Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli

TY - JOUR

T1 - Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli

AU - Nielsen, Morten Thrane

AU - Madsen, Karina M.

AU - Seppälä, Susanna

AU - Riisberg, Lone

AU - Harrison, Scott J.

AU - Møller, Birger Lindberg

AU - Nørholm, Morten Helge Hauberg

PY - 2015/3/20

Y1 - 2015/3/20

N2 - 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.

AB - 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.

U2 - 10.1021/sb500055u

DO - 10.1021/sb500055u

M3 - Journal article

C2 - 24905856

SN - 2161-5063

VL - 4

SP - 274

EP - 282

JO - ACS Synthetic Biology

JF - ACS Synthetic Biology

IS - 3

ER -