-
Biological Chemistry Jul 2023Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an...
Most mitochondrial proteins are nuclear-encoded and imported by the protein import machinery based on specific targeting signals. The proteins that carry an amino-terminal targeting signal (presequence) are imported via the presequence import pathway that involves the translocases of the outer and inner membranes - TOM and TIM23 complexes. In this article, we discuss how mitochondrial matrix and inner membrane precursor proteins are imported along the presequence pathway in with a focus on the dynamics of the TIM23 complex, and further update with some of the key findings that advanced the field in the last few years.
Topics: Protein Transport; Saccharomyces cerevisiae; Mitochondria; Mitochondrial Proteins; Mitochondrial Precursor Protein Import Complex Proteins
PubMed: 37155927
DOI: 10.1515/hsz-2023-0133 -
The EMBO Journal Oct 2023The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly...
The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly of ATG proteins during this process remains incomplete. The Rab GTPase Ypt1/Rab1 is recognized as an essential autophagy regulator. Here, we identify Atg23 and Atg17 as binding partners of Ypt1, with their direct interaction proving crucial for the stepwise assembly of autophagy initiation complexes. Disruption of Ypt1-Atg23 binding results in significantly reduced Atg9 interactions with Atg11, Atg13, and Atg17, thus preventing the recruitment of Atg9 vesicles to the phagophore assembly site (PAS). Likewise, Ypt1-Atg17 binding contributes to the PAS recruitment of Ypt1 and Atg1. Importantly, we found that Ypt1 is phosphorylated by TOR at the Ser174 residue. Converting this residue to alanine blocks Ypt1 phosphorylation by TOR and enhances autophagy. Conversely, the Ypt1 phosphorylation mimic impairs both PAS recruitment and activation of Atg1, thus inhibiting subsequent autophagy. Thus, we propose TOR-mediated Ypt1 as a multifunctional assembly factor that controls autophagy initiation via its regulation of the stepwise assembly of ATG proteins.
Topics: Autophagy; Autophagy-Related Proteins; Phagosomes; Phosphorylation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37635626
DOI: 10.15252/embj.2022112814 -
Nucleic Acids Research Aug 2023During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strands can displace the parental strand to generate single-strand DNAs (ssDNAs). Many...
During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strands can displace the parental strand to generate single-strand DNAs (ssDNAs). Many programmed DSBs and thus many ssDNAs occur during meiosis. However, it is unclear how these ssDNAs are removed for the complete repair of meiotic DSBs. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) results in an abundant accumulation of RPA and an expansion of RPA from DSBs to broader regions in Saccharomyces cerevisiae. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, Dna2 induction at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreases and increases RPA accumulation in dna2-md, respectively. In addition, blocking DNA synthesis during meiotic recombination dramatically decreases RPA accumulation in dna2-md. Together, our findings show that meiotic DSB repair requires Dna2 to remove ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.
Topics: DNA; DNA Repair; DNA, Single-Stranded; DNA-Binding Proteins; Meiosis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37351599
DOI: 10.1093/nar/gkad537 -
Microbial Cell Factories Oct 2023"ATP wasting" has been observed in C metabolic flux analyses of Saccharomyces cerevisiae, a yeast strain commonly used to produce ethanol. Some strains of S. cerevisiae,...
BACKGROUND
"ATP wasting" has been observed in C metabolic flux analyses of Saccharomyces cerevisiae, a yeast strain commonly used to produce ethanol. Some strains of S. cerevisiae, such as the sake strain Kyokai 7, consume approximately two-fold as much ATP as laboratory strains. Increased ATP consumption may be linked to the production of ethanol, which helps regenerate ATP.
RESULTS
This study was conducted to enhance ethanol and 2,3-butanediol (2,3-BDO) production in the S. cerevisiae strains, ethanol-producing strain BY318 and 2,3-BDO-producing strain YHI030, by expressing the fructose-1,6-bisphosphatase (FBPase) and ATP synthase (ATPase) genes to induce ATP dissipation. The introduction of a futile cycle for ATP consumption in the pathway was achieved by expressing various FBPase and ATPase genes from Escherichia coli and S. cerevisiae in the yeast strains. The production of ethanol and 2,3-BDO was evaluated using high-performance liquid chromatography and gas chromatography, and fermentation tests were performed on synthetic media under aerobic conditions in batch culture. The results showed that in the BY318-opt_ecoFBPase (expressing opt_ecoFBPase) and BY318-ATPase (expressing ATPase) strains, specific glucose consumption was increased by 30% and 42%, respectively, and the ethanol production rate was increased by 24% and 45%, respectively. In contrast, the YHI030-opt_ecoFBPase (expressing opt_ecoFBPase) and YHI030-ATPase (expressing ATPase) strains showed increased 2,3-BDO yields of 26% and 18%, respectively, and the specific production rate of 2,3-BDO was increased by 36%. Metabolomic analysis confirmed the introduction of the futile cycle.
CONCLUSION
ATP wasting may be an effective strategy for improving the fermentative biosynthetic capacity of S. cerevisiae, and increased ATP consumption may be a useful tool in some alcohol-producing strains.
Topics: Saccharomyces cerevisiae; Ethanol; Metabolic Engineering; Fermentation; Adenosine Triphosphatases; Adenosine Triphosphate
PubMed: 37807050
DOI: 10.1186/s12934-023-02221-z -
Trends in Plant Science Aug 2023Arsenic (As) is harmful to all living organisms, including humans and plants. To limit As uptake and avoid its toxicity, plants employ systems that regulate the uptake... (Review)
Review
Arsenic (As) is harmful to all living organisms, including humans and plants. To limit As uptake and avoid its toxicity, plants employ systems that regulate the uptake of As from the soil and its translocation from roots to grains. Ubiquitination, a highly conserved post-translational modification (PTM) in all eukaryotes, plays crucial roles in modulating As detoxification mechanisms in budding yeast (Saccharomyces cerevisiae), but little is known about its roles in As tolerance and transport in plants. In this opinion article we review recent findings and suggest that ubiquitination plays a crucial role in regulating As transport in plants. We also propose ideas for future research to explore the importance of ubiquitination for enhancing As tolerance in crops.
Topics: Humans; Arsenic; Plants; Ubiquitination; Biological Transport; Saccharomyces cerevisiae
PubMed: 37002000
DOI: 10.1016/j.tplants.2023.03.008 -
ACS Synthetic Biology Nov 2023Advancements in synthetic biology have provided new opportunities in biosensing, with applications ranging from genetic programming to diagnostics. Next generation...
Advancements in synthetic biology have provided new opportunities in biosensing, with applications ranging from genetic programming to diagnostics. Next generation biosensors aim to expand the number of accessible environments for measurements, increase the number of measurable phenomena, and improve the quality of the measurement. To this end, an emerging area in the field has been the integration of DNA as an information storage medium within biosensor outputs, leveraging nucleic acids to record the biosensor state over time. However, slow signal transduction steps, due to the time scales of transcription and translation, bottleneck many sensing-DNA recording approaches. DNA polymerases (DNAPs) have been proposed as a solution to the signal transduction problem by operating as both the sensor and responder, but there is presently a lack of DNAPs with functional sensitivity to many desirable target ligands. Here, we engineer components of the Pol δ replicative polymerase complex of to sense and respond to Ca, a metal cofactor relevant to numerous biological phenomena. Through domain insertion and binding site grafting to Pol δ subunits, we demonstrate functional allosteric sensitivity to Ca. Together, this work provides an important foundation for future efforts in the development of DNAP-based biosensors.
Topics: DNA-Directed DNA Polymerase; DNA Replication; DNA; Saccharomyces cerevisiae; Protein Domains; Biosensing Techniques
PubMed: 37856140
DOI: 10.1021/acssynbio.3c00302 -
ACS Synthetic Biology Nov 2023Synthetic biology toolkits are one of the core foundations on which the field has been built, facilitating and accelerating efforts to reprogram cells and organisms for...
Synthetic biology toolkits are one of the core foundations on which the field has been built, facilitating and accelerating efforts to reprogram cells and organisms for diverse biotechnological applications. The yeast , an important model and industrial organism, has benefited from a wide range of toolkits. In particular, the MoClo Yeast Toolkit (YTK) enables the fast and straightforward construction of multigene plasmids from a library of highly characterized parts for programming new cellular behavior in a more predictable manner. While YTK has cultivated a strong parts ecosystem and excels in plasmid construction, it is limited in the extent and flexibility with which it can create new strains of yeast. Here, we describe a new and improved toolkit, the Multiplex Yeast Toolkit (MYT), that extends the capabilities of YTK and addresses strain engineering limitations. MYT provides a set of new integration vectors and selectable markers usable across common laboratory strains, as well as additional assembly cassettes to increase the number of transcriptional units in multigene constructs, CRISPR-Cas9 tools for highly efficient multiplexed vector integration, and three orthogonal and inducible promoter systems for conditional programming of gene expression. With these tools, we provide yeast synthetic biologists with a powerful platform to take their engineering ambitions to exciting new levels.
Topics: Saccharomyces cerevisiae; CRISPR-Cas Systems; Ecosystem; Biotechnology; Plasmids
PubMed: 37930278
DOI: 10.1021/acssynbio.3c00423 -
ACS Synthetic Biology Sep 2023A fundamental challenge of metabolic engineering involves assembling and screening vast combinations of orthologous enzymes across a multistep biochemical pathway....
A fundamental challenge of metabolic engineering involves assembling and screening vast combinations of orthologous enzymes across a multistep biochemical pathway. Current pathway assembly workflows involve combining genetic parts and assembling one pathway configuration per tube or well. Here, we present CRAPS, hromosomal-epair-ssisted athway huffling, an pathway engineering technique that enables the self-assembly of one pathway configuration per cell. CRAPS leverages the yeast chromosomal repair pathway and utilizes a pool of inactive, chromosomally integrated orthologous gene variants corresponding to a target multistep pathway. Supplying gRNAs to the CRAPS host activates the expression of one gene variant per pathway step, resulting in a unique pathway configuration in each cell. We deployed CRAPS to build more than 1000 theoretical combinations of a four-step carotenoid biosynthesis network. Sampling the CRAPS pathway space yielded strains with distinct color phenotypes and carotenoid product profiles. We anticipate that CRAPS will expedite strain engineering campaigns by enabling the generation and sampling of vast biochemical spaces.
Topics: Saccharomyces cerevisiae; Carotenoids; Metabolic Engineering; CRISPR-Cas Systems
PubMed: 37584634
DOI: 10.1021/acssynbio.3c00170 -
Microbiology Spectrum Apr 2024(baker's yeast, budding yeast) is one of the most important model organisms for biological research and is a crucial microorganism in industry. Currently, a huge number...
(baker's yeast, budding yeast) is one of the most important model organisms for biological research and is a crucial microorganism in industry. Currently, a huge number of genome sequences are available at the public domain. However, these genomes are distributed at different websites and a large number of them are released without annotation information. To provide one complete annotated genome data resource, we collected 2,507 genome assemblies and re-annotated 2,506 assemblies using a custom annotation pipeline, producing a total of 15,407,164 protein-coding gene models. With a custom pipeline, all these gene sequences were clustered into families. A total of 1,506 single-copy genes were selected as marker genes, which were then used to evaluate the genome completeness and base qualities of all assemblies. Pangenomic analyses were performed based on a selected subset of 847 medium-high-quality genomes. Statistical comparisons revealed a number of gene families showing copy number variations among different organism sources. To the authors' knowledge, this study represents the largest genome annotation project of so far, providing rich genomic resources for the future studies of the model organism and its relatives.IMPORTANCE (baker's yeast, budding yeast) is one of the most important model organisms for biological research and is a crucial microorganism in industry. Though a huge number of genome sequences are available at the public domain, these genomes are distributed at different websites and most are released without annotation, hindering the efficient reuse of these genome resources. Here, we collected 2,507 genomes for , performed genome annotation, and evaluated the genome qualities. All the obtained data have been deposited at public repositories and are freely accessible to the community. This study represents the largest genome annotation project of so far, providing one complete annotated genome data set for , an important workhorse for fundamental biology, biotechnology, and industry.
Topics: Saccharomyces cerevisiae; Genome, Fungal; DNA Copy Number Variations; Genomics; Molecular Sequence Annotation
PubMed: 38488392
DOI: 10.1128/spectrum.03582-23 -
Nature Chemical Biology Dec 2023Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications,...
Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.
Topics: Saccharomyces cerevisiae; Monoterpenes; Indole Alkaloids; Plants; Pharmaceutical Preparations; Catharanthus; Plant Proteins
PubMed: 37932529
DOI: 10.1038/s41589-023-01430-2