YeastFab: the design and construction of standard biological parts for metabolic engineering in<i>Saccharomyces cerevisiae</i>

TLDR

Metabolic engineering routinely requires introducing multicomponent pathways into heterologous hosts, but this process can take weeks to months because standardized genetic tools are lacking. The authors present a method for designing and constructing biological parts derived from native Saccharomyces cerevisiae genes and regulatory elements. They developed YeastFab Assembly, a highly efficient single‑tube protocol that synthesizes standardized parts, calibrates promoter function with reporter assays, and enables rapid assembly of transcription units or multi‑gene pathways targeting genomic loci or plasmids. Applying the method to a β‑carotene biosynthesis pathway, they demonstrated that a pathway can be constructed and tested in days and its production optimized through combinatorial assembly of hundreds of regulatory parts.

Abstract

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts.