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Applied and Environmental Microbiology, June 2007, p. 3877-3886, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00200-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Engineering Central Metabolic Pathways for High-Level Flavonoid Production in Escherichia coli{triangledown} ,{ddagger}

Effendi Leonard,{dagger} Kok-Hong Lim,{dagger} Phan-Nee Saw, and Mattheos A. G. Koffas*

Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, New York 14260

Received 25 January 2007/ Accepted 22 April 2007

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.

* Corresponding author. Mailing address: Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, 904 Furnas Hall, Buffalo, NY 14260. Phone: (716) 645-2911, ext. 2221. Fax: (716) 645-3822. E-mail: mkoffas{at}buffalo.edu

{triangledown} Published ahead of print on 27 April 2007.

{ddagger} Supplemental material for this article may be found at http://aem.asm.org/.

{dagger} These authors contributed equally to this work.

Applied and Environmental Microbiology, June 2007, p. 3877-3886, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00200-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Fowler, Z. L., Gikandi, W. W., Koffas, M. A. G. (2009). Increased Malonyl Coenzyme A Biosynthesis by Tuning the Escherichia coli Metabolic Network and Its Application to Flavanone Production. Appl. Environ. Microbiol. 75: 5831-5839 [Abstract] [Full Text]  
  • Koffas, M. A. G. (2009). Expanding the repertoire of biofuel alternatives through metabolic pathway evolution. Proc. Natl. Acad. Sci. USA 106: 965-966 [Full Text]  
  • Leonard, E., Koffas, M. A. G. (2007). Engineering of Artificial Plant Cytochrome P450 Enzymes for Synthesis of Isoflavones by Escherichia coli. Appl. Environ. Microbiol. 73: 7246-7251 [Abstract] [Full Text]  


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