Part:BBa_K4947036
Daidzein Biosynthetic Pathway Module A112
This composite part is a gene assembly that produces isoliquiritigenin from p-coumaric acid. It contains the first three genes involved in the biosynthetic pathway for daidzein. This device contains At4Cl1 (https://parts.igem.org/Part:BBa_K4947021), GmCHS7 (https://parts.igem.org/Part:BBa_K4947023), and GuCHR1 (https://parts.igem.org/Part:BBa_K4947025). 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 isoliquiritigenin from p-coumaric acid. It contains the first three genes involved in the biosynthetic pathway for daidzein. This device contains At4Cl1 (https://parts.igem.org/Part:BBa_K4947021), GmCHS7 (https://parts.igem.org/Part:BBa_K4947023), and GuCHR1 (https://parts.igem.org/Part:BBa_K4947025). 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. 4CL converts p-coumaric acid into p-coumaroyl-CoA. CHS and CHR convert p-coumaroyl-CoA into isoliquiritigenin. 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
4CL. 4-Coumarate-CoA ligase (4CL), a promiscuous enzyme, can convert p-coumaric acid to p-coumaroyl-CoA. In the first step, p-coumarate and adenosine triphosphate (ATP) become ligated to form p-coumaroyl-AMP (AKA p-coumaroyl-adenylate). In the second step, in between the coumarate body and AMP body, CoA is ligated to form p-coumaroyl-CoA. 4CL also works on other substrates, including, but not limited to, ferulic acid, caffeic acid, and most notably, cinnamic acid. It is unclear whether substrate specificity or affinity changes when cinnamic acid is cis or trans. 4CL acts upon cinnamic acid to produce cinnamoyl-CoA, which is a precursor for pinocembrin (PIN). PIN is an adjacent flavonoid which biosynthesis involves the 4CL, CHS, CHR, and CHI enzymes. For the purpose of producing DZN, PIN represents an unwanted byproduct. Another unwanted byproduct near this step is phloretin, which is created when p-coumaroyl-CoA is acted upon by a carbon double bond reductase (CDBR) to form dihydro-p-coumaroyl-CoA. This is then used by CHS to form phloretin. CDBR is an important promiscuous common enzyme in many organisms, present endogenously in E. coli. 4CLs exist in many isoforms which can have distinct functions and localizations in plants. They are primarily used for (class I) lignin production, usually for wound response, or (class II) flavonoid production and p-coumaric acid regulation, usually UV-activated. Among homologs, the AMP binding site is highly conserved. 4CL is Mg+-dependent, and ascorbate improves enzyme and substrate stability.
CHS.. 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.
CHR. Chalcone reductase (CHR) is an NADPH-dependent enzyme that directs the flavonoid pathway toward DZN production. CHR acts with CHS to produce ISO, diverting away from 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, CHIs, and IFS/2-HISs. Cross-species metabolons are generally weak. [1]
Usage
At4CL was selected because it worked best in yeast, outperforming Pl4CL1, Gm4CL3, At4CL2, Ph4CL1, Pc4CL2, and a mutant At4CL1m to produce daidzein [2]. GmCHS7 was selected because in E. coli, it outperformed PcCHS, AtCHS, GuCHS, and appeared to be more specific for daidzein biosynthetic pathway as opposed to the genistein biosynthetic pathway [3]. GuCHR1 outperformed MsCHR and GmCHR5 and was more specific for the daidzein pathway in E. coli [5]. These are the reasons why they were 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 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 A112-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 start production of daidzein. It is also an important device for production for isoliquiritigenin, an expensive and understudied intermediate. 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. 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
5. Yan, Y. (2020). De novo biosynthesis of liquiritin in Saccharomyces cerevisiae. NCBI. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161706/
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 908
Illegal EcoRI site found at 1064
Illegal XbaI site found at 298
Illegal XbaI site found at 2465
Illegal XbaI site found at 3933
Illegal XbaI site found at 5189
Illegal SpeI site found at 1
Illegal SpeI site found at 706
Illegal PstI site found at 1137
Illegal PstI site found at 1220
Illegal PstI site found at 5048 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 908
Illegal EcoRI site found at 1064
Illegal NheI site found at 2562
Illegal SpeI site found at 1
Illegal SpeI site found at 706
Illegal PstI site found at 1137
Illegal PstI site found at 1220
Illegal PstI site found at 5048
Illegal NotI site found at 233 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 908
Illegal EcoRI site found at 1064
Illegal BglII site found at 4968
Illegal BglII site found at 5052
Illegal BglII site found at 5100
Illegal XhoI site found at 4559 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 908
Illegal EcoRI site found at 1064
Illegal XbaI site found at 298
Illegal XbaI site found at 2465
Illegal XbaI site found at 3933
Illegal XbaI site found at 5189
Illegal SpeI site found at 1
Illegal SpeI site found at 706
Illegal PstI site found at 1137
Illegal PstI site found at 1220
Illegal PstI site found at 5048 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 908
Illegal EcoRI site found at 1064
Illegal XbaI site found at 298
Illegal XbaI site found at 2465
Illegal XbaI site found at 3933
Illegal XbaI site found at 5189
Illegal SpeI site found at 1
Illegal SpeI site found at 706
Illegal PstI site found at 1137
Illegal PstI site found at 1220
Illegal PstI site found at 5048
Illegal NgoMIV site found at 1835
Illegal NgoMIV site found at 2167
Illegal NgoMIV site found at 2246
Illegal NgoMIV site found at 4777
Illegal AgeI site found at 322
Illegal AgeI site found at 944
Illegal AgeI site found at 1400 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
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| proteins | -NA- |
//function/biosynthesis
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