Efficient Production Of 19,20-Epoxycytochalasin Q And Pyrroloquinoline Quinone With Marine Microorganisms

19,20-Epoxycytochalasin Q (B5A) and pyrroloquinoline quinone (PQQ) were bioactive natural products both could be isolated from marine microorganisms. B5A derived from fungus Xylaria was a cytochalasan possessing a variety of biological activities, such as inhibiting HIV virus, anti-malaria and so on; while PQQ was a specific cofactor produced by Gram-negative bacteria, which not only could stimulate the cell growth, but also had anti-aging activity. The main contents and conclusions in this program were listed as follows:Firstly, systematic studies were carried out to produce B5A with marine fungus Xylaria sp. sofll. Through the morphological characteristics and the 18S rDNA sequence analysis, the B5A producing strain Xylaria sp. sofll was further identified as the candidate of the genus Xylaria. Traditional analytical methods including HPLC, thin layer chromatography and LC-MS were applied to detect and analyze B5A efficiently. Using Sabouraud medium as the original medium, single factor experiments were executed. The results showed that disaccharides and polysaccharides were prefered carbon sources, while fish meal and corn meal were the most suitable nitrogen sources. Three important factors including sucrose, fishmeal and medium volume were screened out by Plackett-Burman experiments. The optimum producing medium was finally formulized by surface analysis method. The final B5A yield was reached 443.1 mg/L, approximately 4.4-fold higher than that in the original medium.The production of B5A was scaled up in a 10 L fermenter by batch and fed-batch fermentation. When the dissolved oxygen was controlled more than 40%, and the pH was set at 6.5, the titer of B5A reached 528 mg/L with the feeding of sucrose and fish meal, which was further increased for about 19.2% comparing to that of shaking flask experiments.The subsequent separation and purification route of B5A was also established, the major steps including the extraction of B5A from Xylaria sp. sofl 1 mycelia, the isolation of B5A using silica gel column chromatography and C18 semi-preparative chromatography. After the optimization of isolation procedure, more than 98% pure B5A was achieved with the recovery rate of 76%.Secondly, systematic investigations were done about how to produce PQQ efficiently with marine strain Methylophaga sp. ZJU414. A PQQ producing strain was screened out from the mud of the South China Sea, which was nominated as Methylophaga sp. ZJU414. Through the shaking flask experiments, we found that the carbon source has a huge impact on the biosynthesis of PQQ, while DO and nitrogen source only exert minor effects on the production of PQQ. Methylophaga sp. ZJU414 was fermentated in a 3 L fermentor using methanol as the substrate.The PQQ titer was improved up to 12.9 mg/L with the feeding of methanol.A separation route using one-step anion ion-exchange resin adsorption was applied for PQQ purification, which was characterized by several aspects:1) Among the nine tested resins, LKA98 displayed the best adsorption and desorption performance; 2) The adsorption equilibrium data fitted the Langmuir equation quite well and the adsorption kinetic data under different temperature followed the pseudo-second order model; 3) The effects of PQQ initial concentration, feed flow rate and bed heights on the adsorption efficiency were evaluated, and the data well fitted the Thomas, Yoon-Nelson and Adams-Bohart models. After the optimization of ion exchange conditions, the recovery yield of PQQ was reached to 87.3%, and the purity can be increased to 93.8%; 4) The crystallization process was also improved, and the crystallization yield and purity were 94.8% and 97.5%, respectively.Through our work, the production and purification of two important marine natural products B5A and PQQ were greatly improved, which shall facilitate their further biosynthetically and biotechnological studies at both theoretical and practical levels.