Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes.

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.

Effects of orphan histidine kinases on clostridial sporulation progression and metabolism.

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.

Active-Site Controlled, Jahn-Teller Enabled Regioselectivity in Reductive S-C Bond Cleavage of S-Adenosylmethionine in Radical SAM Enzymes.

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe-4S]+ to SAM, generating an R3S0 radical that undergoes regioselective homolytic reductive cleavage of the S-C5' bond to generate the 5'-dAdo· radical. However, cryogenic photoinduced S-C bond cleavage has regioselectively yielded either 5'-dAdo· or ·CH3, and indeed, each of the three SAM S-C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S-C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S-C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5'-dAdo·, and in another it forms ·CH3. The identity of the cleaved S-C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe-4S] cluster. We have recognized the reduced-SAM R3S0 radical is a (2E) state with its antibonding unpaired electron in an orbital doublet, which renders R3S0 Jahn-Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S-C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S-C5' bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S-C5' for cleavage. This motion also can explain how 5'-dAdo· subsequently forms the organometallic intermediate Ω.

Ferredoxin-linked flavoenzyme defines a family of pyridine nucleotide-independent thioredoxin reductases.

Ferredoxin-dependent thioredoxin reductase was identified 35 y ago in the fermentative bacterium Clostridium pasteurianum [Hammel KE, Cornwell KL, Buchanan BB (1983) Proc Natl Acad Sci USA 80:3681-3685]. The enzyme, a flavoprotein, was strictly dependent on ferredoxin as reductant and was inactive with either NADPH or NADH. This early work has not been further pursued. We have recently reinvestigated the problem and confirmed that the enzyme, here designated ferredoxin-dependent flavin thioredoxin reductase (FFTR), is a flavoprotein. The enzyme differs from ferredoxin-thioredoxin reductase (FTR), which has a signature [4Fe-4S] cluster, but shows structural similarities to NADP-dependent thioredoxin reductase (NTR). Comparative amino acid sequence analysis showed that FFTR is present in a number of clostridial species, some of which lack both FTR and an archetypal NTR. We have isolated, crystallized, and determined the structural properties of FFTR from a member of this group, Clostridium acetobutylicum, both alone and in complex with Trx. The structures showed an elongated FFTR homodimer, each monomer comprising two Rossmann domains and a noncovalently bound FAD cofactor that exposes the isoalloxazine ring to the solvent. The FFTR structures revealed an alternative domain organization compared with NTR that enables the enzyme to accommodate Fdx rather than NADPH. The results suggest that FFTR exists in a range of conformations with varying degrees of domain separation in solution and that the stacking between the two redox-active groups for the transfer of reducing equivalents results in a profound structural reorganization. A mechanism in accord with the findings is proposed.

A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792.

Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.

Generating dihydrogen by tethering an [FeFe]hydrogenase via a molecular wire to the A1A/A1B sites of photosystem I.

Photosystem I complexes from the menB deletion mutant of Synechocystis sp. PCC 6803 were previously wired to a Pt nanoparticle via a molecular wire consisting of 15-(3-methyl-1,4-naphthoquinone-2-yl)]pentadecyl sulfide. In the presence of a sacrificial electron donor and an electron transport mediator, the PS I-NQ(CH2)15S-Pt nanoconstruct generated dihydrogen at a rate of 44.3 µmol of H2 mg Chl-1 h-1 during illumination at pH 8.3. The menB deletion strain contains an interruption in the biosynthetic pathway of phylloquinone, which results in the presence of a displaceable plastoquinone-9 in the A1A/A1B sites. The synthesized quinone contains a headgroup identical to the native phylloquinone along with a 15-carbon long tail that is terminated in a thiol. The thiol on the molecular wire is used to bind the Pt nanoparticle. In this short communication, we replaced the Pt nanoparticle with an [FeFe]H2ase variant from Clostridium acetobutylicum that contains an exposed iron on the distal [4Fe-4S] cluster afforded by mutating the surface exposed Cys97 residue to Gly. The thiol on the molecular wire is then used to coordinate the corner iron atom of the iron-sulfur cluster. When all three components are combined and illuminated in the presence of a sacrificial electron donor and an electron transport mediator, the PS I-NQ(CH2)15S-[FeFe]H2ase nanoconstruct generated dihydrogen at a rate of 50.3 ± 9.96 µmol of H2 mg Chl-1 h-1 during illumination at pH 8.3. This successful in vitro experiment sets the stage for assembling a PS I-NQ(CH2)15S-[FeFe]H2ase nanoconstruct in vivo in the menB mutant of Synechocystis sp. PCC 6803.

Biochemical characterization of an esterase from Clostridium acetobutylicum with novel GYSMG pentapeptide motif at the catalytic domain.

Gene CA_C0816 codes for a serine hydrolase protein from Clostridium acetobutylicum (ATCC 824) a member of hormone-sensitive lipase of lipolytic family IV. This gene was overexpressed in E. coli strain BL21and purified using Ni2+-NTA affinity chromatography. Size exclusion chromatography revealed that the protein is a dimer in solution. Optimum pH and temperature for recombinant Clostridium acetobutylicum esterase (Ca-Est) were found to be 7.0 and 60 °C, respectively. This enzyme exhibited high preference for p-nitrophenyl butyrate. KM and kcat/KM of the enzyme were 24.90 µM and 25.13 s-1 µM-1, respectively. Sequence analysis of Ca-Est predicts the presence of catalytic amino acids Ser 89, His 224, and Glu 196, presence of novel GYSMG conserved sequence (instead of GDSAG and GTSAG motif), and undescribed variation of HGSG motif. Site-directed mutagenesis confirmed that Ser 89 and His 224 play a major role in catalysis. This study reports that Ca-Est is hormone-sensitive lipase with novel GYSMG pentapeptide motif at a catalytic domain.

Genetic sensor-regulators functional in Clostridia.

This study addressed the functionality of genetic circuits carrying natural regulatory elements of Clostridium acetobutylicum ATCC 824 in the presence of the respective inducer molecules. Specifically, promoters and their regulators involved in diverse carbon source utilization were characterized using mCherryOpt or beta-galactosidase as a reporter. Consequently, most of the genetic circuits tested in this study were functional in Clostridium acetobutylicum ATCC 824 in the presence of an inducer, leading to the expression of reporter proteins. These genetic sensor-regulators were found to be transferable to another Clostridium species, such as Clostridium beijerinckii NCIMB 8052. The gradual expression of reporter protein was observed as a function of the carbohydrates of interest. A xylose-inducible promoter allows a titratable and robust expression of a reporter protein with stringency and efficacy. This xylose-inducible circuit was seen to enable induction of the expression of reporter proteins in the presence of actual sugar mixtures incorporated in woody hydrolysate wherein glucose and xylose are present as predominant carbon sources.

H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG.

[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H2 at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe]H) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]F), we have heterologously expressed HydF in the presence of HydE alone (HydFE) or HydG alone (HydFG), and characterized the resulting purified HydFE and HydFG using UV-visible, EPR, and FTIR spectroscopies and biochemical assays. The iron-sulfur clusters on HydF are modified by co-expression with HydE or HydG, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydFE is capable of activating HydAΔEFG to a limited extent (~ 1% of WT) even though the normal source of CO and CN- ligands of [2Fe]H (HydG) was absent. Activation assays performed with HydFG, in contrast, exhibit no ability to mature HydAΔEFG. It appears that in the case of HydFE, trace diatomics from the cellular environment are incorporated into a [2Fe]F-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe]H, is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.

Multi-modular engineering for renewable production of isoprene via mevalonate pathway in Escherichia coli.

AIMS: To establish the biotechnology platforms for production of bio-based chemicals in various micro-organisms is considered as a promising target to improve renewable production of isoprene. METHODS AND RESULTS: In this study, we heterologously expressed the mevalonate (MVA) isoprene biosynthesis pathway, and explored three strategies of increasing isoprene production in Escherichia coli. We first manipulated the expression levels of the MVA pathway genes through changing the gene cassettes and promoters. To introduce cofactor engineering, we then overexpressed NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene from Clostridium acetobutylicum to supply available NADPH. To reduce the inhibitory by-product accumulation, we finally knocked out acetate-producing genes, phosphate acetyl transferase and pyruvate oxidase B in E. coliJM109 (DE3), decreasing acetate accumulation 89% and increasing isoprene production 39%. The strategies described here finally increased the isoprene titre to 92 mg l-1 in two-gene deletion strain JMAB-4T7P1Trc, increasing 2·6-fold comparing to strain JM7T7. CONCLUSION: The multimodularly engineering approaches including promoter engineering, cofactor engineering and by-product reducing could be used to improve isoprene production in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic strategies in this study show us directions for further studies to promote transformation of renewable sources to isoprene.

Microbial production of short-chain alkanes.

Increasing concerns about limited fossil fuels and global environmental problems have focused attention on the need to develop sustainable biofuels from renewable resources. Although microbial production of diesel has been reported, production of another much in demand transport fuel, petrol (gasoline), has not yet been demonstrated. Here we report the development of platform Escherichia coli strains that are capable of producing short-chain alkanes (SCAs; petrol), free fatty acids (FFAs), fatty esters and fatty alcohols through the fatty acyl (acyl carrier protein (ACP)) to fatty acid to fatty acyl-CoA pathway. First, the ß-oxidation pathway was blocked by deleting the fadE gene to prevent the degradation of fatty acyl-CoAs generated in vivo. To increase the formation of short-chain fatty acids suitable for subsequent conversion to SCAs in vivo, the activity of 3-oxoacyl-ACP synthase (FabH), which is inhibited by unsaturated fatty acyl-ACPs, was enhanced to promote the initiation of fatty acid biosynthesis by deleting the fadR gene; deletion of the fadR gene prevents upregulation of the fabA and fabB genes responsible for unsaturated fatty acids biosynthesis. A modified thioesterase was used to convert short-chain fatty acyl-ACPs to the corresponding FFAs, which were then converted to SCAs by the sequential reactions of E. coli fatty acyl-CoA synthetase, Clostridium acetobutylicum fatty acyl-CoA reductase and Arabidopsis thaliana fatty aldehyde decarbonylase. The final engineered strain produced up to 580.8 mg l(-1) of SCAs consisting of nonane (327.8 mg l(-1)), dodecane (136.5 mg l(-1)), tridecane (64.8 mg l(-1)), 2-methyl-dodecane (42.8 mg l(-1)) and tetradecane (8.9 mg l(-1)), together with small amounts of other hydrocarbons. Furthermore, this platform strain could produce short-chain FFAs using a fadD-deleted strain, and short-chain fatty esters by introducing the Acinetobacter sp. ADP1 wax ester synthase (atfA) and the E. coli mutant alcohol dehydrogenase (adhE(mut)).

Anaerobic butanol production driven by oxygen-evolving photosynthesis using the heterocyst-forming multicellular cyanobacterium Anabaena sp. PCC 7120.

Cyanobacteria are oxygen-evolving photosynthetic bacteria. Established genetic manipulation methods and recently developed gene-regulation tools have enabled the photosynthetic conversion of carbon dioxide to biofuels and valuable chemicals in cyanobacteria, especially in unicellular cyanobacteria. However, the oxygen sensitivity of enzyme(s) introduced into cyanobacteria hampers productivity in some cases. Anabaena sp. PCC 7120 is a filamentous cyanobacterium consisting of a few hundred of vegetative cells, which perform oxygenic photosynthesis. Upon nitrogen deprivation, heterocysts, which are specialized cells for nitrogen fixation, are differentiated from vegetative cells at semiregular intervals. The micro-oxic environment within heterocysts protects oxygen-labile nitrogenase from oxygen. This study aimed to repurpose the heterocyst as a host for the production of chemicals with oxygen-sensitive enzymes under photosynthetic conditions. Herein, Anabaena strains expressing enzymes of 1-butanol synthetic pathway from the anaerobe Clostridium acetobutylicum within heterocysts were created. A strain that expressed a highly oxygen-sensitive Bcd/EtfAB complex produced 1-butanol even under photosynthetic conditions. Furthermore, the 1-butanol production per heterocyst cell of a butanol-producing Anabaena strain was fivefold higher than that per cell of unicellular cyanobacterium with the same set of 1-butanol synthetic pathway genes. Thus, our study showed the usefulness of Anabaena heterocysts as a chassis for anaerobic production driven by oxygen-evolving photosynthesis.

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.

CO-Bridged H-Cluster Intermediates in the Catalytic Mechanism of [FeFe]-Hydrogenase CaI.

The [FeFe]-hydrogenases ([FeFe] H2ases) catalyze reversible H2 activation at the H-cluster, which is composed of a [4Fe-4S]H subsite linked by a cysteine thiolate to a bridged, organometallic [2Fe-2S] ([2Fe]H) subsite. Profoundly different geometric models of the H-cluster redox states that orchestrate the electron/proton transfer steps of H2 bond activation have been proposed. We have examined this question in the [FeFe] H2ase I from Clostridium acetobutylicum (CaI) by Fourier-transform infrared (FTIR) spectroscopy with temperature annealing and H/D isotope exchange to identify the relevant redox states and define catalytic transitions. One-electron reduction of Hox led to formation of HredH+ ([4Fe-4S]H2+-FeI-FeI) and Hred' ([4Fe-4S]H1+-FeII-FeI), with both states characterized by low frequency µ-CO IR modes consistent with a fully bridged [2Fe]H. Similar µ-CO IR modes were also identified for HredH+ of the [FeFe] H2ase from Chlamydomonas reinhardtii (CrHydA1). The CaI proton-transfer variant C298S showed enrichment of an H/D isotope-sensitive µ-CO mode, a component of the hydride bound H-cluster IR signal, Hhyd. Equilibrating CaI with increasing amounts of NaDT, and probed at cryogenic temperatures, showed HredH+ was converted to Hhyd. Over an increasing temperature range from 10 to 260 K catalytic turnover led to loss of Hhyd and appearance of Hox, consistent with enzymatic turnover and H2 formation. The results show for CaI that the µ-CO of [2Fe]H remains bridging for all of the "Hred" states and that HredH+ is on pathway to Hhyd and H2 evolution in the catalytic mechanism. These results provide a blueprint for designing small molecule catalytic analogs.

A synthetic enzymatic pathway for extremely thermophilic acetone production based on the unexpectedly thermostable acetoacetate decarboxylase from Clostridium acetobutylicum.

One potential advantage of an extremely thermophilic metabolic engineering host (T opt ≥ 70°C) is facilitated recovery of volatile chemicals from the vapor phase of an active fermenting culture. This process would reduce purification costs and concomitantly alleviate toxicity to the cells by continuously removing solvent fermentation products such as acetone or ethanol, a process we are calling "bio-reactive distillation." Although extremely thermophilic heterologous metabolic pathways can be inspired by existing mesophilic versions, they require thermostable homologs of the constituent enzymes if they are to be utilized in extremely thermophilic bacteria or archaea. Production of acetone from acetyl-CoA and acetate in the mesophilic bacterium Clostridium acetobutylicum utilizes three enzymes: thiolase, acetoacetyl-CoA: acetate CoA transferase (CtfAB), and acetoacetate decarboxylase (Adc). Previously reported biocatalytic pathways for acetone production were demonstrated only as high as 55°C. Here, we demonstrate a synthetic enzymatic pathway for acetone production that functions up to at least 70°C in vitro, made possible by the unusual thermostability of Adc from the mesophile C. acetobutylicum, and heteromultimeric acetoacetyl-CoA:acetate CoA-transferase (CtfAB) complexes from Thermosipho melanesiensis and Caldanaerobacter subterraneus, composed of a highly thermostable α-subunit and a thermally labile ß-subunit. The three enzymes produce acetone in vitro at temperatures of at least 70°C, paving the way for bio-reactive distillation of acetone using a metabolically engineered extreme thermophile as a production host.

Phototrophic hydrogen production from a clostridial [FeFe] hydrogenase expressed in the heterocysts of the cyanobacterium Nostoc PCC 7120.

The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H2. As a result, their H2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H2 production activities, but their sensitivity to O2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O2. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 µmol-H2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H2 production by an O2-sensitive hydrogenase.

Novel polycondensed biopolyamide generated from biomass-derived 4-aminohydrocinnamic acid.

Biomass plastics are expected to contribute to the establishment of a carbon-neutral society by replacing conventional plastics derived from petroleum. The biomass-derived aromatic amine 4-aminocinnamic acid (4ACA) produced by recombinant bacteria is applied to the synthesis of high-performance biopolymers such as polyamides and polyimides. Here, we developed a microbial catalyst that hydrogenates the α,ß-unsaturated carboxylic acid of 4ACA to generate 4-aminohydrocinnamic acid (4AHCA). The ability of 10 microbial genes for enoate and xenobiotic reductases expressed in Escherichia coli to convert 4ACA to 4AHCA was assessed. A strain producing 2-enoate reductase from Clostridium acetobutylicum (ca2ENR) reduced 4ACA to 4AHCA with a yield of > 95% mol mol-1 and reaction rates of 3.4 ± 0.4 and 4.4 ± 0.6 mM h-1 OD600-1 at the optimum pH of 7.0 under aerobic and anaerobic conditions, respectively. This recombinant strain reduced caffeic, cinnamic, coumaric, and 4-nitrocinnamic acids to their corresponding propanoic acid derivatives. We polycondensed 4AHCA generated from biomass-derived 4ACA by dehydration under a catalyst to form high-molecular-weight poly(4AHCA) with a molecular weight of M n = 1.94 MDa. This polyamide had high thermal properties as indicated by a 10% reduction in weight at a temperature of T d10 = 394 °C and a glass transition temperature of T g = 240 °C. Poly(4AHCA) derived from biomass is stable at high temperatures and could be applicable to the production of high-performance engineering plastics.

The effect of a C298D mutation in CaHydA [FeFe]-hydrogenase: Insights into the protein-metal cluster interaction by EPR and FTIR spectroscopic investigation.

A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid causes significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.

Activation Thermodynamics and H/D Kinetic Isotope Effect of the Hox to HredH+ Transition in [FeFe] Hydrogenase.

Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The Hox→HredH+ reduction step measured by Fourier-transform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol-1 and a ∼2.5-fold kinetic isotope effect. Overall, these results support electron injection from CdSe into CaI involving F-clusters, and that the Hox→HredH+ step of catalytic proton reduction in CaI proceeds by a proton-dependent process.

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum.

With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO2, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms.IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO2 reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. 13C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO2 to CO and oxidize CO during growth on glucose.