Part:BBa_K343006

Expresses B-carotene monooxygenase on a constitutive promotor

This generator expresses a beta-carotene 15,15'-monooxygenase from Drosophila melanogaster.

Beta-carotene monooxygenase

β,β-carotene-15,15′-monooxygenase is an enzyme that cleaves beta-carotene into two molecules of retinal via the following reaction: Beta-carotene + O(2) <=> 2 retinal [1].

Beta-carotene monooxygenase plays an important role in animal vision, as retinal forms the chemical basis for vision in animals [2]. Animals cannot synthesize retinal de novo and thus relies on beta-carotene monooxygenase to transform carotenoids (beta-carotene, alpha-carotene, gamma-carotene, and beta-cryptoxanthin) into retinal [3].

Here we present a BioBrick containing the gene encoding this enzyme, and show that it produces retinal both when beta-carotene is added directly to bacteria containing this BioBrick; and in bacteria double transformed with this BioBrick and the Cambridge 2009 BBa_K274210 (CrtEBIY under constitutive promoter) BioBrick [4] which produces beta-carotene in vivo.

Background

Retinal synthesis pathway

Only two mechanisms for collecting light energy and converting it into chemical energy have been found in nature so fare. The first mechanism depends upon photochemical reaction centers (a multisubunit protein complex containing chlorophylls or bacteriochlorophylls, in which light energy is transduced into redox chemistry). The second mechanism uses rhodopsins, retinal-binding proteins that respond to light stimuli [5].

The latter mechanism is found extensively througout nature, which has been speculated to be because of the ease, with which this system works; lateral transfer of rhodopsin-based photosystems requires only the genes encoding the rhodopsin apoprotein and a carotenoid oxygenase that produces retinal [5].

The use of rhodopsin photosystes could be of great use in synthetic biology, but this also requires a retinal-forming brick, which we present here.

Beta-carotene monooxygenase gene

The beta-carotene monooxygenase gene (also called "neither inactivation nor afterpotential B" or NinaB for short) was cloned from Drosophila melanogaster cDNA [7]. This gene was the first beta-carotene monooxygenase to be molecularly identified and studied for its function [8]. Formerly, this enzyme has been called beta-carotene dioxygenase, but it is now known that only one atom of the dioxygen is incorporated into retinal, hence the name change [9].

The enzyme requires Fe(II) and bile salts as co-factors. In the presence of FeSO4/ascorbate and using varying amounts of substrate of beta-caroten (800 pmol to 100 pmol), the apparent Km value of the enzyme was estimated to be 5 mM [8].

β,β-carotene-15,15′-monooxygenase cleaves beta-carotene into two molecules of retinal via the following reaction: Beta-carotene + O(2) <=> 2 retinal [1].

The reaction proceeds in three stages, epoxidation of the 15,15′-double bond, hydration of the double bond leading to ring opening, and oxidative cleavage of the diol formed [9].

Protein structure

The protein has a calculated molecular weight of 69.94 kilodaltons. The protein structure of beta-carotene monooxygenase is not known.

Retinal

Retinal

In the presence of beta-carotene, this BioBrick creates retinal. Retinal (also called retinaldehyde, vitamin A aldehyde or RAL) has the molecular formular C20H28O and weighs 284.43572 [g/mol]. C 84.45%, H 9.92%, O 5.63%. Melting point at 61-64 degrees celcius. The molecule can exist in four stereoisomeric forms [6].

The appearance of the molecule is orange crystals (in petr ether). UV max is 373 nm (in cyclohexane). Soluble in ethanol, chloroform, cyclohexane, petr ether and oils [6].

Usage and parameters

Usage

This brick needs beta-carotene to function. In experiments, addition of FeSO4 and ascorbat was shown to increase the activity of the enzyme [8]. We speculate that normal strains of E. coli and the media the bacteria are grown in contain these cofactors.

In double transformed bacteria with this BioBrick along with the beta-carotene-producing BioBrick, BBa_K274210, we have observed that the retinal and beta-carotene production is more active in the stationary phase then in the exponential growth phase in TOP 10 and MG1655 strains.

Performance

Response time: No measurable amounts of retinal have been produced, thus rendering a response time measurement impossible. The HPLC determination for the part can be seen [http://2010.igem.org/Team:SDU-Denmark/project-p#HPLC_determination_of_beta-carotene_and_retinal_production here]

Production rate: No measurable amounts of retinal have been produced, thus rendering a production rate measurement impossible. The HPLC determination for the part can be seen [http://2010.igem.org/Team:SDU-Denmark/project-p#HPLC_determination_of_beta-carotene_and_retinal_production here]

Plasmid stability: A stability assay has been performed. The data can be accessed [http://2010.igem.org/Team:SDU-Denmark/project-p#Stability_assay_3 here].
Almost all of the bacteria had shed the plasmid after 20 generations, suggesting that the plasmid is only stable within the cell for a few generations (<20). This is presumably due to the strain brought upon the bacteria by the plasmid. When the bacteria are carrying a high-copy plasmid like pSB1C3-K343006, it is plausible that the bacteria will quickly shed the burdening plasmid when no longer exposed to a selection pressure.

Growth rate: A growth assay has been performed. The data can be accessed [http://2010.igem.org/Team:SDU-Denmark/project-p#Growth_assay_3 here]
From our data we see no significant difference between the plasmid carrying bacteria and the wild type. This can be said to be quite contradictory to our results obtained from the stability assay. The transitory stability of pSB1C3-K343006 suggests that it is highly unfavorable for the bacteria. Therefore it might be expected that the growth of the bacteria containg this plasmid would be affected. Thus, however much a disadvantage the plasmid pose to the bacteria, their growth are not significantly influenced by the plasmid. The added reproduction load due to the plasmids might also prolong the lag phase of the bacteria. Whether this is the case cannot be concluded based on this experiment as no lag phase was observed in this experiment.

Compatibility

This brick has been tested in the following plasmids and strains:

Chassis: E. coli TOP10, E. coli MG1655.

Plasmids: PSB1C3 (high-copy), PSB3C5 (low-copy).

Devices: Device has been shown to work with BBa_K274210.

Risk-assessment

A general risk assessment of our used E. coli stains can be found at our team [http://2010.igem.org/Team:SDU-Denmark/safety-b#Monooxygenase_.28Part_K343001.29 homepage]

General use

This BioBrick poses no treat to the welfare of people working with it, as long as this is done in at least a level 1 safety laboratory by trained people. No special care is needed when working with this BioBrick.

Potential pathogenicity

We do not recommend using this BioBrick for any type of system in humans or animals for the following reasons:

1. Retinoic acid, which retinal can degrade into, can affect gene expression and function of almost any cell, including cells of the immune system; it also plays a fundamental role in cellular functions by activating nuclear receptors [11].
2. Vitamin A toxicity can lead to hepatic congestion and fibrosis [12].
3. Vitamin A and its derivatives have been implicated as chemopreventive and differentiating agents in a variety of cancers [13].

These effects have been observed in humans. Please see references for more information.

Environmental impact

Beta-carotene monooxygenase is found in a wide variety of different bacteria, insects and animals [5]. As such, we would be cautious as to letting a system containing this BioBrick into the wild, since it's function might conflict with existing systems. On the other hand, one might argue that since its function is already available in nature, the function is widely available.

The product of the BioBrick, retinal, also plays an important function in nature and animals. For this reason we believe that the BioBrick could be used in controlled settings, but not in the wild.

Results

UV-Vis spectroskopy and HPLC analysis of organic extractions of the bacteria containing K343006 didn't give conclusive results conserning the Retinal production.
The stability of pSB1C3-K343006 is most likely <20 generations.
The growth assay showed no significant difference between the wild type and the cells containing pSB1C3-K343006.
For further information, raw data and background of the assay see [http://2010.igem.org/Team:SDU-Denmark/K343006 characterization of K343006] on our team wiki

References

1. ENZYME entry 1.14.99.36 [Internet]. [cited 2010 Oct 13];Available from: http://www.expasy.org/cgi-bin/nicezyme.pl?1.14.99.36
2. von Lintig J, Dreher A, Kiefer C, Wernet MF, Vogt K. Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2001 Jan 30;98(3):1130 -1135.
3. Retinal - Wikipedia, the free encyclopedia [Internet]. [cited 2010 Oct 13];Available from: http://en.wikipedia.org/wiki/Retinal
4. Part:BBa K274210 - parts.igem.org [Internet]. [cited 2010 Oct 13];Available from: https://parts.igem.org/Part:BBa_K274210
5. Bryant DA, Frigaard N. Prokaryotic photosynthesis and phototrophy illuminated. Trends Microbiol. 2006 Nov;14(11):488-496.
6. Retinaldehyde - PubChem Public Chemical Database [Internet]. [cited 2010 Oct 13];Available from: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1070
7. ninaB neither inactivation nor afterpotential B [Drosophila melanogaster] - Gene result [Internet]. [cited 2010 Oct 13];Available from: http://www.ncbi.nlm.nih.gov/gene/41678
8. von Lintig J, Vogt K. Filling the Gap in Vitamin A Research. Journal of Biological Chemistry. 2000 Apr 21;275(16):11915 -11920.
9. ENZYME: 1.14.99.36 [Internet]. [cited 2010 Oct 13];Available from:http://www.genome.jp/dbget-bin/www_bget?ec:1.14.99.36
10. Kelley LA & Sternberg MJE. Protein structure prediction on the web: a case study using the Phyre server. Nature Protocols. 4, 363 - 371 (2009).
11. Spiegl N, Didichenko S, McCaffery P, Langen H, Dahinden CA. Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid. Blood. 2008 Nov 1;112(9):3762-71.
12. Russell RM. The vitamin A spectrum: from deficiency to toxicity. American Journal of Clinical Nutrition, Vol. 71, No. 4, 878-884, April 2000.
13. Pasquali D, Thaller C, Eichele G. Abnormal level of retinoic acid in prostate cancer tissues. J Clin Endocrinol Metab. 1996 Jun;81(6):2186-91.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 765
  Illegal BamHI site found at 500
  Illegal BamHI site found at 1757
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1361
  Illegal BsaI site found at 1809