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Recently, a hyphae-dispersed type of filamentous fungus Aspergillus oryzae was constructed via genetic engineering, and industrial applications are expected due to the ease of handling and to the level of protein production properties. In this study, we constructed cellulase-expressing strains using wild-type and hyphae-dispersed strains to investigate the correlation between protein productivity and metabolism. Compared with the original strain, the hyphae-dispersed cellulase-expressing strain showed elevated cellulase activity, rapid glucose consumption, increased mycelial dry weight, an increased expression of cellulase genes, and activated respiration activity. Comparative metabolomic analysis showed fewer metabolites in the glycolysis and TCA cycles in the dispersed strains than in the original strains. These results indicate that the flux of carbohydrate metabolism in the hyphae-dispersed strains is smoother than that in the original strains. Such efficient metabolic flux would contribute to efficient energy conversion and to sufficient energy supply to anabolisms, such as mycelial growth and protein production. Our findings suggest that the hyphae-dispersed strains could be a useful host not only for protein production but also for the biological production of various chemicals such as organic acids.

Cytochrome c′ is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c′ (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.

Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals. We successfully improved productivity by redirecting pyruvate metabolism in the aerobic filamentous fungus Aspergillus oryzae via the deletion of two genes that encode pyruvate decarboxylase and mitochondrial pyruvate carriers. Production of ethanol as a major byproduct was completely inhibited, and the limited translocation of pyruvate into the mitochondria shifted the metabolism from respiration for energy conversion to the effective production of lactate or 2,3-butandiole, even under aerobic conditions. Metabolomic and transcriptomic analyses showed an emphasis on glycolysis and a repressed TCA cycle. Although the dry mycelial weights of the deletion mutants were reduced compared with those of wild type, the titer and yields of the target products were drastically increased. In particular, the redirection of pyruvate metabolism shifted from anabolism for biomass production to catabolism for the production of target chemicals. Conclusively, our results indicate that the redirection of pyruvate metabolism is a useful strategy in the metabolic engineering of aerobic microorganisms.

Methanogens capable of accepting electrons from Fe

Cytochrome c′ (CP) is a gas-binding homo-dimeric heme protein. Mesophilic Allochromatium vinosum CP (AVCP) and thermophilic Hydrogenophilus thermoluteolus CP (PHCP) have high sequence and structure similarities. AVCP is known to exhibit a dimer–monomer transition upon CO binding/dissociation, whereas detailed CO-binding properties of PHCP remain unrevealed. Here, we found that the CO-binding affinity of wild-type PHCP is lower than that of AVCP, and the PHCP dimer does not dissociate to monomers under CO-saturated reduced conditions. The CO-binding affinity of PHCP increased by mutations in the subunit–subunit interface (F11T, T18F, or F71D). The T18F, F71D, and T18F/F71D PHCP variants exhibited similar dimer–monomer transitions upon CO binding/dissociation to that of AVCP, although the F11T variant did not. The simulated structures of the PHCP variants revealed that the T18F and F71D mutations caused rearrangement in the subunit–subunit interface, whereas the F11T mutation did not, indicating that the effective dimer–monomer transitions upon CO binding/dissociation are induced by the rearrangement in the subunit–subunit interface. The present results indicate that subunit–subunit interface mutation of oligomeric proteins is a useful approach in the adjustment of protein stability and ligand binding affinity, leading to a change in the quaternary structure.

Hydrogenophilus thermoluteolus, Thermochromatium tepidum, and Allochromatium vinosum, which grow optimally at 52, 49, and 25 °C, respectively, have homologous cytochromes c′ (PHCP, TTCP, and AVCP, respectively) exhibiting at least 50% amino acid sequence identity. Here, the thermal stability of the recombinant TTCP protein was first confirmed to be between those of PHCP and AVCP. Structure comparison of the 3 proteins and a mutagenesis study on TTCP revealed that hydrogen bonds and hydrophobic interactions between the heme and amino acid residues were responsible for their stability differences. In addition, PHCP, TTCP, and AVCP and their variants with altered stability similarly bound nitric oxide and carbon oxide, but not oxygen. Therefore, the thermal stability of TTCP together with PHCP and AVCP can be tuned through specific interactions around the heme without affecting their gas-binding function. These cytochromes c′ will be useful as specific gas sensor proteins exhibiting a wide thermal stability range.

Purpose To develop an automated analytic module for calculation of tumor growth rate from serial CT scans and to apply the module and evaluate reproducibility in a pilot cohort of advanced NSCLC patients with EGFR mutations treated with EGFR tyrosine kinase inhibitors. Materials and methods The module utilized a commercially available image-processing workstation equipped with a validated tumor volume measurement tool. An automated analytic software module was programmed with the capability to record and display serial tumor volume changes and to calculate tumor volume growth rate over time and added to the workstation. The module was applied to evaluate the tumor growth rate in a pilot cohort of 24 EGFR-mutant patients treated with EGFR inhibitors, and reproducibility references as tested by two independent thoracic radiologists. Results The module analyzed chest CT scans from 24 patients (5 males, 19 females; median age: 61) with a median of 8 scans per patient, totaling 227 scans and provided a graphical display with an automated and instant calculation of tumor growth rate after the nadir volume for each patient. High inter and intraobserver agreements were noted for tumor growth rates, with concordance correlation coefficients of 0.9323 and 0.9668, respectively. Interpretation of slow versus fast tumor growth using previously identified threshold of ≤0.15/month had a perfect interobserver agreement (κ = 1.00), and an excellent intraobserver agreement (κ = 0.895). Conclusions The present study describes the development of an image analytic module for assessing tumor growth rate and the data demonstrates the functionality and reproducibility of the module in a pilot cohort of EGFR-mutant NSCLC patients treated with EGFR-TKI. The image analytic module is an initial step for clinical translation of the tumor growth rate approach to guide cancer treatment in precision oncology.

Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe0), are largely unknown. In this study, Fe0-corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe0-corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed “MIC island”, encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe0, and oxidize it, generating hydrogen gas: Fe0 + 2 H+ → Fe2+ + H2. Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis.

Filamentous fungi exhibit versatile abilities, including organic acid fermentation, protein production, and secondary metabolism, amongst others, and thus have applications in the medical and food industries. Previous genomic analyses of several filamentous fungi revealed their further potential as host microorganisms for bioproduction. Recent advancements in molecular genetics, marker recycling, and genome editing could be used to alter transformation and metabolism, based on optimized design carbolated with computer science. In this review, we detail the current applications of filamentous fungi and describe modern molecular genetic tools that could be used to expand the role of these microorganisms in bioproduction. The present review shed light on the possibility of filamentous fungi as host microorganisms in the field of bioproduction in the future.

Reversible denaturation of Pseudomonas aeruginosa cytochrome c551 (PAc551) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc551, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.

This work was performed under the Cooperative Research Program of the “Network Joint Research Center for Materials and Devices”.

Microbiologically influenced corrosion (MIC) involves two types of microorganisms; one causes corrosion by directly consuming an electron from a metal, while the other produces corrosive substances, such as acids or sulfides, thereby indirectly inducing corrosion. In this chapter, the electron flow in the direct type of MIC and the possibility of its application is discussed, and the predicted mechanisms of such corrosion will also be described. Since an iron-corrosive methanogen produces methane and hydrogen during corrosion, and not any corrosive substances, the electron consumption can easily be estimated from gas production linked with energy conversion. Moreover, the adhesion of cells to the metal surface is essential, and notably, it accelerates the electron flow rate compared to abiotic chemical corrosion. Furthermore, when iron-corrosive methanogens are in the presence of sulfate-reducing bacteria (SRB), the electron flow rate is further accelerated; therefore, SRB play a role as enhancers. These findings suggest that electron-consuming methanogens could be useful for microbial electrosynthesis from renewable energy sources; therefore, the identification of truly corrosive microorganisms is crucial.

The phenomenon of microorganisms affecting corrosion of metal materials is well-known and refers to microbiologically influenced corrosion (MIC). MIC has been related to considerable corrosion costs and causes environmental pollution via the leakage of chemicals and petroleum oil into natural environments. Although sulfate-reducing bacteria are the most widely known corrosive microorganisms, novel types have been recently reported. Notably, it has been reported that microorganisms can use electrons directly from metal materials, which is a novel corrosion mechanism. Because almost of natural environments are nutrient-poor environments, the corrosion by such electron-consuming microorganism may affect to actively the corrosion under nutrient-poor environments. Based on the novel knowledge on the types of iron-corrosive microorganisms, it is imperative that we heed the potential effects these microorganisms can have on our environment and attempt to prevent them. Unfortunately, little is known about MIC, no rapid and accurate diagnosis method exists, and it is uncertain whether anticorrosion measures and post-corrosion treatment are sufficient to mitigate the effects. To overcome this situation, there is an urgent need to elucidate the broad spectrum of corrosive microorganisms and establish diagnostic techniques. In this chapter, I will describe a brief history of MIC research, introduce latest knowledge of novel corrosive microorganisms, and propose further research and endeavors for the future.

Recent microbiology research, particularly studies of electrochemically active microorganisms, has greatly advanced in the field of electromicrobiology. Studies of living organisms and electrons have a long history, and electron transport chains in respiration and photosynthesis have been widely examined. In addition, “electron-based bioscience” such as extracellular electron transfer and electrotrophy and “electron-based biotechnology” such as microbial fuel cells and microbial electrosynthesis have been developed in modern microbiology. However, it is necessary to move past traditional culture technologies and accumulate raw biochemical data that does not depend only on genomic analysis information for the future development of (electro)microbiology. In this chapter, I provide a brief history of electromicrobiology and the technologies that has supported the development of this field, as well as suggest remaining issues in modern microbiology.

Monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea (SVcytc5) was stable against heat and denaturant compared with the homologous protein from shallow-sea piezo-sensitive Shewanella livingstonensis (SLcytc5). Here, the SVcytc5 crystal structure revealed that the Lys-50 side chain on the flexible loop formed a hydrogen bond with heme whereas that of corresponding hydrophobic Leu-50 could not form such a bond in SLcytc5, which appeared to be one of possible factors responsible for the difference in stability between the two proteins. This structural insight was confirmed by a reciprocal mutagenesis study on the thermal stability of these two proteins. As SVcytc5 was isolated from a deep-sea piezophilic bacterium, the present comparative study indicates that adaptation of monomeric SVcytc5 to high pressure environments results in stabilization against heat.

Deep-sea Shewanella violacea 5′-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions. Abbreviations: NTase: 5′-nucleotidase; SANTase: Shewanella amazonensis 5′-nucleotidase; SVNTase: Shewanella violacea 5′-nucleotidase; CD: circular dichroism

The stability of dimeric cytochrome c' from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit-subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c' from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c' from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure.

A variety of extreme environments, characterized by extreme values of various physicochemical parameters (temperature, pressure, salinity, pH, and so on), are found on Earth. Organisms that favorably live in such extreme environments are called extremophiles. All living organisms, including extremophiles, must acquire energy to maintain cellular homeostasis, including extremophiles. For energy conversion in harsh environments, thermodynamically useful reactions and stable biomolecules are essential. In this review, I briefly summarize recent studies of extreme environments and extremophiles living in these environments and describe energy conversion processes in various extremophiles based on my previous research. Furthermore, I discuss the correlation between the biological system of electrotrophy, a third biological energy acquisition system, and the mechanism underlying microbiologically influenced corrosion. These insights into energy conversion in extremophiles may improve our understanding of the “limits of life”. Abbreviations: PPi: pyrophosphate; PPase: pyrophosphatase; ITC: isothermal titration microcalorimetry; SVNTase: Shewanella violacea 5ʹ-nucleotidase; SANTase: Shewanella amazonensis 5ʹ-nucleotidase

Microbiologically influenced corrosion (MIC) of metallic materials imposes a heavy economic burden. The mechanism of MIC of metallic iron (Fe 0 ) under anaerobic conditions is usually explained as the consumption of cathodic hydrogen by hydrogenotrophic microorganisms that accelerates anodic Fe 0 oxidation. In this study, we describe Fe 0 corrosion induced by a nonhydrogenotrophic nitrate-reducing bacterium called MIC1-1, which was isolated from a crude-oil sample collected at an oil well in Akita, Japan. This strain requires specific electron donor-acceptor combinations and an organic carbon source to grow. For example, the strain grew anaerobically on nitrate as a sole electron acceptor with pyruvate as a carbon source and Fe 0 as the sole electron donor. In addition, ferrous ion and l -cysteine served as electron donors, whereas molecular hydrogen did not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MIC1-1 was a member of the genus Prolixibacter in the order Bacteroidales . Thus, Prolixibacter sp. strain MIC1-1 is the first Fe 0 -corroding representative belonging to the phylum Bacteroidetes . Under anaerobic conditions, Prolixibacter sp. MIC1-1 corroded Fe 0 concomitantly with nitrate reduction, and the amount of iron dissolved by the strain was six times higher than that in an aseptic control. Scanning electron microscopy analyses revealed that microscopic crystals of FePO 4 developed on the surface of the Fe 0 foils, and a layer of FeCO 3 covered the FePO 4 crystals. We propose that cells of Prolixibacter sp. MIC1-1 accept electrons directly from Fe 0 to reduce nitrate.

AVCP cytochrome c′ from mesophilic Allochromatium vinosum exhibits lower stability than a thermophilic counterpart, Hydrogenophilus thermoluteolus cytochrome c′ (PHCP), in which the six specific amino acid residues that are not conserved in AVCP are responsible for its stability. Here we measured the stability of AVCP variants carrying these specific residues instead of the original AVCP ones. Among the six single AVCP variants, all of which formed a dimeric structure similar to that of the wild-type, three were successfully stabilized compared with the wild-type, while one showed lower stability than the wild-type. In addition, the most stabilized and destabilized AVCP variants could bind CO, similar to the wild-type. These results indicated that mesophilic AVCP could be stabilized through specific three mutations modeled by the thermophilic counterpart, PHCP, without changing the CO binding ability.

Two cytochromes c5 (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c551 (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation. Deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.

Across the Earth, a variety of organisms inhabit both mild and extreme environments wherever liquid water is available. Among these, extremophilic microorganisms, termed extremophiles, favorably live in extreme environments by adapting their physiological properties. Such extremophiles must acquire energy in order to maintain their cell homeostasis, which is functionally similar to organisms living in mild environments. Numerous enzyme proteins from extremophiles such as thermophiles, psychrophiles, piezophiles, and halophiles have been investigated to date, revealing both unity and diversity in their biochemical and structural biological features through comparison with their homologous counterpart enzymes from organisms living in mild environments. In this chapter, we aim to summarize the biochemical and thermodynamic aspects of enzymes related to the energy conversion that occurs in extremophiles. The obtained insights into extremophilic enzymes related to energy conversion thereby allow us to decipher the mechanistic fundamentals of these protein machineries.

Reversible denaturation of Pseudomonas aeruginosa cytochrome c

Microorganisms form biofilms on various surfaces in different environments and cause diverse problems such as biofouling and biocorrosion. Biofilm formation includes at least three steps: initial adhesion, colonisation, and development. In this study, we enriched bacterial populations capable of adhering to a metallic iron surface. The community structure of the enriched populations differed from the structures of those without enrichment. From the enriched populations, we isolated six strains capable of adhering to the surface of metallic iron. A Marinobacter hydrocarbonoclasticus strain M1 showed the highest adhesion rate of approximately 4×106 cells cm−2 min−1. Atomic force microscopy showed that a high density monolayer of cells was formed within a few minutes of adherence of single cells of this strain to the surface of metallic iron.

Apo-cytochomes c without heme are usually unstructured. Here we showed that apo-form of thermophilic Hydrogenophilus thermoluteolus cytochrome c’ (PHCP) was a monomeric protein with high helix content. Apo-PHCP was thermally stable, possibly due to the hydrophobic residues and ion pairs. PHCP is the first example of a structured apo-cytochrome c’, which will expand our view of hemoprotein structure formation.

In this study, it was shown that how rheology and fluid dynamics effected the behavior of cell growth and L-Lactic acid productivity during cultivation of filamentous fungi (Aspergillus oryzae) in a stirred-tank. Submerged culture of A. oryzae was a shear-thinning fluid with complex rheology and morphology. It is a shear-sensitive fluid. The scope of this study, was enhancing the gas-liquid flow of fermentation fluid to improve the liquid-gas mass transfer, L-lactic acid production and stagnant zone reduction at low energy consumption by effective shear rate generation using multi-large blade agitator with global axial pattern of mixing. Due to this reason, the advantage of Fullzone® (FZ), and impeller was investigated. Experimental results when using multi-large and multi-radial impeller, showed that the same volumetric power consumption of the dissolved oxygen concentration, Re, macro and micro-morphology size, biomass and L-lactic acid production when using multi large impeller was higher and more stable than that of the multi radial impeller. Most of above positive properties were due to the reduction in viscosity and apparent consistence index of fermented fluid.

The soluble protein fraction of the extremely halophilic archaeon Haloarcula japonica exhibits substantial inorganic pyrophosphate (PPi) hydrolysis activity in the presence of 2–4 M NaCl (Wakai et al, J Biol Chem 288:29247–29251, 2013), which provides high ionic strength (2–4). In this study, much higher PPi hydrolysis activity was unexpectedly detected, even with 0 M NaCl in the presence of 100–200 mM MgSO4, providing a much lower ionic strength of 0.4–0.8, in the same protein fraction. Na+ and Mg2+ ions were required for activity under high and low ionic strength conditions, respectively. A recombinant H. japonica pyrophosphatase (HjPPase) exhibited PPi hydrolysis activity with the same broad ionic strength range, indicating that the activity associated with such a broad ionic strength range could be attributed to a single enzyme. Thus, we concluded that the broad ionic strength range of HjPPase may contribute to adaptation for both Na+ and Mg2+ which are abundant but variable in the unstable living environments of H. japonica.

Summary Hydrogenophilus is a thermophilic, facultative chemoautotroph, which lives prevalently in high temperature geothermal niches. Despite the environmental distribution, little is known about its oxidative phosphorylation. Here, we show that inverted membrane vesicles derived from Hydrogenophilus thermoluteolus cells autotrophically cultivated with H2 formed a proton gradient on the addition of succinate, dl-lactate, and NADH, and exhibited oxidation activity toward these three organic compounds. These indicate the capability of mixotrophic growth of this bacterium. Biochemical analysis demonstrated that the same vesicles contained an F-type ATP synthase. The F1 sector of the ATP synthase purified from H. thermoluteolus membranes exhibited optimal ATPase activity at 65°C. Transformed Escherichia coli membranes expressing H. thermoluteolus F-type ATP synthase exhibited the same temperature optimum for the ATPase. These findings shed light on H. thermoluteolus oxidative phosphorylation from the aspects of membrane bioenergetics and ATPase biochemistry, which must be fundamental and advantageous in the biogeochemical cycles occurred in the high temperature geothermal niches.

Hydrogenophilus thermoluteolus cytochrome c′ (PHCP) has typical spectral properties previously observed for other cytochromes c′, which comprise Ambler’s class II cytochromes c. The PHCP protein sequence (135 amino acids) deduced from the cloned gene is the most homologous (55% identity) to that of cytochrome c′ from Allochromatium vinosum (AVCP). These findings indicate that PHCP forms a four-helix bundle structure, similar to AVCP. Strikingly, PHCP with a covalently bound heme was heterologously synthesized in the periplasm of Escherichia coli strains deficient in the DsbD protein, a component of the System I cytochrome c biogenesis machinery. The heterologous synthesis of PHCP by aerobically growing E. coli also occurred without a plasmid carrying the genes for Ccm proteins, other components of the System I machinery. Unlike Ambler’s class I general cytochromes c, the synthesis of PHCP is not dependent on the System I machinery and exhibits similarity to that of E. coli periplasmic cytochrome b562, a 106-residue four-helix bundle. Database The sequence data reported here have been deposited in the DDBJ database under accession no. AB617519.

Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A. ferrooxidans genome, A. thiooxidans and A. caldus had a single copy of form I cbbL gene in their genomes. A phylogenetic analysis based on deduced amino acid sequences from cbbL genes detected in the ARD treatment plant and their close relatives revealed that 89% of the total clones were affiliated with A. ferrooxidans. Clones loosely affiliated with the cbbL from A. thiooxidans NB1-3 or Thiobacillus denitrificans was also detected in the treatment plant. cbbL gene sequences of iron- or sulfur-oxidizing bacteria isolated from the ARD and the ARD treatment plant were not detected in the cbbL libraries from the treatment plant, suggesting the low frequencies of isolates in the samples.

A variety of pollutants are present in natural and artificial environments. These pollutants are divided into two groups, chemical and biological pollutants. They cause various problems such as metal corrosion, environmental damage, and human health issues. Chemical pollutants contain heavy metals, hydrocarbons, exhaust gases, and endocrine disruptors. Such pollutants may possibly be detected by using a biosensor. On the other hand, the main biological pollutants are microorganisms. Microorganisms in environments can be detected by using culture-dependent and molecular biology-dependent methods. In particular, this chapter focuses on the molecular biology-dependent evaluation for biological pollution. First I introduce some methods of gene analysis as molecular biology-dependent evaluation techniques. Then I discuss gene analysis for endocrine disruptor detection and the application of a biosensor. Materials may be exposed to various pollutions in their respective environments so a variety of evaluation techniques for different pollutants exist. Thus, the adoption of a proper evaluation technique would be useful for maintaining the quality of materials.

Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent K(m) values for sulfide and ubiquinone were 42 and 14 muM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells.

Background Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. Results Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. Conclusions We successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0600-4) contains supplementary material, which is available to authorized users.

Kraft pulp is a promising feedstock for bioproduction. The efficiency of kraft pulp saccharification was improved by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae. Application of the cellulase cocktail was demonstrated by simultaneous saccharification and fermentation, using kraft pulp and non-cellulolytic yeast. Such application would make possible to do an efficient production of other chemicals from kraft pulp.