Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803

TY - JOUR

T1 - Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803

AU - Wlodarczyk, Artur Jacek

AU - Gnanasekaran, Thiyagarajan

AU - Nielsen, Agnieszka Janina Zygadlo

AU - Nokolunga, Nodumo

AU - Mellor, Silas Busck

AU - Manja, Luckner

AU - Frederik Bang Thøfner, Jens

AU - Olsen, Carl Erik

AU - Motawie, Mohammed Saddik

AU - Burow, Meike

AU - Pribil, Mathias

AU - Feussner, Ivo

AU - Møller, Birger Lindberg

AU - Jensen, Poul Erik

PY - 2016/1/1

Y1 - 2016/1/1

N2 - Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66. mg/L of p-hydroxyphenylacetaldoxime and 5. mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3. mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.

AB - Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66. mg/L of p-hydroxyphenylacetaldoxime and 5. mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3. mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation.

U2 - 10.1016/j.ymben.2015.10.009

DO - 10.1016/j.ymben.2015.10.009

M3 - Journal article

C2 - 26548317

SN - 1096-7176

VL - 33

SP - 1

EP - 11

JO - Metabolic Engineering

JF - Metabolic Engineering

ER -