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

Abstract

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to 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 without p-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.

Exploring engineering strategies that enhance de novo production of exotic cyclopropane fatty acids in Saccharomyces cerevisiae

“…The details of yeast transformation and colony PCR were described in our previous study. [ 35 ] Yeast transformation was performed by the lithium acetate (LiOAc) protocol, [ 36 ] and yeast transformants were selected and grown in appropriate synthetic complete minimal medium (SC medium) based on the specific auxotrophic requirements of the transformants. The composition of the SC medium consisted of a 6.7 g L −1 yeast nitrogen base without amino acids, 20 g L −1 of glucose, and an appropriate yeast synthetic drop‐out medium supplement.…”

A molecular toolkit of cross-feeding strains for engineering synthetic yeast communities

“…Cell culture (300 μl) was mixed with an equal volume of ethanol by incubating at 700 r.p.m. at 30 °C for 5 min, then centrifuging at 2,500 g for 30 min before loading the supernants into a 96-well sample plate for LC–MS analysis as previously described 61 , 62 . An Agilent 1290 Infinity system was used to analyse these prepared samples with an online diode array detector in combination with an Agilent 6500 Q-ToF mass spectrometer.…”

Development of a co-culture system for green production of caffeic acid from sugarcane bagasse hydrolysate

“…Co-culture engineering provides a powerful method for expanding microbial synthesis capabilities by leveraging the unique functionality of different microbial platforms and reducing the metabolic burden by distributing complex biosynthetic pathways across different strains ( Johnston et al, 2020 ; He et al, 2023 ). The advantages and capabilities of different strains are fully utilized to improve the economics of the target product ( Liu et al, 2018 ; Yan et al, 2022 ; Peng et al, 2023 ). Recently, several co-culture systems embracing different genetically engineered microorganisms have been reported for the production of high value-added chemicals.…”