海洋灰绿曲霉发酵生产灰绿霉素及真菌聚酮合成酶基因Tspks1的异源表达
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摘要
灰绿霉素A是中国海洋大学从海洋丝状真菌灰绿曲霉(Aspergillus glaucus HB1-19, CCTCC M 206022)代谢产物中分离出来的一种具有良好抗肿瘤活性、结构新颖的蒽醌内酯衍生物,由聚酮途径合成。本课题旨在通过代谢调控、发酵优化及放大来提高灰绿霉素A产量,促进后续的细胞和动物等药学实验、临床研究及其他构效关系研究,同时为较为缺乏的海洋微生物发酵研究提供参考。此外,鉴于近年来真菌聚酮合成酶基因克隆及功能鉴定的研究发展迅速,但有关真菌宿主异源表达真菌聚酮合成酶基因的研究较少,本文研究了米曲霉M-2-3表达系统异源表达Talaromyces stipitatus ATCC 10500聚酮合成酶基因Tspksl。
本文首先研究了柠檬酸对灰绿曲霉发酵的代谢调控及对生长、发育的影响。在固体平板培养中,添加15 mmol/L柠檬酸使灰绿曲霉HB1-19生长降低31.5%,灰绿霉素A产量降低23.0%,无性生殖即分生孢子的数量降低84.8%,但有性生殖即子囊果的数量却提高了360%。在液体深层培养中,第3天和第4天两次等量添加终浓度40 mmol/L的柠檬酸时,灰绿霉素A产量提高了80%,且菌体量增加了16.7%,糖利用率增加了10%。通过pH对照及发酵液有机酸测定可知,柠檬酸和低pH可以显著促进丙酮酸积累并抑制琥珀酸和延胡索酸的积累。低pH条件下,柠檬酸有较高的利用率。丙酮酸的积累及柠檬酸的高效利用有利于乙酰辅酶A的积累及向胞质的转移,促进了灰绿霉素A的生产。
培养基开发是发酵优化的重要组成部分。本文采用正交设计、Plackett-Burman设计和响应面设计成功设计了一种灰绿霉素A高产培养基,使产量达到71.2 mg/L,是原始培养基中产量的4.22倍。元素分析结果表明,培养基碳氮比由原始的20.1：1提高到86.6：1。高碳氮比有利于积累乙酰辅酶A,提高灰绿霉素A产量。尽管如此,5L双层六平叶涡轮桨反应器发酵中灰绿霉素A产量却不高于6.0 mg/L。摇瓶培养中剪切和溶解氧定性研究结果表明,灰绿曲霉生产灰绿霉素A对剪切环境非常敏感,且生长期相对较高而生产期相对较低的溶解氧环境有利于灰绿霉素A的生产。因此,本研究在5L生物反应器发酵中采用六平叶涡轮桨和改进的三扇形斜叶桨的不同组合,以优化剪切和供氧环境。不同桨叶组合方式下,菌体生长、代谢及形态呈现很大不同。上斜叶下平叶涡轮桨效果最好,菌体量达到13.8g/L,灰绿霉素A产量达到19.3 mg/L。灰绿曲霉发酵过程起泡问题较严重,通气量受限。本文通过添加氧载体解决氧供应问题,添加0.35%正十二烷使灰绿霉素A产量提高至25.3 mg/L,但菌体生长有所下降。过量氧载体添加对灰绿霉素A生产及菌体生长均不利。
发酵条件优化的结果表明,接种量14%、磷酸盐浓度1%、消泡剂总添加浓度2%、初始pH 6.5、过程pH 6.0-6.5有利于灰绿霉素A的生产。等组分比例不同浓度的培养基及间歇性补加碳源实验证明分批发酵对灰绿霉素A生产较为合理。结合5L反应器剪切及溶解氧研究,采用桨叶末端线速度相似的发酵放大策略、控制生产期pH 6.0-7.0,并结合溶解氧强度及菌丝形态相似的原则,成功将灰绿曲霉发酵放大到30 L和500 L。在30 L和500 L发酵中,灰绿霉素A最高产量分别达到37 mg/L和30 mg/L以上。
在柄篮状菌聚酮合成酶基因的异源表达研究中,首先通过D-TOPO克隆和酵母重组技术成功克隆了聚酮合成酶基因Tspks1(8060 bp),并通过LR重组实验构建了Tspksl真菌表达质粒。然后,利用该质粒转化米曲霉M-2-3原生质体并以精氨酸营养缺陷型培养基筛选转化子。以基因组DNA为模板的PCR结果表明,表达质粒成功整合入米曲霉基因组。RT-PCR结果表明Tspksl在米曲霉中成功转录,且部分转化子的内含子得以正确剪切修饰。在Tspksl末端连接增强型绿色荧光蛋白基因egfp,荧光结果表明90%转化子基因得以正确转录和翻译。然而,液质联用分析结果表明所得阳性转化子无目的化合物生产。通过成功移除表达质粒中Tspksl的内含子并转化米曲霉,使100%转化子产生绿色荧光,说明egfp基因得以正确转录和翻译。液质联用分析结果表明,一株阳性转化子生产两种新聚酮化合物,其中一种化合物保留时间、紫外吸收及分子量与目的产物3-methylorcinaldehyde基本相同,核磁共振氢谱结果进一步证明该化合物为3-methylorcinaldehyde。根据核磁共振氢谱、异核单量子相关谱和异核多量子相关谱分析结果推断,另一种化合物为3,6-二甲基-4-羟基-2-吡喃酮,是中间释放的三酮链产物。该结果表明Tspksl的确负责聚酮化合物3-methylorcinaldehyde的合成,且可释放短碳链中间产物。本研究表明,即使在真菌表达宿主中,内含子也会对真菌聚酮合成酶基因的异源表达产生较大影响,不同表达宿主对真菌聚酮合成酶基因的内含子识别机制可能不同。
Aspergiolide A, a structurally novel anthraquinone derivative isolated from cultures of a marine-derived fungus Aspergillus glaucus HB1-19 by Ocean University of China, was proven to hold striking anti-tumor activities and be synthesized via polyketide pathway. However, aspergiolide A production from the original cultures was quite low (< 4 mg/L). In present work, we aim to enhance aspergiolide A production using metabolic regulation, process optimization and fermentation scale-up to provide sufficient quantity of compound for further pharmacy studies of animal experiments, clinic research and structure activity relationship. We are also dedicated to introduce some useful information to marine microorganism fermentations owing to the lack of systematic culture techniques of marine microbes. In addition, cloning and function analysis of fungal polyketide synthase (PKS) gene have developed rapidly in recent years. By contrast, heterologous expression of fungal PKS gene is still in its infancy. Here we attempted to heterologously express Tspks1 from Talaromyces stipitatus ATCC 10500.
Aspergiolide A production enhancement by citrate and its effects on growth and sexual development of marine-derived fungus Aspergillus glaucus HB1-19 were first investigated. In agar plate culture,15 mmol/L citric acid decreased colony radial growth and aspergiolide A production by 31.5% and 23.0%, respectively. It also improved sexual cleistothecium formation by 360% but depressed asexual conidiospore generation by 84.8%. In submerged culture, adding 40 mmol/L citric acid finally promoted aspergiolide A production by 80.0%, which accompanied with 16.7% increase of biomass and 10.0% enhancement of sugar utilization. Citrate and low pH could significantly improve pyruvate accumulation but inhibit succinate and fumarate production. Moreover, low pH was favorable to citrate utilization.
Medium development is important for the whole fermentation optimization. Statistical methodologies including orthogonal design, Plackett-Burman design and response surface methodology were used to develop new medium to facilitate aspergiolide A production. Under the proposed optimized conditions, the experimental aspergiolide A production reached 71.2 mg/L, which increased 4.22 times compared to that in original medium. Elemental analysis was finally conducted, and carbon-nitrogen ratio in the medium increased from 20.1:1 to 86.6:1. High carbon-nitrogen ratio facilitates acetyl-CoA accumulation and further improves aspergiolide A production.
Even with metabolic regulation and high yield medium, aspergiolide A production in 5 L stirred-tank bioreactor is still too low (<6.0 mg/L). Study about shear stress and dissolved oxygen tension in shaking flask cultures revealed that the marine-derived fungus Aspergillus glaucus HB1-19 is typically shear sensitive and mechanical shear stress severely damaged mycelia and diminished aspergiolide A production. Moreover, aspergiolide A biosynthesis favored high dissolved oxygen tension in the growth phase but low one in the production period. Afterwards, cultures with different impeller combinations were conducted. Growth, production and morphology differed greatly among these batches. The combination of upper three-sector-blade pitched blade turbine impeller and lower six-flat-blade disc turbine impeller led to the maximum dry biomass (13.8 g/L) and aspergiolide A production (19.3 mg/L). On another hand, feeding 0.35% (v/v) n-dodecane further improved the production by 31.0%, i.e.,25.3 mg/L in the bioreactor despite the overall cell growth was decreased.
Results of further optimization of fermentation conditions showed that inoculation ratio of 14%, phosphate concentration of 1%, total antifoam addition of 2%, original pH of 6.5 and process pH between 6.0 and 6.5 are preferable to aspergiolide A production. Cultures with media of different nutrition concentration but same composition or intermittent carbon source feeding strategy showed no production improvement. Therefore, batch culture is suitable for aspergiolide A production by Aspergillus glaucus HB1-19. By combined use of the scale-up strategy, i.e., similar impeller linear velocity together with similar dissolved oxygen tension and fungus morphology, the fermentation was successfully scaled up to 30 L and 500 L finally. Aspergiolide A production in 30 L and 500 L cultures reached up to more than 37 mg/L and 30 mg/L, respectively.
As for heterologous expression of Tspksl from T. stipitatus, gateway technics were adopted to construct the expression vector. D-TOPO cloning and yeast recombination were first applied to clone the intact Tspksl gene. After that, the Tspksl gene was transferred into the expression vector by LR recombination. Afterwards, the expression vectors were transformed into protoplasts of Aspergillus orazye M-2-3, and arginine defective medium was used for transformant screening. PCR experiments towards genomic DNA of the transformants indicated the vectors were effectively integrated into their genomic DNA. From the RT-PCR assays, Tspksl transcription worked well. The sequencing results showed that the intron was correctly removed in some cases. However, none of the cultivated transformants produced any positive desired compound. To investigate whether or not the translation processed correctly, the egfp gene was linked to the end of Tspksl by removing the stop coden of Tspksl. About 90% of the new transformants showed green fluorescence, which indicated the translation frame was mostly correct. Although dozens of the strongly fluorescent colonies were analyzed, none of these transformants produced desired compounds. The intron was then removed from Tspksl in the expression vector, by which means 100% of the new intron deleted transformants presented green fluorescence. LC-MS results showed positive transformants produced two new compounds 3-methylorcinaldehyde and 3,6-dimethyl-4-hydroxy-2-pyrone, which was a triketide early released from TSPKS1. These results showed that intron made great effects on heterologous expression of fungal polyketide synthase genes even in fungi host strain. Moreover, different host strains might own different intron identification mechanisms of the fungal polyketide synthase genes.
本文首先研究了柠檬酸对灰绿曲霉发酵的代谢调控及对生长、发育的影响。在固体平板培养中,添加15 mmol/L柠檬酸使灰绿曲霉HB1-19生长降低31.5%,灰绿霉素A产量降低23.0%,无性生殖即分生孢子的数量降低84.8%,但有性生殖即子囊果的数量却提高了360%。在液体深层培养中,第3天和第4天两次等量添加终浓度40 mmol/L的柠檬酸时,灰绿霉素A产量提高了80%,且菌体量增加了16.7%,糖利用率增加了10%。通过pH对照及发酵液有机酸测定可知,柠檬酸和低pH可以显著促进丙酮酸积累并抑制琥珀酸和延胡索酸的积累。低pH条件下,柠檬酸有较高的利用率。丙酮酸的积累及柠檬酸的高效利用有利于乙酰辅酶A的积累及向胞质的转移,促进了灰绿霉素A的生产。
培养基开发是发酵优化的重要组成部分。本文采用正交设计、Plackett-Burman设计和响应面设计成功设计了一种灰绿霉素A高产培养基,使产量达到71.2 mg/L,是原始培养基中产量的4.22倍。元素分析结果表明,培养基碳氮比由原始的20.1：1提高到86.6：1。高碳氮比有利于积累乙酰辅酶A,提高灰绿霉素A产量。尽管如此,5L双层六平叶涡轮桨反应器发酵中灰绿霉素A产量却不高于6.0 mg/L。摇瓶培养中剪切和溶解氧定性研究结果表明,灰绿曲霉生产灰绿霉素A对剪切环境非常敏感,且生长期相对较高而生产期相对较低的溶解氧环境有利于灰绿霉素A的生产。因此,本研究在5L生物反应器发酵中采用六平叶涡轮桨和改进的三扇形斜叶桨的不同组合,以优化剪切和供氧环境。不同桨叶组合方式下,菌体生长、代谢及形态呈现很大不同。上斜叶下平叶涡轮桨效果最好,菌体量达到13.8g/L,灰绿霉素A产量达到19.3 mg/L。灰绿曲霉发酵过程起泡问题较严重,通气量受限。本文通过添加氧载体解决氧供应问题,添加0.35%正十二烷使灰绿霉素A产量提高至25.3 mg/L,但菌体生长有所下降。过量氧载体添加对灰绿霉素A生产及菌体生长均不利。
发酵条件优化的结果表明,接种量14%、磷酸盐浓度1%、消泡剂总添加浓度2%、初始pH 6.5、过程pH 6.0-6.5有利于灰绿霉素A的生产。等组分比例不同浓度的培养基及间歇性补加碳源实验证明分批发酵对灰绿霉素A生产较为合理。结合5L反应器剪切及溶解氧研究,采用桨叶末端线速度相似的发酵放大策略、控制生产期pH 6.0-7.0,并结合溶解氧强度及菌丝形态相似的原则,成功将灰绿曲霉发酵放大到30 L和500 L。在30 L和500 L发酵中,灰绿霉素A最高产量分别达到37 mg/L和30 mg/L以上。
在柄篮状菌聚酮合成酶基因的异源表达研究中,首先通过D-TOPO克隆和酵母重组技术成功克隆了聚酮合成酶基因Tspks1(8060 bp),并通过LR重组实验构建了Tspksl真菌表达质粒。然后,利用该质粒转化米曲霉M-2-3原生质体并以精氨酸营养缺陷型培养基筛选转化子。以基因组DNA为模板的PCR结果表明,表达质粒成功整合入米曲霉基因组。RT-PCR结果表明Tspksl在米曲霉中成功转录,且部分转化子的内含子得以正确剪切修饰。在Tspksl末端连接增强型绿色荧光蛋白基因egfp,荧光结果表明90%转化子基因得以正确转录和翻译。然而,液质联用分析结果表明所得阳性转化子无目的化合物生产。通过成功移除表达质粒中Tspksl的内含子并转化米曲霉,使100%转化子产生绿色荧光,说明egfp基因得以正确转录和翻译。液质联用分析结果表明,一株阳性转化子生产两种新聚酮化合物,其中一种化合物保留时间、紫外吸收及分子量与目的产物3-methylorcinaldehyde基本相同,核磁共振氢谱结果进一步证明该化合物为3-methylorcinaldehyde。根据核磁共振氢谱、异核单量子相关谱和异核多量子相关谱分析结果推断,另一种化合物为3,6-二甲基-4-羟基-2-吡喃酮,是中间释放的三酮链产物。该结果表明Tspksl的确负责聚酮化合物3-methylorcinaldehyde的合成,且可释放短碳链中间产物。本研究表明,即使在真菌表达宿主中,内含子也会对真菌聚酮合成酶基因的异源表达产生较大影响,不同表达宿主对真菌聚酮合成酶基因的内含子识别机制可能不同。
Aspergiolide A, a structurally novel anthraquinone derivative isolated from cultures of a marine-derived fungus Aspergillus glaucus HB1-19 by Ocean University of China, was proven to hold striking anti-tumor activities and be synthesized via polyketide pathway. However, aspergiolide A production from the original cultures was quite low (< 4 mg/L). In present work, we aim to enhance aspergiolide A production using metabolic regulation, process optimization and fermentation scale-up to provide sufficient quantity of compound for further pharmacy studies of animal experiments, clinic research and structure activity relationship. We are also dedicated to introduce some useful information to marine microorganism fermentations owing to the lack of systematic culture techniques of marine microbes. In addition, cloning and function analysis of fungal polyketide synthase (PKS) gene have developed rapidly in recent years. By contrast, heterologous expression of fungal PKS gene is still in its infancy. Here we attempted to heterologously express Tspks1 from Talaromyces stipitatus ATCC 10500.
Aspergiolide A production enhancement by citrate and its effects on growth and sexual development of marine-derived fungus Aspergillus glaucus HB1-19 were first investigated. In agar plate culture,15 mmol/L citric acid decreased colony radial growth and aspergiolide A production by 31.5% and 23.0%, respectively. It also improved sexual cleistothecium formation by 360% but depressed asexual conidiospore generation by 84.8%. In submerged culture, adding 40 mmol/L citric acid finally promoted aspergiolide A production by 80.0%, which accompanied with 16.7% increase of biomass and 10.0% enhancement of sugar utilization. Citrate and low pH could significantly improve pyruvate accumulation but inhibit succinate and fumarate production. Moreover, low pH was favorable to citrate utilization.
Medium development is important for the whole fermentation optimization. Statistical methodologies including orthogonal design, Plackett-Burman design and response surface methodology were used to develop new medium to facilitate aspergiolide A production. Under the proposed optimized conditions, the experimental aspergiolide A production reached 71.2 mg/L, which increased 4.22 times compared to that in original medium. Elemental analysis was finally conducted, and carbon-nitrogen ratio in the medium increased from 20.1:1 to 86.6:1. High carbon-nitrogen ratio facilitates acetyl-CoA accumulation and further improves aspergiolide A production.
Even with metabolic regulation and high yield medium, aspergiolide A production in 5 L stirred-tank bioreactor is still too low (<6.0 mg/L). Study about shear stress and dissolved oxygen tension in shaking flask cultures revealed that the marine-derived fungus Aspergillus glaucus HB1-19 is typically shear sensitive and mechanical shear stress severely damaged mycelia and diminished aspergiolide A production. Moreover, aspergiolide A biosynthesis favored high dissolved oxygen tension in the growth phase but low one in the production period. Afterwards, cultures with different impeller combinations were conducted. Growth, production and morphology differed greatly among these batches. The combination of upper three-sector-blade pitched blade turbine impeller and lower six-flat-blade disc turbine impeller led to the maximum dry biomass (13.8 g/L) and aspergiolide A production (19.3 mg/L). On another hand, feeding 0.35% (v/v) n-dodecane further improved the production by 31.0%, i.e.,25.3 mg/L in the bioreactor despite the overall cell growth was decreased.
Results of further optimization of fermentation conditions showed that inoculation ratio of 14%, phosphate concentration of 1%, total antifoam addition of 2%, original pH of 6.5 and process pH between 6.0 and 6.5 are preferable to aspergiolide A production. Cultures with media of different nutrition concentration but same composition or intermittent carbon source feeding strategy showed no production improvement. Therefore, batch culture is suitable for aspergiolide A production by Aspergillus glaucus HB1-19. By combined use of the scale-up strategy, i.e., similar impeller linear velocity together with similar dissolved oxygen tension and fungus morphology, the fermentation was successfully scaled up to 30 L and 500 L finally. Aspergiolide A production in 30 L and 500 L cultures reached up to more than 37 mg/L and 30 mg/L, respectively.
As for heterologous expression of Tspksl from T. stipitatus, gateway technics were adopted to construct the expression vector. D-TOPO cloning and yeast recombination were first applied to clone the intact Tspksl gene. After that, the Tspksl gene was transferred into the expression vector by LR recombination. Afterwards, the expression vectors were transformed into protoplasts of Aspergillus orazye M-2-3, and arginine defective medium was used for transformant screening. PCR experiments towards genomic DNA of the transformants indicated the vectors were effectively integrated into their genomic DNA. From the RT-PCR assays, Tspksl transcription worked well. The sequencing results showed that the intron was correctly removed in some cases. However, none of the cultivated transformants produced any positive desired compound. To investigate whether or not the translation processed correctly, the egfp gene was linked to the end of Tspksl by removing the stop coden of Tspksl. About 90% of the new transformants showed green fluorescence, which indicated the translation frame was mostly correct. Although dozens of the strongly fluorescent colonies were analyzed, none of these transformants produced desired compounds. The intron was then removed from Tspksl in the expression vector, by which means 100% of the new intron deleted transformants presented green fluorescence. LC-MS results showed positive transformants produced two new compounds 3-methylorcinaldehyde and 3,6-dimethyl-4-hydroxy-2-pyrone, which was a triketide early released from TSPKS1. These results showed that intron made great effects on heterologous expression of fungal polyketide synthase genes even in fungi host strain. Moreover, different host strains might own different intron identification mechanisms of the fungal polyketide synthase genes.
引文
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