Exploring the potential of the bacterial carotene desaturase CrtI to increase the β-carotene content in Golden Rice

Abstract

To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.

Transcriptional-Metabolic Networks in β-Carotene-Enriched Potato Tubers: The Long and Winding Road to the Golden Phenotype

“…3A). The more pronounced phenotype reported here might be attributed to species-specific differences between tomato/tobacco and potato, or it may be the result of the codon-optimized CrtI gene used in this study (Al-Babili et al, 2006), probably leading to higher CrtI protein amounts. However, western-blot experiments were unable to reveal any CrtI protein in leaves of the expressor lines (data not shown).…”

Essential oil production: relationship with abundance of glandular trichomes in aerial surface of plants

“…In one study, increased carotenoid accumulation was reported in Escherichia coli by replacing the native promoter of the chromosomal isoprenoid genes with the strong bacteriophage T5 promoter (Yuan et al 2006). In a second study (Babili et al 2006), a synthetic CrtI (carotene desaturase) gene was generated under the control of the endospermspecific glutelin B1 promoter that is important for increasing the b-carotene content in Golden Rice. Chen et al (2003) showed the promoter activity of terpenoid volatiles in Arabidopsis.…”

A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes

“…In 'Golden Rice' (Ye et al, 2000), for instance, the effectiveness of PSY (Paine et al, 2005), and not that of the bacterial carotene desaturase CrtI (Al-Babili et al, 2006), was decisive for increased carotenoid levels. Similarly, the overexpression of a bacterial PSY in canola (Brassica napus) seeds led to increased b-carotene Figure 1.…”