Produzione di composti ad alto valore aggiunto da sottoprodotti rinnovabili mediante un sistema multienzimatico a cascata

The efficient valorization of the lignocellulosic biomass components, and in particular the lignin fraction, could serve as a starting point for the establishment of a circular bioeconomy model aimed at the recycling and reutilization of industrial by-products over the exploitation of virgin feedstock. From lignocellulosic biomass both fermentable carbohydrate and aromatics can be obtained, which can be used for the production of biofuels and bioplastics thus reducing the dependence on petroleum-based feedstocks. The present Ph.D. thesis focuses on the development of novel biotechnological processes aimed at the extraction of aromatics from lignocellulosic by-products and their conversion to value-added products using whole-cell biocatalytic approaches. Firstly, I developed an efficient and green process to produce ccMA from renewable feedstocks (i.e. kraft lignin and wheat bran) based on: a) the optimization of the extraction procedures of vanillin from lignin and of ferulic acid from wheat bran; b) the genetic engineering of an E. coli strain to modulate the expression of up to seven recombinant enzymes. In detail, vanillin was recovered from kraft lignin (4.5 mg vanillin/g kraft lignin) by an enzymatic treatment using the recombinant Bacillus licheniformis laccase, and ferulic acid from wheat bran (3.0 mg ferulic acid/g wheat bran) by a thermo-enzymatic method using the Ultraflo®XL commercial enzyme. The whole-cell biocatalyst used to convert vanillin into ccMA expresses the dehydrogenase LigV, the demethylase VanAB, the decarboxylase AroY and the dioxygenase C12O; meanwhile the whole-cell biocatalyst to convert ferulic acid to ccMA expresses all the above-mentioned enzymes plus the decarboxylase Fdc and the dioxygenase Ado. The engineered strains converted >95% of lignin-derived vanillin in 30 minutes, obtaining the production of 4.2 mg ccMA/g of kraft lignin. Starting from the wheat bran-derived ferulic acid, ccMA was produced with a >95% conversion yield in 10 hours, corresponding to 0.73 g ccMA/g ferulic acid, and 2.2 mg ccMA/g wheat bran biomass. To further evaluate the capabilities of the whole-cell biocatlyst, the scaled-up production of ccMA from vanillin using the engineered E. coli growing cells was studied. The bioconversion reaction was carried out in a fermenter, providing improved control of the reaction conditions such as pH, dissolved oxygen and substrate pulse-feed rate, streamlining the biocatalytic process and enhancing scalability. The optimized growth medium composition (0.5 g/L glucose and 2 g/L lactose) and substrate addition strategy (1 mmol/h pulse-feed) enabled the engineered strain to produce 5.2 ± 0.36 g/L of ccMA in 48 hours, corresponding to 0.86 g ccMA/g vanillin. The purification of the produced ccMA from the fermentation broth was achieved through crystallization, yielding 2.58 ± 0.07 g per liter of broth, corresponding to a ≈50% purification yield. Lastly, a preliminary analysis of a one-pot process for the production of 4-vinylguaiacol from wheat bran was conducted. The process involves the extraction of ferulic acid from the wheat bran using the three-step thermo-enzymatic protocol utilized previously and the simultaneous conversion of ferulic acid into 4-vinylguaicol using an engineered E. coli strain expressing the decarboxylase Fdc. The novelty of this process arises from the employment of the wheat bran crude extract as an auto-inducing growth medium, based on the presence of several fermentable carbohydrates and the utilization of a hybrid phenol-inducible promoter for the induction of Fdc expression, making the wheat bran-derived ferulic acid both the inducer and the substrate of the enzyme. The unoptimized process produced 1.8 mg 4-vinylguaiacol per gram of wheat bran, which correspond to the conversion of ≈75% of the ferulic acid extracted using the thermo-enzymatic method and ≈64% of the alkaline extractable ferulic acid present in the wheat bran.