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Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9

  • Metabolic Engineering and Synthetic Biology
  • Published:
Journal of Industrial Microbiology & Biotechnology

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.

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Acknowledgments

We would like to thank the following DuPont colleagues for their assistance with the work described here. We thank Xiaochun Fan, Brian Paul, William Rutherford, Pamela Sharpe, Melissa Bosak, Wonchul Suh, Bhaska Bhadra and Zhixiong Xue for sharing yeast strains, plasmids and protocols. We thank John Kozlowski and Beth Ann Fenner for performing HPLC analysis of β-carotene. Arle Kruckeberg, Raymond Seung-Pyo Hong, Mick Ward, and Zhixiong Xue are appreciated for editing the manuscript. We also thank Ethel Jackson, Ryan Frisch, and Xiaochun Fan for valuable discussions. In addition, we would like to thank Wayne Alphin and Eva Nagel for their help with imaging.

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Authors and Affiliations

  1. Industrial Biosciences, E. I. Du Pont Company, Wilmington, DE, USA

    Steve F. EauClaire, Jianzhong Zhang, Corban Gregory Rivera & Lixuan L. Huang

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  1. Steve F. EauClaire
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  2. Jianzhong Zhang
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Correspondence to Lixuan L. Huang.

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EauClaire, S.F., Zhang, J., Rivera, C.G. et al. Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9. J Ind Microbiol Biotechnol 43, 1001–1015 (2016). https://doi.org/10.1007/s10295-016-1776-0

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