A New Type of Asymmetrically Acting β-Carotene Ketolase Is Required for the Synthesis of Echinenone in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

We have isolated, based on the knowledge of the complete genomic sequence of the cyanobacterium Synechocystis sp. PCC 6803, an open reading frame (slr0088) similar to known bacterial carotene desaturases and have analyzed the function of the encoded protein. Surprisingly, this protein has no detectable desaturase activity with phytoene, hydroxyneurosporene, or -carotene as substrates, but is rather a ␤-carotene ketolase that acts asymmetrically introducing a keto group on only one of the two ␤-ionone rings of ␤-carotene to generate echinenone. This is in contrast to the so far characterized ␤-carotene ketolases that act symmetrically, producing the di-keto carotenoid canthaxanthin from ␤-carotene without significant accumulation of echinenone. We have designated this new gene crtO. The function of the crtO gene product has been demonstrated by 1) the biosynthesis of echinenone when the crtO gene is expressed in an Escherichia coli strain able to accumulate ␤-carotene, 2) the in vitro biosynthesis of echinenone from ␤-carotene with cell free extracts from E. coli cells that express the crtO gene, and 3) the absence of echinenone in a Synechocystis strain in which the crtO gene has been insertionally inactivated. The primary structure of the Synechocystis asymmetric ketolase bears no similarity with the known ␤-carotene ketolases. crtO is not required for normal growth under standard or high light conditions, neither is the photosynthetic activity of the crtO-deficient strain affected.

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

“…The crtZ gene product (a b-carotene hydroxylase) converts b-carotene into zeaxanthin in two hydroxylation steps at the 3 and 3 0 positions of the b rings. Synthesis of the echinenone cofactor was accomplished by means of the pACCAR25DcrtXZcrtO plasmid, in which the crtZ gene was inactivated by cloning the crtO gene encoding a b-carotene ketolase (31,32) into the HpaI cloning site of the crtZ gene. After transformation, cells were selected on plates containing 50 mg/mL ampicillin and 34 mg/mL chloramphenicol and incubated overnight at 37 C. Starter cultures of 10 mL Luria-Bertani (LB) medium with the corresponding antibiotics were inoculated from single colonies and grew overnight at 37 C at 200 rpm in an orbital shaker (infors HT, Bottmingen, Switzerland).…”

The zeaxanthin biosynthesis enzyme ²‐carotene hydroxylase is involvedin myxoxanthophyll synthesis in Synechocystis sp. PCC 6803

“…This procedure allows a gentle exposure of transformants to increasing kanamycin concentration. Gene names for biosynthetic enzymes that have been identified in the genome are in parentheses next to the enzyme that they encode: crtB [19], crtP [20], crtQ [21], crtO [22] and crtR [9]. Dotted arrow indicates pathways that remain hypothetical.…”

Control of light-dependent keto carotenoid biosynthesis inNostoc7120 by the transcription factor NtcA

“…Most keto carotenoid-synthesizing cyanobacteria utilize two different ketolases. One is encoded by the bacterial CrtW gene [12], the other one by crtO which is found only in cyanobacteria and which in Synechocystis encodes a mono ketolase specific for the synthesis of echinenone [13]. In Nostoc PCC 7120 [14] and also in Nostoc PCC 73102 [10] both types of ketolase genes are present simultaneously.…”