The Primary Studies On A Key Enzyme-7-β-Xylosyltaxanes Glycoside Hydrolases In Taxol Synthesis From Cockroach Intestinal Bacteria

Taxol is a diterpenoid anticancer compound that has been widely used in clinical practice for the treatment of advanced ovarian cancer,breast cancer,and non-small cell lung cancer.Taxol is derived from the bark of an endangered species-Taxus species,and its yield is extremely limited,which results in a high price for taxol.Semisynthesis is currently the main route for the industrial production of paclitaxel,which is produced by a 10-step chemical reaction using 10-deacetylbaccatin III（10-DAB）as a precursor.7-β-xylosyl-10-deacetylpaclitaxel（7-XDT）and other 7-xylose taxanes are the parent nucleus structural analogues of paclitaxel,which are mainly found in the roots,stems,and leaves of yew and contain more than five times the amount of taxol,but are not utilized.7-β-xylosyltaxanes glycoside hydrolases（7-β-xyl）is aβ-xylosidase that converts 7-XDT to 10-deacetylpaclitaxel（10-deacetylpaclitaxel,10-DAT）and 10-DAB.Although 10-DAT is not currently available for the semisynthesis of paclitaxel,it can be used for the direct biosynthesis of paclitaxel via 10-deacetylbaccatin III-10-β-O-acetyltransferase（DBAT）,suggesting that 7-β-xyl is a key enzyme in paclitaxel synthesis.Currently,there are few sources of 7-β-xyl.Therefore,the research on new 7-β-xyl resources has practical significance.Cockroach are omnivorous and have diverse intestinal flora,which can produce xylanase.However,there are no reports of 7-β-xyl being found in the cockroach intestinal bacteria.Therefore,this study intends to investigate the 7-β-xyl-producing ability of 178 strains of cockroach intestinal bacterium.On this basis,strain WA11-1-1 was used as the research object,the enzymatic properties of7-β-xyl were preliminarily analyzed,and the conditions for producing7-β-xyl enzyme were optimized.The whole genome sequencing was used to mine the 7-β-xyl gene,and molecular cloning,expression and activity detection of the gene were carried out.The main research contents and results of this paper are listed below:1.Screening of 7-β-xyl producing strains from cockroach gutUsing the Congo red transparent circle method and fluorescent color method,55 of the 178 strains of cockroach intestinal bacterium were found to have xylanase producing ability.On the basis of this,the thin layer chromatography and high performance liquid chromatography was used to further find that 8 strains of Streptomyces and 2 strains of Cupriavidus can produce 7-β-xyl,and Streptomyces WA11-1-1 had better activity.Therefore,strain WA11-1-1 was selected as the follow-up research objects.2.Identification of strain WA11-1-1 and determination of the efficiency of taxol intermediate 10-DAT catalysis synthesized by 7-β-xylThe Strain WA11-1-1 was identified as Streptomyces rochei by bacterial morphology,physiological and biochemical experiments,combined with 16S r RNA gene sequence analysis.The 16S r RNA gene sequence was uploaded to Gen Bank database to obtain the entry number OK047707.The crude extracts of fermentation broth from strain WA11-1-1 could convert 7-XDT into 10-DAT and 10-DAB.The results of high performance liquid chromatography showed that the conversion rate of 10-DAT was 23.48±2.30%.The optimum temperature of the7-β-xyl crude enzyme solution derived from strain WA11-1-1 was 35°C and the optimum p H was 6.It had better stability in temperature of20℃～30℃and p H close to 7.Na⁺、K⁺、Mg²⁺had a promotive effect on the enzyme activity,with Na⁺having the most significant effect,while Ca²⁺,Fe³⁺,and Zn²⁺had an inhibitory effect.3.Optimization of 7-β-xyl-producing fermentation conditions for strain WA11-1-1In order to further improve the efficiency of strain WA11-1-1 in converting 7-XDT to 10-DAT,the 7-β-xyl-producing fermentation by strain WA11-1-1 were optimized by single factor experiment and orthogonal test.The optimal combination of fermentation conditions was obtained as follows:xylan concentration 0.8%,fermentation time 4 d,fermentation temperature 28℃,and liquid volume 250 m L.Under these conditions,the conversion of 10-DAT reached 40.72±2.71%,which was1.73 times higher than that before optimization.4.Whole genome sequencing of strain WA11-1-1 and cloning and expression of 7-β-xyl geneAfter the whole genome sequencing of strain WA11-1-1,data analysis revealed that it has 16β-xylosidase genes.Combining the literature research and the results of the isozyme analysis of 7-β-xyl crude enzyme solution,it was speculated that 5 genes could encode 7-β-xyl（GE65,GE77,GE79,GE84 and GE87）.Prokaryotic and eukaryotic expression of the above genes were carried out respectively,and the correct expression of 5 proteins in both prokaryotic and eukaryotic expression systems was verified by induced expression and immunofluorescence staining.However,due to the limitation of research time,the activity assay of recombinant protein and recombinant yeast hydrolyzing 7-XDT has not been performed.In summary,a total of 10 7-β-xyl-producing strains were isolated from 178 strains of cockroach intestinal bacterium,which were 8 strains of Streptomyces and 2 strains of Cupriavidus.Among them,the conversion rate of 10-DAT by Streptomyces rochei WA11-1-1 reached23.48±2.30%,the optimum temperature of 7-β-xyl was 35°C and the optimum p H was 6.Under optimal fermentation conditions（xylan concentration 0.8%,fermentation time 4 d,fermentation temperature 28℃,and liquid volume 250 m L）,the conversion of 10-DAT reached40.72±2.71%,which was 1.73 times higher than that before optimization.Whole genome sequencing of strain WA11-1-1 was performed,the five potential 7-β-xyl genes were mined,and successfully cloned and expressed.