CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Mengwan Li; Xuye Lang; Marcos Moran Cabrera; Sawyer De Keyser; Xiyan Sun; Nancy Da Silva; Ian Wheeldon

doi:10.1186/s13068-020-01852-3

CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Biotechnol Biofuels. 2021 Jan 6;14(1):3. doi: 10.1186/s13068-020-01852-3.

Authors

Mengwan Li¹, Xuye Lang¹, Marcos Moran Cabrera¹, Sawyer De Keyser¹, Xiyan Sun¹, Nancy Da Silva², Ian Wheeldon^{3

4}

Affiliations

¹ Department of Chemical and Environmental Engineering, University of California Riverside, Riverside, CA, 92521, USA.
² Department of Chemical and Biomolecular Engineering, University of California Irvine, Irvine, CA, 92697, USA.
³ Department of Chemical and Environmental Engineering, University of California Riverside, Riverside, CA, 92521, USA. iwheeldon@engr.ucr.edu.
⁴ Center for Industrial Biotechnology, University of California Riverside, Riverside, CA, 92527, USA. iwheeldon@engr.ucr.edu.

Abstract

Background: 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material.

Results: We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 3³-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4^K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7^G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures.

Conclusions: The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

Keywords: Expression regulation; Flavors and fragrances; Metabolic engineering; Thermotolerance.

Abstract

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