Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9
| Autor: | Lixuan L Huang, Jianzhong Zhang, Corban G. Rivera, Steven F. EauClaire |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0106 biological sciences
0301 basic medicine Saccharomyces cerevisiae Proteins Saccharomyces cerevisiae Bioengineering DNA Fragmentation 01 natural sciences Applied Microbiology and Biotechnology Metabolic engineering Fungal Proteins 03 medical and health sciences Genome editing 010608 biotechnology Gene Expression Regulation Fungal Gene biology Base Sequence Cas9 Protein engineering biology.organism_classification beta Carotene Yeast Biosynthetic Pathways Culture Media Metabolic pathway 030104 developmental biology Biochemistry Genetic Loci Amino Acid Transport Systems Basic Genome Fungal Gene Deletion Biotechnology RNA Guide Kinetoplastida |
| Zdroj: | Journal of industrial microbiologybiotechnology. 43(7) |
| ISSN: | 1476-5535 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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