| Synthetic biology which was first appeared in a review of the medical journal"The Lancet" in1911goes into a booming period since2006. It brings a lot of directbenefits through the transformation of existing organisms, helps to solve a lot ofimportant issues that human faced, so it brings the dawn in energy, health and theenvironment. Bioenergy, microbial synthetic drugs and renewable chemicals aremore promising areas currently. We hope to design, restructure and optimize thebiosynthetic pathway and regulate the metabolism and the associated traits ofmicroorganisms using the knowledge of synthetic biology. Then the targetmetabolites were earned effectively. Though synthetic biology is developing rapidly,some assident issues have emerged. While it has just started in this field, a deal ofcore technology is far from satisfied the needs of synthetic biology, which restrictsthe development of synthetic biology in China.Natural product drugs, as secondary metabolites of microorganisms play animportant role in health care and anti-cancer activity. The production was attainedfrom natural extracts or semi-synthetic chemical which was the main production waybefore. It provides a new mode of production to scientists. If using microbialfermentation combining with biosynthetic pathway, the object production wasincreased through optimizing ermentation conditions and medium composition.Maytansine, which is a macrolide antibiotic, was first isolated from the plantMaytenus. It has a very good inhibitory activity to a variety of tumor cells.Andansamitocin was a maytansinoid antibiotics which isolated from microbes-Actinosynnema pretiosum ssp.auranticum ATCC31565. It differs from Maytansine ingroups at C-3. Ansamitocin is usually used to produce maytansine as a precursor compound because the biosynthetic pathway of maytansine is unclear currently.Therefore, the aim of this paper is earning maytansine with synthetic biologytechniques.We will construct the engineered strains by knocking out and alternativemethod. Then we will prepare the maytansine derivative through the microbialfermentation method and achieve the transition from ansamitocin to maytansine, andprovide the foundation for the large-scale production of maytansine. The same time,it will provide the foundation in natural products drug research. The main contentsare summarized as follows:1、The genomic DNA was extracted from Yunnan Maytenus fresh leaves using anon-cylindrical centrifugal extraction method. Then the gene clusters that weredivided into multiple fragments were amplified using the genomic DNA as atemplate. The amplified genes were sequenced in order to find the mechanismsof maytansine synthesis. It is showed that the amplified sequence was similar tothe ATCC31565and one of the bases was mutation. It tells us it may contain thekey gene in the genome of Maytenus.2、We obtained14.26Gb Clean Data by High-throughput sequencing technology toYunnan Maytenus blades.Q30base percentage were at88.62percent andabove.A total of58,574pieces Unigene were obtained.The average length ofUnigene is700.75bp.N50length is1,275bp.The sequencing results eventuallywon the29,977Unigene of annotation information.The length of more than300bases in several Unigene has21,667, the length greater than the1,000baseshas10,290.In the COG database, Unigene number was9,616. In GO database,Unigene number was22,886.In KEGG database, Unigene number was7,415.InSwiss-Prot database, Unigene number was18,661.In NR database, Unigenenumber was29,854.The expressed genes were classified.In the secondarymetabolic pathway genes of COG, the results by qRT-PCR validation showed itwas consistent with the transcriptome.3、We have analyzed the biosynthetic gene cluster of ansamitocin and found that ithad two clusters, the full-length of cluster Ⅰ83kb, cluster Ⅱ12kb, twoclusters were separated from30kb, the GC content was reached to70%. The biosynthesis was formed ansamitocin precursors proansamitocin first withAHBA as a starting unit and polyketide synthase I as catalytic enzyme.Followed by a series of post-modification reaction, active ansamitocin P-3wasformed. Wherein, asm19, as3-O-acyltransferase gene, which had acyltransferasefunction, esterified for the C-3side chain. Asm25, N-glycosyltransferase,catalyzed C-4hydroxyl group to carbamylation. We will knockout asm25toreduce offshoot reaction product and then knockout asm19to detect itsfermentation product. Ultimately, the blocking expression vector of asm19andasm25have been constructed, which can be transfered to Actinosynnemapretiosum ssp.auranticum by recombination. And the asm19and asm25deletionmutants strain will be constructed.4、We have found three homologous genes of asm19by searching the literature:AcyA, MdmB and Rif20. Therefore, the expression vectors of asm19homologous genes were constructed and the asm19will be replaced by itshomologous genes. Then the fermentation products and activities will bedetected.5、The detection method of the actinomycetes fermentation broth was established.There are: high-performance liquid chromatography, thin layer chromatographyand mass spectrometry detection. First we have detected the AP-3offermentation broth using the three methods. Then the fermentation broth wasextracted with ethyl acetate and was purified using XAD-16macroporous resin,and finally ethanol was distillation. The concentration of AP-3was improved byHPLC.6、We amplified the regulatory genes and unknown genes of ansamitocin syntheticgene cluster by ordinary PCR and picked the single band genes.Then the singleband genes can be amplified by real-time quantitative PCR.It is showed thatasm2and asm36expression is positive correlation to AP-3yield, it may play apositive role in the regulation ansamitocin synthesis.7、We use UV mutagenesis method for screening high-yield strains of ansamitocin.First single spore suspension was irradiated by UV mutagenesis, and ultimately the exposure time was determined to50s. Then the yield of the mutant strainwhich is higher than the original strain was detected by HPLC after fermentationof mutagenic strains.8、We extracted total protein of Maytenus leaf through direct extraction andimproved acetone precipitation. PVPP was added which can reduce the influenceof polysaccharide and polyphenols substance and protease inhibitor was addedwhich can prevent protein degradation during the extraction process. Followedthe protein after ultrafiltration interacted with AP-3with a certain percentage wasdetermined by HPLC and TLC. It is known that it may occur some kind ofinteraction between the protein and AP-3.In summary, we have mastered the method of knockout and replacement inactinomycetes genome by synthetic biology, and achieve ansamitocin detection,have a preliminary understanding of the biosynthetic of maytansine. It willprovide the foundation for natural products drug research by synthetic biology. |
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