A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly
Reads0
Chats0
TLDR
A versatile engineering platform for yeast, which contains both a rapid, modular assembly method and a basic set of characterized parts, and genome-editing tools for making modifications directly to the yeast chromosomes, which the authors find preferable to plasmids due to reduced variability in expression.Abstract:
Saccharomyces cerevisiae is an increasingly attractive host for synthetic biology because of its long history in industrial fermentations. However, until recently, most synthetic biology systems have focused on bacteria. While there is a wealth of resources and literature about the biology of yeast, it can be daunting to navigate and extract the tools needed for engineering applications. Here we present a versatile engineering platform for yeast, which contains both a rapid, modular assembly method and a basic set of characterized parts. This platform provides a framework in which to create new designs, as well as data on promoters, terminators, degradation tags, and copy number to inform those designs. Additionally, we describe genome-editing tools for making modifications directly to the yeast chromosomes, which we find preferable to plasmids due to reduced variability in expression. With this toolkit, we strive to simplify the process of engineering yeast by standardizing the physical manipulations and...read more
Citations
More filters
Journal ArticleDOI
Engineering Cellular Metabolism
TL;DR: How new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation is discussed.
Journal ArticleDOI
Synthetic biology strategies for microbial biosynthesis of plant natural products
TL;DR: This review describes recent developments in metabolic engineering at the level of host, pathway, and enzyme, and discusses how the field is approaching ever more complex biosynthetic opportunities.
Journal ArticleDOI
Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications.
TL;DR: This review summarizes the recent development of metabolic engineering approaches to modulate yeast metabolism with representative examples and highlights new tools for biosynthetic pathway optimization and genome engineering to advance metabolic engineering in yeast.
Journal ArticleDOI
Continuous evolution of base editors with expanded target compatibility and improved activity
Benjamin W. Thuronyi,Benjamin W. Thuronyi,Benjamin W. Thuronyi,Luke W. Koblan,Luke W. Koblan,Jonathan M. Levy,Jonathan M. Levy,Wei-Hsi Yeh,Wei-Hsi Yeh,Christine R. Zheng,Gregory A. Newby,Gregory A. Newby,Christine D. Wilson,Christine D. Wilson,Mantu Bhaumik,Olga Shubina-Oleinik,Jeffrey R. Holt,David R. Liu,David R. Liu,David R. Liu +19 more
TL;DR: Phage-assisted continuous evolution of base editors (BE–PACE) is developed to improve their editing efficiency and target sequence compatibility and data from evolved CBEs are used to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation.
Journal ArticleDOI
A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae
Amanda Reider Apel,Amanda Reider Apel,Leo d'Espaux,Leo d'Espaux,Maren Wehrs,Maren Wehrs,Daniel Sachs,Daniel Sachs,Rachel Li,Rachel Li,Gary J. Tong,Gary J. Tong,Megan E. Garber,Megan E. Garber,Oge Nnadi,Oge Nnadi,W. Zhuang,Nathan J. Hillson,Nathan J. Hillson,Nathan J. Hillson,Jay D. Keasling,Aindrila Mukhopadhyay +21 more
TL;DR: A cloning-free toolkit is built that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility, and is demonstrated by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase.
References
More filters
Journal ArticleDOI
Enzymatic assembly of DNA molecules up to several hundred kilobases
Daniel G. Gibson,Lei Young,Ray-Yuan Chuang,J. Craig Venter,Clyde A. Hutchison,Hamilton O. Smith +5 more
TL;DR: An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase is described.
Journal ArticleDOI
Functional profiling of the Saccharomyces cerevisiae genome.
Guri Giaever,Angela M. Chu,Li Ni,Carla Connelly,Linda Riles,Steeve Veronneau,Sally Dow,Ankuta Lucau-Danila,Keith Anderson,Bruno André,Adam P. Arkin,Anna Astromoff,Mohamed El Bakkoury,Rhonda Bangham,Rocío Benito,Sophie Brachat,Stefano Campanaro,Matt Curtiss,Karen Davis,Adam M. Deutschbauer,K. D. Entian,Patrick Flaherty,Françoise Foury,David J. Garfinkel,Mark Gerstein,Deanna Gotte,Ulrich Güldener,Johannes H. Hegemann,Svenja Hempel,Zelek S. Herman,Daniel F. Jaramillo,Diane E. Kelly,Steven L. Kelly,Peter Kötter,Darlene LaBonte,David C. Lamb,Ning Lan,Hong Liang,Hong Liao,Lucy Y. Liu,Chuanyun Luo,Marc Lussier,Rong Mao,Patrice Menard,Siew Loon Ooi,José L. Revuelta,Christopher J. Roberts,Matthias Rose,Petra Ross-Macdonald,Bart Scherens,Greg Schimmack,Brenda Shafer,Daniel D. Shoemaker,Sharon Sookhai-Mahadeo,Reginald Storms,Jeffrey N. Strathern,Giorgio Valle,Marleen Voet,Guido Volckaert,Ching Yun Wang,Teresa R. Ward,Julie Wilhelmy,Elizabeth A. Winzeler,Yonghong Yang,Grace Yen,Elaine M. Youngman,Kexin Yu,Howard Bussey,Jef D. Boeke,Michael Snyder,Peter Philippsen,Ronald W. Davis,Mark Johnston +72 more
TL;DR: It is shown that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment, and less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal Growth in four of the tested conditions.
Journal ArticleDOI
Production of the antimalarial drug precursor artemisinic acid in engineered yeast
Dae-Kyun Ro,Eric M. Paradise,Mario Ouellet,Karl Fisher,Karyn L. Newman,John M. Ndungu,Ho Kimberly,Eachus Rachel,Timothy S. Ham,James Kirby,Michelle C. Y. Chang,Sydnor T. Withers,Yoichiro Shiba,Richmond Sarpong,Jay D. Keasling +14 more
TL;DR: The engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l-1) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase from A. annua that performs a three-step oxidation of amorpha-4,11-diene to art Artemisinic acid.
Journal ArticleDOI
A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance.
TL;DR: Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a positive selection, based on the loss of orotidine-5′-phosphate decarboxylase activity, and seems applicable to a variety of eucaryotic and procaryotic cells.
Journal ArticleDOI
Systematic Genetic Analysis with Ordered Arrays of Yeast Deletion Mutants
Amy Hin Yan Tong,Marie Evangelista,Ainslie B. Parsons,Hong Xu,Gary D. Bader,Gary D. Bader,Nicholas Pagé,Mark D. Robinson,Sasan Raghibizadeh,Christopher W. V. Hogue,Christopher W. V. Hogue,Howard Bussey,Brenda J. Andrews,Mike Tyers,Mike Tyers,Charles Boone,Charles Boone +16 more
TL;DR: A method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of ∼4700 deletion mutants is developed, which should produce a global map of gene function.