Automated Yeast Transformation Protocol to Engineer Saccharomyces cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames That Express Proteins Binding to Xylose Isomerase Identified Using a Robotic Two-Hybrid Screen

Abstract The nib 1 allele of yeast confers a sensitivity to an endogenous plasmid, 2 mu DNA, in that nib 1 strains bearing 2 mu DNA (cir+) exhibit a reduction in division potential. In the present study, the reduction in division potential characteristic of nib 1 cir+ strains is shown to be dependent on the simultaneous presence of both the A and the D open reading frames of 2 mu DNA as well as on the presence of an unidentified extrachromosomal element other than 2 mu DNA. Furthermore, in nib 1 strains, an uncharacterized extrachromosomal element can cause a less severe reduction of division potential in the absence of intact 2 mu DNA. Thus, the nib 1 allele may confer a generalized sensitivity to extrachromosomal elements.

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Far3 and Five Interacting Proteins Prevent Premature Recovery from Pheromone Arrest in the Budding Yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.23.5.1750-1763.2003 ◽

2003 ◽

Vol 23 (5) ◽

pp. 1750-1763 ◽

Cited By ~ 77

Author(s):

Hilary A. Kemp ◽

George F. Sprague,

Keyword(s):

Cell Cycle ◽

Budding Yeast ◽

Open Reading Frames ◽

Velocity Sedimentation ◽

Interacting Proteins ◽

Yeast Saccharomyces Cerevisiae ◽

Cycle Arrest ◽

Mutant Cells ◽

Two Hybrid ◽

Reading Frames

ABSTRACT In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G1 arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.

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Influence of key variables on the simultaneous isomerization and fermentation (SIF) of xylose by a native Saccharomyces cerevisiae strain co-encapsulated with xylose isomerase for 2G ethanol production

Biomass and Bioenergy ◽

10.1016/j.biombioe.2018.09.016 ◽

2018 ◽

Vol 119 ◽

pp. 277-283 ◽

Cited By ~ 8

Author(s):

Thais Suzane Milessi ◽

Patricia M. Aquino ◽

Cláudia R. Silva ◽

Guilherme S. Moraes ◽

Teresa C. Zangirolami ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Production ◽

Xylose Isomerase ◽

Key Variables

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Optimized xylose isomerase uptake and expression level in Saccharomyces cerevisiae for improving ethanol production

Applied Environmental Biotechnology ◽

10.26789/aeb.2018.01.007 ◽

2019 ◽

Vol 3 (1) ◽

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Production ◽

Xylose Isomerase ◽

Expression Level

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Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.01419-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5708-5716 ◽

Cited By ~ 96

Author(s):

Sun-Mi Lee ◽

Taylor Jellison ◽

Hal S. Alper

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Production ◽

Directed Evolution ◽

Ethanol Yield ◽

Xylose Isomerase ◽

Mutant Enzyme ◽

Xylose Utilization ◽

Xylose Consumption ◽

Content Type ◽

Consumption Rates

ABSTRACTThe heterologous expression of a highly functional xylose isomerase pathway inSaccharomyces cerevisiaewould have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways inS. cerevisiaesuffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of thePiromycessp. xylose isomerase (encoded byxylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing agre3knockout andtal1andXKS1overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

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Divergent overlapping transcripts at the PET122 locus in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3027-3035.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3027-3035

Author(s):

J D Ohmen ◽

K A Burke ◽

J E McEwen

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Sequence ◽

Cytochrome C Oxidase ◽

Genomic Dna ◽

Transcription Unit ◽

Open Reading Frames ◽

Base Pairs ◽

Opposite Strand ◽

Overlapping Transcripts ◽

Reading Frames

PET122 is one of three nuclear genes specifically required for translation of the mitochondrial mRNA for cytochrome c oxidase subunit III in Saccharomyces cerevisiae. The nucleotide sequence of 2,862 base pairs (bp) of yeast genomic DNA encompassing the PET122 locus shows very close spacing between the PET122 gene (254 codons) and two unidentified open reading frames, termed ORF2 and ORF3. ORF2 is encoded by the same strand of DNA as PET122 and is located 53 bp downstream of PET122, while ORF3 is encoded on the opposite strand and is located 215 bp upstream of PET122. Five transcripts, with sizes of 2.9, 2.3, 2.1, 1.5, and 1.4 kilobases (kb), are produced from this locus. The 2.1- and 1.4-kb transcripts encode ORF3, the 1.5-kb transcript encodes ORF2, and the 2.9- and 2.3-kb transcripts encode PET122. A particularly interesting feature of the ORF3-PET122-ORF2 transcription unit is a 535-base overlap between the 2.3-kb PET122 transcript produced from one strand and a 2.1-kb ORF3 transcript produced from the opposite strand. Similarly, the 2.9-kb PET122 transcript overlaps the 2.1-kb ORF3 transcript by more than 900 bases and the 1.5-kb ORF3 transcript by at least 200 bases. Hence, these pairs of transcripts are antisense to one another and have the potential to regulate, in an interdependent fashion, the posttranscriptional expression of ORF3 and PET122.

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Regulation of transcription by Saccharomyces cerevisiae 14-3-3 proteins

Biochemical Journal ◽

10.1042/bj20031885 ◽

2004 ◽

Vol 382 (3) ◽

pp. 867-875 ◽

Cited By ~ 20

Author(s):

Astrid BRUCKMANN ◽

H. Yde STEENSMA ◽

M. Joost TEIXEIRA de MATTOS ◽

G. Paul H. van HEUSDEN

Keyword(s):

Saccharomyces Cerevisiae ◽

Stress Response ◽

Open Reading Frames ◽

Ergosterol Biosynthesis ◽

Temperature Sensitive ◽

Regulation Of Transcription ◽

Mrna Levels ◽

Transcription Analysis ◽

A Genome ◽

Reading Frames

14-3-3 proteins form a family of highly conserved eukaryotic proteins involved in a wide variety of cellular processes, including signalling, apoptosis, cell-cycle control and transcriptional regulation. More than 150 binding partners have been found for these proteins. The yeast Saccharomyces cerevisiae has two genes encoding 14-3-3 proteins, BMH1 and BMH2. A bmh1 bmh2 double mutant is unviable in most laboratory strains. Previously, we constructed a temperature-sensitive bmh2 mutant and showed that mutations in RTG3 and SIN4, both encoding transcriptional regulators, can suppress the temperature-sensitive phenotype of this mutant, suggesting an inhibitory role of the 14-3-3 proteins in Rtg3-dependent transcription [van Heusden and Steensma (2001) Yeast 18, 1479–1491]. In the present paper, we report a genome-wide transcription analysis of a temperature-sensitive bmh2 mutant. Steady-state mRNA levels of 60 open reading frames were increased more than 2.0-fold in the bmh2 mutant, whereas those of 78 open reading frames were decreased more than 2.0-fold. In agreement with our genetic experiments, six genes known to be regulated by Rtg3 showed elevated mRNA levels in the mutant. In addition, several genes with other cellular functions, including those involved in gluconeogenesis, ergosterol biosynthesis and stress response, had altered mRNA levels in the mutant. Our data show that the yeast 14-3-3 proteins negatively regulate Rtg3-dependent transcription, stimulate the transcription of genes involved in ergosterol metabolism and in stress response and are involved in transcription regulation of multiple other genes.

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