Part:BBa_K4947023

G. max Chalcone Synthase 7 Codon-Optimized CDS

This part is the gene coding for Chalcone Synthase 7 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 Synthase 7 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 CHS was chosen rationally after thorough literature review. CHS is an enzyme involved in the biosynthetic pathway of daidzein, converting p-coumaroyl-CoA into isoliquiritigenin with the help of Chalcone 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

Chalcone synthase (CHS) is an important first step in flavonoid biosynthesis, usually called the first dedicated step toward production. It is a promiscuous enzyme that converts p-coumaroyl-CoA to a chalcone, notably either naringenin chalcone (NAC) or isoliquiritigenin (ISO) without or with the presence of CHR, respectively. This reaction utilizes the p-coumaroyl-CoA and three molecules of malonyl-CoA to produce the chalcone, four molecules of CoA and three carbon dioxide molecules. Somewhere along the way, the intermediate p-coumaroylcyclohexantrione is produced and consumed. As the substrates are larger molecules, there is a propensity for derailing to occur during the reaction, especially under unstable conditions. When only a single malonyl-CoA molecule is used, bis-noryangonin (NYG) is produced. When two malonyl-CoA molecules are used, p-coumaroyltriacetic acid lactone (CTAL) is produced. The probability of derailment is likely low. As well, CHILs have been shown to reduce CTAL production (with limited NYG production effect) when co-expressed, implying the enzyme-stabilizing function of CHILs. CHS can form multimers (dimers, trimers, tetramers, etc.), and this function may be important to its catalytic activity. As well, it can be involved in metabolons, transient multi-protein complexes of sequential enzymes that mediate substrate channeling, that involve CHRs, CHIs, and IFS/2-HISs. Cross-species metabolons are generally weak. CHS’s active site is buried. [2]

Usage

In E. coli, this gene homolog outperformed PcCHS, AtCHS, GuCHS, and appeared to be more specific for daidzein biosynthetic pathway as opposed to the genistein biosynthetic pathway [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 second lane, the amplicon for this part is clear and distinct.

Significance

CHS is one of the first major enzymes involved in many flavonoids’ biosynthetic pathways. 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/NP_001340309.1

2. View our contributions page (https://2023.igem.wiki/yale/contribution) for a spreadsheet of all our sources!

3. Li, J., et al. (2021, September 25). Diversion of metabolic flux towards 5-deoxy(iso)flavonoid production via enzyme self-assembly in escherichia coli. Metabolic Engineering Communications. https://www.sciencedirect.com/science/article/pii/S2214030121000250#bib64

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

protein

-NA-