紫外线诱变深黄被孢霉选育花生四烯酸高产菌株

为获得生长活力较强, 产花生四烯酸能力强的菌株, 以微生物油脂产量和花生四烯酸产量为评价指标, 采用2轮紫外线诱变的方法, 利用单因素试验确定紫外线照射时间, 并采用气相色谱分析花生四烯酸含量。试验结果表明: 紫外灯功率20 W, 照射距离30 cm, 照射时间80 s, 其致死率为76.4%。经过诱变及菌种筛选, 获得1株高产菌株Z80s2-109, 其总油脂含量为16 g/L, 花生四烯酸产量为2.34 g/L, 花生四烯酸产量比原始对照菌株提高377%, 并且遗传性能稳定。     

微波等离子体溅射诱变选育花生四烯酸高产菌及补料工艺研究

采用微波等离子（N^ ,15w,3min)溅射高山被孢霉T105,筛选到高产菌R254,该菌最高生物量29．3g/L,油脂11．5g/L,花生四烯酸4．20g/L,花生四烯酸产量是原出发菌株1．35倍。通过对突变株R245的代谢情况、继代稳定性以及脂肪酸组成分析,认为微波等离子溅射诱变育种是获得花生四烯酸高产菌株的有效方法。并采用补糖工艺可进一步提高其产量,花生四烯酸产量为7．43g/L,是未补糖时花生四烯酸产量的1．76倍,且为出发菌株花生四烯酸最高产量的2．40倍。     

YAG激光照射对高山被孢霉花生四烯酸产量的影响

花生四烯酸产生菌高山被孢霉的孢子在时间为8min,距离为10cm的YAG激光剂量照射下,其致死率为75％－80％,以该剂量反复进行照射及菌种筛选,得到其最高生物量28.6g/L,油脂11.04g/L,3.10g/L的突变株T105,比对照菌株的AA产量及油脂量分别提高了2．95倍和1．71倍。通过对突变株T105的形态,代谢情况以及继代稳定性进行分析,认为YAG激光诱变育种是获得茶花生四烯酸高产菌株的有效方法。     

高产DHA破囊壶菌Aurantiochytrium sp. PKU#SW7诱变株的筛选

【目的】对野生菌株Aurantiochytrium sp.PKU#SW7诱变育种,筛选高产DHA突变株。【方法】采用UV诱变和化学药物胁迫筛选方式,以菌株的生物量、油脂产量、DHA产量作为筛选指标,获得高产DHA突变株。【结果】经鉴定获得一株DHA高产突变株PKU#PM003,该菌株传代4次后仍保持较好的遗传稳定性。摇瓶发酵后,PKU#PM003生物量产量高达6.62 g/L,比原始菌株5.95 g/L提高了11.26%,脂肪酸含量高达4.01 g/L,比原始菌株3.18 g/L提高了26.1%,DHA在脂肪酸中所占比例由29.97%增加到33.43%,产量提高了41.01%,油脂突变效果显著。【结论】突变株PKU#PM003可作为性状优良的工业化发酵生产菌种,并在DHA产量提升上仍具有巨大的空间。     

紫外线和硫酸二乙酯复合诱变选育PHB高产菌株

【目的】获得高产聚β-羟基丁酸酯(Poly-β-hydroxybutyricacid,PHB)菌株。【方法】以假单胞菌属Pseudomonas koreensis PK3菌株为出发菌株,采用硫酸二乙酯和紫外线相结合的诱变方法进行多轮诱变。【结果】经过初筛和复筛得到一株高产PHB突变菌株,命名为Pseudomonas koreensis UVCN-18。连续传代9次后,发酵28 h条件下,PHB产量达到15.94 g/L,占细胞干重69.54%,较原出发菌株Pseudomonas koreensis PK3(4.42 g/L)提高了2.61倍,并且该菌株具有良好的遗传稳定性。【结论】采用硫酸二乙酯和紫外线相结合的诱变方法,成功获得了一株高产PHB突变菌株。     

高产不饱和油脂海鞘真菌桔青霉Asc-2-4的诱变选育

诱变育种是获得高产菌株,实现微生物工业化生产油脂的重要措施。以前期获得的高产不饱和油脂菌株桔青霉（Penicillium citrinum）Asc-2-4为出发菌株,利用丙二酸建立快速筛选高产不饱和脂肪酸突变菌的方法,通过紫外线 氯化锂复合诱变得到1株高产油脂突变菌Asc-2-4-1,油脂含量比出发菌株提高了92.98%。经过初步的培养基无机盐优化,其油脂得率和不饱和脂肪酸产量达到了7.10 g/L和3.84 g/L,与Asc-2-4相比,分别提高了84.42%和77.78%。结果表明,通过复合诱变选育技术可选育出高产突变菌株,选育的Asc-2-4-1可望作为产油微生物被开发利用。     

花生四烯酸高产突变株的选育及其发酵调控

以拉曼被孢霉（Mortierellaramanniana）SM541为原始菌株,经过紫外线复合氯化锂诱变处理,得到突变株SM541－9,其生物量由126g／L提高到28．8g／L,油脂含量由5．8g／L提高到15.7g／L。花生四烯酸含量由321mg／L增加到623mg／L。传代实验表明,SM541－9具有良好的遗传稳定性,以葡萄糖为碳源,硝酸钾为氮源的最适培养条件下,10L罐发酵,生物量和花生四烯酸含量较原始菌株分别提高了45．2％和109.5％。在液体发酵过程中,菌丝老化、增大溶氧量、间歇流加葡萄糖都有利于花生四烯酸的合成。     

深黄被孢霉△5-脱饱和酶基因在花生四烯酸合成中的作用

【目的】研究△5-脱饱和酶基因的mRNA表达量与花生四烯酸产量之间的关系。【方法】实验利用荧光定量PCR方法检测了△5-脱饱和酶在五株深黄被孢霉的同一培养时间及菌株YZ-124在不同发酵阶段的mRNA表达水平,同时利用气相色谱仪测定其花生四烯酸含量。【结果】结果表明:不同菌株的△5-脱饱和酶基因的mRNA表达水平不同,原始菌株As3.3410最低,诱变菌株YZ-124最高;深黄被孢霉YZ-124不同发酵阶段的△5-脱饱和酶基因mRNA表达量随菌龄的增加逐渐增加。【结论】结合ARA的得率显示,△5-脱饱和酶基因mRNA表达量与培养物油脂中ARA含量呈一定的正相关关系。     

花生四烯酸油脂高产菌株的选育

以高山被孢霉为出发菌株,抗氧化剂——没食子酸辛酯为筛选剂,经过紫外-LiCl复合诱变处理,筛选出抗脂肪酸脱氢酶抑制剂的菌株。将筛选出的菌株经过摇瓶发酵复筛,筛选到1株生产性能优于出发菌株的突变株R807。与原始菌株相比,该菌株的油脂组成脂肪酸分布中C18系列脂肪酸相对较少,花生四烯酸(ARA)占总脂肪酸的含量保持在40%(质量分数)以上。其菌体生物量达到39.2 g/L,油脂产量达到16.3 g/L,ARA占总脂肪酸含量为41.72%(质量分数),ARA产量达6.81 g/L。各数值比原始菌株分别提高了22.9%、3.2%、35.1%和39.8%。连续传代多次,其产量性状无显著变化。     

原生质体紫外诱变选育可利用廉价碳源的高产油新菌株

【目的】研究并建立利用原生质体紫外诱变技术选育可利用廉价碳源发酵的高产油新菌株的方法。【方法】采用1.5%蜗牛酶和1.0%纤维素酶混合液水解去除细胞壁得到2A00015(近平滑假丝酵母,Candida parapsilosis)的原生质体,将其放于紫外灯下诱变及再生壁培养,筛选获得可利用廉价碳源发酵的高产油酵母,并采用气相色谱质谱联用法(GC-MS)测定其脂肪酸组成。【结果】突变效果最好的突变菌株2A00015/25用葡萄糖发酵培养7 d后,其生物量、油脂产率和产油量分别为17.77 g/L、58.12%和10.32 g/L,较原始菌株分别提高了12.45%、23.32%和38.68%;利用废糖蜜发酵培养,其生物量、油脂产率和产油量分别为18.54 g/L、49.44%和9.17 g/L,较原始菌株分别提高了9.09%、21.16%和32.18%。利用废糖蜜培养其产油效率虽低于利用葡萄糖培养,但从环境保护及原材料成本的角度考虑,用废糖蜜作为碳源发酵培养产生油脂更具优势。诱变菌株利用废糖蜜发酵后产生油脂经检测含有8种脂肪酸,其脂肪酸组成与植物油近似,其中不饱和脂肪酸含量占脂肪酸总量的82.4%。【结论】通过利用原生质体紫外诱变技术,成功选育出一株新的可利用廉价碳源的高产油海洋菌株,产油率达到49.4%,提高了21.2%。     

谷氨酸对花生四烯酸产生菌被孢霉发酵的影响

研究了不同浓度谷氨酸对被孢霉生产花生四烯酸的影响,发现当加入谷氨酸浓度为0.8g/L时总油脂和花生四烯酸产量达最高,选择0.8g/L谷氨酸进行花生四烯酸产生菌被孢霉发酵动力学研究的结果表明,在培养基中加入0.8g/L谷氨酸可以明显促进被孢霉的生长,加速基质代谢,提高单位被孢霉中的油脂和花生四烯酸产量,尤其在发酵第7d时生物量、油脂和花生四烯酸产量达到最大,分别为24.43、9.21、1.41g/L,分别是对照组第7d所得的1.13、1.15和1.69倍。     

Production of γ-linolenic acid by Mortierella isabellina grown on hexadecanol

AIMS: To optimize the production of linolenic acid by Mortierella isabellina grown on hexadecanol. METHODS AND RESULTS: Effects of culture conditions such as culture time, pH of medium, hexadecanol concentration, incubation temperature and ageing of mycelia on production of linolenic acid were studied. The production of gamma-linolenic acid reached 2.44 mg ml-1 (271 mg g-1 dry cells) when Mortierella isabellina was cultivated in a medium consisting of 2% hexadecanol and 1% yeast extract at 23 degrees C for 120 h and then the mycelia, after removal of medium by suction filtration, were allowed to stand for a further 15 d at 5 degrees C. CONCLUSION: Ageing of mycelia and incubation temperature showed predominant effects on the increased linolenic acid production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights effective conditions for increasing linolenic acid production by Mortierella isabellina grown on hexadecanol.     

Rapid screening and cultivation of oleaginous microorganisms

Oleaginous microbial strains were cultivated to identify the best oil-producing strain amongst Yarrowia lipolytica (CGMCC 2.1398), Lipomyces starkeyi (CGMCC 2.1608), Rhodosporidium toruloides (CGMCC 2.1389), Mortierella isabellina (CGMCC 3.3410), Cunninghamella blakeleana (CGMCC 3.970), and Mycobacterium QJ311. A method for rapid determination of oil content and fatty acid composition was established to identify the optimum oil-producing strains. This method had a relative standard deviation of 4.09%, an average recovery ratio of 97.09% and a detection limit of 0.1-1.0 g. Mortierella isabellina CGMCC 3.3410 was identified as the best oil-producing strain amongst the six strains tested, with a total biomass of 75 g/10 L and a lipid content of 35%. A rapid screening method of oleaginous microorganisms is discussed for the first time.     

深黄被孢霉△~6-脂肪酸脱氢酶基因的克隆及序列分析

γ－亚麻酸（ＧＬＡ,Ｃ１８：３△６,９,１２）是由△６－脂肪酸脱氢酶以亚油酸（ＬＡ,Ｃ１８：２△９,１２）为底物,在Ｃ６位脱氢形成的。由于在人体中,γ－亚麻酸是花生四烯酸、前列腺素类和白三烯类等生理活性物质的前体物,而深黄被孢霉是目前用于微生物发酵生产γ－亚麻酸的主要菌株。本文根据脂肪酸脱氢酶的保守区设计引物,利用反转录聚合酶链式反应从丝状真菌深黄被孢霉中克隆了编码△６－脂肪酸脱氢酶的ｃＤＮＡ,全长为１３７４个核苷酸,编码４５７ 个氨基酸,但与其他位点的脂肪酸脱氢酶不同的是, △６－脂肪酸脱氢酶在其序列的 Ｎ 端特有细胞色素 ｂ５（Ｃｙｔｂ５）区。这是国际上对深黄被孢霉△６－脂肪酸脱氢酶基因的首次报道。     

Production of arachidonic acid byMortierella alpina ATCC 32222

Summary WhenMortierella alpina ATCC 32222 was incubated in a glucose salts medium at 25°C the biomass (17.5 g/l) contained 9.62% arachidonic acid which amounted to 54% (w/w) of total biomass lipids. When the glucose concentration in the medium was varied from 0 to 150 g/l, the percentage of arachidonic acid in biomass and in lipids was highest at a glucose concentration of 30 g/l, but highest yield of arachidonic acid per litre of culture broth was observed at a glucose concentration of 100 g/l. While production of biomass reached a plateau of 17 g/l after a 3-day incubation at 25°C, the percentage of arachidonic acid in lipids and biomass increased dramatically from 3 to 6 days with a concurrent arachidonic acid yield increase from 0.89 to 1.63 g/l. Optimum initial culture pH for arachidonic acid production was in the range 6.0–6.7. By increasing the concentration of the glucose salts medium three-fold, yields of biomass and arachidonic acid were increased to 35.8 g/l and 3.73 g/l, respectively.     

深黄被孢霉利用不同碳源产油脂比较

本研究主要探讨深黄被孢霉M2菌株对生物质全糖的利用,考察其碳源同化能力、不同碳源下产脂情况以及对玉米皮渣的利用能力。研究结果表明,M2菌株能够利用葡萄糖、木糖、阿拉伯糖和甘露糖进行生长和油脂积累。M2菌株以6%糖浓度的玉米皮渣水解液为底物发酵培养,油脂微生物生物量达18.2g/L,干菌体油脂含量45.7%,单位体积发酵液油脂产量为8.3g/L。     

高产PUFAs深黄被孢霉菌株的筛选

以深黄被孢霉(Mortierella isabellina As3.3410)为出发菌株,经微波诱变和紫外诱变,乙酰水杨酸与低温(15℃)相结合的筛选方法,获得1株高产多不饱和脂肪酸菌株A35-4,其生物量为17.9 g/L,油脂含量为67.8%,油脂产量为12.12 g/L,PUFAs含量为20.3%,PUFAs产量为2.46 g/L,上述指标比原始菌株A0分别增加32.6%、49.8%、98.69%、14.0%和125.7%。连续斜面传代培养证实该菌株具有较好的遗传稳定性。本研究为直接利用该菌株生产PUFAs以及克隆高效PUFAs相关基因,创造高含PUFAs转基因植物材料奠定基础。     

Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media

AIMS: To study patterns of reserve lipid biosynthesis and turnover (degradation) in two oleaginous Zygomycetes, namely Cunninghamella echinulata and Mortierella isabellina under various growth conditions. Fatty acid composition of the reserve lipid of both strains was also studied in all growth steps. METHODS AND RESULTS: Cunninghamella echinulata and Mortierella isabellina were grown in carbon-excess batch cultures. In the investigated strains, accumulation of reserve lipid occurred only when the activity of both NAD(+)-isocitrate dehydrogenase (ICDH) and NADP(+)-ICDH were not detectable in the cell-free extract. Specifically, in C. echinulata, NAD(+)-ICDH activity was detected even after depletion of ammonium nitrogen in the medium, resulting in a delay of the initiation of lipid accumulation period. On the contrary, in M. isabellina, lipid accumulation occurred simultaneously with ammonium nitrogen exhaustion in the growth medium, as the activity of both NAD(+)- and NADP(+)-ICDH were not detectable after nitrogen depletion. In C. echinulata reserve lipid was not degraded after glucose had been exhausted. Supplementations of the medium with Fe(3+), yeast extract or Mg(2+) induced, however, reserve lipid breakdown and formation of lipid-free material. In M. isabellina after glucose exhaustion, notable lipid degradation occurred, accompanied by a significant lipid-free material biosynthesis. Nevertheless, in multiple-limited media, in which Mg(2+) or yeast extract, besides carbon and nitrogen, were limiting nutrients, reserve lipid breakdown was repressed. In both strains, the quantity of gamma-linolenic acid (GLA) in the reserve lipids [varying between 9 and 16% (w/w) in C. echinulata and 1.5-4.5% (w/w) in M. isabellina] was proportional to lipid-free biomass. CONCLUSIONS: Lipid accumulation period in Zygomycetes is initiated by the attenuation of ICDH activity in the mycelium while the regulation of ICDH from ammonium nitrogen is strain specific. While a single nitrogen limitation was enough to induce lipid accumulation, however, multiple limitations were needed in order to repress lipid turnover in oleaginous Zygomycetes. As for GLA, its biosynthesis in the mycelium seemed proportional to lipid-free biomass synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Several nutrients are indispensable for functioning the mechanisms involved in the mobilization of reserve lipid in oleaginous moulds. Therefore, reserve lipid turnover in oleaginous moulds could be repressed in multiple-limited media.