**4. Enhancing carotenoid content in transgenic potato**

Carotenoids are yellow to red pigments that play an essential role in human nutrition, with the most important carotenoid being β-carotene, a major source of provitamin A. Deficiency of vitamin A is a major global micronutrient problem that causes blindness and weakens the immune system [71, 72]. Carotenoids enhance may improve the immune system, reduce cardiovascular disease and cancer and help prevent atherosclerosis [73–75]. For these reasons, there is considerable interest in developing potatoes with increased levels of carotenoids [76–79].

Xanthophylls lutein, zeaxanthin, violaxanthin and neoxanthin are the major carotenoids present in the tubers of cultivated potato while that of β-carotene is found in low levels [80, 81]. The Candidate genes used in the modification of carotenoid content in transgenic potatoes are highlighted in **Figure 4** and **Table 3**. Lycopene is produced from phytoene by phytoene desaturase (CRTI), and cyclized by lycopene β-cyclase (LCY-β) to form β-carotene. While α-carotene is produced by both LCY-β and LCY-ε). The hydroxylation of α-carotene yields lutein. Two subsequent hydroxylations of β-carotene, catalyzed by carotenoid β-hydroxylase (CHYB) produce zeaxanthin. Zeaxanthin can be epoxidized by zeaxanthin epoxidase (ZEP) to form violaxanthin, which can be used by violaxanthin de-epoxidase (VDE) to regenerate zeaxanthin. The final step in carotenoid biosynthesis is the conversion of violaxanthin

#### **Figure 4.**

*Carotenoid biosynthesis pathway. The representative enzymes are PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTISO, carotenoids isomerase; LCY-e, lycopene e-cyclase; LCY-b, lycopene β-cyclase; CHB/BCH, β-carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NXS, neoxanthin synthase; CCD, carotenoid cleavage dioxygenase; Crt-ketolase. The figure is adapted and modified from Zhou et al. [31].*


#### **Table 3.**

*Genes used in the modification of carotenoid content in transgenic potatoes.*

to neoxanthin by neoxanthin synthase [92]. Introduction of various carotenogenesisrelated genes has resulted in increased production of specific carotenoids and total carotenoid content using overexpression [84, 93], co-transformation with more than two genes [83], antisense [94], and RNAi technology [75, 87].

Phytoene synthase (PSY) is the rate-limiting step in the carotenoid biosynthetic pathway [82] and manipulation of *PSY* expression resulted in enhanced carotenoid synthesis in tubers [84]. The hydroxylation of β-carotene is a second important regulatory step in carotenogenesis [95]. Tuber-specific overexpression of the bacterial phytoene synthase (*CrtB*) gene caused a 7-fold increase in total carotenoids [84]. Expression of three genes from the bacterium *Erwinia herbicol* encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene betacyclase (CrtY), under the tuber-specific patatin promoter resulted with deep yellow flesh and increased levels of β-carotene, α-carotene, lutein and violaxanthin [83]. Silencing of LCY-e resulted in increased carotenoid levels, with up to 14-fold more β-carotene in tubers [85]. Silencing of the genes encoding β-carotene hydroxylases CHY1 and CHY2 using the tuber-specific patatin promoter increased β-carotene levels along with increased levels of phytofluene, violaxanthin, neoxanthin, lutein and total carotenoids [86]. Both the overexpression and silencing of the major genes in carotenoid biosynthesis pathway produced increased carotenoids in transgenic potato tubers.

Zeaxanthin have become increasingly important due to their benefits in the prevention of degenerative diseases [96]. Zeaxanthin is an immediate biochemical derivative of β-carotene thus tubers that accumulate high levels of zeaxanthin produce β-carotene that subsequently serves as the substrate for zeaxanthin synthesis [75]. RNAi was used to silence the β-carotene hydroxylase gene (*BCH*/*CHB*), which converts β-carotene to zeaxanthin under the control of GBSS or CaMV35S promoters [75]. Transgenic lines with silenced *CHB* expression showed altered carotenoid profiles. Transformants derived from the GBSS promoter contained more β-carotene than CaMV35S transformants, demonstrating that silencing *CHB* has the potential to increase the content of carotenoids in potato for mitigating vitamin A deficiency [75]. Oxidative cleavage of carotenoids is catalyzed by carotenoid cleavage dioxygenases

#### *Transgenic Approaches for Nutritional Enhancement of Potato DOI: http://dx.doi.org/10.5772/intechopen.106898*

(CCDs). Down-regulation of *CCD4* though RNAi resulted in a 2-5-fold higher carotenoid content due to elevated violaxanthin content. Down-regulation of zeaxanthin epoxidase under the control of GBSS promoter resulted in zeaxanthin-rich potato lines, increased total carotenoids, and reduced the amount of lutein [94]. Thus, RNAi is a useful strategy to improve the carotenoid content in potato tubers thereby alleviating the vitamin A deficiency.

Astaxanthin, is an important ketocarotenoid associated with the reduction in oral cancer and mammary tumor growth [97, 98] and increasing astaxanthin and other ketocarotenoids levels has been studied in potato [88]. Astaxanthin is derived from β-carotene by 3-hydroxylation and 4-ketolation at both ionone end groups [99]. The hydroxylation reaction is widespread in many organisms, but ketolation is restricted to a few bacteria, fungi, and some unicellular green algae [100]. Previous studies used the transgenic expression of ketolase genes to produce ketocarotenoids in potato [88] as well as other Solanaceae members such as tomato [101] and tobacco [102]. A transgenic potato cultivar that accumulates increased zeaxanthin due to inactivated zeaxanthin epoxidase was co-transformed with the *ketolase* (*crtO β-carotene*) gene from the cyanobacterium *Synechocystis* under the control of a constitutive promoter. The resulting transgenic potato plants accumulated more ketocarotenoids in leaves, as well as more 3′-hydroxyechinenone, 4-ketozeaxanthin and astaxanthin in the tuber [88]. Likewise, overexpression of the *crtW* gene from the marine bacterium *Brevundimonas* under the control of GBSS promoter resulted in enhanced astaxanthin content in transgenic potato tubers [101, 103]. These and other studies reveal that transgenic potato lines can be produced with increased carotenoid content using bacterial and algal genes.

In another approach, increased carotenoid content was observed in tubers when cauliflower *Or* gene was overexpressed in potato [89, 90]. Lu et al. [89] showed that the cauliflower *Or* gene encoding a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulation can be used to increase total carotenoid and β-carotene levels in potato tubers. Overexpression of the *Or* gene induced the formation of chromoplasts and resulted in high levels of carotenoids in transgenic tubers [90, 91, 103, 104]. This increase was found to be associated with the Or-regulated stability of PSY protein in these tubers, thus facilitating continuous carotenoid synthesis in the transgenic tubers [104]. Thus, overall studies indicated that genetic manipulation of *Or* genes can be an effective strategy to achieve increased carotenoid content and high quality potato tubers.

Overall, these studies indicate that the suppression and overexpression of various genes involved in carotenoid biosynthesis under different promoters resulted in altered carotenoid content in transgenic potato tubers. Although the transgenic strategy may be an effective way for increasing carotenoids production in potato, CRISPR/ Cas9 gene editing might be challenging in tailoring efficient and non-transgenic potato cultivars with improved nutritional quality.
