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Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae.

ABSTRACT: Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.

SUBMITTER: Wu H

PROVIDER: S-EPMC1287709 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae.

Wu Hong H Ito Kiyoshi K Shimoi Hitoshi H

Applied and environmental microbiology 20051101 11

Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeast ...[more]

PMID: 16269718

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Project description:Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1 The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.

Project description:It is essential that cells orchestrate gene expression for the specific niche that they occupy, and this often requires coordination of the expression of large sets of genes. There are multiple regulatory systems that exist for modulation of gene expression, including the adjacent-gene coregulation of the rRNA and ribosome biogenesis and ribosomal protein families. Both gene families exhibit a nonrandom genomic distribution, often clustered directly adjacent to another member of the same family, which results in a tighter transcriptional coordination among adjacent paired genes than that of the unpaired genes within each regulon and can result in a shared promoter that coordinates expression of the pairs. This nonrandom genomic distribution has been seen in a few functionally related gene families, and many of these functional pairings are conserved across divergent fungal lineages. To date, the significance of these observations has not been extended in a systematic way to characterize how prevalent the role of adjacent-gene coregulation is in transcriptional regulation. In the present study, we systematically analyzed the transcriptional coherence of the functional pairs compared to the singletons within all gene families defined by the Gene Ontology Slim designation, using Saccharomyces cerevisiae as a model system, finding that clusters exhibit a tighter transcriptional correlation under specific contexts. We found that the longer a functional pairing is conserved the tighter its response to broad stress and nutritional responses, that roughly 25% of gene families exhibit a nonrandom genomic distribution, and that many of these clusters are conserved. This suggests that adjacent-gene coregulation is a widespread, yet underappreciated, transcriptional mechanism.IMPORTANCE The spatial positioning of genes throughout the genome arrangement can alter their expression in many eukaryotic organisms. Often this results in a genomic context-specific effect on transcription. One example of this is through the clustering of functionally related genes, which results in adjacent-gene coregulation in the budding yeast Saccharomyces cerevisiae In the present study, we set out to systematically characterize the prevalence of this phenomenon, finding the genomic organization of functionally related genes into clusters is a characteristic of myriad gene families. These arrangements are found in many evolutionarily divergent fungi and thus represent a widespread, yet underappreciated, layer of transcriptional regulation.

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Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae.

Publications

Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae.

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