为解决化学合成法生产间苯三酚环境污染严重的问题，本研究设计2 对不同的扩增间苯三酚合成关键基因phlD 的引物，对前期分离得到的270 余株假单胞菌进行筛选。采用LC-MS 法对筛选出的目标菌株进行发酵产物检测，并对菌株进行种属鉴定及系统发育树构建；采用HPLC 法测定其间苯三酚的产量，再利用正交试验优化菌株的发酵培养条件。结果显示，引物2 在筛选产间苯三酚菌株方面效果较好，经LC-MS 检测验证，筛选得到的海洋生境假单胞菌LBF-96 发酵产物为间苯三酚，其经生理生化鉴定及16S 核糖体RNA(16S rRNA) 基因序列分析，确定为芸薹假单胞菌(Pseudomonas brassicacearum)。该菌株在28 ℃、150 r/min 条件下发酵培养至72 h 时，间苯三酚的产量为0.23 g/L，经正交试验优化发酵培养条件后，菌株在30 ℃、pH 7.0、200 r/min 条件下间苯三酚的产量最高，比优化前提高了39％。这是关于芸薹假单胞菌产间苯三酚的首次报道，为间苯三酚的生物合成提供了新的种质资源。
Abstract
Two pairs of different primers for amplifying the key gene phlD for synthesis of phloroglucinol were designed to screen more than 270 Pseudomonas strains isolated in the earlier stage, which was in order to solve the problem of serious environmental pollution in the production of phloroglucinol by chemical synthesis. The LC-MS method was used to detect the fermentation products of the selected target strains, identify the strains and construct the phylogenetic tree. The HPLC method was used to determine the output of phloroglucinol, and the fermentation conditions of the strain were optimized by orthogonal experiment. The results showed that primer 2 was effective in screening of phloroglucinol producing strains. The LC-MS test verified that the fermentation product of Pseudomonas LBF-96 screened in marine habitat was phloroglucinol, and the strain was identified as Pseudomonas brassicacearum through physiological and biochemical identification and 16S ribosomal RNA(16S rRNA) gene sequence analysis. When the strain was cultured at 28 ℃ and 150 r/min for 72 h, the yield of phloroglucinol was 0.23 g/L. After optimizing the fermentation conditions by the orthogonal experiment, the strain produced the highest amount of phloroglucinol under the conditions of 30 ℃, pH 7.0 and 200 r/min, which increased by 39％ compared with that before optimization. The production of phloroglucinol by Pseudomonas brassicacearum was reported for the first time in this paper, which provided a new germplasm resource for the biosynthesis of phloroglucinol.
关键词
间苯三酚 /
phlD基因 /
液相色谱-质谱 /
芸薹假单胞菌 /
发酵
{{custom_keyword}} /
Key words
phloroglucinol /
phlD gene /
LC-MS /
Pseudomonas brassicacearum /
fermentation
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] SU J C, WANG S, CHENG W, et al．Phloroglucinol derivatives with unusual skeletons from Cleistocalyx operculatus and their in vitro antiviral activity [J]．J Org Chem, 2018, 83(15): 8522-8532．
[2] KIM R K, UDDIN N, HYUN J W, et al．Novel anticancer activity of phloroglucinol against breast cancer stem-like
cells [J]．Toxicol Appl Pharmacol, 2015, 286(3): 143-150．
[3] 咸 漠, 刘 炜, 袁 珊, 等．一种可提高间苯三酚生物合成产量并降低生产成本的基因工程菌及其构建方法与应用与流程: 中国, 109943513A [P]．2019-06-28．
[4] 王蓉华, 孙秀娟, 李 倩, 等．海洋功能分子间苯三酚的研究进展[J]．中国生物工程杂志, 2015, 35(10): 115-121．
[5] 李永军, 葛忠学, 刘 鸿．间苯三酚的合成研究进展[J]．合成化学, 2004, 12(6): 555-558．
[6] 张汝兵, 咸 漠, 曹玉锦, 等．一种高间苯三酚合成能力的基因工程菌及构建方法和应用与流程: 中国, 106929527B[P]．2017-07-07．
[7] 高海军, 肖 丹, 杨永政, 等．荧光假单胞菌phlD基因的克隆, 表达及功能[J]．北京理工大学学报, 2009, 29(5): 465-470．
[8] CAO Y J, XIAN M．Production of phloroglucinol by Escherichia coli using a stationary-phase promoter [J]．Biotechnol Lett, 2011, 33(9): 1853-1858．
[9] YANG F, CAO Y．Biosynthesis of phloroglucinol compounds in microorganisms [J]．Appl Microbiol Biotechnol, 2012, 93(2): 487-495．
[10] PAULSEN I T, PRESS C M, RAVEL J, et al．Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5 [J]．Nat Biotechnol, 2005, 23(7): 873-878．
[11] SINGH I P, BHARATE S B．Phloroglucinol compounds of natural origin [J]．Nat Prod Rep, 2006, 23(4): 558-591．
[12] ACHKAR J, XIAN M, ZHAO H, et al．Biosynthesis of phloroglucinol [J]．J Am Cheml Soc, 2005, 127(15): 5332-5233．
[13] 魏海雷, 周洪友, 张力群, 等．抗生素2,4-二乙酰基间苯三酚作为荧光假单胞菌2P24菌株生防功能因子的实证分析[J]．微生物学报, 2004, 44(5): 663-666．
[14] 廖昌军, 凌保东, 王 茜, 等．间苯三酚栓剂在家兔体内的药动学研究[J]．中国测试, 2017, 43(8): 55-59．
[15] 郝羚竹, 潘书洋, 李超生．HPLC法测定铁冬青总皂苷中间苯三酚的含量[J]．中国药品标准, 2012, 13(6): 430-432．
[16] 东秀珠, 蔡妙英．常见细菌系统鉴定手册[M]．北京: 科学出版社, 2001: 349-398．
[17] 陈忠翔, 房志家, 陈 婷, 等．一种简单高效的酵母单菌落PCR方法[J]．生物技术通讯, 2013, 24(2): 225-229．
[18] 冯语嫣, 刘晓凡, 范 青, 等．葡萄抑菌附生细菌筛选鉴定及其结构表征[J]．食品与发酵工业, 2019, 45(5): 31-37．
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家自然科学基金项目(51804021)、2019年山东省高等学校青创人才引育计划(133)、2018年度山东省高校科研计划项目 (J18KB062、J18KB083)
{{custom_fund}}
PDF(3116 KB)
