Construction Of Multifunctional Pesticides Degrading Genetically Engineered Microorganism And Its Risk Assessment For Environmental Release

The intensive use of chemical pesticides has caused environmental problems. Bacterialenzymatic detoxification of pesticides has become the focus of many studies because it iseconomical and effective. Sphingomonas species are characterized by versatile capabilitiesof environmental pollutants degradation. For this reason, there is an increasing interest inusing them in bioremediation of environmental contaminants. Because of the various kindsof pesticide-residues in environment, multifunctional genetically engineeredmicroorganisms (GEMs) are needed to clean up these pollutants. Construction GEMs usingbroad-host-range plasmids is restricted for its less stability, self-transferity and thepossibility of bringing resistant gene into recipients. Hence, integrative systems are neededto finally get self-cloned strains devoid of any antibiotic markers. Although integrationthrough recombination mediated by an insertion sequence (IS) can success, integration viahomologous recombination may have many advantages. On the other hand, due to thecomplexity and uncertainty of the GEM cells, the biosafety of the release of the GEM cellsis mostly concerned. Risk assessment is a key task in developing GEMs intended forrelease into the environment.Ribosomal RNA (rDNA) genes of bacteria have high degree of sequence conservationand have some obvious advantages when used as homologous recombination target site forinsertion exogenous genes. In this study, we described for the first time to userRNA-encoding region of Sphingomonas species as target site for integration of exogenousmethyl parathion hydroalse gene (mpd) to construct various multifunctionalpesticides-degrading GEMs. Homologous recombination vectors with the recipient's 16SrRNA (rDNA) as homologous recombination directing sequence (HRDS) and sacB gene asdouble crossover recombinants counter selectable marker were firstly constructed. The mpdgene was inserted into the16S rDNA site of the carbofuran degrading strain Sphingomonasagrestis CDS-1 by homologous recombination single crossover in the level of about 3.7×10^-7～6.8×10^-7. Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed.The homologous recombination events were confirmed by PCR and Southern blot methods.The obtained GEMs were genetically stable and could degrade methyl parathion andcarbofuran simultaneously. The insertion of mpd gene into one rrn site did not have anysignificant effect on recipient's physiological and original degrading characteristics. Themethyl parathion hydrolase (MPH) was expressed at a relatively high level in therecombinants and the recombinant MPH specific activities in cell lysate were higher thanthat of original bacterium (DLL-1) in every growth phase tested. The highest recombinantMPH specific activity was 6.22 mu/μg. The constructed multifunctional pesticidesdegrading GEMs are promising for developing bioremediation strategies for thedecontamination of pesticides polluted soils.To investigate the key parameters controlling the exogenous mpd targeting frequencyat the rrn site of Sphingomonas species, targeting vectors with different homology lengthsand recipient target DNA with different homology identities were used to investigate theparameters. The highest targeting frequency was obtained when homologous sequenceswere 493 and 778bp on each flank. A decrease in the length of homology on both flanksgreatly reduced the targeting frequency. With homologous sequences of 154 and 130bp oneach flank, the targeting frequency was diminished by 76.3-fold compared withhomologous sequences of 317 and 538bp. The minimal size for normal homologousrecombination was＞100bp. Homologous recombination could succeed even if there were3-4% mismatches. However, there was a significant decrease in the targeting frequencywith increasing sequence divergence. In instances where the genomic target DNAcontained 3-4% mismatches with the donor DNA, integration frequency was reduced atleast 3.3-to 35.7-fold relative to recombination between identical sequences. The Redrecombination system could increase the targeting frequency to some extent. Utilization ofthe Red system increased the targeting frequency of pWSMKR-U to 5.1±0.59×10^-10compared with that of the pWSMK-U vector (no exconjugants were found).In order to improve the exprssion level of MPH, one to four copies of mpd genes wereintegrated into the rrn sites of P. putida KT2440.The growth curves of recombinants withone to four copies of rrn disrupted were similar with their original bacterium KT2440. Thehighest specfic MPH activity of KT2440-4mpd was 12.9 mU/μg protein, which was3.2-fold of that of DLL-1.Solid carriers were screened as the best carrier for the conservation of GEM Sphingomonas CDS-mpd. Based on the shelf life and the activities of GEM cells, peat andvermiculite were selected as the best carriers among solid carriers tested. With the peat asthe carrier, the shelf life was extended from 20d to 120d compared with liquid solution.Liquid solution was not suitable for the production of Sphingomonas inoculant since thehigh death ratio. Add 5% molasses, 10% wheat bran or 10% minimal salt medium canonly slightly improve the shelf life of solid bacterium. The optimal inoculation amount ofGEM cells was 10⁹CFU/g peat. After conservation with peat for 120d, the inoculation of7.1×10⁷CFU/g dry soil of GEM cells showed good abilities to completely degrade50mg/kg MP and 25 mg/kg carbofuran in sterilized and non-sterlized soil in pot trials. Insterilized and non-sterlized soil, the inoculated GEM cells could not be detected in 15d and20d respectively.In the risk assessment of GEM Sphingomonas sp. CDS-mpd for environmental release,1.01×10⁷CFU/g dry soil of GEM cells inoculated into field could degrade both10.71mg/kg MP and 1.29mg/kg carbofuran in 30d. Based on the method of plate counting,GEM cells declined quickly with time. The method of monitoring of the GEM cells wasimproved by the molecular method MPN-PCR, the GEM cell number in 4d, 15d detectedby MPN-PCR was 2.15±0.98×10⁶ CFU/g dry soil and 3.70±4.66×10⁴ CFU/g dry soil,respectively. After 30d, the GEM cells could not be detected anywhere, indicating thatGEM cells would not be the permanence populations in the soil, and would not escapefrom the fields for release. The GEM cells in soil could even be specifictly detected by themethod of Fluorescent in situ Hybridization (FISH). The effect of release of GEM cells onthe community structure and diversity of soil microorganisms was also studied by platecounting of culturable bacteria, fugi and actinomycete in soil as well as the PCR-DGGEmethod of bacteria 16S rDNA V3 region. The use of pesticides changed the communitystructure based on the analysis of PCR-DGGE profiles, however, the community structurewas recoveried in 60d. The inoculation of GEM cells also affected the structure of soilmicroorganisms in prophase. In 4d, 11d and 30d, the similarities of DGGE profiles of GEMcells released soil were 49.57%, 38.3% and 83.3%, respectively, compared with controls. In60d, DGGE profiles of all treats had high similarities, indicating that the communitystructures of soil microorganisms were similar, and the release of the GEM cells did notaffect the community and structure in the long term.