Fermentative carotenoid production

Details Fermentative carotenoid production Fermentative carotenoid production

1997-12-01

2001-09-18

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing a carotene nucleus

C435S132000, C435S155000, C435S252330, C536S023200

Reexamination Certificate

active

06291204

ABSTRACT:

BACKGROUND OF THE INVENTION
Over 600 different carotenoids have been described from carotenogenic organisms found among bacteria, yeast, fungi and plants. Currently only two of them, &bgr;-carotene and astaxanthin are commercially produced in microorganisms and used in the food and feed industry. &bgr;-carotene is obtained from algae and astaxanthin is produced in Pfaffia strains which have been generated by classical mutation. However, fermentation in Pfaffia has the disadvantage of long fermentation cycles and recovery from algae is cumbersome. Therefore it is desirable to develop production systems which have better industrial applicability, e.g. can be manipulated for increased titers and/or reduced fermentation times.
Two such systems using the biosynthetic genes form
Erwinia herbicola
and
Erwinia uredovora
have already been described in WO 91/13078 and EP 393 690, respectively. Furthermore, three &bgr;-carotene ketolase genes (&bgr;-carotene &bgr;-4-oxygenase) of the marine bacteria
Agrobacterium aurantiacum
and Alcaligenes strain PC-1 (crtW) [Misawa, 1995, Biochem. Biophys. Res. Com. 209, 867-876][Misawa, 1995, J. Bacteriology 177, 6575-6584] and from the green algae
Haematococcus pluvialis
(bkt) [Lotan, 1995, FEBS Letters 364, 125-128][Kajiwara, 1995, Plant Mol. Biol. 29, 343-352] have been cloned.
E. coli
carrying either the carotenogenic genes (crtE, crtB, crtY and crtI) of
E. herbicola
[Hundle, 1994, MGG 245, 406-416] or of
E. uredovora
and complemented with the crtW gene of
A. aurantiacum
[Misawa, 1995] or the bkt gene of
H. pluvialis
[Lotan, 1995][Kajiwara, 1995] resulted in the accumulation of canthaxanthin (&bgr;,&bgr;-carotene-4,4′-dione), originating from the conversion of &bgr;-carotene, via the intermediate echinenone (&bgr;,&bgr;-carotene-4-one).
Introduction of the above mentioned genes (crtW or bkt) into
E. coli
cells harbouring besides the carotenoid biosynthesis genes mentioned above also the crtZ gene of
E. uredovora
[Kajiwara, 1995][Misawa, 1995], resulted in both cases in the accumulation of astaxanthin (3,3′-dihydroxy-&bgr;,&bgr;-carotene-4,4′-dione). The results obtained with the bkt gene, are in contrast to the observation made by others [Lotan, 1995], who using the same experimental set-up, but introducing the
H. pluvialis
bkt gene in a zeaxanthin (&bgr;,&bgr;-carotene-3,3′-diol) synthesising
E. coli
host harbouring the carotenoid biosynthesis genes of
E. herbicola,
a close relative of the above mentioned
E. uredovora
strain, did not observe astaxanthin production.
SUMMARY OF THE INVENTION
Novel proteins of microorganism E-396 (FERM BP-4283) and the DNA sequences which encode these proteins have been discovered which provide an improved biosynthetic pathway from farnesyl pyrophosphate and isopentyl pyrophosphate to various carotenoids, especially zeaxanthin, astaxanthin, adonixanthin and canthaxanthin.


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Pasamontes, et al., “Isolation and characterization of the carotenoid biosynthesis genes ofFlavobacteriumsp. strain R1534,”Gene, vol. 185, pp. 35-41 (1997).
Misawa et al., “Canthaxanthin Biosynthesis by the Conversion of Methylene to Keto Groups in a Hydrocarbon &bgr;-Carotene by a Singele Gene”, Biochemical and Biophysical Research, vol. 209, No. 3, pp. 867-876 (1995).
Misawa et al., “Structure and Functional Analysis of a Marine Bacterial Carotenoid Biosynthesis Gene Cluster and Astaxanthin Biosynthetic Pathway Proposed at the Gene Level”, Journal of Bacteriology, vol. 177, No. 22., pp. 6575-6584 (Nov. 1995).
Kajiwara et al., “Isolation and functional identification of a novel cDNA for astaxathin biosynthesis fromHaematococcus pluvialis, and astaxathin synthesis inEscherichia coli”, Plant Molecular Biology, 29:343-352 (1995).
Hundle et al., “Functional assignment of Erwinia herbicola Eho 10 carotenoid genes expressed inEscherichia coli”, Mol. Gen. Genet., 245:406-416 (1994).

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