Study On Estrogen Degrading Gene And Related Functions Of Lysinibacillus Sphaericus DH-B01

Estradiol(E2)in natural estrogen has extremely high estrogen activity and is also a major environmental endocrine disruptor.Long-term exposure to the environment contaminated with estrogen may destroy the animal's sexual development,and have an adverse effect on the ecological balance and humans.To date,the use of microbial biodegradation to remove estrogen from the environment is one of the most effective strategies.At present,a series of microorganisms with estrogen degradation ability have been isolated,such as Sphingomonas,Pseudomonas,Acinetobacter,Rhodococcus and Bacillus.There have been few reports on the degradation of estrogen by Bacillus sphaericus.Therefore,for the newly isolated estrogen-degrading bacteria Lysinibacillus sphaericus DH-B01,16 S rDNA and DNA-DNA hybridization methods were used to determine the strain's genus classification;through condition optimization experiments,L.sphaericus DH-B01 was determined to degrade estrogen.The best conditions;through L.sphaericus DH-B01 whole gene sequencing,and annotate the genome of L.sphaericus DH-B01,select relevant functional genes for further research;by using liquid chromatography and liquid chromatography-mass spectrometry to determine the efficiency of L.sphaericus DH-B01 in degrading estrogen,and analysis of the resulting metabolites;verify the expression of L.sphaericus DH-B01 estrogen degrading enzyme by RT-PCR technology,and may play a key role in metabolism The structure and composition of 3?-hydroxysteroid dehydrogenase and catechol 2,3 dioxygenase were analyzed.This study provides theoretical support for the practical application of subsequent Bacillus sphaericus and enriches the types of estrogen-degrading bacteria.The main findings are as follows:16S rDNA sequencing showed that the bacterium had a genetic relationship with Lysinibacillus sphaericus.DNA-DNA hybridization verified that the bacterium belonged to Bacillus sphaericus and Lysinibacillus sphaericus,so it was named Lysinibacillus sphaericus DH-B01(L.sphaericus DH-B01).Deposited at the China General Microbial Culture Collection Management Center(Accession no.CCTCC M 2019039).L.sphaericus DH-B01 grows and reproduces itself and degrades estrogen at a temperature of 30°C,pH 8,3 mL of bacterial solution,and a shaker of 120 r/min.The E2 degradation efficiency of 30 mg/L is highest,24-48 hours.The degradation rate is the fastest,and more than 90% of E2 can be degraded in 48 hours.Using liquid chromatography-mass spectrometry to detect the molecular weight of the metabolite of L.sphaericus DH-B01 degrading E2,it is speculated that E2 is hydroxylated to E1 by the enzyme of L.sphaericus DH-B01,and E1 generates 4-OH E1 eventually enters the ring to crack,and the cracked product eventually loses estrogen activity.L.sphaericus DH-B01 contains one chromosome,which is 4,602,887 bp in length;GC content is 37.27%;a total of 4395 genes are predicted to be encoded;and 4278 are proteins.107 tRNA,37 rRNA(including 13 5s rRNA,12 16 s rRNA,and 12 23 s rRNA),64 tandem repeats,the total length of the repeats is 18874 bp,accounting for 0.49% of the total genome length.The number of gene annotations is 2289 for KEGG,4244 for COG,and 3581 for GO.L.sphaericus DH-B01 degraded E2 for 48 hours,and verified the degradation of alcohol dehydrogenase,enoyl-CoA hydratase,3-hydroxyacyl-CoA dehydrogenase,catechol 2 by RT-PCR technology,3 dioxygenase,acyl COA dehydrogenase,3?-hydroxysteroid dehydrogenase and short chain dehydrogenase are all expressed.Among them,3?-hydroxysteroid dehydrogenase gene3855 and catechol 2,3 dioxygenase gene2159 have important significance.It is speculated that gene3855 dehydrogenates CH-OH at the C-17 position of the D ring of E2 to generate C=O,and gene2159 cleaves the benzene ring of E2 and enters the ring cleavage.In conclusion,L.sphaericus DH-B01 is the only few Lysinibacillus sphaericus in the current study that has been proven to efficiently degrade estrogen.At the same time,as a genus of heavy metal tolerance and bioremediation potential,L.sphaericus DH-B01 can be specifically combined in future research and applied to complex environments under extreme conditions Microbial remediation of the environment also provides special bacterial vectors for the construction of recombinant bacteria.