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

Abstract

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.

Keywords: Synechocystis sp. PCC 6803; astaxanthin; canthaxanthin; keto-carotenoids; metabolic engineering.

Figure 1

Whole cells absorption spectra. (a) Comparison of absorption spectra of wild-type (wt, black line) and engineered Synechocystis strains constitutively expressing Brevundimonas CrtW (C-W, pink line), CrtZ (C-Z, orange line), CrtW and CrtZ in tandem (C-WZ, purple line), CrtZ and CrtW in tandem (C-ZW, violet line). All spectra are normalised at their maximal Qy absorbance (680 nm). (b) Comparison of the difference spectra “engineered strain minus wild-type” in the 425–605 nm window, colour coding as for the engineered strains in panel (a). The main spectral features of the difference spectra are marked by vertical solid lines and the wavelength indicated.

Figure 2

Simplified scheme of the astaxanthin biosynthetic pathway from endogenous β-carotene (β-car) in engineered Synechocystis strains, resulting from the heterologous expression of Brevundimonas CrtW and CrtZ. The endogenous enzymes CrtO (β-car ketolase) and CrtR (β-car hydroxylase) are shown in black. Parentheses and the dashed arrow indicate weak or possible catalytic function. Brevundimonas β-car ketolase (CrtW) and β-car hydroxylase (CrtZ) are indicated in pink and orange letters, respectively. Ech, echinenone; Can, canthaxanthin; Zea, zeaxanthin.

Figure 3

Relative distribution of the main carotenoids in the different engineered Synechocystis strains with respect to the wild-type. (a) Engineered strains constitutively expressing Brevundimonas CrtW and CrtZ (C-W, C-Z, C-WZ and C-ZW) grown at 28 °C. (b) and (c) Engineered strains expressing Brevundimonas CrtW and CrtZ in a temperature-controlled manner (TI-W, TI-Z, TI-WZ and TI-ZW) after two days induction at (b) 33 °C or (c) 39 °C. β-car, β-carotene (orange); Ech, echinenone (peach); Zea, zeaxanthin (yellow); Can, canthaxanthin (light magenta); Asx, astaxanthin (pink). wt, wild-type. Error bars indicate SE.

Figure 4

Absolute amounts of total exogenous keto-carotenoids in the engineered constitutive (C-) and temperature-inducible (TI-) Synechocystis strains. Also shown is the individual contribution of the carotenoids (Car) canthaxanthin (Can, light magenta) and astaxanthin (Asx, pink) to the total. Values are expressed as mg Car/g dry cell weight (DCW). Error bars indicate SE.

Figure 5

Relative distribution of the main carotenoids in the C-W strain constitutively expressing Brevundimonas CrtW shortly after its generation (“initial” phenotype), after several months of continuous sub-culturing in fresh medium (“silenced” phenotype) and grown to late stationary phase (“un-silenced” phenotype). Also shown is the carotenoid pattern of an exponentially growing wild-type (wt) culture for comparison. Ech, echinenone (peach); Zea, zeaxanthin (yellow); Can, canthaxanthin (light magenta); Asx, astaxanthin (pink). Error bars indicate SE.