Vanillin production using metabolically engineered Escherichia coli under non-growing conditions

Background: Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details. Results: Effect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 mu moles/g x min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass. Conclusion: Ferulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products, using E. coli JM109 cells expressing genes from the ferulic acid-degrader Pseudomonas fluorescens BF13. Optimization of culture conditions and bioconversion parameters, together with the reuse of the biomass, leaded to a final production of 2.52 g of vanillin per liter of culture, which is the highest found in the literature for recombinant strains and the highest achieved so far applying such strains under resting cells conditions.