Adaptive laboratory evolution of β-caryophyllene producing Saccharomyces cerevisiae

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.

Microbial Adaptation to Enhance Stress Tolerance

“…Furthermore, adaptation has been applied to microbes to overcome other stresses. The engineered yeast was adapted with oxidative stress to improve the tolerance and the production of β - caryophyllene ( Godara and Kao, 2021 ). Adaptation of E. coli with the formic acid decreased the doubling time from 70 to 8 h ( Kim et al, 2020 ).…”

Advances in Metabolic Engineering Paving the Way for the Efficient Biosynthesis of Terpenes in Yeasts

“…Using the same theoretical basis, the team isolated five mutants that overproduce the FPP-derived extracellular product β-caryophyllene by ALE due to a positive correlation between colony size and yield and revealed the crucial mutation affecting a threonine of STE6 by genome sequencing. When the mutant α-factor STE6 T1025N responsible for β-caryophyllene transport was overexpressed, the enhanced recombinant S. cerevisiae produced up to 13.8 mg/g DCW of β-caryophyllene in tube culture, which was four times higher than in the parental strain . Additionally, ALE provided key technology for increasing triterpene accumulation in S. cerevisiae to obtain robust strains with high squalene production …”

“…When the mutant α-factor STE6 T1025N responsible for β-caryophyllene transport was overexpressed, the enhanced recombinant S. cerevisiae produced up to 13.8 mg/g DCW of β-caryophyllene in tube culture, which was four times higher than in the parental strain. 127 Additionally, ALE provided key technology for increasing triterpene accumulation in S. cerevisiae to obtain robust strains with high squalene production. 128 ALE was also applied to improve strain tolerance to inhibitory products to accelerate the generation of robust microbial cell factories.…”

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β‐alanine metabolism

“…Moreover, it can be used as an environment‐friendly material for ozone removal (Parshintsev et al ., 2008), or as the direct precursor of other important terpenoids such as β‐caryophyllene oxide‐natural compounds (Fidyt et al ., 2016). Microbial synthetic β‐caryophyllene has great importance for its wide applications (Godara and Kao, 2015; Yang and Nie, 2016).…”