Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme

Pantaleoni, Laura; Longoni, Paolo; Ferroni, Lorenzo; Baldisserotto, Costanza; Leelavathi, Sadhu; Reddy, Vanga Siva; Pancaldi, Simonetta; Cella, Rino

doi:10.1007/s00709-013-0564-1

Cited by 17 publications

(13 citation statements)

References 34 publications

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“…CelK1 leaf extract was used in combination with a homogenate obtained from either a previously described line overexpressing a thermostable xylanase (BSX) [15, 18] or a Pga2 transformed line. Since the final protein concentration in each assay was 0.1 mg/mL, the amount of reducing sugars released from poplar wood when assayed at pH 7 with a mixture of CelK1 and BSX was synergistic as compared to the action of each enzyme alone (Figure 6(a)).…”

Section: Resultsmentioning

confidence: 99%

“…Enzymes used for industrial applications, among which biofuel production is found, are currently produced via microbial fermentation even if the process requires high investment, production, and maintenance costs. Several studies show that protein/enzyme production by plant molecular farming might offer some advantages over microorganisms, as plants have both eukaryotic (nuclear) and prokaryotic (chloroplast) expression systems [15–18] that can be used singly or in combination. Transgenic plants were shown to be a valuable system for the production of a variety of antibodies, proteins/enzymes, and vaccines [19].…”

Section: Introductionmentioning

confidence: 99%

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Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification

Longoni

Leelavathi

Doria

et al. 2015

BioMed Research International

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification

Longoni

Leelavathi

Doria

et al. 2015

BioMed Research International

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“…The development of chloroplast derived cocktails of enzymes for production of fermentable sugars from different ligno-cellulosic biomass become major fresh breakthrough in biofuels research. Different enzymes from bacteria or fungi, namely β - 1,4 - endoglucanase , Beta glucosidase, Swollenin, esterase , cutinase , endoglucanases , exoglucanase , pectate lyases , xylanase , lipase , acetyl , Acetyl xylan esterase and xylan were expressed in tobacco chloroplasts for production of fermentable sugars [107–111]. …”

Section: Introductionmentioning

confidence: 99%

“…β-Mannanase enzyme from Trichoderma reesei showed sixfold to sevenfold higher enzyme activity than E. coli . β-Mannanase enzyme cocktail with chloroplast derived mannanse yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase [111]. Catalytic activity of chloroplast produced Xylanase was detected with birch wood xylan as substrate [112].…”

Section: Introductionmentioning

confidence: 99%

Recent achievements obtained by chloroplast transformation

2017

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Chloroplasts play a great role for sustained wellbeing of life on the planet. They have the power and raw materials that can be used as sophisticated biological factories. They are rich in energy as they have lots of pigment-protein complexes capable of collecting sunlight, in sugar produced by photosynthesis and in minerals imported from the plant cell. Chloroplast genome transformation offers multiple advantages over nuclear genome which among others, include: integration of the transgene via homologus recombination that enables to eliminate gene silencing and position effect, higher level of transgene expression resulting into higher accumulations of foreign proteins, and significant reduction in environmental dispersion of the transgene due to maternal inheritance which helps to minimize the major critic of plant genetic engineering. Chloroplast genetic engineering has made fruit full progresses in the development of plants resistance to various stresses, phytoremediation of toxic metals, and production of vaccine antigens, biopharmaceuticals, biofuels, biomaterials and industrial enzymes. Although successful results have been achieved, there are still difficulties impeding full potential exploitation and expansion of chloroplast transformation technology to economical plants. These include, lack of species specific regulatory sequences, problem of selection and shoot regeneration, and massive expression of foreign genes resulting in phenotypic alterations of transplastomic plants. The aim of this review is to critically recapitulate the latest development of chloroplast transformation with special focus on the different traits of economic interest.

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“…In the latter case, chloroplast metabolic engineering is used to synthesize low-cost enzyme cocktails for biomass hydrolysis and especially for the digestion of lignocellulosic biomass in order to generate fermentable sugars for ethanol production. However, the stability of the recombinant enzymes under variable field conditions, e.g., abiotic stressors, such as light, temperature, etc., as well as during extraction and storage also has to be maintained (Pantaleoni et al 2014). An example for bioplastic production is the introduction of complex metabolic pathways such as polyhydroxybutyrate (PHB) synthesis into the plastids (Nakashita et al 2001;Lössl et al 2003Lössl et al , 2005.…”

Section: Molecular Pharming-accumulation Of Recombinant Proteinsmentioning

confidence: 99%

Transplastomic plants for innovations in agriculture. A review

Wani

Sah

Sági

et al. 2015

Agron. Sustain. Dev.

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Food production has to be significantly increased in order to feed the fast growing global population estimated to be 9.1 billion by 2050. The Green Revolution and the development of advanced plant breeding tools have led to a significant increase in agricultural production since the 1960s. However, hundreds of millions of humans are still undernourished, while the area of total arable land is close to its maximum utilization and may even decrease due to climate change, urbanization, and pollution. All these issues necessitate a second Green Revolution, in which biotechnological engineering of economically and nutritionally important traits should be critically and carefully considered. Since the early 1990s, possible applications of plastid transformation in higher plants have been constantly developed. These represent viable alternatives to existing nuclear transgenic technologies, especially due to the better transgene containment of transplastomic plants. Here, we present an overview of plastid engineering techniques and their applications to improve crop quality and productivity under adverse growth conditions. These applications include (1) transplastomic plants producing insecticidal, antibacterial, and antifungal compounds. These plants are therefore resistant to pests and require less pesticides. (2) Transplastomic plants resistant to cold, drought, salt, chemical, and oxidative stress. Some pollution tolerant plants could even be used for phytoremediation. (3) Transplastomic plants having higher productivity as a result of improved photosynthesis. (4) Transplastomic plants with enhanced mineral, micronutrient, and macronutrient contents. We also evaluate field trials, biosafety issues, and public concerns on transplastomic plants. Nevertheless, the transplastomic technology is still unavailable for most staple crops, including cereals. Transplastomic plants have not been commercialized so far, but if this crop limitation were overcome, they could contribute to sustainable development in agriculture.

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Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme

Cited by 17 publications

References 34 publications

Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification

Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification

Recent achievements obtained by chloroplast transformation

Transplastomic plants for innovations in agriculture. A review

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