Biopolymers are attractive “green” alternatives to conventional petroleum-based plastics, however, their sustainable exploitation is hampered by the high production costs. In this PhD thesis, an Escherichia coli recombinant system (LipoA) was constructed to allow production of Polyhydroxyalkanoates (PHAs). The system was engineered with a newly isolated PHAs biosynthetic operon from Bacillus cereus 6E/2 and tested for PHAs production on different carbon sources. Results highlighted the LipoA peculiar specificity to drive the incorporation of 3-hydroxyhexanoate monomers (>40%), whatever was the supplied fatty acid. To increase polymer production, media optimization and system engineering were applied. In this frame, two new PHA producing systems were developed considering i) the expression levels of the recombinant PHA bio-synthetic proteins (LipoB) and ii) the "host metabolic background" (LipoC) yielding to a 6-fold increment of mcl-PHA yields. Polymers were characterized revealing a low grade of crystallinity and hydrophobic features. To enhance biopolymer properties and expand fields of applicability, PHAs were enzymatically functionalized. Commercial lipase B from Candida antarctica (CaLB) was able to catalyse coupling of PHA with dimethyl itaconate (DMI) as well as with polyethylene glycol (PEG). The obtained functional hydrophilic biopolymers open new perspectives for application of PHAs in the biomedical field thanks to the possibility of easy coupling of bioactive compounds on the lateral C=C of DMI and to the enhanced hydrophilicity conferred by the PEG moieties. The enzymatic strategy was also applied for sustainable synthesis of oligoesters. Catalytic potential of immobilized Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) was investigated. Three different carriers, linked to the enzyme using a novel nontoxic His-tag method based on chelated Fe(III) ions, were tested. Selectivity chain (diols-diesters) and recyclability studies in solvent-free environment were conducted. Results not only revealed a peculiar substrates specificity but also a retention of activity >94 % over 24 h reaction cycle claiming the potentiality of new immobilization strategy. In addition, degrading capabilities of this enzyme against aromatic (PET) and aliphatic biopolyesters (PBS, PHBV, PLA) were investigated. Two glycosylation sites knock out mutants, recombinantly expressed in Pichia pastoris, were tested in comparison to wild-type (wt) enzyme. Data claimed that rThc_Cut1 and its mutants hydrolyse aromatic and aliphatic polyester powders at different rates. The best performances were observed against PBS with concentration of released products 10-fold higher than PHBV and PLA. It is worth of note that one mutant was found to be significantly more active on both powder and PBS films than the wt. These results together with the high activity of variants of rThc_Cut1 on PET provide a significant contribution toward enzymatic degradation of polyesters. To make PHA production environmental sustainable and economically competitive, the use of inexpensive substrates was also investigated: waste frying oils (WFOs) were tested as substrates. The commercial value of WFO is depending on several factors and the free fatty acids (FFAs) content strongly decrease the price since it strongly affects the yield of the biodiesel production process. Microbial PHA production alternative to acid pre-treatment of FFAs-rich WFO was explored. The introduction of an upstream microbial fermentation step of ad hoc systems achieves the 2-fold purpose of reduction of the FFAs content of the waste and of producing added-value products. Lab-scale results proved the exploitability of the proposed bioprocess both with recombinant and native PHA producing cell factories. In the case of native microorganism, the effect of extracellular lipase in biopolymers production and FFAs reduction was also investigated. Moreover, to re-introduce into the production flow a by-product of biodiesel production, glycerol was tested as C-source for fermentation. This substrate was applied for boosting PHAs production in above-mentioned bioprocess as well as main C-source for properly designed recombinant E. coli strains (Omni strains). The investigation of these systems on media supporting production of different PHA co-polymers laid the foundation for the selection and enhancement of enzymes and metabolic background contributing to biopolymers production
