Part:BBa_K4947029
C. roseus Cytochrome P450 Reductase Codon-Optimized CDS
This part is the gene coding for Cytochrome P450 Reductase from C. roseus. It is codon-optimized and domesticated for SalI, EcoRV, KpnI, PvuII, SphI, MluI, and SpeI restriction sites.
Introduction
This part is the gene coding for Cytochrome P450 Reductase from C. roseus. It is codon-optimized and domesticated for SalI, EcoRV, KpnI, PvuII, SphI, MluI, and SpeI restriction sites. This gene homolog that encodes for CPR was chosen rationally after thorough literature review. CPR is an enzyme involved in the biosynthetic pathway of daidzein, helping convert liquiritigenin into 2,7,4’-trihydroxyisoflavanone with the help of isoflavone synthase. The gene sequence was sourced from NCBI GenBank [1], and produced by Twist Bioscience. The codon optimization and domestication was done to improve recombinant expression in E. coli and enable restriction enzyme-based swapping of promoters and terminators, respectively.
Description
NADPH–cytochrome P450 reductase (CPR) is an NADPH-dependent CYP450 enzyme. It assists IFS/2-HIS to convert flavanones into 6’-deoxychalcones. These products are generally unstable. CPR has an N-terminus hydrophobic region (that has solubility effects) that is used for membrane anchorage. At the membrane, the CPR transfers an electron to the IFS/2-HIS by reducing NADPH while IFS/2-HIS is acting upon the flavanone substrate. This produces 6’-deoxychalcone. The reaction usually performs best under optimal reduction conditions. Like ATR, CPR likely exists naturally in plants in much lower concentrations compared to its enzymatic partner (IFS/2-HIS in this case). [2]
Usage
This gene homolog performed moderately in yeast to produce genistein [3], but in E. coli, it performed the best out of all tested CPRs to produce daidzein [5]. This is why it was selected for, in terms of optimizing the production of daidzein through recombinant expression of its pathway in E. coli . The sequence was codon-optimized using the CAD-SGE algorithm developed by Jaymin Patel in Farren Isaacs’ lab at Yale University [4]. This DNA was synthesized from Twist Bioscience, as an in-kind donation. There were no problems with gene synthesis. Problems encountered during amplification, plasmid construction, and everything else in the cloning process was not due to the gene sequence or source itself. This DNA is of biosafety level 1.
Experience
We amplified these genes using high-fidelity PCR with primers designed to anneal at each end (Figure 1). We then DpnI-digested and purified these amplicons. Subsequently, we performed Golden Gate assembly using NEBridge® Golden Gate Assembly Kit (which was also donated in-kind) and their specified protocol to build plasmids using this part. We electroporated TransforMax EC100D pir+ electrocompetent E. coli with the assembled DNA, and plated on selective media. Then, we ran diagnostic colony PCR that amplified parts of the plasmid to check for the presence of successful junctions, which indicate successful assembly. Of the colonies that had positive results, some were inoculated, plasmid-purified (using QIAGEN mini-prep kit and protocol), and sent for whole plasmid sequencing, a service purchased from Plasmidsaurus. Finally, whole plasmid sequencing results confirmed success or failure. This is the general procedure we recommend for using and characterizing this part, as it was successful for us.
Characterization
Figure 1. In the lane labeled 109, the amplicon for this part is clear and distinct.
Significance
CPR is a main rate-determining enzyme in the biosynthesis of daidzein. It is important for efficient production of daidzein. Optimizing for a specific flavonoid, daidzein in this case, is a great first step to improving production. This part specifically is important for optimal daidzein production, when being produced recombinantly by E. coli. Take a look at the rest of our wiki (https://2023.igem.wiki/yale/index.html) for how this part connects to human health, economics, and more!
References
1. https://www.ncbi.nlm.nih.gov/protein/Q05001.1
2. View our contributions page (https://2023.igem.wiki/yale/contribution) for a spreadsheet of all our sources!
3. Liu, Q., Liu, Y., Li, G., Savolainen, O., Chen, Y., & Nielsen, J. (2021, October 19). De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories. Nature News. https://www.nature.com/articles/s41467-021-26361-1
4. Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome. Patel JR, Oh J, Wang S, Crawford JM, Isaacs FJ. Cell. 2022 Apr 28;185(9):1487-1505.e14. doi: 10.1016/j.cell.2022.03.008. Epub 2022 Apr 1. 10.1016/j.cell.2022.03.008 PubMed 35366417
5. Chemler, et al., J. (2010). A Versatile Microbial System for Biosynthesis of Novel Polyphenols with Altered Estrogen Receptor Binding Activity. Cell. https://www.cell.com/cell-chemical-biology/pdf/S1074-5521(10)00121-3.pdf
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1408
Illegal PstI site found at 821
Illegal PstI site found at 1465
Illegal PstI site found at 1629 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1408
Illegal PstI site found at 821
Illegal PstI site found at 1465
Illegal PstI site found at 1629 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1408
Illegal BamHI site found at 1299
Illegal XhoI site found at 6 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1408
Illegal PstI site found at 821
Illegal PstI site found at 1465
Illegal PstI site found at 1629 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1408
Illegal PstI site found at 821
Illegal PstI site found at 1465
Illegal PstI site found at 1629
Illegal NgoMIV site found at 981
Illegal NgoMIV site found at 1674 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| protein | -NA- |
//cds/biosynthesis
//cds/enzyme
//function/biosynthesis
| protein |