RESUMO
Resveratrol is a unique, natural polyphenolic compound with diverse health benefits. In the present study, we attempted to improve resveratrol biosynthesis in yeast by different methods of metabolic engineering. We first mutated and then re-synthesized tyrosine ammonia lyase (TAL) by replacing the bacteria codons with yeast-preferred codons, which increased translation and improved p-coumaric acid and resveratrol biosynthesis drastically. We then demonstrated that low-affinity, high-capacity bacterial araE transporter could enhance resveratrol accumulation, without transporting resveratrol directly. Yeast cells carrying the araE gene produced up to 2.44-fold higher resveratrol than control cells. For commercial applications, resveratrol biosynthesis was detected in sucrose medium and fresh grape juice using our engineered yeast cells. In collaboration with the Chaumette Winery of Missouri, we were able to produce resveratrol-containing white wines, with levels comparable to the resveratrol levels found in most red wines.
Assuntos
Amônia-Liases/biossíntese , Proteínas de Bactérias/biossíntese , Proteínas de Transporte de Monossacarídeos/biossíntese , Organismos Geneticamente Modificados/metabolismo , Saccharomyces cerevisiae/metabolismo , Estilbenos/metabolismo , Amônia-Liases/genética , Proteínas de Bactérias/genética , Transporte Biológico Ativo/genética , Ácidos Cumáricos/metabolismo , Meios de Cultura/farmacologia , Proteínas de Transporte de Monossacarídeos/genética , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/crescimento & desenvolvimento , Propionatos , Resveratrol , Rhodobacter sphaeroides/enzimologia , Rhodobacter sphaeroides/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sacarose/farmacologia , Vinho/microbiologiaRESUMO
Resveratrol, an interesting plant phenolic compound, is found in red wine but is not widely distributed in other common food sources. Health benefits of resveratrol include prevention of cardiovascular diseases and cancers, and--as discovered more recently--promotion of longevity in several animal systems. The pathway and enzymes for resveratrol biosynthesis are well characterized. Furthermore, metabolic engineering of this compound has been achieved in plants, microbes and animals. This review attempts to summarize current understanding of resveratrol pathway-engineering in various systems, to outline the challenges in commercial applications and to identify future opportunities for resveratrol bioengineering.
Assuntos
Animais Geneticamente Modificados/metabolismo , Suplementos Nutricionais , Previsões , Melhoramento Genético/métodos , Plantas Geneticamente Modificadas/metabolismo , Estilbenos/metabolismo , Animais , ResveratrolRESUMO
The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.