RESUMO
BACKGROUND: Acetogens, a diverse group of anaerobic autotrophic bacteria, are promising whole-cell biocatalysts that fix CO2 during their growth. However, because of energetic constraints, acetogens exhibit slow growth and the product spectrum is often limited to acetate. Enabling acetogens to form more valuable products such as volatile fatty acids during autotrophic growth is imperative for cementing their place in the future carbon neutral industry. Co-cultivation of strains with different capabilities has the potential to ease the limiting energetic constraints. The lactate-mediated co-culture of an Acetobacterium woodii mutant strain, capable of lactate production, with the Clostridium drakei SL1 type strain can produce butyrate and hexanoate. In this study, the preceding co-culture is characterized by comparison of monocultures and different co-culture approaches. RESULTS: C. drakei grew with H2 + CO2 as main carbon and energy source and thrived when further supplemented with D-lactate. Gas phase components and lactate were consumed in a mixotrophic manner with acetate and butyrate as main products and slight accumulation of hexanoate. Formate was periodically produced and eventually consumed by C. drakei. A lactate-mediated co-culture of the A. woodii [PbgaL_ldhD_NFP] strain, engineered for autotrophic lactate production, and C. drakei produced up to 4 ± 1.7 mM hexanoate and 18.5 ± 5.8 mM butyrate, quadrupling and doubling the respective titers compared to a non-lactate-mediated co-culture. Further co-cultivation experiments revealed the possible advantage of sequential co-culture over concurrent approaches, where both strains are inoculated simultaneously. Scanning electron microscopy of the strains revealed cell-to-cell contact between the co-culture partners. Finally, a combined pathway of A. woodii [PbgaL_ldhD_NFP] and C. drakei for chain-elongation with positive ATP yield is proposed. CONCLUSION: Lactate was proven to be a well-suited intermediate to combine the high gas uptake capabilities of A. woodii with the chain-elongation potential of C. drakei. The cell-to-cell contact observed here remains to be further characterized in its nature but hints towards diffusive processes being involved in the co-culture. Furthermore, the metabolic pathways involved are still speculatory for C. drakei and do not fully explain the consumption of formate while H2 + CO2 is available. This study exemplifies the potential of combining metabolically engineered and native bacterial strains in a synthetic co-culture.
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Acetobacterium , Processos Autotróficos , Clostridium , Técnicas de Cocultura , Ácidos Graxos Voláteis , Ácido Láctico , Ácido Láctico/metabolismo , Acetobacterium/metabolismo , Acetobacterium/crescimento & desenvolvimento , Acetobacterium/genética , Ácidos Graxos Voláteis/metabolismo , Clostridium/metabolismo , Clostridium/genética , Clostridium/crescimento & desenvolvimento , Dióxido de Carbono/metabolismo , Acetatos/metabolismoRESUMO
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.
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Eubacterium , Engenharia Metabólica , Eubacterium/genética , Metanol/metabolismo , Filogenia , Butiratos/metabolismoRESUMO
Lactate has various uses as industrial platform chemical, poly-lactic acid precursor or feedstock for anaerobic co-cultivations. The aim of this study was to construct and characterise Acetobacterium woodii strains capable of autotrophic lactate production. Therefore, the lctBCD genes, encoding the native Lct dehydrogenase complex, responsible for lactate consumption, were knocked out. Subsequently, a gene encoding a D-lactate dehydrogenase (LDHD) originating from Leuconostoc mesenteroides was expressed in A. woodii, either under the control of the anhydrotetracycline-inducible promoter Ptet or under the lactose-inducible promoter PbgaL. Moreover, LDHD was N-terminally fused to the oxygen-independent fluorescence-activating and absorption-shifting tag (FAST) and expressed in respective A. woodii strains. Cells that produced the LDHD fusion protein were capable of lactate production of up to 18.8 mM in autotrophic batch experiments using H2 + CO2 as energy and carbon source. Furthermore, cells showed a clear and bright fluorescence during exponential growth, as well as in the stationary phase after induction, mediated by the N-terminal FAST. Flow cytometry at the single-cell level revealed phenotypic heterogeneities for cells expressing the FAST-tagged LDHD fusion protein. This study shows that FAST provides a new reporter tool to quickly analyze gene expression over the course of growth experiments of A. woodii. Consequently, fluorescence-based reporters allow for faster and more targeted optimization of production strains.Key points â¢Autotrophic lactate production was achieved with A. woodii. â¢FAST functions as fluorescent marker protein in A. woodii. â¢Fluorescence measurements on single-cell level revealed population heterogeneity.
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Dióxido de Carbono , Ácido Láctico , Acetatos/metabolismo , Acetobacterium , Dióxido de Carbono/metabolismo , FluorescênciaRESUMO
A strictly anaerobic bacterial strain designated EA1T was isolated from an enrichment culture inoculated with biogas reactor content. Cells of strain EA1T are spore-forming rods (1-3×0.4-0.8 µm) and stain Gram-negative, albeit they possess a Gram-positive type of cell-wall ultrastructure. Growth of strain EA1T was observed at 30 and 37 °C and within a pH range of pH 5-9. The major components recovered in the fatty acid fraction were C14:0, C16:0, C16:0 DMA (dimethyl acetal) and C16:1 ω7c. Strain EA1T fermented several mono- and disaccharides. Metabolic end products from fructose were acetate, butyrate, caproate and lactate. Furthermore, ethanol, CO2 and H2 were identified as products. The genome consists of a chromosome (3.9 Mbp) with 3797 predicted protein-encoding genes and a G+C content of 51.25 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EA1T represents a novel taxon within the family Oscillospiraceae. The most closely related type strains of EA1T, based on 16S rRNA gene sequence identity, are Caproiciproducens galactitolivorans BS-1T (94.9 %), [Clostridium] leptum DSM 753T (93.8 %), [Clostridium] sporosphaeroides DSM 1294T (91.7 %) and Ruminococcus bromii ATCC 27255T (91.0 %). Further phenotypic characteristics of strain EA1T differentiate it from related, validly described bacterial species. Strain EA1T represents a novel genus and novel species within the family Oscillospiraceae. The proposed name is Caproicibacter fermentans gen. nov., sp. nov. The type strain is EA1T (DSM 107079T=JCM 33110T).
Assuntos
Reatores Biológicos/microbiologia , Caproatos/metabolismo , Clostridiales/classificação , Filogenia , Bactérias Anaeróbias/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , Clostridiales/isolamento & purificação , DNA Bacteriano/genética , Ácidos Graxos/química , Fermentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
The advantage of using acetogens such as Acetobacterium woodii as biocatalysts converting the cheap substrate and greenhouse gas carbon dioxide (CO2) into value-added chemicals comes together with the disadvantage of a low overall ATP gain due to the bioenergetics associated with the Wood-Ljungdahl pathway. Expanding the product spectrum of recombinant A. woodii strains to compounds with high ATP-demanding biosynthesis is therefore challenging. As a least invasive strategy for improved ATP generation, the exploitation of the arginine deiminase pathway (ADI) was examined under native conditions and via using heterologously expressed genes in A. woodii. Several promoters were analyzed for application of different gene expression levels in A. woodii using ß-glucuronidase assays. Heterologous expression of the ADI pathway genes from Clostridium autoethanogenum was controlled using either the constitutive pta-ack promoter from Clostridium ljungdahlii or a tightly regulated tetracycline-inducible promoter Ptet. Unlike constitutive expression, only induced expression of the ADI pathway genes led to a 36% higher maximal OD600 when using arginine (OD600 3.4) as nitrogen source and a 52% lower acetate yield per biomass compared to cells growing with yeast extract as nitrogen source (OD600 2.5). In direct comparison, a 69% higher maximal OD600 and about 60% lower acetate yield per biomass in induced to non-induced recombinant A. woodii cells was noticed when using arginine. Our data suggests the application of the ADI pathway in A. woodii for expanding the product spectrum to compounds with high ATP-demanding biosynthesis.
Assuntos
Acetobacterium/enzimologia , Acetobacterium/crescimento & desenvolvimento , Expressão Gênica , Hidrolases/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Proteínas Recombinantes/metabolismo , Acetatos/metabolismo , Acetobacterium/genética , Arginina/metabolismo , Clostridium/enzimologia , Clostridium/genética , Hidrolases/genética , Nitrogênio/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Ativação TranscricionalRESUMO
Anaerobic production of the biopolymer poly(3-hydroxybutyrate) (PHB) and the monomer 3-hydroxybutyrate (3-HB) was achieved using recombinant clostridial acetogens supplied with syn(thesis) gas as the sole carbon and energy source. 3-HB production was successfully accomplished by a new synthetic pathway containing the genes thlA (encoding thiolase A), ctfA/B (encoding CoA-transferase A/B), and bdhA (encoding (R)-3-hydroxybutyrate dehydrogenase). The respective recombinant Clostridium coskatii [p83_tcb] strain produced autotrophically 0.98 ± 0.12 mM and heterotrophically 21.7 ± 0.27 mM 3-HB. As a proof of concept, production of PHB was achieved using recombinant C. coskatii and Clostridium ljungdahlii strains expressing a novel synthetic PHB pathway containing the genes thlA (encoding thiolase A), hbd (encoding 3-hydroxybutyryl-CoA dehydrogenase), crt (encoding crotonase), phaJ (encoding (R)-enoyl-CoA hydratase), and phaEC (encoding PHA synthase). The strain C. coskatii [p83_PHB_Scaceti] synthesized heterotrophically 3.4 ± 0.29% PHB per cell dry weight (CDW) and autotrophically 1.12 ± 0.12% PHB per CDW.
Assuntos
Ácido 3-Hidroxibutírico/biossíntese , Bactérias Anaeróbias/metabolismo , Clostridium/metabolismo , Hidroxibutiratos/química , Poliésteres/química , Ácido 3-Hidroxibutírico/química , Processos Autotróficos , Bactérias Anaeróbias/química , Clostridium/química , Gases/química , Gases/metabolismo , Hidroxibutiratos/síntese química , Poliésteres/síntese químicaRESUMO
Anaerobic bacterial gas fermentation gains broad interest in various scientific, social, and industrial fields. This microbial process is carried out by a specific group of bacterial strains called acetogens. All these strains employ the Wood-Ljungdahl pathway but they belong to different taxonomic groups. Here we provide an overview of the metabolism of acetogens and naturally occurring products. Characteristics of 61 strains were summarized and selected acetogens described in detail. Acetobacterium woodii, Clostridium ljungdahlii, and Moorella thermoacetica serve as model organisms. Results of approaches such as genome-scale modeling, proteomics, and transcriptomics are discussed. Metabolic engineering of acetogens can be used to expand the product portfolio to platform chemicals and to study different aspects of cell physiology. Moreover, the fermentation of gases requires specific reactor configurations and the development of the respective technology, which can be used for an industrial application. Even though the overall process will have a positive effect on climate, since waste and greenhouse gases could be converted into commodity chemicals, some legislative barriers exist, which hamper successful exploitation of this technology.
Assuntos
Bactérias Anaeróbias/metabolismo , Produtos Biológicos/metabolismo , Dióxido de Carbono/metabolismo , Gases/metabolismo , Hidrogênio/metabolismo , Anaerobiose , Fermentação , Perfilação da Expressão Gênica , Redes e Vias Metabólicas , Metaboloma , Proteoma/análiseRESUMO
Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_actthlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters PthlA and Ppta-ack. Batch fermentations using A. woodii [pMTL84151_actthlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high Km value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_actthlA] was increased from 1.2mgL(-1)h(-1) in bottle fermentation to 26.4mgL(-1)h(-1) in continuous gas fermentation.
Assuntos
Acetatos/metabolismo , Acetobacterium/fisiologia , Acetona/metabolismo , Vias Biossintéticas/fisiologia , Dióxido de Carbono/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/fisiologia , Acetona/isolamento & purificação , Melhoramento Genético/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Biofilm-based reactors are effectively used for wastewater treatment but are not common in biogas production. This study investigated biofilm dynamics on biofilm carriers incubated in batch biogas reactors at high and low organic loading rates for sludge from meat industry dissolved air flotation units. Biofilm formation and dynamics were studied using various microscopic techniques. Resulting micrographs were analysed for total cell numbers, thickness of biofilms, biofilm-covered surface area, and the area covered by extracellular polymeric substances (EPS). Cell numbers within biofilms (10(11) cells ml(-1)) were up to one order of magnitude higher compared to the numbers of cells in the fluid reactor content. Further, biofilm formation and structure mainly correlated with the numbers of microorganisms present in the fluid reactor content and the organic loading. At high organic loading (45 kg VS m(-3)), the thickness of the continuous biofilm layer ranged from 5 to 160 µm with an average of 51 µm and a median of 26 µm. Conversely, at lower organic loading (15 kg VS m(-3)), only microcolonies were detectable. Those microcolonies increased in their frequency of occurrence during ongoing fermentation. Independently from the organic loading rate, biofilms were embedded completely in EPS within seven days. The maturation and maintenance of biofilms changed during the batch fermentation due to decreasing substrate availability. Concomitant, detachment of microorganisms within biofilms was observed simultaneously with the decrease of biogas formation. This study demonstrates that biofilms of high cell densities can enhance digestion of organic waste and have positive effects on biogas production.
Assuntos
Archaea/metabolismo , Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Metano/biossíntese , Consórcios Microbianos/fisiologia , Anaerobiose , Archaea/ultraestrutura , Bactérias/ultraestrutura , Carga Bacteriana , Técnicas de Cultura Celular por Lotes , Biocombustíveis , Reatores Biológicos , Fermentação , Produtos da Carne , Pressão , Temperatura , ResíduosRESUMO
We report on the closed genome sequences of the acetogen Blautia hydrogenotrophica S5a33T (DSM 10507T) and of Blautia coccoides CLC-1T (DSM 935T). The B. hydrogenotrophica S5a33T genome harbors a chromosome (3,590,609 bp) and a plasmid (7,176 bp). The B. coccoides CLC-1T genome consists of a single chromosome (6,097,890 bp).
RESUMO
The establishment of sustainable processes for the production of commodity chemicals is one of today's central challenges for biotechnological industries. The chemo-autotrophic fixation of CO2 and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium A. woodii WP1 as the feeder strain for growth and the recombinant production of P. aeruginosa PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain P. putida KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of P. putida in cell-containing A. woodii culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360-400 mg/L for both cell-free and cell-containing A. woodii spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by P. putida KT2440.
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Acetogenic bacteria employing the Wood-Ljungdahl pathway can be used as biocatalysts in syngas fermentation for the production ofbiofuels such as ethanol or butanol as well as biocommodities such as acetate, lactate, butyrate, 2,3 butanediol, and acetone. The potential of such processes can be projected by the global syngas output, which was 70,817 megawatts thermal in 2010 and is expected to increase up to 72% in 2016. To date, different acetogens are used as commercial production strains for industrial syngas fermentations in pilot or demonstration plants (Coskata, INEOS Bio, LanzaTech) and first commercial units are expected to launch operation in the near future (INEOS Bio, LanzaTech). Considerations on potential yields are quite promising for fermentative production. New methods for metabolic engineering were established to construct novel recombinant acetogenic biocatalysts. Synthetic biology will certainly play a major role in constructing strains for commercial operations. This way, a cheap and abundant carbon source most probably replace, processes based on crude oil or sugar in the near future.
Assuntos
Bactérias/metabolismo , Biocombustíveis , Acetona/metabolismo , Bactérias/química , Biotecnologia , Dióxido de Carbono/metabolismo , Monóxido de Carbono/metabolismo , Fermentação , Hidrogênio/metabolismo , Microbiologia IndustrialRESUMO
Syngas fermentation processes with acetogens represent a promising process for the reduction of CO2 emissions alongside bulk chemical production. However, to fully realize this potential the thermodynamic limits of acetogens need to be considered when designing a fermentation process. An adjustable supply of H2 as electron donor plays a key role in autotrophic product formation. In this study an anaerobic laboratory scale continuously stirred tank reactor was equipped with an All-in-One electrode allowing for in-situ H2 generation via electrolysis. Furthermore, this system was coupled to online lactate measurements to control the co-culture of a recombinant lactate-producing Acetobacterium woodii strain and a lactate-consuming Clostridium drakei strain to produce caproate. When C. drakei was grown in batch cultivations with lactate as substrate, 1.6 g·L-1 caproate were produced. Furthermore, lactate production of the A. woodii mutant strain could manually be stopped and reinitiated by controlling the electrolysis. Applying this automated process control, lactate production of the A. woodii mutant strain could be halted to achieve a steady lactate concentration. In a co-culture experiment with the A. woodii mutant strain and the C. drakei strain, the automated process control was able to dynamically react to changing lactate concentrations and adjust H2 formation respectively. This study confirms the potential of C. drakei as medium chain fatty acid producer in a lactate-mediated, autotrophic co-cultivation with an engineered A. woodii strain. Moreover, the monitoring and control strategy presented in this study reinforces the case for autotrophically produced lactate as a transfer metabolite in defined co-cultivations for value-added chemical production.
RESUMO
Acetogenic bacteria are of high interest for biotechnological applications as industrial platform organisms, however, acetogenic strains from the genus Terrisporobacter have hitherto been neglected. To date, three published type strains of the genus Terrisporobacter are only covered by draft genome sequences, and the genes and pathway responsible for acetogenesis have not been analyzed. Here, we report complete genome sequences of the bacterial type strains Terrisporobacter petrolearius JCM 19845T, Terrisporobacter mayombei DSM 6539T and Terrisporobacter glycolicus DSM 1288T. Functional annotation, KEGG pathway module reconstructions and screening for virulence factors were performed. Various species-specific vitamin, cofactor and amino acid auxotrophies were identified and a model for acetogenesis of Terrisporobacter was constructed. The complete genomes harbored a gene cluster for the reductive proline-dependent branch of the Stickland reaction located on an approximately 21 kb plasmid, which is exclusively found in the Terrisporobacter genus. Phylogenomic analysis of available Terrisporobacter genomes suggested a reclassification of most isolates as T. glycolicus into T. petrolearius.
Assuntos
Clostridium , Genoma , Filogenia , Plasmídeos , Clostridium/genética , Análise de Sequência de DNA , RNA Ribossômico 16S/genética , DNA Bacteriano/genética , Técnicas de Tipagem BacterianaRESUMO
Acetobacterium woodii is known to produce mainly acetate from CO2 and H2, but the production of higher value chemicals is desired for the bioeconomy. Using chain-elongating bacteria, synthetic co-cultures have the potential to produce longer-chained products such as caproic acid. In this study, we present first results for a successful autotrophic co-cultivation of A. woodii mutants and a Clostridium drakei wild-type strain in a stirred-tank bioreactor for the production of caproic acid from CO2 and H2 via the intermediate lactic acid. For autotrophic lactate production, a recombinant A. woodii strain with a deleted Lct-dehydrogenase complex, which is encoded by the lctBCD genes, and an inserted D-lactate dehydrogenase (LdhD) originating from Leuconostoc mesenteroides, was used. Hydrogen for the process was supplied using an All-in-One electrode for in situ water electrolysis. Lactate concentrations as high as 0.5 g L-1 were achieved with the AiO-electrode, whereas 8.1 g L-1 lactate were produced with direct H2 sparging in a stirred-tank bioreactor. Hydrogen limitation was identified in the AiO process. However, with cathode surface area enlargement or numbering-up of the electrode and on-demand hydrogen generation, this process has great potential for a true carbon-negative production of value chemicals from CO2.
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BACKGROUND: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. RESULTS: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth. CONCLUSIONS: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.
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Two different isobutanol synthesis pathways were cloned into and expressed in the two model acetogenic bacteria Acetobacterium woodii and Clostridium ljungdahlii. A. woodii is specialized on using CO2 + H2 gas mixtures for growth and depends on sodium ions for ATP generation by a respective ATPase and Rnf system. On the other hand, C. ljungdahlii grows well on syngas (CO + H2 + CO2 mixture) and depends on protons for energy conservation. The first pathway consisted of ketoisovalerate ferredoxin oxidoreductase (Kor) from Clostridium thermocellum and bifunctional aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum. Three different kor gene clusters are annotated in C. thermocellum and were all tested. Only in recombinant A. woodii strains, traces of isobutanol could be detected. Additional feeding of ketoisovalerate increased isobutanol production to 2.9 mM under heterotrophic conditions using kor3 and to 1.8 mM under autotrophic conditions using kor2. In C. ljungdahlii, isobutanol could only be detected upon additional ketoisovalerate feeding under autotrophic conditions. kor3 proved to be the best suited gene cluster. The second pathway consisted of ketoisovalerate decarboxylase from Lactococcus lactis and alcohol dehydrogenase from Corynebacterium glutamicum. For increasing the carbon flux to ketoisovalerate, genes encoding ketol-acid reductoisomerase, dihydroxy-acid dehydratase, and acetolactate synthase from C. ljungdahlii were subcloned downstream of adhA. Under heterotrophic conditions, A. woodii produced 0.2 mM isobutanol and 0.4 mM upon additional ketoisovalerate feeding. Under autotrophic conditions, no isobutanol formation could be detected. Only upon additional ketoisovalerate feeding, recombinant A. woodii produced 1.5 mM isobutanol. With C. ljungdahlii, no isobutanol was formed under heterotrophic conditions and only 0.1 mM under autotrophic conditions. Additional feeding of ketoisovalerate increased these values to 1.5 mM and 0.6 mM, respectively. A further increase to 2.4 mM and 1 mM, respectively, could be achieved upon inactivation of the ilvE gene in the recombinant C. ljungdahlii strain. Engineering the coenzyme specificity of IlvC of C. ljungdahlii from NADPH to NADH did not result in improved isobutanol production.
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Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO2 footprint by converting industrial emissions containing CO2 , CO and H2 into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO2 + H2 , CO and CO + CO2 + H2 (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood-Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD+ provider for CO2 + H2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fdred ) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fdred formation when CO is used as substrate, whereas Fdred is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO2 + H2 . Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.
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Dióxido de Carbono , Elétrons , Monóxido de Carbono , Clostridium , HumanosRESUMO
Caproiciproducens galactitolivorans BS-1T is an anaerobic bacterium that produces acetate, butyrate, and caproate. The genome has a size of 2.57 Mbp and harbors 2,439 predicted protein-coding genes.
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Biogas production is a biotechnological process realized by complex bacterial, archaeal and likely fungal communities. Their composition was assessed in nine full-scale biogas plants with distinctly differing feedstock input and process parameters. This study investigated the actually active microbial community members by using a comprehensive sequencing approach based on ribosomal 16S and 28S rRNA fragments. The prevailing taxonomical units of each respective community were subsequently linked to process parameters. Ribosomal rRNA of bacteria, archaea and fungi, respectively, showed different compositions with respect to process parameters and supplied feedstocks: (i) bacterial communities were affected by the key factors temperature and ammonium concentration; (ii) composition of archaea was mainly related to process temperature; and (iii) relative abundance of fungi was linked to feedstocks supplied to the digesters. Anaerobic digesters with a high methane yield showed remarkably similar bacterial communities regarding identified taxonomic families. Although archaeal communities differed strongly on genus level from each other, the respective digesters still showed high methane yields. Functional redundancy of the archaeal communities may explain this effect. 28S rRNA sequences of fungi in all nine full-scale anaerobic digesters were primarily classified as facultative anaerobic Ascomycota and Basidiomycota. Since the presence of ribosomal 28S rRNA indicates that fungi may be active in the biogas digesters, further research should be carried out to examine to which extent they are important players in anaerobic digestion processes.