Insights into CO ₂ Fixation Pathway of Clostridium autoethanogenum by Targeted Mutagenesis

Abstract

The future sustainable production of chemicals and fuels from nonpetrochemical resources and reduction of greenhouse gas emissions are two of the greatest societal challenges. Gas fermentation, which utilizes the ability of acetogenic bacteria such as Clostridium autoethanogenum to grow and convert CO 2 and CO into low-carbon fuels and chemicals, could potentially provide solutions to both. Acetogens fix these single-carbon gases via the Wood-Ljungdahl pathway. Two enzyme activities are predicted to be essential to the pathway: carbon monoxide dehydrogenase (CODH), which catalyzes the reversible oxidation of CO to CO 2 , and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which combines with CODH to form a CODH/ACS complex for acetyl-CoA fixation. Despite their pivotal role in carbon fixation, their functions have not been confirmed in vivo. By genetically manipulating all three CODH isogenes (acsA, cooS1, and cooS2) of C. autoethanogenum, we highlighted the functional redundancies of CODH by demonstrating that cooS1 and cooS2 are dispensable for autotrophy. Unexpectedly, the cooS1 inactivation strain showed a significantly reduced lag phase and a higher growth rate than the wild type on H 2 and CO 2 . During heterotrophic growth on fructose, the acsA inactivation strain exhibited 61% reduced biomass and the abolishment of acetate production (a hallmark of acetogens), in favor of ethanol, lactate, and 2,3-butanediol production. A translational readthrough event was discovered in the uniquely truncated (compared to those of other acetogens) C. autoethanogenum acsA gene. Insights gained from studying the function of CODH enhance the overall understanding of autotrophy and can be used for optimization of biotechnological production of ethanol and other commodities via gas fermentation.IMPORTANCE Gas fermentation is an emerging technology that converts the greenhouse gases CO 2 and CO in industrial waste gases and gasified biomass into fuels and chemical commodities. Acetogenic bacteria such as Clostridium autoethanogenum are central to this bioprocess, but the molecular and genetic characterization of this microorganism is currently lacking. By targeting all three of the isogenes encoding carbon monoxide dehydrogenase (CODH) in C. autoethanogenum, we identified the most important CODH isogene for carbon fixation and demonstrated that genetic inactivation of CODH could improve autotrophic growth. This study shows that disabling of the Wood-Ljungdahl pathway via the inactivation of acsA (encodes CODH) significantly impairs heterotrophic growth and alters the product profile by abolishing acetate production. Moreover, we discovered a previously undescribed mechanism for controlling the production of this enzyme. This study provides valuable insights into the acetogenic pathway and can be used for the development of more efficient and productive strains for gas fermentation.

Energy Conservation and Carbon Flux Distribution During Fermentation of CO or H2/CO2 by Clostridium ljungdahlii

“…Clostridium ljungdahlii, a close relative of "Clostridium autoethanogenum, " is used as a model organism for studying the production of ethanol and acetate from syngas, which is a gas mixture mainly composed of carbon monoxide (CO), carbon dioxide (CO 2 ), and hydrogen (H 2 ) (Köpke et al, 2010(Köpke et al, , 2011aAdamberg et al, 2015;Dürre and Eikmanns, 2015;Liew et al, 2016a). Both CO and H 2 can act as energy sources for growth and metabolism of C. ljungdahlii during gas fermentation.…”

CO2-Fixation Strategies in Energy Extremophiles: What Can We Learn From Acetogens?

“…Because syngas (H 2 /CO 2 /CO) is the main source of carbon and energy for hydrogenotrophic methanogens and acetogens, they are excellent "bio-converters." For instance, acetogens turn industrial waste gases, rich in H 2 , CO and CO 2 , to butanediol, ethanol or acetate, potential biofuels or starting points for new chemical synthesis (Wang et al, 2013;Mock et al, 2015;Liew et al, 2016). With the discovery of genetically tractable acetogens (Liew et al, 2016;Basen et al, 2018) the possibilities for biocompound synthesis, and bioremediation are expanding.…”

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

“…Nevertheless, plasmid-based gene expression methods developed for Clostridium species have been shown to be functional in other acetogens. In addition, many metabolic engineering efforts have recently been made using homologous recombination (HR) [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ], ClosTron [ 28 , 36 , 37 , 38 , 39 ] and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) along with its CRISPR-associated (Cas) protein (CRISPR-Cas) system [ 32 , 40 , 41 , 42 , 43 , 44 , 45 ] to improve the production of value-added biochemicals from C1 gases.…”

“…Furthermore, selection markers simplified mutant isolation based on acquisition of antibiotic resistance (e.g., clarithromycin and thiamphenicol). Among acetogens, this tool has been implemented in C. autoethanogenum [ 28 , 36 , 37 , 38 ] and C. ljungdahlii [ 39 ]. Most of these studies utilized ClosTron to disrupt the genes involved in carbon fixation and energy conservation, which are the keys to understanding the basic physiology of acetogens.…”

“…While cooS1 or cooS2 inactivation mutants exhibited no growth retardation effect under CO or H 2 /CO 2 , the acsA mutant could not grow on both autotrophic conditions. This result indicated that acsA is essential for carbon fixation in C. autoethanogenum [ 38 ]. Although the ClosTron system is a valuable tool for generating deletion mutants, its use is limited by the length of an integrated DNA sequence, as the efficiency decreases when the sequence length exceeds 1 kb [ 70 ] and its use leaves a scar of the inserted intron in the genome [ 68 ].…”