Bilirubin oxidase expression and activity enhancement from Myrothecium verrucaria in Aspergillus species.

We constructed a new Aspergillus expression vector (pSENSU2512nid) under the control of the enolase promoter with 12 tandem repeats of cis-acting elements (region III) and the heat shock protein 12 (Hsp12) 5' untranslated region (UTR). Bilirubin oxidase (EC: 1.3.3.5) from Myrothecium verrucaria, which catalyzes the oxidation of bilirubin to biliverdin, was overexpressed in Aspergillus oryzae and A. niger. The productivity was estimated to be approximately 1.2 g/L in the culture broth, which was approximately 6-fold higher than that of recombinant bilirubin oxidase (BOD) expressed in Pichia pastoris (Komagataella phaffii). BOD was purified using hydrophobic interaction chromatography, followed by ion exchange chromatography. The specific activity of the purified BOD against 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate was 57.6 U/mg and 66.4 U/mg for A. oryzae and A. niger, respectively. l-Ascorbic acid (4 mM) addition and storage under deoxygenated conditions for 3-7 d increased the specific activity of these Aspergillus-expressed BODs approximately 2.3-fold (154.1 U/mg). The BOD specific activity was enhanced by incubation at higher temperature (30-50 °C). Further characterization of the enzyme catalytic efficiency revealed that the Km value remained unchanged, whereas the kcat value improved 3-fold. In conclusion, this high-level of BOD expression meets the requirements for industrial-level production. Additionally, we identified an effective method to enhance the low specific activity during expression, making it advantageous for industrial applications.

Mechanisms of production and control of acetate esters in yeasts.

Acetate esters, such as isoamyl acetate and ethyl acetate, are major aroma components of alcoholic beverages. They are produced through synthesis from acetyl CoA and the corresponding alcohol by alcohol acetyltransferase (AATase) with specific control of reaction factors, including unsaturated fatty acids and precursors, the percentage of nitrogen, and oxygen. However, the mechanisms by which these specific reaction factors affect acetate ester production remain largely unknown. The cellular mechanisms underlying the effects of these factors on acetate ester production were examined by purifying AATase from yeast, characterizing it, and cloning the ATF gene encoding AATase from sake yeast and bottom-fermenting yeast. Genetic and biochemical studies suggested that the decrease in acetate production with the addition of oxygen and unsaturated fatty acids was due to a decrease in enzyme synthesis resulting from transcriptional repression of the ATF1 gene, which is responsible for most of the AATase activity. Furthermore, these results suggest that expression of the ATF1 gene is intricately regulated by a number of transcriptional regulatory genes such as ROX1 and RAP1. Based on these results, the mechanism of ester regulation by oxygen, unsaturated fatty acids and precursors, and ratio of nitrogen source are becoming clearer from a molecular biological point of view. The physiological significance of ester production by yeast is then discussed. In this review, we summarize the studies on AATase, ATF gene, regulation of ester production, and physiological significance of acetate ester.

Effects of ethyl-α-d-glucoside on human dermal fibroblasts.

Ethyl α-d-glucoside (α-EG) is a glycoside present in sake, Japanese rice wine. Previous studies have reported that α-EG suppresses skin roughness after ultraviolet B irradiation, transepidermal water loss, and hepatic function disorder, and has a skin moisturizing effect. In this study, 0.48 µM of α-EG was found to increase the proliferation of normal human dermal fibroblasts (NHDF) by 121.0%, and the amount of collagen I produced by NHDF increased by 159.6% at an α-EG concentration of 0.048 µM, compared to those in cells cultured without α-EG. In NHDF cultured in α-EG-supplemented medium, the expression of fibroblast growth factor I and VII mRNA increased by 148.8 and 153.1%, at an α-EG concentration of 4.8 and 0.048 µM, respectively, as measured by a quantitative reverse transcription-polymerase chain reaction. Transcript levels of type I collagen genes, COL1A1 and COL1A2, increased by 152.4 and 129.7%, respectively, and that of a type III collagen gene, COL3A1, increased by 131.8% at an α-EG concentration of 0.48 µM. These findings supported the possibility that α-EG was involved in the maintenance and improvement of skin homeostasis and moisturizing functions.

Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalyst highly expressing Candida antarctica lipase B.

For enzymatic biodiesel production from plant oil hydrolysates, an Aspergillus oryzae whole-cell biocatalyst that expresses Candida antarctica lipase B (r-CALB) with high esterification activity was developed. Each of soybean and palm oils was hydrolyzed using Candida rugosa lipase, and the resultant hydrolysates were subjected to esterification where immobilized r-CALB was used as a catalyst. In esterification, r-CALB afforded a methyl ester content of more than 90% after 6 h with the addition of 1.5 M equivalents of methanol. Favorably, stepwise additions of methanol and a little water were unnecessary for maintaining the lipase stability of r-CALB during esterification. During long-term esterification in a rotator, r-CALB can be recycled for 20 cycles without a significant loss of lipase activity, resulting in a methyl ester content of more than 90% even after the 20th batch. Therefore, the presented reaction system using r-CALB shows promise for biodiesel production from plant oil hydrolysates.

Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae.

In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5' untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL#1) and double copies (pSENSU-FHL#2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL#1 and #2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL#2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time.

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.