Part:BBa_K5327009:Design
Cytochrome P450 83A1
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 911
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 246
Illegal PstI site found at 911 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 641
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 911
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 911
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The design of the Cytochrome P450 83A1 (CYP83A1) gene utilizes the coding sequence (CDS) from Arabidopsis thaliana, optimized for codon usage in Saccharomyces cerevisiae (S288C) to ensure efficient expression in yeast. CYP83A1 plays a crucial role in the metabolism of aliphatic and aromatic aldoximes, participating in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates.[1]In this study, CYP83A1 metabolizes (E)-aldoximes derived from chain-elongated methionine, such as dihomomethionine. The gene is expressed in yeast using the PGI1 promoter (PGI1pBBa_K5327017) and HXT7 terminator (HXT7tBBa_K5327019) to ensure high-level expression and mRNA stability. The optimized gene was inserted into a vector and introduced into Saccharomyces cerevisiae S288C via homologous recombination, followed by screening and expression validation using a defective strain. This method aims to enhance the synthesis of aliphatic glucosinolates in yeast and optimize yeast as a metabolic engineering platform.
Plasmid
- Fig 1. The plasmid expression of cytochrome P450 83A1
Source
Arabidopsis thaliana
References
- ↑ NAUR P, PETERSEN B L, MIKKELSEN M D, et al. CYP83A1 and CYP83B1, Two Nonredundant Cytochrome P450 Enzymes Metabolizing Oximes in the Biosynthesis of Glucosinolates in Arabidopsis [J]. Plant physiology, 2003, 133(1): 63-72.