CO2-Induced Transcriptional Reorganization: Molecular Basis of Capnophillic Lactic Fermentation in Thermotoga neapolitana

Abstract

Capnophilic lactic fermentation (CLF) is a novel anaplerotic pathway able to convert sugars to lactic acid (LA) and hydrogen using CO 2 as carbon enhancer in the hyperthermophilic bacterium Thermotoga neapolitana. In order to give further insights into CLF metabolic networks, we investigated the transcriptional modification induced by CO 2 using a RNA-seq approach. Transcriptomic analysis revealed 1601 differentially expressed genes (DEGs) in an enriched CO 2 atmosphere over a total of 1938 genes of the T. neapolitana genome. Transcription of PFOR and LDH genes belonging to the CLF pathway was up-regulated by CO 2 together with 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydratase (EDD) of the Entner-Doudoroff (ED) pathway. The transcriptomic study also revealed up-regulation of genes coding for the flavin-based enzymes NADH-dependent reduced ferredoxin:NADP oxidoreductase (NFN) and NADferredoxin oxidoreductase (RNF) that control supply of reduced ferredoxin and NADH and allow energy conservation-based sodium translocation through the cell membrane. These results support the hypothesis that CO 2 induces rearrangement of the central carbon metabolism together with activation of mechanisms that increase availability of the reducing equivalents that are necessary to sustain CLF. In this view, this study reports a first rationale of the molecular basis of CLF in T. neapolitana and provides a list of target genes for the biotechnological implementation of this process.

Effect of Cultivation Parameters on Fermentation and Hydrogen Production in the Phylum Thermotogae

“…In dark fermentation, hydrogen and lactic acid levels competed for a common pool of reducing power. Whereas, in CLF, the H 2 level remained high, probably due to additional sources of reductants to sustain NADH-dependent pathways ( Figure 1 ) [ 118 , 119 , 120 ]. Recently, an additional increase in lactic acid production occurred in a T. neapolitana mutant that was isolated from a culture adapted to continuous exposure to CO 2 [ 62 ].…”

Fermentation of Biodegradable Organic Waste by the Family Thermotogaceae

“…Moreover, some members of the Thermotogaceae family possess an unprecedented anaplerotic mechanism, called capnophilic lactic fermentation (CLF), that represents the first example of biological non-autotrophic sequestration of CO 2 in hyperthermophilic bacteria, more advantageous than classical dark fermentation regarding the production of hydrogen through degradation of carbon substrates [32,33,[55][56][57]. This process is activated during glucose fermentation under CO 2 sparging, and it is based on the coupling of acetate and CO 2 derived from glycolysis to produce enantiopure L-lactic acid without affecting H 2 yields [32,33,55,56,[58][59][60]. This mechanism was extensively studied in Thermotoga neapolitana, and only a few members of the Thermotoga and Pseudothermotoga genera operated this CO 2 recycling mechanism [34].…”

“…This mechanism was extensively studied in Thermotoga neapolitana, and only a few members of the Thermotoga and Pseudothermotoga genera operated this CO 2 recycling mechanism [34]. Under CLF conditions, the bacteria also shift their glucose utilization through downregulation of EMP, activation of ED and/or OPP pathways, and the upregulation of some bifurcating enzymes that could supply NADH in these metabolic processes [60].…”

“…1.1.1.44) [54]. Interestingly, these pathways showed an environmental-dependent activation mechanism in T. neapolitana, because the insufflation of CO 2 instead of N 2 induced the upregulation of the genes involved in ED and OPP [60]. Another peculiarity of some Thermotogales members (e.g., T.maritima) is the presence of an unconventional triosephosphate isomerase (TIM, E.C.…”

“…In this context, it is not surprising to find in Thermotogaceae genomes the entire regulons for monosaccharide metabolism and the ABC transporters for the import/export of simple and complex sugars [76]. The fine regulation of these mechanisms leads to efficient catabolism of the sugar substrates [60,163,171].…”