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Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases

Michener, Joshua K. and Nielsen, Jens and Smolke, Christina D. (2012) Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases. Proceedings of the National Academy of Sciences of the United States of America, 109 (47). pp. 19504-19509. ISSN 0027-8424. PMCID PMC3511110. doi:10.1073/pnas.1212287109.

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Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.

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URLURL TypeDescription DOIArticle CentralArticle
Smolke, Christina D.0000-0002-5449-8495
Additional Information:© 2012 National Academy of Sciences. Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved October 7, 2012 (received for review July 18, 2012). Published online before print November 5, 2012. We thank R. Kumar for assistance with the microarray experiments and J. L. M. Ruiz and L. Liu, who provided the HEM-overexpression plasmids. This work was supported by the National Science Foundation and Vetenskapsrådet through a Nordic Research Fellowship (to J.K.M.), National Science Foundation Grant CBET-0917638, and an Alfred P. Sloan Foundation fellowship (to C.D.S.). Author contributions: J.K.M., J.N., and C.D.S. designed research; J.K.M. performed research; J.K.M., J.N., and C.D.S. analyzed data; and J.K.M., J.N., and C.D.S. wrote the paper.
Funding AgencyGrant Number
Nordic Research Fellowship VetenskapsrådetUNSPECIFIED
Alfred P. Sloan Foundation fellowshipUNSPECIFIED
Subject Keywords:systems biology; yeast; CYP102A1; cytochrome P450
Issue or Number:47
PubMed Central ID:PMC3511110
Record Number:CaltechAUTHORS:20130104-104959337
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:36170
Deposited By: Jason Perez
Deposited On:04 Jan 2013 19:26
Last Modified:09 Nov 2021 23:20

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