Nowadays, the yeastSaccharomyces cerevisiaeis the platform of choice for demonstrating the proof of concept
of the production of metabolites with a complex structure. However,
introducing heterologous genes and rewiring the endogenous metabolism
is still not standardized enough, affecting negatively the readiness-to-market
of such metabolites. We developed the Easy Modular Integrative fuSion-ready
Expression (Easy-MISE) toolkit, which is a novel combination of synthetic
biology tools based on a single Golden Gate multiplasmid assembly
meant to further ameliorate the rational predictability and flexibility
of the process of yeast engineering. Thanks to an improved cloning
screening strategy, double and independent transcription units are
easily assembled and subsequently integrated into previously characterized
loci. Moreover, the devices can be tagged for localization. This design
allows for a higher degree of modularity and increases the flexibility
of the engineering strategy. We show with a case study how the developed
toolkit accelerates the construction and the analysis of the intermediate
and the final engineered yeast strains, leaving space to better characterize
the heterologous biosynthetic pathway in the final host and, overall,
to improve the fermentation performances. DifferentS. cerevisiaestrains were built harboring different
versions of the biochemical pathway toward glucobrassicin (GLB) production,
an indolyl-methyl glucosinolate. In the end, we could demonstrate
that in the tested conditions the best-producing strain leads to a
final concentration of GLB of 9.80 ± 0.267 mg/L, a titer 10-fold
higher than the best result previously reported in the literature.