The Construction Of Gene Library Of Ammonia Oxidase And The Screening Of High Activity Ammonia-oxidizing Engineered Bacteria

With the rapid development of modern industry,agriculture and aquaculture,environmental pollution is becoming increasingly serious.According to the China State of the Environment Bulletin 2018,nitrogen pollution is the number one polluter.The disturbance of human activities on the nitrogen cycle has greatly accelerated the changes in the earth's ecological environment,causing serious adverse effects such as nitrogen cycle imbalances,intensified nitrogen pollution,and increased greenhouse gas emissions.It is urgent to resolve the nitrogen pollution problem in a timely and effective manner.At present,microorganisms have played an important role in the control of nitrogen pollution.Ammonia oxidation is an important part of nitrogen pollution control.The ammonia oxidizing bacteria currently researched and applied are mainly isolated and purified strains from the environment,or clone the ammonia of a single microorganism.An oxidase gene(amoA)constructs its engineered bacteria.In order to further improve the screening efficiency of ammonia-oxidizing bacteria(AOB),an amoA gene library of autotrophic ammonia-oxidizing bacteria from environmental samples was constructed in this paper,and the high activity AmoA was obtained through the screening of recombinant activity.Furthermore,a directed evolution technology was used to construct an amoA mutation library The highly active AmoA recombinant was screened,and the amoA and the hydroxylamine oxidase gene(hao)were co-expressed in the host strain in order to obtain a highly active AOB.The main results of this study are as follow:(1)Establishment of the initial screening system of ammonia monoooxygenase(AmoA): A method for in situ determination of AmoA activity was established using Pseudomonas(P.)Aeruginosa ATCC9027 amoA to construct engineering bacteria E.coli BL21(amoA).The recombinant strain was selected and cultured in 96-well plates for 12 h.This method has good repeatability(RSD is 4.8%)and is easy to realize highthroughput screening,which lays a foundation for gene library method to screen highly active AmoA recombinants.(2)Construction of amo A library and selection of recombinants: A degenerationdegeneration-primer was designed based on the full-length sequence of amoA of 34 strains of autotrophic ammonia-oxidizing bacteria of 3 genera and 15 species,and amoA gene was amplified to construct a gene library using the genomic DNA of environmental samples as a template.After primary screening and secondary screening,4 highly active recombinant vectors were obtained,among which UA146 was the most active.E.coli BL21 was used as host bacteria to co-express the amoA gene and hao gene of the screened highly active recombinants,and a highly active ammox recombinant strain UA146-H10 was selected.The strain was used in 100 mg/L ammonia nitrogen determination system for 60 h,and the removal rate of ammonia nitrogen was 75.8%,and the accumulation of nitrate nitrogen was significantly detected,up to 0.11 mg/L.(3)Construction of amoA mutant library and screening of highly active amoA:the use of recombinant bacteria UA146 amoA,through error-prone PCR directed evolution technique,build amoA gene mutant library,select highly active ammonia single oxygenase amoA,again will highly active amoA cloning and hao also transferred into E.coli BL21,1 strain screened strongest recombinant bacteria,ammonia oxidation capacity Numbers for Er-UA1090-H19,the strain in ammonia nitrogen concentration of 100 mg/L,36 h after reaction of ammonia nitrogen removal rate is maximum,at 97.95%,1.28 times before the removal rate of mutation.In the presence of ethanol,sodium acetate,lactose,glucose and other small molecular organic carbon sources,the mutant strain could effectively remove ammonia nitrogen and accumulate a small amount of nitrate.When glucose was used as carbon source,the removal rate of ammonia nitrogen(100 mg/L)was the highest,up to 96.1%.In the presence of organic nitrogen sources such as urea or peptone with high concentration,the strain had a good effect on ammonia nitrogen removal,while high concentration of yeast extract had an inhibitory effect on ammonia nitrogen removal.In conclusion,this study established a screening system for the high-throughput activity of ammonia monooxygenase.By constructing gene libraries and using directional evolution technology,a strain of Engineering E.coli Er-UA1090-H19 and amoA genes with high ammonia-oxidizing ability was screened out.This study provided reference for the further selection of highly active denitrifying bacteria or genes.