Metabolic engineering of added-value compounds

The plant kingdom is enormously rich in bioactive molecules from a wide variety of compound classes (e.g. flavonoids, terpenoids, alkaloids, etc). Only a minute fraction of these molecules are currently applied for manufacturing food ingredients, pharmaceuticals, cosmetics, etc. Systematic exploration of this chemical diversity is complicated by the complexity and costs of both purification of relevant compounds from plant biomass and their de novo chemical synthesis. Industrial microorganisms can be grown at scales ranging from < 0.1 ml (high throughput screening) to > 100 m3 (industrial production). Moreover, industrial microorganisms are unsurpassed in terms of genetic accessibility and detailed knowledge on regulation of their central metabolism, which provides the precursors for product formation.

₂ Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. In an early work, Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations. 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 80-fold increase of extracellular naringenin titers (to approximately 400 μM) in aerated, pH controlled batch reactors. We demonstrated 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. This engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions.

People: Jasmijn Hassing, Mario Beck (start October 2017), research associate position.

_{2 Schematic representation of the engineered naringenin production pathway in S. cerevisiae. Six A. thaliana genes were overexpressed: PAL1 (phenylalanine ammonia lyase), C4H (Cinnamate 4-hydroxylase), CPR1 (cytochrome P450 reductase), 4CL3 (4-coumaric acid-CoA ligase), CHS3 (chalcone synthase) and CHI1 (chalcone isomerase), and one gene from R. capsulatus; Tal1 (tyrosine ammonia lyase). Dashed lines indicate feedback inhibition. Grey arrows indicate the S. cerevisiae pathway for phenylethanol production. Bold dark grey arrows indicate the naringenin production pathway as described for A. thaliana. Aro3/Aro4: 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, Pdc1, 5, 6; pyruvate decarboxylases, Aro10; phenylpyruvate decarboxylase.}