紫外诱变选育巴弗洛霉素A1高产菌株及其培养基优化

doi:10.13560/j.cnki.biotech.bull.1985.2024-0898

摘要/Abstract

摘要：

目的巴弗洛霉素是由灰褐色链霉菌（Streptomyces griseobrunneus）发酵产生的一类大环内酯化合物，对多种植物真菌具有拮抗作用，其代表性化合物为巴弗洛霉素A1（Baf A1）。为了进一步提升链霉菌（Streptomyces sp.） FIM-B0711合成Baf A1的发酵产量，降低生产成本，以其为出发菌株选育Baf A1高产菌株，并对其发酵工艺进行优化。方法采用紫外诱变选育高产突变株，单因素试验、最陡爬坡试验和响应面法进行摇瓶发酵工艺优化。结果紫外诱变菌株UV-21的Baf A1产量达到397.99 mg/L，较原始菌株提高了23.60%。单因素试验表明，该菌株发酵的最佳碳源、氮源和无机盐分别为麦芽糊精、大豆蛋白胨和碳酸钙，最佳培养条件为pH 7.0、接种量3%（体积分数）、装液量30 mL/250 mL、发酵培养时间96 h。最陡爬坡试验表明，在麦芽糊精45 g/L、大豆蛋白胨15 g/L、碳酸钙0.5 g/L时相对效价达到最高，为147.18%。响应面优化试验表明，该菌株的最佳发酵工艺：麦芽糊精45.98 g/L、大豆蛋白胨14.96 g/L、碳酸钙0.48 g/L、缬氨酸3 g/L，初始pH 7.0、接种量3%（体积分数）、装液量30 mL/250 mL、发酵培养时间96 h。优化后高产菌株UV-21摇瓶发酵产量为627.58 mg/L，较初始工艺提高了57.69%。结论基于紫外诱变及培养基的响应面优化试验获得1株Baf A1产量为627.58 mg/L的高产菌株，较原始菌株FIM-B0711产量提高了57.69%。

关键词: 巴弗洛霉素, 链霉菌, 响应面试验, 发酵优化, 紫外诱变, 培养条件, 工艺参数

Abstract:

Objective Bafilomycins are a class of macrolide compounds produced by the fermentation of Streptomyces griseobrunneus, which have antagonistic effect on a variety of plant fungi, and its representative compound is bafilomycin A1 (Baf A1). In order to further enhance the yield of Baf A1 in Streptomyces sp. FIM-B0711 and reduce the production cost, the high-yielding strain of Baf A1 was selected and its fermentation process was optimized. Method High-yield mutant strains were generated through ultraviolet mutagenesis, followed by optimization of the fermentation process using single factor experiment, the steepest ascent experiment, and response surface methodology. Result The Baf A1 yield of the UV-21 strain reached 397.99 mg/L, representing a 23.60% increase compared to the original strain. Single factor experiment identified that maltodextrin, soybean peptone, and calcium carbonate were as the optimal carbon source, nitrogen source, and inorganic salt for strain UV-21, respectively. The optimal culture conditions were determined to be pH 7.0, an inoculum concentration of 3% (volume fraction), a liquid volume of 30 mL/250 mL, and a fermentation time of 96 h. The steepest ascent experiment revealed that the highest relative titer of 147.18% was achieved with a carbon source concentration of 45 g/L, nitrogen source concentration of 15 g/L, and inorganic salt concentration of 0.5 g/L. The response surface optimization demonstrated that the optimal fermentation process for the strain involved maltodextrin at 45.98 g/L, soybean peptone at 14.96 g/L, calcium carbonate at 0.48 g/L, valine at 3 g/L, an initial pH of 7.0, an inoculum concentration of 3% (volume fraction), a liquid volume of 30 mL/250 mL, and a fermentation time of 96 h. Under these optimized conditions, the fermentation titer reached 627.58 mg/L, a 57.69% increase compared to the initial process. Conclusion A high-yield Baf A1 strain with 627.58 mg/L is obtained based on ultraviolet mutagenesis and medium response surface optimization experiment, which increases the yield by 57.69% compared to the original strain FIM-B0711.

Key words: bafilomycins, Streptomyces, response surface test, fermentation optimization, ultraviolet mutagenesis, cultivation condition, process parameters

陈海敏, 孙菲, 袁源, 吴佳雯, 江红, 周剑. 紫外诱变选育巴弗洛霉素A1高产菌株及其培养基优化[J]. 生物技术通报, 2025, 41(3): 51-61.

CHEN Hai-min, SUN Fei, YUAN Yuan, WU Jia-wen, JIANG Hong, ZHOU Jian. The High-yield Bafilomycin A1 Strain Obtained from UV Mutagenesis and Its Medium Optimization[J]. Biotechnology Bulletin, 2025, 41(3): 51-61.

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参考文献 30

1	Intra B, Euanorasetr J, Nihira T, et al. Characterization of a gamma-butyrolactone synthetase gene homologue (stcA) involved in bafilomycin production and aerial mycelium formation in Streptomyces sp. SBI034 [J]. Appl Microbiol Biotechnol, 2016, 100(6): 2749-2760.
2	Xie X, Lu SS, Pan XY, et al. Antiviral bafilomycins from a feces-inhabiting Streptomyces sp [J]. J Nat Prod, 2021, 84(2): 537-543.
3	Shacka JJ, Klocke BJ, Roth KA. Autophagy, bafilomycin and cell death: the "a-B-cs" of plecomacrolide-induced neuroprotection [J]. Autophagy, 2006, 2(3): 228-230.
4	黄议莹, 李喆, 潘信利, 等. 产体外抗鼻咽癌物质红树林土壤细菌筛选及其活性成分分析 [J]. 广西科学, 2022, 29(5): 846-853.
	Huang YY, Li Z, Pan XL, et al. Screening of mangrove soil bacteria producing in vitro anti-nasopharyngeal carcinoma substance and analysis of its active components [J]. Guangxi Sci, 2022, 29(5): 846-853.
5	陈锋龙, 陈金龙, 张弋. 巴佛洛霉素A1抑制胶质瘤迁移、侵袭及血管生成拟态的生物机制研究 [J]. 立体定向和功能性神经外科杂志, 2022, 35(2): 75-80.
	Chen FL, Chen JL, Zhang Y. Biological mechanism of bafilomycin A1 inhibiting glioma migration, invasion and vasculogenic-mimicry [J]. Chin J Stereotact Funct Neurosurg, 2022, 35(2): 75-80.
6	魏秉洁, 张翠玲, 尚超, 等. 巴弗洛霉素A1体外广谱抗冠状病毒作用研究 [J]. 中国病原生物学杂志, 2023, 18(3): 260-264, 270.
	Wei BJ, Zhang CL, Shang C, et al. Broad-spectrum antiviral effect of bafilomycin A1 on coronavirus [J]. J Pathog Biol, 2023, 18(3): 260-264, 270.
7	Xu J, Cheng T, Feng HT, et al. Structure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis [J]. Histol Histopathol, 2007, 22(4): 443-454.
8	杨巍民, 斯聪聪, 杨星, 等. 海洋放线菌Y-0117农用活性代谢产物的研究 [J]. 化学与生物工程, 2013, 30(1): 24-27.
	Yang WM, Si CC, Yang X, et al. Study of agro-active metabolites of marine actinomycete Y-0117 [J]. Chem Bioeng, 2013, 30(1): 24-27.
9	Kretschmer A, Dorgerloh M, Deeg M, et al. The structures of novel insecticidal macrolides: bafilomycins D and E, and oxohygrolidin [J]. Agricultural and Biological Chemistry, 1985, 49(8): 2509-2511.
10	Moon SS, Hwang WH, Chung YR, et al. New cytotoxic bafilomycin C1-amide produced by Kitasatospora cheerisanensis [J]. J Antibiot, 2003, 56(10): 856-861.
11	魏刚, 苏超, 张道敬, 等. 海洋放线菌Y12-26代谢产物中bafilomycins分离纯化及结构鉴定 [J]. 中国抗生素杂志, 2011, 36(8): 571-575.
	Wei G, Su C, Zhang DJ, et al. Isolation, purification and structure identification of secondary metabolites produced by marine actinomycete Y12-26 [J]. Chin J Antibiot, 2011, 36(8): 571-575.
12	Lee DW, Ng BG, Kim BS. Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin biosynthesis and branched-chain α-keto acid dehydrogenase complex expression [J]. J Ind Microbiol Biotechnol, 2015, 42(11): 1507-1517.
13	李盛英, 李众, 张伟, 等. 一种巴弗洛霉素高产工程菌及其构建和应用: CN111378610B [P]. 2022-06-14.
	Li SY, Li Z, Zhang W, et al. Construction and application of a high-yield bafilomycin engineering strain: CN111378610B [P]. 2022-06-14.
14	周剑, 方志锴, 孙菲, 等. 一株链霉菌的鉴定及其产bafilomycin A1的发酵工艺研究 [J]. 食品与发酵工业, 2019, 45(6): 30-35.
	Zhou J, Fang ZK, Sun F, et al. Identification of a Streptomyces isolate and fermentation process of bafilomycin A1 production [J]. Food Ferment Ind, 2019, 45(6): 30-35.
15	周剑, 孙菲, 方志锴, 等. 小分子前体物对巴弗洛霉素A1生物合成的影响 [J]. 生物技术通报, 2019, 35(6): 125-130.
	Zhou J, Sun F, Fang ZK, et al. Effects of small molecule precursors on bafilomycin A1 biosynthesis [J]. Biotechnol Bull, 2019, 35(6): 125-130.
16	Nisamedtinov I, Kevvai K, Orumets K, et al. Metabolic changes underlying the higher accumulation of glutathione in Saccharomyces cerevisiae mutants [J]. Appl Microbiol Biotechnol, 2011, 89(4): 1029-1037.
17	卢承蓉, 叶美芝, 上官文丹, 等. 高产胞外多糖乳酸菌的诱变育种及其益生特性 [J]. 食品与发酵工业, 2020, 46(12): 14-20.
	Lu CR, Ye MZ, Shangguan WD, et al. Mutation breeding for high-yield exopolysaccharide lactic acid bacteria and evaluation of its probiotic properties [J]. Food Ferment Ind, 2020, 46(12): 14-20.
18	Xu L, Yuan N, Liu H, et al. Bafilomycin A1 targets patient-derived CD34⁺CD19⁺ leukemia stem cells [J]. Haematologica, 2020, 105(1): e17-e21.
19	Xu ZJ, Liu RY, Ke HY, et al. ATP6V1D drives hepatocellular carcinoma stemness and progression via both lysosome acidification-dependent and-independent mechanisms [J]. Autophagy, 2024: 1-17.
20	Sabino C, Basic M, Bender D, et al. Bafilomycin A1 and U18666A efficiently impair ZIKV infection [J]. Viruses, 2019, 11(6): 524.
21	de Lima Júnior AA, de Sousa EC, de Oliveira THB, et al. Genus Streptomyces: Recent advances for biotechnological purposes [J]. Biotechnol Appl Biochem, 2023, 70(4): 1504-1517.
22	Lee N, Hwang S, Lee Y, et al. Synthetic biology tools for novel secondary metabolite discovery in Streptomyces [J]. J Microbiol Biotechnol, 2019, 29(5): 667-686.
23	Yang ZJ, He JQ, Wei X, et al. Exploration and genome mining of natural products from marine Streptomyces [J]. Appl Microbiol Biotechnol, 2020, 104(1): 67-76.
24	Schwarz J, Hubmann G, Rosenthal K, et al. Triaging of culture conditions for enhanced secondary metabolite diversity from different bacteria [J]. Biomolecules, 2021, 11(2): 193.
25	梅柏杨, 李宇诗, 孙国刚, 等. 暹罗芽孢杆菌N2对茄立枯丝核菌发酵培养基及其发酵条件的优化[J/OL].吉林农业大学学报, 2024. .
	Mei BY, Li YS, Sun GG, et al. Optimization of fermentation medium and fermentation conditions of Bacillus siamensis N2 for Solanicum [J]. J Jilin Agric Univ, 2024. .
26	Mondal S, Rai VR. Molecular profiling of endophytic Streptomyces cavourensis MH16 inhabiting Millingtonia hortensis Linn. and influence of different culture media on biosynthesis of antimicrobial metabolites [J]. Naturwissenschaften, 2019, 106(9/10): 51.
27	Wu K, Ding LJ, Zhu P, et al. Application of the response surface methodology to optimize the fermentation parameters for enhanced docosahexaenoic acid (DHA) production by Thraustochytrium sp. ATCC 26185 [J]. Molecules, 2018, 23(4): 974.
28	宋健, 张海剑, 丰硕, 等. 对韭菜迟眼蕈蚊高活性的苏云金芽胞杆菌JQD117发酵培养基及摇瓶发酵条件优化 [J]. 中国生物防治学报, 2022, 38(2): 333-341.
	Song J, Zhang HJ, Feng S, et al. Optimization of fermentation culture medium and flask fermentation conditions for Bacillus thuringiensis strain JQD117 with high toxicity against Bradysia odoriphaga [J]. Chin J Biol Contr, 2022, 38(2): 333-341.
29	Abdel-Mageed HM, Barakat AZ, Bassuiny RI, et al. Biotechnology approach using watermelon rind for optimization of α‍-amylase enzyme production from Trichoderma virens using response surface methodology under solid-state fermentation [J]. Folia Microbiol, 2022, 67(2): 253-264.
30	王大红, 张颖, 郑迎莹, 等. L-缬氨酸对Streptomyces natalensis HW-2合成纳他霉素的影响 [J]. 精细化工, 2019, 36(4): 708-714.
	Wang DH, Zhang Y, Zheng YY, et al. Effect of L-valine on biosynthesis of natamycin by Streptomyces natalensis HW-2 [J]. Fine Chem, 2019, 36(4): 708-714.

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分组 Group	麦芽糊精 Maltodextrin/（g·L^-1）	大豆蛋白胨 Soybean peptone/（g·L^-1）	碳酸钙 CaCO₃/（g·L^-1）	相对效价 Relative titer/%
1	25	5	0.1	93.79
2	35	10	0.3	131.41
3	45	15	0.5	147.18
4	55	20	0.7	122.96
5	65	25	0.9	119.62

分组 Group	麦芽糊精 Maltodextrin/（g·L^-1）	大豆蛋白胨 Soybean peptone/（g·L^-1）	碳酸钙 CaCO₃/（g·L^-1）	相对效价 Relative titer/%
1	25	5	0.1	93.79
2	35	10	0.3	131.41
3	45	15	0.5	147.18
4	55	20	0.7	122.96
5	65	25	0.9	119.62

分组 Group	A：麦芽糊精 Maltodextrin/（g·L^-1）	B：大豆蛋白胨 Soybean peptone/（g·L^-1）	C：碳酸钙 CaCO₃/（g·L^-1）	Y：相对效价 Relative titer/%
1	1	1	0	96.78
2	0	0	0	154.74
3	-1	-1	0	77.81
4	1	0	-1	97.22
5	0	0	0	156.93
6	0	0	0	153.96
7	0	1	1	63.76
8	0	0	0	162.08
9	-1	1	0	71.85
10	1	-1	0	94.26
11	1	0	1	88.37
12	-1	0	-1	91.74
13	0	1	-1	96.08
14	0	-1	-1	85.91
15	-1	0	1	84.93
16	0	-1	1	82.55
17	0	0	0	162.35

分组 Group	A：麦芽糊精 Maltodextrin/（g·L^-1）	B：大豆蛋白胨 Soybean peptone/（g·L^-1）	C：碳酸钙 CaCO₃/（g·L^-1）	Y：相对效价 Relative titer/%
1	1	1	0	96.78
2	0	0	0	154.74
3	-1	-1	0	77.81
4	1	0	-1	97.22
5	0	0	0	156.93
6	0	0	0	153.96
7	0	1	1	63.76
8	0	0	0	162.08
9	-1	1	0	71.85
10	1	-1	0	94.26
11	1	0	1	88.37
12	-1	0	-1	91.74
13	0	1	-1	96.08
14	0	-1	-1	85.91
15	-1	0	1	84.93
16	0	-1	1	82.55
17	0	0	0	162.35

来源 Source	平方和 Sum of squares	自由度 Df	均方 Mean square	F	P
模型 Model	19 374.19	9	2 152.69	60.56	<0.000 1
A-麦芽糊精	316.26	1	316.26	8.90	0.020 4
B-大豆蛋白胨	18.18	1	18.18	0.511 5	0.497 7
C-碳酸钙	329.47	1	329.47	9.27	0.018 7
AB	17.98	1	17.98	0.505 8	0.500 0
AC	1.04	1	1.04	0.029 3	0.869 0
BC	209.67	1	209.67	5.90	0.045 5
A²	4 358.46	1	4 358.46	122.61	< 0.000 1
B²	6 962.19	1	6 962.19	195.86	< 0.000 1
C²	5 238.82	1	5 238.82	147.38	< 0.000 1
失拟 Lack of fit	185.16	3	61.72	3.88	0.111 7
残差误差 Residual error	248.82	7	35.55	R²=0.987 3
纯误差 Pure error	63.66	4	15.92	Adj R²=0.971 0
合计 Total	19 623.01	16