Part:BBa_K4588039

4-hydroxy phenylacetate 3-monooxygenase reductase component C (S. cerevisiae), HpaC

This part encodes 4-hydroxy phenylacetate 3-monooxygenase reductase component C from Lysinibacillus capsici

Biology

The organism this gene is initially expressed in is L. capsici.

HpaC works with HpaB together to catalyze the oxidation of 4-hydroxyphenylacetate in the presence of NADH and FAD [1]. HpaC is a coupling factor that enhances NADH oxidation for HpaB [1].

Design

This BioBrick contains a T7 promoter (BBa_I712074) for strong expression and a strong ribosome binding site (BBa_B0034) for optimal translation initiation. The composite BioBrick for HpaB consists of the coding region HpaC (BBa_K4588024) and is codon-optimized according to Escherichia coli codon bias for its expression in this bacteria. Following the coding region, a 6x His Tag (BBa_K1223006) was inserted for protein expression detection. The strong double terminator (BBa_B0015) was chosen to terminate gene transcription completely.

Usage

This enzyme is implemented in the synthesis pathway to produce rosmarinic acid in the Saccharomyces cerevisiae culture. HpaC, in complex with HpaB, is part of the two-step process of converting p-coumaric acid into caffeic acid, which adds a hydroxyl group onto the phenyl ring [2].

References

1. Xun, L., & Sandvik, E. R. (2000). Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase. Applied and environmental microbiology, 66(2), 481–486. https://doi.org/10.1128/AEM.66.2.481-486.2000

2. 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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]