Part:BBa_K4588034

Lactate dehydrogenase(E. coli), DLDH

This part encodes the enzyme lactate dehydrogenase from Escherichia coli

Biology

The organism this gene is initially expressed in is Escherichia coli

E. coli uses DLDH to convert pyruvate into D-lactate using nicotinamide adenine dinucleotide- hydrogen (NADH) under anaerobic conditions and low pH [1]. Electrons from D-lactate oxidation are transferred to the ubiquinone/cytochrome electron transfer chain, providing energy for the active transport of amino acids and sugars across cell membranes [2].

Design

The L-rhamnose inducible promoter (BBa_K914003) allows for moderate upregulation of recombinant protein expression in the presence of rhamnose. Following the promoter is a strong ribosome binding site (BBa_B0034) for optimal translation initiation. The coding region consists of the BioBrick basic part BBa_K4588022 that was codon optimized. 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 4-dihydroxy phenyllactate by replacing a carbon double-bonded oxygen with a carbon-bonded hydroxyl group. In the presence of 3,4-dihydroxy phenylpyruvate, DLDH will convert it to salvianic acid A by replacing a carbon double-bonded oxygen with a carbon-bonded hydroxyl group [3].

This part can be used in a Bio-Safety Level 1 (BLS1) laboratory and was obtained by DNA synthesis.

References

1. Bunch, P. K., Mat-Jan, F., Lee, N., & Clark, D. P. (1997). The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology (Reading, England), 143 ( Pt 1), 187–195. https://doi.org/10.1099/00221287-143-1-187

2. P06149 · DLD_ECOLI. UniProt. (n.d.). https://www.uniprot.org/uniprotkb/P06149/entry

3. 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]