Part:BBa_K4947042
Daidzein Biosynthetic Pathway Module C211
This composite part is a gene assembly that produces daidzein from liquiritigenin. It contains the last three genes involved in the biosynthetic pathway for daidzein. This device contains CrCPR (https://parts.igem.org/Part:BBa_K4947029), TpIFS (https://parts.igem.org/Part:BBa_K4947032), and GmHID (https://parts.igem.org/Part:BBa_K4947033). Each of these genes are codon-optimized and domesticated for SalI, EcoRV, KpnI, PvuII, SphI, MluI, and SpeI restriction sites. Each gene has a respective synthetic yeast promoter that is low-to-moderately constitutively expressed in E. coli.
Introduction
This composite part is a gene assembly that produces daidzein from liquiritigenin. It contains the last three genes involved in the biosynthetic pathway for daidzein. This device contains GmCPR (https://parts.igem.org/Part:BBa_K4947030), Ge2-HIS (https://parts.igem.org/Part:BBa_K4947031), and GmHID (https://parts.igem.org/Part:BBa_K4947033). Each of these genes are codon-optimized and domesticated for SalI, EcoRV, KpnI, PvuII, SphI, MluI, and SpeI restriction sites. Each gene has a respective synthetic yeast promoter that is low-to-moderately constitutively expressed in E. coli. These gene homologs were chosen rationally after thorough literature review. CPR and IFS/2-HIS convert liquiritigenin into 2,7,4’-trihydroxyisoflavanone. HID then converts 2,7,4’-trihydroxyisoflavanone into daidzein. The gene sequences were sourced from NCBI GenBank (view references on the individual parts’ pages!), 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. Domestication also allowed BioBrick standardization, enabling Golden Gate assembly.
Description
IFS/2-HIS. 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.
CPR. 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). HID. 2-hydroxyisoflavanone dehydratase (HID) is a promiscuous enzyme that catalyzes the dehydration of 6’-deoxychalcones to produce isoflavones, namely TeHIF to GEN and TriHIF to DZN. The dehydration step of the unstable 6’-deoxychalcones is a spontaneous reaction and proceeds relatively quickly, especially in acidic conditions. HID, however, enhances speed and even yield of isoflavone biosynthesis and works optimally in basic pHs. Among the isoflavone biosynthesis pathway enzymes, HID is by far the least characterized. The product of interest, DZN, may spontaneously dehydrate to form isoformomentin. [1]
Usage
GmCPR performed the best in yeast to produce genistein [3]. Ge2-HIS performed the best in yeast to produce genistein [3] and daidzein [2]. GmHID was the best at producing genistein and daidzein in both yeast and E. coli<i/> [2, 3, 4]. These are the reasons why they were selected for, in terms of optimizing the production of daidzein through recombinant expression of its pathway in <i>E. coli. The sequence was codon-optimized using the CAD-SGE algorithm developed by Jaymin Patel in Farren Isaacs’ lab at Yale University [5]. 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 the initial parts using high-fidelity PCR with primers designed to anneal at each end. 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 (Figure 1). 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 lanes labeled C211-M, amplicons of length 3 kb are clear and distinct. An amplicon of this size was expected if assembly was successful. Successful assembly with no mutations was confirmed among these colonies using whole plasmid sequencing.
Significance
This device is crucial to finish the production of daidzein. This composite 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 device connects to human health, economics, and more!
References
1. View our contributions page (https://2023.igem.wiki/yale/contribution) for a spreadsheet of relevant sources!
2. 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
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. Akashi, et al., T. (2005). Molecular and Biochemical Characterization of 2-Hydroxyisoflavanone Dehydratase. Involvement of Carboxylesterase-Like Proteins in Leguminous Isoflavone Biosynthesis. Plant Physiology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1065389/
5. 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 1689
Illegal EcoRI site found at 4337
Illegal XbaI site found at 298
Illegal XbaI site found at 2830
Illegal XbaI site found at 4677
Illegal XbaI site found at 5895
Illegal SpeI site found at 1
Illegal SpeI site found at 706
Illegal PstI site found at 1260
Illegal PstI site found at 2142
Illegal PstI site found at 3060
Illegal PstI site found at 3374 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1689
Illegal EcoRI site found at 4337
Illegal NheI site found at 1971
Illegal NheI site found at 2929
Illegal SpeI site found at 1
Illegal SpeI site found at 706
Illegal PstI site found at 1260
Illegal PstI site found at 2142
Illegal PstI site found at 3060
Illegal PstI site found at 3374
Illegal NotI site found at 233 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1689
Illegal EcoRI site found at 4337
Illegal BglII site found at 761
Illegal BglII site found at 4390
Illegal XhoI site found at 4660 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1689
Illegal EcoRI site found at 4337
Illegal XbaI site found at 298
Illegal XbaI site found at 2830
Illegal XbaI site found at 4677
Illegal XbaI site found at 5895
Illegal SpeI site found at 1
Illegal SpeI site found at 706
Illegal PstI site found at 1260
Illegal PstI site found at 2142
Illegal PstI site found at 3060
Illegal PstI site found at 3374 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1689
Illegal EcoRI site found at 4337
Illegal XbaI site found at 298
Illegal XbaI site found at 2830
Illegal XbaI site found at 4677
Illegal XbaI site found at 5895
Illegal SpeI site found at 1
Illegal SpeI site found at 706
Illegal PstI site found at 1260
Illegal PstI site found at 2142
Illegal PstI site found at 3060
Illegal PstI site found at 3374
Illegal NgoMIV site found at 4013
Illegal AgeI site found at 322
Illegal AgeI site found at 917
Illegal AgeI site found at 1725
Illegal AgeI site found at 2235 - 1000COMPATIBLE WITH RFC[1000]
//function/biosynthesis
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