Avinash Godara1, Katy C Kao2,3. 1. Department of Chemical Engineering, Texas A&M University, College Station, TX, USA. 2. Department of Chemical Engineering, Texas A&M University, College Station, TX, USA. katy.kao@sjsu.edu. 3. Department of Chemical and Materials Engineering, San Jose State University, One Washington Sq, San Jose, CA, 95192, USA. katy.kao@sjsu.edu.
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.
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.
Authors: Robert E Reinsvold; Robert E Jinkerson; Randor Radakovits; Matthew C Posewitz; Chhandak Basu Journal: J Plant Physiol Date: 2010-12-23 Impact factor: 3.549
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Authors: Mohammad A Asadollahi; Jérôme Maury; Kasper Møller; Kristian Fog Nielsen; Michel Schalk; Anthony Clark; Jens Nielsen Journal: Biotechnol Bioeng Date: 2008-02-15 Impact factor: 4.530