Microbial-derived aromatics provide a sustainable and
renewable
alternative to petroleum-derived chemicals. In this study, we used
the model yeastSaccharomyces cerevisiaeto produce aromatic molecules by exploiting the concept of modularity
in synthetic biology. Three different modular approaches were investigated
for the production of the valuable fragrance raspberry ketone (RK),
found in raspberry fruits and mostly produced from petrochemicals.
The first strategy used was modular cloning, which enabled the generation
of combinatorial libraries of promoters to optimize the expression
level of the genes involved in the synthesis pathway of RK. The second
strategy was modular pathway engineering and involved the creation
of four modules, one for product formation: RK synthesis module (Mod.
RK); and three for precursor synthesis: aromatic amino acid synthesis
module (Mod. Aro),p-coumaric acid synthesis module
(Mod.p-CA), and malonyl-CoA synthesis module (Mod.
M-CoA). The production of RK by combinations of the expression of
these modules was studied, and the best engineered strain produced
63.5 mg/L RK from glucose, which is the highest production described
in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported
in any organism withoutp-coumaric acid supplementation.
The third strategy was the use of modular cocultures to explore the
effects of division of labor on RK production. Two two-member communities
and one three-member community were created, and their production
capacity was highly dependent on the structure of the synthetic community,
the inoculation ratio, and the culture media. In certain conditions,
the cocultures outperformed their monoculture controls for RK production,
although this was not the norm. Interestingly, the cocultures showed
up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone,
the direct precursor of RK, which can be used for the semi-synthesis
of RK. This study illustrates the utility of modularity in synthetic
biology tools and their applications to the synthesis of products
of industrial interest.