Mining Characterization Of Stable Genetic Elements And Research On Biodegradation Of Algal Toxins

The eutrophication of water makes cyanobacteria bloom frequently in lakes.The cyanobacteria produce algal toxin when lytic and dying,which causes secondary pollution.Among the cyanobacterial toxins,microcystins(MCs)is widely distributed and more toxic than the others.Moreover,the LR subtype(MC-LR)is the most toxic in all microcystins.Firstly,degradation of MC-LR by using physical or chemical strategies is costly and environmental unfriendly.Secondly,screening wildtype strains which can degrade MC-LR from water takes a long time.Lastly,construction of engineered strains is a promising strategy to solve MC-LR pollution but resistant bacteria need antibiotics to ensure the stable of strains.In this study,we constructed a stable genetic element substituting for antibiotics and a genetically engineered strain to degrade MC-LR.The stable genetic element was constructed basing on Maz E-Maz F,which is one of the Escherichia coli toxin-antitoxin(TA)systems and also regulates programmed cell apoptosis.The strains died when Maz E was moved and unable to form complexes with Maz F.By studying the inhibition degree of maz E gene deletion on cell growth and verifying the effect of the expression intensity of maz E gene on the product yield and passage yield stability by exploiting gallic acid biosynthesis,we elucidated the regulatory mechanism of Maz E-Maz F and confirmed that the adaptation of Maz E-Maz F expression intensity between plasmid and genome was the key factor affecting the stability of plasmid passage.Based on this,a stable genetically screening element was constructed and its efficiency was comparable to that of antibiotic screening after 60 passages.Then,we screened three MC-LR degradation genes mlr A 、 mlr B and mlr C from Sphingomonas ACM3962 by literature reading and characterized the catalytic capacity of these three proteins.Finally,we used Mlr A,Mlr B,Mlr C and the transporter protein Mlr D to construct MC-LR degradation pathway in E.coli.The engineered E.coli could degrade MC-LR to the basically non-toxic Adda,and the degradation proficiency was9 times higher than the reported wild strain ACM3962.In conclusion,a stable genetic element was mined and charactered to avoid the addition of antibiotics and its proficiency was compatible with that of antibiotic screening.Moreover,a genetically engineered strain was constructed to degrade highly toxic MC-LR to the basically non-toxic Adda.The construction of genetic element and the engineered strain laid a theoretical foundation for the degradation of microcystins in water.