响应面法优化暹罗芽孢杆菌产大环内酯的发酵培养条件

doi:10.13560/j.cnki.biotech.bull.1985.2024-0040

摘要/Abstract

摘要：

【目的】大环内酯（MLNs）是一类由聚酮链环化而成的新型化合物，具有显著的抗菌、抗病毒和抗肿瘤活性，但因传统发酵的大环内酯产量较低而限制了其广泛应用。旨在提高暹罗芽孢杆菌K53合成MLNs的产量，以期为MLNs的批量生产提供科学依据。【方法】基于前期研究发现，通过单因素试验研究碳源、氮源、氨基酸等营养成分对菌株K53中MLNs发酵产量的影响，确定关键营养因子，运用响应面分析法对关键营养因子进行优化，获得发酵MLNs的最佳工艺参数。【结果】单因素试验中，菌株K53的发酵培养基中葡萄糖为最优碳源，酵母提取物和(NH₄)₂SO₄分别为最优有机氮源和无机氮源，额外添加组氨酸和缬氨酸可提高MLNs的产量。响应面优化中，菌株K53的最佳发酵培养基：22.0 g/L葡萄糖、3.0 g/L（NH₄）₂SO₄、1.0 g/L酵母提取物、6.1 g/L缬氨酸和5.9 g/L组氨酸，其中缬氨酸和组氨酸为合成MLNs的前体物质。在该培养基中MLNs的产量达到5.37×10² μg/mL，与理论预测的最大值（5.46×10² μg/mL）非常接近，与优化前的菌株合成MLNs产量（1.31×10² μg/mL）相比提高4.10倍。【结论】响应面法优化后的发酵培养基有利于提高菌株K53的MLNs产量，为菌株MLNs的后续开发应用提供参考。

关键词: 大环内酯, 暹罗芽孢杆菌, 发酵培养基, 前体物质, 响应面优化

Abstract:

【Objective】Macrolactins（MLNs）are a class of 24-membered macrolides formed by cyclization of polyketones, which have a variety of biological activities such as antibacterial, antifungal, antiviral, and anti-tumor. However, due to the low yield of macrolides in traditional fermentation, its development and application are limited. Therefore, this article aims to increase the yield of MLNs synthesized by Bacillus siamensis K53, in order to provide scientific basis for the mass production of MLNs.【Method】Based on previous research, single factor experiments was used to study the effects of carbon, nitrogen and amino acids on the MLNs yield of strain K53 and to determine the key nutritional factors. Response surface analysis was used to optimize the key nutritional factors for obtaining the optimal parameters in fermenting MLNs.【Result】In the single factor experiment, glucose was the optimal carbon source in the fermentation medium of strain K53. The yeast extract and (NH₄)₂SO₄ were the optimal organic and inorganic nitrogen sources, respectively. The addition of histidine and valine increased the yield of MLNs. In response surface optimization, the optimal fermentation medium for strain K53 was 22.0 g/L glucose, 3.0 g/L (NH₄)₂SO₄, 1.0 g/L yeast extract, 6.1 g/L valine, and 5.9 g/L histidine. The valine and histidine were precursors for the synthesis of MLNs. The MLNs yield in this medium reached 5.37×10² μg/mL, which was very close to the maximum value（5.46×10² μg/mL）predicted by theory, and 4.10 times higher than that in the non-optimized medium（1.31×10² μg/mL）.【Conclusion】The optimized medium formula by response surface methodology is conducive to the MLNs yield of the strain K53，and the results provide theoretical bases for the consequent development and application of the strain K53.

Key words: macrolactins, Bacillus siamensis, fermentation medium, precursor substance, optimization via response surface

张梦菲, 余炼, 李菲, 李喆, 苏芯莹, 蓝彩碧, 朱虎, 秦莹. 响应面法优化暹罗芽孢杆菌产大环内酯的发酵培养条件[J]. 生物技术通报, 2024, 40(8): 299-308.

ZHANG Meng-fei, YU Lian, LI Fei, LI Zhe, SU Xin-ying, LAN Cai-bi, ZHU Hu, QIN Ying. Optimization of Fermentation Culture Conditions for Macrolactins Yield of Bacillus siamensis Using Response Surface Methodology[J]. Biotechnology Bulletin, 2024, 40(8): 299-308.

图/表 9

参考文献 37

[1]	桑建伟, 杨扬, 陈奕鹏, 等. 内生解淀粉芽孢杆菌BEB17脂肽类和聚酮类化合物的抑菌活性分析[J]. 植物病理学报, 2018, 48(3): 402-412.
	Sang JW, Yang Y, Chen YP, et al. Antibacterial activity analysis of lipopeptide and polyketide compounds produced by endophytic bacteria Bacillus amyloliquefaciens BEB17[J]. Acta Phytopathol Sin, 2018, 48(3): 402-412.
[2]	Regmi SC, Park SY, Kim SJ, et al. The anti-tumor activity of succinyl macrolactin A is mediated through the β-catenin destruction complex via the suppression of tankyrase and PI3K/akt[J]. PLoS One, 2015, 10(11): e0141753.
[3]	Tareq FS, Kim JH, Lee MA, et al. Antimicrobial gageomacrolactins characterized from the fermentation of the marine-derived bacterium Bacillus subtilis under optimum growth conditions[J]. J Agric Food Chem, 2013, 61(14): 3428-3434.
[4]	Mojid Mondol MA, Tareq FS, Kim JH, et al. Cyclic ether-containing macrolactins, antimicrobial 24-membered isomeric macrolactones from a marine Bacillus sp[J]. J Nat Prod, 2011, 74(12): 2582-2587. doi: 10.1021/np200487k pmid: 22133265
[5]	Schneider K, Chen XH, Vater J, et al. Macrolactin is the polyketide biosynthesis product of the pks2 cluster of Bacillus amyloliquefaciens FZB42[J]. J Nat Prod, 2007, 70(9): 1417-1423. pmid: 17844999
[6]	Srihari P, Sayini R. Studies on the total synthesis of antibiotic macrolactin S: a conventional approach for the synthesis of the C1-C9 and C10-C24 fragments[J]. Synthesis, 2018, 50(3): 663-675.
[7]	Chen H, Wu MB, Chen ZJ, et al. Enhancing production of a 24-membered ring macrolide compound by a marine bacterium using response surface methodology[J]. J Zhejiang Univ Sci B, 2013, 14(4): 346-354.
[8]	Zhang LY, Jin MS, Shi X, et al. Macrolactin metabolite production by Bacillus sp. ZJ318 isolated from marine sediment[J]. Appl Biochem Biotechnol, 2022, 194(6): 2581-2593.
[9]	杜瑜欣, 江蕾, 余炼, 等. 前体调控对Bacillus sp. 108菌株发酵合成抑制甘蔗鞭黑粉菌活性物质产量的影响[J]. 南方农业学报, 2019, 50(4): 755-760.
	Du YX, Jiang L, Yu L, et al. Effects of precursor regulation on inhibition of production of active substances in sugarcane Sporisorium scitamineum by Bacillus sp. 108 fermentation and synthesis[J]. J South Agric, 2019, 50(4): 755-760.
[10]	Li BC, Cai DB, Chen SW. Metabolic engineering of central carbon metabolism of Bacillus licheniformis for enhanced production of poly-γ-glutamic acid[J]. Appl Biochem Biotechnol, 2021, 193(11): 3540-3552.
[11]	Sha YY, Sun T, Qiu YB, et al. Investigation of glutamate dependence mechanism for poly-γ-glutamic acid production in Bacillus subtilis on the basis of transcriptome analysis[J]. J Agric Food Chem, 2019, 67(22): 6263-6274.
[12]	杨代凯, 陈守文, 黎煊. 解淀粉芽孢杆菌ZM9液体发酵伊枯草菌素A培养基优化[J]. 应用化工, 2015, 44(3): 428-430.
	Yang DK, Chen SW, Li X. Optimization of culture medium for iturin A submerged fermentation by Bacillus amyloliquefaciens ZM9[J]. Appl Chem Ind, 2015, 44(3): 428-430.
[13]	Li CL, Li M, Zhang WG, et al. Accelerating the menaquinone-7 production in Bacillus amyloliquefaciens by optimization of the biosynthetic pathway and medium components[J]. Syst Microbiol Biomanuf, 2023, 3(4): 776-791.
[14]	方舟, 孟慧云, 杨渊, 等. 前体物质对Glarea lozoyensis发酵产生纽莫康定B0的影响[J]. 中国抗生素杂志, 2018, 43(4): 437-443.
	Fang Z, Meng HY, Yang Y, et al. Precursors supplement effects on the level of pneumocandin B0 produced by Glarea lozoyensis[J]. Chin J Antibiot, 2018, 43(4): 437-443.
[15]	薛欣怡, 张翼, 廖清楠, 等. 添加前体对2株真菌次级代谢产物及其生物活性的影响[J]. 广西科学, 2022, 29(5): 854-862.
	Xue XY, Zhang Y, Liao QN, et al. Effects of adding precursors on secondary metabolites and their activities of two fungal strains[J]. Guangxi Sci, 2022, 29(5): 854-862.
[16]	Zhang B, Zhang YH, Chen Y, et al. Enhanced AmB production in Streptomyces nodosus by fermentation regulation and rational combined feeding strategy[J]. Front Bioeng Biotechnol, 2020, 8: 597. doi: 10.3389/fbioe.2020.00597 pmid: 32760700
[17]	张宪花. 螺旋霉素发酵过程转录组分析及发酵优化[D]. 上海: 华东理工大学, 2021.
	Zhang XH. Transcriptomics analysis and optimization of spiramycin fermentation process[D]. Shanghai: East China University of Science and Technology, 2021.
[18]	倪婕. 大环内酯Macrolactin高产菌株的诱变选育及对草莓灰葡萄孢菌的抑制作用[D]. 南宁: 广西大学, 2020.
	Ni J. Breeding high-yield macrolactin strains with mutation and antibacterial activity of macrolactin against Botrytis cinerea[D]. Nanning: Guangxi University, 2020.
[19]	Yu L, Li F, Ni J, et al. UV-ARTP compound mutagenesis breeding improves macrolactins production of Bacillus siamensis and reveals metabolism changes by proteomic[J]. J Biotechnol, 2024, 381: 36-48.
[20]	Mondol MA, Kim JH, Lee HS, et al. Macrolactin W, a new antibacterial macrolide from a marine Bacillus sp[J]. Bioorg Med Chem Lett, 2011, 21(12): 3832-3835. doi: 10.1016/j.bmcl.2010.12.050 pmid: 21570834
[21]	李永红, 刘波, 赵宗保, 等. 圆红冬孢酵母菌发酵产油脂培养基及发酵条件的优化研究[J]. 生物工程学报, 2006, 22(4): 650-656.
	Li YH, Liu B, Zhao ZB, et al. Optimized culture medium and fermentation conditions for lipid production by Rhodosporidium toruloides[J]. Chin J Biotechnol, 2006, 22(4): 650-656.
[22]	代志凯, 张翠, 阮征. 试验设计和优化及其在发酵培养基优化中的应用[J]. 微生物学通报, 2010, 37(6): 894-903.
	Dai ZK, Zhang C, Ruan Z. The application of experimental design and optimization techniques in optimization of microbial medium[J]. Microbiol China, 2010, 37(6): 894-903.
[23]	Krivoruchko A, Zhang YM, Siewers V, et al. Microbial acetyl-CoA metabolism and metabolic engineering[J]. Metab Eng, 2015, 28: 28-42. doi: S1096-7176(14)00147-5 pmid: 25485951
[24]	Brobst SW, Townsend CA. The potential role of fatty acid initiation in the biosynthesis of the fungal aromatic polyketide aflatoxin B₁[J]. Can J Chem, 1994, 72(1): 200-207.
[25]	Jiang LF. Optimization of fermentation conditions for pullulan production by Aureobasidium pullulan using response surface methodology[J]. Carbohydr Polym, 2010, 79(2): 414-417.
[26]	许正宏, 窦文芳, 王霞, 等. 氮源及其添加模式对钝齿棒杆菌JDN28-75合成L-精氨酸的影响[J]. 应用与环境生物学报, 2006, 12(3): 381-385.
	Xu ZH, Dou WF, Wang X, et al. Effects of nitrogen source and its supply manner on production of L-arginine by Corynebacterium crenatum JDN28-75[J]. Chin J Appl Environ Biol, 2006, 12(3): 381-385.
[27]	李永辉. 芳香族氨基酸生物合成代谢途径调控研究[D]. 北京: 中国人民解放军军事医学科学院, 2003.
	Li YH. Study on the regulation of metabolic pathway in aromatic amino acids biosynthesis[D]. Beijing: Academy of Military Medical Sciences of Chinese People's Liberation Army, 2003.
[28]	朱峰, 乔建军. 聚酮合成酶底物专一性的研究进展[J]. 中国抗生素杂志, 2006, 31(11): 641-645, 664.
	Zhu F, Qiao JJ. Progress in substrate specificity of polyketide synthase[J]. Chin J Antibiot, 2006, 31(11): 641-645, 664.
[29]	夏梦雷. 代谢物轮廓分析提高筑波链霉菌FK506产量[D]. 天津: 天津大学, 2013.
	Xia ML. Enhanced FK506 production in Streptomyces tsukubaensis by comparative metabolic profiling analysis[D]. Tianjin: Tianjin University, 2013.
[30]	Kunioka M. Biodegradable water absorbent synthesized from bacterial poly(amino acid)S[J]. Macromol Biosci, 2004, 4(3): 324-329. pmid: 15468223
[31]	王明璐. 一种新型大环内酯类抗真菌抗生素的发酵工艺及稳定性研究[D]. 杭州: 浙江大学, 2013.
	Wang ML. Fermentation process and stability research of a novel acrolide antifungal-antibiotic[D]. Hangzhou: Zhejiang University, 2013.
[32]	Rahman MS, Ano T, Shoda M. Second stage production of iturin A by induced germination of Bacillus subtilis RB14[J]. J Biotechnol, 2006, 125(4): 513-515.
[33]	王永菲, 王成国. 响应面法的理论与应用[J]. 中央民族大学学报: 自然科学版, 2005, 14(3): 236-240.
	Wang YF, Wang CG. The application of response surface methodology[J]. J Cent Univ Natl Nat Sci Ed, 2005, 14(3): 236-240.
[34]	李莉, 张赛, 何强, 等. 响应面法在试验设计与优化中的应用[J]. 实验室研究与探索, 2015, 34(8): 41-45.
	Li L, Zhang S, He Q, et al. Application of response surface methodology in experiment design and optimization[J]. Res Explor Lab, 2015, 34(8): 41-45.
[35]	郝学财, 余晓斌, 刘志钰, 等. 响应面方法在优化微生物培养基中的应用[J]. 食品研究与开发, 2006, 27(1): 38-41.
	Hao XC, Yu XB, Liu ZY, et al. The applicationof response surface methodology in optimization of microbial media[J]. Food Res Dev, 2006, 27(1): 38-41.
[36]	Gouveia ER, Baptista-Neto A, Badino Jr AC, et al. Optimisation of medium composition for clavulanic acid production by Streptomyces clavuligerus[J]. Biotechnol Lett, 2001, 23(2): 157-161.
[37]	汪敦飞, 朱胜男, 肖清铁, 等. 基于响应面法的耐镉假单胞菌TCd-1培养条件优化[J]. 浙江农林大学学报, 2020, 37(5): 914-921.
	Wang DF, Zhu SN, Xiao QT, et al. Optimization of culture conditions of Cd-tolerant strain Pseudomonas TCd-1 based on response surface methodology[J]. J Zhejiang A F Univ, 2020, 37(5): 914-921.

因素Factor/（g·L^-1）	低水平Low level（-1）	高水平High level（1）
葡萄糖（X₁）	10	20
酵母提取物（X₂）	0.6	1.0
(NH₄)₂SO₄（X₃）	1.0	3.0
缬氨酸（X₄）	2.0	6.0
组氨酸（X₅）	2.0	6.0

因素Factor/（g·L^-1）	低水平Low level（-1）	高水平High level（1）
葡萄糖（X₁）	10	20
酵母提取物（X₂）	0.6	1.0
(NH₄)₂SO₄（X₃）	1.0	3.0
缬氨酸（X₄）	2.0	6.0
组氨酸（X₅）	2.0	6.0

水平 Level	葡萄糖Glucose X_1/（g·L^-1）	酵母提取物Yeast extract X₂/（g·L^-1）	缬氨酸Valine X₄/（g·L^-1）	组氨酸Histidine X₅/（g·L^-1）
-1	15	0.8	5.0	5.0
0	20	1.0	6.0	6.0
1	25	1.2	7.0	7.0

水平 Level	葡萄糖Glucose X_1/（g·L^-1）	酵母提取物Yeast extract X₂/（g·L^-1）	缬氨酸Valine X₄/（g·L^-1）	组氨酸Histidine X₅/（g·L^-1）
-1	15	0.8	5.0	5.0
0	20	1.0	6.0	6.0
1	25	1.2	7.0	7.0

来源Source	自由度Freedom	离均差平方和Sum of squares of deviation from mean	均方差Standard deviation	F值 F value	P值P value
模型	5	0.170	0.034	15.76	0.002 1
X₁	1	0.014	0.014	6.28	0.046 2
X₂	1	0.089	0.089	40.88	0.000 7
X₃	1	0.001	0.001	0.64	0.453 0
X₄	1	0.047	0.047	21.65	0.003 5
X₅	1	0.021	0.021	9.37	0.022 2
残差	6	0.013	0.002
总离差	11	0.190