RESUMEN
Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.
Asunto(s)
Alquenos/metabolismo , Carboxiliasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Mononucleótido de Flavina/química , Glucosa/metabolismo , Alquenos/química , Biocatálisis , Carboxiliasas/genética , Crotonatos/metabolismo , Evolución Molecular Dirigida/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentación , Mononucleótido de Flavina/metabolismo , Glucosa/química , Hypocreales/enzimología , Hypocreales/genética , Ácido Mevalónico/metabolismo , PrenilaciónRESUMEN
The promiscuous activities of a recursive, generalist enzyme provide raw material for the emergence of metabolic pathways. Here, we use a synthetic biology approach to recreate such an evolutionary setup in central metabolism and explore how cellular physiology adjusts to enable recursive catalysis. We generate an Escherichia coli strain deleted in transketolase and glucose 6-phosphate dehydrogenase, effectively eliminating the native pentose phosphate pathway. We demonstrate that the overexpression of phosphoketolase restores prototrophic growth by catalyzing three consecutive reactions, cleaving xylulose 5-phosphate, fructose 6-phosphate, and, notably, sedoheptulose 7-phosphate. We find that the activity of the resulting synthetic pathway becomes possible due to the recalibration of steady-state concentrations of key metabolites, such that the in vivo cleavage rates of all three phosphoketolase substrates are similar. This study demonstrates our ability to rewrite one of nature's most conserved pathways and provides insight into the flexibility of cellular metabolism during pathway emergence.