Anti-Allergic Effects of the Subcritical Water Extract Powder of Citrus unshiu in Mouse and Guinea Pig Models.

We evaluated the effects of unripe mandarin orange (Citrus unshiu) extract powder (unripe mandarin extract powder [UMEP]) treated with subcritical water on allergic diseases by using animal models. High performance liquid chromatography (HPLC) analysis revealed that subcritical water is a more effective solvent than alcohol and hot water, as it quickly extracted approximately 90% of the functional compounds narirutin (1) and hesperidin (2) from whole fruits. Repeated oral administration of UMEP significantly reversed the peripheral blood flow decline observed during the promotion of allergies after sensitization with the antigen, hen egg-white lysozyme (HEL). UMEP also significantly inhibited compound 48/80-induced scratching behavior in HEL-sensitized mice, which are more sensitive to itching stimuli than are normal mice, without suppressing locomotor activity. In addition, repeated oral administration of UMEP in ovalbumin-challenged guinea pigs significantly suppressed the late phase of nasal airway resistance. This study provides evidence that the subcritical water extract powder of unripe C. unshiu fruit is an effective anti-allergic functional food.

5' Untranslated region of the Hsp12 gene contributes to efficient translation in Aspergillus oryzae.

We describe a 5' untranslated region (5'UTR) that dramatically increases the expression level of an exogenous gene in Aspergillus oryzae. Using a series of 5'UTR::GUS (uidA) fusion constructs, we analyzed the translation efficiency of chimeric mRNAs with different 5'UTRs at different temperatures. We found that the 5'UTR of a heat-shock protein gene, Hsp12, greatly enhanced the translation efficiency of the chimeric GUS mRNA at normal temperature (30 degrees C). Moreover, at high temperature (37 degrees C), the translation efficiency of the mRNA containing the Hsp12 5'UTR was far superior to that of mRNAs containing nonheat-shock 5'UTRs, resulting in much more efficient expression of GUS protein (about 20-fold higher GUS activity compared to the control construct). This 5'UTR can be used in combination with various strong promoters to enhance the expression of foreign proteins in A. oryzae.

High expression of a synthetic gene encoding potato alpha-glucan phosphorylase in Aspergillus niger.

We describe the successful heterologous expression of the Solanum tuberosum alpha-glucan phosphorylase (GP) gene in Aspergillus niger. Special attention was paid to the influence of different codon usage and A+T content in the coding region on GP protein expression. Use of A. niger-preferred codon usage and lower A+T content in a synthetic gene (GP-syn) resulted in a significant improvement in the level of the GP mRNA and a dramatic increase in the quantity of GP protein produced such that it accounted for approximately 10% of the total soluble protein. We suggest that redesigning the primary DNA sequence encoding a desired protein product can be an extremely effective method for improving heterologous protein production in filamentous fungi.

Improvement of the Aspergillus oryzae enolase promoter (P-enoA) by the introduction of cis-element repeats.

We constructed a protein expression vector with an improved enoA promoter that harbored 12 tandem repeats of the cis-acting element (region III) of Aspergillus oryzae. The improved promoter yielded reporter beta-glucuronidase (GUS) activity approximately 30-fold of the original promoter. Northern blot analysis confirmed that GUS expression was increased at the transcriptional level. The transformant harboring seven copies of the novel vector showed more than 100,000 U/mg GUS protein, which was approximately 30% of all the cell-free soluble proteins.

Expression of the Fusarium oxysporum lactonase gene in Aspergillus oryzae: molecular properties of the recombinant enzyme and its application.

The lactonase gene of Fusarium oxysporum was expressed in Aspergillus oryzae for optical resolution of DL-pantoyl lactone. When the chromosomal gene encoding the full-length form of the lactonase, which has its own NH2-terminal signal peptide, was introduced in the host cells, the resulting transformant produced an enzyme of 46,600 Da, which corresponded to the wild-type enzyme. In contrast, A. oryzae transformed with the cDNA coding the mature enzyme produced a protein of 41,300 Da. Deglycosylation analysis with an endoglycosidase revealed that the difference in molecular mass arose from the different sugar contents of the recombinant enzymes. The mycelia of the transformant were used as a catalyst for asymmetric hydrolysis of DL-pantoyl lactone. The initial velocity of the asymmetric hydrolysis reaction catalyzed by the transformant was estimated to be 30 times higher than that by F. oxysporum. When the mycelia of the transformant were incubated with a 20% DL-pantoyl lactone solution for 4 h, 49.9% of the racemic mixture was converted to D-pantoic acid (>95% ee).

Translation efficiency mediated by the 5' untranslated region greatly affects protein production in Aspergillus oryzae.

We demonstrate that the 5' untranslated region (5'UTR) plays an important role in determining translation efficiency in Aspergillus oryzae, using a model beta-glucuronidase (GUS) expression system. Alterations in the 5' UTR resulted in an increase in GUS activity of up to eight-fold, without affecting mRNA levels. Moreover, using the most effective 5'UTR construct, we could achieve remarkable intracellular overproduction of GUS protein; and the GUS level reached more than 50% of the total soluble protein. This is the first experimental evidence indicating the feasibility of improving recombinant protein yield by promoting translation initiation in filamentous fungi.