Mucor circinelloides serves as a model organism to investigate the lipid metabolism in oleaginous microorganisms. It is considered as an important producer of γ-linolenic acid (GLA) that has vital medicinal benefits. In this study, we used WJ11, a high lipid-producing strain of M. circinelloides (36% w/w lipid, cell dry weight, CDW), to examine the role in lipid accumulation of two mitochondrial malic enzyme (ME) genes malC and malD. The homologous overexpression of both malC and malD genes enhanced the total lipid content of WJ11 by 41.16 and 32.34%, respectively. In parallel, the total content of GLA was enhanced by 16.73 and 46.76% in malC and malD overexpressing strains, respectively, because of the elevation of total lipid content. The fact that GLA content was enhanced more in the strain with lower lipid content increase and vice versa, indicated that engineering of mitochondrial MEs altered the fatty acid profile. Our results reveal that mitochondrial ME plays an important role in lipid metabolism and suggest that future approaches may involve simultaneous overexpression of distinct ME genes to boost lipid accumulation even further.
Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P) + to NAD(P)H. The NADP + -dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP + as the cofactor. The Km values for L-malate and NADP + were 2.19±0.01 and 0.38±0.02 mM, respectively, while the Vmax values were 147±2 and 302±14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn 2+ ,M g 2+ ,C o 2+ ,N i 2+ , and low concentrations of Zn 2+ rather than Ca 2+ ,C u 2+ , or high concentrations of Zn 2+ . Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their Ki values were 0.08 and 0.6 mM, respectively.
Although multiple roles of lipases have been reported in yeasts and microalgae, the functions of lipases have not been studied in oleaginous filamentous fungi. Lipase Lip6 has been reported in the oleaginous filamentous fungus Mucor circinelloides with the consensus lipase motif GXSXG and the typical acyltransferase motif of H-(X)4-D. To demonstrate that Lip6 might play dual roles as a lipase and an acyltransferase, we performed site-directed mutagenesis in the lipase motif and the acyltransferase motif of Lip6. Mutation in the lipase motif increased cell biomass by 12%–18% and promoted lipid accumulation by 9%–24%, while mutation in the acyltransferase motif induced lipid degradation. In vitro, purified Lip6 had a slight lipase activity but had a stronger phospholipid:DAG acyltransferase activity. Enzyme activity assays in vivo and phospholipid synthesis pathway analysis suggested that phosphatidyl serine and phosphatidyl ethanolamine can be the supplier of a fatty acyl moiety to form TAG in M. circinelloides.
In order to obtain the optimal control point for single-cell lipid production with fatty acid profile similar to cocoa butter, this study explored the effects of carbon source, nitrogen source, temperature, sterculic acid methyl ester, cobalt on cell growth and lipid accumulation of Yarrowia lipolytica. Glycerol and ammonium tartrate-yeast extract were chosen as carbon source and nitrogen source for production of cocoa butter equivalent. Gradual increase in temperature (20-35°C) resulted in growth reduction, while increased in lipid content per Cell Dry Weight (CDW) (10.13 to 19.7%, w/w) and Saturated Fatty Acids (SFA). Our results suggests that the optimum conditions for Y. lipolytica to synthesize cocoa butter equivalent was 30°C with 0.6 mg L-1 of CoCl2.6H2O or 0.03 mL L-1 sterculic acid methyl ester in the medium with glycerol and ammonium tartrate-yeast extract as carbon source and nitrogen source.
Thioesterases play an essential role in the metabolism of fatty acids since they are considered one of the key enzymes to change the total amount and composition of fatty acid in an organism’s cells. Acyl-coenzyme A thioesterase 8 (ACOT8) exhibits substrate specificity mainly for short- to long-chain acyl-CoA. To identify and characterize the ACOT8 enzyme’s superfamily in Mucor circinelloides, three genes were characterized and homologously expressed in M. circinelloides WJ11 which has been used as a model organism to investigate the mechanism of lipid accumulation. Multiple sequence alignment showed that McACOT8s had significant conserved motifs in the ACOT8 family. Experimental data indicated that the biomass of the three recombinant strains (McACOT8a, McACOT8b and McACOT8c) was slightly lower than that of the control strain Mc2075, but the total fatty acid (TFA) content was significantly increased by 30.3, 21.5 and 23.9%, respectively, and the free fatty acid production increased from 12.9% (control strain) to 19.2, 25.2 and 26.2% (recombinant types), respectively, with a maximum increase of 103.1% in McACOT8c. The three strains did not show any significant improvement in fatty acid composition compared to the control strain. However, in terms of fatty acid composition of free fatty acid, McACOT8b and McACOT8c contained γ-linolenic acid (C18:3), which was not detected in the control strain or in McACOT8a, indicating that ACOT8b and ACOT8c had substrate specificity for C18:3. These results displayed that ACOT8 can increase TFA accumulation and that it may be an important target of genetic manipulation for microbial oil production.
Abstract In the present study, antioxidant activities and functional properties of cowhide collagen antioxidant peptides (CCAPs) with different molecular weight (MW) were investigated. The optimum preparation conditions of CCAPs were hydrolysis time of 1.53 hr, temperature of 54.9 °C, pH 7.38, and neutral enzyme to trypsin ratio of 0.048 g: 0.016 g according to single factor test and response surface methodology (RSM). Three fractions (CCAP‐I, CCAP‐II, and CCAP‐III) were obtained by ultrafiltration and lyophilization. Antioxidant activities revealed that CCAP‐III had high reducing power activity (0.323 ± 0.035) and scavenging effect on 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radicals (64.30 ± 5.99%), 2,2‐azino‐bis‐(3‐ethylbenzothiazoline)‐6‐sulfonic acid (ABTS) radicals (75.25 ± 3.14%), and hydroxyl radicals (68.26 ± 6.74%) compared to the other fractions. In addition, LC‐MS/MS analysis showed that Ala‐Gly‐Glu‐Arg, Gly‐Ile‐Ala‐Gly‐Glu‐Arg, Gly‐Pro‐Ala‐Gly‐Pro‐Ala‐Gly‐Pro‐Arg, Gly‐Val‐Val‐Gly‐Pro‐Glu‐Gly‐Ala‐Arg and Gly‐Phe‐Ser‐Gly‐Leu‐Asp‐Gly‐Ala‐Lys were the major peptides of CCAP‐III. CCAP‐III showed good hygroscopicity (HYG), water holding capacity (WHC), and oil holding capacity (OHC) when compared with CCAP‐I and CCAP‐II. However, CCAP‐II has great emulsifying properties, and CCAP‐I has excellent foaming properties. Therefore, CCAPs can be used as a promising source of functional peptides with antioxidant properties. Practical Application This study demonstrated the peptides of cowhide collagen has superior antioxidant and functional properties. This study provided a scientific basis for the preparation of antioxidant peptides from cowhide collagen.
Human liver cancer has emerged as a serious health concern in the world, associated with poorly available therapies. The Berberis genus contains vital medicinal plants with miraculous healing properties and a wide range of bioactivities. In this study, different crude extracts of B. lycium Royle were prepared and screened against Human Hepatocarcinoma (HepG2) cell lines. The water/ethanolic extract of B. lycium Royle (BLE) exhibited significant antiproliferative activity against the HepG2 cancer cell line with an IC50 value of 47 μg/mL. The extract decreased the clonogenic potential of HepG2 cells in a dose-dependent manner. It induced apoptotic cell death in HepG2 cells that were confirmed by cytometric analysis and microscopic examination of cellular morphology through DAPI-stained cells. Biochemical evidence of apoptosis came from elevating the intracellular ROS level that was accompanied by the loss of mitochondrial membrane potential. The mechanism of apoptosis was further confirmed by gene expression analysis using RT-qPCR that revealed the decline in Bcl-2 independent of p53 mRNA and a rise in CDK1 while downregulating CDK5, CDK9, and CDK10 mRNA levels at 48 h of BLE treatment. The most active fraction was subjected to HPLC which indicated the presence of berberine (48 μg/mL) and benzoic acid (15.8 μg/mL) as major compounds in BLE and a trace amount of luteolin, rutin, and gallic acid. Our study highlighted the importance of the most active BLE extract as an excellent source of nutraceuticals against Human Hepatocarcinoma that can serve as an herbal natural cure against liver cancer.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)