解脂耶罗维亚酵母产油脂的研究进展

单细胞油脂是生产生物柴油最理想的原料,随着化石能源的日益枯竭,单细胞油脂的生产受到广泛关注。解脂耶罗维亚酵母是生产单细胞油脂的最佳菌株,它能够利用诸多廉价底物作为碳源,在工业上有极大的应用前景。其遗传背景清晰,全基因组测序已完成,基因表达系统已构建。在此基础上对油脂累积途径进行了深入研究,多株油脂含量更高的菌株被构建。深入了解解脂耶罗维亚酵母的基因表达及油脂代谢系统,对日后对其进一步的代谢改造具有重要意义。     

代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯

中长链聚羟基脂肪酸酯(mcl-PHA)是一大类由微生物合成的天然生物聚酯,因具有可再生性和生物降解性越来越受到人们的关注。Mcl-PHA可由一些假单胞菌类利用自身的脂肪酸合成途径或β-氧化途径来合成。耶氏解脂酵母具有很好的脂/脂肪酸分解代谢能力,但是它体内缺乏PHA合成酶不能合成mcl-PHA。采用代谢工程策略构建重组解脂酵母,外源表达来自铜绿假单胞菌PAO1(Pseudomonas aeruginosa PAO1)的PHA合成酶。在PHA合成酶的C端添加PTS1过氧化物酶体定位信号序列,使其在过氧化物酶体内发挥功能,并对其编码基因PhaC1进行密码子优化得到oPhaC1。利用pINA1312载体构建表达框,借助载体上的zeta序列元件将oPhaC1基因表达框整合至酵母基因组,完成基因的稳定表达。重组菌PSOC在葡萄糖为唯一碳源的培养基中几乎不产PHA,添加0.5%的油酸时可合成占细胞干重0.67%的mcl-PHA。在含三油酸甘油酯的培养基中发酵72h产生1.51% mcl-PHA(wt%)。实验结果充分证明重组解脂酵母作为有潜力的微生物细胞工厂可以用于生产mcl-PHA,也为将来利用富含油脂和其他营养的餐厨垃圾水解液等廉价资源生产mcl-PHA打下基础。     

脂肪酸延长酶缺陷对酵母细胞脂质代谢及油酸胁迫响应的影响

生物医学证据表明,过量的油脂特别是脂肪酸(fatty acids,FA)在非脂肪组织累积会引起脂代谢障碍,引起细胞功能紊乱或坏死。脂肪酸延长酶家族参与脂肪酸代谢,具有真核生物的高度保守性,且与膜脂的代谢密切相关。但脂肪酸延长酶与细胞脂毒效应的关系并不清楚。该文利用模式生物酿酒酵母在脂类代谢研究中性状易于表征、遗传操作便利的优势,通过对比脂肪酸延长酶缺陷型elo1Δ、elo2Δ和elo3Δ与野生型酵母(wild-type,WT)对不同脂肪酸胁迫的响应,发现极长链脂肪酸延长酶基因ELO2和ELO3缺陷后对油酸(oleic acid,OLA)高度敏感;细胞脂滴及中性脂质的代谢对维持细胞脂类平衡起关键作用。研究结果显示,长链脂肪酸的合成缺陷或油酸处理均促进细胞脂滴的形成,同时显著提高细胞中性油脂(TAG)和甾醇酯(SE)合成;采用气相色谱–质谱联用技术分析脂肪酸组成,结果显示,ELO3缺陷,C_(26)脂肪酸基本检测不到,而C_(20)与C_(22)脂肪酸会累积;ELO2缺失后,C_(26)脂肪酸的含量也明显降低。而油酸的处理会增加BY4741胞内总的极长链脂肪酸的比例;elo2Δ和elo3Δ的不饱和脂肪酸与饱和脂肪酸的比例增大;相反,过表达脂肪酸延长酶基因,与野生型菌株相比能显著降低细胞油酸的含量。模式生物脂肪酸延长酶对细胞脂质代谢及油酸胁迫响应的研究,为医学脂代谢障碍及细胞脂毒效应研究提供了基础数据。     

新型可再生工业用油脂的代谢工程

植物种子油是一种可再生资源,亦用作生物燃油和化学工业原料. 一些野生植物能高水平合成积累羟化、环氧化和共轭脂肪酸等具有重要工业应用价值的特异脂肪酸.催化这些特异脂肪酸合成的酶主要是类脂肪酸去胞和酶2(类FAD2). 由特异脂肪酸合成到三酰基甘油脂 (TAG) 形成还需要酰基转移酶 (如DGAT) 的参与. 在油料作物种子中表达类FAD2酶及其相关基因(如DGAT),已培育出了能合成积累一定含量特异脂肪酸的工程油料品系,为基于农作物生产高附加值工业用油脂开辟了新途径. 本文论述了参与特异脂肪酸生物合成途径的关键酶基因、油料作物代谢工程策略,以及应用工程油料作物大规模生产重要工业用脂肪酸的研究进展、存在问题和应用前景等.     

植物脂肪酸的生物合成与基因工程

植物脂肪酸既具重要生理功能,又有巨大食用和工业价值。其生物合成途径较为复杂,涉及乙酰－ＣｏＡ羟化酶、脂肪酸合成酶、脂肪酸去饱和酶和脂肪酸延长酶等一系列酶。近年来,对脂肪酸生物合成途径进行了大量研究,克隆出许多相关基因,初步阐明了脂肪酸合成规律,并在此基础上开展了利用基因工程技术调控脂肪酸合成研究,取得可喜进展。本文详细介绍了植物饱和脂肪酸、不饱和脂肪酸和超长链脂肪酸的生物合成与基因工程研究的新结果     

利用a凝集素在酿酒酵母表面展示解脂耶氏酵母脂肪酶Lip2

[目的]将解脂耶氏酵母胞外脂肪酶Lip2展示在酿酒酵母表面,构建全细胞催化剂.[方法]采用PCR方法扩增得到解脂耶氏酵母胞外脂肪酶Lip2成熟肽编码基因LIP2,将其连接到AGA2基因的下游构建表面展示载体pCTLIP2.分别以橄榄油、三丁酸甘油酯和对硝基苯酚棕榈酸酯(pNPP)为底物检测展示的脂肪酶酶活.在此基础上,对野生菌及工程菌的酶学性质进行比较.[结果]展示Lip2的酿酒酵母重组菌株在半乳糖的诱导下,表现出水解橄榄油、三丁酸甘油脂以及pNPP的活性,20℃诱导72h时酶活达到最高,为182 U/g干细胞.对展示的Lip2的酶学性质研究表明,其最适温度为40℃,最适pH为8.0,温度稳定性比自由酶有所提高,50℃温浴4 h后残余酶活为其最大酶活的23.2%.以不同碳链长度的对硝基苯酚酯为底物检测其底物特异性,结果显示其水解C8,C12,C16对硝基苯酚酯活性相近,均远高于对硝基苯酚丁酸酯(C4)的水解酶活.[结论]对于Lip2,a凝集素系统是一个有效的展示系统,利用该系统成功将Lip2展示在酿酒酵母表面,从而构建了酿酒酵母全细胞催化剂,该全细胞催化剂具有良好的潜在应用前景.     

△12-脂肪酸去饱和酶FAD2的基本特性及其在胁迫中的功能

脂肪酸去饱和酶(fatty acid desaturase,FAD)催化与载体结合的饱和脂肪酸或不饱和脂肪酸在脂酰链上形成双键.脂肪酸去饱和酶可以分为脂酰ACP去饱和酶、脂酰CoA去饱和酶和脂酰脂去饱和酶三类.而脂酰脂去饱和酶中的△12-脂肪酸去饱和酶(△12 fatty acid desaturase,FAD2)是催化脂肪酸链第12位碳原子形成双键的去饱和酶类,控制着油酸、亚油酸和其他多种不饱和脂肪酸的合成和含量.主要从△12-脂肪酸去饱和酶FAD2的基本特性和在胁迫中的功能进行了综述,并对相关研究领域的未来研究方向进行了展望.     

解脂耶氏酵母表达调控工具的开发及天然产物合成的研究进展

解脂耶氏酵母是一种重要的产油酵母,由于其能利用多种疏水性底物,具有良好的耐酸、耐盐等胁迫耐受性,具有高通量的三羧酸循环,可提供充足的乙酰辅酶A前体等特点,被认为是生产萜类、聚酮类和黄酮类等天然产物的理想宿主,在代谢工程领域有着广泛的应用。近年来,越来越多的基因编辑、表达和调控工具被逐渐开发,这促进了解脂耶氏酵母合成各种天然产物的研究。文中综述了近年来解脂耶氏酵母中基因表达和天然产物合成方面的研究进展,并探讨了在该酵母中异源合成天然产物所面临的挑战和可能的解决方案。     

昆虫脂肪酸合成通路关键基因的研究进展

脂肪酸对昆虫生长、发育、繁殖、信息交流起到重要的作用。主要介绍脂肪酸合成通路中的5个关键基因,乙酰辅酶A羧化酶基因(ACC)、脂肪酸合成酶基因(FAS)、超长链脂肪酸延伸酶基因(ELO)、去饱和酶基因(desat)及脂酰辅酶A还原酶基因(FAR)在昆虫中的研究进展。     

黑曲霉中内切菊粉酶基因及其全合成基因在解脂耶氏酵母中表达的比较

利用酵母密码子偏爱性将黑曲霉(Aspergillus niger)中的内切菊粉酶(Endoinu linase)基因通过基因全合成的方式合成为酵母密码子偏爱性的内切菊粉酶基因。然后将原始和全合成的内切菊粉酶基因克隆到解脂耶氏酵母表达载体PINA1296上,得重组解脂耶氏酵母表达载体pHBM2020、pHBM2021,将两种质粒分别转化解脂耶氏酵母(Yarrowia lipolytica)CLIB725,筛选得到重组解脂耶氏酵母CLIB725(pHBM2020)、CLIB725(pHBM2021),将两种重组酵母摇瓶培养,经SDS-PAGE、测酶活检测表明两种基因在解脂耶氏酵母中都有表达,全合成菊粉酶比原始菊粉酶酶活要高。     

Production of extracellular higher fatty acids by yeasts grown on hexadecanoic acid

Production of exocellular higher fatty acids by Candida yeasts was studied during their growth in a mineral medium with hexadecane. The qualitative and quantitative composition of exocellular higher fatty acids was investigated during cultivation of Candida lipolytica VCM Y 2378(695) under the conditions of different aeration (5 and 80% saturation of the medium with oxygen). Palmitic (C16:0), palmitooleic (C16:1), oleic (C18:1) and linoleic (C18:2) acids predominated among other higher fatty acids. The overall amount of fatty acids increased and the content of unsaturated fatty acids decreased when the yeast growth was limited with oxygen.     

Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana

Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Delta12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.     

Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves

The seeds of many nondomesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered seeds remains low and is often hampered by their inefficient exclusion from phospholipids. Recent studies have established the feasibility of increasing triacylglycerol content in plant leaves, which provides a novel approach for increasing energy density of biomass crops. Here, we determined whether the fatty acid composition of leaf oil could be engineered to accumulate unusual fatty acids. Eleostearic acid (ESA) is a conjugated fatty acid produced in seeds of the tung tree (Vernicia fordii) and has both industrial and nutritional end‐uses. Arabidopsis thaliana lines with elevated leaf oil were first generated by transforming wild‐type, cgi‐58 or pxa1 mutants (the latter two of which contain mutations disrupting fatty acid breakdown) with the diacylglycerol acyltransferases (DGAT1 or DGAT2) and/or oleosin genes from tung. High‐leaf‐oil plant lines were then transformed with tung FADX, which encodes the fatty acid desaturase/conjugase responsible for ESA synthesis. Analysis of lipids in leaves revealed that ESA was efficiently excluded from phospholipids, and co‐expression of tung FADX and DGAT2 promoted a synergistic increase in leaf oil content and ESA accumulation. Taken together, these results provide a new approach for increasing leaf oil content that is coupled with accumulation of unusual fatty acids. Implications for production of biofuels, bioproducts, and plant–pest interactions are discussed.     

Incorporation of Very-Long-Chain Fatty Acids into Sphingolipids of Cultured Neural Cells

We examined effects of exogenous very-long-chain fatty acids on lipids of cultured chick neurons and astrocytes. When chick neurons were incubated in chemically defined medium containing 10 microM nervonic acid (C24:1) for 7 days, it was found that a major fatty acid moiety of gangliosides and sphingomyelin was nervonic acid itself, which was not normally detected in the sphingolipid fraction. This alteration in the fatty acid composition apparently occurred in each ganglioside species. Under these experimental conditions, nervonic acid was not found in the glycerophospholipid fraction, and the amounts of triacylglycerol and free nervonic acid increased. Addition of behenic acid (C22:0) or erucic acid (C22:1) also induced changes in the fatty acid composition of gangliosides. When chick astrocytes were incubated in the presence of 10 microM nervonic acid for 7 days, no significant change was observed in the fatty acid composition of gangliosides. These studies indicate that the manipulation of the fatty acid moiety of sphingolipids in cultured neurons is possible.     

Elongation of mitochondrial fatty acids during brain development

Abstract— Elongation of mitochondrial fatty acids was studied in whole brain samples from rats before, during and after the period of myelination. The mitochondria were isolated by centrifugation in a discontinuous sucrose gradient and incubated under N₂ in a medium containing NADH, NADPH, ATP and acetyl-[1-¹⁴C]coenzyme A. Fatty acids were extracted, methylated and analysed by gas-liquid chromatography. A distinct pattern emerged in which brain mitochondria from rats undergoing myelination synthesized longer chain fatty acids preferentially, particularly C_22:4. Mitochondria from brains of mature rats synthesized shorter chain fatty acids preferentially, mainly C_18:0 and C_20:4. We suggest that eicosamonoenoic acid (C_22:1) is a precursor in vivo of nervonic acid (C_24:1).     

Synthesis and characterization of poly(3-hydroxyalkanoates) from Brassica carinata oil with high content of erucic acid and from very long chain fatty acids

Pseudomonas aeruginosa produced medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) when grown on substrates containing very long chain fatty acids (VLCFA, C>20). Looking for low cost carbon sources, we tested Brassica carinata oil (erucic acid content 35-48%) as an intact triglyceride containing VLCFA. Oleic (C18:1), erucic (C22:1), and nervonic (C24:1) acids were also employed for mcl-PHA production as model substrates. The polymers obtained were analyzed by GC of methanolyzed samples, GPC, 1H and 13C NMR, ESI MS of partially pyrolyzed samples, and DSC. The repeating units of such polymers were saturated and unsaturated, with a higher content of the latter in the case of the PHA obtained from B. carinata oil. Statistical analysis of the ion intensity in the ESI mass spectra showed that the PHAs from pure fatty acids are random copolymers, while the PHA from B. carinata oil is either a pure polymer or a mixture of polymers. Weight-average molecular weight varied from ca. 56,000 g/mol for the PHA from B. carinata oil and oleic acid, to about 120,000 g/mol for those from erucic and nervonic acids. The PHAs from erucic and nervonic acids were partially crystalline, with rubbery characteristics and a melting point (Tm) of 50°C, while the PHAs from oleic acid and from B. carinata oil afforded totally amorphous materials, with glass transition temperatures (Tg) of -52°C and -47°C, respectively.     

Metabolism of saturated and polyunsaturated very-long-chain fatty acids in fibroblasts from patients with defects in peroxisomal beta-oxidation.

The metabolism of [1-14C]lignoceric acid (C24:0) and [1-14C]tetracosatetraenoic acid (C24:4, n-6) was studied in normal skin fibroblast cultures and in cultures from patients with defects in peroxisomal beta-oxidation (but normal peroxisomal numbers). Cells from X-linked adrenoleukodystrophy (ALD) patients with a presumed defect in a peroxisomal acyl-CoA synthetase, specific for fatty acids of carbon chain lengths greater than 22 (very-long-chain fatty acids; VLCFA), showed a relatively normal production of radiolabelled CO2 and water-soluble metabolites from [1-14C]C24:0. However, the products of synthesis from acetate de novo (released by beta-oxidation), i.e. C16 and C18 fatty acids, were decreased, and carbon chain elongation of the fatty acid was increased. In contrast, cell lines from two patients with an unidentified lesion in peroxisomal beta-oxidation (peroxisomal disease, PD) showed a marked deficiency in CO2 and water-soluble metabolite production, a decreased synthesis of C16 and C18 fatty acids and an increase in carbon chain elongation. The relatively normal beta-oxidation activity of ALD cells appears to be related to low uptake of substrate, as a defect in beta-oxidation is apparent when measurements are performed on cell suspensions under high uptake conditions. Oxidation of [1-14C]C24:4 was relatively normal in ALD cells and in the cells from one PD patient but abnormal in those from the other. Our data suggest that, despite the deficiency in VLCFA CoA synthetase, ALD cells retain a near normal ability to oxidize both saturated and polyunsaturated VLCFA under some culture conditions. However, acetate released by beta-oxidation of the saturated VLCFA and, to a much lesser degree, the polyunsaturated VLCFA, appears to be used preferentially for the production of CO2 and water-soluble products, and acetate availability for fatty acid synthesis in other subcellular compartments is markedly decreased. It is likely that the increased carbon chain elongation of the saturated VLCFA which is also observed reflects the increased availability of substrate (C24:0) and/or an increase in microsomal elongation activity in ALD cells.     

Inhibition of hyaluronidases and chondroitinases by fatty acids

The inhibitory effects of various fatty acids on three hyaluronidases (h-ST, h-SH and h-SD) and four chondroitinases (c-ABC, c-B, c-ACI and c-ACII) were examined, and their structure-activity relationships and mechanism of action were studied. The fatty acids used in this experiment showed various inhibitory activities against the enzymes. None of the fatty acids did not inhibit h-ST and h-SH. The saturated fatty acids (C10:0 to C22:0) showed very weak or no inhibition against h-SD, c-ABC, c-B, c-ACI and c-ACII but the unsaturated fatty acids (C14:1 to C24:1) with one double bond strongly inhibited the enzymes, and the inhibitory potency increased with increase in carbon chain length of the fatty acids. In contrast, the increase in number of double bonds caused a decrease in inhibitory potency against the enzymes. The position of the double bond and the stereochemistry of the cis-trans form of oleic acid (C18:1) did not influence the inhibitory potency against the enzymes. Carboxyl and hydroxyl groups in the fatty acid molecule were concerned in the inhibition of c-ACI. Among the fatty acids, eicosatrienoic acid (C20:3) generally inhibited h-SD, c-ABC, c-B and c-ACI, and nervonic acid (C24:1) was a potent inhibitor of c-ACII, and the fatty acids inhibited the enzymes in a noncompetitive manner.     

Control of fatty-acid synthetase levels by exogeneous long-chain fatty acids in the yeasts Candida lipolytica and Saccharomyces cerevisiae.

Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long-chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose-containing growth medium. From fatty-acid-grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross-reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty-acid-induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty-acid-free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants.     

Inhibition of plant fatty acid synthesis by nitroimidazoles.

1. The effect of the addition of a number of nitroimidazoles was tested on fatty acid synthesis by germinating pea seeds, isolated lettuce chloroplasts and a soluble fraction from pea seeds. 2. All the compounds tested had a marked inhibition on stearate desaturation by lettuce chloroplasts and on the synthesis of very-long-chain fatty acids by pea seeds. 3. In contrast, the effect of the drugs on total fatty acid synthesis from [14C]acetate in chloroplasts was related to the compound's electron reduction potentials. 4. Of the compounds used, only metronidazole had a marked inhibition on palmitate elongation in the systems tested. 5. The mechanism of inhibition of plant fatty acid synthesis by nitroimidazoles is discussed and the possible relevance of these findings to their neurotoxicity is suggested.