Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis

Abstract

It is proposed that the lytB gene encodes an enzyme of the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A mutant of the cyanobacterium Synechocystis strain PCC 6803 with an insertion in the promoter region of lytB grew slowly and produced greenish-yellow, easily bleached colonies. Insertions in the coding region of lytB were lethal. Supplementation of the culture medium with the alcohol analogues of IPP and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely alleviated the growth impairment of the mutant. The Synechocystis lytB gene and a lytB cDNA from the flowering plant Adonis aestivalis were each found to significantly enhance accumulation of carotenoids in Escherichia coli engineered to produce these colored isoprenoid compounds. When combined with a cDNA encoding deoxyxylulose-5-phosphate synthase (dxs), the initial enzyme of the DOXP pathway, the individual salutary effects of lytB and dxs were multiplied. In contrast, the combination of lytB and a cDNA encoding IPP isomerase (ipi) was no more effective in enhancing carotenoid accumulation than ipi alone, indicating that the ratio of IPP and DMAPP produced via the DOXP pathway is influenced by LytB.

FIG. 1

The DOXP pathway for biosynthesis of the isoprenoid precursors IPP and DMAPP in E. coli. Gene products implicated in the pathway are boxed. The order of the reactions catalyzed by the products of ygbB, ygbP, and ychB is uncertain. Later reaction steps in the pathway remain to be determined. Note that a gene encoding IPI, although present in E. coli, is not routinely found in organisms that utilize the DOXP pathway (Table 1). The plasmid pAC-LYC (10), shown to the lower right, encodes enzymes (products of the crtE, crtB, and crtI genes) that lead to the synthesis of the pink isoprenoid lycopene from IPP and DMAPP. Cm, chloramphenicol resistance gene; G-3-P, d-glyceraldehyde-3-phosphate; GAPD, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 2

Schematic illustration of Synechocystis strain PCC 6803 genomic DNA encompassing the lytB gene. A kanamycin resistance gene (Kan^r) was inserted into the NheI site immediately upstream of the lytB gene.

FIG. 3

PCR amplification of lytB in Synechocystis PCC6803 using genomic DNA obtained from the wild-type strain and from a mutant with a Kan^r insertion in the NheI site immediately upstream of lytB. The ca. 3.7-kb PCR product from the insertion mutant lytB(NheI)::Kan^r, displayed in the right lane of this 1% agarose gel, is the expected 1.2 kb larger than the wild-type product of about 2.5 kb (middle lane). No trace of the wild-type (i.e., uninterrupted) gene is discernable in the mutant lane. The PCR primers were those used for the initial amplification of lytB and flanking regions (see Fig. 2 and Materials and Methods) and lie outside the BamHI and SmaI sites that define the cloned DNA fragment used to transform Synechocystis. The standards lane (left lane) contains DNA fragments of the indicated sizes.

FIG. 4

Impaired growth of Synechocystis strain PCC 6803 lytB(NheI)::Kan^r mutant strain on BG-11 agar plates containing 50 μg of kanamycin per ml (upper panel) is much improved by supplementation of the growth medium with 2.5 mM (each) 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol (lower panel). The dark blue-green color of the colonies in the lower panel is comparable to that displayed by the wild type grown on BG-11 agar plates lacking kanamycin and alcohols.

FIG. 5

A lycopene-accumulating strain of E. coli that contains a plasmid with an Adonis aestivalis cDNA encoding LytB (pAplytB; see Materials and Methods) yields colonies much darker (the dark pink colonies) and containing much more of this pink isoprenoid than colonies produced by cells in which the second plasmid is the empty cloning vector (pale pink colonies).

FIG. 6

Alignment of amino acid sequences deduced from a bacterial (Escherichia coli; Ec), a cyanobacterial (Synechocystis strain PCC 6803; Sy), and a plant (Adonis aestivalis; Aa) lytB gene or cDNA. Residues identical in a given position for the three sequences are in white text on a black background. Where two of the three are identical, residues are in black text on a grey background. A pound sign (#) below the alignment denotes a position at which the amino acid residues are identical in more than 90% and/or similar in 100% of 22 sequences deduced from lytB homologues for which the complete coding regions are currently available in GenBank (5 April 2000). GenBank accession numbers are as follows: A. aestivalis, AF270978:27-1421; E. coli, AE000113:5618-6568; and Synechocystis strain PCC 6803, D64000:46364-47584.