Yuping Bi

Overexpression of Peanut Diacylglycerol Acyltransferase 2 in Escherichia coli

Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14’ using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a–GST, or AhDGAT2b–GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a–GST and AhDGAT2b–GST proteins increased the sizes of the host cells by 2.4–2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a–GST and AhDGAT2a–GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

Cloning of Acyl-ACP Thioesterase FatA from Arachis hypogaea L. and Its Expression in Escherichia coli

In this study, a full-length cDNA of the acyl-ACP thioesterase, AhFatA, was cloned from developing seeds of Arachis hypogaea L. by 3′-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50–70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed that AhFatA was expressed in all tissues of A. hypogaea L., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression of AhFatA in Escherichia coli affected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition of E. coli.

A Comprehensive Alanine-Scanning Mutagenesis Study Reveals Roles for Salt Bridges in the Structure and Activity of Pseudomonas aeruginosa Elastase

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (k cat/K m), half-lives (t 1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.

Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

Microalgal Oil from Schizochytrium sp. Prevents HFD-Induced Abdominal Fat Accumulation in Mice

ABSTRACT Objective: Dietary n-3 polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acids (EPA) and docosahexaenoic acid (DHA), are proved to be effective in obesity reduction. Microalgal oil (MO) is an important alternative source of n-3 PUFAs that effectively alleviates obesity. The aim of the present study was to explore the anti-obesity effects of microalgal oil from Schizochytrium sp. (SMO) and to compare the effects of 2 SMOs (SMO1 and SMO2) with different levels of purity of n-3 PUFAs on high fat diet (HFD)-induced obesity in male C57BL/6J mice. Methods: Mice were randomly divided into 5 groups: (1) regular chow (RC); (2) HFD; (3) HFD + fish oil (FO); (4) HFD + SMO1; and (5) HFD + SMO2. Body weight and food intake were weekly monitored. After 16 weeks of treatment, a glucose tolerance test (GTT) and an insulin tolerance test (ITT) were performed. Serum lipid profile, morphological changes in the liver and epididymal white adipose tissue (eWAT), and the mRNA expression of lipid metabolism–related genes were also examined. Results: SMO treatment significantly decreased HFD-induced abdominal fat accumulation, lowered the levels of triglycerides, cholesterol, and low-density lipoprotein, as did the positive control treated with FO. Morphological examination revealed a remarkable reduction in lipid droplet formation in the liver tissue and the particle size of eWAT. An alleviation of inflammation infiltration in eWAT caused by a high-fat diet was also observed. Real-time reverse transcription–polymerase chain reaction analysis examination confirmed that microalgal oil inhibited the gene expression of fatty acid synthase, sterol responsive element-binding protein-1c, and acetyl-CoA carboxylase but promoted that of hormone-sensitive lipase and lipoprotein lipase, carnitine palmitoyltransferase-1, and uncoupling proteins in the liver and eWAT. Moreover, similar anti-obesity effects were obtained with the same dosage but different purity of n-3 PUFAs. Conclusions: As an alternative n-3 PUFAs resource, dietary intake of SMO might be beneficial to prevent HFD-induced abdominal fat accumulation.

Antioxidant activity of alcohol aqueous extracts of Crypthecodinium cohnii and Schizochytrium sp.

Crypthecodinium cohnii (dinoflagellate) and Schizochytrium sp. (thraustochytrid) are the main sources for docosahexaenoic acid (DHA). The present study aimed to evaluate the antioxidant activity of petroleum ether, ethyl acetate, n-butanol, and water fractions of alcohol aqueous extracts of these two microalgae and to provide a theoretical basis for comprehensive utilization. The antioxidant activity was determined by total antioxidant capacity (TAC) determination, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, ferrous ion-chelating ability (FICA) assay, and reducing power (RP) assay. The total phenolic content (TPC) and total flavonoid content (TFC) were also measured by the Folin-Ciocalteu and spectrophotometry methods, respectively. The results indicated that the extracts from these two microalgae possessed good antioxidant capacity. Analysis showed that most antioxidant performance indicators (TAC, DPPH, and RP) were positively correlated with the TPC of the extracts, suggesting that the phenolics might be the major components in C. cohnii and Schizochytrium sp., contributing to their antioxidative function. Therefore, the polar fractions of C. cohnii and Schizochytrium sp. could be further examined and considered for application in health products or cosmetics.中文概要目的评价隐甲藻和裂殖壶藻醇水提物的石油醚萃取 相、乙酸乙酯萃取相、正丁醇萃取相和水相的抗 氧化活性水平,为综合利用两种微藻提供理论依 据。创新点隐甲藻和裂殖壶藻是二十二碳六烯酸(DHA)的 重要原料,然而提取DHA 后剩余的藻渣未得到 充分利用。本文首次对两种微藻的极性提取物的 不同极性部位进行抗氧化活性评价,并初步确定 多酚类化合物是其发挥抗氧化作用的主要物质成 分,为综合利用两种微藻提供了理论依据和参考。方法用70%乙醇浸提隐甲藻和裂殖壶藻藻粉,所得粗 提物分别使用石油醚、乙酸乙酯、正丁醇和水依 次萃取,得到不同极性组分萃取物。采用总抗氧 化能力、2,2-二苯基-1-三硝基苯肼(DPPH)自由 基清除能力、亚铁离子螯合能力及总还原力等方 法对不同极性组分的萃取物进行抗氧化活性评 价。采用Folin-Ciocalteu 方法测定样品中总多酚 含量,采用分光光度法测定样品中总黄酮含量。结论本实验结果显示,隐甲藻和裂殖壶藻醇水提物的 不同极性组分具有较好的抗氧化作用(图2~5)。 相关性分析结果表明萃取物中的多酚类化合物 与其抗氧化水平显著相关(表2 和表3),因此, 我们推测多酚类化合物是两种微藻的主要抗氧 化成分。综上所述,隐甲藻和裂殖壶藻具有综合 开发利用潜力,可深入研究。