Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation
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 purif...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
KeAi Communications Co., Ltd.
2016-03-01
|
| Series: | Synthetic and Systems Biotechnology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X15300028 |
| _version_ | 1827279235083927552 |
|---|---|
| author | Xiangyang Liu Hui Zhu Sreya Biswas Yi-Qiang Cheng |
| author_facet | Xiangyang Liu Hui Zhu Sreya Biswas Yi-Qiang Cheng |
| author_sort | Xiangyang Liu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T08:15:42Z |
| format | Article |
| id | doaj.art-0f3402a9bfca48f9a1530e1c1edfad9c |
| institution | Directory Open Access Journal |
| issn | 2405-805X |
| language | English |
| last_indexed | 2024-04-24T08:15:42Z |
| publishDate | 2016-03-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj.art-0f3402a9bfca48f9a1530e1c1edfad9c2024-04-17T03:40:33ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2016-03-0111343810.1016/j.synbio.2016.02.002Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentationXiangyang Liu0Hui Zhu1Sreya Biswas2Yi-Qiang Cheng3UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX, USAUNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX, USADepartment of Biological Sciences, University of Wisconsin–Milwaukee, Milwaukee, WI, USAUNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX, USAThailanstatin 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.http://www.sciencedirect.com/science/article/pii/S2405805X15300028FermentationMetabolic engineeringNatural productProductionThailanstatin |
| spellingShingle | Xiangyang Liu Hui Zhu Sreya Biswas Yi-Qiang Cheng Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation Synthetic and Systems Biotechnology Fermentation Metabolic engineering Natural product Production Thailanstatin |
| title | Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation |
| title_full | Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation |
| title_fullStr | Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation |
| title_full_unstemmed | Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation |
| title_short | Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation |
| title_sort | improved production of cytotoxic thailanstatins a and d through metabolic engineering of burkholderia thailandensis msmb43 and pilot scale fermentation |
| topic | Fermentation Metabolic engineering Natural product Production Thailanstatin |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X15300028 |
| work_keys_str_mv | AT xiangyangliu improvedproductionofcytotoxicthailanstatinsaanddthroughmetabolicengineeringofburkholderiathailandensismsmb43andpilotscalefermentation AT huizhu improvedproductionofcytotoxicthailanstatinsaanddthroughmetabolicengineeringofburkholderiathailandensismsmb43andpilotscalefermentation AT sreyabiswas improvedproductionofcytotoxicthailanstatinsaanddthroughmetabolicengineeringofburkholderiathailandensismsmb43andpilotscalefermentation AT yiqiangcheng improvedproductionofcytotoxicthailanstatinsaanddthroughmetabolicengineeringofburkholderiathailandensismsmb43andpilotscalefermentation |