Engineering Plants to Improve Vitamin E Content

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Various groups have reported engineering tocopherol content composition in Arabidopsis leaves and seeds by overexpression of various pathway enzymes (Table 18.3) [29, 32, 47, 59, 64, 74, 91, 92].

Seed-specific expression of Arabidopsis VTE3 and VTE4 alone or together did not significantly alter the total level of tocopherols in transgenic soybean seed but had a dramatic impact on tocopherol composition [92]. Overexpression of VTE3 alone increases soybean seed y- and a-tocopherol levels and correspondingly reduces the levels of 5- and P-tocopherols. Like overexpression of VTE4 in Arabidopsis seed [29], VTE4 overexpression in soybean seed converts y-tocopherol almost completely to a-tocopherol, a sevenfold increase to 75% of the total, and 5-tocopherol almost completely to P-tocopherol, a tenfold increase to 25% of the total. Overexpression of VTE3 and VTE4 together shifted the tocopherol composition of soybean seeds from only 10% of a-tocopherol to about 90% a-tocopherol.

Sense expression of the barley HPD gene, under control of the 35S promoter, resulted in an up to twofold increase in the tocochromanol content of tobacco seeds. A similar increase was obtained by sense expression of the gene encoding HPT from Synechocystis PCC6803 under the control of the seed-specific napin promoter [64]. Dufourmantel et al. have expressed a sensitive bacterial HPPD gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean. HPPD accumulates to approx. 5% of the total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the overexpression of HPPD had no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively.

Overexpression of CHL P in tobacco plants resulted in a four- to sixfold and a two- to threefold increase in tocopherol level in the leaves and seeds, respectively, compared to the wild-type plants. Interestingly, the transgenic progeny plants had higher tocopherol content than the nontransformed control [62].

Table 18.3 Metabolic engineering and the potential level of vitamin E in plants

Gene source

Promoter used

Gene

Plant species engineered

Product level

Referern

A. thaliana

35S

VTE1

A. thaliana leaf

7

[102]

A. thaliana, Zea

Napin

VTE1

B.napus seed

0.28

[80]

mays

0.18

A. thaliana

35S

VTE1

N.tabacum leaf

10.2

[96]

A. thaliana

35S

HPPD

Synechocystis

7

[68]

A. thaliana,

Lac

At-HPPD,

A. thaliana

1.78

[93]

E.herbicola

Eh-tyrA

B.campestris

2.44

[68]

G.max

2.58

A. thaliana, Sac-

35S

At-HPPD &

N.tabacum leaf

10

[91]

charomyces

Sc-PDH

cerevisiae

Synechocystis sp.

35S

At-HPPD &

Synechocystis sp.

16.5

[68]

PCC6803

Eh-tyrA

PCC6803

At-VTE2 &

G. max seed

14.7

At-GGH

A. thaliana

35S

HPPD, tyrA

B.campestris

3.7

[68]

VTE2

seed

A. thaliana

35S

HPPD, tyrA,

A. thaliana

5

[68]

VTE2

35S

HPPD & tyrA

A. thaliana seed

1.8

[68]

G. max seed

2.6

A. thaliana

Napin

HPPD

1.0-1.2

[60]

A. thaliana

35S

HPPD

A. thaliana

0.28

[59]

0.96-1.1

[68]

A. thaliana

35S

HPPD

Synechocystis sp.

7

[68]

PCC6803 seed

A. thaliana

DC3

HPPD

A. thaliana seed

1.28

[59]

H.vulgare

35S

HPPD

N.tabacum seed

2

[60]

H.vulgare

35S

HGGT

A. thaliana leaf

10-15

[68]

Z.mays seed

6

Zea mays

35S

HGGT

Zea mays seed

8

[68]

A. thaliana

Napin

HPT

A. thaliana seed

2

[22]

Synechocystis

sp. PCC6803

A. thaliana

35S

HPT

A. thaliana leaf

3-4.4

[93]

A. thaliana seed

0.4

A. thaliana

35S

HPT & y-TMT

A. thaliana seed

12

[93]

A. thaliana

35S

y-tmt

A. thaliana seed

10

[93]

E.herbicola

35S

TYRA

Synechocystis

1.6

[68]

Napin

A. thaliana

1.53

B.campestris

2.37

G.max

1.11

E.herbicola

Napin

TYRA

1-1.4

[60]

E. herbicola

35S

TYRA

0.72-1.14

[60]

A. thaliana

Napin

GGH

0.94-1.12

[68]

A. thaliana

35S

GGH

0.9-1.06

[68]

18 Vitamin E Metabolic Modulation in Plants Table 18.3 (continued)

Gene source

Promoter used Gene

Plant species engineered

Product level

Referent

A. thaliana

35S

VTE2

A. thaliana leaf

4.4

[93]

A. thaliana

35S

VTE2

G. max seed

1.4

[93]

A. thaliana seed

1.8

[64]

[68]

A. thaliana

Napin

VTE2

1.29-1.53

[64]

Synechocystis sp.

Napin

VTE2

1.2-1.52

[64]

PCC 6803

Synechocystis sp.

35S

VTE2

0.9-1.05

[68]

PCC 6803

A. thaliana

35S

Y-TMT

B.campestris

2

[98]

canola

2

A. thaliana

35S

y-tmt

Latuca sativa L.

2

[99]

A. thaliana

Napin

mpbq-mt&y-

G. max seed

5

[74]

TMT

Perillafrutrscens

35S

y-tmt

G. max seed

4.8

[88]

Solanum

35S

Zeaxanthin

Solanum

2- to 3-fold a-T

[113]

tuberosum

epoxidase

tuberosum

Synthetic

35S

ZFP-TFs

A. thaliana

20-fold a-T

[106]

N.tabacum

35S

CHLP

N.tabacum leaf

4-6

[62]

N.tabacum seed

2-3

In overexpression studies, a tenfold increase in HPT activity translated into a 4.4-fold increase in leaf tocopherol levels, relative to wild-type plants [93]. Similar results were obtained in seeds, but the magnitude of the tocopherol increase was lower, ranging from 0.4- to twofold compared with wild-type levels [93, 94]. Both HPT1 and y-TMT overexpressed in seeds resulted in a total tocopherol content 12fold higher than wild types.

The maize HGGT gene was overexpressed in maize seeds, leading to a 20-fold increase in tocotrienol levels, which translated into an 8-fold increase in total to-cols (tocopherols and tocotrienols) [47]. This result is the largest increase in to-col production ever observed in plants and significantly increases the antioxidant potential of corn. Unfortunately, because dietary tocotrienols are not absorbed as well as a-tocopherol, the large increase in tocotrienol levels observed in the HGGT overexpressing maize seeds did not add much to the vitamin E nutritional value of these plants. However, because tocotrienols have superior in vitro antioxidant activity [3], transgenic plants with elevated tocotrienol levels could be used as sources of chemical antioxidants for industrial applications, such as oxidative stabilizers for paints, coatings, and other lipophilic products. Furthermore, it has been reported that tocotrienols might have a therapeutic role in decreasing the cholesterol level in humans [95].

Overexpression of genes for TCs from Arabidopsis in maize and canola plants led to an 18% and 28% increase of the total tocochromanol content in the seed oil, respectively. The average 5-tocopherol content increased up to 1.6-fold and

2.7-fold, respectively [80]. Overexpression of tocopherol cyclase (ATPT2 sequence) in Arabidopsis resulted in a 50% increase in total tocopherol levels and over a threefold increase in 5-tocopherol levels in the seed (Table 18.3). The 5-tocopherol content increased by the conversion of 2-methyl-6-phytyl-1,4-hydroquinol (MPQ) to 5-tocopherol in overexpressing lines. Our results showed that overexpression of At-VTE1 in tobacco increased 10.2-fold, compared with that in the control [96].

In a recent report by Van Eenennaam et al. [74], the genes encoding y-TMT and MPBQMT were overexpressed in soybean seeds to improve this important dietary source of vitamin E. The overexpression of the two tocopherol methyltransferases resulted in a 95% conversion of these lesser forms of vitamin E to a-tocopherol, which translated into a fivefold increase in vitamin E content. To put this into a real-world perspective, although four tablespoons of soybean oil from wild-type plants contains only 13 international units (IU) of vitamin E, the same volume of oil from the methyltransferase-overexpressing lines contain 65 IU of vitamin E. Because 100 IU is the recommended minimum therapeutic dose to decrease the risk of heart disease, the work of Van Eenennaam et al. [74] has done much to increase the nutraceutical potential of plant-derived vitamin E.

The enzymes catalyzing the reactions at the flux control points have been over-expressed with the aim of increasing the total tocopherol levels. The studies include the overexpression of hydroxyphenyl pyruvate dioxygenase by Tsegaye et al. [59] and Falk et al. [60], of deoxyxylulose phosphate synthase by Estevez et al. [97], and of homogentisate phytyl transferase by Collakova and DellaPenna [93] and Savidge et al. [64]. The success rates in these studies have varied, but not very drastically. Methylphytylbenzoquinone methyl transferase, tocopherol cyclase, and y-TMT are the enzymes important in determining the tocopherol composition [14]. The overexpression of MBPQMT and y-TMT in soybean seeds resulted in an increase of a-tocopherol by greater than 8-fold, at the expense of 5-, P-, and y-tocopherols [98], while the overexpression of y-TMT in the model plant A. thaliana increased the seed a-tocopherol levels by 80-fold [29]. In lettuce it led to a more than twofold elevation [99]. The overexpression of y-TMT gene increased the a-tocopherol levels by more than sixfold [100]. The y-TMT gene isolated from Perilla frutescens was overexpressed in soybean using a seed-specific promoter, and vitamin E content in T2 seeds was 4.8-fold higher than that in the wild type [88].

Single-gene engineering strategies for more complex approaches involved the simultaneous overexpression and/or suppression of multiple genes. The use of regulatory factors to control the abundance or activity of several enzymes is also becoming more widespread. In combination with emerging methods to model metabolic pathways, this should facilitate the enhanced production of natural products and the synthesis of novel materials in a predictable and useful manner [101].

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