Sviluppo di una piattaforma di prototipizzazione, basata su approcci di Biologia Sintetica, per la produzione di Bioplastiche

Polyhydroxyalkanoates (PHAs) are biopolyesters produced in an array of wild-type organisms and due to their similar characteristics to oil-derived plastics; PHAs have attracted widespread attention as sustainable alternative to traditional plastics. In contrast to many polyolefins, PHAs are also biodegradable and biocompatible. PHAs production is also currently more expensive compared to polyolefins and therefore more efficient PHAs production processes, including the use of low-cost substrates, such as whey permeate, a waste by-product of cheese production, would be desirable. Cell-free systems, based on the endogenous transcription-translation machinery of the cellular extracts have been described to be useful for providing a prototyping environment for the rapid characterisation of DNA bioparts and biosynthetic pathways. To this end, I developed an E. coli-based cell-free system, characterised its transcription-translation activity by expressing gfpmut3b and used these as a prototyping platform to characterise bioplastic-producing operons that were engineered in this study. Special attention was given to pH and sulfhydryl groups variations during cell-free bioplastic reactions as these can relate to cell-free performance and bioplastic pathway activities. Also, LC-MS metabolomics analysis profiles revealed the presence of S-3HB monomers in cell-free bioplastic reactions. Furthermore, I obtained an eight-fold higher transcription-translation activity by using bacterial haemoglobin (VHb) engineered E. coli for cell-free systems generation. Additionally, to increase in vivo PHAs production yields I generated several novel bioplastic operon designs, containing phaCAB genes from Ralstonia eutropha H16 and transformed them into E. coli MG1655. Using these strains I characterised PHAs production using media containing either glucose, propionic acid or whey permeate. Qualitative and quantitative analyses were carried out using Nile Red-based assays (plates and flow cytometry), bioplastic purification and GC-MS analysis. The most productive phaCAB engineered E. coli strain, C104_VHb consisting of J23104 Anderson promoter, B0034 Ribosome Binding Site and Vitreoscilla haemoglobin design, produced P(3HB-co-3HV) content (wt % of cell dry weight) of 44.42 ± 3.52 from propionic acid and P(3HB) content of 78.36 ± 6.13 from whey permeate. GC-MS analysis of purified bioplastic confirmed that P(3HB-co-3HV) co-polymer and P(3HB) were produced using propionic acid and whey permeate, respectively. With this pilot study I was able to assess that cell-free systems coupled with biochemical and LC-MS analysis are useful prototyping environments for PHAs production characterisation. Finally, using the phaCAB engineered E. coli strains, generated in this and in a previous study, I obtained high levels of P(3HB-co-3HV) co-polymer from propionic acid and of P(3HB) from glucose and whey permeate, an inexpensive carbon source that could further decrease PHAs production costs.