*4.2. Industrial Enzymes and Biomaterials*

The chloroplast genome has been repeatedly engineered to produce industrial enzymes and biomaterials. Polyhydroxyalkanoates (PHAs) are a large class of biodegradable polyesters biopolymers naturally synthesized by many microorganisms that can be used as an alternative to petroleum-based plastics [137]. The first described and most well-studied PHA is polyhydroxybutyrate (PHB). Various systems have been adapted for the production of PHB, including microbial cells and various plant tissues. To date, however, the highest level of PHB accumulation was achieved in tobacco plastids, with levels of 18.8% of dry weight (DW). The tobacco system was based on an operon extension strategy to synthesize high PHB levels by introducing a bacterial operon, consisting of three genes encoding enzymes necessary for PHB biosynthesis, into the tobacco chloroplast genome [145]. The high amounts of PHB produced in this system stems from the high flux of the PHB biosynthetic precursor acetyl-CoA released during fatty acid biosynthesis [146]. Typical examples of industrial enzymes and biomaterials obtained through plastid transformation are given in Table 6 [8,97,145,147–151].



DW: dry weight; FW: fresh weight; NR: not recorded; TCP: total cellular protein; TSP: total soluble protein.

#### *4.3. Phytoremediation*

Mercury (Hg), especially in its organic form, is a highly toxic pollutant that affects humans, animals, and plants alike. At present, phytoremediation is a cost-effective method to remove heavy metals from contaminated soils by using plants to clean up contaminated environments by taking up the desired pollutant [152]. In plants, Hg mainly targets chloroplasts, where it impairs electron transport and photosynthesis. Therefore, chloroplasts would be ideal sites in which to increase resistance to organic and inorganic Hg and repair damage resulting from Hg exposure [153].

Transgenic tobacco plants with engineered chloroplasts exhibited enhanced uptake of inorganic Hg, accumulating about 100-fold more than in untransformed plants [154]. An operon containing the bacterial genes *merA* (mercuric ion reductase gene) and *merB* (organomercurial lyase gene), expressed in tobacco chloroplasts, significantly improved plant tolerance to organic compounds [102]. Integrating the murine *Metallothionein* gene (*MT1*) into the tobacco chloroplast genome allowed high Hg accumulation within tobacco cells. These transplastomic lines were resistant up to 20 μM Hg and remained healthy with normal chlorophyll content and biomass [101]. Plastid transformation may also increase tolerance to high concentrations of copper, and sustain higher growth rates [91].

#### *4.4. Biofuels Production*

At present, biofuels research mainly focuses on the production of liquid fuel using sugars and lignocellulose from cassava (*Manihot esculenta*), sweet sorghum and other starchy or sugary non-grain crops as raw materials. The most important step in biofuels production is the hydrolysis of lignocellulose [155], with enzymatic digestion being the most efficient and environmentally friendly method, although fungi or bacteria producing the necessary cellulase make the process less efficient and more expensive. A chloroplast-based cellulolytic enzyme has been applied as an industrially pretreated feedstock (*Arundo donax*) for biofuel production [8]. The high levels and compartmentalization of toxic

proteins possible within chloroplasts can however protect transgenic plants from multidirectional effects, turning the many chloroplasts within each cell into ideal bioreactors for industrial enzyme production [156]. Enzymes from various fungi and bacteria have been successfully produced in plant chloroplasts: for example, β-glucosidase [8,157], β-1,4-endoglucanase [158], cutinase, exoglucanase, pectinase, xylanase, lipase and acetyl xylan esterase were expressed in tobacco chloroplasts to produce fermentable sugars [111,159–162].

The enzymes derived from transplastomic plants have high activities for further applications. The β-glucosidase BglC and the endoglucanase Cel6A from *Thermobifida fusca* were highly active against synthetic test substrates when expressed in tobacco chloroplasts [157,163]. Treatment of cotton fiber with chloroplast-derived cutinase resulted in enlarged segments and the irreversible unwinding of intertwined inner fibers due to the expansion activity of cutinase. Transgenic plants accumulating cutinase also exhibited esterase and lipase activities [164]. A cocktail of these enzymes efficiently promoted sugar release from filter paper, pine wood and citrus peel [111]. The β-1,4-endoglucanase EGPh from the Archaeon *P. horikoshiiwere*, expressed in tobacco chloroplasts, can hydrolyze carboxymethyl cellulose (CMC) equally well in dry and fresh leaves. Furthermore, the inactive form of EGPh in mature leaves is easily removed by heat treatment [158]. The expression of endo-β-mannanase from *Trichoderma reesei* reached 25 units per gram of leaf (fresh weight), and the activity of endo-β-mannanase from chloroplast extracts was 6–7 fold higher than in *Echerichia coli* extracts, while also having higher temperature stability (40 ◦C to 70 ◦C) and wider pH optimum (pH 3.0 to 7.0) [149]. These reports provide convincing evidence that chloroplast-made enzymes have better temperature stability and a wider pH optimum range than those made in other systems [8,111,162].

The contributions of different elements of plastid transformation vectors can be assessed by comparing different efforts to produce the same protein. Xylanase is an important enzyme for lignocellulosic biomass fermentation and sugar release that has been transgenically expressed in tobacco. The xylanase genes used to date have been selected from different fungi or bacteria, expressed using different promoters, and inserted to the different sites in the chloroplast genome (Table 7). A comparison shows that the *Prrn* promoter drives the highest accumulation among these *xyn* transplastomic tobacco plants, although the *Prrn*-driven xylanase still has variable accumulation levels that might arise from the insertion site or source of the gene [8,111,150,161,165]. Kolotilin et al. tested different expression cassettes with *Prrn* or *psbA* promoter to express xylanase in tobacco [150]. Using *Prrn* as the promoter did indeed generate the most transcript in tobacco leave; however, it caused growth retardation in the transgenic plants, and ultimately produced similar protein accumulation levels as the construct using *psbA* as the promoter. These results indicate that mRNA accumulation can be too high in some cases and might even be lethal to the plant.

Another factor to consider is that different sources of genes will have dissimilar codon usage. As the amount of foreign protein expressed in the chloroplast is related to both the promoter and its codon usage preference, codon optimization of the gene of interest provides an alternative way to increase protein expression [166].


**Table 7.** Xylanse produced by tobacco chloroplasts.

DW: dry weight; TSP: total soluble protein.
