[Research progress in bioconversion of C1 gases into oleochemicals].

The utilization of C1 gases (CH4, CO2, and CO) for the production of oleochemicals applied in the energy and platform chemicals through microbial engineering has emerged as a promising approach to reduce greenhouse gas emissions and decrease dependence on fossil fuel. C1 gas-utilizing microorganisms, such as methanotrophs, microalgae, and acetogens, are capable of converting C1 gases as the sole substrates for cell growth and oleochemical synthesis with different carbon-chain lengths, garnering considerable attention from both scientific community and industry field for sustainable biomanufacturing. This paper comprehensively reviews recent advancements in the development of engineered cell factories utilizing C1 gases for the production of oleochemicals, elucidating the key metabolic pathways of biosynthesis. Furthermore, this paper highlights the research progress and prospects in optimizing gene expression, metabolic pathway reconstruction, and fermentation conditions for efficient oleochemical production from C1 gases. This review provides valuable insights and guidance for the efficient utilization of C1 gases and the development of carbon cycling-based bioeconomy.

Synthetic biology approaches and bioseparations in syngas fermentation.

Fossil fuel use drives greenhouse gas emissions and climate change, highlighting the need for alternatives like biomass-derived syngas. Syngas, mainly H2 and CO, is produced via biomass gasification and offers a solution to environmental challenges. Syngas fermentation through the Wood-Ljungdahl pathway yields valuable chemicals under mild conditions. However, challenges in scaling up persist due to issues like unpredictable syngas composition and microbial fermentation contamination. This review covers advancements in genetic tools and metabolic engineering to expand product range, highlighting crucial enabling technologies that expedite strain development for acetogens and other non-model organisms. This review paper provides an in-depth exploration of syngas fermentation, covering microorganisms, gas composition effects, separation techniques, techno economic analysis, and commercialization efforts.

Industrial biotechnology goes thermophilic: Thermoanaerobes as promising hosts in the circular carbon economy.

Transitioning away from fossil feedstocks is imperative to mitigate climate change, and necessitates the utilization of renewable, alternative carbon and energy sources to foster a circular carbon economy. In this context, lignocellulosic biomass and one-carbon compounds emerge as promising feedstocks that could be renewably upgraded by thermophilic anaerobes (thermoanaerobes) via gas fermentation or consolidated bioprocessing to value-added products. In this review, the potential of thermoanaerobes for cost-efficient, effective and sustainable bioproduction is discussed. Metabolic and bioprocess engineering approaches are reviewed to draw a comprehensive picture of current developments and future perspectives for the conversion of renewable feedstocks to chemicals and fuels of interest. Selected bioprocessing scenarios are outlined, offering practical insights into the applicability of thermoanaerobes at a large scale. Collectively, the potential advantages of thermoanaerobes regarding process economics could facilitate an easier transition towards sustainable bioprocesses with renewable feedstocks.

Simultaneous Formate and Syngas Conversion Boosts Growth and Product Formation by Clostridium ragsdalei.

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20-50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min-1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei.

Biochar facilitated Biological CO2 conversion to C2-C6 alcohols and fatty acids.

Microbial CO2 utilization reduces the carbon footprint, providing economic potential. Biochar, rich in minerals and trace metals, can enhance microbial activity. This study investigates poultry litter and switchgrass biochars produced at 350 and 700 °C (PLB350, PLB700, SGB350 and SGB700, respectively) affect CO2 conversion to C2-C6 alcohols and acids by Clostridium muellerianum P21, C. ragsdalei P11 and C. carboxidivorans P7. Fermentations were in 250-mL bottles containing H2:CO2:N2 (60:20:20) shaken at 125 rpm and 37 °C. SGB350 increased alcohol titers by 1.1-2.1 fold, and PLB350 enhanced acid concentrations by 1.2-1.7 fold compared to the control without biochar. About 2.0-3.3 fold more ethanol was formed by strain P11 compared to strains P7 and P21 with SGB350. However, strain P21 produced 2.4-fold more butanol than strain P7 with SGB350, including unique hexanol production. These results highlight the potential of biochar in enhancing C2-C6 alcohol production from CO2, thereby boosting process feasibility.

Refining and illuminating acetogenic Eubacterium strains for reclassification and metabolic engineering.

BACKGROUND: The genus Eubacterium is quite diverse and includes several acetogenic strains capable of fermenting C1-substrates into valuable products. Especially, Eubacterium limosum and closely related strains attract attention not only for their capability to ferment C1 gases and liquids, but also due to their ability to produce butyrate. Apart from its well-elucidated metabolism, E. limosum is also genetically accessible, which makes it an interesting candidate to be an industrial biocatalyst. RESULTS: In this study, we examined genomic, phylogenetic, and physiologic features of E. limosum and the closest related species E. callanderi as well as E. maltosivorans. We sequenced the genomes of the six Eubacterium strains 'FD' (DSM 3662T), 'Marburg' (DSM 3468), '2A' (DSM 2593), '11A' (DSM 2594), 'G14' (DSM 107592), and '32' (DSM 20517) and subsequently compared these with previously available genomes of the E. limosum type strain (DSM 20543T) as well as the strains 'B2', 'KIST612', 'YI' (DSM 105863T), and 'SA11'. This comparison revealed a close relationship between all eleven Eubacterium strains, forming three distinct clades: E. limosum, E. callanderi, and E. maltosivorans. Moreover, we identified the gene clusters responsible for methanol utilization as well as genes mediating chain elongation in all analyzed strains. Subsequent growth experiments revealed that strains of all three clades can convert methanol and produce acetate, butyrate, and hexanoate via reverse ß-oxidation. Additionally, we used a harmonized electroporation protocol and successfully transformed eight of these Eubacterium strains to enable recombinant plasmid-based expression of the gene encoding the fluorescence-activating and absorption shifting tag (FAST). Engineered Eubacterium strains were verified regarding their FAST-mediated fluorescence at a single-cell level using a flow cytometry approach. Eventually, strains 'FD' (DSM 3662T), '2A' (DSM 2593), '11A' (DSM 2594), and '32' (DSM 20517) were genetically engineered for the first time. CONCLUSION: Strains of E. limosum, E. callanderi, and E. maltosivorans are outstanding candidates as biocatalysts for anaerobic C1-substrate conversion into valuable biocommodities. A large variety of strains is genetically accessible using a harmonized electroporation protocol, and FAST can serve as a reliable fluorescent reporter protein to characterize genetically engineered cells. In total eleven strains have been assigned to distinct clades, providing a clear and updated classification. Thus, the description of respective Eubacterium species has been emended, improved, aligned, and is requested to be implemented in respective databases.

Functional similarity, despite taxonomical divergence in the millipede gut microbiota, points to a common trophic strategy.

BACKGROUND: Many arthropods rely on their gut microbiome to digest plant material, which is often low in nitrogen but high in complex polysaccharides. Detritivores, such as millipedes, live on a particularly poor diet, but the identity and nutritional contribution of their microbiome are largely unknown. In this study, the hindgut microbiota of the tropical millipede Epibolus pulchripes (large, methane emitting) and the temperate millipede Glomeris connexa (small, non-methane emitting), fed on an identical diet, were studied using comparative metagenomics and metatranscriptomics. RESULTS: The results showed that the microbial load in E. pulchripes is much higher and more diverse than in G. connexa. The microbial communities of the two species differed significantly, with Bacteroidota dominating the hindguts of E. pulchripes and Proteobacteria (Pseudomonadota) in G. connexa. Despite equal sequencing effort, de novo assembly and binning recovered 282 metagenome-assembled genomes (MAGs) from E. pulchripes and 33 from G. connexa, including 90 novel bacterial taxa (81 in E. pulchripes and 9 in G. connexa). However, despite this taxonomic divergence, most of the functions, including carbohydrate hydrolysis, sulfate reduction, and nitrogen cycling, were common to the two species. Members of the Bacteroidota (Bacteroidetes) were the primary agents of complex carbon degradation in E. pulchripes, while members of Proteobacteria dominated in G. connexa. Members of Desulfobacterota were the potential sulfate-reducing bacteria in E. pulchripes. The capacity for dissimilatory nitrate reduction was found in Actinobacteriota (E. pulchripes) and Proteobacteria (both species), but only Proteobacteria possessed the capacity for denitrification (both species). In contrast, some functions were only found in E. pulchripes. These include reductive acetogenesis, found in members of Desulfobacterota and Firmicutes (Bacillota) in E. pulchripes. Also, diazotrophs were only found in E. pulchripes, with a few members of the Firmicutes and Proteobacteria expressing the nifH gene. Interestingly, fungal-cell-wall-degrading glycoside hydrolases (GHs) were among the most abundant carbohydrate-active enzymes (CAZymes) expressed in both millipede species, suggesting that fungal biomass plays an important role in the millipede diet. CONCLUSIONS: Overall, these results provide detailed insights into the genomic capabilities of the microbial community in the hindgut of millipedes and shed light on the ecophysiology of these essential detritivores. Video Abstract.

Genomic insights into clostridia in bioenergy production: Comparison of metabolic capabilities and evolutionary relationships.

Bacteria from diverse genera, including Acetivibrio, Bacillus, Cellulosilyticum, Clostridium, Desulfotomaculum, Lachnoclostridium, Moorella, Ruminiclostridium, and Thermoanaerobacterium, have attracted significant attention due to their versatile metabolic capabilities encompassing acetogenic, cellulolytic, and C1-metabolic properties, and acetone-butanol-ethanol fermentation. Despite their biotechnological significance, a comprehensive understanding of clostridial physiology and evolution has remained elusive. This study reports an extensive comparative genomic analysis of 48 fully sequenced bacterial genomes from these genera. Our investigation, encompassing pan-genomic analysis, central carbon metabolism comparison, exploration of general genome features, and in-depth scrutiny of Cluster of Orthologous Groups genes, has established a holistic whole-genome-based phylogenetic framework. We have classified these strains into acetogenic, butanol-producing, cellulolytic, CO2-fixating, chemo(litho/organo)trophic, and heterotrophic categories, often exhibiting overlaps. Key outcomes include the identification of misclassified species and the revelation of insights into metabolic features, energy conservation, substrate utilization, stress responses, and regulatory mechanisms. These findings can provide guidance for the development of efficient microbial systems for sustainable bioenergy production. Furthermore, by addressing fundamental questions regarding genetic relationships, conserved genomic features, pivotal enzymes, and essential genes, this study has also contributed to our comprehension of clostridial biology, evolution, and their shared metabolic potential.

Heterologous Production of Isopropanol Using Metabolically Engineered Acetobacterium woodii Strains.

The depletion of fossil fuel resources and the CO2 emissions coupled with petroleum-based industrial processes present a relevant issue for the whole of society. An alternative to the fossil-based production of chemicals is microbial fermentation using acetogens. Acetogenic bacteria are able to metabolize CO or CO2 (+H2) via the Wood-Ljungdahl pathway. As isopropanol is widely used in a variety of industrial branches, it is advantageous to find a fossil-independent production process. In this study, Acetobacterium woodii was employed to produce isopropanol via plasmid-based expression of the enzymes thiolase A, CoA-transferase, acetoacetate decarboxylase and secondary alcohol dehydrogenase. An examination of the enzymes originating from different organisms led to a maximum isopropanol production of 5.64 ± 1.08 mM using CO2 + H2 as the carbon and energy source. To this end, the genes thlA (encoding thiolase A) and ctfA/ctfB (encoding CoA-transferase) of Clostridium scatologenes, adc (encoding acetoacetate decarboxylase) originating from C. acetobutylicum and sadH (encoding secondary alcohol dehydrogenase) of C. beijerinckii DSM 6423 were employed. Since bottlenecks in the isopropanol production pathway are known, optimization of the strain was investigated, resulting in a 2.5-fold increase in isopropanol concentration.

Adaptive laboratory evolution of a thermophile toward a reduced growth temperature optimum.

Thermophily is an ancient trait among microorganisms. The molecular principles to sustain high temperatures, however, are often described as adaptations, somewhat implying that they evolved from a non-thermophilic background and that thermophiles, i.e., organisms with growth temperature optima (TOPT) above 45°C, evolved from mesophilic organisms (TOPT 25-45°C). On the contrary, it has also been argued that LUCA, the last universal common ancestor of Bacteria and Archaea, may have been a thermophile, and mesophily is the derived trait. In this study, we took an experimental approach toward the evolution of a mesophile from a thermophile. We selected the acetogenic bacterium T. kivui (TOPT 66°C) since acetogenesis is considered ancient physiology and cultivated it at suboptimal low temperatures. We found that the lowest possible growth temperature (TMIN) under the chosen conditions was 39°C. The bacterium was subsequently subjected to adaptive laboratory evolution (ALE) by serial transfer at 45°C. Interestingly, after 67 transfers (approximately 180 generations), the adapted strain Adpt45_67 did not grow better at 45°C, but a shift in the TOPT to 60°C was observed. Growth at 45°C was accompanied by a change in the morphology as shorter, thicker cells were observed that partially occurred in chains. While the proportion of short-chain fatty acids increased at 50°C vs. 66°C in both strains, Adpt45_67 also showed a significantly increased proportion of plasmalogens. The genome analysis revealed 67 SNPs compared to the type strain, among these mutations in transcriptional regulators and in the cAMP binding protein. Ultimately, the molecular basis of the adaptation of T. kivui to a lower TOPT remains to be elucidated. The observed change in phenotype is the first experimental step toward the evolution of thermophiles growing at colder temperatures and toward a better understanding of the cold adaptation of thermophiles on early Earth.

[Genetic modification of acetogens and optimization of fermentation process in C1-gas bioconversion].

The current linear economy model relies on fossil energy and increases CO2 emissions, which contributes to global warming and environmental pollution. Therefore, there is an urgent need to develop and deploy technologies for carbon capture and utilization to establish a circular economy. The use of acetogens for C1-gas (CO and CO2) conversion is a promising technology due to high metabolic flexibility, product selectivity, and diversity of the products including chemicals and fuels. This review focuses on the physiological and metabolic mechanisms, genetic and metabolic engineering modifications, fermentation process optimization, and carbon atom economy in the process of C1-gas conversion by acetogens, with the aim to facilitate the industrial scale-up and carbon negative production through acetogen gas fermentation.

Acetate Production from Syngas Produced from Lignocellulosic Biomass Materials along with Gaseous Fermentation of the Syngas: A Review.

Biotransformation of lignocellulose-derived synthetic gas (syngas) into acetic acid is a promising way of creating biochemicals from lignocellulosic waste materials. Acetic acid has a growing market with applications within food, plastics and for upgrading into a wide range of biofuels and bio-products. In this paper, we will review the microbial conversion of syngas to acetic acid. This will include the presentation of acetate-producing bacterial strains and their optimal fermentation conditions, such as pH, temperature, media composition, and syngas composition, to enhance acetate production. The influence of syngas impurities generated from lignocellulose gasification will further be covered along with the means to alleviate impurity problems through gas purification. The problem with mass transfer limitation of gaseous fermentation will further be discussed as well as ways to improve gas uptake during the fermentation.

Microorganisms Involved in Hydrogen Sink in the Gastrointestinal Tract of Chickens.

Hydrogen sink is a beneficial process, which has never been properly examined in chickens. Therefore, the aim of this study was to assess the quantity and quality of microbiota involved in hydrogen uptake with the use of real-time PCR and metagenome sequencing. Analyses were carried out in 50 free-range chickens, 50 commercial broilers, and 54 experimental chickens isolated from external factors. The median values of acetogens, methanogens, sulfate-reducing bacteria (SRB), and [NiFe]-hydrogenase utilizers measured in the cecum were approx. 7.6, 0, 0, and 3.2 log10/gram of wet weight, respectively. For the excreta samples, these values were 5.9, 4.8, 4, and 3 log10/gram of wet weight, respectively. Our results showed that the acetogens were dominant over the other tested groups of hydrogen consumers. The quantities of methanogens, SRB, and the [NiFe]-hydrogenase utilizers were dependent on the overall rearing conditions, being the result of diet, environment, agrotechnical measures, and other factors combined. By sequencing of the 16S rRNA gene, archaea of the genus Methanomassiliicoccus (Candidatus Methanomassiliicoccus) were discovered in chickens for the first time. This study provides some indication that in chickens, acetogenesis may be the main metabolic pathway responsible for hydrogen sink.

The two-electron reduced A cluster in acetyl-CoA synthase: Preparation, characteristics and mechanistic implications.

Acetyl-CoA synthase (ACS) is a central enzyme in the carbon and energy metabolism of certain anaerobic species of bacteria and archaea that catalyzes the direct synthesis and cleavage of the acetyl CC bond of acetyl-CoA by an unusual enzymatic mechanism of special interest for its use of organonickel intermediates. An Fe4S4 cluster associated with a proximal, reactive Nip and distal spectator Nid comprise the active site metal complex, known as the A cluster. Experimental and theoretical methods have uncovered much about the ACS mechanism, but have also opened new unanswered questions about the structure and reactivity of the A cluster in various intermediate forms. Here we report a method for large scale isolation of ACS with its A cluster in the acetylated state. Isolated acetyl-ACS and the two-electron reduced ACS, produced by acetyl-ACS reaction with CoA, were characterized by UV-visible and EPR spectroscopy. Reactivity with electron acceptors provided an assessment of the apparent Em for two-electron reduction of the A cluster. The results help to distinguish between alternative electronic states of the reduced cluster, provide evidence for a role of the Fe/S cluster in catalysis, and offer an explanation of why one-electron reductive activation is observed for a reaction cycle involving 2-electron chemistry.

Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO.

BACKGROUND: Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates. RESULTS: We engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae, were expressed in C. autoethanogenum. Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (< 0.01 mM) for strains expressing Eat1. Supplementation of ethanol was investigated as potential boost for ethyl acetate production but resulted only in a 1.5-fold increase (0.3 mM ethyl acetate). Besides ethyl acetate, C. autoethanogenum expressing Atf1 could produce 4.5 mM of butyl acetate when 20 mM butanol was supplemented to the growth medium. CONCLUSIONS: This work offers for the first time a proof-of-principle that autotrophic short chain ester production from C1-carbon feedstocks is possible and offers leads on how this approach can be optimized in the future.

Methanogen Levels Are Significantly Associated with Fecal Microbiota Composition and Alpha Diversity in Healthy Adults and Irritable Bowel Syndrome Patients.

Hydrogenotrophic microbes, primarily including the three functional groups methanogens, sulfate-reducing bacteria, and reductive acetogens, use hydrogen as an energy source and play an important role in maintaining the hydrogen balance in gut ecosystems. A distorted hydrogen balance has been associated with irritable bowel syndrome (IBS). However, the role of hydrogenotrophic microbes in overall microbiota composition and function remains largely unknown. This study aims to assess the distribution and stability of hydrogenotrophic functional groups in healthy adults (HAs) and IBS patients and their association with overall microbiota composition and IBS symptoms. A two-time-point study with 4 weeks in between was performed with 27 HAs and 55 IBS patients included. Our observations revealed that methanogens showed a bimodal distribution across samples. A high-level methanogen microbiota was consistently associated with higher alpha diversity, and its composition was significantly different from that of individuals with a low-level methanogen microbiota. In general, these associations were more pronounced in IBS patients than in HAs. The differences in the copy numbers of genes indicative of total bacteria and acetogens between HAs and IBS patients and their correlations with IBS symptom severity, anxiety, depression, and quality of life (QoL) were sampling time dependent. Hydrogenotrophic functional groups did not show negative abundance correlations with each other in HAs and IBS patients. These findings suggest that methanogen levels in the gut have a pronounced association with microbiota alpha diversity and composition, and the interactions between hydrogenotrophic functional groups are complex in gut ecosystems. IMPORTANCE Hydrogenotrophic microbes play an essential role in the disposal of hydrogen and the maintenance of the hydrogen balance in gut ecosystems. Their abundances vary between individuals and have been reported to be associated with human gut disorders such as irritable bowel disease. This study confirms that methanogen levels show a bimodal distribution. Moreover, a high-level methanogen microbiota was associated with higher alpha diversity, and its composition was different from that of individuals with a low-level methanogen microbiota. These associations are more pronounced in IBS patients than in healthy subjects. In addition, associations between hydrogenotrophic microbes and IBS symptom scores vary over time, which argues for the use of longitudinal study designs. Last but not least, this study suggests that the different hydrogenotrophic microbes coexist with each other and do not necessarily compete for hydrogen in the gut. The findings in this study highlight the impact of methanogens on overall microbiota composition and function.

The Potential of Sequential Fermentations in Converting C1 Substrates to Higher-Value Products.

Today production of (bulk) chemicals and fuels almost exclusively relies on petroleum-based sources, which are connected to greenhouse gas release, fueling climate change. This increases the urgence to develop alternative bio-based technologies and processes. Gaseous and liquid C1 compounds are available at low cost and often occur as waste streams. Acetogenic bacteria can directly use C1 compounds like CO, CO2, formate or methanol anaerobically, converting them into acetate and ethanol for higher-value biotechnological products. However, these microorganisms possess strict energetic limitations, which in turn pose limitations to their potential for biotechnological applications. Moreover, efficient genetic tools for strain improvement are often missing. However, focusing on the metabolic abilities acetogens provide, they can prodigiously ease these technological disadvantages. Producing acetate and ethanol from C1 compounds can fuel via bio-based intermediates conversion into more energy-demanding, higher-value products, by deploying aerobic organisms that are able to grow with acetate/ethanol as carbon and energy source. Promising new approaches have become available combining these two fermentation steps in sequential approaches, either as separate fermentations or as integrated two-stage fermentation processes. This review aims at introducing, comparing, and evaluating the published approaches of sequential C1 fermentations, delivering a list of promising organisms for the individual fermentation steps and giving an overview of the existing broad spectrum of products based on acetate and ethanol. Understanding of these pioneering approaches allows collecting ideas for new products and may open avenues toward making full use of the technological potential of these concepts for establishment of a sustainable biotechnology.

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

BACKGROUND: The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging. RESULTS: Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L-1 butanol and 133 mg L-1 hexanol from fructose in complex medium, and 174 mg L-1 butanol and 15 mg L-1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L-1) at the expense of butanol (158 mg L-1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L-1 butanol and 393 mg L-1 hexanol. CONCLUSIONS: We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes.

Mixotrophic co-utilization of glucose and carbon monoxide boosts ethanol and butanol productivity of continuous Clostridium carboxidivorans cultures.

In this study, continuous cultivations of C.carboxidivorans to study heterotrophic and mixotrophic conversion of glucose and H2, CO2, and CO were established. Glucose fermentations at pH 6 showed a high ratio of alcohol-to-acid production of 2.79 mol mol-1. While H2 or CO2 were not utilized together with glucose, CO feeding drastically increased the combined alcohol titer to 9.1 g l-1. Specifically, CO enhanced acetate (1.9-fold) and ethanol (1.7-fold) production and triggered chain elongation to butanol (1.5-fold) production but did not change the alcohol:acid ratio. Flux balance analysis showed that CO served both as a carbon and energy source, and CO mixotrophy displayed a carbon and energy efficiency of 45 and 77%, respectively. This study expands the knowledge on physiology and metabolism of C.carboxidivorans and can serve as the starting point for rational engineering and process intensification to establish efficient production of alcohols and acids from carbon waste.