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

Abstract

The autotrophic acetogen Clostridium ljungdahlii has emerged as a major candidate in the biological conversion of one-carbon gases (CO 2 /CO) to bulk chemicals and fuels. Nevertheless, the regulatory pathways and downstream metabolic changes responsible for product formation and distribution in this bacterium remain minimally explored. Protein lysine acetylation (PLA), a prevalent posttranslational modification, controls numerous crucial cellular functions. Herein, we revealed a novel cross-regulatory mechanism that uses both the PLA system and transcription factors to regulate the carbon flow distribution for product formation in C. ljungdahlii . The dominant acetylation/deacetylation system (At2/Dat1) in C. ljungdahlii was found to regulate the ratio of two major products, acetic acid and ethanol. Subsequent genetic and biochemical analyses revealed that the activities of Pta and AdhE1, two crucial enzymes responsible for acetic acid and ethanol synthesis, respectively, were greatly affected by their levels of PLA. We found that the acetylation statuses of Pta and AdhE1 underwent significant dynamic changes during the fermentation process, leading to differential synthesis of acetic acid and ethanol. Furthermore, the crucial redox-sensing protein Rex was shown to be regulated by PLA, which subsequently altered its transcriptional regulation on genes responsible for acetic acid and ethanol formation and distribution. Based on our understanding of this cross-regulatory module, we optimized the ethanol synthetic pathway by modifying the acetylation status (deacetylation-mimicked mutations of crucial lysine residues) of the related key enzyme, achieving significantly increased titer and yield of ethanol, an important chemical and fuel, by C. ljungdahlii in gas fermentation.

Absolute Proteome Quantification in the Gas-Fermenting Acetogen Clostridium autoethanogenum

“…Further research is needed to identify which mechanism from posttranslational protein modification, allosteric regulation, or substrate concentration change is responsible for the posttranslational regulation of fluxes. Interestingly, recent work demonstrates the relevance of both protein acetylation and intracellular metabolite levels for the regulation of acetogen metabolism ( 20 , 67 , 68 ).…”

Thermal and alkaline pre-treatments of inoculum halt methanogenesis and enables cheese whey valorization by batch acidogenic fermentation

“…This was in opposition to all other conditions tested. It is likely that in such operational conditions, acetate served as the substrate for fermentative bacteria to form iso-butyric acid and ethanol, as already described by Thatikayala et al (2021) and ThatikayalaLiu et al (2022). Both chemicals are of great industrial interest (i.e., pharmaceutical, feed, chemical, biofuels).…”

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO2 and H2

“…The mRNA of AOR [ 73 ] as well as the enzymes AOR and ADH [ 45 ] were found to be highly abundant in C. ljungdahlii growing on syngas. However, it was found just recently, that ethanol and acetate formation in C. ljungdahlii is also regulated by posttranslational modification ‘protein lysine acetylation’ of AdhE1 and Pta, influencing the enzyme activities [ 74 ].…”