title: Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9.
abstract: The yeast Saccharomyces cerevisiae is an important industrial platform for the production of grain and cellulosic ethanol, isobutanol, butanediol, isoprenoids, and other chemicals. The construction of a successful production strain usually involves multiple gene knockouts and chromosomal integration of expression cassettes to redirect the metabolic fluxes for the conversion of sugars and other feed stocks into the desired product. RNA-guided Cas9 based genome editing has been demonstrated in many prokaryotic and eukaryotic hosts including S. cerevisiae, in which it has been additionally exploited as a tool for metabolic engineering. To extend the utilization of RNA-guided Cas9 as a metabolic pathway building tool, we demonstrated the direct assembly and chromosomal integration of up to 17 overlapping DNA fragments encoding the beta-carotene biosynthetic pathway. Furthermore, we generated a combinatorial strain library for the beta-carotene biosynthetic pathway, directly integrated into the yeast genome to create a diverse library of strains. This enabled the screening of combinatorial libraries in stable chromosomally integrated strains for rapid improvements of product titers. This combinatorial approach for pathway assembly will significantly accelerate the current speed of metabolic engineering for S. cerevisiae as an industrial platform, and increase the number of strains that can be simultaneously evaluated for enzyme screening, expression optimization and protein engineering to achieve the titer, rate and yield necessary for the commercialization of new industrial fermentation products.
authors.affiliation: Industrial Biosciences, E. I. Du Pont Company, Wilmington, DE, USA.
authors.forename: Steve F
authors.initials: SF
authors.lastname: EauClaire
authors.affiliation: Industrial Biosciences, E. I. Du Pont Company, Wilmington, DE, USA.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Industrial Biosciences, E. I. Du Pont Company, Wilmington, DE, USA.
authors.forename: Corban Gregory
authors.initials: CG
authors.lastname: Rivera
authors.affiliation: Industrial Biosciences, E. I. Du Pont Company, Wilmington, DE, USA. Lisa.L.Huang@dupont.com.
authors.forename: Lixuan L
authors.initials: LL
authors.lastname: Huang
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D053898:Biosynthetic Pathways / Q000235:genetics*; D003470:Culture Media / Q000737:chemistry; D053938:DNA Fragmentation; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D056426:Genetic Loci; D016681:Genome, Fungal; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D019207:beta Carotene / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D026923:Amino Acid Transport Systems, Basic; D003470:Culture Media; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019207:beta Carotene; D000094704:RNA, Guide, CRISPR-Cas Systems; C502212:CAN1 protein, S cerevisiae
keywords: Combinatorial pathway assembly; Metabolic engineering; RNA-guided Cas9; S. cerevisiae; β-Carotene biosynthesis