Mulan Jiang

Identification and characterization of PtDGAT2B, an acyltransferase of the DGAT2 acyl‐Coenzyme A: Diacylglycerol acyltransferase family in the diatom Phaeodactylum tricornutum

Diacylglycerol acyltransferase (DGAT) plays a pivotal role in triacylglycerol (TAG) formation in some oleaginous organisms. We describe here the identification of a type 2 DGAT (PtDGAT2B) in the diatom Phaeodactylum tricornutum that contains four putative type 2 acyl‐CoA:DGATs, sharing little sequence similarity with each other. TAG synthesis and lipid body formation could be completely restored in a Saccharomyces cerevisiae TAG‐deficient quadruple mutant by expressing PtDGAT2B. Up‐regulation of PtDGAT2B precedes the accumulation of TAG. Functional analysis of enzyme activity in vivo demonstrated that expression of PtDGAT2B can increase the proportion of unsaturated C16 and C18 fatty acids in yeast TAG.

Triacylglycerol accumulation and change in fatty acid content of four marine oleaginous microalgae under nutrient limitation and at different culture ages.

Alteration of lipid biosynthesis is one of important biochemical changes when oleaginous microalgae grow under varied environmental conditions. The effects of culture age and nutrient limitation on triacylglycerol (TAG) accumulation and fatty acid content were investigated in four eicosapentaenoic acid (EPA)‐rich marine microalgae. The amounts of TAGs in Chaetoceros sp., Phaeodactylum tricornutum and Nannochloropsis oculata increased sharply from day 4 to day 11, and then the former two remained nearly unchanged while the latter declined gradually during the batch culture. In contrast, no marked increase in TAG accumulation was observed in Pavlova viridis during the culture. Changes in total fatty acid (TFA) content mirrored those observed for TAG accumulation, while the EPA content reached a maximum generally at day 7 or 11 in the range of 11 − 32 mg g−1 dry cell weight (DCW) and then declined. Nitrogen limitation led to a gradual increase in the amounts of TAGs from N. oculata pronouncedly but almost no change in other three species. The TFA content of the cultures after 5 days of nitrogen limitation was nearly twice that after 1 day in Chaetoceros sp., P. tricornutum and P. viridis, while the lowest increase (220 − 283 mg g−1 DCW) was observed in N. oculata. TAGs increased gradually under phosphorus limitation in all four species but not sharply compared with that under nitrogen limitation in N. oculata. The TFA content increased gradually under phosphorus limitation and after 5 days of phosphorus limitation it was 1.5 − 2 times that after 1 day. The EPA content was generally not significantly affected by nitrogen or phosphorus limitation. Culture age and nutrient limitation could be useful variables for optimizing TAG accumulation and fatty acid content with potential for biodiesel production.

Expression of the Laccase Gene from a White Rot Fungus in Pichia pastoris Can Enhance the Resistance of This Yeast to H2O2-Mediated Oxidative Stress by Stimulating the Glutathione-Based Antioxidative System

ABSTRACT Laccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus in Pichia pastoris can significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H2O2 and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression in Pichia pastoris could increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene in Pichia pastoris was found to be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2 stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, including PpYAP1, PpGPX1, PpPMP20, PpGLR1, and PpGSH1. Taken together, these results suggest that the expression of the laccase gene in Pichia pastoris can enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage.

Enhancing the Laccase Production and Laccase Gene Expression in the White-Rot Fungus Trametes velutina 5930 with Great Potential for Biotechnological Applications by Different Metal Ions and Aromatic Compounds

Laccase is useful for various biotechnological and industrial applications. The white-rot fungus Trametes velutina 5930 and its laccase, isolated from the Shennongjia Nature Reserve in China by our laboratory, has great potential for practical application in environmental biotechnology. However, the original level of laccase produced by Trametes velutina 5930 was relatively low in the absence of any inducer. Therefore, in order to enhance the laccase production by Trametes velutina 5930 and make better use of this fungus in the field of environmental biotechnology, the regulation of laccase production and laccase gene expression in Trametes velutina 5930 were investigated in this study. Different metal ions such as Cu2+ and Fe2+ could stimulate the laccase synthesis and laccase gene transcription in Trametes velutina 5930. Some aromatic compounds structurally related to lignin, such as tannic acid, syringic acid, cinnamic acid, gallic acid and guaiacol, could also enhance the level of laccase activity and laccase gene transcription. We also found that there existed a positive synergistic effect of aromatic compound and metal ion on the laccase production and laccase gene transcription in Trametes velutina 5930. Taken together, our study may contribute to the improvement of laccase productivity by Trametes velutina 5930.

Isolation and characterization of the diatom Phaeodactylum Δ5-elongase gene for transgenic LC-PUFA production in Pichia pastoris.

The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host.

Aqueous Enzymatic Extraction of Rapeseed Oil and Protein from Dehulled Cold-pressed Double-low Rapeseed Cake

Abstract The oil and protein of dehulled cold-pressed double-low rapeseed cake was extracted by an aqueous enzymatic process. The rapeseed cake was treated by the chosen combined enzymes of Viscozyme L and Alcalase 2.4L (VLA,1:1,w/w). Preliminary experiments and Response Surface Methodology (RSM) were used to study the effects of enzyme concentration, incubation time and water-to-cake ratio on the extraction yield of oil and protein. This is how the desirable conditions were obtained. Transmissive electron microscope photo showed that after cold-pressing the cell structure of rapeseed was partly damaged while dehulling had little effect on the cell structure of rapeseed. In RSM experiments water-to-cake ratio showed significant effects on the extraction of oil and protein (P<0.05),while incubation time only showed significant effects on protein yield (P<0.05).The desirable conditions were as follows: 1.0% concentration (w/w) of VLA; water-to-cake ratio(w/w),6:1; 80 min incubation time. Under this condition, the extraction yield of protein and oil were 82.10% and 71.89%, respectively. Through combining both the cold-press and the aqueous enzymatic processes together, the total oil yield reached 91.6%, which is higher than the normal cold-press process or the aqueous enzymatic extraction process alone.

Improvement of omega-3 docosahexaenoic acid production by marine dinoflagellate Crypthecodinium cohnii using rapeseed meal hydrolysate and waste molasses as feedstock.

Rapeseed meal and waste molasses are two important agro-industrial by-products which are produced in large quantities. In this study, solid state fermentation and fungal autolysis were performed to produce rapeseed meal hydrolysate (RMH) using fungal strains of Aspergillus oryzae, Penicillium oxalicum and Neurospora crassa. The hydrolysate was used as fermentation feedstock for heterotrophic growth of microalga Crypthecodinium cohnii that produce docosahexaenoic acid (DHA). The addition of waste molasses as a supplementary carbon source greatly increased the biomass and DHA yield. In the batch fermentations using media composed of diluted RMH (7%) and 1-9% waste molasses, the highest biomass concentration and DHA yield reached 3.43 g/L and 8.72 mg/L, respectively. The algal biomass produced from RMH and molasses medium also had a high percentage of DHA (22-34%) in total fatty acids similar to that of commercial algal biomass. RMH was shown to be rich in nitrogen supply comparable to the commercial nitrogen feedstock like yeast extract. Using RMH as sole nitrogen source, waste molasses excelled other carbon sources and produced the highest concentration of biomass. This study suggests that DHA production of the marine dinoflagellate C. cohnii could be greatly improved by concomitantly using the cheap by-products rapeseed meal hydrolysate and molasses as alternative feedstock.

Characterization of the Factors that Influence Sinapine Concentration in Rapeseed Meal during Fermentation

We analyzed and compared the difference in sinapine concentration in rapeseed meal between the filamentous fungus, Trametes sp 48424, and the yeast, Saccharomyces cerevisiae, in both liquid and solid-state fermentation. During liquid and solid-state fermentation by Trametes sp 48424, the sinapine concentration decreased significantly. In contrast, the liquid and solid-state fermentation process by Saccharomyces cerevisiae just slightly decreased the sinapine concentration (P ≤ 0.05). After the solid-state fermented samples were dried, the concentration of sinapine in rapeseed meal decreased significantly in Saccharomyces cerevisiae. Based on the measurement of laccase activity, we observed that laccase induced the decrease in the concentration of sinapine during fermentation with Trametes sp 48424. In order to eliminate the influence of microorganisms and the metabolites produced during fermentation, high moisture rapeseed meal and the original rapeseed meal were dried at 90°C and 105°C, respectively. During drying, the concentration of sinapine in high moisture rapeseed meal decreased rapidly and we obtained a high correlation coefficient between the concentration of sinapine and loss of moisture. Our results suggest that drying and enzymes, especially laccase that is produced during the solid-state fermentation process, may be the main factors that affect the concentration of sinapine in rapeseed meal.

Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana

In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid acyltransferases from N. benthamiana.