TY - JOUR
T1 - Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases
AU - Michener, Joshua K.
AU - Nielsen, Jens
AU - Smolke, Christina D.
PY - 2012/11/20
Y1 - 2012/11/20
N2 - 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 fromconditions of either high or lowstress, 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.
AB - 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 fromconditions of either high or lowstress, 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.
KW - CYP102A1
KW - Cytochrome P450
KW - Systems biology
KW - Yeast
UR - http://www.scopus.com/inward/record.url?scp=84869744652&partnerID=8YFLogxK
U2 - 10.1073/pnas.1212287109
DO - 10.1073/pnas.1212287109
M3 - Article
C2 - 23129650
AN - SCOPUS:84869744652
SN - 0027-8424
VL - 109
SP - 19504
EP - 19509
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 47
ER -