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.

The cytoplasmic membrane of a prokaryotic cell consists of a lipid bilayer or a monolayer that shields the cellular content from the environment. In addition, the membrane contains proteins that are responsible for transport of proteins and metabolites as well as for signalling and energy transduction. Maintenance of the functionality of the membrane during changing environmental conditions relies on the cell’s potential to rapidly adjust the lipid composition of its membrane. Despite the fundamental chemical differences between bacterial ester lipids and archaeal ether lipids, both types are functional under a wide range of environmental conditions. We here provide an overview of archaeal and bacterial strategies of changing the lipid compositions of their membranes. Some molecular adjustments are unique for archaea or bacteria, whereas others are shared between the two domains. Strikingly, shared adjustments were predominantly observed near the growth boundaries of bacteria. Here, we demonstrate that the presence of membrane spanning ether-lipids and methyl branches shows a striking relationship with the growth boundaries of archaea and bacteria.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-017-0939-x) contains supplementary material, which is available to authorized users.

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Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a P-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein.The native P-glucosidase has a molecular mass of 230 -+ 20 m a , composed of 58 ? 2-kDa subunits. The enzyme has a pl of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH5.0 and 102-105°C. From Lineweaver-Burk plots, V,,, values of 470U/mg and 700U/mg were found for cellobiose (K, = 20 mM) andp-nitrophenyl-P-D-glucopyranoside ( K , = 0.15 mM), respectively. The purified enzyme also exhibits high j?-galactosidase activity and P-xylosidase activity, but shows no activity towards a-linked disaccharides or P-linked polymers, like cellulose.The purified P-glucosidase shows a remarkable thermostability with a half life of 85 h at 100°C and 13 h at 110°C.Hyperthermophilic microorganisms have their optimal growth temperature at 80-110°C [l]. With a few exceptions, they all belong to the domain of the Archaea [2]. The majority of these extreme thermophiles are strictly anaerobic and depend on the reduction of elemental sulfur (So) for growth. Due to its rapid growth and relatively high cell yields, Pyrococcusfuriosus is one of the best studied representatives of this group. It grows by a fermentative type of metabolism, using yeast extract, peptone, casein, starch, maltose or pyruvate as substrate. Acetate, H, and CO, are reported as the major end products [3,4]. In the presence of s", H,S is formed instead of H,. This reduction of So to H,S is apparently required only as a means to eliminate inhibition In order to utilize the different polymers, P furiosus harbours several hydrolytic enzyme activities. As many as 13 active proteinases have been demonstrated, both extracellular and intracellular [5-71. Furthermore, depending on the type of carbohydrates present in the growth medium, a-amylase, pullulanase or a-glucosidase activity has been detected [ 8,

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.

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.

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 lignocelluloseutilizing, biofuel-producing bacterium.Microbial hydrogen production from biomass has been recognized as an important source of renewable energy (15, 47). High-temperature microorganisms are well suited for production of biohydrogen from plant polysaccharides, as anaerobic fermentation is thermodynamically favored at elevated temperatures (17, 43). The extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903, a fermentative anaerobe initially isolated from wood in the flow of a thermal spring in New Zealand, first received attention because of its capacity to utilize cellulose at its optimal growth temperature, 70°C (37). Further work showed that C. saccharolyticus (i) can utilize a wide range of plant materials, including cellulose, hemicellulose, starch, and pectin, (ii) has a very high hydrogen yield (almost 4 mol of H 2 per mol of glucose) (14,20,48), and (iii) can ferment C 5 and C 6 sugars simultaneously. These features have led to the development of bioprocessing schemes based on C. saccharolyticus. For example, H 2 production is now being investigated using a two-step process in which H 2 and acetate are generated from biomass hydrolysates in one bioreactor and the acetate is fed to a second bioreactor and used by phototrophic organisms (Rhodobacter spp.) to produce additional H 2 in the presence of...

Coutilization of hexoses and pentoses derived from lignocellulose is an attractive trait in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H 2 -producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus growing on individual monosaccharides (arabinose, fructose, galactose, glucose, mannose, and xylose), mixtures of these sugars, as well as on xylan and xylogluco-oligosacchrides. C. saccharolyticus grew at approximately the same rate (t d , ϳ95 min) and to the same final cell density (1 ؋ 10 8 to 3 ؋ 10 8 cells/ml) on all sugars and sugar mixtures tested. In the monosaccharide mixture, although simultaneous consumption of all monosaccharides was observed, not all were utilized to the same extent (fructose > xylose/arabinose > mannose/glucose/galactose). Transcriptome contrasts for monosaccharide growth revealed minimal changes in some cases (e.g., 32 open reading frames [ORFs] changed >2-fold for glucose versus galactose), while substantial changes occurred for cases involving mannose (e.g., 353 ORFs changed >2-fold for glucose versus mannose). Evidence for catabolite repression was not noted for either growth on multisugar mixtures or the corresponding transcriptomes. Based on the whole-genome transcriptional response analysis and comparative genomics, carbohydrate specificities for transport systems could be proposed for most of the 24 putative carbohydrate ATP-binding cassette transporters and single phosphotransferase system identified in C. saccharolyticus. Although most transporter genes responded to individual monosaccharides and polysaccharides, the genes Csac_0692 to Csac_0694 were upregulated only in the monosaccharide mixture. The results presented here affirm the broad growth substrate preferences of C. saccharolyticus on carbohydrates representative of lignocellulosic biomass and suggest that this bacterium holds promise for biofuel applications.Cellulose and hemicellulose comprise ϳ70% (dry basis) of plant biomass (23) and, as such, these complex carbohydrates are promising targets for alternative energy feedstocks. For biofuel production, lignocellulose must be deconstructed into fermentable sugars by various combinations of physical, physical-chemical, chemical, and biological processing steps (33). The more efficiently and economically that this is accomplished, the more attractive this material becomes for alternative fuels (25,28,37,44). For this reason, there is great interest in identifying microorganisms that can minimize or eliminate the need for physical and chemical pretreatment of plantbased biomass and are at the same time capable of simultaneously fermenting the resulting pentoses and hexoses to biofuels (17).In environmental settings, microbial survival can be based on the capacity to utilize a variety of available carbohydrates as carbon and energy sources (19,22). When complex polysaccharides are involved, as is the case for plant-based biomass, extracellular glycoside hydrolases depolymerize potential...