裂殖壶菌高密度发酵生产DHA

本实验从海水中筛选分离了一株裂殖壶菌,通过摇瓶实验对其发酵产二十二碳六烯酸的培养基、发酵参数进行了优化,在此基础上对裂殖壶菌进行了50 L发酵罐流加补料发酵,并成功放大培养到500 L发酵罐。在500 L发酵罐经过96 h的培养,生物量、油脂含量和二十二碳六烯酸含量分别达到110.7、43.5、22.1 g/L,初步达到了工业化生产的水平。     

重组酿酒酵母生物合成菜油甾醇

菜油甾醇作为甾体药物（孕酮、雄烯二酮、氢化可的松等）的重要合成前体已受到国内外研究学者的广泛关注。首先通过生物信息学分析,筛选了10种不同来源的7-脱氢胆固醇还原酶DHCR7,并采用CRISPR/Cas9基因编辑技术将酿酒酵母（Saccharomyces cerevisiae）内源的ERG5基因替换成不同来源的DHCR7基因,构建了菜油甾醇合成菌株。结果发现整合来源于Pangasianodon hypophthalmus DHCR7的菌株Zw507表现出最高的菜油甾醇的产量216.93 mg/L。进一步筛选了10种酵母内源启动强度较强的启动子来与PhDHCR7基因进行组合,结果显示以TEF1p为启动子时菜油甾醇的产量最高可达253.35 mg/L。为了进一步提高菜油甾醇产量,增加了DHCR7表达盒在酵母基因组上的拷贝数。当拷贝数为3个时,菜油甾醇的产量达到最高302.27 mg/L。最终,通过5 L发酵罐进行补料分批发酵,实现了916.88 mg/L菜油甾醇产量。该菌株可作为后续甾体药物生物合成的优良底盘细胞。     

微生物发酵法生产DHA的菌种系统优化

本研究采用单因素实验等方法对裂殖壶菌生产二十二碳六烯酸(DHA)的培养基进行了配比选择与优化,提高初始培养基中的葡萄糖浓度(6.0%)有利于菌体生长与产品的合成;以及适合的pH(4.0~6.0)有利于菌体的生长与产品的积累。通过添加缓冲剂Ca CO3控制发酵过程中的pH在6.0,用廉价易得的玉米浆干粉与硫酸铵可以替代昂贵的酵母膏,可以大幅度的降低DHA的生产制造成本(降低8.7%)。使用裂殖壶菌生产DHA的发酵工艺中,控制适当的供氧有利于菌体繁殖以及产品的合成。     

破囊壶菌发酵生产DHA的研究进展

二十二碳六烯酸（DHA）是一种非常重要的多不饱和脂肪酸,能参与人及动物体内多个生理过程。破囊壶菌具有生长迅速、细胞内DHA含量高的特点,是工业化生产DHA的潜力菌。本文主要介绍破囊壶菌DHA代谢通路、影响破囊壶菌生产DHA的因素以及破囊壶菌中试发酵的研究现状。首先,对破囊壶菌合成DHA的两个代谢途径,即脂肪酸合成酶（fatty acid synthesis pathway,FAS）途径和聚酮合酶（polyketide synthesis pathway,PKS）途径进行总结和描述;其次,对影响破囊壶菌发酵生产DHA的三个主要因素（碳氮源、溶氧和温度）进行综述;随后,阐述了破囊壶菌发酵生产DHA的中试放大工艺的研究现状;最后,提出破囊壶菌发酵生产DHA过程中存在的问题,并指出进一步分离获得优质的破囊壶菌菌株、对其代谢途径和关键酶的研究以及中试放大工艺的研究是下一步研究的重点。通过对上述一系列问题进行综述,旨在为利用破囊壶菌工业化生产DHA提供一定的参考。     

Klebsiella sp.WL1316乙醇脱氢酶基因过表达提高乙醇生产效率

为获得能用于纤维素乙醇高效生产的基因重组菌株,文中针对能利用木质纤维素水解液的Klebsiella sp.WL1316,对其乙醇脱氢酶adh基因进行了克隆和同源过表达。成功构建adh-pET-28a-Klebsiella sp.WL1316重组菌株,通过对菌株发酵稻草水解液生产乙醇过程的监测确定该重组菌适宜的发酵时间为48 h。结果表明：进一步通过单因素和响应面试验获得菌株乙醇发酵生产的优化工艺参数为IPTG诱导浓度0.89 mmol/L,发酵起始pH值7.59,起始还原糖质量浓度60.60 g/L。在此优化工艺条件下进行验证实验,乙醇产量达(8.28±0.55)g/L,与模型的理论预测值较接近,说明建立的模型是切实可行的,并且,该乙醇产量为同等发酵条件下野生菌的2.26倍,说明adh基因的过表达促进了乙醇产量的显著提高。     

ARTP选育ε-聚赖氨酸高产菌株及其发酵条件优化

ε-聚赖氨酸(ε-PL)是由25~35个L-赖氨酸单体组成的一种天然聚合物,在食品、医学、农药等领域有很大的应用潜力。目前,微生物法生产ε-PL存在生产强度低、发酵周期长、工艺不稳定等问题。为此,本研究以S.albulus FMME-545为出发菌株,通过常温常压等离子诱变(ARTP)结合核糖体工程选育了一株具有利福霉素抗性的高产菌株S.albulus FMME-545RX,其ε-PL产量达到2.44 g/L,相较于出发菌株提升了105%。为了进一步提高ε-聚赖氨酸的产量,在5 L发酵罐中通过分批补料的方式对碳源的调控策略、pH调控方法、DO控制水平进行了系统的研究。结果表明,采用葡萄糖-蔗糖双碳源调控策略有助于提高菌体代谢强度;在发酵过程中添加柠檬酸钠能有效帮助菌体抵御酸性环境;产物合成所需的最适pH值和DO值分别为3.80和30%。经过192 h的分批补料发酵,ε-PL的产量、生产强度、单位细胞合成能力分别达到了53.0 g/L, 6.63 g/(L?d), 0.88 g/g,相比于原始菌株分别提高了130%, 131%, 118%。上述研究结果为ε-聚赖氨酸工业化生产提供了有益的借鉴。     

产蒎烯人工酵母细胞的构建

蒎烯可衍生为高能量密度燃料,但在酿酒酵母中的全生物合成却未见报道。酿酒酵母由于拥有强大的蛋白表达和翻译后修饰系统以及完整的内膜系统,相比于大肠杆菌等原核生物更适于P450等蛋白的表达,因此将酿酒酵母作为宿主细胞,对于蒎烯或者其他物质实现如“疯狂碳环”的高能量化是至关重要的。本研究在酿酒酵母底盘中表达内源焦磷酸香叶酯合成酶（ERG20）的突变体ERG20^ww和火炬松来源的蒎烯合酶（PtPS）构建了蒎烯的合成路径。通过截短PtPS N端2～51位氨基酸残基（tPtPS）,蒎烯产量较初始产量（0.329 mg·L^-1）提高了2.23倍。在过表达异戊二烯焦磷酸异构酶（IDI1）和RNA聚合酶Ш负调控因子（MAF1）的基础上,表达ERG20^ww和tPtPS的融合蛋白,蒎烯产量进一步提高了5.16倍。通过将内源基因ERG20启动子原位替换为弱启动子HXT1,下调ERG20的转录,蒎烯的产量提高了26.0%。最终通过调节发酵过程中的培养基pH使蒎烯产量达11.7 mg·L^-1,较初始产量提高了34.5倍。本研究在酿酒酵母中实现蒎烯的从头合成,并获得已知蒎烯摇瓶水平的最高产量。     

工业废弃合成气厌氧发酵产己醇研究进展

己醇作为高附加值中链醇,其生物合成越来越受到研究人员的广泛关注。利用工业废弃合成气制备生物己醇能够降低原料成本,然而,生物己醇的低产率严重限制了其广泛应用。本文基于生物己醇发酵的新近报道,首次对合成气发酵制备生物己醇的研究进展进行了分析与综述。文中指出目前能够直接利用合成气合成生物己醇的菌株仅有Clostridium carboxidivorans一例,但其更倾向合成乙酸、乙醇、丁酸和丁醇等,其最高己醇产量仅为1.06g/L。己醇发酵性能受合成气组分、抑制物浓度、气液传质效率、温度和pH等影响,优化并调控关键环境因子有利于提高己醇产量。采用混菌发酵是生物己醇合成的另一种可替代途径,其关键三步反应为：①合成气向乙酸乙醇转化;②乙酸乙醇链延伸向己酸转化;③己酸还原为己醇,相关菌株包括C. ljungdahlii、A. bacchi和C. kluyveri等,然而混合发酵的经济性能有待进一步研究。文中指出未来生物己醇的研究将重点解决己醇产量低的“瓶颈”问题,通过合成生物学或基因工程手段改造并获得高产己醇菌未来可期。     

黄色短杆菌ilvN基因定点突变和ilvBN、ilvC串联表达对L-缬氨酸产量的影响

由ilvBN、ilvC基因编码的乙酰羟酸合成酶（AHAS）和乙酰羟酸异构还原酶（AHAIR）是L-缬氨酸合成途径的两个关键酶。本实验以黄色短杆菌Brevibacterium flavum MD515为出发菌株,通过PCR技术扩增其ilvBN和ilvC基因,对调节亚基ilvN进行定点突变,获得抗反馈抑制突变型编码基因ilvBN^rC;然后将其插入穿梭表达载体pZ8-1中,构建串联表达质粒pZ8-1-ilvBN^rC并转化出发菌株,筛选获得工程菌株B.flavum MD515/pZ8-1-ilvBN^rC。摇瓶发酵该工程菌株L-缬氨酸产量达29.5 g·L^-1,较出发菌株提高27.7%,同时生长速度和生物量也比出发菌株有所提高,丙氨酸含量降低,L-亮氨酸及L-异亮氨酸含量提高。在30 L发酵罐连续补料发酵60 h后L-缬氨酸产量达61.7 g·L^-1,糖酸转化率为39.2%。菌株MD515/pZ8-1-ilvBN^rC发酵液透光率较出发菌株高且蛋白含量低,这些特性有利于发酵液后期的分离提取。     

安丝菌素P-3生产菌株Actinosynnema pretiosum ssp. auranticum的诱变育种及其发酵培养基优化

为了提高安丝菌素P-3（AP-3）的发酵产量,通过紫外诱变并运用紫外-分光光度法建立96孔板高通量快速筛选体系对原始菌株（Actinosynnema pretiosum ssp.auranticum）进行育种,筛选获得了一株产量较高的突变菌株B24-13。发酵7天后,其发酵液中AP-3的含量为112.5mg/L,是原始菌株的2.03倍。随后通过单因素优化与正交实验设计对突变菌株B24-13进行发酵培养基优化,得到最优发酵培养基组分：蔗糖25g/L,甘油15g/L,玉米浆25g/L,CaCO₃ 7g/L,异丁醇2g/L,缬氨酸0.5g/L,MgSO₄·7H₂O 0.5g/L,FeSO₄·7H₂O 0.01g/L。对优化结果进行验证实验,发酵7天后AP-3的产量为（127.5±6.3） mg/L。实验结果表明：通过对生产菌株的诱变育种与发酵培养基优化可有效的提高AP-3的发酵产量,也从侧面验证了该研究所建立的96孔板高通量快速筛选体系用于AP-3高产菌株的初筛是可行的。     

Production of eicosapentaenoic acid bySaprolegnia sp. 28YTF-1

Saprolegnia sp. 28YTF-1, isolated from a freshwater sample, is a potent producer of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). The fungus used various kinds of carbon sources, such as starch, dextrin, sucrose, glucose, and olive oil for growth, and olive oil was the best carbon source for EPA production. The EPA content reached 17 mg/g dry mycelium (0.25 mg/L) when the fungus was grown in a medium that contained 2.5% olive oil and 0.5% yeast extract, at pH 6.0 and 28°C for 6 d with shaking. Accompanying production of arachidonic acid (AA; 3.2 mg/g dry mycelia, EPA/AA = 5.1) and other ω6 polyunsaturated fatty acids was low. Both EPA content and EPA/AA ratio increased in parallel by lowering growth temperature. Triglyceride was the major mycelial lipid (ca. 84%), but EPA comprised only 2.2% of the total fatty acids of this lipid. About 40% of the EPA produced was found in polar lipids, such as phosphatidylethanolamine (EPA content, 28.2%), phosphatidylcholine (13.6%), and phosphatidylserine (21.2%).     

Metabolism and synthesis of lipids in the polyunsaturated fatty acid-producing fungus Mortierella alliacea

The fungal strain Mortierella alliacea YN-15 is a promising industrial producer of polyunsaturated fatty acids (PUFAs), in particular arachidonic acid. In order to more efficiently produce PUFAs, the metabolism of an externally supplied plant oil, α-linolenic acid (ALA)-rich linseed triacylglycerol (TAG), was examined, and time-dependent changes in the composition of its lipid and fatty acid metabolites were traced. Addition of linseed TAG to growing cultures resulted in a transient increase in extracellular 1,2-diacylglycerol (DAG), and even more so of 1,3-DAG, in the mycelia. This was followed by a decrease in both DAGs and an increase in TAG. Eicosapentaenoic acid (EPA), a desaturated and elongated product of ALA, accumulated to a greater extent in cellular phospholipids than in neutral lipids. Moreover, the addition of ALA in free fatty acid form to the culture led to the generation of EPA. However, EPA production was not observed upon addition of ALA-rich 1,2- or 1,3-DAG, indicating that fatty acids released from exogenous lipids were used for resynthesis of mycelial TAG. These results suggested that TAG might be hydrolyzed by extracellular lipases, whereas its synthesis might be catalyzed by intracellular enzymes. Appropriate regulation of such enzymes might be an effective strategy to enhance PUFA production under plant oil supplementation.     

Increase of plasma fatty acids without changes in n-6/n-3-PUFA ratio in asymptomatic obese subjects

Obesity is associated with a low grade inflammation which contributes to the development of insulin resistance and diabetes. The aim of this study was to assess the total saturated (SFAs), monounsaturated (MUFAs) and polyunsaturated fatty acids (PUFAs) in plasma from asymptomatic obese subjects and to determine the arachidonic/eicosapentanoic acid ratio [ARA/EPA] as a marker of inflammation, and its eventual association with ultrasensitive CRP. Fourteen obese (34.4 +/- 11.1y.; BMI: 36.0 +/- 4,5 kg/m2) and 12 normal-weight (30.6 +/- 7.8y.; BMI: 23,6 +/- 2,4 kg/m2) subjects were recruited and their plasma fatty acids were determined by gas chromatography. usCRP was higher in the obese subjects (p = 0.01) and correlates with their body fat content. The percentages of SFAs, MUFAs, PUFAs were not affected in the obese subjects but their concentrations were increased, compared with the control group. However, no differences in the long chain PUFAs (DHA and EPA) concentrations or in the plasmatic ARA/EPA ratio were observed in these subjects. These observations do not support a relation between the ARA/EPA ratio and the presence of low grade inflammation evaluated by plasma usCRP in this group of asymptomatic obese subjects.     

Comparison between extraction of lipids and fatty acids from microalgal biomass

Seven solvent mixtures have been used to extract the lipid fraction of lyophilized biomass ofIsochrysis galbana. Six of them were composed of biocompatible solvents. Each method was carried out under relaxed operating conditions (i.e., one hour at room temperature) with extraction in a nitrogen atmosphere to prevent autooxidation and degradation of polyunsaturated fatty acids (PUFAs). Apart from the well-established Bligh and Dyer method [Can. J. Biochem. Physiol. 37:911 (1959)] (Cl₃CH/MeOH/H₂O, 1∶2∶0.8, vol/vol/vol), which rendered the highest yield of lipids (93.8%), ethanol (96%) and hexane/ethanol (96%), 1∶2.5 vol/vol produced the best results (84.4 and 79.6%, respectively). To obtain free fatty acids, KOH was added to the solvent mixtures used to extract the total lipids, except for Cl₃CH/MeOH/H₂O, and direct saponification was carried out at 60°C for 1 h or at room temperature for 8 h. The highest yields obtained by direct saponicification were 81% with hexane/ethanol (96%), 1∶2.5, vol/vol and 79.8% with ethanol (96%). Partial yields of the mainn-3 PUFAs found inI. galbana, stearidonic acid (SA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were calculated for both extraction methods. For lipid extraction with ethanol (96%), yields of 91, 82 and 83% were obtained for SA, EPA and DHA, respectively. When direct saponification was used, hexane/ethanol (96%; 1∶2.5, vol/vol) produced the best yields of (91, 79 and 69% for SA, EPA and DHA, respectively).     

Inhibition of gonadotropin-stimulated ovarian steroid production by polyunsaturated fatty acids in teleost fish

The effects of the polyynsaturated fatty acids (PUFAs)—eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA)—onin vitro steroid production by full-grown prematurational ovarian follicles from goldfish and rainbow trout were investigated. EPA and DHA inhibited gonadotropin-stimulated testosterone production in a dose-related manner, but AA was inhibitory only at the highest dose tested (400 μM). AA alone stimulated testosterone production by increasing cAMP production, but the effects of other PUFAs alone were marginal. The inhibitory actions by PUFAs were not restricted to long-chain PUFAs, as linoleic and linolenic acids had similar actions in the goldfish. The inhibitory action of EPA on testosterone production was reversible upon removal of the PUFA from medium. Testosterone production stimulated by the addition of the cAMP analogues, dibutyryl cAMP, and 8-bromo cAMP, was attenuated by PUFAs, suggesting that they act at a site distal to cAMP formation. A post-cAMP site regulating cholesterol availability may be involved as testosterone production induced by addition of 25OH-cholesterol was not affected by the PUFAs in either fish species. Together, these findings underscore the importance of lipids in ovarian physiology and suggest that PUFAs may participate in the regulation of ovarian steroidogenesis in teleost fish.     

Production of eicosapentaenoic acid from marine bacteria

A marine bacterium, judged as a new species close toShewanella putrefaciens, was isolated from the intestinal contents of the Pacific mackerel. The isolated strain SCRC-2378 produced eicosapentaenoic acid (EPA) as the sole polyunsaturated fatty acid, which amounted to 24–40% of the total fatty acid in the cell, which corresponded to 2% of dry cell weight. Under the optimal growth conditions (pH 7.0, 20°C, and grown aerobically for 12–18 h), the yield of SCRC-2738 reached 15 g of dry cells/L or 2×10¹⁰ viable cells/mL. EPA existed as phospholipid and was found in thesn-2 position of phosphatidylethanolamine and phosphatidylglycerol. The 38 kbp (1,000 base pairs) genome DNA fragment was cloned from SCRC-2738 and expressed inEscherichia coli, which resulted in the production of EPA. The nucleotide sequence of the 38 kbp DNA fragment was determined. The DNA fragment contains eight open reading frames, and three of them posses homology with enzymes involved in fatty acid synthesis. Thus, it may be possible that these EPA biosynthesis genes are applied for EPA production in yeasts or higher plants, and offer a new method for EPA synthesis as new foods containing EPA.     

Serum and diet long-chain omega-3 fatty acid nutritional status in Chinese elite athletes

Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) are essential for improving the health and performance of athletes. The present study aimed to evaluate the nutritional status of omega-3 PUFAs in Chinese elite athletes by both dietary intake analysis and serum biomarker detection. A cross-sectional analysis of data from 54 elite athletes (24 men and 30 women) from Shanghai professional sports teams was conducted. A food frequency questionnaire (FFQ) was employed to analyze dietary intake, and gas chromatography–mass spectrometry (GC–MS/MS) was conducted to measure serum biomarkers of PUFAs. Correlation analysis was performed to investigate the relationships of PUFA biomarkers with diet, inflammation and oxidative stress. The results showed that the median intake of EPA + DHA among athletes was 132 mg/d, which is lower than the minimum value recommended by dietary guidelines (250 mg/d). The average serum EPA + DHA was 4.0 ± 1.1%, and the ratio of omega-6/omega-3 was 7.7 ± 1.7. Most (96.3%) of the athletes were below the targeted value of serum EPA + DHA, which is associated with a reduction in cardiovascular risk. Correlation analysis showed that the serum EPA + DHA was positively correlated with the long-term dietary intake of EPA + DHA and negatively correlated with inflammatory markers. In conclusion, the serum circulating EPA + DHA and omega-6/omega-3 ratio are effective biomarkers reflecting the nutritional status of PUFAs in athletes. Omega-3 PUFAs have a potential effect on inhibiting inflammatory markers. Hence, it is necessary for Chinese athletes to improve their suboptimal nutritional status of PUFAs through dietary intervention.     

Chemometric Characterization and Classification of Selected Freshwater and Marine Fishes from Turkey Based on their Fatty Acid Profiles

The fatty acid (FA) profiles of edible muscle of selected commercially important freshwater and marine fish species from Turkey were investigated. The fatty acid compositions of freshwater fish species were 23.00–29.60% saturated (SFA), 25.70–36.50% monounsaturated (MUFAs), and 26.59–31.92% polyunsaturated acids (PUFAs), whereas the fatty acid compositions of marine fish consisted of 21.08–36.90% (SFA), 18.03–51.45% MUFAs, and 20.92–53.17% PUFAs. There was a wide variation and significant (P < 0.05) differences among the fatty acid profiles of the freshwater and marine fish samples, including differences in the SFA, MUFA, PUFA, EPA, DHA, DHA/EPA, total n-3 PFAs, total n-6 PUFAs and n-3/n-6 values. In addition, the cheap marine fish species such as anchovy and European pilchard, bogue are better dietary sources of n-3 PUFAs than more expensive species such as bluefish, Atlantic mackerel, sea bream and sea bass. Through the application of two multivariate statistical methods, Principal Component and Hierarchical Analysis, fish species from Turkey waters were classified according to the geographical locations categorized in terms of fatty acid profiles. Clustering by fish species also gave rise to defined groups.     

甲醇溶剂法分离蚕蛹油多不饱和脂肪酸

采用甲醇溶剂法分离蚕蛹油多不饱和脂肪酸。通过单因素实验探讨了温度、甲醇浓度、甲醇脂肪比、时间对多不饱和脂肪酸分离效果的影响,然后通过正交实验确定了多不饱和脂肪酸分离的较佳条件。结果表明,适宜的分离条件为：温度-10℃,甲醇浓度90%（w）,甲醇脂肪比2.5,结晶时间30min,在上述条件下多不饱和脂肪酸得率为62.3%,含量由71.0%提高到95.8%。     

Metabolic Effects of Krill Oil are Essentially Similar to Those of Fish Oil but at Lower Dose of EPA and DHA,in Healthy Volunteers

The purpose of the present study is to investigate the effects of krill oil and fish oil on serum lipids and markers of oxidative stress and inflammation and to evaluate if different molecular forms, triacylglycerol and phospholipids, of omega-3 polyunsaturated fatty acids (PUFAs) influence the plasma level of EPA and DHA differently. One hundred thirteen subjects with normal or slightly elevated total blood cholesterol and/or triglyceride levels were randomized into three groups and given either six capsules of krill oil (N = 36; 3.0 g/day, EPA + DHA = 543 mg) or three capsules of fish oil (N = 40; 1.8 g/day, EPA + DHA = 864 mg) daily for 7 weeks. A third group did not receive any supplementation and served as controls (N = 37). A significant increase in plasma EPA, DHA, and DPA was observed in the subjects supplemented with n-3 PUFAs as compared with the controls, but there were no significant differences in the changes in any of the n-3 PUFAs between the fish oil and the krill oil groups. No statistically significant differences in changes in any of the serum lipids or the markers of oxidative stress and inflammation between the study groups were observed. Krill oil and fish oil thus represent comparable dietary sources of n-3 PUFAs, even if the EPA + DHA dose in the krill oil was 62.8% of that in the fish oil.