title: Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli.
abstract: Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing, and collaborative efforts such as the iGEM competition. All of these attributes facilitate exploitation of the wealth of genetic information made available by genome and RNA sequencing. Standardization also comes at the cost of reduced flexibility. We addressed this paradox by formulating a set of design principles aimed at maximizing standardization while maintaining high flexibility in choice of cloning technique and minimizing the impact of standard sequences. The design principles were applied to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable and simplifies the experimental design of projects aimed at biosynthetic pathway construction or engineering.
authors.affiliation: †Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
authors.forename: Morten T
authors.initials: MT
authors.lastname: Nielsen
authors.affiliation: †Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
authors.forename: Karina M
authors.initials: KM
authors.lastname: Madsen
authors.affiliation: †Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
authors.forename: Susanna
authors.initials: S
authors.lastname: Seppälä
authors.affiliation: †Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
authors.forename: Ulla
authors.initials: U
authors.lastname: Christensen
authors.affiliation: †Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
authors.forename: Lone
authors.initials: L
authors.lastname: Riisberg
authors.affiliation: †Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Harrison
authors.affiliation: †Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
authors.affiliation: †Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, DK-2970 Hørsholm, Denmark.
authors.forename: Morten H H
authors.initials: MH
authors.lastname: Nørholm
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D060847:Metabolic Engineering; D011166:Porphyrins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D011166:Porphyrins; D004247:DNA
keywords: biobricks; molecular cloning; porphyrin bioengineering; synthetic biology; uracil-excision cloning