Produciton Of THAILANDEPSIN A With Continuous Absorption To Hp-20 Resin By Burkholderia Thailandensis E264

Burkholderia thailandensis E264 was isolated from the rice field in Thailand. It can produce an antitumor drug of histone deacetylase inhibitors (HDACi) type: Thailandepsin A. As a structural analogue of anti-cancer drug FK228, Thailandepsin A possesses distinctive selective inhibition towards different type of HDAC, thus especially demonstrates significant impact towards T-cell lymphoma. Despite of its prospect as a potential effective anti-cancer drug, the bottleneck lies in its low production capacity. Currently, Thailandepsin A yield produced by wild-type strain was very low (10mg/L) . Under optimization on culture medium and cultivation condition, The production capacity increased somewhat. In this research, we attempted to further employ fermentation medium optimization on this basis, and then scale-up the fermentation process to 5L fermentor and applied an absorption-coupled fermentation process, to remove product inhibition and thus increase the productivity of Thailandepsin AFirstly, at the shake flask level, with the one-factor-at-a-time screening method, we investigated impact on Thailandepsin A yield of amino acid precursor, compound carbon source and nitrogen source, and trace elements of different kinds and concentration. The results were: with a compound addition ofα-alanine, L-cysteine, L-phenylalanine and Glycine under optimal concentration in culture meidum, Thailandepsin A yield increased by 65.9%. With the application of compound carbon source of glucose and glycerol, maximum Thailandepsin A yield increased by 54.48%. With the application of compound nitrogen source of trypton and ammonium sulfate/ ammonium chloride, Thailandepsin A yield decreased on the contrary. With the addition of FeSO₄·7H₂O/ FeCl₃ (1mg/L), ZnSO₄·7H₂O(2mg/L), MnCl₂·4H₂O(0.1mg/L) in culture meidum, Thailandepsin A yield could increase more or less.Secondly, we investigated different fed-batch cultivation strategy on Thailandepsin A yield. By a progressive glucose fed-batch strategy, maximum Thailandepsin A yield increased to 205.29mg/L, which was 2.28-fold compared to control and 20.5-fold compared to initial yield, thus found an effective way to increase Thailandepsin A production capacity and lay down a foundation for further scale-up fermentation.Thirdly, in a laboratory scale 5L stirred tank bioreactor with a working volume of 3 L, Thailandepsin A fermentation was preliminarily optimized by the investigation of cultivation condition of ventilation conditions, seed source conditions and absorption of HP-20 resin. On this basis, we designed and made an external absorbitor column to connect with bioreactor, thus realized the process of continuous absorption-coupled fermentation. With the investigation of certain parameters such as circulative velocities, initial absorption time and fed-batch strategy, Thailandepsin A yield increased significantly.The maximum Thailandepsin A yield cultivation strategy was : (1) inoculum volume 1%; three-level seed source; (2) agitation speed 150rpm; air flow rate 1.3L/min; (3) cultivation pressure 0.01MPa; temperature 28℃; (4) coupled to continuous absorption with 4% pretreated HP-20 resin, initial absorption time 36h and circulative velocities 20ml/min; (5) glucose fed-batch 1g/L per 12h start from 60h; (6) fermentation end determined to 24h after dry cell weight reaches peak. The fermentation experiment under above condition demonstrated a Thailandepsin A yield of 224.11mg/L, which was 5.6-fold compared to control and 22.4-fold compared to initial yield; Thailandepsin A productivity of 1.10 mg/L*h^-1, which increased by 65% compared to control and 6.6-fold compared to initial productivity; thus lay down solid foundation for further research on Thailandepsin A fermentation.