Increased production of zeaxanthin and other pigments by application of genetic engineering techniques to Synechocystis sp. strain PCC 6803

Abstract

The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis in Synechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and the Synechocystis beta-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP and crtB, respectively) were introduced into Synechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, and crtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the beta-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the beta-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtO gene, encoding beta-carotene ketolase, which converts beta-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

FIG. 1

Simplified biosynthesis pathway for the carotenoids (boxed) that significantly accumulate in Synechocystis sp. strain PCC 6803. Gene names for biosynthetic enzymes that have been identified in the genome are in parentheses next to the enzymes that they encode. Arrowheads indicate pathways that remain hypothetical. DMAPP, dimethylallyl diphosphate; GGPP, geranylgeranyl diphosphate.

FIG. 2

Integration platform and its use to overexpress any gene in Synechocystis sp. strain PCC 6803. (A) Construction of the integration platform by replacement of the psbAII coding region with two selectable markers: the sacB gene, encoding a levan sucrase, and aphX, a gene conferring kanamycin resistance. (B) Use of the integration platform to express a generic gene, Y, in the Synechocystis sp. strain PCC 6803 genome, in lieu of psbAII; an NdeI site at the translation start site allows a simple in-frame introduction of gene Y into the integration platform, under the control of the psbAII promoter.

FIG. 3

Segregation of Synechocystis sp. strain PCC 6803 transformants. (A) Organization of the genomic DNA at the psbAII and crtO loci in the different strains. (B) PCR products indicating complete segregation of the psbAII-KS, crtPB², and crtR² strains at the psbAII locus and the presence of an intact wild-type copy of crtP, crtB, and crtR in the crtPB² and crtR² strains. (C) PCR products indicating segregation of the appropriate strains at the crtO locus. The numbers 1/2, 3/4, 5/6, and 7/8 stand for the four different sets of primers used to amplify genes and check for complete segregation of the different strains. Primers are as follows: 1, 5′psbAII_up; 2, 3′psbAII_down; 3, 5′crtPB; 4, 3′crtPB; 5, 5′crtR; 6, 3′crtR; 7, 5′crtO; and 8, 3′crtO (Table 2). The hybridization locations of these primers are indicated in panel A.

FIG. 4

Northern blot analysis of crtR and crtPB. Probes were prepared by PCR with the cloned genes as templates. (A) Steady-state level of the crtR transcript in the wild type and the crtR² strain, determined by using a crtR gene-internal 400-bp fragment as a probe. (B) Northern blot analysis performed on the wild type and the crtPB² strain by using a crtB gene-internal 400-bp fragment as a probe. (C) Northern blot analysis performed on the wild type and the crtPB² strain by using a 400-bp fragment inside crtP as a probe.

FIG. 5

Detection of the ipi transcript and IPP isomerase. (A) Steady-state level of the yeast ipi transcript in the wild type and the ipi^Sc strain. The probe was prepared by PCR with an ipi gene-internal 400-bp fragment as a template. (B) SDS-PAGE analysis of the soluble cell fraction of the wild type and the ipi^Sc strain. This fraction, prepared by breaking cells and collecting the supernatant, was loaded (20 μg protein per lane) on an SDS gel containing 10% acrylamide. Proteins were visualized by staining the gel with Coomassie brilliant blue. Arrows show two proteins that appear to be present in the ipi^Sc strain and not in the wild type.

FIG. 6

HPLC analysis of pigments extracted from wild-type and crtR² cells of Synechocystis sp. strain PCC 6803. Chromatograms were recorded as a function of the absorbance at 440 nm. m, myxoxanthophyll; z, zeaxanthin; chl, chlorophyll a; e, echinenone; β-car, β-carotene.