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Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry. [ABSTRACT FROM AUTHOR]

The high cost of recombinant enzymes for the production of biofuel from ligno-cellulosic biomass is a crucial factor affecting the economic sustainability of the process. The use of plants as biofactories for the production of the suitable recombinant enzymes might be an alternative to microbial fermentation. In the case of enzyme accumulation in chloroplasts, it is fundamental to focus on the issue of full photosynthetic efficiency of transplastomic plants in the field where they might be exposed to abiotic stress such as high light intensity and high temperature. Xylanases (EC 3.2.1.8), a group of enzymes that hydrolyse linear polysaccharides of beta-1,4-xylan into xylose, find an application in the biofuel industry favouring biomass saccharification along with other cell-wall degrading enzymes. In the present study, we analysed how a high level of accumulation of a thermostable xylanase in tobacco chloroplasts does not impact on photosynthetic performance of transplastomic plants grown outdoors. The recombinant enzyme was found to be stable during plant development, ex planta and after long-term storage. [ABSTRACT FROM AUTHOR]

Chlorophyll b is synthesized by the oxidation of a methyl group on the B ring of a tetrapyrrole molecule to a formyl group by chlorophyllide a oxygenase (CAO). The full-length CAO from Arabidopsis (Arabidopsis thaliana) was overexpressed in tobacco (Nicotiana tabacum) that grows well at light intensities much higher than those tolerated by Arabidopsis. This resulted in an increased synthesis of glutamate semialdehyde, 5-aminolevulinic acid, magnesium-porphyrins, and chlorophylls. Overexpression of CAO resulted in increased chlorophyll b synthesis and a decreased chlorophyll a/b ratio in low light-grown as well as high light-grown tobacco plants; this effect, however, was more pronounced in high light. The increased potential of the protochlorophyllide oxidoreductase activity and chlorophyll biosynthesis compensated for the usual loss of chlorophylls in high light. Increased chlorophyll b synthesis in CAO-overexpressed plants was accompanied not only by an increased abundance of light-harvesting chlorophyll proteins but also of other proteins of the electron transport chain, which led to an increase in the capture of light as well as enhanced (40%-80%) electron transport rates of photosystems I and II at both limiting and saturating light intensities. Although the quantum yield of carbon dioxide fixation remained unchanged, the light-saturated photosynthetic carbon assimilation, starch content, and dry matter accumulation increased in CAO-overexpressed plants grown in both low- and high-light regimes. These results demonstrate that controlled up-regulation of chlorophyll b biosynthesis comodulates the expression of several thylakoid membrane proteins that increase both the antenna size and the electron transport rates and enhance carbon dioxide assimilation, starch content, and dry matter accumulation. [ABSTRACT FROM AUTHOR]

High level expression and efficient recovery of recombinant protein are two main critical factors that determine the use of transgenic plants as natural bioreactors to produce foreign proteins for industrial applications. We demonstrate here the potential of a new strategy involving chloroplast transformation, GUS-fusions and affinity-tag based chromatography to overexpress and purify a human therapeutic protein, interferon gamma (IFN-g) in tobacco plants. Our results show that IFN-g accumulation reaches up to 6% of total soluble protein when expressed as a GUS-fusion protein in tobacco chloroplasts. Addition of His-tag simplified the downstream process and the recombinant protein yields were considerably high (∼360 μg/g fresh leaf tissue). Further we demonstrate the use of GUS-fusions to identify recombinant protein containing fractions very rapidly (< 5 minutes) through simple GUS assay, an important consideration for those proteins that are highly labile during lengthy and harsh downstream processing conditions. The chloroplast-produced IFN-g is biologically as active as the same protein obtained through E. coli expression without any involvement of refolding procedure. Our results demonstrate that the new strategy has tremendous potential for large scale production of proteins from heterologous source, independent of their physio-chemical and biological properties, using plants as ‘natural bioreactors’. [ABSTRACT FROM AUTHOR]

Overproduction of cellulolytic enzymes through conventional nuclear transformation approaches posed a major challenge as they can potentially degrade the cell wall components and thereby affect transgenic plant growth and development. In this study, we have tested the possibility to over produce an alkali-thermostable xylanase gene from Bacillus sp. Strain NG-27 in tobacco plants through chloroplast expression. Our results showed that the xylanase expression can reach up to 6% of the total soluble protein, a value comparable to high level expression reported for several non-cellulolytic proteins in tobacco chloroplasts. The chloroplast-expressed xylanase retained its activity even when the leaves were dried under sun or at 42 °C, offering flexibility in the agricultural system in transport and storage. The recombinant enzyme was purified to homogeneity using single step chromatography with more than 85% recovery. Most importantly, transgenic plants were indistinguishable from the control untransformed plants in their morphology, growth and in seed setting. These results open up new avenues for large scale production of several other industrially useful cellulolytic enzymes through chloroplast expression. [ABSTRACT FROM AUTHOR]