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 ␤-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 ␤-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 ␤-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 ␤-carotene ketolase, which converts ␤-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.

A photoactive carotenoid protein acting as light intensity sensor

“…S2 A). To obtain a strain overexpressing the OCP, the genome region containing the slr1963 and slr1964 genes was cloned in the pPSBA2 ampicillin-resistant vector (31). Nucleotides encoding for 6 His were added in the 3 Ј side of the slr1963 gene and a 1.3-kb kanamycin resistance gene was inserted.…”

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

“…Hom2 stretches from chromosomal position 2301292 to 2301835 and was cloned into the XhoI and KpnI sites of pBluescript II SK(ϩ). The kanamycin resistance cassette (aphX), originating with the E. coli transposon Tn903, was amplified from pPSBA2KS (23) and cloned into the SalI site of pBluescript II SK(ϩ). To ease further cloning, the remaining multiple-cloning site (MCS) of the integration plasmid was designed to be compatible with the BioBrick restriction sites.…”

Flavodiiron Protein Flv2/Flv4-Related Photoprotective Mechanism Dissipates Excitation Pressure of PSII in Cooperation with Phycobilisomes in Cyanobacteria

“…The Dflv4 strain was described by Zhang et al (2012). To obtain flv4-2/OE, the operon fragment was amplified by PCR with the 1Fw primer (59-AATACCATATGGTTACCCTAATTGATTCTCC-39) and the 2Rv primer (59-AGTTCGCTAGCCTAATATTGTCCCCCCGATTTG-39), and then inserted in the NdeI-NheI sites of the modified pPSBA2KS plasmid (Lagarde et al, 2000) containing both spectinomycin-and kanamycin-resistance cassettes. Insertion of the flv4-2 operon at the NdeI-NheI sites resulted in placing of the operon under control of the psbA2 promoter and deletion of the kanamycinresistance cassette.…”