Part:BBa_K4588033

4-hydroxy phenylacetate 3-monooxygenase reductase component B (E. coli), HpaB

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

Biology

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

HpaB is a reduced FAD (FADH2)-utilizing monooxygenase. This enzyme component uses FADH2 and O2 to oxidize 4-hydroxyphenylacetate [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 HpaB (BBa_K4588023) 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 salvianic acid A in an E. coli culture. This enzyme converts 4-hydroxyphenylpyruvate into 3,4-dihydroxy phenylpyruvate by adding a hydroxyl group to the benzene 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. Bloch, S. E., & Schmidt‐Dannert, C. (2014). Construction of a chimeric biosynthetic pathway for the de novo biosynthesis of rosmarinic acid in Escherichia coli. ChemBioChem, 15(16), 2393–2401. https://doi.org/10.1002/cbic.201402275

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]