一株链霉菌的鉴定及其产格尔德霉素的发酵工艺研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-1215

摘要/Abstract

摘要：

【目的】 对链霉菌Streptomyce sp. FIM18-0592进行菌种鉴定，并对其胞外格尔德霉素产量进行发酵工艺优化，旨在提高产量并降低发酵成本。【方法】 通过形态特征、培养特征和生理生化特性，结合16S rDNA序列分析构建系统发育树进行菌种鉴定；采用单因素试验优化培养条件及培养基配方，进一步使用最陡爬坡试验和响应面试验优化培养基配方的含量。【结果】 通过对链霉菌FIM18-0592的形态特征、培养特征及生理生化特征进行初步培养观察发现，其在ISP2等培养基上生长较好，气生菌丝旺盛，产黑色素。结合16S rDNA分子鉴定，确定该菌为格尔德霉素链霉菌（Streptomyces geldanamycininus）。通过单因素试验对发酵条件以及培养基配方进行优化，得到最适宜菌株发酵的培养条件为转速140 r/min、装液量12%（体积分数）、接种量7%（体积分数）、培养时间144 h。最佳的碳源、氮源、无机盐分别为葡萄糖、黄豆饼粉和硫酸铵。采用最陡爬坡试验和响应面优化试验确定了其最优的发酵培养基为：葡萄糖10.42%、黄豆饼粉1.68%、硫酸铵0.3%、乳酸0.3%、甘油4%、硫酸镁0.1%、碳酸钙0.4%，在此条件下，格尔德霉素的发酵效价达到2 887 μg/mL，较原始发酵工艺效价提高了66%。【结论】 链霉菌FIM18-0592为格尔德霉素链霉菌，通过对其发酵工艺进行优化显著提高格尔德霉素的产量，为格尔德霉素及其衍生物的开发和利用奠定基础。

关键词: 格尔德霉素, 菌种鉴定, 发酵优化, 单因素优化, 响应面试验, 形态特征, 培养特性

Abstract:

【Objective】 Streptomyces sp. FIM18-0592 was identified and its fermentation process was optimized for extracellular geldanamycin yield with the aim of increasing the yield and reducing the fermentation cost.【Method】 Strain identification was carried out by morphological features, culture characteristics and physiological and biochemical properties, combined with 16S rDNA sequence analysis to construct a phylogenetic tree; a one-factor test was used to optimize the culture conditions and media formulations, and the contents of the media formulations were further optimized by the steepest-climbing test and the response surface test. 【Result】 Through preliminary culture observation of the morphological characteristics, culture characteristics and physiological and biochemical characteristics of Streptomyces geldanamycininus FIM18-0592, we found that it grew well on ISP2 and other media, with vigorous aerial mycelium and melanin production. Combined with 16S rDNA molecular identification, the Streptomyces sp. was identified as Streptomyces geldanamycininus. The fermentation conditions and medium formulation were optimized by one-factor test, and the most optimal culture conditions for the fermentation of the strain were speed of 140 r/min, liquid content of 12%, inoculum concentration of 7%（V/V）, and fermentation time of 144 h. The optimal carbon source, nitrogen source, and mineral salt were glucose, soybean cake powder and NH₄HPO₄, respectively. The optimal fermentation medium was determined by steepest-climbing test and response surface optimization test as follows: Glucose 10.42%, soybean cake powder 1.68%,(NH₄)₂SO₄ 0.3%, lactic acid 0.3%, glycerol 4%, magnesium sulphate 0.1%, and calcium carbonate 0.4%. Under these conditions, the fermentation titer of geldanamycin reached 2 887 μg/mL, which was 66% higher than the original fermentation process. 【Conclusion】 Streptomyces sp. FIM18-0592 belongs to Streptomycete geldanamycininus, whose fermentation process is optimized to significantly increase the yield of geldanamycin, laying the foundation for the development and utilization of geldanamycin and its derivatives.

Key words: geldanamycin, strain identification, optimization of fermentation, one-factor optimization, response surface test, morphological characteristics, culture characteristics

杨鹭, 袁源, 方志锴, 林如, 江红, 周剑. 一株链霉菌的鉴定及其产格尔德霉素的发酵工艺研究[J]. 生物技术通报, 2024, 40(6): 299-309.

YANG Lu, YUAN Yuan, FANG Zhi-kai, LIN Ru, JIANG Hong, ZHOU Jian. Identification of a Streptomyces Strain and Study on the Fermentation Process of Geldanamycin Production[J]. Biotechnology Bulletin, 2024, 40(6): 299-309.

图/表 11

参考文献 33

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培养基类型 Type of culture medium	培养基配方 Component of culture medium
种子培养基Seed medium	葡萄糖1.8%；麦芽糊精1.0%；黄豆饼粉2.25%；酵母粉0.5%；MgSO₄·7H₂O 0.1%；K₂HPO₄·3H₂O 0.1%（pH 7.0）
发酵培养基Fermentation medium	淀粉4.0%；黄豆饼粉4.0%；氯化钴0.001%；豆油0.5%；碳酸钙1.0%（pH 7.2）
胰蛋白胨-酵母膏琼脂ISP1	胰蛋白胨0.5%；酵母膏0.3%；琼脂2%（pH 7.0-7.4）
酵母膏-麦芽膏琼脂ISP2	酵母膏0.4%；麦芽膏1%；葡萄糖0.4%；琼脂2%（pH 7.3）
燕麦粉琼脂ISP3	燕麦粉2%；微盐0.1%；琼脂2%（pH 7.3）
无机盐-淀粉琼脂ISP4	可溶性淀粉1%；K₂HPO₄·3H₂O 0.1%；MgSO₄·7H₂O 0.1%；NaCl 0.1%；(NH₄)₂SO₄ 0.2%；CaCO₃ 0.2%；琼脂2%
甘油-天冬酰胺琼脂ISP5	L-天冬酰胺0.%；K₂HPO₄·3H₂O 0.1%；甘油1%；微盐0.1%；琼脂2%
蛋白胨酵母提取物铁琼脂ISP6	Bacto蛋白胨铁琼脂3.6%；酵母膏0.1%（pH 7.0-7.2）
酪氨酸琼脂ISP7	酵母膏0.1%；L-酪氨酸0.1%；NaCl 0.85%；琼脂2%（pH 7.5）
高氏1号培养基G1	可溶性淀粉2%；KNO₃ 0.1%；K₂HPO₄·3H₂O 0.05%；MgSO₄·7H₂O 0.05%；NaCl 0.05%；FeSO₄ 0.001%；琼脂2%（pH 7.2-7.4）
马铃薯培养基PDA	马铃薯20%；葡萄糖2%；琼脂2%（pH 6-6.5）
蛋白胨-甘油培养基CM008	蛋白胨0.5%；牛肉提取物0.3%；甘油2%；琼脂1.5%（pH 7.0）
葡萄糖-天门冬素培养基CM0011	葡萄糖1%；天门冬素0.05%；K₂HPO₄·3H₂O 0.05%；琼脂1.5%（pH 7.2-7.4）
查氏-蔗糖Charlery - sucrose	蔗糖3%；K₂HPO₄·3H₂O 0.1%；MgSO₄·7H₂O 0.05%；KCl 0.05%；NaNO₃ 0.2%；琼脂2%（pH 7.2-7.4）

培养基类型 Type of culture medium	培养基配方 Component of culture medium
种子培养基Seed medium	葡萄糖1.8%；麦芽糊精1.0%；黄豆饼粉2.25%；酵母粉0.5%；MgSO₄·7H₂O 0.1%；K₂HPO₄·3H₂O 0.1%（pH 7.0）
发酵培养基Fermentation medium	淀粉4.0%；黄豆饼粉4.0%；氯化钴0.001%；豆油0.5%；碳酸钙1.0%（pH 7.2）
胰蛋白胨-酵母膏琼脂ISP1	胰蛋白胨0.5%；酵母膏0.3%；琼脂2%（pH 7.0-7.4）
酵母膏-麦芽膏琼脂ISP2	酵母膏0.4%；麦芽膏1%；葡萄糖0.4%；琼脂2%（pH 7.3）
燕麦粉琼脂ISP3	燕麦粉2%；微盐0.1%；琼脂2%（pH 7.3）
无机盐-淀粉琼脂ISP4	可溶性淀粉1%；K₂HPO₄·3H₂O 0.1%；MgSO₄·7H₂O 0.1%；NaCl 0.1%；(NH₄)₂SO₄ 0.2%；CaCO₃ 0.2%；琼脂2%
甘油-天冬酰胺琼脂ISP5	L-天冬酰胺0.%；K₂HPO₄·3H₂O 0.1%；甘油1%；微盐0.1%；琼脂2%
蛋白胨酵母提取物铁琼脂ISP6	Bacto蛋白胨铁琼脂3.6%；酵母膏0.1%（pH 7.0-7.2）
酪氨酸琼脂ISP7	酵母膏0.1%；L-酪氨酸0.1%；NaCl 0.85%；琼脂2%（pH 7.5）
高氏1号培养基G1	可溶性淀粉2%；KNO₃ 0.1%；K₂HPO₄·3H₂O 0.05%；MgSO₄·7H₂O 0.05%；NaCl 0.05%；FeSO₄ 0.001%；琼脂2%（pH 7.2-7.4）
马铃薯培养基PDA	马铃薯20%；葡萄糖2%；琼脂2%（pH 6-6.5）
蛋白胨-甘油培养基CM008	蛋白胨0.5%；牛肉提取物0.3%；甘油2%；琼脂1.5%（pH 7.0）
葡萄糖-天门冬素培养基CM0011	葡萄糖1%；天门冬素0.05%；K₂HPO₄·3H₂O 0.05%；琼脂1.5%（pH 7.2-7.4）
查氏-蔗糖Charlery - sucrose	蔗糖3%；K₂HPO₄·3H₂O 0.1%；MgSO₄·7H₂O 0.05%；KCl 0.05%；NaNO₃ 0.2%；琼脂2%（pH 7.2-7.4）

培养基 Medium	生长状态 Growth state	表面颜色 Surface color	背面颜色 Back color	气生菌丝 Aerial mycelium	基质菌丝 Substrate mycelium	产孢情况 Spore production situation	可溶性色素 Soluble pigment	质地 Texture
ISP1	+++	绿白色	绿白色	浅绿灰色	绿白色	适中	无	圆形，波浪边缘，表面不光滑
ISP2	++++	绿灰色	中等黄褐色	绿灰色	绿白色	较厚	有	圆形，波浪边缘，表面不光滑，较多褶皱
ISP3	++++	绿灰色	浅绿灰色	绿灰色	绿白色	较厚	无	圆形，锯形边缘，表面不光滑
ISP4	++++	灰黄绿	黄灰色	灰黄绿	绿白色	较厚	有	圆形，锯形边缘，表面不光滑
ISP5	++++	浅绿灰色	绿白色	浅绿灰色	绿白色	较厚	无	圆形，锯形边缘，表面不光滑
ISP6	+++	绿白色	深黄色	绿白色	绿白色	较薄	无	圆形，波浪边缘，表面较光滑
ISP7	++++	浅灰黄色	深黄褐色	浅灰黄色	浅灰黄色	适中	有	圆形，波浪边缘，表面光滑
G1	++++	灰黄绿	浅绿灰色	灰黄绿	淡黄绿色	较厚	有	圆形，波浪边缘，表面光滑
PDA	++++	绿灰色	绿灰色	绿灰色	绿白色	较厚	有	圆形，波浪边缘，表面不光滑
CM008	+++	绿白色	中等黄色	浅绿灰色	绿白色	适中	无	圆形，波浪边缘，表面不光滑，较多褶皱
CM0011	++++	灰黄棕色	绿白色	灰黄棕色	黄白色	较厚	无	圆形，波浪边缘，表面较光滑
查氏-蔗糖 Charlery - sucrose	++	绿白色	淡绿色	绿白色	绿白色	较薄	无	圆形，波浪边缘，表面较光滑

培养基 Medium	生长状态 Growth state	表面颜色 Surface color	背面颜色 Back color	气生菌丝 Aerial mycelium	基质菌丝 Substrate mycelium	产孢情况 Spore production situation	可溶性色素 Soluble pigment	质地 Texture
ISP1	+++	绿白色	绿白色	浅绿灰色	绿白色	适中	无	圆形，波浪边缘，表面不光滑
ISP2	++++	绿灰色	中等黄褐色	绿灰色	绿白色	较厚	有	圆形，波浪边缘，表面不光滑，较多褶皱
ISP3	++++	绿灰色	浅绿灰色	绿灰色	绿白色	较厚	无	圆形，锯形边缘，表面不光滑
ISP4	++++	灰黄绿	黄灰色	灰黄绿	绿白色	较厚	有	圆形，锯形边缘，表面不光滑
ISP5	++++	浅绿灰色	绿白色	浅绿灰色	绿白色	较厚	无	圆形，锯形边缘，表面不光滑
ISP6	+++	绿白色	深黄色	绿白色	绿白色	较薄	无	圆形，波浪边缘，表面较光滑
ISP7	++++	浅灰黄色	深黄褐色	浅灰黄色	浅灰黄色	适中	有	圆形，波浪边缘，表面光滑
G1	++++	灰黄绿	浅绿灰色	灰黄绿	淡黄绿色	较厚	有	圆形，波浪边缘，表面光滑
PDA	++++	绿灰色	绿灰色	绿灰色	绿白色	较厚	有	圆形，波浪边缘，表面不光滑
CM008	+++	绿白色	中等黄色	浅绿灰色	绿白色	适中	无	圆形，波浪边缘，表面不光滑，较多褶皱
CM0011	++++	灰黄棕色	绿白色	灰黄棕色	黄白色	较厚	无	圆形，波浪边缘，表面较光滑
查氏-蔗糖 Charlery - sucrose	++	绿白色	淡绿色	绿白色	绿白色	较薄	无	圆形，波浪边缘，表面较光滑

分组Group	A:葡萄糖Glucose/（g·L^-1）	B:黄豆饼粉Soybean cake powder/（g·L^-1）	C:硫酸铵(NH₄)₂SO₄/（g·L^-1）	相对效价Relative titer（Y）
1	1	1	0	145.79±37.27
2	0	0	0	163.10±10.45
3	-1	-1	0	146.09±11.86
4	1	0	-1	134.75±13.15
5	0	0	0	165.58±1.63
6	0	0	0	153.91±16.84
7	0	1	1	25.25±0.59
8	0	0	0	158.94±5.90
9	-1	1	0	37.32±1.45
10	1	-1	0	138.31±1.15
11	1	0	1	135.49±7.29
12	-1	0	-1	153.49±1.61
13	0	1	-1	146.57±13.94
14	0	-1	-1	140.41±2.33
15	-1	0	1	49.91±8.92
16	0	-1	1	148.60±12.41
17	0	0	0	154.04±1.46