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Clostridium ljungdahlii represents a microbial production platform based on syngas.

ABSTRACT: Clostridium ljungdahlii is an anaerobic homoacetogen, able to ferment sugars, other organic compounds, or CO(2)/H(2) and synthesis gas (CO/H(2)). The latter feature makes it an interesting microbe for the biotech industry, as important bulk chemicals and proteins can be produced at the expense of CO(2), thus combining industrial needs with sustained reduction of CO and CO(2) in the atmosphere. Sequencing the complete genome of C. ljungdahlii revealed that it comprises 4,630,065 bp and is one of the largest clostridial genomes known to date. Experimental data and in silico comparisons revealed a third mode of anaerobic homoacetogenic metabolism. Unlike other organisms such as Moorella thermoacetica or Acetobacterium woodii, neither cytochromes nor sodium ions are involved in energy generation. Instead, an Rnf system is present, by which proton translocation can be performed. An electroporation procedure has been developed to transform the organism with plasmids bearing heterologous genes for butanol production. Successful expression of these genes could be demonstrated, leading to formation of the biofuel. Thus, C. ljungdahlii can be used as a unique microbial production platform based on synthesis gas and carbon dioxide/hydrogen mixtures.

SUBMITTER: Kopke M

PROVIDER: S-EPMC2919952 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Clostridium ljungdahlii represents a microbial production platform based on syngas.

Köpke Michael M Held Claudia C Hujer Sandra S Liesegang Heiko H Wiezer Arnim A Wollherr Antje A Ehrenreich Armin A Liebl Wolfgang W Gottschalk Gerhard G Dürre Peter P

Proceedings of the National Academy of Sciences of the United States of America 20100702 29

Clostridium ljungdahlii is an anaerobic homoacetogen, able to ferment sugars, other organic compounds, or CO(2)/H(2) and synthesis gas (CO/H(2)). The latter feature makes it an interesting microbe for the biotech industry, as important bulk chemicals and proteins can be produced at the expense of CO(2), thus combining industrial needs with sustained reduction of CO and CO(2) in the atmosphere. Sequencing the complete genome of C. ljungdahlii revealed that it comprises 4,630,065 bp and is one of ...[more]

PMID: 20616070

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Project description:BackgroundClostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.ResultsWe have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.ConclusionsThe developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

Project description:There is a growing interest in the use of microbial fermentation for the generation of high-demand, high-purity chemicals using cheap feedstocks in an environmentally friendly manner. One example explored here is the production of isoprene (C5H8), a hemiterpene, which is primarily polymerized to polyisoprene in synthetic rubber in tires but which can also be converted to C10 and C15 biofuels. The strictly anaerobic, acetogenic bacterium Clostridium ljungdahlii, used in all of the work described here, is capable of glycolysis using the Embden-Meyerhof-Parnas pathway and of carbon fixation using the Wood-Ljungdahl pathway. Clostridium-Escherichia coli shuttle plasmids, each bearing either 2 or 3 different heterologous genes of the eukaryotic mevalonic acid (MVA) pathway or eukaryotic isopentenyl pyrophosphate isomerase (Idi) and isoprene synthase (IspS), were constructed and electroporated into C. ljungdahlii These plasmids, one or two of which were introduced into the host cells, enabled the synthesis of mevalonate and of isoprene from fructose and from syngas (H2, CO2, and CO) and the conversion of mevalonate to isoprene. All of the heterologous enzymes of the MVA pathway, as well as Idi and IspS, were shown to be synthesized at high levels in C. ljungdahlii, as demonstrated by Western blotting, and were enzymatically active, as demonstrated by in vivo product synthesis. The quantities of mevalonate and isoprene produced here are far below what would be required of a commercial production strain. However, proposals are made that could enable a substantial increase in the mass yield of product formation.IMPORTANCE This study demonstrates the ability to synthesize a heterologous metabolic pathway in C. ljungdahlii, an organism capable of metabolizing either simple sugars or syngas or both together (mixotrophy). Syngas, an inexpensive source of carbon and reducing equivalents, is produced as a major component of some industrial waste gas, and it can be generated by gasification of cellulosic biowaste and of municipal solid waste. Its conversion to useful products therefore offers potential cost and environmental benefits. The ability of C. ljungdahlii to grow mixotrophically also enables the recapture, should there be sufficient reducing equivalents available, of the CO2 released upon glycolysis, potentially increasing the mass yield of product formation. Isoprene is the simplest of the terpenoids, and so the demonstration of its production is a first step toward the synthesis of higher-value products of the terpenoid pathway.

Project description:BackgroundThe production of ethanol through the biochemical conversion of syngas, a mixture of H2, CO and CO2, has been typically studied using pure cultures. However, mixed microbial consortia may offer a series of benefits such as higher resilience and adaptive capacity, and non-sterile operation, all of which contribute to reducing the utility consumption when compared to pure culture-based processes. This work focuses on the study of strategies for the enrichment of mixed microbial consortia with high ethanologenic potential, investigating the effect of the operational conditions (pH and yeast extract addition) on both the ethanol yield and evolution of the microbial community along the enrichment process. The pH was selected as the main driver of the enrichment as it was expected to be a crucial parameter for the selection of carboxydotrophic bacteria with high ethanologenic potential. Additionally, a thermodynamic analysis of the network of biochemical reactions carried out by syngas-converting microbial consortia was performed and the potential of using thermodynamics as a basis for the selection of operational parameters favoring a specific microbial activity was evaluated.ResultsAll enriched consortia were dominated by the genus Clostridium with variable microbial diversity and species composition as a function of the enrichment conditions. The ethanologenic potential of the enriched consortia was observed to increase as the initial pH was lowered, achieving an ethanol yield of 59.2 ± 0.2% of the theoretical maximum in the enrichment at pH 5. On the other hand, yeast extract addition did not affect the ethanol yield, but triggered the production of medium-chain fatty acids and alcohols. The thermodynamic analysis of the occurring biochemical reactions allowed a qualitative prediction of the activity of microbial consortia, thus enabling a more rational design of the enrichment strategies targeting specific activities. Using this approach, an improvement of 22.5% over the maximum ethanol yield previously obtained was achieved, reaching an ethanol yield of 72.4 ± 2.1% of the theoretical maximum by increasing the initial acetate concentration in the fermentation broth.ConclusionsThis study demonstrated high product selectivity towards ethanol using mixed microbial consortia. The thermodynamic analysis carried out proved to be a valuable tool for interpreting the metabolic network of microbial consortia-driven processes and designing microbial-enrichment strategies targeting specific biotransformations.

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Clostridium ljungdahlii represents a microbial production platform based on syngas.

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Clostridium ljungdahlii represents a microbial production platform based on syngas.

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