Chromosomal integration of the pSOL1 megaplasmid of Clostridium acetobutylicum for continuous and stable advanced biofuels production.

Biofuel production by Clostridium acetobutylicum is compromised by strain degeneration due to loss of its pSOL1 megaplasmid. Here we used engineering biology to stably integrate pSOL1 into the chromosome together with a synthetic isopropanol pathway. In a membrane bioreactor continuously fed with glucose mineral medium, the final strain produced advanced biofuels, n-butanol and isopropanol, at high yield (0.31 g g-1), titre (15.4 g l-1) and productivity (15.5 g l-1 h-1) without degeneration.

Improvement of the Genome Editing Tools Based on 5FC/5FU Counter Selection in Clostridium acetobutylicum.

Several genetic tools have been developed for genome engineering in Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. In our group, a method based on the integration, by single crossing over, of a suicide plasmid (pCat-upp) followed by selection for the second crossing over using a counter-selectable marker (the upp gene and 5FU resistance) was recently developed for genome editing in C. acetobutylicum. This method allows genome modification without leaving any marker or scar in a strain of C. acetobutylicum that is ∆upp. Unfortunately, 5FU has strong mutagenic properties, inducing mutations in the strain's genome. After numerous applications of the pCat-upp/5FU system for genome modification in C. acetobutylicum, the CAB1060 mutant strain became entirely resistant to 5FU in the presence of the upp gene, resulting in failure when selecting on 5FU for the second crossing over. It was found that the potential repressor of the pyrimidine operon, PyrR, was mutated at position A115, leading to the 5FU resistance of the strain. To fix this problem, we created a corrective replicative plasmid expressing the pyrR gene, which was shown to restore the 5FU sensitivity of the strain. Furthermore, in order to avoid the occurrence of the problem observed with the CAB1060 strain, a preventive suicide plasmid, pCat-upp-pyrR*, was also developed, featuring the introduction of a synthetic codon-optimized pyrR gene, which was referred to as pyrR* with low nucleotide sequence homology to pyrR. Finally, to minimize the mutagenic effect of 5FU, we also improved the pCat-upp/5FU system by reducing the concentration of 5FU from 1 mM to 5 µM using a defined synthetic medium. The optimized system/conditions were used to successfully replace the ldh gene by the sadh-hydG operon to convert acetone into isopropanol.

Exploitation of a Type 1 Toxin-Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen Eubacterium limosum.

Targeted mutations in the anaerobic methylotroph Eubacterium limosum have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from Eubacterium callanderi was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in Eubacterium limosum B2. The genes targeted in this study were those encoding the histidine biosynthesis gene hisI, the methanol methyltransferase and corrinoid protein mtaA and mtaC, and mtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within hisI brought about the expected histidine auxotrophy, and deletions of mtaA and mtaC both abolished autotrophic growth on methanol. Deletion of mtcB was shown to abolish the growth of E. limosum on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of E. limosum.

Genome Sequence of Eubacterium limosum B2 and Evolution for Growth on a Mineral Medium with Methanol and CO2 as Sole Carbon Sources.

Eubacterium limosum is an acetogen that can produce butyrate along with acetate as the main fermentation end-product from methanol, a promising C1 feedstock. Although physiological characterization of E. limosum B2 during methylotrophy was previously performed, the strain was cultured in a semi-defined medium, limiting the scope for further metabolic insights. Here, we sequenced the complete genome of the native strain and performed adaptive laboratory evolution to sustain growth on methanol mineral medium. The evolved population significantly improved its maximal growth rate by 3.45-fold. Furthermore, three clones from the evolved population were isolated on methanol mineral medium without cysteine by the addition of sodium thiosulfate. To identify mutations related to growth improvement, the whole genomes of wild-type E. limosum B2, the 10th, 25th, 50th, and 75th generations, and the three clones were sequenced. We explored the total proteomes of the native and the best evolved clone (n°2) and noticed significant differences in proteins involved in gluconeogenesis, anaplerotic reactions, and sulphate metabolism. Furthermore, a homologous recombination was found in subunit S of the type I restriction-modification system between both strains, changing the structure of the subunit, its sequence recognition and the methylome of the evolved clone. Taken together, the genomic, proteomic and methylomic data suggest a possible epigenetic mechanism of metabolic regulation.

Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum.

Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP+ reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD+ reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.

Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology-several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.

Physicochemical and metabolic constraints for thermodynamics-based stoichiometric modelling under mesophilic growth conditions.

Metabolic engineering in the post-genomic era is characterised by the development of new methods for metabolomics and fluxomics, supported by the integration of genetic engineering tools and mathematical modelling. Particularly, constraint-based stoichiometric models have been widely studied: (i) flux balance analysis (FBA) (in silico), and (ii) metabolic flux analysis (MFA) (in vivo). Recent studies have enabled the incorporation of thermodynamics and metabolomics data to improve the predictive capabilities of these approaches. However, an in-depth comparison and evaluation of these methods is lacking. This study presents a thorough analysis of two different in silico methods tested against experimental data (metabolomics and 13C-MFA) for the mesophile Escherichia coli. In particular, a modified version of the recently published matTFA toolbox was created, providing a broader range of physicochemical parameters. Validating against experimental data allowed the determination of the best physicochemical parameters to perform the TFA (Thermodynamics-based Flux Analysis). An analysis of flux pattern changes in the central carbon metabolism between 13C-MFA and TFA highlighted the limited capabilities of both approaches for elucidating the anaplerotic fluxes. In addition, a method based on centrality measures was suggested to identify important metabolites that (if quantified) would allow to further constrain the TFA. Finally, this study emphasised the need for standardisation in the fluxomics community: novel approaches are frequently released but a thorough comparison with currently accepted methods is not always performed.

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

Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.

Trends in Systems Biology for the Analysis and Engineering of Clostridium acetobutylicum Metabolism.

Clostridium acetobutylicum has received renewed interest worldwide as a promising producer of biofuels and bulk chemicals such as n-butanol, 1,3-propanediol, 1,3-butanediol, isopropanol, and butyrate. To develop commercial processes for the production of bulk chemicals via a metabolic engineering approach it is necessary to better characterize both the primary metabolism and metabolic regulation of C. acetobutylicum. Here, we review the history of the development of omics studies of C. acetobutylicum, summarize the recent application of quantitative/integrated omics approaches to the physiological analysis and metabolic engineering of this bacterium, and provide directions for future studies to address current challenges.

Author Correction: The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l-Homoserine to 4-Hydroxy-2-ketobutyrate in a Glycerol-Independent Pathway of 1,3-Propanediol Production from Glucose.

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4-hydroxy-2-ketobutyrate, a key reaction in producing 1,3-propanediol (1,3-PDO) from glucose in a novel glycerol-independent metabolic pathway. To this end, a computation-based rational approach is used to change the substrate specificity of SerC from l-phosphoserine to l-homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerCR42W/R77W , is successfully improved by 4.2-fold in comparison to the wild type when l-homoserine is used as the substrate, while its activity toward the natural substrate l-phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3-PDO production, the "homoserine to 1,3-PDO" pathway is constructed in E. coli by coexpression of SerCR42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L-1 1,3-PDO in fed-batch fermentation, which is 13-fold higher than the wild-type strain and represents an important step forward to realize the promise of the glycerol-independent synthetic pathway for 1,3-PDO production from glucose.

An efficient method for markerless mutant generation by allelic exchange in Clostridium acetobutylicum and Clostridium saccharobutylicum using suicide vectors.

BACKGROUND: Clostridium acetobutylicum and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of their genomes has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals. RESULTS: The method used in this paper to knock-out, knock-in, or edit genes in C. acetobutylicum and C. saccharobutylicum combines an improved electroporation method with the use of (i) restrictionless Δupp (which encodes uracil phosphoribosyl-transferase) strains and (ii) very small suicide vectors containing a markerless deletion/insertion cassette, an antibiotic resistance gene (for the selection of the first crossing-over) and upp (from C. acetobutylicum) for subsequent use as a counterselectable marker with the aid of 5-fluorouracil (5-FU) to promote the second crossing-over. This method was successfully used to both delete genes and edit genes in both C. acetobutylicum and C. saccharobutylicum. Among the edited genes, a mutation in the spo0A gene that abolished solvent formation in C. acetobutylicum was introduced in C. saccharobutylicum and shown to produce the same effect. CONCLUSIONS: The method described in this study will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.

Reviving the Weizmann process for commercial n-butanol production.

Developing a commercial process for the biological production of n-butanol is challenging as it needs to combine high titer, yield, and productivities. Here we engineer Clostridium acetobutylicum to stably and continuously produce n-butanol on a mineral media with glucose as sole carbon source. We further design a continuous process for fermentation of high concentration glucose syrup using in situ extraction of alcohols by distillation under low pressure and high cell density cultures to increase the titer, yield, and productivity of n-butanol production to the level of 550 g/L, 0.35 g/g, and 14 g/L/hr, respectively. This process provides a mean to produce n-butanol at performance levels comparable to that of corn wet milling ethanol plants using yeast as a biocatalyst. It may hold the potential to be scaled-up at pilot and industrial levels for the commercial production of n-butanol.

Roles of the F-domain in [FeFe] hydrogenase.

The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_ΔDA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H+/H2 potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.

Metabolic flexibility of a butyrate pathway mutant of Clostridium acetobutylicum.

Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg-1) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.

Mechanism of O2 diffusion and reduction in FeFe hydrogenases.

FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

Cap0037, a Novel Global Regulator of Clostridium acetobutylicum Metabolism.

An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2 A CA_P0037::int (189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int (189/190s) mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032), or coding for the main flavodoxin (CA_C0587) were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037::int (189/190s) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach. IMPORTANCE: Clostridium acetobutylicum is well-known for its ability to produce solvents, especially n-butanol. Understanding the regulatory network of C. acetobutylicum will be crucial for further engineering to obtain a strain capable of producing n-butanol at high yield and selectivity. This study has discovered that the Cap0037 protein is a novel regulator of C. acetobutylicum that drastically affects metabolism under both acidogenic and alcohologenic fermentation conditions. This is pioneering work for further determining the regulatory mechanism of Cap0037 in C. acetobutylicum and studying the role of proteins homologous to Cap0037 in other members of the phylum Firmicutes.

Reactivity of the Excited States of the H-Cluster of FeFe Hydrogenases.

FeFe hydrogenases catalyze H2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe2(CO)3(CN)2(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the use of hydrogenase for the photoproduction of H2. Here, we explore in great detail the reactivity of the excited states of the H-cluster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This strategy could be used for probing other aspects of the photoreactivity of the H-cluster.

Elucidation of the roles of adhE1 and adhE2 in the primary metabolism of Clostridium acetobutylicum by combining in-frame gene deletion and a quantitative system-scale approach.

BACKGROUND: Clostridium acetobutylicum possesses two homologous adhE genes, adhE1 and adhE2, which have been proposed to be responsible for butanol production in solventogenic and alcohologenic cultures, respectively. To investigate their contributions in detail, in-frame deletion mutants of each gene were constructed and subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic, and alcohologenic chemostat cultures. RESULTS: Under solventogenesis, compared to the control strain, only ΔadhE1 mutant exhibited significant changes showing decreased butanol production and transcriptional expression changes in numerous genes. In particular, adhE2 was over expressed (126-fold); thus, AdhE2 can partially replace AdhE1 for butanol production (more than 30 % of the in vivo butanol flux) under solventogenesis. Under alcohologenesis, only ΔadhE2 mutant exhibited striking changes in gene expression and metabolic fluxes, and butanol production was completely lost. Therefore, it was demonstrated that AdhE2 is essential for butanol production and thus metabolic fluxes were redirected toward butyrate formation. Under acidogenesis, metabolic fluxes were not significantly changed in both mutants except the complete loss of butanol formation in ΔadhE2, but numerous changes in gene expression were observed. Furthermore, most of the significantly up- or down-regulated genes under this condition showed the same pattern of change in both mutants. CONCLUSIONS: This quantitative system-scale analysis confirms the proposed roles of AdhE1 and AdhE2 in butanol formation that AdhE1 is the key enzyme under solventogenesis, whereas AdhE2 is the key enzyme for butanol formation under acidogenesis and alcohologenesis. Our study also highlights the metabolic flexibility of C. acetobutylicum to genetic alterations of its primary metabolism.