolidation strategy to finish Sc2.0
Multiple synthetic chromosomes in Sc2.0 were consolidated together into a single yeast strain, revealing combinational genetic interactions through CRISPR D-BUGS.
Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of ...Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.
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•Designed, built, and characterized a neochromosome with 275 tRNA genes in yeast•Additional tRNA genes burden the host cell, causing deletions or increased ploidy•Chemical tRNA neochromosome extraction enables transplantation into new yeast strains•Functional analysis reveals insights into tRNA and chromosomal biology
A designer tRNA neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae reveals unexpected genomic plasticity and insights into both tRNA and chromosome biology.
The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.0 consortium has achieved several significant ...milestones by synthesizing and characterizing all 16 nuclear chromosomes of the yeast Saccharomyces cerevisiae, as well as a 17thde novo neochromosome containing all nuclear tRNA genes. In this commentary, we discuss the recent technological advances achieved in this project and provide a perspective on how they will impact the emerging field of synthetic genomics in the future.
The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.0 consortium has achieved several significant milestones by synthesizing and characterizing all 16 nuclear chromosomes of the yeast Saccharomyces cerevisiae, as well as a 17thde novo neochromosome containing all nuclear tRNA genes. In this commentary, we discuss the recent technological advances achieved in this project and provide a perspective on how they will impact the emerging field of synthetic genomics in the future.
MicroRNAs (miRNAs) are short non-coding RNAs predicted to regulate one third of protein coding genes via mRNA targeting. In conjunction with key transcription factors, such as the repressor REST (RE1 ...silencing transcription factor), miRNAs play crucial roles in neurogenesis, which requires a highly orchestrated program of gene expression to ensure the appropriate development and function of diverse neural cell types. Whilst previous studies have highlighted select groups of miRNAs during neural development, there remains a need for amenable models in which miRNA expression and function can be analyzed over the duration of neurogenesis.
We performed large-scale expression profiling of miRNAs in human NTera2/D1 (NT2) cells during retinoic acid (RA)-induced transition from progenitors to fully differentiated neural phenotypes. Our results revealed dynamic changes of miRNA patterns, resulting in distinct miRNA subsets that could be linked to specific neurodevelopmental stages. Moreover, the cell-type specific miRNA subsets were very similar in NT2-derived differentiated cells and human primary neurons and astrocytes. Further analysis identified miRNAs as putative regulators of REST, as well as candidate miRNAs targeted by REST. Finally, we confirmed the existence of two predicted miRNAs; pred-MIR191 and pred-MIR222 associated with SLAIN1 and FOXP2, respectively, and provided some evidence of their potential co-regulation.
In the present study, we demonstrate that regulation of miRNAs occurs in precise patterns indicative of their roles in cell fate commitment, progenitor expansion and differentiation into neurons and glia. Furthermore, the similarity between our NT2 system and primary human cells suggests their roles in molecular pathways critical for human in vivo neurogenesis.
We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within
(
), ...encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the
transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome.
Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair ...chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in
under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.
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