Aumento della produzione eterologa di 2-feniletanolo nel cianobatterio Synechococcus elongatus PCC 7942 tramite overespressione della Shikimato Chinasi endogena e addizione di L-fenilalanina.

2-Phenylethanol (2-PE) is a valuable aromatic compound highly requested for its rose-like scent. Its global market is driven by cosmetics, pharmaceuticals, food and beverage industries, and is projected to reach 351 Million USD by 2027. Many flowers (e.g., rose) synthetize 2-PE via the Shikimate Pathway, the biological route dedicated to aromatics synthesis, but its extraction from petals is expensive and low-yielding. As a consequence, most of 2-PE produced today derives from chemical synthesis, which employs petroleum as feedstock and generates unwanted byproducts. Considering the increasing need for “green processes” and the public demand for natural products, a promising alternative could be represented by biotechnological production systems. So far, different microorganisms have been studied and engineered for 2-PE biotechnological production with particular emphasis on yeast fermentation; but fewer studies have focused on autotrophic microorganisms. Among them, cyanobacteria represent ideal microbial factories thanks to their ability to photosynthetically convert CO2 into valuable compounds, their minimal nutritional requirements, high photosynthetic rate and the availability of genetic and bioinformatics tools. In this work, a heterologous pathway for 2-PE biosynthesis was integrated into Synechococcus elongatus PCC 7942 genome (7942_p120) to create a sustainable microbial factory able to couple CO2 fixation with target aromatic compound bioproduction. Strain 7942_p120 overexpresses four exogenous proteins involved in 2-PE biosynthesis, i.e., DAHP-synthase-fbr (feedback-inhibition-resistant), chorismate mutase/prephenate dehydratase-fbr, phenylpyruvate decarboxylase and alcohol dehydrogenase A. This mutant strain was physiologically characterized for growth and 2-PE titre either in standard BG-11 growth medium or in presence of different aromatic compounds, in order to assess possible physiological effects of the heterologous pathway expression on the mutant’s metabolism. The most interesting result was obtained with 0.3 g/L L-phenylalanine doping, which doubled 7942_p120 strain’s 2-PE yield compared with standard BG-11 growth medium. In addition, from 7942_p120 mutant, a Shikimate Kinase (SK) overexpressing mutant strain (7942_p120_SK) was created. The SK catalyzes the ATP-dependent phosphorylation of shikimate to shikimate-3-phosphate in the shikimate pathway, and has been reported as possible bottleneck of this biological route in some model microorganisms. Thus, 7942_p120 and 7942_p120_SK mutants reached a maximum 2-PE titre of ~ 90 mg/L and ~ 280 mg/L respectively, in BG-11 medium doped with 0.3 g/L L-phenylalanine, showing a 2.3-fold increment compared with literature for 7942_p120 strain. Overexpression of the shikimate kinase significantly incremented 2-PE maximum yield from ~ 127 mg per g Dry cell Weight to ~ 305 mg/gDW, and also maintained a steady 2-PE average productivity of 20 mg/L per day for two weeks. Also, L-phe consumption kinetics showed a significant slowdown in 7942_p120_SK compared to 7942_p120, suggesting a metabolic remodeling caused by shikimate kinase overexpression. This thesis, by combining two different strategies, namely metabolite doping and shikimate pathway engineering, leveraged the potential of cyanobacteria as photoautotrophic microbial factories for aromatics synthesis by successfully conceiving and experimentally validating a 2-PE cyanobacterial bioproduction processes of extreme interest.