Efficient Strain Development Strategies for Production of Terpenes in Saccharomyces Cerevisiae

Abstract

Adaptive laboratory evolution (ALE) is a powerful tool used to increase strain fitness in the presence of environmental stressors. Apart from growth improvement, ALE can also be used for improving productivity if production and fitness is coupled. However, extensive metabolic engineering is generally required to ensure growth-coupling of product formation based on computational predictions; however, such in silico predictions typically result in cells with lower than expected growth and/or production. In this work, we develop a generalized ALE strategy to improve production of compounds with antioxidant potential. Terpene are the biggest class of natural products produced by plants with various uses such as flavors, fragrances, colorants, vitamins, commodity chemicals and pharmaceuticals. We used ALE for improving production of two terpenes with antioxidant potential, one produced intracellularly and another extracellularly in yeast. Productivity of terpenes in yeast was improved using oxidative stress over short evolution experiments. Evolved population resulting from evolution experiments were further screened to select for hyperproducers with improved production. Using these hyperproducers, we also aim to gain a deeper understanding of the genotype-to-phenotype correlation of casual mutations responsible for improved production of terpenes in order to develop chassis strains with high productivity of terpenes. Next generation sequencing (NGS) analysis of hyperproducers revealed multiple unique mutations in genes which are not previously known to be related to terpene biosynthesis in yeast.