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Gas fermentation using acetogenic bacteria offers a promising route for the sustainable production of low carbon fuels and commodity chemicals from abundant, inexpensive C1 feedstocks including industrial waste gases, syngas, reformed methane or methanol. Clostridium autoethanogenum is a model gas fermenting acetogen that produces fuel ethanol and 2,3-butanediol, a precursor for nylon and rubber. Acetogens have already been used in large scale industrial fermentations, they are ubiquitous and known to play a prominent role in the global carbon cycle. Still, they are considered to live on the thermodynamic edge of life and potential energy constraints when growing on C1 gases pose a major challange for the commercial production of fuels and chemicals. We have developed a systematic platform to investigate acetogenic energy metabolism, exemplified here by experiments contrasting heterotrophic and autotrophic metabolism. The platform is built from complete omics technologies, augmented with genetic tools and complemented by a manually curated genome-scale mathematical model. Together the tools enable the design and development of new, energy efficient pathways and strains for the production of chemicals and advanced fuels via C1 gas fermentation. As a proof-of-platform, we investigated heterotrophic growth on fructose versus autotrophic growth on gas that demonstrate the role of the Rnf complex and Nfn complex in maintaining growth using the Wood–Ljungdahl pathway. Pyruvate carboxykinase was found to control the rate-limiting step of gluconeogenesis and a new specialized glyceraldehyde-3-phosphate dehydrogenase was identified that potentially enhances anabolic capacity by reducing the amount of ATP consumed by gluconeogenesis. The results have been confirmed by the construction of mutant strains. [ABSTRACT FROM AUTHOR]

Many plant genes have been shown to be induced by water stress and function in stress tolerance. The erd1 gene has been shown to be upregulated in response to both water stress and etiolation. Promoter studies using the erd1 promoter region fused to the luciferase (LUC) reporter gene in Arabidopsis thaliana were performed to identify the putative cis elements involved. Results indicated that the cis elements, responsible for gene expression during dehydration and etiolation, are separately located in two discrete portions of the erd1 promoter. Base substitution analysis showed that a 14-bp region from -599 to -586, and a myc recognition motif from -466 to -461 are necessary for the induction of LUC activity in dehydrated plants. On the other hand, base substitution analysis revealed that both an abscisic acid responsive element (ABRE)-like sequence (from -199 to -195) and an ACGT sequence (from -155 to -152) are required for an etiolation-induced increase in LUC activity. LUC activity measurements from etiolated transgenic plants incubated in either water, N6-benzyleadenine (BA), or a 1% sucrose solution found that while BA was able to delay the increase in LUC activity seen in water-treated plants, no increase in LUC activity was seen in plants incubated in sucrose. These results indicate that the erd1 promoter contains two different regulatory systems that are involved in upregulation by dehydration stress and dark-induced senescence. [ABSTRACT FROM AUTHOR]

Introduction: Quantification of tetrahydrofolates (THFs), important metabolites in the Wood-Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.Objective: To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.Methods: Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC-MS/MS was used for quantification.Results: Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.Conclusion: Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards. [ABSTRACT FROM AUTHOR]

2,3-butanediol (23BD) is a high value chemical usually produced petro-chemically, but which can also be synthesized by some bacteria. To date, the best microbial 23BD production rates have been observed using pathogenic bacteria in fermentation systems that depend on sugars as the carbon and energy source for product synthesis. Here we present evidence of 23BD production by three non-pathogenic acetogenic Clostridium species C. autoethanogenum, C. ljungdahlii, and C. ragsdalei using carbon monoxide containing industrial waste gases or syngas as the sole source of carbon and energy. Through an analysis of the C. ljungdahlii genome the complete pathway from carbon monoxide to 23BD has been proposed. Homologues of the genes involved in this pathway were also confirmed in the other two species investigated. A gene expression study demonstrates a correlation between mRNA accumulation from 23BD biosynthetic genes and the onset of 23BD production, while a broader expression study of Wood-Ljungdahl pathway genes provides a transcriptional level view of one of the oldest existing biochemical pathways. [ABSTRACT FROM AUTHOR]

Clostridium autoethanogenum is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predicted to encode seven open-reading frames and estimated to be a low-copy number plasmid present at approximately 12 copies per chromosome. RNA-seq analyses indicate that pCA genes are transcribed at low levels and two proteins, CAETHG_05090 (putative replication protein) and CAETHG_05115 (hypothetical, a possible Mob protein), were detected at low levels during batch gas fermentations. Thiolase ( thlA ), CoA-transferase ( ctfAB ), and acetoacetate decarboxylase ( adc ) genes were introduced into a vector for isopropanol production in C. autoethanogenum using the native plasmid origin of replication. The availability of the pCA sequence will facilitate studies into its physiological role and could form the basis for genetic tool optimization., Competing Interests: LanzaTech has an interest in commercializing gas fermentation with C. autoethanogenum. SN, VR, RN, CL, WM, MK, SS and SB are employees of LanzaTech. MJ consults for and has joint funding with LanzaTech. MK is co-inventors on granted US patent 9,365,868 (assigned to LanzaTech) related to production of acetone and isopropanol by fermentation of a gaseous substrate. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nogle, Nagaraju, Utturkar, Giannone, Reynoso, Leang, Hettich, Mitchell, Simpson, Jewett, Köpke and Brown.)

Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO 2 and H 2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 C-mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO 2 and H 2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO 2 , and enhanced CO 2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO 2 and CO. Furthermore, the addition of CO led to superior CO 2 -valorizing characteristics (9.7 ± 0.4 g/L ethanol with a 66 ± 2 C-mol% distribution, and 540 ± 20 mmol CO 2 /gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO 2 and H 2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO 2 gas fermentation, which can contribute to mitigating climate change., (Copyright © 2020 Heffernan, Valgepea, de Souza Pinto Lemgruber, Casini, Plan, Tappel, Simpson, Köpke, Nielsen and Marcellin.)

Autotrophic acetogenic bacteria are able to capture carbon (CO or CO 2 ) through gas fermentation, allowing them to grow on a spectrum of waste gases from industry (e.g., steel manufacture and oil refining, coal, and natural gas) and to produce ethanol. They can also consume syn(thesis) gas (CO and H 2 ) made from the gasification of renewable/sustainable resources, such as biomass and domestic/agricultural waste. Acetogenic gas fermentation can, therefore, produce ethanol in any geographic region without competing for food or land. The commercialization of the process is now at an advanced stage. The real potential of acetogens, however, resides in their capacity to produce chemicals and fuels other than ethanol. This requires the redesign and implementation of more efficient metabolic pathways, adapting them to high performing manufacturing processes. Respective species, their bioenergetics, the genetic tools developed for their metabolic engineering, culture techniques and fermenter set-ups, as well as the commercialization, are comprehensively described and discussed in this chapter.

Unlabelled: 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 CO2 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 CO2, 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 H2 and CO2 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 CO2 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., (Copyright © 2016 Liew et al.)