Induction of Oxidative Stress in Sirtuin Gene-Disrupted Ashbya gossypii Mutants Overproducing Riboflavin.

AgHST1 and AgHST3 genes encode sirtuins that are NAD+-dependent protein deacetylases. According to previous reports, their disruption leads to the overproduction of riboflavin in Ashbya gossypii. In this study, we investigated the potential causes of riboflavin overproduction in the AgHST1Δ and AgHST3Δ mutant strains of A. gossypii. The generation of reactive oxygen species was increasd in the mutants compared to in WT. Additionally, membrane potential was lower in the mutants than in WT. The NAD+/NADH ratio in AgHST1Δ mutant strain was lower than that in WT; however, the NAD+/NADH ratio in AgHST3Δ was slightly higher than that in WT. AgHST1Δ mutant strain was more sensitive to high temperatures and hydroxyurea treatment than WT or AgHST3Δ. Expression of the AgGLR1 gene, encoding glutathione reductase, was substantially decreased in AgHST1Δ and AgHST3Δ mutant strains. The addition of N-acetyl-L-cysteine, an antioxidant, suppressed the riboflavin production in the mutants, indicating that it was induced by oxidative stress. Therefore, high oxidative stress resulting from the disruption of sirtuin genes induces riboflavin overproduction in AgHST1Δ and AgHST3Δ mutant strains. This study established that oxidative stress is an important trigger for riboflavin overproduction in sirtuin gene-disrupted mutant strains of A. gossypii and helped to elucidate the mechanism of riboflavin production in A. gossypii.

Spectroscopic and Theoretical Studies on Conformational Stability of Benzyl Methyl Ether.

Conformer-selected electronic and vibrational spectra of benzyl methyl ether and its terminal methyl group-substituted derivative in a supersonic jet have been measured using ultraviolet (UV)-UV hole burning and fluorescence-detected infrared spectroscopy to investigate the conformational stability of flexible molecules. Various quantum chemical calculations as well as experimental observations reveal the coexistence of three conformers with different CCOC dihedral angles and side-chain orientations relative to the benzene ring plane. Vibrational analysis in the excited state with time-dependent density functional theory and IR simulations containing anharmonic coupling sufficiently reproduce the experimental results, suggesting that these three conformers can be distinguished into one gauche-conformer and two trans-ones with respect to the CCOC dihedral angle. We also observe that the gauche conformer exhibits higher-frequency CH2 modes. The natural bond orbital analysis indicates that this phenomenon is attributed to the electron delocalization from the non-bonding orbitals and the C-O orbitals associated with the neighboring oxygen atom, which leads to a conformer dependence of the methylene C-H bond strength.

Involvement of a flavoprotein, acetohydroxyacid synthase, in growth and riboflavin production in riboflavin-overproducing Ashbya gossypii mutant.

BACKGROUND: Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria. RESULTS: In the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain. CONCLUSION: The significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii.

Effects of sirtuins on the riboflavin production in Ashbya gossypii.

This study focuses on sirtuins, which catalyze the reaction of NAD+-dependent protein deacetylase, for riboflavin production in A. gossypii. Nicotinamide, a known inhibitor of sirtuin, made the color of A. gossypii colonies appear a deeper yellow at 5 mM. A. gossypii has 4 sirtuin genes (AgHST1, AgHST2, AgHST3, AgHST4) and these were disrupted to investigate the role of sirtuins in riboflavin production in A. gossypii. AgHST1∆, AgHST3∆, and AgHST4∆ strains were obtained, but AgHST2∆ was not. The AgHST1∆ and AgHST3∆ strains produced approximately 4.3- and 2.9-fold higher amounts of riboflavin than the WT strain. The AgHST3∆ strain showed a lower human sirtuin 6 (SIRT6)-like activity than the WT strain and only in the AgHST3∆ strain was a higher amount of acetylation of histone H3 K9 and K56 (H3K9ac and H3K56ac) observed compared to the WT strain. These results indicate that AgHst3 is SIRT6-like sirtuin in A. gossypii and the activity has an influence on the riboflavin production in A. gossypii. In the presence of 5 mM hydroxyurea and 50 µM camptothecin, which causes DNA damage, especially double-strand DNA breaks, the color of the WT strain colonies turned a deeper yellow. Additionally, hydroxyurea significantly led to the production of approximately 1.5 higher amounts of riboflavin and camptothecin also enhanced the riboflavin production even through the significant difference was not detected. Camptothecin tended to increase the amount of H3K56ac, but the amount of H3K56ac was not increased by hydroxyurea treatment. This study revealed that AgHst1 and AgHst3 are involved in the riboflavin production in A. gossypii through NAD metabolism and the acetylation of H3, respectively. This new finding is a step toward clarifying the role of sirtuins in riboflavin over-production by A. gossypii.Key points• Nicotinamide enhanced the riboflavin production in Ashbya gossypii.• Disruption of AgHST1 or AgHST3 gene also enhanced the riboflavin production in Ashbya gossypii.• Acetylation of H3K56 led to the enhancement of the riboflavin production in Ashbya gossypii.