Publication Date

12-1-2021

Document Type

Article

Publication Title

Microbial Cell Factories

Volume

20

Issue

1

DOI

10.1186/s12934-021-01598-z

Abstract

Background: β-Caryophyllene is a plant terpenoid with therapeutic and biofuel properties. Production of terpenoids through microbial cells is a potentially sustainable alternative for production. Adaptive laboratory evolution is a complementary technique to metabolic engineering for strain improvement, if the product-of-interest is coupled with growth. Here we use a combination of pathway engineering and adaptive laboratory evolution to improve the production of β-caryophyllene, an extracellular product, by leveraging the antioxidant potential of the compound. Results: Using oxidative stress as selective pressure, we developed an adaptive laboratory evolution that worked to evolve an engineered β-caryophyllene producing yeast strain for improved production within a few generations. This strategy resulted in fourfold increase in production in isolated mutants. Further increasing the flux to β-caryophyllene in the best evolved mutant achieved a titer of 104.7 ± 6.2 mg/L product. Genomic analysis revealed a gain-of-function mutation in the a-factor exporter STE6 was identified to be involved in significantly increased production, likely as a result of increased product export. Conclusion: An optimized selection strategy based on oxidative stress was developed to improve the production of the extracellular product β-caryophyllene in an engineered yeast strain. Application of the selection strategy in adaptive laboratory evolution resulted in mutants with significantly increased production and identification of novel responsible mutations.

Funding Number

CBET-1605347

Funding Sponsor

National Science Foundation

Keywords

Adaptive laboratory evolution, CRISPR–Cas9, Saccharomyces cerevisiae, Selective pressure, β-Caryophyllene

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.

Department

Chemical and Materials Engineering

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