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Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

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Abstract
Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (similar to 570 +/- 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.
Keywords
natural products, oxygenated taxanes, metabolic engineering, P450, Taxol, NATURAL-PRODUCTS, GENE-EXPRESSION, METABOLIC PATHWAY, CYTOCHROMES P450, BIOSYNTHESIS, BIOLOGY, DIVERSIFICATION, ARTEMISININ, TAXADIENE, CHEMICALS

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MLA
Biggs, Bradley Walters et al. “Overcoming Heterologous Protein Interdependency to Optimize P450-mediated Taxol Precursor Synthesis in Escherichia Coli.” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113.12 (2016): 3209–3214. Print.
APA
Biggs, B. W., Lin, C. G., Saglinani, K., Shankar, S., Stephanopoulos, G., De Mey, M., & Ajikumar, P. K. (2016). Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 113(12), 3209–3214.
Chicago author-date
Biggs, Bradley Walters, Chin Giaw Lin, Kristen Saglinani, Smriti Shankar, Gregory Stephanopoulos, Marjan De Mey, and Parayil Kumaran Ajikumar. 2016. “Overcoming Heterologous Protein Interdependency to Optimize P450-mediated Taxol Precursor Synthesis in Escherichia Coli.” Proceedings of the National Academy of Sciences of the United States of America 113 (12): 3209–3214.
Chicago author-date (all authors)
Biggs, Bradley Walters, Chin Giaw Lin, Kristen Saglinani, Smriti Shankar, Gregory Stephanopoulos, Marjan De Mey, and Parayil Kumaran Ajikumar. 2016. “Overcoming Heterologous Protein Interdependency to Optimize P450-mediated Taxol Precursor Synthesis in Escherichia Coli.” Proceedings of the National Academy of Sciences of the United States of America 113 (12): 3209–3214.
Vancouver
1.
Biggs BW, Lin CG, Saglinani K, Shankar S, Stephanopoulos G, De Mey M, et al. Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 2016;113(12):3209–14.
IEEE
[1]
B. W. Biggs et al., “Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli,” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 113, no. 12, pp. 3209–3214, 2016.
@article{7084692,
  abstract     = {Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (similar to 570 +/- 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.},
  author       = {Biggs, Bradley Walters and Lin, Chin Giaw and Saglinani, Kristen and Shankar, Smriti and Stephanopoulos, Gregory and De Mey, Marjan and Ajikumar, Parayil Kumaran},
  issn         = {0027-8424},
  journal      = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
  keywords     = {natural products,oxygenated taxanes,metabolic engineering,P450,Taxol,NATURAL-PRODUCTS,GENE-EXPRESSION,METABOLIC PATHWAY,CYTOCHROMES P450,BIOSYNTHESIS,BIOLOGY,DIVERSIFICATION,ARTEMISININ,TAXADIENE,CHEMICALS},
  language     = {eng},
  number       = {12},
  pages        = {3209--3214},
  title        = {Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli},
  url          = {http://dx.doi.org/10.1073/pnas.1515826113},
  volume       = {113},
  year         = {2016},
}

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