Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering

Abstract

SummaryThe natural pigment astaxanthin has attracted much attention because of its beneficial effects on human health, despite its expensive market price. In order to produce astaxanthin, transgenic plants have so far been generated through conventional genetic engineering of Agrobacterium-mediated gene transfer. The results of trials have revealed that the method is far from practicable because of low yields, i.e. instead of astaxanthin, large quantities of the astaxanthin intermediates, including ketocarotenoids, accumulated in the transgenic plants. In the present study, we have overcome this problem, and have succeeded in producing more than 0.5% (dry weight) astaxanthin (more than 70% of total caroteniods) in tobacco leaves, which turns their green color to reddish brown, by expressing both genes encoding CrtW (b-carotene ketolase) and CrtZ (b-carotene hydroxylase) from a marine bacterium Brevundimonas sp., strain SD212, in the chloroplasts. Moreover, the total carotenoid content in the transplastomic tobacco plants was 2.1-fold higher than that of wild-type tobacco. The tobacco transformants also synthesized a novel carotenoid 4-ketoantheraxanthin. There was no significant difference in the size of the aerial part of the plant between the transformants and wild-type plants at the final stage of their growth. The photosynthesis rate of the transformants was also found to be similar to that of wild-type plants under ambient CO 2 concentrations of 1500 lmol photons m )2 s )1 light intensity.

Synthetic Botany

“…The high efficiency of homologous recombination in the chloroplast (Blowers et al 1989) also allows the simultaneous introduction of modifications to several sites of the chloroplast genome by means of cotransformation (Kindle et al 1991), encouraging the implementation of phenotypic traits based on multiple foreign genes. Multiple genes may be conveniently organized in operon-like polycistronic units (Hasunuma et al 2008), which can be processed into more efficiently translated monocistronic transcripts by the incorporation of intercistronic expression elements (Lu et al 2013). Previous studies have also highlighted the activity of chloroplast promoters in bacteria (Brixey et al 1997) and of bacterial promoters in the chloroplast (Newell et al 2003).…”

“…No gene silencing has been observed in chloroplasts despite such high accumulation of foreign transcripts (169 times higher than in nuclear transgenic plants, Lee et al 2003) or foreign protein (46% of total leaf protein [De Cosa et al 2001]). Metabolic engineering for the production of bioplastic monomers (Bohmert-Tatarev et al 2011) and compounds of nutritional relevance (Craig et al 2008;Hasunuma et al 2008;Apel and Bock 2009) has also been applied to chloroplasts. The high biosynthetic capacity of the chloroplast is closely linked to the polyploid nature of the system: At 10 -100 chloroplasts per cell, and 10 -1000 genomes per chloroplast (Bendich 1987), stable integration of a transgene into the chloroplast genome enables a substantial amplification in transgene copy number.…”

Pathway engineering of plants toward astaxanthin production

“…Moreover, the total carotenoid amount in the transplastomic tobacco was 2.1-fold higher than that of wild-type tobacco , suggesting up-regulation for carotenoid biosynthesis probably due to the stream from the plant-original carotenoids to the ketocarotenoid formation. Hasunuma et al (2008) also found that the simultaneous high expression of crtZ (SD212) in addition to crtW (SD212) was necessary for the efficient synthesis of astaxanthin, i.e., a transplastomic tobacco plant expressing only the crtW (SD212) gene produced much lower level of astaxanthin (1.88 mg g Ϫ1 dry weight) in the leaves (Table 1).…”

“…Jayaraj et al (2008) expressed the bkt1 gene in carrot roots using the double CaMV 35S promoter, and found that the transgenic roots were able to accumulate large amounts of ketocarotenoids (236 mg g Ϫ1 fresh weight; 68% of total carotenoids) containing 91.6 mg g Ϫ1 fresh weight of astaxanthin, 57.0 mg g Ϫ1 fresh weight of adonirubin and 50.1 mg g Ϫ1 fresh weight of canthaxanthin (Table 1). Recently, Hasunuma et al (2008) introduced the genes [crtW (SD212) and crtZ (SD212)] encoding the CrtW and CrtZ proteins of Asterisks indicate data for dry weight of tissue.…”

Transplastomic plants for innovations in agriculture. A review

“…Similarly to Kumar et al (2012), a general increment in isoprenoid content (chlorophyll a, phytol, β-carotene, lutein, antheraxanthin, solanesol, and β-sitosterol) was observed when the enzyme responsible for the first committed step of the plastidial nonmevalonate pathway, i.e., 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) from Synechocystis was overexpressed in transplastomic tobacco plants (Hasunuma et al 2008a). …”