Part:BBa_K4588040

4-coumaroyl CoA-ligase(Saccharomyces cerevisiae), 4Cl

This part encodes 4-coumaroyl CoA-ligase from Arabidopsis thaliana

Biology

The organism this gene is initially expressed in is A. thaliana

Four isoforms of 4-coumaroyl CoA ligase exist in A. thaliana. 4CL is an important enzyme in the biosynthesis pathway of lignin, flavonoids, and hydroxycinnamoyl esters that play roles as plant metabolites and cell wall polymers. 4CL catalyzes the last step of the phenylpropanoid pathway, which produces major specialized metabolites. Lignin allows plants to stand upright and transport water long distances and is the first mechanical defense against pathogens [1]. The enzyme follows a two-step reaction mechanism where the carboxylate substrate first undergoes adenylation by ATP, then a thioesterification in the presence of CoA to produce CoA thioesters [2].

Design

This BioBrick contains a GAL1 promoter (BBa_K2637059) that supports strong inducible expression in the presence of galactose. Following this promoter, the sequence contains a Kozak sequence (BBa_J63003) for optimal ribosome binding. The protein coding sequence (BBa_K4588029) was optimized for S. cerevisiae expression. The coding region also contains an HA tag (BBa_K1150016) for protein detection. The strong ADH1 terminator (BBa_K1486025) completely stops protein expression.

Usage

This enzyme is implemented in the synthesis pathway to produce rosmarinic acid in the S. cerevisiae culture. This enzyme converts caffeic acid into caffeoyl CoAy attaching a SCoA to the terminal carbon [3]

References

1. Li, Y., Kim, J. I., Pysh, L., & Chapple, C. (2015). Four Isoforms of Arabidopsis 4-Coumarate:CoA Ligase Have Overlapping yet Distinct Roles in Phenylpropanoid Metabolism. Plant physiology, 169(4), 2409–2421. https://doi.org/10.1104/pp.15.00838

2. Q42524 · 4CL1_ARATH https://www.uniprot.org/uniprotkb/Q42524/entry

3. Babaei, M., Borja Zamfir, G. M., Chen, X., Christensen, H. B., Kristensen, M., Nielsen, J., & Borodina, I. (2020). Metabolic engineering of saccharomyces cerevisiae for rosmarinic acid production. ACS Synthetic Biology, 9(8), 1978–1988. https://doi.org/10.1021/acssynbio.0c0004

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 551
  Illegal BglII site found at 2128
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]