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.
Highlights
Introduction of chromosomal gene alterationPlasmids and oligonucleotide primers used in this study are listed in Tables 2 and 3, respectively
Selection of naringenin biosynthetic genes from A. thaliana As a first step towards heterologous expression of naringenin in S. cerevisiae, flavonoid biosynthetic genes were selected from A. thaliana
In previous studies on microbial biotransformation of aromatic precursors to naringenin, the core biosynthetic pathway for naringenin synthesis was, in most cases, reconstituted by co-expressing genes from different plants and/or bacteria [17,18,19,27,28]. One of those studies used naringenin biosynthetic genes originating from a single donor organism (A. thaliana), which resulted in the first demonstration of de novo naringenin biosynthesis in E. coli [20]
Summary
Introduction of chromosomal gene alterationPlasmids and oligonucleotide primers used in this study are listed in Tables 2 and 3, respectively. PK 2.1C using primers ARO3 KO for and ARO3 KO rev as described above In this resulting strain, the chromosomal ARO4 allele was replaced by the feed-backinsensitive ARO4G226S allele [38,39]. Plant flavonoids, which comprise a family of over 9000 compounds, have attracted a tremendous increase in research interest [1,2,3] This interest is mainly attributed to highly promising human health applications of specific flavonoids [4,5,6,7,8]. The identified mechanisms of action include scavenging of oxygen radicals, anti-inflammatory, antiviral and antitumor activities [15,16] Both for health related research and commercial nutritional applications, availability of sufficient amounts of defined flavonoid preparations is important. Flavonoids can be produced chemically, efficient production of flavonoids by organic synthesis is severely hindered by the complexity of the molecules, as well as by the necessity of utilizing toxic chemicals and extreme reaction conditions [13,14]
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