Accumulation Of One Key Intermediate Cumic Acid From The Biodegradation Of ?-Pinene By Pseudomonas Veronii

?-Pinene is a typical Volatile Organic Compounds,it is harmful for human body and environment.Microbial technique is a kind of useful methods for?-pinene degradation.The key genes about environmental pollutant for bacteria could be identified through the metabolic pathway reconstruction under pollutants.The key genes could be malfunction and the metabolic pathway could be remaked through ultraviolet mutagenesis and transposon mutagenesis,Which could provide the basis for further research on resource reuse about pollutants and metabolism for bacteria.An approach combining traditional biological methods and modern molecular biological techniques was used for investigating the stress response of strain Pseudomonas veronii ZW upon the?-pinene challenge,which was screened by our laboratory years ago.At the same time,4-isopropylbenzoic acid?KMS?,as a by-product,was accumulation.The KMS was detected by Gas Chromatography-Mass Spectrometer?GC-MS?as one of the by-products for?-pinene degradation strain ZW,which had improved the metabolic pathway of?-pinene degradation by strain ZW:?-pinene was converted to p-cymene,then the p-cymene was converted to p-cumic alcohol and p-cumic alcohol was converted to p-isopropylbenzaldehyde,p-isopropylbenzaldehyde was converted to KMS.At last,the KMS would form low molecular weight organic acids as2-methyl-1,4-butanedioic acid,oxalic acid,acetic acid etc.,then enter into TAC cycle.Strain ZW-3 was obtained by ultraviolet mutagenesis,which had lost the ampicillin antibiotic resistance ability,its morphology and the metabolic pathway about?-pinene basically consistent with the normal strain ZW,both of them could degrade?-pinene?100 mg/L?completely in 36 h.That could provide a receptor strain for transposon mutagenesis technology.We have got a large number of mutants through the combining strain ZW-3 and plasmid pSC123 by Tn5 mutagenesis,mutant strain ZW-A was one of them,which could utilize?-pinene only to KMS,and do not to carry on.The degradation rate of?-pinene was 80%and the accumulation rate of KMS was 20%.and the mutant gene orfA was cloned and analyzed from mutant ZW-A by SEFA-PCR,it was high homology of rulAB,which was reported having the ability to related aromatic compounds degradation.Then,the orfA could have the same function as rulAB.We have found a kind of inorganic salt by optimization of inorganic salt,which could sensitive to?-pinene degradation.The optimal value concentration of this inorganic salt was picked up by changing the concentration gradient,which could make the accumulation rate maximum,the inorganic salt was MgSO₄ and the optimal value concentration was 0.307 mg/L.The degradation rate of?-pinene was 85%and the accumulation rate of KMS was raised from 20%to 24.5%for mutant strain ZW-A under those conditions.