Enhancement of the productivity of free dihomo-γ-linolenic acid via co-overexpression of elongase and two desaturase genes in Aspergillus oryzae.

Free dihomo-γ-linolenic acid (DGLA), a polyunsaturated free fatty acid (FFA), is a precursor of the eicosanoid prostaglandin E1 and is expected to be a source material for artificial production. We previously constructed the Aspergillus oryzae mutant strain ARA1 that produced free DGLA from the disruptant of faaA, an acyl-CoA synthetase gene, where FFA productivity increased by 9.2-fold compared with that of the wild-type strain. Here, we aimed to achieve enhancement of free DGLA productivity. Because saturated FFAs, such as palmitic acid and stearic acid, accounted for about 45% and 25% of total FFAs produced by ARA1, respectively, we used a strategy to facilitate elongation and desaturation of these FFAs to oleic acid and linoleic acid by overexpressing genes encoding elongase, Δ9-desaturase, and Δ12-desaturase originally expressed in A. oryzae. Ten genes were predicted to encode desaturases, and their overexpression DNA constructs were introduced into ARA1. AO090001000224 and AO090011000488 facilitated Δ12-desaturation and Δ9-desaturation most, respectively, following overexpression. Next, ARA1 strain was modified to DGLA1cre strain for producing free DGLA as a final product, and co-overexpression of these two desaturase genes was then introduced to DGLA1cre. Moreover, single overexpression of two genes predicted to encode elongases was additionally introduced, and only AO090003000572 facilitated elongation. Consequently, in the co-overexpression mutant of AO090001000224, AO090011000488, and AO090003000572, free DGLA content ratio increased by 1.8-fold from ARA1 to 14.5%, and the productivity also increased by 1.8-fold to 0.086 mmol/g-dry-cell-weight. The yield was 284 mg/L. These findings provided insights into strategies for improving microbial production of polyunsaturated FFAs.

Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation.

Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.

Systemic AA amyloidosis in a patient with lung metastasis from renal cell carcinoma.

AA amyloidosis occurs in patients with high levels of serum amyloid A protein (SAA), which is produced by liver cells in response to signals from several pro-inflammatory cytokines. Chronic inflammatory disease is a major cause of AA amyloidosis; however, malignant neoplasms are rarely reported to be associated with AA amyloidosis. We report herein a case of a solitary lung metastasis of renal cell carcinoma associated with systemic AA amyloidosis. Pathological specimens of the resected lung tumor demonstrated renal cell carcinoma, and the presence of IL-1ß, IL-6, and TNF-α in the lymphocytes and plasma cells surrounding the tumor cells, and AA amyloid in the vascular area, but not in metastatic clear cells. Four weeks after surgery, serum IL-6, SAA, and CRP levels normalized. Although this case is very rare, it is full of interesting suggestions about the pathogenesis of malignancy-related systemic amyloidosis.

Proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA) positive IgG4-related retroperitoneal fibrosis: utility of PET-CT with 18F-fluorodeoxy glucose (FDG).

A 55-year-old man showed a serum creatinine level of 1.51 mg/dL, CRP of 0.79 mg/dL, and proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA) of 43.9 EU (normal range: below 10). The serum levels and ratios of IgG1, IgG2, IgG3, and IgG4 to total IgG were 1,570 mg/dL (49%), 1,190 mg/dL (37%), 82 mg/dL (3%), and 351 mg/dL (11%), respectively. Positron emission tomography and CT with (18)F-fluorodeoxyglucose (PET-CT) demonstrated retroperitoneal fibrosis. After a diagnosis of IgG4-related retroperitoneal fibrosis with PR3-ANCA was made, oral prednisolone improved serum creatinine and the titer of PR3-ANCA to normal levels, with no abnormal findings on PET-CT.

Hepatoprotective effect of syringic acid and vanillic acid on CCl4-induced liver injury.

The mycelia of the edible mushroom Lentinula edodes can be cultured in solid medium containing lignin, and the hot-water extracts (L.E.M.) is commercially available as a nutritional supplement. During the cultivation, phenolic compounds, such as syringic acid and vanillic acid, were produced by lignin-degrading peroxidase secreted from L. edodes mycelia. Since these compounds have radical scavenging activity, we examined their protective effect on oxidative stress in mice with CCl(4)-induced liver injury. We examined the hepatoprotective effect of syringic acid and vanillic acid on CCl(4)-induced chronic liver injury in mice. The injection of CCl(4) into the peritoneal cavity caused an increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intravenous administration of syringic acid and vanillic acid significantly decreased the levels of the transaminases. Four weeks of CCl(4) treatment caused a sufficiently excessive deposition of collagen fibrils. An examination of Azan-stained liver sections revealed that syringic acid and vanillic acid obviously suppressed collagen accumulation and significantly decreased the hepatic hydroxyproline content, which is the quantitative marker of fibrosis. Both of these compounds inhibited the activation of cultured hepatic stellate cells, which play a central role in liver fibrogenesis, and maintained hepatocyte viability. These data suggest that the administration of syringic acid and vanillic acid could suppress hepatic fibrosis in chronic liver injury.

Hepatoprotective effect of syringic acid and vanillic acid on concanavalin a-induced liver injury.

The edible mushroom Lentinula edodes (shiitake) contains many bioactive compounds. In the present study, we cultivated L. edodes mycelia in solid medium and examined the hot-water extract (L.E.M.) for its suppressive effect on concanavalin A (ConA)-induced liver injury in mice. ConA injection into the tail vein caused a great increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intraperitoneal administration of L.E.M. significantly decreased the levels of the transaminases. L.E.M. contains many bioactive substances, including polysaccharides and glucan, which could be immunomodulators. Since ConA-induced liver injury is caused by the activation of T cells, immunomodulating substances might be responsible for the suppressive effect of L.E.M. L.E.M. also contains phenolic compounds that are produced from lignocellulose by mycelia-derived enzymes. The major phenolics in L.E.M., syringic acid and vanillic acid, were intraperitoneally injected into mice shortly before the ConA treatment. Similar to L.E.M., the administration of syringic acid or vanillic acid significantly decreased the transaminase activity and suppressed the disorganization of the hepatic sinusoids. In addition, the inflammatory cytokines tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6 in the serum increased rapidly, within 3 h of the ConA administration, but the administration of syringic acid or vanillic acid significantly suppressed the cytokine levels. Together, these findings indicate that the phenolic compounds in L.E.M. are hepatoprotective through their suppression of immune-mediated liver inflammation.

Identification of the neuroblastoma-amplified gene product as a component of the syntaxin 18 complex implicated in Golgi-to-endoplasmic reticulum retrograde transport.

Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.

Fucoidan partly prevents CCl4-induced liver fibrosis.

Fucoidan, a sulfated polysaccharide extracted from brown algae, has a wide range of biological activities, including anti-inflammatory, anti-viral, and anti-tumor activities. In the present study, we investigated the effects of fucoidan on CCl4-induced liver fibrosis. Administration of fucoidan reduced CCl4-induced acute and chronic liver failure. Hepatic fibrosis induced by CCl4 was also attenuated by injection of fucoidan. Damage to hepatocytes and activation of hepatic stellate cells are key events in liver fibrosis, and, interestingly, treatment of hepatocytes with fucoidan prevented CCl4-induced cell death and inhibited the proliferation hepatic stellate cells. These results indicate that fucoidan might be a promising anti-fibrotic agent possessing dual functions, namely, protection of hepatocytes and inhibition of hepatic stellate cell proliferation.