A Comparison of Constitutive and Inducible Non-Endogenous Keto-Carotenoids Biosynthesis in Synechocystis sp. PCC 6803

The model cyanobacterium Synechocystis sp. PCC 6803 has gained significant attention as an alternative and sustainable source for biomass, biofuels and added-value compounds. The latter category includes keto-carotenoids, which are molecules largely employed in a wide spectrum of industrial applications in the food, feed, nutraceutical, cosmetic and pharmaceutical sectors. Keto-carotenoids are not naturally synthesized by Synechocystis, at least in any significant amounts, but their accumulation can be induced by metabolic engineering of the endogenous carotenoid biosynthetic pathway. In this study, the accumulation of the keto-carotenoids astaxanthin and canthaxanthin, resulting from the constitutive or temperature-inducible expression of the CrtW and CrtZ genes from Brevundimonas, is compared. The benefits and drawbacks of the two engineering approaches are discussed.

: Schematic representation of generated plasmids. Sequences corresponding to Synechocystis genomic regions located immediately up-and down-stream of the psbAII gene, which promote homologous recombination between the plasmid and the Synechocystis psbAII gene, are indicated by grey boxes. psbAII upstream region comprises the strong native psbAII promoter. Also indicated are the Km R gene (light grey arrow), the 3xFLAG tag (blue box) and the ribosome binding site (green box).   Transcript abundance was quantified using qRT-PCR and expression levels were normalised to rnpB (wild-type was used as calibrator). Data are the means of three qRT-PCR technical replicates for each biological duplicate, error bars indicate SE.
qRT-PCR analysis of the C-strains indicates a general lack of correlation between the transcript levels of CrtW and CrtZ and the carotenoid accumulation pattern. CrtZ is highly expressed in C-Z, but the observed increase in Zea content is relatively low (a ~30% increment with respect to the wild-type). CrtW is expressed at rather lower levels in C-W, despite being under the control of the same strong promoter as CrtZ in C-Z, but the accumulation of Can was the highest (more than 60% of the total pool). In C-WZ the relative abundance of the CrtZ transcript is several folds lower than the one of CrtW. If there were to be a simple correlation between gene transcription and product accumulation, this would explain the very similar carotenoid pattern observed in C-WZ and C-W. However the CrtW expression level in C-W was rather low.
Moreover, in C-ZW, despite CrtW transcription being several fold lower than CrtZ, the resulting carotenoid pattern phenotype was completely different from that of C-Z, and indeed C-ZW is amongst the strains accumulating the larger Asx amounts. It is arguable that the low expression of CrtW in this strain is sufficient for the production of exogenous keto-carotenoids, but at the same time highlights the need for further detailed investigations at the protein level, to achieve a better understanding of the observed phenotypes.
Experimental procedure: total RNA was extracted from each strain in the presence of NucleoZOL (Macherey-Nagel) following the manufacturer instructions. RNA integrity was checked by agarose gel electrophoresis.
The absence of contaminant DNA was checked by PCR using primer specific to the control gene rnpB (RT-rnpB_F 5'-CGTTAGGATAGTGCCACAG-3' and RT-rnpB_R 5'-CGCTCTTACCGCACCTTTG-3'), which was also used as endogenous control in qRT-PCR. cDNA synthesis was performed from 1.3 μg of DNA-free RNA. Reactions were run in the 7300 RealTime PCR System (Applied Biosystems) and data analysed using the 7300 System Software (Applied Biosystems). The following primers were used: for CrtW, RT-CrtW_F 5'-ATTGCCTTATTTGGGTTGGG-3' and RT-CrtW_R 5'-CAGGTGCCAAAGGTAAACAA-3'; for CrtZ, RT-CrtZ_F 5'-GGATTATGCACCGTTATGTG-3' and RT-CrtZ_R 5'-CACGGCAAATAAATCATTCT-3'. For each gene, cDNA dilution curves were generated (cDNA dilutions: 1/3, 1/9, 1/27 and 1/81) and used to calculate the individual real-time PCR efficiencies. Reactions were manually assembled and contained 0.25 M of each primer, 10 L of iTaq Universal SYBR Green Super mix (Bio-Rad) and 2 L of template cDNA (dilution 1/27). The PCR program consisted of: 3 min at 95°C followed by 40 cycles of 30 s at 95°C, 30 s at 56°C and 30 s at 72°C. Dissociation analysis was performed at the end of each run to confirm the specificity of the reaction. Quantitative variation was evaluated by the 2 −ΔΔCT method.