| Polyketide synthases (PKS) catalyze certain substrates to producestructurally different polyketides and their vast analogues. Some of themhave important pharmaceutical activities. In biotechnological drug research,it is important to isolate and express new PKSs which produce bioactivecompound. Because of its structure characteristics, PKSs have become idealmaterials for research and development of combinatorial biosynthetic drugs.In this study we isolatedthe genome DNA of Bacillus amyloliquefaciensF201721, and we also construct its bacterial artificial chromosome library,and then we designed degenerate primers to amplify and stitch the codingsequence of PKS, we finally got a sequence about531835base pair. Weanalyzed this gene through the databaseandbioinformaticssoftware： Itdividing into9modules, encodes41enzymes which catalyzed the synthesisof polyketides.Compared with the PKS extracted from one of bacillus amyloliquefacienswhich derived from land, the gene PKS’s promoter regionof F201721deleteabout25base pairs nucleotides. Predicted by professional software, these25base pairs are connected with the activity of PKS or they can determine theproducts. Then, we identified these25base pairs can be attached by specific protein using EMSA, this protein is about35KD. Combined withbacteriostatic ring experiment, we thought the protein can attached to thissequence only afterLogarithmic growth phase, which means the protein caninhibits the expression of PKS. Comparison between these two stains, atLogarithmic growth phase, this protein can attached to the sequence in both,because the diameter of bacteriostatic ring are both about2cm, but whenreached to the late stage of logarithmic growth phase, in stain FZB42, thisprotein can still attached to this sequence, but it’s not in stain F202721, ourexperiment also shows that the size of bacteriostatic ring of FZB42is obviousbigger than F201721, the result shows that this protein can down regulationthe expression of PKS. At the late state of logarithmic growth phase, thisspecific protein attached to the DNA fragment to inhibit the expression ofPKS, which leads to the PKS expressed more in FZB42compared withF201721. All this evidences indicates that this25base pairs sequence cancontrol the expression of PKS,and also the regulation mechanism of PKSexpression is different between these two stains.Part I: Construction of metagenomic DNA library of stain F201721This bacteria stain F201721is conserved in our lab. We extracted the genomeDNA of this stain using the method of low melting point agarose, finally wegot a genome about200kb, then we used BamH1to digest it about10min toget DNA fragments whose length are about100bp, after purified the DNA,we construct BAC library using Epicentre kit. We got the positive clonescontaining appropriate size of fragment through blue and white, then by sequencing this fragment though BAC ending sequence, we got the positiveclone which contains the coding sequence of PKS. Finally, we find out thefragment length is150kb, it is inconvenient for the experimental operation.Part II: Analyze the sequence and function of obtained PKS codingsequenceAccording the PKS coding sequence of FZB42, using BLAST we find outthe conserved domains of PKS. According to these conserved regions, wedesigned14pairs of primers, the length between primers is6kb-10kb, theannealing temperature is between65℃-73℃, the annealing temperaturedifference between upstreamprimerand reverse primer is less than5℃, thelength of primers are25bp. We use the extracted DNA using Axygen bacteriagenome extraction kit as models and amplified sequence using Lataq enzymeto PCR. The production of PCR issequenced by inverchgen, at last we get thePKS’ whole coding sequence mln, it’s length is531895bp, analyzed it’ssequence and function using the database of NCBI, we get the codingsequence of stain F201721’s PKS. It is divided into9modules, contains41enzymes genes related to the synthesis of polyketides. These sequences arehomology with the PKS sequence of FZB42: the lowest similarity ratio is97%, the highest can reach to100%. The amino acids sequence highestsimilarity ratio is99%,and the lowest is96%. When analyzed the positivefunctions of these sequence, we find out that compared with the PKS codingsequence of stain derived from land, the stain derived from marine’ is deleted25bp at the promoter region. We used different kinds of promoter predict software to analyze this deleted region, the results all show that this region isessential for the regulation of expression of PKS.Part III: Verify the regulation difference of PKS’s expression caused bythe sequence difference between the two bacteria stainsWe find that the deleted sequence is located at promoter region usingpromoter predict software analyze the difference, furthermore in the stains ofbacillus subtilis, the secondary metabolites’ produce is controlled by differentσfactors。We find out that there is protein can attached to the deletion regionat stabilization period. When the specific protein attached to the sequence thebacteriostatic ring is become bigger, it indicates that this protein participatesin the expression of Macrolactins, it caused the production of Macrolactinsgrowth at stabilization period. It indicates that the deletion sequenceparticipates in the regulation of Macrolactins’ expression. It is a functionalgene. It also shows that this coding sequences can catalyzed the synthesis ofMacrolactins indeed. |
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