Analysis Of Alkane Degradation Related Genes In An Acinetobacter Sp.KJ-1

With the rapid development of the social economy,the demand for petroleum products such as diesel and petrol is increasing,and environmental pollution is becoming more and more serious.Good progress has been made in screening strains including Acinetobacter spp.,which can utilize diesel as a carbon and energy source.However,there have been no reports on the degradation of diesel by Acinetobacter vivianii.In this study,the strain KJ-1,which isolated because of the ability to degrade petroleum hydrocarbon efficiently,is used as research object.Its growth and diesel degradation conditions were analyzed,and the functional genes related to alkane degradation were explored.It provides important information for understanding the mechanism of diesel degradation at the molecular level.The major results obtained in this study are the following:（1）The strain KJ-1 isolated in the laboratory was re-identified and determined as Acinetobacter vivianii.Through single factor experiment,it is found that A.vivianii KJ-1 was rapidly growing on basal salt medium with diesel as the sole carbon source.Over40%degradation of diesel was obtained at 14 d at an initial diesel concentration of12418.5 mg/L.The optimal conditions for growth and diesel degradation for A.vivianii KJ-1 were confirmed to be 35°C and p H 7.5.The concentrations of NH₄NO₃ had no significant effect on the growth of A.vivianii KJ-1 and the degradation of diesel.（2）The whole genome sequence of A.vivianii KJ-1 was analyzed.The genome of this strain only contains a 3927757 bp long chromosome,with a GC content of 41.5%and a total of 3740 coding sequences.According to the annotation information,more than50 genes related to alkane degradation were excavated,including two alkane 1-monooxygenase genes alk B1,a long-chain alkane monooxygenase gene lad A,a flavin-binding monooxygenase gene alm A and some intermediate hydroxylase encoding genes.By analyzing the fatty acid metabolism pathway,it was found that two alk B1 with another25 genes in the A.vivianii KJ-1 genome were involved in the fatty acid metabolism pathway.（3）By whole genome comparison with three strains of other species of Acinetobacter,it was found that the genome size of four Acinetobacter spp.was between 3.93-4.15 Mb,the GC content ranged from 38.72%to 41.50%,and the number of CDSs was concentrated between 3648-3879.The pan-genome and core-genome of the four strains contained 5998 genes and 2203 genes,respectively.The genes related to alkane degradation in the 4 genomes were excavated,and alk B1＿1,alk B1＿2,lad A,and alm A genes were found in the genome of the four strains of Acinetobacter.By comparing all the alkane 1-monooxygenase sequences of the 4 strains with the Alk B1 sequences of other strains downloaded from NCBI,it was found that the two Alk B1 in the A.vivianii KJ-1had the highest homology with the two Alk B proteins of Acinetobacter sp.YK3.The alkane 1-monooxygenases of the above four Acinetobacter strains contained six transmembrane helix regions,one HYG-motif and three Hist motifs,and the conserved motifs were located inside the cells.（4）The strain KJ-1 was cultured with diesel oil（D）and n-hexadecane（C）as the sole carbon source,respectively,and transcriptome analysis was performed,sodium acetate（Y）was used as the substrate for the control.The results showed that there were 1009 and1275 differentially expressed genes in the transcripts of n-hexadecane treatment（C＿VS＿Y）and diesel treatment（D＿VS＿Y）,respectively.Most of them are concentrated in molecular function,followed by the cell component,and the number of genes enriched in the biological process category was the least.The genes involved in alkane hydroxylation were expressed in all treatments.Gene alk B1＿1 and alk B1＿2 were significantly up-regulated in groups C＿VS＿Y and D＿VS＿Y.The expression difference of lad A in groups C＿VS＿Y and D＿VS＿Y was not significant and gene alm A was significantly up-regulated in the D＿VS＿Y group,the expression difference in the C＿VS＿Y group was not significant.Additionally,multiple genes encoding alcohol dehydrogenase and aldehyde dehydrogenase showed significant differences in expression in transcripts treated with the same carbon source.（5）Based on the results of genomic and transcriptomic data analysis,the alk B1＿1and alk B1＿2 gene fragments were obtained by PCR amplification.The recombinant plasmid p SUMO1-alk B1＿1 was obtained by ligation with the expression vector p SUMO1,and transformed into E.coli BL21（DE3）to obtain recombinant strain E.coli BL-alk B1＿1.E.coli BL-alk B1＿1 was induced to be expressed and the protein was purified,testing the enzymatic activity of the recombinant protein and finding that the optimum conditions for the recombinant protein were 35°C and p H 7.5.Another degradation experiment with E.coli BL-alk B1＿1 proved that recombinant bacteria play a role in the degradation of alkanes.