Draft Genome Sequence of NYR20, a Red Pigment-Secreting Mutant of Saccharomyces cerevisiae.

A Saccharomyces cerevisiae mutant strain, NYR20, produces a red pigment owing to adenine auxotrophy. Unlike other yeast adenine biosynthetic mutants, this strain not only produces but also secretes this pigment. Here, we report the NYR20 draft genome sequence, thereby advancing our understanding of pigment secretion mechanisms.

Draft Genome Sequence of Glycoside Hydrolase-Producing Trichoderma asperellum Strain IC-1.

Glycoside hydrolases capable of degrading lignocellulose are important for effectively utilizing cellulosic biomass as a next-generation chemical resource. Trichoderma asperellum IC-1 produces various glycoside hydrolases. Here, we report a draft genome sequence of T. asperellum IC-1 to better understand its gene structures and gene regulatory mechanisms.

Draft Genome Sequence of Saccharomyces cerevisiae Strain P-684, Isolated from Prunus verecunda.

Saccharomyces cerevisiae strain P-684 is a yeast isolated from the flowers of Prunus verecunda 'Antiqua,' producing high quantities of malic and succinic acids in sake brewing. Here, we report the draft genome sequence of P-684, enlightening the mechanisms of biosynthesis of these organic acids by this strain.

Draft Genome Sequence of the Aspergillus terreus High-Itaconic-Acid-Productivity Strain IFO6365.

Itaconic acid is an important organic acid used in the chemical industry. Aspergillus terreus strain IFO6365 is one of the highest-yielding itaconic acid-producing wild-type strains. Here, we report the draft genome sequence of IFO6365, enhancing the understanding of the role and biosynthesis of itaconic acid in this fungus.

Effect of Escherichia coli on phospholipid monolayers: surface tensiometry and Brewster angle microscopy measurements.

The effect of Escherichia coli (E. coli) cells on two phospholipids [dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC)] monolayers at the surface of a 1.5 wt% NaCl salt solution has been investigated using surface tension measurement and Brewster angle microscopy. The results showed that a DPPC monolayer that has an elastic structure was changed in morphology by interaction with E. coli cells, whereas a DMPC monolayer that has an expandable structure did not change in morphology. In particular, the morphology changed significantly around the liquid-expanded (LE)-liquid-condensed (LC) phase transition point for the DPPC monolayer. It was found that the LE-LC phase transition range in a DPPC monolayer was sensitive to influence from the outside of the monolayer such as the action of E. coli cells. Such a monolayer has the potential for application as a membrane sensor for detecting a small amount of bacteria in a short time.

Draft Genome Sequence of Aspergillus terreus High-Itaconic-Acid-Productivity Mutant TN-484.

Itaconic acid is an important organic acid used in the chemical industry. Aspergillus terreus strain TN-484 is a high-itaconic-acid-productivity mutant derived from strain IFO6365. Here, we report the draft genome sequence of strain TN-484, advancing the understanding of the biosynthesis of itaconic acid in filamentous fungi.

Draft Genome Sequence of Saccharomyces cerevisiae Strain Pf-1, Isolated from Prunus mume.

Saccharomyces cerevisiae strain Pf-1 is a yeast isolated from Prunus mume; it potentially can be used to produce wine and traditional Japanese sake. Here, we report the draft genome sequence of this strain. The genomic information will provide a deeper understanding of the brewing characteristics of this strain.

Draft Genome Sequence of Saccharomyces cerevisiae Strain Hm-1, Isolated from Cotton Rosemallow.

Saccharomyces cerevisiae strain Hm-1 is a yeast isolated from the flower of cotton rosemallow. This yeast is used for the production of Seishu, a traditional Japanese refined sake. Here, we report the strain's draft genome sequence. With this genomic information, the brewing characteristics of the strain can be better understood.

Improved reaction pattern of an endoglycanase from Paenibacillus cookii for chitosan oligosaccharide production.

The reaction pattern of an endoglycanase from Paenibacillus cookii SS-24 (Pgl8A) was improved to facilitate chitosan oligosaccharide production. Based on the sequence alignment with chitosanase of a known structure, we performed site-directed mutagenesis of possible substrate-binding residues in Pgl8A. The mutants were expressed in Escherichia coli cells, and their cellulase and chitosanase activities were then characterised. Our results indicated that three amino acid residues (W139, W208 and E285) were important for the substrate specificity of Pgl8A. D156 and Y390 were also essential for the cellulase and chitosanase activities of Pgl8A. The products of chitosan degradation by W139A, W208A and E285Q mutants were different from those by the wild type. A chitosan pentamer accumulated following chitosan degradation by W139A, W208A and E285Q mutants. Thus, the mutants obtained in this study are potentially useful for the production of biofunctional chitosan oligosaccharides.

Improvement of the transcriptional strength of baculovirus very late polyhedrin promoter by repeating its untranslated leader sequences and coexpression with the primary transactivator.

Modified polyhedrin promoter (Ppolh) was designed by repeating burst sequences (BSs) and adopted to overexpress rat α2,6-sialyltransferase (ST6Gal I) in silkworm. Modified Ppolh of five BSs with VLF-1 coexpression yielded 2.9 U/ml ST6Gal I activity and 32.5 mU/mg specific activity, which was 1.7- and 2.3-fold higher, respectively compared to Ppolh.

XlnR-independent signaling pathway regulates both cellulase and xylanase genes in response to cellobiose in Aspergillus aculeatus.

The expression levels of the cellulase and xylanase genes between the host strain and an xlnR disruptant were compared by quantitative RT-PCR (qPCR) to identify the genes controlled by XlnR-independent signaling pathway. The cellulose induction of the FI-carboxymethyl cellulase (cmc1) and FIb-xylanase (xynIb) genes was controlled by XlnR; in contrast, the cellulose induction of the FIII-avicelase (cbhI), FII-carboxymethyl cellulase (cmc2), and FIa-xylanase (xynIa) genes was controlled by an XlnR-independent signaling pathway. To gain deeper insight into the XlnR-independent signaling pathway, the expression profile of cbhI was analyzed as a representative target gene. Cellobiose together with 1-deoxynojirimycin (DNJ), a glucosidase inhibitor, induced cbhI the most efficiently among disaccharides composed of ß-glucosidic bonds. Furthermore, cellobiose with DNJ induced the transcription of cmc2 and xynIa, whereas cmc1 and xynIb were not induced. GUS reporter fusion analyses of truncated and mutated cbhI promoters revealed that three regions were necessary for effective cellulose-induced transcription, all of which contained the conserved sequence 5'-CCGN(2)CCN(7)G(C/A)-3' within the CeRE, which has been identified as the upstream activating element essential for expression of eglA in A. nidulans (Endo et al. 2008). The data therefore delineate a pathway in which A. aculeatus perceives the presence of cellobiose, thereby activating a signaling pathway that drives cellulase and hemicellulase gene expression under the control of the XlnR-independent regulation through CeRE.

Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme.

An endoglycanase gene of Paenibacillus cookii SS-24 was cloned and sequenced. This Pgl8A gene had an open reading frame of 1,230 bp that encoded a putative signal sequence (31 amino acids) and mature enzyme (378 amino acids: 41,835 Da). The enzyme was most homologous to a ß-1,3-1,4-glucanase of Bacillus circulans WL-12 with 84% identity. The recombinant enzyme hydrolyzed carboxymethyl cellulose, swollen celluloses, chitosan and lichenan but not Avicel, chitin powder or xylan. With chitosan as the substrate, the optimum temperature and hydrolysis products of the recombinant enzyme varied at pH 4.0 and 8.0. This is the first report that characterizes chitosanase activity under different pH conditions.

Enhanced gene expression in insect cells and silkworm larva by modified polyhedrin promoter using repeated Burst sequence and very late transcriptional factor-1.

The Burst of expression from polyhedrin (polh) promoter during very late phase of baculovirus infection requires a sequence located between TAAG and the translation initiation site, typically referred to as burst sequence (BS). The expression of polh promoter is stimulated by specifically binding of very late transcriptional factor 1 (VLF-1) to BS. In order to enhance the production of recombinant proteins the polh promoter was modified via a multiple BS bacmid system in which the number of BSs was increased. Compared to an expression from a normal polh promoter, ß-glucuronidase (GUS) activity in High Five insect cells was three times higher with a modified polh promoter containing two BSs. Using a modified polh promoter that contains nine BSs in silkworm expression system, ß1-3-N-acetylglucosaminyltransferase 2 (ß3GnT2) activity per larva was 6.8-fold higher than control. Furthermore, the co-expression of modified promoters along with VLF-1-enhanced ß3GnT activity. Thus, an increased optimal number of BS and its co-expression with VLF-1 leads to the production of higher level of gene expression in insect cells and silkworm larvae. This new modified promoter engineered in the current study is the strongest promoter for overexpressing foreign proteins in an eukaryotic cell and system, thus leading a progress in baculovirus-insect cell and silkworm biotechnology.

Biotechnological production of itaconic acid and its biosynthesis in Aspergillus terreus.

More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields--dental, ophthalmic, and drug delivery--to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.

Molecular chaperone-assisted production of human alpha-1,4-N-acetylglucosaminyltransferase in silkworm larvae using recombinant BmNPV bacmids.

In this study, human alpha-1,4-N-acetylglucosaminyltransferase (alpha4GnT) fused with GFP(uv) (GFP(uv)-alpha4GnT) was expressed using both a transformed cell system and silkworm larvae. A Tn-pXgp-GFP(uv)-alpha4GnT cell line, isolated after expression vector transfection, produced 106 mU/ml of alpha4GnT activity in suspension culture. When Bombyx mori nucleopolyhedrovirus containing a GFP(uv)-alpha4GnT fusion gene (BmNPV-CP (-)/GFP(uv)-alpha4GnT) bacmid was injected into silkworm larvae, alpha4GnT activity in larval hemolymph was 352 mU/ml, which was 3.3-fold higher than that of the Tn-pXgp-GFP(uv)-alpha4GnT cell line. With human calnexin (CNX) or human immunoglobulin heavy chain-binding protein (BiP, GRP78) coexpressed under the control of the ie-2 promoter, alpha4GnT activity in larval hemolymph increased by 1.4-2.0-fold. Moreover, when BmNPV-CP (-)/GFP(uv)-alpha4GnT bacmid injection was delayed for 3 h after BmNPV-CP (-)/CNX injection, the alpha4GnT activity increased significantly to 922 mU/ml, which was 8.7-fold higher than that of the Tn-pXgp-GFP(uv)-alpha4GnT cell line. Molecular chaperone assisted-expression in silkworm larvae using the BmNPV bacmid is a promising tool for recombinant protein production. This system could lead to large-scale production of more complex recombinant proteins.

Development of a homologous transformation system for Aspergillus aculeatus based on the sC gene encoding ATP-sulfurylase.

A homologous transformation system was developed using the endogenous ATP-sulfurylase gene, AasC, as a selectable marker in Aspergillus aculeatus. Spontaneous mutation was proved to be beneficial in isolating AasC-deficient mutants. Molecular analysis of sC(+) transformants revealed that the frequency of single copy integration at ATP-sulfurylase locus was more than 40%.

Importance of malate synthase in the glyoxylate cycle of Ashbya gossypii for the efficient production of riboflavin.

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.

Structure of Cu/Zn superoxide dismutase from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6.

The deep-sea yeast Cryptococcus liquefaciens strain N6 shows high tolerance towards heavy metals, and can grow in the presence of high concentrations of copper ions. Enzymatic analysis indicated that copper ions induced the Cu/Zn superoxide dismutase activity of strain N6 (Cl-SOD1). In this study, the 1.2A resolution crystal structure of Cl-SOD1 has revealed several significant residue substitutions compared to the other Cu/Zn SODs. In the electrostatic loop, notably, His135 and Pro136 replace the well-conserved linear residues, while Thr133 substitutes a highly conserved glycine. The electrostatic loop has been shown to be involved in the copper uptake process, and these substitutions have caused an inward dragging of the turn region of the loop. As the introduction of proline and abolishment of glycine decrease loop flexibility, this structural reorganization may have helped stabilize the loop conformation, possibly resulting in more efficient copper uptake and a more stabilized copper-bound form.

Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus.

A filamentous fungus Aspergillus terreus produces itaconic acid, which is predicted to be derived from cis-aconitic acid via catalysis by cis-aconitic acid decarboxylase (CAD) in the carbon metabolism of the fungus. To clarify the enzyme's function and a pathway for itaconic acid biosynthesis, we cloned a novel gene encoding the enzyme. The open reading frame of this gene (CAD1) consists of 1,529 bp encoding 490 amino acids and is interrupted by a single intron. Among the identified proteins in the database, the primary structure of the protein encoded by CAD1 shared high identity with the MmgE/PrpD family of proteins, including a number of 2-methylcitrate dehydratases of bacteria. The cloned gene excluding an intron was introduced into the expression plasmid pAUR-CAD1 controlled by the ADH1 promoter. The CAD activity in Saccharomyces cerevisiae was confirmed by directly detecting itaconic acid as a product from cis-aconitic acid as a substrate. This result reveals for the first time that this gene encodes CAD, which is essential for itaconic acid production in A. terreus.