Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts

Schmidt, Jennifer A.; McGrath, Justin M.; Hanson, Maureen R.; Long, Stephen P.; Ahner, Beth A.

doi:10.1038/s41477-019-0467-z

Cited by 23 publications

(31 citation statements)

References 40 publications

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“…Moreover, culturing microalgae in non-axenic conditions, by phosphite selective nutrition, may significantly reduce the current cost of algal biomass production. The combined use of (i) PTXD together with (ii) a cheaper growth medium (i.e.T10APhi medium) and (iii) a low-light demand for optimal CWDEexpression (50-100 µE, Figure 2b-c) can reduce production cost of PBR-grown microalgae to 3.2-3.8 € kg/DW as argued by Tredici et al (2016) and Slade and Bauen (2013), a value close to that reported for field-grown tobacco plants (2 € kg/DW; Maksymowicz, and Palmer 1997;Schmidt et al 2019). Similarly, transgenic tobacco production is low cost in open field and under optimal conditions, which unfortunately could only be achieved in countries with no restriction on GMO cultivation, while production cost is significantly higher for greenhouse-grown plants (6 € kg/DW; Fa e et al 2017).…”

Section: Discussionsupporting

confidence: 67%

“…Compartmentalized expression of CWDEs in the chloroplast may enhance the yield of recombinant protein since chloroplast expression is not prone to gene silencing (Li et al 2019); nevertheless, the use of endogenous promoters and other cis-acting elements to drive chloroplast expression of transgenes may be subjected to the host regulation, requiring the optimization of specific growth conditions in order to enhance recombinant protein yield (Fields et al 2018, Figure 2). Transplastomic tobacco plants that accumulate high level of GH5, GH6 and GH9 endoglucanases and pectin lyases have been reported (Fa e et al 2017;Schmidt et al 2019;Verma et al 2010). Moreover, transplastomic tobacco plants expressing the GH3 b-glucosidase Bgl1 from Trichoderma reesei had an increased biomass yield and an improved resistance towards aphids than wild-type plants (Jin et al 2011).…”

Section: Discussionmentioning

confidence: 99%

“…(2016) and Slade and Bauen (2013), a value close to that reported for field‐grown tobacco plants (2 € kg/DW; Maksymowicz, and Palmer 1997; Schmidt et al . 2019). Similarly, transgenic tobacco production is low cost in open field and under optimal conditions, which unfortunately could only be achieved in countries with no restriction on GMO cultivation, while production cost is significantly higher for greenhouse‐grown plants (6 € kg/DW; Faè et al .…”

Section: Discussionmentioning

confidence: 99%

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A microalgal‐based preparation with synergistic cellulolytic and detoxifying action towards chemical‐treated lignocellulose

Benedetti

Barera

Longoni

et al. 2020

Plant Biotechnology Journal

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High-temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the b-glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non-axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline-treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory byproducts released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose-derived sugars by C. vulgaris under mixotrophic growth.

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A microalgal‐based preparation with synergistic cellulolytic and detoxifying action towards chemical‐treated lignocellulose

Benedetti

Barera

Longoni

et al. 2020

Plant Biotechnology Journal

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“…A recent study with plastidengineered tobacco plants further demonstrated the viability of plant-based protein production in a field setting. The results showed that field-grown tobacco expressing a recombinant protein were capable of achieving an order of magnitude reduction in costs compared to traditional cell culture methods [44]. In addition, as the glycosylation state of antibody therapeutics is crucial for their function [45,46], plants are uniquely suited to produce RIC vaccines.…”

Section: Discussionmentioning

confidence: 99%

Codelivery of improved immune complex and virus-like particle vaccines containing Zika virus envelope domain III synergistically enhances immunogenicity

Diamos

Pardhe

Sun

et al. 2020

Vaccine

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a b s t r a c tZika virus (ZIKV) reemergence poses a significant health threat especially due to its risks to fetal development, necessitating safe and effective vaccines that can protect pregnant women. Zika envelope domain III (ZE3) has been identified as a safe and effective vaccine candidate, however it is poorly immunogenic. We previously showed that plant-made recombinant immune complex (RIC) vaccines are a robust platform to improve the immunogenicity of weak antigens. In this study, we altered the antigen fusion site on the RIC platform to accommodate N-terminal fusion to the IgG heavy chain (N-RIC), and thus a wider range of antigens, with a resulting 40% improvement in RIC expression over the normal C-terminal fusion (C-RIC). Both types of RICs containing ZE3 were efficiently assembled in plants and purified to >95% homogeneity with a simple one-step purification. Both ZE3 RICs strongly bound complement receptor C1q and elicited strong ZE3-specific antibody titers that correlated with ZIKV neutralization. When either N-RIC or C-RIC was codelivered with plant-produced hepatitis B core (HBc) virus-like particles (VLP) displaying ZE3, the combination elicited 5-fold greater antibody titers (>1,000,000) and more strongly neutralized ZIKV than either RICs or VLPs alone, after only two doses without adjuvant. These findings demonstrate that antigens that require a free N-terminus for optimal antigen display can now be used with the RIC system, and that plant-made RICs and VLPs are highly effective vaccines targeting ZE3. Thus, the RIC platform can be more generally applied to a wider variety of antigens.

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“…However, these systems are often expensive to establish, labor-intensive to maintain, prone to contamination, and inflexible to changing market demands [2]. With wellestablished cultivation practices, a genetically engineered (GE) biomass crop, like tobacco, may represent a lowcost alternative [3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins

Schmidt

Richter

Condoluci

et al. 2021

Biotechnol Biofuels

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Background The global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth. Results Here, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants’ metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant−1) by 50–70%. Conclusions The work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.

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Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts

Cited by 23 publications

References 40 publications

A microalgal‐based preparation with synergistic cellulolytic and detoxifying action towards chemical‐treated lignocellulose

A microalgal‐based preparation with synergistic cellulolytic and detoxifying action towards chemical‐treated lignocellulose

Codelivery of improved immune complex and virus-like particle vaccines containing Zika virus envelope domain III synergistically enhances immunogenicity

Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins

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