Improvement of the nitrogenase activity in Escherichia coli that expresses the nitrogen fixation-related genes from Azotobacter vinelandii.

The transfer of nitrogen fixation (nif) genes from diazotrophs to non-diazotrophic hosts is of increasing interest for engineering biological nitrogen fixation. A recombinant Escherichia coli strain expressing Azotobacter vinelandii 18 nif genes (nifHDKBUSVQENXYWZMF, nifiscA, and nafU) were previously constructed and showed nitrogenase activity. In the present study, we constructed several E. coli strain derivatives in which all or some of the 18 nif genes were additionally integrated into the fliK locus of the chromosome in various combinations. E. coli derivatives with the chromosomal integration of nifiscA, nifU, and nifS, which are involved in the biosynthesis of the [4Fe-4S] cluster of dinitrogenase reductase, exhibited enhanced nitrogenase activity. We also revealed that overexpression of E. coli fldA and ydbK, which encode flavodoxin and flavodoxin-reducing enzyme, respectively, enhanced nitrogenase activity, likely by facilitating electron transfer to dinitrogenase reductase. The additional expression of nifM, putatively involved in maturation of dinitrogenase reductase, further enhanced nitrogenase activity and the amount of soluble NifH. By combining these factors, we successfully improved nitrogenase activity 10-fold.

Re-discovery of MS-347a as a fungicide candidate through a new drug discovery platform with a multidrug-sensitive Saccharomyces cerevisiae screening system and the introduction of a global secondary metabolism regulator, laeA gene.

We found that the culture broth of fungi showed anti-fungal activity against multidrug-sensitive budding yeast. However, we could not identify the anti-fungal compound due to the small quantity. Therefore, we attempted to increase the productivity of the target compound by the introduction of a global secondary metabolism regulator, laeA to the strain, which led to the successful isolation of 10-folds greater amount of MS-347a (1) than Aspergillus sp. FKI-5362. Compound 1 was not effective against Candida albicans and the detailed anti-fungal activity of 1 remains unverified. After our anti-fungal activity screening, 1 was found to inhibit the growth of broad plant pathogenic fungal species belonging to the Ascomycota. It is noteworthy that 1 showed little insecticidal activity against silkworms, suggesting its selective biological activity against plant pathogenic fungi. Our study implies that the combination strategy of multidrug-sensitive yeast and the introduction of laeA is useful for new anti-fungal drug discovery.

Modification of substrate specificity of L-arginine oxidase for detection of L-citrulline.

Enzymatic detection of citrulline, a potential biomarker for various diseases, is beneficial. However, determining citrulline levels requires expensive instrumental analyses and complicated colorimetric assays. Although L-amino acid oxidase/dehydrogenase is widely used to detect L-amino acids, an L-citrulline-specific oxidase/dehydrogenase has not been reported. Therefore, in this study, we aimed to develop an L-citrulline-specific enzyme by introducing a mutation into L-arginine oxidase (ArgOX) derived from Pseudomonas sp. TPU 7192 to provide a simple enzymatic L-citrulline detection system. The ratio of the oxidase activity against L-arginine to that against L-citrulline (Cit/Arg) was 1.2%, indicating that ArgOX could recognize L-citrulline as a substrate. In the dehydrogenase assay, the specific dehydrogenase activity towards L-arginine was considerably lower than the specific oxidase activity. However, the specific dehydrogenase activity towards L-citrulline was only slightly lower than the oxidase activity, resulting in improved substrate specificity with a Cit/Arg ratio of 49.5%. To enhance the substrate specificity of ArgOX, we performed site-directed mutagenesis using structure-based engineering. The 3D model structure indicated that E486 interacted with the L-arginine side chain. By introducing the E486 mutation, the specific dehydrogenase activity of ArgOX/E486Q for L-citrulline was 3.25 ± 0.50 U/mg, which was 3.8-fold higher than that of ArgOX. The Cit/Arg ratio of ArgOX/E486Q was 150%, which was higher than that of ArgOX. Using ArgOX/E486Q, linear relationships were observed within the range of 10-500 µM L-citrulline, demonstrating its suitability for detecting citrulline in human blood. Consequently, ArgOX/E486Q can be adapted as an enzymatic sensor in the dehydrogenase system.

Heterologous production of ascofuranone and ilicicolin A in Aspergillus sojae.

Ascofuranone and its precursor, ilicicolin A, are secondary metabolites with various pharmacological activities that are produced by Acremonium egyptiacum. In particular, ascofuranone strongly inhibits trypanosome alternative oxidase and represents a potential drug candidate against African trypanosomiasis. However, difficulties associated with industrial production of ascofuranone by A. egyptiacum, specifically the co-production of ascochlorin, which inhibits mammalian respiratory chain complex III at low concentrations, has precluded its widespread application. Therefore, in this study, ascofuranone biosynthetic genes (ascA-E and H-J) were heterologously expressed in Aspergillus sojae, which produced very low-levels of endogenous secondary metabolites under conventional culture conditions. As a result, although we obtained transformants producing both ilicicolin A and ascofuranone, they were produced only when an adequate concentration of chloride ions was added to the medium. In addition, we succeeded in increasing the production of ilicicolin A, by enhancing the expression of the rate-determining enzyme AscD, using a multi-copy integration system. The heterologous expression approach described here afforded the production of both ascofuranone and ilicicolin A, allowing for their development as therapeutics.

High-level heterologous protein production using an attenuated selection marker in Aspergillus sojae.

Filamentous fungi, including Aspergillus sojae, are essential for the industrial production of enzymes. Although multi-copy introduction of a gene encoding the protein of interest is useful for increasing protein production, this method has not been established in the case of filamentous fungi. In this study, we aimed to establish an efficient system for multi-copy chromosomal integration and high-level expression of a heterologous gene in A. sojae using an attenuated selectable marker. Consequently, by truncating the promoter region of selectable markers, we efficiently introduced multiple copies of a heterologous gene and enhanced the rate of high-level protein-production in the strains. Since the multi-copy strains obtained in this study maintained high productivity even in a non-selective medium, this system could be applicable for industrial protein production.

Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization.

FAD-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is an enzyme utilized industrially in glucose sensors. Previously, FAD-GDH isolated from Mucor prainii (MpGDH) was demonstrated to have high substrate specificity for glucose. However, MpGDH displays poor thermostability and is inactivated after incubation at 45 °C for only 15 min, which prevents its use in industrial applications, especially in continuous glucose monitoring (CGM) systems. Therefore, in this study, a chimeric MpGDH (Mr144-297) was engineered from the glucose-specific MpGDH and the highly thermostable FAD-GDH obtained from Mucor sp. RD056860 (MrdGDH). Mr144-297 demonstrated significantly higher heat resistance, with stability at even 55 °C. In addition, Mr144-297 maintained both high affinity and accurate substrate specificity for D-glucose. Furthermore, eight mutation sites that contributed to improved thermal stability and increased productivity in Escherichia coli were identified. Collectively, chimerization of FAD-GDHs can be an effective method for the construction of an FAD-GDH with greater stability, and the chimeric FAD-GDH described herein could be adapted for use in continuous glucose monitoring sensors.

Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae.

Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels.

Use of yttrium phosphate as a coprecipitant for separation/concentration of lanthanoids.

A useful method to separate/concentrate lanthanoids was developed based on a rapid coprecipitation technique using yttrium phosphate. Lanthanoids, which were quantitatively coprecipitated at pH 3 with yttrium phosphate, could be readily determined by inductively coupled plasma atomic emission spectrometry with indium used as an internal standard element. The detection limits ranged from 0.0003 microg (Yb, Lu) to 0.0099 microg (Er) in 100 mL of sample solutions. The proposed method was applicable to the separation/concentration of lanthanoids in NIST SRM 1515 (apple leaves).

Coprecipitation with yttrium phosphate as a separation technique for iron(III), lead, and bismuth from cobalt, nickel, and copper matrices.

The coprecipitation behavior of 44 elements (47 ions because of chromium(III,VI), arsenic(III,V), and antimony(III,V)) with yttrium phosphate was investigated at various pHs. Yttrium phosphate could quantitatively coprecipitate iron(III), lead, bismuth, and indium over a wide pH range; however, 18 ions, including alkali metals and oxo anions, such as vanadium(V), chromium(VI), molybdenum(VI), tungsten(VI), germanium(IV), arsenic(III,V), selenium(IV), and tellurium(VI), were scarcely collected. In addition, 19 ions, including cobalt, nickel, and copper(II), were hardly coprecipitated at pHs below about 3. Based on these results, the separation of iron(III), lead, and bismuth from cobalt, nickel, and copper(II) matrices was investigated. Iron(III), lead, and bismuth ranging from 0.5 to 25mug could be separated effectively from a solution containing 0.5g of cobalt, nickel, or copper at pH 3.0. The separated iron(III), lead, and bismuth could be determined by inductively coupled plasma atomic emission spectrometry using internal standardization. The detection limits (3sigma, n=7) of iron(III), lead, and bismuth were 0.008, 0.137, and 0.073mug, respectively. The proposed method was applied to the analyses of metals and chlorides of cobalt, nickel, and copper.

Application of internal standardization to rapid coprecipitation technique using lanthanum phosphate for flame atomic absorption spectrometric determination of iron and lead.

By applying an internal standardization, we could use a rapid coprecipitation technique using lanthanum phosphate as a coprecipitant for preconcentration of iron(III) and lead in their flame atomic absorption spectrometric determination. Indium as an internal standard was added to the initial sample solution together with lanthanum and phosphoric acid; the coprecipitation of iron(III) and lead was then carried out at pH about 3. After measuring the atomic absorbances of iron, lead, and indium in the final sample solution, we determined the contents of iron(III) and lead in the original sample solution by using the internal standardization with indium. In this method, complete collection of the precipitate was not required after the coprecipitation of iron(III), lead, and indium, because the ratio of the recovery of iron(III) or lead to that of indium was almost constant regardless of the recovery of the precipitate. This method was simple and rapid, and was available for the determination of 2-300 micrograms L-1 of iron(III) and 5-400 micrograms L-1 of lead in some water samples.