Part:BBa_K4947027
G. uralensis Chalcone Isomerase 1 Codon-Optimized CDS
This part is the gene coding for Chalcone Isomerase 1 from M. sativa. 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 Isomerase 1 from G. uralensis. It is codon-optimized and domesticated for SalI, EcoRV, KpnI, PvuII, SphI, MluI, and SpeI restriction sites. This gene homolog that encodes for CHI was chosen rationally after thorough literature review. CHI is an enzyme involved in the biosynthetic pathway of daidzein, converting isoliquiritigenin into liquiritigenin, both stable intermediates. 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 isomerase (CHI) is a promiscuous enzyme that catalyzes the isomerization of ISO to LIG and/or NAC to naringenin (NAG), depending on the type of CHI. There are four types of CHI: Type I are bona fine and only convert NAC to NAG; type II convert both NAC to NAG and ISO to LIG; type III are involved in fatty acid metabolism; type IV enhance flavonoid production without performing catalytic activity directly. Type IV CHIs are thusly named chalcone isomerase-like enzymes (CHIL). Specific motifs exist in the types that can give the enzymes their respective functions. The isomerization of the chalcone to the respective flavanone is a spontaneous step. The chalcone tends to be more stable under physiological conditions, as the rate of reaction is especially affected by pH. At lower pHs, the spontaneous speed increases. The spontaneous step produces a racemic mixture of (2RS)-flavanone and is slowed down in the presence of high concentrations of proteins. With CHI, the step proceeds much faster (10E7 increase in speed) and becomes stereoselective, only producing (2S)-flavanone, and the step works more efficiently at higher pHs. Only the S stereomer is able to be used in subsequent reactions for flavonoid biosynthesis. Among flavanone-producing enzymes, CHI is seen as the rate-determining conversion step. CHILs enhance flavonoid production by improving the rate of reaction of the CHI step as well as improving CHS stability, preventing derailment byproducts CTAL, and to a lesser extent, NYG from forming. This effect may be less pronounced depending on the CHS and CHI enzymes. CHI can be involved in metabolons that involve CHSs, CHRs, and IFS/2-HISs. Cross-species metabolons are generally weak. [2]
Usage
This gene, when overexpressed in E. coli, contributed to good production 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). 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 fourth lane, the amplicon for this part is clear and distinct.
Significance
CHI 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/ABM66533.1
2. View our contributions page (https://2023.igem.wiki/yale/contribution) for a spreadsheet of all our sources!
3. Yan, Yin., et al. (2020). De novo biosynthesis of liquiritin in Saccharomyces cerevisiae. NCBI. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161706/
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 37
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 37
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 37
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 37
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 37
Illegal NgoMIV site found at 60
Illegal NgoMIV site found at 94 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| protein | -NA- |
//cds/biosynthesis
//cds/enzyme
//function/biosynthesis
| protein |