Part:BBa_K4947021
A. thaliana 4-Coumarate-CoA ligase 1 Codon-Optimized CDS
This part is the gene coding for 4-Coumarate-CoA ligase 1 from A. thaliana. 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 4-Coumarate-CoA ligase 1 from A. thaliana. It is codon-optimized and domesticated for SalI, EcoRV, KpnI, PvuII, SphI, MluI, and SpeI restriction sites. This gene homolog that encodes for 4CL was chosen rationally after thorough literature review. 4CL is an enzyme involved in the biosynthetic pathway of daidzein, converting p-coumaric acid into p-coumaroyl-CoA. 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
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 [2].
Usage
This gene homolog worked best in yeast, outperforming Pl4CL1, Gm4CL3, At4CL2, Ph4CL1, Pc4CL2, and a mutant At4CL1m to produce 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. The first lane represents the amplicon of this part. Despite how faint it is, the product still had sufficient yields for downstream applications.
Significance
4CL represents the first major enzyme 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.uniprot.org/uniprotkb/Q42524/entry
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 148
Illegal EcoRI site found at 304
Illegal PstI site found at 377
Illegal PstI site found at 460 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 148
Illegal EcoRI site found at 304
Illegal PstI site found at 377
Illegal PstI site found at 460 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 148
Illegal EcoRI site found at 304 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 148
Illegal EcoRI site found at 304
Illegal PstI site found at 377
Illegal PstI site found at 460 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 148
Illegal EcoRI site found at 304
Illegal PstI site found at 377
Illegal PstI site found at 460
Illegal NgoMIV site found at 1075
Illegal NgoMIV site found at 1407
Illegal NgoMIV site found at 1486
Illegal AgeI site found at 184
Illegal AgeI site found at 640 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| protein | -NA- |
//cds/biosynthesis
//cds/enzyme
//function/biosynthesis
| protein |