Part:BBa_K3925009

Decompose alcohol

Decompose alcohol

Alcohol dehydrogenase gene (adh), Production of acetaldehyde from ethanol dehydrogenation Aldehyde dehydrogenase gene (ald) Dehydrogenation of acetaldehyde to acetic acid

Characterization of Sortase BBa_K3925009 (and comparison to BBa_K857000)

pSB-BBa_K857000 only contains the alcohol dehydrogenase of BBa_K857000. In order to improve its enzyme activity, we modified BBa_K3925009. In addition to the alcohol dehydrogenase of BBa_K857000, pSB-BBa_K3925009 also has BBa_K3925005-NAD synthetase gene (nadE) and BBa_K3925007-NADH oxidase gene (nox).

In order to more intuitively reflect the expression of functional genes of the engineered strains E.coli pSB-BBa_K857000 and E.coli pSB-BBa_K3925009, we tested the enzymatic activity of various exogenous enzymes and the coenzyme NAD+ under the conditions of in vitro culture of different strains. content.

The results are shown in the figure. In the crude enzyme extracts of bacteria, E. coli 1917 basically did not show alcohol dehydrogenase, acetaldehyde dehydrogenase and NADH oxidase activities, and the content of the coenzyme NAD+ was extremely low; E. The ADH activity of coli pSB-BBa_K857000 was 47.91 ± 3.12 U/mL, the ALDH activity was 33.57 ± 2.59 U/mL, and NADH oxidase activity was not detected; while the ADH activity of E. coli pSB-BBa_K3925009 was 81.41 ± 3.64 U/mL mL, ALDH activity is 57.56 ± 1.48 U/mL, nox activity is 14.4 ± 2.29 U/mL, bacterial expression of ADH and ALDH enzyme activity is greatly increased, and the content of coenzyme NAD+ is also greatly increased compared with strain E.coli pSB-BBa_K857000. It shows that the expression of NAD synthase gene nadE and NADH oxidase gene nox can help increase the content of dehydrogenase coenzyme NAD in bacterial cells, thereby enhancing the degradation ability of alcohol dehydrogenase and acetaldehyde dehydrogenase.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 349
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 765
  
• 1000
  COMPATIBLE WITH RFC[1000]