Part:BBa_K1604021

J23100 + β-carotene 15,15’-dioxygenase (blh gene)

blh encodes for β-carotene 15,15’-dioxygenase, that cleaves β-carotene into retinal

Usage and Biology

β-carotene 15,15’-dioxygenase is involved in retinal biosynthesis and catalizes the last step of the pathway by cleaving one molecule of β-carotene into two molecules of retinal. Blh is the gene that encodes for this enzyme, and was first isolated in the uncultured bacteria SAR86 (ref). Blh is present in other organisms (i.e. cyanobacteria and algae) however enzymes involved in the cleavage of β-carotene that involve different mechanisms are found in many other organisms.

The enzyme requires oxygen for the cleavage of β-carotene, which was demonstrated to be provided by water (ref). This data are consistent with the fact the bacteria lives in deep marine waters. It requires Fe2+ that is coordinated by 4 His residues that are highly conserved.

This device is composed of a constitutive promoter a strong RBS and blh. It was characterized by the Unitn Trento 2015 team in the composite part BBa_K1604022 which contains the enzymes necessary for β-carotene biosynthesis.

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FIGURE 1. Loss of β-carotene. NEBβ cells were cotransformed with BBa_K1604021 (in puC57) and BBa_ K1604020 (aracpBad-β carotene). The cells were grown up to an OD of 0.6 and induced with 5 mM of arabinose for 24 hours. Co-trasformation of BBa_K1604021 and BBa_ K1604020 (B), with 5 mM arabinose (C), and with 5 mM arabinose, 5 uM FeSO4 and 10 mM of ascorbate (D); empty cells (E). Expression of blh causes the loss of the typical orange colored pellet of β-carotene expressing cells. </b>

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FIGURE 2. Extraction of β-carotene and retinal. NEBβ cells were cotransformed with BBa_K1604021 (in puC57) and BBa_ K1604020 (aracpBad-β carotene). BBa_K1604020 only was used for positive control. The cells were grown in 100 mL of LB and induced as described in figure 1. After 24 hours the cells were span down and the supernatant was discarded. The pellets were incubated with 2.5 mL of acetone for 10 min at 50C. Afterward they were centrifuged to recover the extracted pigments. The samples were diluted 1:7 in acetone and the spectra were taken with UV-VIS Agilent Cary 8454 UV-Vis equipped with a deuterium and tungsten lamps. The spectra were acquired between 300 and 800 nm and blanked with acetone. UV-Vis spectra: BBa_K1604020 (β-carotene producer cells) with arabinose Fe²⁺ and ascorbate (purple), BBa_K1604020 (β carotene) + BBa_K1604021 (blh) with Fe and ascorbate. </b>

Our data show that there is a loss of β carotene when the cells express blh. The UV_Vis analysis confirmed the loss of β carotene (452 nm), but did not evidenced the presence of retinal (373 nm). For a more detailed characterization please check BBa_K1604022.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 245
  
• 1000
  COMPATIBLE WITH RFC[1000]