Esercizio, Nunzia (2022) Characterization of the Capnophilic Lactic Fermentation pathway and molecular approaches in the hyperthermophilic bacterium Thermotoga neapolitana. [Tesi di dottorato]

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Abstract

Capnophilic lactic fermentation (CLF) is a novel anaplerotic pathway recently identified and patented in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana. The CO2-activated mechanism enables a non-competitive synthesis of hydrogen and L-lactic acid at high yields from sugar-based waste and renewable feedstocks, which makes it economically attractive for development of sustainable biotechnological processes. This research work investigates the transcriptional modification induced by CO2 in T.neapolitana cultures using an RNA-seq approach, showing a massive metabolic rearrangement of the central carbon metabolism, but also the activation of side mechanisms that could increase the availability of reducing equivalents necessary to support the concomitant production of hydrogen and lactic acid by CLF pathway. The analysis of the differentially expressed genes allowed the selection of possible candidates for the genetic manipulation of T.neapolitana. The development of a specific transformation strategy allowed to the heterologous expression of the acs gene, by which the mutant strain ACS03 is able to boost the flow from CO2 and acetyl-CoA to produce lactic acid, thus increasing CLF fixation rate. The study of hyperthermophilic transformation techniques also showed that Thermotoga sp. RQ7 is the closest parental strain of T.neapolitana, and the only naturally competent strain of the genus Thermotoga. In order to improve the molecular investigation of the CLF pathway, CLF activation has been described in T. sp. strain RQ7 as a peculiar pathway activated by CO2. Transcriptomic analysis also revealed the possible switch from the classical Embden-Meyerhof-Parnas pathway to the alternative Entner-Dourdoroff or oxidative pentose phosphate pathway, and retro-biosynthetic analysis conducted in this PhD thesis using NMR and GC-MS techniques highlighted the possible activation of the pentose phosphate pathway in T.neapolitana under CO2. Lastly, the effect of sulfured source on the CLF performance, as well as the effect of yeast extract and tryptone deprivation have been evaluated in T.neapolitana, in order to optimize fermentation yields.

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