Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation.
Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation.
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DOI：
10.1016/j.synbio.2016.02.002
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发表时间：
2016-03
影响因子：
4.8
通讯作者：
Cheng YQ
中科院分区：
文献类型：
--
作者：
Liu X;Zhu H;Biswas S;Cheng YQ
Thailanstatin A（TST-A）是本研究组通过基因组指导的方法从Burkholderia thailandensis MSMB 43中发现的一种有效的抗增殖天然产物。由于TST-A在细菌发酵中的低滴度、适度的稳定性和纯化过程中非常低的回收率，其有限的供应阻碍了TST-A作为抗癌候选药物的研究。在这里，我们报告了显着的产量提高TST-A和它的直接前体，thailanstatin D（TST-D），通过代谢工程的thailanstatin生物合成途径在MSMB 43。缺失tstP（其编码参与将TST-A转化为下游产物（包括FR 901464（FR））的双加氧酶）分别导致发酵液中TST-A滴度增加58%至144.7 ± 2.3 mg/L和TST-D滴度增加132%至14.6 ± 0.5 mg/L。缺失tstR（其编码参与将TST-D转化为TST-A的细胞色素P450）导致发酵液中TST-D滴度增加超过7倍至53.2 ± 12.1 mg/L。在120-L发酵罐中进行tstP缺失突变体的90 L中试规模补料分批发酵导致制备714 mg纯度大于98.5%的TST-A。发现TST-D在磷酸盐缓冲液中的半衰期至少为202 h，显著长于TST-A或FR，表明其具有优越的上级稳定性。然而，TST-D对代表性人癌细胞系的IC 50值被确定为大于TST-A的IC 50值，表明较弱的抗增殖活性。这项工作使我们能够为我们正在进行的转化研究制备足够量的TST-A和TST-D。
Thailanstatin A (TST-A) is a potent antiproliferative natural product discovered by our group from Burkholderia thailandensis MSMB43 through a genome-guided approach. The limited supply of TST-A, due to its low titer in bacterial fermentation, modest stability and very low recovery rate during purification, has hindered the investigations of TST-A as an anticancer drug candidate. Here we report the significant yield improvement of TST-A and its direct precursor, thailanstatin D (TST-D), through metabolic engineering of the thailanstatin biosynthetic pathway in MSMB43. Deletion of tstP, which encodes a dioxygenase involved in converting TST-A to downstream products including FR901464 (FR), resulted in 58% increase of the TST-A titer to 144.7 ± 2.3 mg/L and 132% increase of the TST-D titer to 14.6 ± 0.5 mg/L in the fermentation broth, respectively. Deletion of tstR, which encodes a cytochrome P450 involved in converting TST-D to TST-A, resulted in more than 7-fold increase of the TST-D titer to 53.2 ± 12.1 mg/L in the fermentation broth. An execution of 90 L pilot-scale fed-batch fermentation of the tstP deletion mutant in a 120-L fermentor led to the preparation of 714 mg of TST-A with greater than 98.5% purity. The half-life of TST-D in a phosphate buffer was found to be at least 202 h, significantly longer than that of TST-A or FR, suggesting superior stability. However, the IC50 values of TST-D against representative human cancer cell lines were determined to be greater than those of TST-A, indicating weaker antiproliferative activity. This work enabled us to prepare sufficient quantities of TST-A and TST-D for our ongoing translational research.
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影响因子：
3.3
作者：
MILLERWIDEMAN, M;MAKKAR, N;STONARD, R
通讯作者：
STONARD, R
影响因子：
--
作者：
Wang, Cheng;Wesener, Shane R.;Cheng, Yi-Qiang
通讯作者：
Cheng, Yi-Qiang
影响因子：
5.1
作者：
Wang, Cheng;Henkes, Leonhard M.;Cheng, Yi-Qiang
通讯作者：
Cheng, Yi-Qiang
DOI：
10.1038/ja.2013.38
发表时间：
2013-09
期刊：
The Journal of antibiotics
影响因子：
--
作者：
通讯作者：
--
影响因子：
14.8
作者：
Kaida, Daisuke;Motoyoshi, Hajime;Yoshida, Minoru
通讯作者：
Yoshida, Minoru