Study on New Genes Associated with Dexamethasone Degradation

doi:10.13523/j.cb.20180102

China Biotechnology

2018, Vol. 38

Issue (1): 15-24 DOI: 10.13523/j.cb.20180102

Orginal Article

Study on New Genes Associated with Dexamethasone Degradation

Jin ZHANG¹,Dan SI¹,Zhi-bang YANG¹,Yu-xia XIONG¹,Lian-ju MA²,Jin-yang LI³,Ren-ju JIANG¹(

)

1 Department of Pathogenic Biology, Basic Medical College, Chongqing Medical University, Chongqing 400016,China
2 Center of Pharmacy Experimental Teaching, College of Pharmacy, Chongqing Medical University, Chongqing 400016, China
3 Clinical Medical College, Chongqing Medical University, Chongqing 400016, China

Download:

HTML

PDF(1653KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract Objective:

To investigate the new genes associated with dexamethasone metabolism in order to provide an idea for the construction of an efficient and stable dexamethasone degrading engineering bacterium.

Methods:

Target genes which were suspectly associated with dexamethasone metabolism were screened according to the results of the whole genome sequencing and bioinformatics analysis of the newly discovered dexamethasone degrading bacteria Burkholderia sp.CQ001 (B.CQ001). Total RNA of B.CQ001 was extracted from B.CQ001wasprocessed by RT-PCR to produce cDNA. Using cDNA as a template, the new gene was rapidly cloned subsequently by PCR technology which was verified by real-time fluorescence quantitative PCR (RT-qPCR), then connected with prokaryotic expression vector pET-28a-c (+), after DNA sequencing. The recombinant plasmid pET-28a-Ivd was transformed into B.CQ001. Using high performance liquid chromatography (HPLC) to evaluate the biodegradability of the expressed bacteria for dexmethas one sodium phosphate.

Results:

Three unknown genes were screened out from B.CQ001: ORF05499, ORF05827, ORF06535, and their expressed products were: peroxiredoxin, ketosteroid isomerase, isovaleryl coenzyme A dehydrogenase; Real time quantitative PCR analysis showed that the expression levels of three genes were upregulated in different degrees after induced by dexamethasone ,especially gene ORF06535; HPLC showed that the degradation of dexamethasone sodium phosphate and dexamethasone rate can reach to 89% and 80% after overexpression of ORF06535 in B.CQ001. Compared with the original strain B.CQ001, the degradation ability had improved significantly.

Conclusion:

New gene ORF06535 associated with dexamethasone degradation was discovered in B.CQ001 which had been cloned and functional verified. It provides new genetic information for preparation of dexamethasone and steroid hormone bioremediation.

Key words： Burkholderia Dexamethasone Degradation Gene RT-qPCR

Received: 02 September 2017 Published: 31 January 2018

ZTFLH:

Q78

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Jin ZHANG
	Dan SI
	Zhi-bang YANG
	Yu-xia XIONG
	Lian-ju MA
	Jin-yang LI
	Ren-ju JIANG

Cite this article:

Jin ZHANG,Dan SI,Zhi-bang YANG,Yu-xia XIONG,Lian-ju MA,Jin-yang LI,Ren-ju JIANG. Study on New Genes Associated with Dexamethasone Degradation. China Biotechnology, 2018, 38(1): 15-24.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180102 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I1/15

Table1 Real-time PCR primers of three unknown genes from Burkholderia sp.CQ001

Table 2 Three unknown genes to be validated from Burkholderia sp.CQ001

Fig.1 The arrangement of 3 unknown gene clusters in Burkholderia sp.CQ001 to be verified

Fig.2 The location map of 3 unknown genes in Burkholderia sp.CQ001

Fig.3 The sequence alignment result of ORF06535 amino acid sequence and 5 peptides from 41kDa degradation protein

Table 3 Bioinformatics analysis results of 3 unknown genes from Burkholderia sp.CQ001

Table 3 (continued) Bioinformatics analysis results of 3 unknown genes from Burkholderia sp.CQ001

Fig.4 Different expression levels of 3 genesbetween dexamethasone induced group and sucrose inorganic salt control groupControl group: Sucrose inorganic salt culture group; Induction group: Dexamethasone sodium phosphate culture group

Fig.5 Agarose gel electrophoresis result of gene ORF06535 PCR products1: GeneORF06535; 2: DNA marker DL2000

Fig.6 Agarose gel electrophoresis result of pUCm-Ivd1: DNA marker DL2000; 2 : PCR validation of pUCm-Ivd; 3: pUCm-Ivd; 4: Double enzyme digestion assay of pUCm-Ivd/BamHI+NotI; 5: DNA marker DL15000

Fig.7 Agarose gel electrophoresis result of pET-28a-Ivd1: DNA marker DL2000; 2: PCR validation of pET-28a-Ivd; 3: pET-28a-Ivd; 4: Double enzyme digestion assay of pET-28a-Ivd/BamHI+NotI; 5: DNA marker DL15000

Fig.8 Degradation process HPLC chromatogram map of B.CQ001 and B.CQ002(a),(b) B.CQ001 (c),(d) B.CQ002

Fig.9 Degradation curves of B.CQ001 and B.CQ002dexamethasone sodium phosphate and dexamethasone(a) Degradation curves of B.CQ001 and B.CQ002 on Dexamethasone sodium phosphate (b) Degradation curves of B.CQ001 and B.CQ002 on dexamethasone


[1]	Yabuuchi E, Kosako Y, Oyaizu H, et al.Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiology and Immunology, 1992, 36(12): 1251-1275. doi: 10.1111/j.1348-0421.1992.tb02129.x

[2]	Lemaire B, Chimphango S B M, Stirton C, et al. Biogeographical patterns of legume-nodulating Burkholderia spp.: From African fynbos to continental scales. Applied and Environmental Microbiology, 2016, 82(17): 5099-5115. doi: 10.1128/AEM.00591-16 pmid: 4988186

[3]	Hamid S, Bae W, Kim S, et al.Enhancing co-metabolic degradation of trichloroethylene with toluene using Burkholderia vietnamiensis G4 encapsulated in polyethylene glycol polymer. Environmental Technology, 2014, 35(12): 1470-1477. doi: 10.1080/09593330.2013.871045 pmid: 24701945

[4]	赵奎. 地塞米松磷酸钠注射液在急诊应用的处方分析. 中国医院用药评价与分析, 2011, 11(5): 475-476.

[4]	Zhao K.Prescription analysis of dexamethasone sodium phosphate injection in emergency department . Chinese Hospital Drug Evaluation and Analysis, 2011, 11(5): 475-476.

[5]	Takahashi T, Nakamura Y, Tsuya A, et al.Pharmacokinetics of aprepitant and dexamethasone after administration of chemotherapeutic agents and effects of plasma substance P concentration on chemotherapy-induced nausea and vomiting in Japanese cancer patients. Cancer Chemotherapy and Pharmacology, 2011, 68(3): 653-659. doi: 10.1007/s00280-010-1519-2

[6]	Rathod S S, Motghare V M, Deshmukh V S, et al.Prescribing practices of topical corticosteroids in the outpatient dermatology department of a rural tertiary care teaching hospital. Indian Journal of Dermatology, 2013, 58(5): 342. doi: 10.4103/0019-5154.117293 pmid: 3778770

[7]	Kim E J, Lee J G, Kim J Y, et al. Enhanced immune-modulatory effects of thalidomide and dexamethasone co-treatment on T cell subsets. Immunology, 2017, 152(4): 628-637. doi: 10.1111/imm.12804 pmid: 28758197

[8]	Mushaben E M, Brandt E B, Hershey G K, et al.Differential effects of rapamycin and dexamethasone in mouse models of established allergic asthma. PLoS One, 2013, 8(1): e54426. doi: 10.1371/journal.pone.0054426 pmid: 3547928

[9]	Vander Linden S C, Heringa M B, Man H Y,et al. Detection of multiple hormonal activities in wastewater effluents and surface water, using a panel of steroid receptor CALUX bioassays. Environ Sci Technol, 2008, 42(15): 5814-5820 doi: 10.1021/es702897y pmid: 18754514

[10]	Schriks M, van Leerdam J A, van der Linden S C,et al.High-resolution mass spectrometric identification and quantification of glucocorticoid compounds in various wastewaters in the Netherlands.Environ Sci Technol, 2010, 44(12): 4766-4774. doi: 10.1021/es100013x pmid: 20507090

[11]	Chang H, Hu J, Shao B.Occurrence of natural and synthetic glucocorticoids in sewage treatment plants and receiving river waters.Environ Sci Technol, 2007, 41(10): 3462-3468. doi: 10.1021/es062746o pmid: 17547164

[12]	石中全,周裕珍,杨致邦,等.废水中地塞米松污染的探讨, 中国医药指南,2012, 10(9): 319-321. doi: 10.3969/j.issn.1671-8194.2012.09.258

[12]	Shi Z Q, Zhou Y Z, Yang Z B,et al.Investigation on the pollution of dexamethasone in wastewater. Chinese Medical Guideline,2012, 10(9): 319-321. doi: 10.3969/j.issn.1671-8194.2012.09.258

[13]	Takahashi T,Nakamura Y,Tsuya A,et al.Pharmacokinetics of aprepitant and dexamethasone after administration of chemotherapeutic agents and effects of plasma substance P concentration on chemotherapy-induced nausea and vomiting in Japanese cancer patients.Cancer Chemother Pharmacol, 2011, 68(3): 653-659. doi: 10.1007/s00280-010-1519-2

[14]	韦英杰, 王长梅, 蔡雪婷,等. 地塞米松影响骨骼发育的斑马鱼模型的建立. 药学学报, 2013, 48(2): 255-260.

[14]	Wei Y J, Wang C M, Cai X T, et al.Establishment of zebrafish model of skeletal development affectedby dexamethasone . Journal of Pharmacology, 2013, 48(2): 255-260.

[15]	杨茜, 李晓愚, 斯丹,等. 饮水中地塞米松污染对小鼠肠道菌群的影响. 南方医科大学学报, 2016, 36(2): 238-243.

[15]	Yang Q, Li X Y, Si D, et al.Effects of dexamethasone pollution in drinking water on intestinal microflora in mice . Journal of Southern Medical University, 2016, 36(2): 238-243.

[16]	斯丹, 杨致邦, 蒋任举等.具有高效降解地塞米松能力的伯克霍尔德菌CQ001株的分离及功能基因组学分析. 第三军医大学学报, 2017, 39(13): 1352-1359. doi: 10.16016/j.1000-5404.201612198

[16]	Si D, Yang Z B, Jiang R J, et al.Separation and functional genomics of Burkholderia sp.CQ001with high degradation ability of dexamethasone . Journal of Third Military Medical University, 2017, 39(13): 1352-1359. doi: 10.16016/j.1000-5404.201612198

[17]	Bustin S A, Benes V, Garson J A, et al.The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 2009, 55(4): 611-622. doi: 10.1373/clinchem.2008.112797 pmid: 19246619

[18]	石中全, 杨致邦, 朱黎黎,等. 一株地塞米松磷酸钠降解菌的分离鉴定. 中国微生态学杂志, 2014, 26(11): 1250-1256.

[18]	Shi Z Q, Yang Z B, Zhu L L, et al.Isolation and identification of a strain of dexamethasone sodium phosphate degradation bacteria . Chinese Journal of Microecology, 2014, 26(11): 1250-1256.

[19]	Zhu L, Yang Z, Qian Y, et al.Degradation of dexamethasone by acclimated strain of Pseudomonas Alcaligenes. International Journal of Clinical & Experimental Medicine, 2015, 8(7): 10971.

[20]	Yao K, Wang F Q, Zhang H C, et al.Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum. Metabolic Engineering, 2013, 15(1): 75-87. doi: 10.1016/j.ymben.2012.10.005 pmid: 23164577

[21]	Xiong G, Luo Y, Jin S, et al.Cis- and trans-regulatory elements of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase as biosensor system for steroid determination in the environment. Chemico-Biological Interactions, 2009, 178(3): 215-220. doi: 10.1016/j.cbi.2008.10.012 pmid: 19000658

[22]	Cabrera J E, Paz J L P, Genti-Raimondi S. Steroid-inducible transcription of the 3β/17β-hydroxysteroid dehydrogenase gene(3β/17β-hsd) in Comamonas testosteroni. The Journal of Steroid Biochemistry and Molecular Biology, 2000, 73(3): 147-152. doi: 10.1016/S0960-0760(00)00066-2

[23]	Pandey A K, Sassetti C M.Mycobacterial persistence requires the utilization of host cholesterol. Proceedings of the National Academy of Sciences, 2008, 105(11): 4376-4380. doi: 10.1073/pnas.0711159105 pmid: 18334639

[24]	Bloem A, Sanchez I, Dequin S, et al.Metabolic impact of redox cofactor perturbations on the formation of aroma compounds in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 2015, 82(1): 174-183.

[1]	LI Xiao-jin,LI Yan-meng,LI Zhen-kun,XU An-jian,YANG Xiao-xi,HUANG Jian. *The Mechanism of Copper Accumulation Induced Autophagy in Hepatocytes of ATP7B-deficient Mice Based on RNA-sequencing*[J]. China Biotechnology, 2021, 41(9): 10-19.

[2]	MA Ning,WANG Han-jie. Advances of Optogenetics in the Regulation of Bacterial Production[J]. China Biotechnology, 2021, 41(9): 101-109.

[3]	HUANG Huan-bang,WU Yang,YANG You-hui,WANG Zhao-guan,QI Hao. Progress in Incorporation of Non-canonical Amino Acid Based on Archaeal Tyrosyl-tRNA Synthetase[J]. China Biotechnology, 2021, 41(9): 110-125.

[4]	WU Xiu-zhi,WANG Hong-jie,ZU Yao. *Functional Study of hoxa1a* Regulating Craniofacial Bone Development in Zebrafish**[J]. China Biotechnology, 2021, 41(9): 20-26.

[5]	HE Li-heng,ZHANG Yi,ZHANG Jie,REN Yu-chao,XIE Hong-e,TANG Rui-min,JIA Xiao-yun,WU Zong-xin. Construction of Gene Co-expression Network and Identification of Hub Genes Related to Anthocyanin Biosynthesis Based on RNA-seq and WGCNA in Sweetpotato[J]. China Biotechnology, 2021, 41(9): 27-36.

[6]	CHEN Ya-chao,LI Nan-nan,LIU Zi-di,HU Bing,LI Chun. Metagenomic Mining of Functional Genes Related to Glycyrrhizin Synthesis from Endophytes of Licorice[J]. China Biotechnology, 2021, 41(9): 37-47.

[7]	YANG Liu,MOU Hao,XU Guo-yang,BAI Yun-chuan,YU Yuan-di. Analysis of the Difference in Color Development of Cultured Goatpox Virus Common Cells in X-gal Environment[J]. China Biotechnology, 2021, 41(9): 48-54.

[8]	ZHAO Xiao-yu,XU Qi-ling,ZHAO Xiao-dong,AN Yun-fei. Enhancing Lentiviral Vector Transduction Efficiency for Facilitating Gene Therapy[J]. China Biotechnology, 2021, 41(8): 52-58.

[9]	YANG Wan-bin,XU Yan,ZHUO Shi-xuan,WANG Xin-yi,LI Ya-jing,GUO Yi-fan,ZHANG Zheng-guang,GUO Yuan-yuan. Progress of Long Non-coding RNAs Related Epigenetic Modifications in Cancer[J]. China Biotechnology, 2021, 41(8): 59-66.

[10]	GUO Man-man,TIAN Kai-ren,QIAO Jian-jun,LI Yan-ni. Application of Phage Recombinase Systems in Synthetic Biology[J]. China Biotechnology, 2021, 41(8): 90-102.

[11]	WANG Yu-xuan,CHEN Ting,ZHANG Yong-liang. Research Progress on the Biological Function of MiR-148[J]. China Biotechnology, 2021, 41(7): 74-80.

[12]	Bao-qi FENG,Jiao FENG,Miao ZHANG,Yang LIU,Rui CAO,Han-zhi YIN,Feng-xian QI,Zi-long LI,Shou-liang YIN. Screening of High Avermectin-producing Strains via Tn5 Transposon Mediated Mutagenesis[J]. China Biotechnology, 2021, 41(7): 32-41.

[13]	LI Kai-xiu,SI Wei. Progress in the Treatment of Inflammatory Bowel Diseases by Exosomes Derived from Mesenchymal Stem Cells[J]. China Biotechnology, 2021, 41(7): 66-73.

[14]	LIANG Jin-gang,ZHANG Xu-dong,BI Yan-zhe,WANG Hao-qian,ZHANG Xiu-jie. Development Status and Prospect of Genetically Modified Insect-resistant Maize[J]. China Biotechnology, 2021, 41(6): 98-104.

[15]	HU Xuan,WANG Song,YU Xue-ling,ZHANG Xiao-peng. Construction of a Destabilized EGFP Cell Model for Gene Editing Evaluation[J]. China Biotechnology, 2021, 41(5): 17-26.

Viewed

Full text

Abstract

Cited

Shared

Discussed