المؤلفون: Gossing, Michael, Limeta, Angelo, 1996, Skrekas, Christos, 1990, Wigglesworth, Mark, Davis, Andrew, Siewers, Verena, 1976, David, Florian, 1981

المصدر: ACS Synthetic Biology. 12(8):2271-2277

مصطلحات موضوعية: CRISPR, directed evolution, multiplexing, in vivo mutagenesis, targeted mutagenesis, yeast

الوصف: Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9technology, with its ability to target a specific DNA locus usingguide RNAs (gRNAs), is particularly suited for targeted mutagenesis.The targeted diversification of nucleotides in Saccharomycescerevisiae using a CRISPR-guided error-prone DNA polymerase calledyEvolvR was recently reported. Here, we investigate the effectof multiplexed expression of gRNAs flanking a short stretch of DNAon reversion and mutation frequencies using yEvolvR. Phenotypic assaysdemonstrate that higher reversion frequencies are observed when expressingmultiple gRNAs simultaneously. Next generation sequencing revealsa synergistic effect of multiple gRNAs on mutation frequencies, whichis more pronounced in a mutant with a partially defective DNA mismatchrepair system. Additionally, we characterize a galactose-inducibleyEvolvR, which enables temporal control of mutagenesis. This studydemonstrates that multiplex expression of gRNAs and induction of mutagenesisgreatly improves the capabilities of yEvolvR for generation of geneticlibraries in vivo.

وصف الملف: electronic

URL الوصول: https://research.chalmers.se/publication/536934
https://research.chalmers.se/publication/536934/file/536934_Fulltext.pdf

المؤلفون: Tous Mohedano, Marta, 1995, Mao, Jiwei, 1990, Chen, Yun, 1978

المصدر: ACS Synthetic Biology. 12(1):144-152

مصطلحات موضوعية: flavonoids, yeast, tolerance, pathway optimization, byproduct

الوصف: The flavonoid pinocembrin and its derivatives have gained increasing interest for their benefits on human health. While pinocembrin and its derivatives can be produced in engineered Saccharomyces cerevisiae, yields remain low. Here, we describe novel strategies for improved de novo biosynthesis of pinocembrin from glucose based on overcoming existing limitations in S. cerevisiae. First, we identified cinnamic acid as an inhibitor of pinocembrin synthesis. Second, by screening for more efficient enzymes and optimizing the expression of downstream genes, we reduced cinnamic acid accumulation. Third, we addressed other limiting factors by boosting the availability of the precursor malonyl-CoA, while eliminating the undesired byproduct 2′,4′,6′-trihydroxy dihydrochalcone. After optimizing cultivation conditions, 80 mg/L pinocembrin was obtained in a shake flask, the highest yield reported for S. cerevisiae. Finally, we demonstrated that pinocembrin-producing strains could be further engineered to generate 25 mg/L chrysin, another interesting flavone. The strains generated in this study will facilitate the production of flavonoids through the pinocembrin biosynthetic pathway.

وصف الملف: electronic

URL الوصول: https://research.chalmers.se/publication/533910
https://research.chalmers.se/publication/533910/file/533910_Fulltext.pdf

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المؤلفون: Trivellin, Cecilia, 1993, Olsson, Lisbeth, 1963, Rugbjerg, Peter, 1988

المصدر: ACS Synthetic Biology. 11(4):1686-1691

مصطلحات موضوعية: yeast, Fano factor, robustness quantification, high-throughput, phenomics, bioprocess

الوصف: Stable cell performance in a fluctuating environment is essential for sustainable bioproduction and synthetic cell functionality; however, microbial robustness is rarely quantified. Here, we describe a high-throughput strategy for quantifying robustness of multiple cellular functions and strains in a perturbation space. We evaluated quantification theory on experimental data and concluded that the mean-normalized Fano factor allowed accurate, reliable, and standardized quantification. Our methodology applied to perturbations related to lignocellulosic bioethanol production showed that the industrial bioethanol producing strain Saccharomyces cerevisiae Ethanol Red exhibited both higher and more robust growth rates than the laboratory strain CEN.PK and industrial strain PE-2, while a more robust product yield traded off for lower mean levels. The methodology validated that robustness is function-specific and characterized by positive and negative function-specific trade-offs. Systematic quantification of robustness to end-use perturbations will be important to analyze and construct robust strains with more predictable functions.

وصف الملف: electronic

URL الوصول: https://research.chalmers.se/publication/529224
https://research.chalmers.se/publication/529224/file/529224_Fulltext.pdf

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المؤلفون: Ambri, Francesca, D’Ambrosio, Vasil, Di Blasi, Roberto, Maury, Jerome, Jacobsen, Simo Abdessamad Baallal, McCloskey, Douglas, Jensen, Michael K, Keasling, Jay D

المصدر: ACS Synthetic Biology. 9(2)

مصطلحات موضوعية: Biochemistry and Cell Biology, Biological Sciences, Genetics, Bioengineering, Biotechnology, Generic health relevance, Binding Sites, Biosensing Techniques, Genes, Reporter, Plasmids, Promoter Regions, Genetic, Protein Engineering, Saccharomyces cerevisiae, Transcription Factors, biosensor, transcriptional regulators, operator position, yeast, Medicinal and Biomolecular Chemistry, Biomedical Engineering, Biochemistry and cell biology, Bioinformatics and computational biology

الوصف: Small-molecule binding allosteric transcription factors (aTFs) derived from bacteria enable real-time monitoring of metabolite abundances, high-throughput screening of genetic designs, and dynamic control of metabolism. Yet, engineering of reporter promoter designs of prokaryotic aTF biosensors in eukaryotic cells is complex. Here we investigate the impact of aTF binding site positions at single-nucleotide resolution in >300 reporter promoter designs in Saccharomyces cerevisiae. From this we identify biosensor output landscapes with transient and distinct aTF binding site position effects for aTF repressors and activators, respectively. Next, we present positions for tunable reporter promoter outputs enabling metabolite-responsive designs for a total of four repressor-type and three activator-type aTF biosensors with dynamic output ranges up to 8- and 26-fold, respectively. This study highlights aTF binding site positions in reporter promoters as key for successful biosensor engineering and that repressor-type aTF biosensors allows for more flexibility in terms of choice of binding site positioning compared to activator-type aTF biosensors.

وصف الملف: application/pdf

URL الوصول: https://escholarship.org/uc/item/4d10205b

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المؤلفون: Noskov VN; Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA., Karas BJ, Young L, Chuang RY, Gibson DG, Lin YC, Stam J, Yonemoto IT, Suzuki Y, Andrews-Pfannkoch C, Glass JI, Smith HO, Hutchison CA 3rd, Venter JC, Weyman PD

المصدر: ACS synthetic biology [ACS Synth Biol] 2012 Jul 20; Vol. 1 (7), pp. 267-73. Date of Electronic Publication: 2012 May 21.

نوع المنشور: Journal Article; Research Support, Non-U.S. Gov't

بيانات الدورية: Publisher: American Chemical Society Country of Publication: United States NLM ID: 101575075 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2161-5063 (Electronic) Linking ISSN: 21615063 NLM ISO Abbreviation: ACS Synth Biol Subsets: MEDLINE

مواضيع طبية MeSH: DNA, Bacterial/*chemistry , DNA, Bacterial/*genetics, Base Composition ; Chromosomes, Artificial, Yeast/chemistry ; Chromosomes, Artificial, Yeast/genetics ; Cloning, Molecular ; DNA, Recombinant/chemistry ; DNA, Recombinant/genetics ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Synechococcus/genetics ; Synechococcus/metabolism ; Synthetic Biology

مستخلص: The ability to assemble large pieces of prokaryotic DNA by yeast recombination has great application in synthetic biology, but cloning large pieces of high G+C prokaryotic DNA in yeast can be challenging. Additional considerations in cloning large pieces of high G+C DNA in yeast may be related to toxic genes, to the size of the DNA, or to the absence of yeast origins of replication within the sequence. As an example of our ability to clone high G+C DNA in yeast, we chose to work with Synechococcus elongatus PCC 7942, which has an average G+C content of 55%. We determined that no regions of the chromosome are toxic to yeast and that S. elongatus DNA fragments over ~200 kb are not stably maintained. DNA constructs with a total size under 200 kb could be readily assembled, even with 62 kb of overlapping sequence between pieces. Addition of yeast origins of replication throughout allowed us to increase the total size of DNA that could be assembled to at least 454 kb. Thus, cloning strategies utilizing yeast recombination with large, high G+C prokaryotic sequences should include yeast origins of replication as a part of the design process.

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