Modular Metabolic Engineering and Synthetic Coculture Strategies for the Production of Aromatic Compounds in Yeast

Microbial-derived aromatics provide a sustainable andrenewablealternative to petroleum-derived chemicals. In this study, we usedthe model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularityin synthetic biology. Three different modular approaches were investigatedfor 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 generationof combinatorial libraries of promoters to optimize the expressionlevel of the genes involved in the synthesis pathway of RK. The secondstrategy was modular pathway engineering and involved the creationof four modules, one for product formation: RK synthesis module (Mod.RK); and three for precursor synthesis: aromatic amino acid synthesismodule (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 ofthese modules was studied, and the best engineered strain produced63.5 mg/L RK from glucose, which is the highest production describedin yeast, and 2.1 mg RK/g glucose, which is the highest yield reportedin any organism without p-coumaric acid supplementation.The third strategy was the use of modular cocultures to explore theeffects of division of labor on RK production. Two two-member communitiesand one three-member community were created, and their productioncapacity 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 showedup 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-synthesisof RK. This study illustrates the utility of modularity in syntheticbiology tools and their applications to the synthesis of productsof industrial interest.

Automated summary

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. This study utilized synthetic biology and modular approaches to produce a valuable fragrance compound, raspberry ketone (RK), in the model yeast Saccharomyces cerevisiae. The strategies employed included modular cloning, modular pathway engineering, and modular cocultures, resulting in the production of high levels of RK and its precursor. This research highlights the importance and potential of modularity in synthetic biology for industrial applications.