Screening and Identification of Strains Producing 7-β-xylosyltaxanes Glycoside Hydrolases from the Periplaneta americana Gut

doi:10.13560/j.cnki.biotech.bull.1985.2021-0620

Abstract

Abstract:

The objective of this work is to screen endophytes that can produce 7-β-xylosyltaxanes glycoside hydrolases and to identify its biological characteristics. Taking total 178 strains of intestinal endophytes from Periplaneta americana preserved in the laboratory as the research object,the Congo red transparent circle method and fluorescent color method were used to screen the xylanase-producing strains,and the thin-layer chromatography was further used to screen strains producing the 7-β-xylosyltaxanes glycoside hydrolases. On the basis of this,a strain producing high 7-β-xylosyltaxanes glycoside hydrolases was selected for morphological,physiological,biochemical and molecular biological identification,and the conversion rate of 7-β-xylosyl-10-deacetylpaclitaxel by the strain was measured by high performance liquid chromatography. Among the 178 strains of intestinal endophytes in P. americana,total 55 endophytes produced xylanase,of which 8 strains of Streptomyces and 2 strains of Cupriavidus produced 7-β-xylosyltaxanes glycoside hydrolases. The high-yield strain WA11-2-9 was identified as Streptomyces enissocaesilis by bacterial morphology,physiological and biochemical experiments,combined with 16S rDNA sequence analysis. WA11-2-9 converted 7-β-xylosyl-10-deacetylpaclitaxel to 10-deacetylbaccatin III and 10-deacetylpaclitaxel. The conversion rate of the main product 10-deacetylpaclitaxel was 21.52%. In conclusion,the intestinal endophytes WA11-2-9（GenBank accession number：MZ411692）from P. americana secreted 7-β-xylosyltaxanes glycoside hydrolases,which lays a solid foundation for the subsequent biotransformation of paclitaxel.

Key words: Periplaneta americana gut, 7-β-xylosyltaxanes glycoside hydrolases, 7-β-xylosyl-10-deacetylpac-litaxel, 10-deacetylpaclitaxel

TANG Bin, LIU Wen-bin, LI Xiao-bo, WANG Ning, JIN Xiao-bao. Screening and Identification of Strains Producing 7-β-xylosyltaxanes Glycoside Hydrolases from the Periplaneta americana Gut[J]. Biotechnology Bulletin, 2022, 38(3): 139-148.

Figures/Tables 12

Fig. 1 Distribution of xylanase-producing strains A：Distribution of xylanase-producing actinomycetes. B：Distribution of xylanase-producing endophytes

Fig.2 Congo red transparent circles of xylanase-producing strains

Fig. 3 Fluorescence color image of xylanase-producing strain A to F：WA1-37,WA2-17,WA3-2-36,WA4-65,WA11-2-9 and WA14-2-7. The left is fluorescent color,and the right is self-control

Table 1 Colony diameters,Congo red transparent circles and their ratios of xylanase-producing strains（n=3）

菌株编号 Strain code	菌落直径 Diameter of colony（d/cm）	透明圈直径 Diameter of transparent circle of clony（D/cm）		比值 Ratio（D/d）
菌株编号 Strain code	菌落直径 Diameter of colony（d/cm）	内圈Inner circle	外圈Outer circle	内圈Inner circle	外圈Outer circle
1-37	0.75±0.02	1.67±0.12	3.68±0.18	2.21±0.11	4.88±0.12
2-17	0.83±0.05	1.54±0.05	2.66±0.04	1.87±0.16	3.23±0.17
3-2-36	0.64±0.06	1.33±0.07	2.89±0.02	2.13±0.06	4.63±0.17
4-65	0.68±0.08	1.72±0.12	3.50±0.17	2.56±0.16	5.03±0.13
11-2-9	0.62±0.04	1.33±0.05	3.72±0.04	2.07±0.12	5.55±0.17
14-2-7	0.62±0.04	1.50±0.03	2.92±0.04	2.44±0.10	4.80±0.15

Fig. 4 TLC result of transformed product of strain WA11-2-9 1st to 4th are：WA 10-1-2,WA 11-2-9,WA 11-1-3 and WA 23-1-5 transformation products,the points from 5th to 7th are：7-XDT,10-DAT and 10-DAB standards

Fig. 5 Morphological characteristics of strain WA11-2-9 A：The colony morphology of strain WA11-2-9. B：The gram staining diagram of strain WA11-2-9（100×）

Table 2 Physiological and biochemical characteristics of strain WA11-2-9

检测项目Test item	结果Result	检测项目Test item	结果Result	检测项目Test item	结果Result
淀粉水解	+	H₂S产生	+	葡萄糖	+
明胶液化	+	肌醇	+	阿拉伯糖	+
硝酸盐还原	+	果糖	+	生长温度范围	20-40℃
石蕊牛奶	+	甘露醇	+	生长pH范围	4-9
尿素酶	+	木糖	+	生长耐受NaCl范围	2%-8%

Fig. 6 Molecular biology identification of strain WA11-2-9 A：The 16S rDNA amplified products of strain WA11-2-9. B：The phylogenetic tree of strain WA11-2-9 based on 16S rDNA gene sequence

Fig.7 10-DAT standard curve

Fig. 8 HPLC analyzing spectrum of transformed product of strain WA11-2-9 A：The HPLC spectrogram of standard 7-XDT. B：The HPLC spectrogram of standard 10-DAT. C：The HPLC spectrogram of standard 10-DAB. D：The HPLC spectrogram of strain WA11-2-9. E：The HPLC spectrogram of WA11-2-9 contrast group（no 7-XDT added）

Fig. 9 Optimal reaction temperature and pH of 7-β-xyl A：Optimal reaction temperature. B：Optimal reaction pH

Fig.10 Thermal stability,pH stability and metal ion stab-ility of 7-β-xyl A：Thermal stability. B：pH stability. C：Metal ion stability

References 29

[1]	Yu C, Zhang C, Xu X, et al. Omic analysis of the endangered Taxaceae species Pseudotaxus chienii revealed the differences in taxol biosynjournal pathway between Pseudotaxus and Taxus yunnanensis trees[J]. BMC Plant Biol, 2021, 21(1):104. doi: 10.1186/s12870-021-02883-0 URL
[2]	邱德有, 张彬, 杨艳芳, 等. 紫杉醇生物合成研究历史、现状及展望[J]. 生物技术通报, 2015, 31(4):56-64. doi: 10.13560/j.cnki.biotech.bull.1985.2015.03.004
	Qiu DY, Zhang B, Yang YF, et al. History, current status and the prospects of taxol biosynjournal research[J]. Biotechnol Bull, 2015, 31(4):56-64.
[3]	Xue B, Zhao J, Fan Y, et al. Synjournal of taxol and docetaxel by using 10-deacetyl-7-xylosyltaxanes[J]. Chem Biodivers, 2020, 17(2):e1900631.
[4]	熊亮斌, 唐红菊, 宋新巍, 等. 紫杉醇类抗肿瘤原料药生产的研究进展[J]. 中草药, 2020, 51(15):4042-4049.
	Xiong LB, Tang HJ, Song XW, et al. Recent advances in synjournal of paclitaxel antitumor pharmaceutical raw materials[J]. Chin Tradit Herb Drugs, 2020, 51(15):4042-4049.
[5]	朱凤芝, 程赪, 刘祥胜, 等. 利用响应面法优化巴卡亭III生成10-DAB的工艺条件[J]. 生物技术通报, 2017, 33(4):238-246. doi: 10.13560/j.cnki.biotech.bull.1985.2017.04.031
	Zhu FZ, Cheng C, Liu XS, et al. Optimization of biotransformation technology for 10-DAB production from baccatin III using response surface methodology[J]. Biotechnol Bull, 2017, 33(4):238-246.
[6]	Li BJ, Wang H, Gong T, et al. Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production[J]. Nat Commun, 2017, 8:15544. doi: 10.1038/ncomms15544 URL
[7]	赵俊宏, 樊燕鸽, 王红星, 等. 7-木糖紫杉烷类化合物合成紫杉醇的新工艺研究[J]. 河南师范大学学报:自然科学版, 2020, 48(6):92-98, 105.
	Zhao JH, Fan YG, Wang HX, et al. New technology of synjournal of taxol from 7-xylosyltaxanes[J]. J Henan Norm Univ:Nat Sci Ed, 2020, 48(6):92-98, 105.
[8]	Hao DC, Ge GB, Yang L. Bacterial diversity of Taxus rhizosphere:culture-independent and culture-dependent approaches[J]. FEMS Microbiol Lett, 2008, 284(2):204-212. doi: 10.1111/fml.2008.284.issue-2 URL
[9]	Gales A, Chatellard L, Abadie M, et al. Screening of phytophagous and xylophagous insects guts microbiota abilities to degrade lignocellulose in bioreactor[J]. Front Microbiol, 2018, 9:2222. DOI: 10.3389/fmicb.2018.02222. doi: 10.3389/fmicb.2018.02222 URL
[10]	Lin PB, Shen J, Ou PY, et al. Prodigiosin isolated from Serratia marcescens in the Periplaneta americana gut and its apoptosis-inducing activity in HeLa cells[J]. Oncol Rep, 2019, 41(6):3377-3385.
[11]	曾佳佳, 陈静, 陈振, 等. 复合微生物的筛选及其发酵秸秆的应用效果[J]. 中国酿造, 2021, 40(3):124-128.
	Zeng JJ, Chen J, Chen Z, et al. Screening of compound microorganism and application effect of straw fermentation[J]. China Brew, 2021, 40(3):124-128.
[12]	张正雨, 田继远, 于娟, 等. 黄、东海春季海水胞外酶活性水平分布特征研究[J]. 海洋学报, 2020, 42(4):1-11.
	Zhang ZY, Tian JY, Yu J, et al. Horizontal distribution of extracellular enzyme activities in the Yellow Sea and the East China Sea in spring[J]. Acta Oceanol Sin, 2020, 42(4):1-11.
[13]	Wang XH, Zhang CH, Yang LL, et al. Screening and identification of microbial strains that secrete an extracellular C-7 xylosidase of taxanes[J]. World J Microbiol Biotechnol, 2011, 27(3):627-635. doi: 10.1007/s11274-010-0499-z URL
[14]	中国科学院微生物研究所放线菌分类组. 链霉菌鉴定手册[M]. 北京: 科学出版社, 1975.
	Actinomycetes Classification Group, Institute of Microbiology, Chinese Academy of Sciences. Streptomyces identification handbook[M]. Beijing: Science Press, 1975.
[15]	汤勇, 丁泓皓, 蔡俊. 黑曲霉发酵产木糖苷酶工艺优化[J]. 食品科学, 2020, 41(10):172-179.
	Tang Y, Ding HH, Cai J. Optimization of fermentation conditions for xylosidase production by Aspergillus niger[J]. Food Sci, 2020, 41(10):172-179.
[16]	Rubio MV, Terrasan CRF, Contesini FJ, et al. Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency[J]. Biotechnol Biofuels, 2019, 12:269. doi: 10.1186/s13068-019-1609-2 URL
[17]	Aftab MN, Zafar A, Awan AR. Expression of thermostable β-xylosidase in Escherichia coli for use in saccharification of plant biomass[J]. Bioengineered, 2017, 8(5):665-669. doi: 10.1080/21655979.2016.1267884 URL
[18]	李娜, 张蕊, 黄遵锡, 等. β-木糖苷酶的生物活性物质转化功能研究进展[J]. 微生物学通报, 2020, 47(7):2290-2299.
	Li N, Zhang R, Huang ZX, et al. Research progress in bioactive substances transformation by β-xylosidases[J]. Microbiol China, 2020, 47(7):2290-2299.
[19]	张衡, 甘慧, 吴卓娜, 等. 7-木糖紫杉烷类化合物生物转化[J]. 中国新药杂志, 2013, 22(9):1029-1033.
	Zhang H, Gan H, Wu ZN, et al. Biotransformation of 7-xylose taxanes[J]. Chin J New Drugs, 2013, 22(9):1029-1033.
[20]	区佩渝, 刘凌燕, 陈志宇, 等. 美洲大蠊肠道内生分枝杆菌的分离鉴定及其抑菌活性的初步研究[J]. 中国病原生物学杂志, 2019, 14(5):560-564, 567.
	Ou PY, Liu LY, Chen ZY, et al. Isolation and identification of endophytic Mycobacteria from Periplaneta americana and a preliminary study of its antibacterial activity[J]. J Pathog Biol, 2019, 14(5):560-564, 567.
[21]	刘宇, 郑水, 刘汉博, 等. 利用天蓝色链霉菌转化7-木糖紫杉烷的研究[J]. 氨基酸和生物资源, 2012, 34(4):39-41.
	Liu Y, Zheng S, Liu HB, et al. Study on Transforming 7-xyloyl-taxane by Streptomyces coelicolor[J]. Amino Acids Biotic Resour, 2012, 34(4):39-41.
[22]	李艳, 周剑, 何东贤, 等. 微生物转化在现代中药研发中的应用[J]. 中国抗生素杂志, 2020, 45(5):418-422.
	Li Y, Zhou J, He DX, et al. Application of microbial transformation in the research of modern traditional Chinese medicine[J]. Chin J Antibiot, 2020, 45(5):418-422.
[23]	李勇超, 杨靖, 周修任, 等. 耦合培养法对10-DAB产量的影响[J]. 生物技术, 2016, 26(1):93-97.
	Li YC, Yang J, Zhou XR, et al. Effects of coupling cultivation on the 10-DAB yield[J]. Biotechnology, 2016, 26(1):93-97.
[24]	朱凤芝. 巴卡亭Ⅲ转化生成10-DAB的微生物的筛选、鉴定和工艺优化[D]. 天津:天津科技大学, 2016.
	Zhu FZ. The screening, identification and technology optimization of the strains with the biotransformation ability from baccatin Ⅲ to 10-DAB[D]. Tianjin:Tianjin University of Science & Technology, 2016.
[25]	李建华. 三尖杉宁碱的微生物转化与10-去乙酰紫杉醇的酶法合成[D]. 北京:中国协和医科大学, 2007.
	Li JH. Microbial transformation of cephalomannine and the enzymatic synthesis of 10-deacetyltaxol[D]. Beijing:Peking Union Medical College, 2007.
[26]	李建华. 紫杉醇类似物的生物转化[C]. 吉林:中国植物学会, 2006.
	Li JH. Biotransformation of paclitaxel analogues[C]. Jilin:Chinese Botany Society, 2016.
[27]	张衡. 7-木糖-10-去乙酰紫杉醇的生物转化研究[D]. 南宁:广西医科大学, 2013.
	Zhang H. Biotransformation research of 7-β-xylosyl-10-deacetylpaclitaxel[D]. Nanning:Guangxi Medical University, 2013.
[28]	Li Q, Jiang YJ, Tong XY, et al. Cloning and characterization of the β-xylosidase from Dictyoglomus turgidum for high efficient biotransformation of 10-deacetyl-7-xylosltaxol[J]. Bioorg Chem, 2020, 94:103357. doi: 10.1016/j.bioorg.2019.103357 URL
[29]	Dou TY, Luan HW, Liu XB, et al. Enzymatic hydrolysis of 7-xylosyltaxanes by an extracellular xylosidase from Cellulosimicrobium cellulans[J]. Biotechnol Lett, 2015, 37(9):1905-1910. doi: 10.1007/s10529-015-1867-4 URL