Part:BBa_K4947024
G. max Chalcone Reductase 5 Codon-Optimized CDS
This part is the gene coding for Chalcone Reductase from G. max. 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 Chalcone Reductase 5 from G. max. It is codon-optimized and domesticated for SalI, EcoRV, KpnI, PvuII, SphI, MluI, and SpeI restriction sites. This gene homolog that encodes for CHR was chosen rationally after thorough literature review. CHR is an enzyme involved in the biosynthetic pathway of daidzein, converting p-coumaroyl-CoA into isoliquiritigenin with the help of Chalcone Synthase. It is the enzyme that determines whether the pathway proceeds toward genistein or daidzein as a product. 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
Chalcone reductase (CHR) is an NADPH-dependent enzyme that directs the flavonoid pathway toward daidzein (DZN) production. CHR acts with chalcone synthase (CHS) to produce isoliquiritigenin (ISO), diverting away from naringenin chalcone (NAC) production (which leads to production of genistein (GEN)). CHR has been theorized, incorrectly, to act upon NAC to produce ISO. It actually acts upon p-coumaroylcyclohexantrione, an intermediate of the CHS step. It likely does not interact with CHS’s buried active site. An alternative name for CHR is polyketide reductase (PKR). This is technically more accurate as the enzyme does not actually have a chalcone as its substrate, as the “CHR” name implies. Its other name, while technically correct, makes identification and searching for the enzyme much more difficult given the broad description that is “polyketide reductase,” defeating the purpose of a name. ISO is a yellow compound that is often a precursor to pigments found in yellow flowers of some species. CHR can be involved in metabolons that involve CHSs, chalcone isomerases (CHIs), and isoflavone synthases (IFS/2-HISs). Cross-species metabolons are generally weak. [2]
Usage
In yeast, this gene homolog outperformed PlCHR and MsCHR and was involved in notable combinations of gene homologs that produced high amounts of daidzein [3]. 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, 2). 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 104, the amplicon for this part is clear and distinct.
Figure 2. A clearer picture of the AGE results, showing successful amplifcatiion of the part.
Significance
CHR is the main determining enzyme in whether daidzein or genistein is produced. It is important for specific 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. NCBI Reference Sequence: NP_001304585.2
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
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 427
Illegal PstI site found at 310 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 427
Illegal PstI site found at 310 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 427
Illegal XhoI site found at 337 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 427
Illegal PstI site found at 310 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 427
Illegal PstI site found at 310
Illegal NgoMIV site found at 303
Illegal NgoMIV site found at 555 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| protein | -NA- |
//cds/biosynthesis
//cds/enzyme
//function/biosynthesis
| protein |