Part:BBa_K4947031
G. echinata 2-Hydroxyisoflavanone Synthase Codon-Opt. CDS
This part is the gene coding for 2-Hydroxyisoflavanone Synthase from G. echinata. 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 2-Hydroxyisoflavanone Synthase from G. echinata. It is codon-optimized and domesticated for SalI, EcoRV, KpnI, PvuII, SphI, MluI, and SpeI restriction sites. This gene homolog that encodes for IFS/2-HIS was chosen rationally after thorough literature review. IFS/2-HIS is an enzyme involved in the biosynthetic pathway of daidzein, converting liquiritigenin into 2,7,4’-trihydroxyisoflavanone with the help of cytochrome p450 reductase. 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
Isoflavone synthase (IFS), AKA 2-hydroxyisoflavone synthase (2-HIS), is a promiscuous CYP450s enzyme. Like most CYP450s, it relies on electrostatic and hydrophobic forces to interact with redox partners. It converts flavanones into 6’-deoxychalcones, namely NAG to 2,5,7,4’-tetrahydroxyisoflavanone (TeHIF) or LIG to 2,7,4’-trihydroxyisoflavanone (TriHIF). These products are generally unstable. IFS/2HIS uses the help of an NADPH-dependent cytochrome P450 (CYP450) redox partner CPR for this reaction. IFS/2-HIS 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, an example being glycerol carbon source. For isoflavone production, this reaction step is seen as the rate-determining step. IFS/2-HISs in plants tend to have a substrate preference for LIG instead of NAG (by a ten-fold difference). IFS/2-HIS form quaternary structures that involve heme, an iron-based molecule, in its prosthetic group. The enzyme is feedback-inhibited, mostly by the isoflavone. It is also labile and sacrifices stability for catalytic activity.[2]
Usage
This gene homolog performed the best in yeast to produce genistein [3] and 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 111, the amplicon for this part is clear and distinct.
Significance
IFS/2-HIS 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/nuccore/AB023636.1
2. View our contributions page (https://2023.igem.wiki/yale/contribution) for a spreadsheet of all our sources!
3. 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
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. 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
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1240
Illegal PstI site found at 277 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1240
Illegal PstI site found at 277 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1240
Illegal BglII site found at 1293
Illegal XhoI site found at 1563 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1240
Illegal PstI site found at 277 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1240
Illegal PstI site found at 277
Illegal NgoMIV site found at 916 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| protein | -NA- |
//cds/biosynthesis
//cds/enzyme
//function/biosynthesis
| protein |