Production of artemisinin by hairy rot cultures of Artemisia annua L in bioreactor

Hairy root cultures of Artemisia annua L were cultivated in four different culture systems: a flask, a bubble column, a modified bubble column and a modified inner-loop airlift bioreactor. The artemisinin contents of hairy root cultures in the bubble column and the modified inner-loop airlift bioreactor were higher than that in the modified bubble column. The growth rate and hairy root distribution in the modified inner-loop airlift bioreactor were better than those in other bioreactors, and dry weight and artemisinin production reached to 26.8 g/L and 536 mg/L after 20 days.     

Artemisinin Production by Adventitious Shoots of Artemisia annua in a Novel Mist Bioreactor

A novel ultrasonic inner-loop bioreactor was used for artemisinin production by adventitious shoots in a multiplate culture of Artemisia annua L. The bioreactor was designed to allow the nutrient mist to uprise along a concentric draught-cylinder until it overflows from the top opening and the side-holes of the central tube downward and out of the annulus, so that the nutrient mist can be fulfilled in the bioreactor within 2 ~ 3 minutes. Under the misting cycles of every 3-minute misting in every 90 minute interval, artemisinin production reached totally 46.9 mg DW/L of culture medium at an airflow rate of 0.5 L/min for 25 d of culture in batches. The product amounted 2.9 and 3.2 folds of those obtained from culturing in solid medium and in shaking flasks respectively.     

Studies of Technical Conditions of Somatic Embryogenesis in Cell Suspension Culture of Apium Graveolens

Using hypocotyls (5~10 mm) of Apium graveolens L. as explant, calli were induced in induction medium (MS + 1.0 mg/L 2, 4-D). The embryogenic calli were transformed to differentiation medium (MS+0. 5 mg/L kinetin+ 500 mg/L CH+500 mg/L Prolin) after several subsequent subcultures and selection by replacement of solid and liquid medium. Technical conditions such as the shake rate of the flask, the initial cell density, as well as subsequent the initial pH values during culture were under consideration. With the optimum flask shake rate of about 100~150 r/min, initial cell density of 2.0% (fresh weight) and the initial pH value of 5.5, the authors have obtained 130 normal cotyledon embryos in each mL of cultures.     

Isolation and production of artemisinin and stigmasterol in hairy root cultures of Artemisia annua

Scaled-up hairy root culture of Artemisia annua L. was established in three-liter Erlenmeyer flask. Both artemisinin and stigmasterol that derive from the common precursors of isopentenyl diphosphate and farnesyl pyrophosphate were isolated from hairy roots. The production rate of artemisinin isolated by column chromatography from hairy root cultures was 0.54% (mg.gDW⁻¹). Stigmasterol was identified by mass spectrometry and nuclear magnetic resonance analysis. The production of stigmasterol isolated by column chromatography from hairy root cultures was 108.3% (mg.gDW⁻¹). In hairy root cultures, the production rate of stigmasterol was estimated to be 201 times greater than that of artemisinin. Our results suggest that investigation of secondary metabolites may provide a new insight to study artemisinin production in hairy root cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

乳糖诱导重组尿酸酶基因在大肠杆菌中的表达

对用乳糖替代异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组产朊假丝酵母尿酸酶基因在E.coli JM109(DE3)中表达进行了研究,拟建立一种高效低成本的生产重组尿酸酶的工艺路线。通过摇瓶试验对诱导所采用的乳糖浓度,诱导时机和诱导持续时间进行了优化,并考察在乳糖诱导下的目的产物表达动力学,随后在5 L发酵罐上进行扩大化培养以验证摇瓶优化的结果,进一步将乳糖作为诱导剂应用于高密度发酵过程。实验结果表明乳糖诱导的最佳浓度为5 g/L,最佳诱导时机是对数生长期中后期,诱导持续时间为9~10h;按照优化的条件在摇瓶和5 L发酵罐上进行分批培养,重组尿酸酶最大表达量可达菌体总蛋白的26%左右,可溶性蛋白的36%左右,略高于IPTG的诱导效果;高密度发酵过程菌体终密度达到OD600值40以上,尿酸酶表达量占菌体总蛋白25%左右。     

树状多节孢发酵生产紫杉醇工艺条件的初步研究*

研究了树状多节孢HQD33内生真菌融合子TPF-1摇瓶发酵工艺条件,进行了2.8L和10L通用式机械搅拌罐的发酵试验。结果表明,HQD33适宜发酵工艺条件是:发酵时间16～18d,培养基中蔗糖、苯丙氨酸、醋酸钠、酪氨酸、2,4-二氯苯氧乙酸和亚油酸的加量分别为10g/L、1mg/L、1.5g/L、15mg/L、5mg/L和15mg/L,摇瓶装量为150ml/500ml,在此条件下摇瓶发酵液中紫杉醇平均含量为448.52 g/L; 2.8L和10L罐发酵液中紫杉醇含量达406.95和395.12g/L(平均值)。     

低温脂肪酶产生菌的筛选、产酶发酵及粗酶性质研究

利用含有Tween 80的琼脂平板和摇瓶发酵法,从若尔盖高原土壤中筛选产脂肪酶菌株.通过菌落形态和菌体特征观察初步对菌种进行鉴定,得到一株产低温脂肪酶的适冷菌Pseudomonassp.DL-B,并设计正交试验对该菌株的产酶发酵培养条件进行了优化.摇瓶实验表明,该菌株最适产酶发酵培养基为:蔗糖10 g/L,蛋白胨20 ...     

不同摇瓶条件对侧耳菌生长及漆酶分泌的影响

研究了小试液体摇瓶下不同pH培养条件、摇瓶转速、装液量对侧耳属白腐菌BP漆酶分泌及生长状况、形态变化的影响。结果表明：BP在中、酸性条件下生长较好且表现较高的分泌漆酶能力,用硫酸调pH为5．0时,漆酶活力和生物量分别在第10d和第6d达到最高,分别为742U／mL和8．4g(干重)/L,高于相应缓冲液调节pH体系的结果;天然(pH6．5)培养条件下,最佳转速与装液量分别是150r／min、200mL/500mL三角瓶,此时菌球较小,四周为毛刺状,漆酶酶活和生物量分别在第5d和第6d达到最高,分别为714．2U／ml和9．2gtL。同时,运用SPSS统计软件对各影响因素进行差异显著性检验,获得白腐菌生长及漆酶分泌特性的主要影响因子。     

Enhanced production of artemisinin by Artemisia annua L hairy root cultures in a modified inner-loop airlift bioreactor

Hairy root cultures of Artemisia annua L were cultured in a modified inner-loop airlift bioreactor for achieving maximum artemisinin production. The effects of initial pH, air flow rate, cycle of light irradiation and temperature on growth and artmisinin production in Artemisia annua L hairy root cultures were investigated. Under the optimum conditions, the maximum production of artemisinin reached to 577.5?mg/l after 20 days.     

在大肠杆菌内引入甲羟戊酸途径高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯

以青蒿素为基础的联合药物疗法 (ACTs) 被认为是目前治疗恶性疟疾的最有效方法。然而青蒿素供应不足且价格昂贵,限制了ACTs的广泛使用。采用基因工程手段构建异源类异戊二烯生物合成途径,利用大肠杆菌发酵能高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯。首先在大肠杆菌Escherichia coli DHGT7中引入人工合成的紫穗槐-4,11-二烯合酶基因,利用大肠杆菌内源的法尼基焦磷酸,成功获得了紫穗槐-4,11-二烯。为提高前体供给,引入粪肠球菌的甲羟戊酸途径,紫穗槐-4,11-二烯的产量提高了13     

酿酒酵母突变株J-X25胞内合成GSH的研究

以筛选得高产谷胱甘肽(GSH)产生酵母甲硫氨酸缺陷型变株J-X25为试验菌株。对其培养条件进行研究,结果表明：发酵培养基的最适初始pH值为6.0、最佳发酵温度为30℃、最佳装液量为100ml/500ml、接种量10%、摇床转速为220r/min。在酵母细胞培养到对数期,加入过氧化氢刺激细胞发生应激反应和乳酸钠作为表面活性剂改变细胞通透性,GSH总量达到0.253g/L,比不添加两者情况下的GSH产量高出52％。结果表明优化培养条件后,J-X25胞内积累GSH比出发株提高79％。     

毕赤酵母表达Q8008发酵条件的优化研究

目的对表达巴斯德毕赤酵母的发酵条件进行优化,确定最佳的发酵控制条件,以获得重组textilinin-1(Q8008)最高表达量。方法通过多因素正交实验确定Q8008发酵培养的最适发酵条件。结果表达温度在22℃,pH值为7.0,加入甲醇量为5g/L时为最优发酵条件,诱导表达时间为84~108h。结论优化巴斯德毕赤酵母的发酵条件,可提高Q8008表达量,为开发抗纤溶酶止血药应用于临床具有重要意义。     

内生真菌Preussia sp.液体发酵生产抗肿瘤活性产物Spiropreussione A 的研究

螺光黑壳菌酮A(Spiropreussione A,SP-A)是编号为AS-5的光黑壳属内生真菌Preussia sp.的代谢产物。体外实验表明SP-A对人卵巢癌细胞A2780和人肝癌细胞BEL-7404的半数抑制浓度(IC50)分别为2.4和3.0μmol/L。以SP-A的含量为主要指标,结合AS-5的生物量,通过单因素实验和正交实验,优化确定了适合SP-A积累的AS-5液体发酵培养基和培养条件。研究结果表明:AS-5发酵生产SP-A的最优培养基为葡萄糖2%,麦麸3%,磷酸二氢钾0.3%,硫酸镁0.15%,pH7.0;该菌株最佳摇瓶发酵条件为250mL摇瓶装125mL培养基,接种6片直径9mm的PDA菌片,培养温度25℃,发酵周期16d。在此条件下发酵,SP-A的含量可以达到(25.02±1.02)mg/瓶,比优化前的含量[(17.08±3.24)mg/瓶]提高了46.5%。     

酵母不对称催化制备R-1,2-丙二醇

研究了采用面包酵母还原丙酮醇制备R-1,2-丙二醇的工艺。采用摇瓶对转化条件进行单因素实验,确定最优转化条件：丙酮醇浓度0.3mmol/mL,pH7.0,酵母质量浓度150g/L,乙醇浓度为0.3mmol/mL,转化时间25h。在此条件下,采用分批流加策略进行1.5L规模发酵罐转化试验,转化25h后,发酵液的R-1,2-丙二醇浓度为0.27mmol/mL。     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

多拷贝毕赤酵母重组菌表达GCL摇瓶优化和高密度发酵

目的:构建高效表达白地霉脂肪酶的毕赤酵母重组菌株,并对筛选得到的菌株进行摇瓶发酵条件优化和分批补料高密度发酵工艺研究。方法:将诱导型表达载体pPIC9K-gcl电转化至毕赤酵母GS115。通过橄榄油-罗丹明B平板和摇瓶发酵筛选高脂肪酶活力的重组菌株,运用基于TaqMan探针的实时荧光定量PCR 法确定其拷贝数,并对菌株进行摇瓶发酵条件优化。在此基础上,研究重组菌在3L 发酵罐中的高密度发酵工艺。结果:筛选得到一株具有3 个白地霉脂肪酶基因拷贝的菌株GS115/pPIC9K-gcl 78#,初始酶活力为220 U/ml。当摇瓶发酵条件为甲醇诱导96 h,每24 h甲醇添加量1 %,接种量2 %,培养基初始pH 7.0,500 ml摇瓶装液量50 ml,甲醇诱导温度25℃ 时酶活力达735 U/ml。3L 发酵罐高密度发酵176.5 h,酶活力达到3360 U/ml,总蛋白含量达到4.30 g/L,且发酵过程中细胞活性一直保持在96 % 以上。结论:基因拷贝数与重组菌株的产酶水平呈正相关,摇瓶优化可显著提高重组菌株的产酶能力,为白地霉脂肪酶的工业化生产奠定了技术基础。     

促进黄花蒿发根青蒿素合成的内生真菌诱导子的制备

应用酸解法对黄花蒿(ArtemisiaannuaL.)内生胶孢炭疽菌(Colletotrichumgloeosporioides)菌丝体进行提取,在黄花蒿发根培养系统中比较了各制备提取物的青蒿素诱导活性。活性提取物经过SephadexG25层析后,部分纯化的内生菌寡糖提取物(MW<2500)可显著促进发根青蒿素的合成,培养23d的发根经诱导子(0.4mg/mL)处理4d后,青蒿素产量可达13.51mg/L,比同期对照产量提高51.63%,诱导作用与诱导子浓度、作用时间相关。内生菌寡糖诱导子的制备和使用,在青蒿素生物技术生产研究中为首次应用。     

The improvement of cephalosporin C production by fed-batch culture ofCephalosporium acremonium M25 using rice oil

The objective of this study is to improve cephalosporin C (CPC) production by optimization of medium and culture conditions. A statistical method was introduced to optimize the main culture medium. The main medium for CPC production was optimized using a statistical method. Glucose and corn steep liquor (CSL) were found to be the most effective factors for CPC production. Glucose and CSL were optimized to 2.84 and 6.68%, respectively. CPC production was improved 50% by feeding of 5% rice oil at day 3rd and 5th day during the shake flask culture ofC. acremonium M25. The effect of agitation speeds on CPC production in a 2.5-L bioreactor was also investigated with fed-batch mode. The maximum cell mass (54.5 g/L) was obtained at 600 rpm. However, the maximum CPC production (0.98 g/L) was obtained at 500 rpm. At this condition, the maximum CPC production was improved about 132% compared to the result with batch flask culture.     

High-Level Production of Amorpha-4,11-Diene,a Precursor of the Antimalarial Agent Artemisinin,in Escherichia coli

Background

Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.

Methodology/ Principal Findings

By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.

Conclusions/ Significance

Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.     

Rhamnolipid production in batch and fed-batch fermentation using<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> BYK-2 KCTC 18012P

The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).