Bioengineering

Bioengineering can be defined as the alteration of the gene expression of any biological organism by molecular methodologies. Bioengineering in any metabolic pathway requires that all steps within the pathway be defined, the regulatory points identified, and the stability of the intermediates determined (Scott et al., 2000). Phytochemicals are often differentially expressed both spatially and temporally. For example, the concentration of alkamides differs among plant organs in Echinacea purpurea with the roots having the highest concentration (Perry et al., 1997). To increase harvest efficiency, the production of any phytochemical should be directed to a readily harvestable organ, such as a leaf or seed. Using recombinant technology, expression cassettes can be made to redirect synthesis into different organs (Ye et al., 2000).

Elucidation of metabolic pathways can be accomplished by performing isotopic tracer experiments, screening for mutants, or gene silencing/overexpression of the putative genes in transgeneic plants. Modeling metabolic flux in combination with in vivo analysis must be performed to find control mechanisms. Earlier strategies worked under the premise that there is a rate-limiting enzyme, but there is general agreement that any enzyme in a pathway may affect flux. There are homeostatic genetic and metabolic control mechanisms that keep each enzyme at ideal levels (Jensen and Hammer, 1998); however, over-expression of a single enzyme, y-TMT, increased tocopherol by 80-fold in Arabidopsis seeds, a clear indication that this was the rate-limiting enzyme (Shintani and DellaPenna, 1998).

Metabolic pathways from different organisms can be combined and expressed in E. coli and other organisms (Schmidt-Dannert et al., 2000). A well-reported success of this strategy was the expression of P-carotene in the endosperm of rice (Ye et al., 2000 and references within). P-Carotene is produced in plastids of plant cells by the isoprenoid pathway (Figure 9.2). The genes encoding phytoene synthase, 5-carotene desaturase, and lycopene P-cyclase were introduced into the rice endosperm to enable P-carotene production. The genes for phytoene synthase were taken from daffodil (Narcissus pseudonarcissus), while the latter two genes originated in Erwinia uredovora. These carotene pathway genes were placed into three vectors (Figure 9.3) and placed under control of an endosperm-specific (glutelin; Gt1 p) and a constitutive CaMV (cauliflower mosaic virus; 35S p) promoter. Correct expression of the genes also required that functional transit peptides for import into plastids be part of the construct (crtI, and tp, Figure 9.3). Transferring the pathway and direct-

Geranylgeranyl diphosphate

[Phytoene synthase|

Lycopene |Lycopene ft-cyclase |

O I |ft-Hydroxylase|

Zeaxanthin

Violaxanthin de-epoxidase High light only OH

Lycopene |Lycopene ft-cyclase |

  • Phytol side chain of chlorophyll
  • J . . . ß

|ß-Hydroxylase| |s-Hydroxylase|

Zeaxanthin epoxidase

Violaxanthin de-epoxidase High light only OH

Zeaxanthin epoxidase

<O Antheraxanthin

Zeaxanthin epoxidase

I Violaxanthin de-epoxidase | High light only oh

HO Lutein

<O Antheraxanthin

Zeaxanthin epoxidase

I Violaxanthin de-epoxidase | High light only oh

O Violaxanthin

I Neoxanthin synthase |

Neoxanthin

Figure 9.2 Metabolic pathway for the conversion of geranylgeranyl disphosphate to a- and ft-carotene. Phenylpropanoid pathways have several control points from which many different natural products are synthesized. The metabolic flux between each control point, and within each pathway, may differ with respect to genetic composition of the species, cultivar or variety, the environmental conditions, tissue, and maturity of the plant. (From Buchanan, B.B., Gruissem, W., and Jones, R.L. (eds.) 2000. Biochem, Molecular Biology of Plants, Am. Soc. Plant Physiology, Rockville, MD, 1367 pp. With permission.)

pB19hpc

I-sce I

LB 35S! aphlV 35S p Gt1 p psy nos! 35S p tp crtl nos! npt II RB

pZPsC

I-sce I

Gtlp psy nos! 35Sp tp crtl nos! RB

I-sce I pZLcyH

LB35S! aphIV 35Sp 35S! Icy

Gt1 p RB

Figure 9.3 Vectors used to express phytoene synthase (psy), phytoene desaturase (crtI), and lycopene-P cyclase (lcy) for synthesis of P-carotene in rice endosperm (from Ye et al., 2000). A, Vector B19hpc was engineered to produce lycopene, and was introduced as a single transformant with Agrobacterium. B., C. Vectors pZPsC and pZLcyH were engineered to synthesize P-carotene, and were introduced as cotransformants with Agrobacterium. For proper spatial and temporal expression of the genes, the vectors included both a constitutive promoter (35Sp), and a glutelin promoter (Gtlp) and a transit peptide sequence (tp) for import and expression in endosperm plastids. For additional details concerning vector construction, the reader is referred to Burkhardt et al. (1997) and Ye et al. (2000). (From Ye, X., Al-Babili, S., Kloti, S., Zhang, J., Lucca, P., Beyer, P., and Poltrykus, I. 2000. Engineering the provitamin A (P-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 287:304. With permisssion.)

ing its expression to the endosperm was technically demanding and expensive, reportedly requiring $2.6 million and 7 years (Nash and Robinson, 2000), yet this example demonstrates the potential for interkingdom transfer and expression of metabolic pathways into plants.

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