title: Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli.
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 (∼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.
authors.affiliation: Manus Biosynthesis, Cambridge, MA 02138; Masters in Biotechnology Program, Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208;
authors.forename: Bradley Walters
authors.initials: BW
authors.lastname: Biggs
authors.affiliation: Manus Biosynthesis, Cambridge, MA 02138;
authors.forename: Chin Giaw
authors.initials: CG
authors.lastname: Lim
authors.affiliation: Manus Biosynthesis, Cambridge, MA 02138;
authors.forename: Kristen
authors.initials: K
authors.lastname: Sagliani
authors.affiliation: Manus Biosynthesis, Cambridge, MA 02138;
authors.forename: Smriti
authors.initials: S
authors.lastname: Shankar
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Gregory
authors.initials: G
authors.lastname: Stephanopoulos
authors.affiliation: Manus Biosynthesis, Cambridge, MA 02138; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Laboratory for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000, Belgium marjan.demey@ugent.be pkaji@manusbio.com.
authors.forename: Marjan
authors.identifier: http://orcid.org/0000-0002-8134-9436
authors.initials: M
authors.lastname: De Mey
authors.affiliation: Manus Biosynthesis, Cambridge, MA 02138; marjan.demey@ugent.be pkaji@manusbio.com.
authors.forename: Parayil Kumaran
authors.initials: PK
authors.lastname: Ajikumar
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism*; D017239:Paclitaxel / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D003577:Cytochrome P-450 Enzyme System; D017239:Paclitaxel
keywords: P450; Taxol; metabolic engineering; natural products; oxygenated taxanes