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The Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed.Key Points• The regulatory network governing sporulation initiation varies in solventogenic clostridia.• Media composition and cell density are the main triggers of sporulation.• Spores can be used to improve the fermentation process.

Background Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis. Results This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed 13C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways. Conclusions Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.

Summary Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by‐passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha‐4,11‐diene after cultivation with [4‐13C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance., This work describes the design and implementation of pathway replacement in isoprenoid metabolism in the bacterium Rhodobacter sphaeroides. Integration of a heterologous mevalonate pathway was followed by the inactivation of the endogenous MEP pathway via Cas9 counter‐selection. The resulting strain showed increased amorphadiene yields compared to the parental strain. Therefore replacement of endogenous pathways with non‐native counterparts is suggested for rational design of microbial cell factories.

Background Genetic engineering of microorganisms has become a common practice to establish microbial cell factories for a wide range of compounds. Ethyl acetate is an industrial solvent that is used in several applications, mainly as a biodegradable organic solvent with low toxicity. While ethyl acetate is produced by several natural yeast species, the main mechanism of production has remained elusive until the discovery of Eat1 in Wickerhamomyces anomalus. Unlike other yeast alcohol acetyl transferases (AATs), Eat1 is located in the yeast mitochondria, suggesting that the coding sequence contains a mitochondrial pre-sequence. For expression in prokaryotic hosts such as E. coli, expression of heterologous proteins with eukaryotic signal sequences may not be optimal. Results Unprocessed and synthetically truncated eat1 variants of Kluyveromyces marxianus and Wickerhamomyces anomalus have been compared in vitro regarding enzyme activity and stability. While the specific activity remained unaffected, half-life improved for several truncated variants. The same variants showed better performance regarding ethyl acetate production when expressed in E. coli. Conclusion By analysing and predicting the N-terminal pre-sequences of different Eat1 proteins and systematically trimming them, the stability of the enzymes in vitro could be improved, leading to an overall improvement of in vivo ethyl acetate production in E. coli. Truncated variants of eat1 could therefore benefit future engineering approaches towards efficient ethyl acetate production.

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from 13C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-13C]- and [4-13C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer 13C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.

Background: Ethyl acetate and hydrogen are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products render the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO2. Results: We engineered Escherichia coli, towards improved conversion of formate into hydrogen and CO2 by inactivating the formate hydrogen lyase repressor (hycA), both uptake hydrogenases (hyaAB, hybBC) and/or overexpressing the hydrogen formate lyase activator (fhlA). Initially 10 strains were evaluated in anaerobic serum bottles with respect to growth, after which four strains were further analyzed. Anaerobic co-production of hydrogen and ethyl acetate via heterologous ethanol acyltransferase (Eat1) was achieved in 1.5-L pH controlled bioreactors. Conclusions: We showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70 % of the pathway maximum for ethyl acetate and hydrogen, and propose in-situ product removal via gas stripping as efficient technique to isolate the products of interest.

Background Ethyl acetate (C4H8O2) and hydrogen (H2) are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products renders the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved, but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO2. Results We engineered Escherichia coli towards improved conversion of formate into H2 and CO2 by inactivating the formate hydrogen lyase repressor (hycA), both uptake hydrogenases (hyaAB, hybBC) and/or overexpressing the hydrogen formate lyase activator (fhlA), in an acetate kinase (ackA) and lactate dehydrogenase (ldhA)-deficient background strain. Initially 10 strains, with increasing number of modifications were evaluated in anaerobic serum bottles with respect to growth. Four reference strains ΔldhAΔackA, ΔldhAΔackA p3-fhlA, ΔldhAΔackAΔhycAΔhyaABΔhybBC and ΔldhAΔackAΔhycAΔhyaABΔhybBC p3-fhlA were further equipped with a plasmid carrying the heterologous ethanol acyltransferase (Eat1) from Wickerhamomyces anomalus and analyzed with respect to their ethyl acetate and hydrogen co-production capacity. Anaerobic co-production of hydrogen and ethyl acetate via Eat1 was achieved in 1.5-L pH-controlled bioreactors. The cultivation was performed at 30 °C in modified M9 medium with glucose as the sole carbon source. Anaerobic conditions and gas stripping were established by supplying N2 gas. Conclusions We showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70% of the pathway maximum for ethyl acetate and hydrogen, and propose in situ product removal via gas stripping as efficient technique to isolate the products of interest.

Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment in vitro, accepting a wide range of alcohols (C2–C10) but only a small range of acyl donors (C2–C4). We show that alcoholysis occurs in vivo in several Crabtree negative yeast species but also in engineered Saccharomyces cerevisiae strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters in vivo by overexpressing Eat1 in Clostridium beijerinckii. Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the in vitro alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products.

Acknowledgments : The authors thank the Biomics Platform, C2RT, at the Institut Pasteur supported by France Génomique (ANR-10-INBS-09-09) and IBISA for sequencing the RNA-Seq library, Dr. François Wasels for his help on the genome analysis, Dr. Karel Sedlar for his advice on RNA seq data analysis, Arjen Bader for technical assistance with the fluorescence microscopy analysis and Dr. Eric M. Ransom for the gift of pRAN73 plasmid.; International audience; SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ F , during sporulation. A ΔspoIIE mutant of Clostridium beijerinckii NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the ΔspoIIE mutant are elongated with the presence of multiple septa. This observation suggests that in C. beijerinckii, SpoIIE is necessary for the completion of the sporulation process, as seen in Bacillus and Clostridium acetobutylicum. Moreover, when grown in reactors, the spoIIE mutant produced higher levels of solvents than the wild type strain. The impact of the spoIIE inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ F , σ E , σ G , σ K ) were strongly down-regulated in the mutant. Inactivation of spoIIE also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in C. beijerinckii.

Background Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/T7 and XylS/Pm promoters allowed optimisation of their expression levels. Conclusion Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced 42.8 mM (3.8 g/L) ethyl acetate from glucose with an unprecedented efficiency, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where “normal” proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.

At the junction between the glycolysis and the tricarboxylic acid cycle—as well as various other metabolic pathways—lies the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node (PPO-node). These three metabolites form the core of a network involving at least eleven different types of enzymes, each with numerous subtypes. Obviously, no single organism maintains each of these eleven enzymes; instead, different organisms possess different subsets in their PPO-node, which results in a remarkable degree of variation, despite connecting such deeply conserved metabolic pathways as the glycolysis and the tricarboxylic acid cycle. The PPO-node enzymes play a crucial role in cellular energetics, with most of them involved in (de)phosphorylation of nucleotide phosphates, while those responsible for malate conversion are important redox enzymes. Variations in PPO-node therefore reflect the different energetic niches that organisms can occupy. In this review, we give an overview of the biochemistry of these eleven PPO-node enzymes. We attempt to highlight the variation that exists, both in PPO-node compositions, as well as in the roles that the enzymes can have within those different settings, through various recent discoveries in both bacteria and archaea that reveal deviations from canonical functions., Three metabolites, phosphoenolpyruvate, pyruvate and oxaloacetate together form the PPO-node, and are interconnected by a subset of 11 different enzymes; the current knowledge regarding these enzymes at the heart of metabolism is presented.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium Escherichia coli. By boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viable E. coli strain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and that E. coli has the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20–30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell’s phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.

A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general., Macroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, including Ulva lactuca, the major sugars are l-rhamnose and d-glucose. C. beijerinckii DSM 6423 utilized these sugars in a U. lactuca hydrolysate to produce acetic acid, butyric acid, isopropanol, butanol, and ethanol (IBE), and 1,2-propanediol. d-Glucose was almost completely consumed in diluted hydrolysates, while l-rhamnose or d-xylose was only partially utilized. In this study, the metabolism of l-rhamnose by C. beijerinckii DSM 6423 was investigated to improve its utilization from natural resources. Fermentations on d-glucose, l-rhamnose, and a mixture of d-glucose and l-rhamnose were performed. On l-rhamnose, the cultures showed low growth and sugar consumption and produced 1,2-propanediol, propionic acid, and n-propanol in addition to acetic and butyric acids, whereas on d-glucose, IBE was the major product. On a d-glucose–l-rhamnose mixture, both sugars were converted simultaneously and l-rhamnose consumption was higher, leading to high levels of 1,2-propanediol (78.4 mM), in addition to 59.4 mM butanol and 31.9 mM isopropanol. Genome and transcriptomics analysis of d-glucose- and l-rhamnose-grown cells revealed the presence and transcription of genes involved in l-rhamnose utilization and in bacterial microcompartment (BMC) formation. These data provide useful insights into the metabolic pathways involved in l-rhamnose utilization and the effects on the general metabolism (glycolysis, early sporulation, and stress response) induced by growth on l-rhamnose. IMPORTANCE A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general.

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

Sustainable production of bulk chemicals is one of the major challenges in the chemical industry, particularly due to their low market prices. This includes short and medium chain esters, which are used in a wide range of applications, for example fragrance compounds, solvents, lubricants or biofuels. However, these esters are produced mainly through unsustainable, energy intensive processes. Microbial conversion of biomass-derived sugars into esters may provide a sustainable alternative. This review provides a broad overview of natural ester production by microorganisms. The underlying ester-forming enzymatic mechanisms are discussed and compared, with particular focus on alcohol acyltransferases (AATs). This large and versatile group of enzymes condense an alcohol and an acyl-CoA to form esters. Natural production of esters typically cannot compete with existing petrochemical processes. Much effort has therefore been invested in improving in vivo ester production through metabolic engineering. Identification of suitable AATs and efficient alcohol and acyl-CoA supply are critical to the success of such strategies and are reviewed in detail. The review also focusses on the physical properties of short and medium chain esters, which may simplify downstream processing, while limiting the effects of product toxicity. Furthermore, the esters could serve as intermediates for the synthesis of other compounds, such as alcohols, acids or diols. Finally, the perspectives and major challenges of microorganism-derived ester synthesis are presented.

Rhodobacter sphaeroides is a metabolically versatile bacterium capable of producing terpenes natively. Surprisingly, terpene biosynthesis in this species has always been investigated in complex media, with unknown compounds possibly acting as carbon and nitrogen sources. Here, a defined medium was adapted for R. sphaeroides dark heterotrophic growth, and was used to investigate the conversion of different organic substrates into the reporter terpene amorphadiene. The amorphadiene synthase was cloned in R. sphaeroides, allowing its biosynthesis via the native 2-methyl-d-erythritol-4-phosphate (MEP) pathway and, additionally, via a heterologous mevalonate one. The latter condition increased titers up to eightfold. Consequently, better yields and productivities to previously reported complex media cultivations were achieved. Productivity was further investigated under different cultivation conditions, including nitrogen and oxygen availability. This novel cultivation setup provided useful insight into the understanding of terpene biosynthesis in R. sphaeroides, allowing to better comprehend its dynamics and regulation during chemoheterotrophic cultivation.

Eat1 is a recently discovered alcohol acetyltransferase responsible for bulk ethyl acetate production in yeasts such as Wickerhamomyces anomalus and Kluyveromyces lactis. These yeasts have the potential to become efficient bio-based ethyl acetate producers. However, some fundamental features of Eat1 are still not understood, which hampers the rational engineering of efficient production strains. The cellular location of Eat1 in yeast is one of these features. To reveal its location, Eat1 was fused with yeast-enhanced green fluorescent protein (yEGFP) to allow intracellular tracking. Despite the current assumption that bulk ethyl acetate production occurs in the yeast cytosol, most of Eat1 localized to the mitochondria of Kluyveromyces lactis CBS 2359 Δku80. We then compared five bulk ethyl acetate-producing yeasts in iron-limited chemostats with glucose as the carbon source. All yeasts produced ethyl acetate under these conditions. This strongly suggests that the mechanism and location of bulk ethyl acetate synthesis are similar in these yeast strains. Furthermore, an in silico analysis showed that Eat1 proteins from various yeasts were mostly predicted as mitochondrial. Altogether, it is concluded that Eat1-catalyzed ethyl acetate production occurs in yeast mitochondria. This study has added new insights into bulk ethyl acetate synthesis in yeast, which is relevant for developing efficient production strains. IMPORTANCE Ethyl acetate is a common bulk chemical that is currently produced from petrochemical sources. Several Eat1-containing yeast strains naturally produce large amounts of ethyl acetate and are potential cell factories for the production of bio-based ethyl acetate. Rational design of the underlying metabolic pathways may result in improved production strains, but it requires fundamental knowledge on the function of Eat1. A key feature is the location of Eat1 in the yeast cell. The precursors for ethyl acetate synthesis can be produced in multiple cellular compartments through different metabolic pathways. The location of Eat1 determines the relevance of each pathway, which will provide future targets for the metabolic engineering of bulk ethyl acetate production in yeast.

Esters are essential for the flavor and aroma of fermented products, and are mainly produced by alcohol acyl transferases (AATs). A recently discovered AAT family named Eat (Ethanol acetyltransferase) contributes to ethyl acetate synthesis in yeast. However, its effect on the synthesis of other esters is unknown. In this study, the role of the Eat family in ester synthesis was compared to that of other Saccharomyces cerevisiae AATs (Atf1p, Atf2p, Eht1p, and Eeb1p) in silico and in vivo. A genomic study in a collection of industrial S. cerevisiae strains showed that variation of the primary sequence of the AATs did not correlate with ester production. Fifteen members of the EAT family from nine yeast species were overexpressed in S. cerevisiae CEN.PK2-1D and were able to increase the production of acetate and propanoate esters. The role of Eat1p was then studied in more detail in S. cerevisiae CEN.PK2-1D by deleting EAT1 in various combinations with other known S. cerevisiae AATs. Between 6 and 11 esters were produced under three cultivation conditions. Contrary to our expectations, a strain where all known AATs were disrupted could still produce, e.g., ethyl acetate and isoamyl acetate. This study has expanded our understanding of ester synthesis in yeast but also showed that some unknown ester-producing mechanisms still exist. ispartof: FRONTIERS IN MICROBIOLOGY vol:9 ispartof: location:Switzerland status: published

An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose deconstruction. Our research group has been working on mapping and characterization of glycoside hydrolases produced by C. thermocellum B8, that are critical for lignocellulosic biomass deconstruction. One of the identified genes expressed during growth on sugar cane bagasse and straw is axb8, which encodes a putative cellulosomal GH43_29 α-arabinofuranosidase (EC 3.2.1.55) that has not previously been characterized at the molecular or kinetic levels. The AxB8 predicted amino acid sequence presented GH43 and dockerin domains, as well as a family 6 carbohydrate-binding module (CBM6). Also, it is a close homologue of Firmicutes putatives α-arabinofuranosidases, including cellulosomal proteins. Multiple alignment analysis grouped AxB8 in a cluster with four uncharacterized putative GH43_29 subfamily enzymes, all containing dockerin type I domain and CBM6 modules. Purified heterologously expressed AxB8 showed activity against the synthetic substrates pNPX (p-nytrophenyl-β-D-xylopyranoside) and pNPA (p-nytrophenyl-α-L-arabinofuranoside), as well as against the natural substrate wheat arabinoxylan (WAX), with maximal activity at 50 °C and pH between 5.0 and 6.0. The WAX degradation profile by AxB8 is different from those typically seen for α-arabinofuranosidases, presenting mainly xylose as a hydrolysis product, instead of arabinose. In addition, unlike other GH43_29 enzymes already characterized, AxB8 did not present activity against arabinan. Kinetic parameters using pNPA as a substrate were Km of 23 ± 3 mM and kcat of 104 ± 7 s−1. Despite its activity against pNPX, we did not observe AxB8 saturation with this substrate. AxB8 is the first member in its clade to be characterized regarding kinetic parameters, and together with its closest homologues could represent a large group of glycoside hydrolases with particular properties within the GH43_29 subfamily.

Increased hydrogen (H2) levels are known to inhibit H2 formation in Caldicellulosiruptor saccharolyticus. To investigate this organism's strategy for dealing with elevated H2 levels the effect of the hydrogen partial pressure ( P H 2 ) on fermentation performance was studied by growing cultures under high and low P H 2 in a glucose limited chemostat setup. Transcriptome analysis revealed the upregulation of genes involved in the disposal of reducing equivalents under high P H 2 , like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings are in line with the observed shift in fermentation profiles from acetate production to the production of acetate, lactate and ethanol under high P H 2 . Moreover, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. In addition, presented transcription data provide evidence for the involvement of the redox sensing Rex protein in gene regulation under high P H 2 cultivation conditions.

Caldicellulosiruptor saccharolyticus is one of the most thermophilic cellulolytic organisms known to date. This Gram-positive anaerobic bacterium ferments a broad spectrum of mono-, di- and polysaccharides to mainly acetate, CO2 and hydrogen. With hydrogen yields approaching the theoretical limit for dark fermentation of 4 mol hydrogen per mol hexose, this organism has proven itself to be an excellent candidate for biological hydrogen production. This review provides an overview of the research on C. saccharolyticus with respect to the hydrolytic capability, sugar metabolism, hydrogen formation, mechanisms involved in hydrogen inhibition, and the regulation of the redox and carbon metabolism. Analysis of currently available fermentation data reveal decreased hydrogen yields under non-ideal cultivation conditions, which are mainly associated with the accumulation of hydrogen in the liquid phase. Thermodynamic considerations concerning the reactions involved in hydrogen formation are discussed with respect to the dissolved hydrogen concentration. Novel cultivation data demonstrate the sensitivity of C. saccharolyticus to increased hydrogen levels regarding substrate load and nitrogen limitation. In addition, special attention is given to the rhamnose metabolism, which represents an unusual type of redox balancing. Finally, several approaches are suggested to improve biohydrogen production by C. saccharolyticus.

Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH2-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors. (C) 2016 Elsevier B.V. All rights reserved.

The strong advances in synthetic biology enable the engineering of novel functions and complex biological features in unprecedented ways, such as implementing synthetic autotrophic metabolism into heterotrophic hosts. A key challenge for the sustainable production of fuels and chemicals entails the engineering of synthetic autotrophic organisms that can effectively and efficiently fix carbon dioxide by using sustainable energy sources. This challenge involves the integration of carbon fixation and energy uptake systems. A variety of carbon fixation pathways and several types of photosystems and other energy uptake systems can be chosen and, potentially, modularly combined to design synthetic autotrophic metabolism. Prior to implementation, these designs can be evaluated by the combination of several computational pathway analysis techniques. Here we present a systematic, integrated in silico analysis of photo-electro-autotrophic pathway designs, consisting of natural and synthetic carbon fixation pathways, a proton-pumping rhodopsin photosystem for ATP regeneration and an electron uptake pathway. We integrated Flux Balance Analysis of the heterotrophic chassis Escherichia coli with kinetic pathway analysis and thermodynamic pathway analysis (Max-min Driving Force). The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the proton pumping rhodopsin photosystems and the high ATP maintenance of E. coli. If these factors can be tackled, our analysis indicates the highest growth potential for the natural reductive tricarboxylic acid cycle and the synthetic pyruvate synthase–pyruvate carboxylate -glyoxylate bicycle. Both carbon fixation cycles are very ATP efficient, while maintaining fast kinetics, which also results in relatively low estimated protein costs for these pathways. Furthermore, the synthetic bicycles are highly thermodynamic favorable under conditions analysed. However, the most important challenge identified for improving photo-electro-autotrophic growth is increasing the proton-pumping rate of the rhodopsin photosystems, allowing for higher ATP regeneration. Alternatively, other designs of autotrophy may be considered, therefore the herein presented integrated modeling approach allows synthetic biologists to evaluate and compare complex pathway designs before experimental implementation., PLoS ONE, 11 (6), ISSN:1932-6203

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-β-D-xylopyranoside monoacetates as substrates in a β-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 A resolution, respectively, revealing a classic α/β-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 A, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.

The role of inorganic pyrophosphate (PPi) as an energy carrier in the central metabolism of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was investigated. In agreement with its annotated genome sequence, cell extracts were shown to exhibit PPi-dependent phosphofructokinase and pyruvate phosphate dikinase activity. In addition, membrane-bound pyrophosphatase activity was demonstrated, while no significant cytosolic pyrophosphatase activity was detected. During the exponential growth phase, high PPi levels (approximately 4 +/- 2 mM) and relatively low ATP levels (0.43 +/- 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticus.

The chlorite dismutase (Cld) of Pseudomonas chloritidismutans was purified from the periplasmic fraction in one step by hydroxyapatite chromatography. The enzyme has a molecular mass of 110 kDa and consists of four 31-kDa subunits. Enzyme catalysis followed Michaelis-Menten kinetics, with Vmax and K(m) values of 443 U mg(-1) and 84 microM, respectively. A pyridine-NaOH-dithionite-reduced Cld revealed a Soret peak at 418 nm, indicative for protoheme IX. The spectral data indicate the presence of 1.5 mol protoheme IX mol(-1) tetrameric enzyme while metal analysis revealed 2.2 mol iron mol(-1) tetrameric enzyme. High concentrations of chlorite resulted in the disappearance of the Soret peak, which coincided with loss in activity. Electron paramagnetic resonance analyses showed an axial high-spin ferric iron signal. Cld was inhibited by cyanide, azide, but not by hydroxylamine or 3-amino-1,2,3-triazole. Remarkably, the activity was drastically enhanced by kosmotropic salts, and chaotropic salts decreased the activity, in accordance with the Hofmeister series. Chlorite conversion in the presence of 18O-labeled water did not result in the formation of oxygen with a mass of 34 (16O-18O) or a mass of 36 ((18)O-(18)O), indicating that water is not a substrate in the reaction and that both oxygen atoms originate from chlorite.

Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 °C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO 2 , and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus , consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden–Meyerhof and the Entner–Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.

A Gram-negative, facultatively anaerobic, rod-shaped, dissimilatory chlorate-reducing bacterium, strain AW-1(T), was isolated from biomass of an anaerobic chlorate-reducing bioreactor. Phylogenetic analysis of the 16S rDNA sequence showed 100% sequence similarity to Pseudomonas stutzeri DSM 50227 and 98.6% sequence similarity to the type strain of P. stutzeri (DSM 5190(T)). The species P. stutzeri possesses a high degree of genotypic and phenotypic heterogeneity. Therefore, eight genomic groups, termed genomovars, have been proposed based upon deltaTm values, which were used to evaluate the quality of the pairing within heteroduplexes formed by DNA-DNA hybridization. In this study, DNA-DNA hybridization between strain AW-1(T) and P. stutzeri strains DSM 50227 and DSM 5190(T) revealed respectively 80.5 and 56.5% similarity. DNA-DNA hybridization between P. stutzeri strains DSM 50227 and DSM 5190(T) revealed 48.4% similarity. DNA-DNA hybridization indicated that strain AW-1(T) is not related at the species level to the type strain of P. stutzeri. However, strain AW-1(T) and P. stutzeri DSM 50227 are related at the species level. The physiological and biochemical properties of strain AW-1(T) and the two P. stutzeri strains were compared. A common characteristic of P. stutzeri strains is the ability to denitrify. However, in growth experiments, strain AW-1(T) could use only chlorate or oxygen as an electron acceptor and not nitrate, perchlorate or bromate. Strain AW-1(T) is the first chlorate-reducing bacterium described that does not possess another oxyanion-reduction pathway. Cell extracts of strain AW-1(T) showed chlorate and bromate reductase activities but not nitrate reductase activity. P. stutzeri strains DSM 50227 and DSM 5190(T) could use nitrate or oxygen as an electron acceptor, but not chlorate. Chlorate reductase activity, in addition to nitrate reductase activity, was detected in cell extracts of both P. stutzeri strains. Chlorite dismutase activity was absent in extracts of both P. stutzeri strains but was present in extracts of strain AW-1(T). Based on the hybridization experiments and the physiological and biochemical data, it is proposed that strain AW-1(T) be classified as a novel species of Pseudomonas, Pseudomonas chloritidismutans sp. nov. The type strain is strain AW-1(T) (= DSM 13592(T) = ATCC BAA-443(T)).

In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them ( CBEI_1464 ) showed substantial acetoin reductase activity after expression in Escherichia coli . The purified enzyme ( C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred ( K m , 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the “threonine dehydrogenase and related Zn-dependent dehydrogenases” (COG1063). Metal analysis confirmed the presence of 2 Zn 2+ atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys 37 , His 70 , and Glu 71 , while the structural zinc site is probably composed of Cys 100 , Cys 103 , Cys 106 , and Cys 114 . Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.

The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.

Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an α 3 β 3 composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The K m values for perchlorate and chlorate were 27 and

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.

The current knowledge on microbial reductive dechlorination of chlorinated ethenes (CEs) and its application are discussed. Physiological studies on CEs dechlorinating microorganisms indicate that ...

Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas. Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to cis -1,2-dichloroethene at the optimum temperature of 60 to 65°C. After several transfers, methanogens were eliminated from the culture. Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H 2 , formate, or acetate. Fumarate and l -malate led to the highest dechlorination rate. In the absence of PCE, fumarate was fermented to acetate, H 2 , CO 2 , and succinate. With PCE, less H 2 was formed, suggesting that PCE competed for the reducing equivalents leading to H 2 . PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor. At the optimum dissolved PCE concentration of ∼60 μM, a high dechlorination rate of 1.1 μmol h −1 mg −1 (dry weight) was found, which indicates that the dechlorination is not a cometabolic activity. Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod. Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives. The highest similarity was found with the genus Dehalobacter (90%), represented by the halorespiring organism Dehalobacter restrictus , and with the genus Desulfotomaculum (86%).

A predicted esterase ( EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 angstrom resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 angstrom. There are two molecules in the asymmetric unit, with a VM of 2.9 angstrom(3) Da(-1) and 58% solvent content.

The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.

Anaerobic carbon mineralization is a major regulator of soil methane production, but the relationship between these processes is variable. To explain the dynamics of this relationship a model was developed, which comprises the dynamics of alternative electron-acceptors, of acetate and of methanogenic biomass. Major assumptions are: (i) alternative electron-acceptors suppress methanogenesis and (ii) the rate of electron-acceptor reduction is controlled by anaerobic carbon mineralization. The model was applied to anaerobic incubation experiments with slurried soil samples from a drained and an undrained peat soil in the Netherlands to test the model and to further interpret the data. Three parameters were fitted with a Monte Carlo method, using experimentally determined time series of methane, carbon dioxide and acetate. The fitted parameters were the initial concentration of electron-acceptors, the initial concentration of methanogenic biomass and the maximum relative growth rate of methanogenic biomass. Simulated and measured time courses of methane corresponded reasonably well. The model as such stresses the importance of alternative electron-acceptors. At the drained site initial alternative electron-acceptor concentrations were between 0.3 and 0.8 mol electron equivalents (el. eqv.) kg −1 dw soil, whereas at the undrained site they were between 0.0 and 0.3 mol el. eqv. kg −1 dw soil, depending on the experimental treatments. The sum of measured NO 3 − and SO 4 2− concentrations and estimated maximum Fe 3+ and Mn 4+ concentrations was much lower than the fitted concentrations of alternative electron-acceptors. Apparently, reduction of unknown electron-acceptors consumed a large part of anaerobically-mineralized carbon which, therefore, was not available for methanogenesis.

The biotech industry is continuously seeking for new or improved biocatalysts. The success of these efforts is often hampered by the lack of an efficient screening assay. Thus, to be able to extend the number of enzymes available for industrial applications, high-throughput screening and selection methods are required. In the last few years an impressive range of screening and selection strategies has been developed. In this review, we will mainly focus on in vivo reporter systems in which the activity of a reporter is controlled by the activity of an enzyme of interest. Different mechanisms can be distinguished: (a) binding of the product of the enzymatic reaction to a transcriptional regulator and thereby turning on transcription of the reporter; (b) direct modification of a transcriptional regulator by the enzyme resulting in expression of the reporter; (c) binding of the product to a regulatory riboswitch or ribozyme, resulting in translation of the reporter; and (d) direct modification of the reporter by the enzyme, altering the reporter's activity. The choice for either a selection or a screening strategy depends on the type of reporter, e.g. providing antibiotic resistance (selection) or transmitting a fluorescent signal (screening). Although developing the specificity of each of these reporter-based selection or screening systems towards a certain enzymatic reaction is not yet straightforward, their adjustable modular design appears to be a promise for general applicability in the near future.

The production of biohydrogen from glycerol, by the hyperthermophilic bacterium Thermotoga maritima DSM 3109, was investigated in batch and chemostat systems. T. maritima converted glycerol to mainly acetate, CO2 and H2. Maximal hydrogen yields of 2.84 and 2.41 hydrogen per glycerol were observed for batch and chemostat cultivations, respectively. For batch cultivations: i) hydrogen production rates decreased with increasing initial glycerol concentration, ii) growth and hydrogen production was optimal in the pH range of 7–7.5, and iii) a yeast extract concentration of 2 g/l led to optimal hydrogen production. Stable growth could be maintained in a chemostat, however, when dilution rates exceeded 0.025 h−1 glycerol conversion was incomplete. A detailed overview of the catabolic pathway involved in glycerol fermentation to hydrogen by T. maritima is given. Based on comparative genomics the ability to grow on glycerol can be considered as a general trait of Thermotoga species. The exceptional bioenergetics of hydrogen formation from glycerol is discussed.

Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of D-glucuronic acid to its C5-epimer L-iduronic acid, which is essential for the function of heparan sulfate. Although L-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic D-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with D-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human D-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial D-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min-1 (mg protein)-1. The dismutase obeyed Michaelis-Menten kinetics. The Vmax and Km calculated for chlorite were 2,200 U (mg protein)-1 and 170 microM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30 degrees C.

10.1016/j.egypro.2012.09.036 Given the highly reduced state of carbon in glycerol and its availability as a substantial byproduct of biodiesel production, glycerol is of special interest for sustainable biofuel production. Glycerol was used as a substrate for biohydrogen production using the hyperthermophilic bacterium, Thermotoga maritima and Thermotoga neapolitana. Both species metabolized glycerol to mainly acetate and hydrogen. At glycerol concentrations of 2.5 g/L, hydrogen was produced with a yield of 2.75 and 2.65 mol H2/mol glycerol consumed by T. maritima and T. neapolitana respectively. Additionally, the effect of initial pH (ranging between pH 5.0-8.5) and yeast extract concentrations (0.5, 1, 2, 4 g/L) on glycerol fermentation by T. neapolitana was investigated in batch systems. An initial pH value of around 7 was optimal for hydrogen production by T. neapolitana. Lower concentration of yeast extract resulted in a lower H2 production, however increasing the concentration from 2 to 4 g/L did not affect H2 production.

To gain more insight into the butanol stress response of Clostridium acetobutylicum the transcriptional response of a steady state acidogenic culture to different levels of n -butanol (0.25–1%) was investigated. No effect was observed on the fermentation pattern and expression of typical solvent genes ( aad , ctfA/B , adc , bdhA/B , ptb , buk ). Elevated levels of butanol mainly affected class I heat-shock genes ( hrcA , grpE , dnaK , dnaJ , groES , groEL , hsp90 ), which were upregulated in a dose- and time-dependent manner, and genes encoding proteins involved in the membrane composition ( fab and fad or glycerophospholipid related genes) and various ABC-transporters of unknown specificity. Interestingly, fab and fad genes were embedded in a large, entirely repressed cluster (CAC1988–CAC2019), which inter alia encoded an iron-specific ABC-transporter and molybdenum-cofactor synthesis proteins. Of the glycerophospholipid metabolism, the glycerol-3-phosphate dehydrogenase ( glpA ) gene was highly upregulated, whereas a glycerophosphodiester ABC-transporter ( ugpAEBC ) and a phosphodiesterase ( ugpC ) were repressed. On the megaplasmid, only a few genes showed differential expression, e.g. a rare lipoprotein (CAP0058, repressed) and a membrane protein (CAP0102, upregulated) gene. Observed transcriptional responses suggest that C. acetobutylicum reacts to butanol stress by induction of the general stress response and changing its cell envelope and transporter composition, but leaving the central catabolism unaffected.

The hyperthermophilic archaeon Pyrococcus furiosus was found to form substantial amounts of l-alanine during batch growth on either cellobiose, maltose or pyruvate. Acetate, CO2 and H2 were produced next to alanine. The carbon- and electron balances were complete for all three substrates. Under standard growth conditions (N2/CO2 atmosphere) an alanine/acetate ratio of about 0.3 was found for either substrate. The alanine /acetate ratio was influenced, however, by the hydrogen partial pressure. In the presence of S0 or in coculture with Methanococcus jannaschii this ratio was only 0.07, whereas under a H2/CO2 atmosphere this ratio could amount up to 0.8. Alanine formation was also aflected by the NHinf4sup+concentration, i.e. below 4 mM, NHinf4sup+becomes limiting to alanine formation. Alanine formation was shown to occur via an alanine aminotransferase, which exhibited a specific activity in cell-free extract of up to 6.0 U/mg (90°C; direction of pyruvate formation). The alanine aminotransferase probably cooperates with glutamate dehydrogenase (up to 23 U/mg; 90°C) and ferredoxin: NADP+ oxidoreductase (up to 0.7 U/mg, using methyl viologen; 90°C) to recycle the electron acceptors involved in catabolism. Thus, the existence of this unusual alanine-forming branch enables P. furiosus to adjust its fermentation, depending on the redox potential of the terminal electron acceptor.

Pyrococcus furiosus harbours several hydrolytic enzyme activities that enable growth on a variety of polymeric substrates like proteins and polysaccharides. Recently, the organism was shown to exhibit an extremely high β-glucosidase activity, apparently involved in hydrolysis of cellobiose. The cytoplasmic enzyme was purified to homogeneity and its properties were compared with those of other glycosidases. This comparison can be summarized as follows: i) the β-glucosidase activity in cell-free extracts is at least 100-fold higher than the P. furiosus α-glucosidase activity; ii) the β-glucosidase comprises 5% of total cell protein as to only 0.3% for the α-glucosidase; iii) with respect to substrate specifity, Km, pI, and pH-optimum the P. furiosus β-glucosidase resembles other β-glucosidases from microorganisms from lower temperature ranges; iv) compared to these β-glucosidases the P. furiosus enzyme exhibits an enhanced general stability; v) a high similarity is observed with a β-galacto/glucosidase from...