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Applied and Environmental Microbiology, July 2002, p. 3287-3292, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3287-3292.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Process and Metabolic Strategies for Improved Production of Escherichia coli-Derived 6-Deoxyerythronolide B

Blaine Pfeifer,1 Zhihao Hu,2 Peter Licari,2 and Chaitan Khosla1,3,4*

Departments of Chemical Engineering,,1 Chemistry,,3 Biochemistry, Stanford University, Stanford, California 94305-5025,4 KOSAN Biosciences, Inc., Hayward, California 945452

Received 16 January 2002/ Accepted 12 April 2002

Recently, the feasibility of using Escherichia coli for the heterologous biosynthesis of complex polyketides has been demonstrated. In this report, the development of a robust high-cell-density fed-batch procedure for the efficient production of complex polyketides is described. The effects of various physiological conditions on the productivity and titers of 6-deoxyerythronolide B (6dEB; the macrocyclic core of the antibiotic erythromycin) in recombinant cultures of E. coli were studied in shake flask cultures. The resulting data were used as a foundation to develop a high-cell-density fermentation procedure by building upon procedures reported earlier for recombinant protein production in E. coli. The fermentation strategy employed consistently produced ~100 mg of 6dEB per liter, whereas shake flask conditions generated between 1 and 10 mg per liter. The utility of an accessory thioesterase (TEII from Saccharopolyspora erythraea) for enhancing the productivity of 6dEB in E. coli was also demonstrated (increasing the final titer of 6dEB to 180 mg per liter). In addition to reinforcing the potential for using E. coli as a heterologous host for wild-type- and engineered-polyketide biosynthesis, the procedures described in this study may be useful for the production of secondary metabolites that are difficult to access by other routes.

* Corresponding author. Mailing address: Department of Chemical Engineering, Stanford University, Stanford, CA 94305-5025. Phone: (650) 723-6538. Fax: (650) 723-6538. E-mail: ck{at}chemeng.stanford.edu.

Applied and Environmental Microbiology, July 2002, p. 3287-3292, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3287-3292.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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