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

Abstract

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

Sequencing enabling design and learning in synthetic biology

“…As the field of synthetic biology matures, we expect the development of similar tools tailored to the unique features of living systems. Sequencing is 9 well placed to take on this challenge, especially when combined with complementary analysis methods such as LC-MS [63]. Given these growing capabilities and falling costs, the application of sequencing to synthetic biology is likely to grow rapidly over the next decade, becoming a crucial tool to verify that engineered living systems work precisely as we expect before being used in real-world settings.…”

Building genomes to understand biology

“…This is a key part of the Sc2.0 project, in which loxP sites have been added between each pair of genes on a given synthetic chromosome to allow for random Cre-mediated site-specific recombination 10,[22][23][24][25][26] . Because of these loxP sites, synthetic chromosomes constructed as a part of Sc2.0 facilitate the generation of large libraries of cells with diverse chromosome or genome structures through the induction of synthetic chromosome recombination and modification by LoxP-mediated evolution [44][45][46][47][48][49][50][51] . Analysis of cells produced by SCRaMbLE can reveal how changes in genome structure influence particular phenotypes or can be used to screen for Box 1 | Methods for building and activating chromosomes Assembly of large DNA molecules: Chromosomes are synthesized through the hierarchical assembly of smaller DNA molecules into progressively larger ones.…”

Microbial Cell Factory for Efficiently Synthesizing Plant Natural Products via Optimizing the Location and Adaptation of Pathway on Genome Scale

“…In addition to the issue of leaky expression from plasmids, it is necessary to establish high-throughput screening methods to broaden the application of SCRaMbLE. In order for rapidly screening mutants after SCRaMbLE rearrangement, an ultra-fast LC-MS method using a guard column to substitute a standard analytical column was employed to boost betulinic acid production in S. cerevisiae , reducing the detection time per sample from 5 min to 84 s ( Gowers et al, 2020 ). Ultimately, multiplex nanopore sequencing was utilized to identify the rearrangements in the high-yield strains and establish genotype-phenotype correlation.…”