Interactive Regulation of Formate Dehydrogenase during CO ₂ Fixation in Gas-Fermenting Bacteria

Abstract

Protein lysine acetylation, a prevalent posttranslational modification, regulates numerous crucial biological processes in cells. Nevertheless, how lysine acetylation interacts with other types of regulation to coordinate metabolism remains largely unknown owing to the complexity of the process. Here, using a representative gas-fermenting bacterium, Clostridium ljungdahlii, we revealed a novel regulatory mechanism that employs both the lysine acetylation and transcriptional regulation systems to interactively control CO2 fixation, a key biological process for utilizing this one-carbon gas. A dominant lysine acetyltransferase/deacetylase system, At2/Dat1, was identified and found to regulate FDH1 (formate dehydrogenase responsible for CO2 fixation) activity via a crucial acetylation site (lysine-29). Notably, the global transcription factor CcpA was also shown to be regulated by At2/Dat1; in turn, CcpA could directly control At2 expression, thus indicating an unreported interaction mode between the acetylation system and transcription factors. Moreover, CcpA was observed to negatively regulate FDH1 expression, which, when combined with At2/Dat1, leads to the collaborative regulation of this enzyme. Based on this concept, we reconstructed the regulatory network related to FDH1, realizing significantly increased CO2 utilization by C. ljungdahlii. IMPORTANCE Microbial CO2 fixation and conversion constitute a potential solution to both utilization of greenhouse gas or industrial waste gases and sustainable production of bulk chemicals and fuels. Autotrophic gas-fermenting bacteria play central roles in this bioprocess. This study provides new insights regarding the metabolic regulatory mechanisms underlying CO2 reduction in Clostridium ljungdahlii, a representative gas-fermenting bacterium. A critical formate dehydrogenase (FDH1) responsible for fixing CO2 and a dominant reversible lysine acetylation system, At2/Dat1, were identified. Furthermore, FDH1 was found to be interactively regulated by both the At2/Dat1 system and the global transcriptional factor CcpA, and the two regulatory systems are mutually restricted. Reconstruction of this multilevel metabolic regulatory module led to improved CO2 metabolism by C. ljungdahlii. These findings not only substantively expand our understanding but also provide a potentially useful metabolic engineering strategy for microbial carbon fixation.

Functional dissection and modulation of the BirA protein for improved autotrophic growth of gas‐fermenting Clostridium ljungdahlii

“…In view of that biotin synthesis is a high energy‐consuming process in cells (Feng et al ., 2013), such a BirA‐mediated feedback regulation on biotin synthesis may be crucial for the control of energy expenditure in bacteria. Theoretically, this regulatory mechanism is especially important for gas‐fermenting clostridia because the carbon fixation and assimilation in these autotrophic bacteria are a net energy consumption process (Zhang et al., 2020a,2020b), and excessive energy consumption in biotin synthesis will cause metabolic burden. But interestingly, a modified Cl BirA with only enzymatically active domain led to a better performance of C. ljungdahlii in gas fermentation (Figs 6 and 8), indicating that an uncontrolled overproduction of biotin is not the key point for the autotrophic growth of C. ljungdahlii .…”

“…However, to date, this aspect remains minimally explored due to the challenges in genetic tools. Specific to transcriptional regulators (TFs), to our knowledge, only a global regulator CcpA and a TetR‐family regulator (CAETHG_0459) have been identified and characterized in these autotrophic bacteria (Lemgruber et al ., 2019; Zhang et al., 2020a,2020b). CcpA was found to directly regulate the expression of some WLP genes in Clostridium.…”

“…CcpA was found to directly regulate the expression of some WLP genes in Clostridium. ljungdahlii , and the elimination of CcpA’s inhibition on these genes could significantly enhance CO 2 utilization (Zhang et al., 2020a,2020b). CAETHG_0459, as a transcription activator in Clostridium autoethanogenum , can directly bind to the core enzyme of RNA polymerase and subsequently activate the expression of multiple WLP genes (Lemgruber et al ., 2019).…”

Influence of Pyroligneous Acid on Fermentation Parameters, CO2 Production and Bacterial Communities of Rice Straw and Stylo Silage

“…increased, possibly because some Clostridium spp. are autotrophic acetogenic bacteria that can produce important chemicals and fuels by using CO 2 ( Zhang L. et al, 2020 ). Lachnoclostridium , a newly defined genus under the highly polyphyletic class Clostridia , showed a decreased relative abundance after pyroligneous acid treatment, consistent with that in CO 2 production.…”

Protein acetylation-mediated cross regulation of acetic acid and ethanol synthesis in the gas-fermenting Clostridium ljungdahlii

“…Protein Lysine acetylation (PLA) is known to be a crucial metabolic regulatory mechanism in both prokaryotic and eukaryotic cells, in which multiple PLA-regulated physiological and metabolic processes have been revealed ( 12 , 13 , 14 ). In a previous study on Clostridium ljungdahlii , a representative autotrophic acetogen, we revealed an interactive regulation module that uses both the global transcriptional factor CcpA and acetylation/deacetylation system At2/Dat1 to control carbon fixation ( 15 ). This finding suggested that, in addition to TFs, PLA plays an important role in regulating crucial cellular functions in autotrophic acetogens, leading to the regulation on several levels involving multiple factors.…”