Comparative Genome Analysis Between Aspergillus oryzae Strains Reveals Close Relationship Between Sites of Mutation Localization and Regions of Highly Divergent Genes among Aspergillus Species

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome.     

Acidophilic bacteria and archaea: acid stable biocatalysts and their potential applications

Acidophiles are ecologically and economically important group of microorganisms, which thrive in acidic natural (solfataric fields, sulfuric pools) as well as artificial man-made (areas associated with human activities such as mining of coal and metal ores) environments. They possess networked cellular adaptations to regulate pH inside the cell. Several extracellular enzymes from acidophiles are known to be functional at much lower pH than the cytoplasmic pH. Enzymes like amylases, proteases, ligases, cellulases, xylanases, α-glucosidases, endoglucanases, and esterases stable at low pH are known from various acidophilic microbes. The possibility of improving them by genetic engineering and directed evolution will further boost their industrial applications. Besides biocatalysts, other biomolecules such as plasmids, rusticynin, and maltose-binding protein have also been reported from acidophiles. Some strategies for circumventing the problems encountered in expressing genes encoding proteins from extreme acidophiles have been suggested. The investigations on the analysis of crystal structures of some acidophilic proteins have thrown light on their acid stability. Attempts are being made to use thermoacidophilic microbes for biofuel production from lignocellulosic biomass. The enzymes from acidophiles are mainly used in polymer degradation.     

The archaeon Pyrococcus horikoshii possesses a bifunctional enzyme for formaldehyde fixation via the ribulose monophosphate pathway

Pyrococcus horikoshii OT3, a hyperthermophilic and anaerobic archaeon, was found to have an open reading frame (PH1938) whose deduced amino acid sequence of the N-terminal and C-terminal halves showed significant similarity to two key enzymes of the ribulose monophosphate pathway for formaldehyde fixation in methylotrophic bacteria, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), respectively. The organism constitutively produced the encoded protein and exhibited activity of the sequential HPS- and PHI-mediated reactions in a particulate fraction. The full-length gene encoding the hybrid enzyme, the sequence corresponding to the HPS region, and the sequence corresponding to the PHI region were expressed in Escherichia coli and were found to produce active enzymes, rHps-Phi, rHps, or rPhi, respectively. Purified rHps-Phi and rHps were found to be active at the growth temperatures of the parent strain, but purified rPhi exhibited significant susceptibility to heat, suggesting that thermostability of the PHI moiety of the bifunctional enzyme (rHps-Phi) resulted from fusion with HPS. The bifunctional enzyme catalyzed the sequential reaction much more efficiently than a mixture of rHps and rPhi. These and other biochemical characterizations of the PH1938 gene product suggest that the ribulose monophosphate pathway plays a significant role in the archaeon under extreme environmental conditions.     

Evolutionary process of amino acid biosynthesis in Corynebacterium at the whole genome level

Corynebacterium glutamicum, which is the closest relative of Corynebacterium efficiens, is widely used for the large scale production of many kinds of amino acids, particularly glutamic acid and lysine, by fermentation. Corynebacterium diphtheriae, which is well known as a human pathogen, is also closely related to these two species of Corynebacteria, but it lacks such productivity of amino acids. It is an important and interesting question to ask how those closely related bacterial species have undergone such significant functional differentiation in amino acid biosynthesis. The main purpose of the present study is to clarify the evolutionary process of functional differentiation among the three species of Corynebacteria by conducting a comparative analysis of genome sequences. When Mycobacterium and Streptomyces were used as out groups, our comparative study suggested that the common ancestor of Corynebacteria already possessed almost all of the gene sets necessary for amino acid production. However, C. diphtheriae was found to have lost the genes responsible for amino acid production. Moreover, we found that the common ancestor of C. efficiens and C. glutamicum have acquired some of genes responsible for amino acid production by horizontal gene transfer. Thus, we conclude that the evolutionary events of gene loss and horizontal gene transfer must have been responsible for functional differentiation in amino acid biosynthesis of the three species of Corynebacteria.     

An Improved Cosmid Vector for the Nested Deletion Method Using the Bacteriophage T3 DNA Packaging System

We constructed a new cosmid vector suitable for the previouslydeveloped nested deletion method which used the in vitro DNApackaging system of bacteriophage T3. The first step of thismethod is linearization of a cosmid clone to be packaged, andwe previously introduced cleavage at the cos site using -Terminase,but optimization of the reaction conditions was required forcomplete digestion because of its instability. In the newlyconstructed vector, pAT5, the sites of 4 different restrictionenzymes, Sse8387I, Asc I, Fse I and Pme I, each of which recognizesan 8-bp sequence (8-base cutter) were introduced in the vicinityof the cos site. In addition, the species of restriction sitesfor cloning were increased to broaden its application. The cosmidclone constructed by this new vector could be linearized atone of the 8-base cutter sites which are assumed to rarely occurin the genome, and followed by in vitro packaging, nested deletionclones were successfully prepared.     

Complete Genome Sequence of an Aerobic Hyper-thermophilic Crenarchaeon, Aeropyrum pernix K1

The complete sequence of the genome of an aerobic hyper-thermophiliccrenarchaeon, Aeropyrum pernix K1, which optimally grows at95°C, has been determined by the whole genome shotgun methodwith some modifications. The entire length of the genome was1,669,695 bp. The authenticity of the entire sequence was supportedby restriction analysis of long PCR products, which were directlyamplified from the genomic DNA. As the potential protein-codingregions, a total of 2,694 open reading frames (ORFs) were assigned.By similarity search against public databases, 633 (23.5%) ofthe ORFs were related to genes with putative function and 523(19.4%) to the sequences registered but with unknown function.All the genes in the TCA cycle except for that of alpha-ketoglutaratedehydrogenase were included, and instead of the alpha-ketoglutaratedehydrogenase gene, the genes coding for the two subunits of2-oxoacid:ferredoxin oxidoreductase were identified. The remaining1,538 ORFs (57.1%) did not show any significant similarity tothe sequences in the databases. Sequence comparison among theassigned ORFs suggested that a considerable member of ORFs weregenerated by sequence duplication. The RNA genes identifiedwere a single 16S–23S rRNA operon, two 5S rRNA genes and47 tRNA genes including 14 genes with intron structures. Allthe assigned ORFs and RNA coding regions occupied 89.12% ofthe whole genome. The data presented in this paper are availableon the internet homepage (http://www.mild.nite.go.jp).     

The Sulfolobus tokodaii gene ST1704 codes highly thermostable glucose dehydrogenase

NAD(P)-dependent glucose-1-dehydrogenase (GDH) has been used for glucose determination and NAD(P)H production in bioreactors. Thermostable glucose dehydrogenase exhibits potential advantage for its application in biological processes. The function of the putative GDH gene (ST1704, 360-encoding amino acids) annotated from the total genome analysis of a thermoacidophilic archeaon Sulfolobus tokodaii strain 7 was investigated to develop more effective application of GDH. The gene encoding S. tokodaii GDH was cloned and the activity was expressed in Escherichia coli, which did not originally possess GDH. This shows that the gene (ST1704) codes the sequence of GDH. The enzyme was effectively purified from the recombinant E. coli with three steps containing a heat treatment and two successive chromatographies. The native enzyme (molecular mass: 160 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme utilized both NAD and NADP as the coenzyme. The maximum activity for glucose oxidation in the presence of NAD was observed around pH 9 and 75 °C in the presence of 20 mM Mg²⁺. The enzyme showed broad substrate specificity: several monosaccarides such as 6-deoxy- -glucose, 2-amino-2-deoxy- -glucose and -xylose were oxidized as well as -glucose as the electron donor. -Mannose, -ribose and glucose-6-phosphate were inert as the donor. The enzyme showed high thermostability: remarkable loss of activity was not observed up to 80 °C by incubation for 15 min at pH 8.0. In addition, the enzyme was stable in a wide pH range of 5.0–10.5 by incubation at 37 °C. From the steady-state kinetic analysis, the enzyme reaction of -glucose oxidation proceeds via a sequential ordered Bi–Bi mechanism: NAD and -glucose bind to the enzyme in this order and then -glucono-1,5-lactone and NADH are released from the enzyme in this order. The amino acid sequence alignment showed that S. tokodaii GDH exhibited high homology with the Sulfolobus solfataricus hypothetical glucose dehydrogenase and a Thermoplasma acidophilum one.     

Three Proliferating Cell Nuclear Antigen-Like Proteins Found in the Hyperthermophilic Archaeon Aeropyrum pernix: Interactions with the Two DNA Polymerases

Proliferating cell nuclear antigen (PCNA) is an essential component in the eukaryotic DNA replication machinery, in which it works for tethering DNA polymerases on the DNA template to accomplish processive DNA synthesis. The PCNA also interacts with many other proteins in important cellular processes, including cell cycle control, DNA repair, and an apoptotic pathway in the domain EUCARYA: We identified three genes encoding PCNA-like sequences in the genome of Aeropyrum pernix, a crenarchaeal archaeon. We cloned and expressed these genes in Escherichia coli and analyzed the gene products. All three PCNA homologs stimulated the primer extension activities of the two DNA polymerases, polymerase I (Pol I) and Pol II, identified in A. pernix to various extents, among which A. pernix PCNA 3 (ApePCNA3) provided a most remarkable effect on both Pol I and Pol II. The three proteins were confirmed to exist in the A. pernix cells. These results suggest that the three PCNAs work as the processivity factor of DNA polymerases in A. pernix cells under different conditions. In Eucarya, three checkpoint proteins, Hus1, Rad1, and Rad9, have been proposed to form a PCNA-like ring structure and may work as a sliding clamp for the translesion DNA polymerases. Therefore, it is very interesting that three active PCNAs were found in one archaeal cell. Further analyses are necessary to determine whether each PCNA has specific roles, and moreover, how they reveal different functions in the cells.     

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of Sulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.