length of a leaf, with the PHB concentration increasing from the youngest part of
the leaf (the base) to the oldest (the tip). The rank order of the lines did not change
over time. Moreover, there was a uniform spatiotemporal pattern of relative PHB
accumulation among the lines, despite the fact that they showed marked differences in absolute PHB concentration. Molecular analysis showed that the
expression of the transgenes encoding the PHB biosynthesis enzymes was
apparently coordinated, and that there were good correlations between PHB
concentration and the abundance of the PHB biosynthesis enzymes. The maximum
PHB concentration recorded (1.77 % of leaf dry weight) did not result in agronomic abnormalities. Although moderate PHB concentrations were achieved in
leaves, the maximum total-plant PHB yield was only 0.79 % (11.9 g PHB in
1.51 kg dry weight).
As plant peroxisomes contain the substrate molecules and essential reducing
power for PHB biosynthesis, Tilbrook et al. (2011) generated transgenic sugarcane
with the three-enzyme Ralstonia eutropha PHA biosynthetic pathway targeted at
these cell compartments. PHB accumulated in sugarcane leaves at levels up to
1.6 % dry weight, in both peroxisomes and vacuoles. A small percentage of
total polymer was also identified as the copolymer poly (3-hydroxybutyrateco-3-hydroxyvalerate). As a result of peroxisomal PHA biosynthesis, no obvious
detrimental effect was observed on plants. This study highlights how using
peroxisomal metabolism for PHA biosynthesis could significantly contribute to
reaching commercial production levels of PHAs in crop plants.
Petrasovits et al. (2012) used different plant and viral promoters, in combination
with multigene or single-gene constructs to increase PHB levels in sugarcane.
Promoters tested included the maize and rice polyubiquitin promoters, the maize
chlorophyll A/B-binding protein promoter, and a Cavendish banana streak
badnavirus promoter. At the seedling stage, the highest levels of polymer were
produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the
plants matured. The rice Ubi promoter enabled the production of PHB at levels
similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein
promoter enabled the production of PHB to levels as high as 4.8 % of leaf dry
weight, which is approximately 2.5 times higher than previously reported levels in
sugarcane. However, the highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.
5.4 Sugarcane Biofactory for Protein Products
Regarding pharmaceutical applications, one of the first approaches reported was
done by Holland-Moritz (2003), who transformed sugarcane to produce pharmaceutical-grade human structural proteins for human therapeutics. Later, Wang
et al. (2005) successfully produced the human granulocyte macrophage colonystimulated factor (GM-CSF, used in clinical applications for the treatment of
5 Sugarcane as a Novel Biofactory: Potentialities and Challenges
135
the leaf (the base) to the oldest (the tip). The rank order of the lines did not change
over time. Moreover, there was a uniform spatiotemporal pattern of relative PHB
accumulation among the lines, despite the fact that they showed marked differences in absolute PHB concentration. Molecular analysis showed that the
expression of the transgenes encoding the PHB biosynthesis enzymes was
apparently coordinated, and that there were good correlations between PHB
concentration and the abundance of the PHB biosynthesis enzymes. The maximum
PHB concentration recorded (1.77 % of leaf dry weight) did not result in agronomic abnormalities. Although moderate PHB concentrations were achieved in
leaves, the maximum total-plant PHB yield was only 0.79 % (11.9 g PHB in
1.51 kg dry weight).
As plant peroxisomes contain the substrate molecules and essential reducing
power for PHB biosynthesis, Tilbrook et al. (2011) generated transgenic sugarcane
with the three-enzyme Ralstonia eutropha PHA biosynthetic pathway targeted at
these cell compartments. PHB accumulated in sugarcane leaves at levels up to
1.6 % dry weight, in both peroxisomes and vacuoles. A small percentage of
total polymer was also identified as the copolymer poly (3-hydroxybutyrateco-3-hydroxyvalerate). As a result of peroxisomal PHA biosynthesis, no obvious
detrimental effect was observed on plants. This study highlights how using
peroxisomal metabolism for PHA biosynthesis could significantly contribute to
reaching commercial production levels of PHAs in crop plants.
Petrasovits et al. (2012) used different plant and viral promoters, in combination
with multigene or single-gene constructs to increase PHB levels in sugarcane.
Promoters tested included the maize and rice polyubiquitin promoters, the maize
chlorophyll A/B-binding protein promoter, and a Cavendish banana streak
badnavirus promoter. At the seedling stage, the highest levels of polymer were
produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the
plants matured. The rice Ubi promoter enabled the production of PHB at levels
similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein
promoter enabled the production of PHB to levels as high as 4.8 % of leaf dry
weight, which is approximately 2.5 times higher than previously reported levels in
sugarcane. However, the highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.
5.4 Sugarcane Biofactory for Protein Products
Regarding pharmaceutical applications, one of the first approaches reported was
done by Holland-Moritz (2003), who transformed sugarcane to produce pharmaceutical-grade human structural proteins for human therapeutics. Later, Wang
et al. (2005) successfully produced the human granulocyte macrophage colonystimulated factor (GM-CSF, used in clinical applications for the treatment of
5 Sugarcane as a Novel Biofactory: Potentialities and Challenges
135