Improved betulinic acid biosynthesis using synthetic yeast chromosome recombination and semi-automated rapid LC-MS screening

摘要

Synthetic biology, genome engineering and directed evolution offer innumerable tools toexpedite engineering of strains for optimising biosynthetic pathways. One of the most radicalis SCRaMbLE, a system of inducible in vivo deletion and rearrangement of synthetic yeastchromosomes, diversifying the genotype of millions of Saccharomyces cerevisiae cells in hours.SCRaMbLE can yield strains with improved biosynthetic phenotypes but is limited byscreening capabilities. To address this bottleneck, we combine automated sample preparation,an ultra-fast 84-second LC-MS method, and barcoded nanopore sequencing to rapidlyisolate and characterise the best performing strains. Here, we use SCRaMbLE to optimiseyeast strains engineered to produce the triterpenoid betulinic acid. Our semi-automatedworkflow screens 1,000 colonies, identifying and sequencing 12 strains with between 2- to 7-fold improvement in betulinic acid titre. The broad applicability of this workflow to rapidlyisolate improved strains from a variant library makes this a valuable tool for biotechnology.