Biosynthetic engineering and fermentation media development leads to gram-scale production of spliceostatin natural products in Burkholderia sp. |
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| Affiliation: | 1. State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People''s Republic of China;2. Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, People''s Republic of China;3. Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People''s Republic of China;4. Xi’an Middle School of Shaanxi Province, Xi’an 710021, People''s Republic of China;1. Department of Laboratory Medicine – Microbiology, Manipal Hospital, Bengaluru, Karnataka, India E-mail: bhavana.mv@manipalhospitals.com;1. Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary;2. Department of Inorganic and Analytical Chemistry, University of Debrecen, POB 21, H-4010 Debrecen, Hungary;3. Department of Medical Chemistry, Medical and Health Science Centre, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary;4. Institute of Biology, Medicinal Chemistry and Biotechnology, The National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, GR-116 35 Athens, Greece;1. Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, D-66123 Saarbrücken, Germany;2. ATG:biosynthetics GmbH, D-79249 Merzhausen, Germany;1. Laboratory of Computational Systems Biotechnology (LCSB), Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland;2. Swiss Institute of Bioinformatics, CH-1015, Switzerland |
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| Abstract: | A key challenge in natural products drug discovery is compound supply. Hundreds of grams of purified material are needed to advance a natural product lead through preclinical development. Spliceostatins are polyketide-nonribosomal peptide natural products that bind to the spliceosome, an emerging target in cancer therapy. The wild-type bacterium Burkholderia sp. FERM BP-3421 produces a suite of spliceostatin congeners with varying biological activities and physiological stabilities. Hemiketal compounds such as FR901464 were the first to be described. Due to its improved properties, we were particularly interested in a carboxylic acid precursor analog that was first reported from Burkholderia sp. MSMB 43 and termed thailanstatin A. Inactivation of the iron/α-ketoglutarate-dependent dioxygenase gene fr9P had been shown to block hemiketal biosynthesis. However, a 4-deoxy congener of thailanstatin A was the main product seen in the dioxygenase mutant. We show here that expression of the cytochrome P450 gene fr9R is a metabolic bottle neck, as use of an l-arabinose inducible system led to nearly complete conversion of the 4-deoxy analog to the target molecule. By integrating fermentation media development approaches with biosynthetic engineering, we were able to improve production titers of the target compound >40-fold, going from the starting ~60 mg/L to 2.5 g/L, and to achieve what is predominantly a single component production profile. These improvements were instrumental in enabling preclinical development of spliceostatin analogs as chemotherapy. |
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| Keywords: | Polyketide Biosynthesis Thailanstatin Spliceostatin Splicing inhibitor Antibody drug conjugate |
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