De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

Abstract

BackgroundFlavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules.ResultSaccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations (<5.5 μM). To optimize naringenin titers, a yeast chassis strain was developed. Synthesis of aromatic amino acids was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40-fold increase of extracellular naringenin titers (to approximately 200 μM) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 μM were reached.ConclusionThe results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions.

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

“…To test this, we selected CCM and naringenin, for which highest titers in shake-flask-cultivated haploid yeast of ~1 mM (141 mg/L) and 0.2 mM (54 mg/L), respectively, have recently been reported 25,41 . Also, these two products are of general interest to biotechnology, because CCM is a platform chemical for the production of several valuable consumer bioplastics 24 , and naringenin belongs to a class of secondary metabolites called flavonoids with nutritional and agricultural value 42 .…”

Four isoforms of Arabidopsis thaliana 4-coumarate: CoA ligase (4CL) have overlapping yet distinct roles in phenylpropanoid metabolism

“…is associated with flavonoid gene expression (Koopman et al, 2012). Further, 4cl3 mutants were identified in a large-scale genetic screen to find genes involved in pollen exine formation (Dobritsa et al, 2011).…”

In vivo evolution of metabolic pathways: Assembling old parts to build novel and functional structures

“…These problems could be solved by modifying the causes of the derailment when they are known or suspected, and if they are not critical or essential for cell viability, as recently shown for the flavonoid naringenin. 44 In these cells, the inactivation of genes controlling the synthesis of aromatic amino acids (Aro3 and Aro4) alleviated the feedback inhibition of phenylalanine and tyrosine which optimized their participation in the flavonoid flow.…”