Tentativo di ingegneria metabolica in Clostridium thermocellum per la sovrapproduzione di acido lattico e caratterizzazione del repressore trascrizionale Rex

Nowadays, issues related to environmental safety are of significant interest in most industrial fields. The search for renewable materials that can be used as a starting point to create polymers of interest, bioplastics for example, is an important focus of biotechnological research. A promising molecule for this topic is lactic acid, which is already exploited for several applications: it is used in the food industry, in cosmetics and for the production of polylactic acid, PLA, which is a biodegradable and biocompatible plastic. The research in this field aims at reducing the cost of lactic acid production in terms of energy and money, and, since lactic acid is usually produced via the fermentation of corn, to look for a low-value compound, such as a waste, that can be used as the fermentation feedstock. Therefore, bacteria capable of fermenting lactic acid from other sources, different from food stock, such as lignocellulose, are being investigated. An interesting organism in this subject is Clostridium thermocellum, which is a Gram- positive bacterium, with a high efficiency in degrading cellulose. This ability is due to the presence of the cellulosome, a multi-enzyme structure, that can solubilize and ferment cellulose, and can therefore be exploited in a one-step process of fermentation of lignocellulose which takes the name of CBP, consolidated bioprocessing. In this project, an engineered strain of C. thermocellum, called LL1138, was used and transformed to increase the production of lactic acid, which in this organism is produced by a lactate dehydrogenase enzyme, starting from pyruvate. Normally, the LDH can produce lactate using NADH as a cofactor; in this thesis, a mutated form of the enzyme, LDHI40R, was employed, whose mutation allows to use NADH as well as NADPH as reductant. Strain LL1138 instead shows an atypical metabolism, lacking the pyruvate phosphate dikinase enzyme; therefore, the conversion from PEP to pyruvate can only occur through a pathway called “malate shunt”, which consequently leads to an excess in the production of NADPH. Therefore, because LL1138 produces high quantities of NADPH, the mutated LDH could exploit this excess of cofactor to produce a higher lactic acid yield. The NADH/NAD+ ratio is particularly important in this organism, as it is also involved in the regulation of a transcriptional repressor called Rex. Rex can sense the redox state of the cell and when the NADH/NAD+ ratio is low, Rex binds to a specific consensus sequence of the DNA thus repressing gene transcription; when the ratio is high, it changes its conformation, dissociates from the operator site, and allows the expression of genes. Two gene loci for Rex were identified in C. thermocellum: Clo1313_1799 and Clo1313_2471 which encode for two similar proteins, Rex 1 and Rex 2. In this thesis it was investigated the binding of Rex 2 to different cofactors, NADH and NAD+, as well as NADPH and NADP+, using fluorescence spectroscopy. Rex binding mode to gene promoter regions containing a consensus sequence for the protein was also investigated by using the same technique. Then, proceeding with the characterization of the protein, its stability in the absence and presence of oxidized cofactors, was assessed via Differential Scanning Calorimetry.