Development of next-generation tools for efficient, costeffective, high-throughput engineering of yeast

Commercial scale microbial biosynthesis of chemicals requires repeating the design-build-test (D-B-T) engineering cycle multiple times in order to obtain a production-ready host. The timeline for the D-B-T engineering cycle directly affects the time to market for any new product. At Amyris, we have built the Automated Strain Engineering (ASE) pipeline that accelerates the D-B-T cycle, while simultaneous increasing the throughput of rational strain construction. While the ASE platform has succeeded at lowering the threshold in terms of cost and effort for building and testing strains, there is potential for further process improvements. To enhance the ASE pipeline and to enable the synthetic biology community make faster strain engineering progress, Amyris has continued to develop new molecular biology tools. In this presentation we will report the progress made on the building of some of these tools including:

i) A genome annotation pipeline capable of rapidly processing raw next-generation sequencing data for multiple genomes to standard parts

ii) A modified DNA assembly method capable of assembling large DNA constructs with high efficiency

iii) A next-generation sequencing pipeline for parallel sequencing of thousands of DNA assemblies at low cost

iv) Simultaneous multi-locus marker-free genomic integrations in various yeasts using nucleases

Widespread application of these tools will increase community access to high-throughput rational strain construction, resulting in the generation of Living Foundries at a rate, scale, and cost unprecedented in the field.