Produzione di vanillina “naturale” mediante l’uso di biocatalizzatori microbici
Author(s)
Luziatelli, Francesca
Date Issued
March 14, 2008
Type
Doctoral Thesis
Abstract
This doctoral project deals with the development of metabolically engineered strains for the production of vanillin from ferulic acid. Vanillic aldehyde, or vanillin, is the molecule that gives vanilla its typical aroma and one of the main compounds used in food flavouring. As the natural aroma extracted from Vanilla planifolia pods is very expensive (1400 $/kg) and rare, chemically synthesized vanillin is used almost exclusively in vanilla-flavoured products (including food). “Biotech-vanillin” produced by microorganisms from natural raw materials such as ferulic acid, eugenol or capsaicin, would allow for a natural alternative to both natural vanilla extract and synthetically produced vanillin. To make the biotechnology process economically competitive, it is essential that the biocatalysts used for the production of vanillin are stable and as efficient as possible. Since vanillin is an intermediate product of the ferulic catabolism, a compound phenylpropanoic acid present in significant quantities in the lignocellulosic material, the aim of this thesis was to compare two microbial biocatalysts able to produce vanillin starting from ferulic acid, using two alternative strategies. The first biocatalyst was the Pseudomonas fluorescens strain BF13 which is able to naturally degrade ferulic acid and was previously identified in the biotechnology laboratory at the University of Tuscia. A site-specific mutation was introduced into the gene coding for vanillin dehydrogenase of P. fluorescens strain BF13, resulting in a mutant able to convert ferulic acid into vanillin, but unable to oxidise vanillic acid. The second biocatalyst was obtained from a bacterium (Escherichia coli) unable to use the vanillin and ferulic acid as sources of carbon. Through metabolic engineering, the ability to produce the enzymes needed to transform ferulic acid into vanillin was conferred onto the E. coli strain. The ability of the two microorganisms to efficiently produce vanillin starting from ferulic acid was evaluated in experiments conducted by means of bioconversion with resting cells. Particular attention was paid to the cost of production of biomass which could significantly impact overall costs of the vanillin-production process. Optimal growth conditions (substrate and source of carbon, temperature growth, growth phase) and biomass use (composition of the bioconversion buffer, temperature and pH of the process, pre-treatment of biomass) were identified for both microorganisms.
Successful tests for commercial production of vanillin were carried out for GNOSIS S.p.A., a company interested in the transfer of the process on an industrial scale. Industrial grade raw materials in a 15-L fermentor were use for the preparation of the growth strain and for bioconversion. The best results were obtained by the engineered strain of E. coli, which produced 6.6 g of vanillin/L of culture. This value represents the highest production of vanillin obtained to date using biocatalysts from E . coli.
Additional information
Dottorato di ricerca in Biotecnologie degli alimenti
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