Glucose-based microbial production of the hormone melatonin in yeast <i>Saccharomyces cerevisiae</i>

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N‐acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L‐tryptophan hydroxylase, a 5‐hydroxy‐L‐tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O‐methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co‐factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L(−1) in a 76h fermentation using simulated fed‐batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin

CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae

BACKGROUND: One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers. RESULTS: Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes. CONCLUSIONS: The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0288-3) contains supplementary material, which is available to authorized users

TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability

DNA anaphase bridges are a potential source of genome instability that may lead to chromosome breakage or nondisjunction during mitosis. Two classes of anaphase bridges can be distinguished: DAPI-positive chromatin bridges and DAPI-negative ultrafine DNA bridges (UFBs). Here, we establish budding yeast Saccharomyces cerevisiae and the avian DT40 cell line as model systems for studying DNA anaphase bridges and show that TopBP1/Dpb11 plays an evolutionarily conserved role in their metabolism. Together with the single-stranded DNA binding protein RPA, TopBP1/Dpb11 binds to UFBs, and depletion of TopBP1/Dpb11 led to an accumulation of chromatin bridges. Importantly, the NoCut checkpoint that delays progression from anaphase to abscission in yeast was activated by both UFBs and chromatin bridges independently of Dpb11, and disruption of the NoCut checkpoint in Dpb11-depleted cells led to genome instability. In conclusion, we propose that TopBP1/Dpb11 prevents accumulation of anaphase bridges via stimulation of the Mec1/ATR kinase and suppression of homologous recombination

CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae.

BackgroundOne of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.ResultsHere, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes.ConclusionsThe CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications