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X-ray fluorescence microscopy (XFM) elemental maps for Fe in stem cross sections of extracellular ferritin-expressing and control Arabidopsis plants. The 2-micron-thick cross sections were cut from senesced stems from empty vector (EV) control (a, b) and extracellular ferritin-expressing (FerEX) (c, d) plants. Cell-wall images (a, c) were constructed from binary images of potassium XFM maps. The dashed lines were drawn from the cell-wall images and overlayed on the iron maps (b, d) to more easily distinguish iron intensity inside the cell walls. The intensities in both iron maps (b, d) were scaled the same, and the iron is observed in the FerEx cell walls (d) by noting that the iron intensity in the cell walls is higher than the background iron intensity observed in the empty cell lumina
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Background
Plant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and incre...
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![Table 1 . Composition of lignocellulosic biomass [12].](profile/Aakash-Chawade/publication/325531509/figure/tbl1/AS:667847088951304@1536238562300/Composition-of-lignocellulosic-biomass-12_Q320.jpg)
Bioenergy, biofuels, and a range of valuable chemicals may be extracted from the abundantly available lignocellulosic biomass. To reduce the recalcitrance imposed by the complex cell wall structure, genetic engineering has been proposed over the years as a suitable solution to modify the genes, thereby, controlling the overall phenotypic expression...
Genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance for enhanced biomass enzymatic saccharification and biofuel production in bioenergy crops. Although endo-β-1,4-glucanase (EG II) secreted by fungi has been broadly applied for enzymatic hydrolysis of cellulose, it remains to explore its role in cellu...
Citations
... 102 In addition to this, similar approaches have also been taken by modification of plants at the molecular level to enhance their Fe uptake so that it can catalytically enhance biomass pretreatment results. Several studies [103][104][105] showed the effects of a plant that was molecular engineered to enhance its Fe uptake, leading to increased sugar yield of its biomass conversion. Inoculating the plant seed with plant growth-promoting bacteria also showed an increase in Fe accumulation in the plant. ...
Minerals in biomass have a significant impact on both biofuel quality and yield. This is especially true for current thermochemical biomass conversion processes. However, the roles of plant minerals in biochemical conversion have not been studied extensively, even though they are generally considered to lower the sugar yield because they reduce the feedstock proportion of carbohydrates. A successful strategic solution is thus necessary to overcome the challenges caused by the minerals in biomass, which include (1) decreased quality of biomass feedstocks; (2) reduction of process efficiency; and (3) reduction of the product quality and quantity from biomass conversion. This review summarizes the roles of plant minerals in a biorefinery, focusing on these key challenges. The discussion covers many issues related to plant minerals in biofuel production, including their sources, functions, and distribution in plant biomass, methods of characterizing them, their influence in a biorefinery, and the strategic handling required to manage their occurrence in biomass, based on reported studies. It could inspire better strategies to deal with the variance of mineral content in biomass feedstocks to increase process efficiency and reduce costs while supporting the concept of a circular bioeconomy. © 2023 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd.
... Our results showed enhanced iron accumulation and improved biomass conversion with 20% more glucose and 15% more xylose release than controls [81]. Delivery of ferritin extracellularly into the plant cell wall (referred to as FerEX) resulted in increased biomass yield and even higher pretreatability and digestibility (released 21% and 34% more glucose and xylose, respectively) than the FerIN Arabidopsis plants [82]. Moreover, this in planta iron accumulation is valuable when considering its use for iron biofortification for human nutrition. ...
... Perls' Prussian blue staining used to localize iron in cross-sections of stem tissues [80][81][82] by optical stereomicroscopy. In the EV control, the blue signals cannot be detected either within plant cell or on the cell wall (Fig. 6b, e); in contrast, we observed blue staining in the stem sections of FerIN and FerIN/IBPex (Fig. 6c, d). ...
... First, we modified the SP-CMB-IBP approach used by Yang et al. [81] based on our previous experience from FerEX Arabidopsis [82]. We replaced the secretion signal peptide from SP EXT with rice GRP (SP GRP ), as it had been suggested that the secretory property of rice SP GRP can improve the protein expression of β-glucuronidase (GUS) in monocot plants, which demonstrated the higher intensity of blue coloration in both sorghum and switchgrass [85,86] and higher GUS enzyme activity in apoplastic fluids of transgenic sorghum [86]. ...
Background
Pretreatments are commonly used to facilitate the deconstruction of lignocellulosic biomass to its component sugars and aromatics. Previously, we showed that iron ions can be used as co-catalysts to reduce the severity of dilute acid pretreatment of biomass. Transgenic iron-accumulating Arabidopsis and rice plants exhibited higher iron content in grains, increased biomass yield, and importantly, enhanced sugar release from the biomass.
Results
In this study, we used intracellular ferritin (FerIN) alone and in combination with an improved version of cell wall-bound carbohydrate-binding module fused iron-binding peptide (IBPex) specifically targeting switchgrass, a bioenergy crop species. The FerIN switchgrass improved by 15% in height and 65% in yield, whereas the FerIN/IBPex transgenics showed enhancement up to 30% in height and 115% in yield. The FerIN and FerIN/IBPex switchgrass had 27% and 51% higher in planta iron accumulation than the empty vector (EV) control, respectively, under normal growth conditions. Improved pretreatability was observed in FerIN switchgrass (~ 14% more glucose release than the EV), and the FerIN/IBPex plants showed further enhancement in glucose release up to 24%.
Conclusions
We conclude that this iron-accumulating strategy can be transferred from model plants and applied to bioenergy crops, such as switchgrass. The intra- and extra-cellular iron incorporation approach improves biomass pretreatability and digestibility, providing upgraded feedstocks for the production of biofuels and bioproducts.
... Expression of a fusion protein, comprising several ironbinding peptides with a carbohydratebinding module, secreted into rice and Arabidopsis cell walls, showed significant increases in iron accumulation and saccharification yield compared to WT (93). Site-selective delivery of ferritin catalysts in transgenic lines of Arabidopsis yielded 12 to 18% more shoot biomass and released 20 to 30% more sugar following pretreatment, relative to control plants without iron incorporation (94,95). Delivery of metal catalysts throughout the cell wall structure and creating functionalized sites ready for catalytic transformations increased the effective surface area for catalysis. ...
Lignocellulosic biomass—the lignin, cellulose and hemicellulose that comprise major components of the plant cell well—is a sustainable resource that could be utilized in the United States to displace oil consumption from heavy vehicles, planes and marine-going vessels and commodity chemicals. Biomass-derived sugars can also be supplied for microbial fermentative processing to fuels and chemicals, or chemically deoxygenated to hydrocarbons. However, the economic value of biomass might be amplified by diversifying the range of target products that are synthesized in living plants. Genetic engineering of lignocellulosic biomass has previously focused on changing lignin content or composition to overcome recalcitrance, the intrinsic resistance of cell walls to deconstruction. New capabilities to remove lignin catalytically without denaturing the carbohydrate moiety has enabled the concept of the ‘lignin-first’ biorefinery that includes high-value aromatic products. The structural complexity of plant cell wall components also provides substrates for polymeric and functionalized target products, such as thermosets, thermoplastics, composites, cellulose nanocrystals and nanofibers. With recent advances in design of synthetic pathways, lignocellulosic biomass can be regarded as a substrate at various length scales for liquid hydrocarbon fuels, chemicals and materials. In this review, we describe the architectures of plant cell walls, recent progress in overcoming recalcitrance, and illustrate the potential for natural or engineered biomass to be used in the emerging bioeconomy.
... This challenge is overcome here by using a synchrotron X-ray microprobe to make X-ray fluorescence microscopy (XFM) ion maps of locally applied inorganic ions as they diffuse through wood cell wall layers being conditioned at different RH levels. XFM has been shown to have the sensitivity and spatial resolution to accurately map ions in individual S2 and CML 34,[60][61][62] . In this work, time-dependent concentration profiles of diffusing ions were directly measured from time-lapse XFM ion maps obtained in situ during diffusion. ...
Our future bioeconomy depends on increased utilization of renewable lignocellulosic biomass. Controlling the diffusion of chemicals, such as inorganic ions, within secondary plant cell walls is central to many biomass applications. However, insufficient understanding of intra-cell-wall diffusion within secondary plant cell walls is hindering the advancement of many lignocellulosic biomass applications. In this work, X-ray fluorescence microscopy was used to measure diffusion constants of K⁺, Cu²⁺, and Cl⁻ diffusing through loblolly pine (Pinus taeda) cell wall layers under 70%, 75%, or 80% relative humidity (RH). Results revealed that diffusion constants increased with RH, the larger Cu²⁺ diffused more slowly than the K⁺, and the Cl⁻ diffusion constant was the same as that for the counter cation, indicating cations and anions diffused together to maintain charge neutrality. Comparison with electrical conductivity measurements showed that conductivity is being controlled by ion mobility over these RH. The results further support that intra-cell-wall diffusion of inorganic ions is a Fickian diffusion process occurring through rubbery amorphous polysaccharides, which contradicts previous assertions that intra-cell-wall diffusion is an aqueous process occurring through water pathways. Researchers can now utilize polymer science approaches to engineer the molecular architecture of lignocellulosic biomass to optimize properties for specific end uses.
... Fundamental breakthroughs in this direction include the incorporation of novel chemistries in biomass to facilitate its disassembly (Zhang et al., 2012;Wilkerson et al., 2014), the integration of catalysts which guide the self-deconstruction of biomass feedstocks (Shen et al., 2012;Giessen and Silver, 2016;Donohoe et al., 2017), and metabolic pathway compartmentalization for increased production of valuable metabolites and ease of separation. To illustrate, Lin et al. (2016) recently developed an elegant transgenic strategy to express the iron-storage protein, ferritin, in the model plant species, Arabidopsis thaliana. Transformed plants presented improved enzymatic digestibility of lignocellulose following mild dilute-acid pretreatment. ...
Despite significant progress toward the commercialization of biobased products, today’s biorefineries are far from achieving their intended goal of total biomass valorization and effective product diversification. The problem is conceptual. Modern biorefineries were built around well-optimized, cost-effective chemical synthesis routes, like those used in petroleum refineries for the synthesis of fuels, plastics, and solvents. However, these were designed for the conversion of fossil resources and are far from optimal for the processing of biomass, which has unique chemical characteristics. Accordingly, existing biomass commodities were never intended for modern biorefineries as they were bred to meet the needs of conventional agriculture. In this perspective paper, we propose a new path toward the design of efficient biorefineries, which capitalizes on a cross-disciplinary synergy between plant, physical, and catalysis science. In our view, the best opportunity to advance profitable and sustainable biorefineries requires the parallel development of novel feedstocks, conversion protocols and synthesis routes specifically tailored for total biomass valorization. Above all, we believe that plant biologists and process technologists can jointly explore the natural diversity of plants to synchronously develop both, biobased crops with designer chemistries and compatible conversion protocols that enable maximal biomass valorization with minimum input utilization. By building biorefineries from the bottom-up (i.e., starting with the crop), the envisioned partnership promises to develop cost-effective, biomass-dedicated routes which can be effectively scaled-up to deliver profitable and resource-use efficient biorefineries.
... Liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis of the hemocyanin extract after trypsinolysis revealed that it contained low level contamination with ferritin (1.3%, based on emPAI-derived molar percentages; Supplementary Fig. 6c), which was also detected in the gut fluid proteome (0.5 mol%; PXD009486; MSV000082271). Therefore, we tested the effect of ferritin in pretreatment reactions on willow followed by saccharification with CBH to exclude the possibility that the ferritin co-extracted with hemocyanin in our experiments contributed to the observed digestibility improvement 43 . Ferritin pretreatment had no impact on digestibility of willow when used at a molarity five times higher than the one expected in the hemocyanin extract used in pretreatment reactions (Supplementary Fig. 11). ...
Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining.
... These infiltration problems arise from the binding of iron ions to the biomass surface [42] and the presence of intracellular air-filled voids within cell wall tissues. To more effectively overcome these process limitations, our group and other groups recently used three novel approaches to deliver a metal cocatalyst into the plant cell wall region during plant growth: (1) expressed heterologous iron-storage protein ferritins intracellularly (referred to as FerIN) [43], (2) expressed ferritin extracellularly (referred to as FerEX) [44], and (3) expressed blood iron-binding peptide (BIBP) in Arabidopsis and rice plants [45]. These FerEX transgenic plants accumulated approximately 50-110% more iron under normal growth conditions, compared with the empty-vector control plants. ...
... Remarkably, the harvested biomass of such iron transgenic plants showed enhanced pretreatability and digestibility, releasing approximately 18-21% more glucose and 13-34% more xylose than the empty-vector control plants after dilute-acid pretreatment, or hot water (which can be viewed as a weak acid) pretreatment and co-saccharification [43][44][45]. ...
High-temperature (150–170 °C) pretreatment of lignocellulosic biomass with mineral acids is well established for xylan breakdown. Fe2+ is known to be a cocatalyst of this process although kinetics of its action remains unknown. The present work addresses the effect of ferrous ion concentration on sugar yield and degradation product formation from corn stover for the entire two-step treatment, including the subsequent enzymatic cellulose hydrolysis. The feedstock was impregnated with 0.5% acid and 0.75 mM iron cocatalyst, which was found to be optimal in preliminary experiments. The detailed kinetic data of acid pretreatment, with and without iron, was satisfactorily modelled with a four-step linear sequence of first-order irreversible reactions accounting for the formation of xylooligomers, xylose and furfural as intermediates to provide the values of Arrhenius activation energy. Based on this kinetic modelling, Fe2+ turned out to accelerate all four reactions, with a significant alteration of the last two steps, that is, xylose degradation. Consistent with this model, the greatest xylan conversion occurred at the highest severity tested under 170 °C/30 min with 0.75 mM Fe2+, with a total of 8% xylan remaining in the pretreated solids, whereas the operational conditions leading to the highest xylose monomer yield, 63%, were milder, 150 °C with 0.75 mM Fe2+ for 20 min. Furthermore, the subsequent enzymatic hydrolysis with the prior addition of 0.75 mM of iron(II) increased the glucose production to 56.3% from 46.3% in the control (iron-free acid). The detailed analysis indicated that conducting the process at lower temperatures yet long residence times benefits the yield of sugars. The above kinetic modelling results of Fe2+ accelerating all four reactions are in line with our previous mechanistic research showing that the pretreatment likely targets multiple chemistries in plant cell wall polymer networks, including those represented by the C–O–C and C–H bonds in cellulose, resulting in enhanced sugar solubilization and digestibility.
... Recently, our group has been working on projects expressing biocatalysts [47][48][49] and glycoside hydrolases (GHs) [50] in Arabidopsis and rice plants. In order to extend these activities to bioenergy plants, it is necessary to evaluate and build a simplified and reliable procedure to genetically modify the switchgrass plants for promoting biofuel production. ...
Background
Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection. ResultsAfter evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N6 macronutrient/B5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines. Conclusion
The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.
BACKGROUND
The commercialized Bt (Bacillus thuringiensis) crops accumulate Bt protein within cells, but the intracellular interactions of foreign protein with endogenous protein inevitably result in large or small unintended effects. In this study, the Bt gene Cry1Ca was linked with the sequences of extracellular secretion signal peptide and carbohydrate binding module 11 to constitute a fusion gene SP‐Cry1Ca‐CBM11, and the fusion gene driven by constitutive promoters was used for secreting and anchoring onto the cell wall to minimize unintended effects.
RESULTS
The transient expression in tobacco leaves demonstrated that the fusion protein was anchored on cell walls. The Cry1Ca contents of five homozygous rice transformants of single‐copy insertion were different and descended in the order leaf > root > stem. The maximum content of Cry1Ca was 17.55 μg g⁻¹ in leaves of transformant 21H037. The bioassay results revealed that the transformants exhibited high resistance to lepidopteran pests. The corrected mortality of pink stem borer (Sesamia inferens) and striped stem borer (Chilo suppressalis) ranged from 96.33% to 100%, and from 83.32% to 100%, respectively, and the corrected mortality of rice leaf roller (Cnaphalocrocis medinalis) was 92.53%. Besides, the agronomic traits of the five transformants were normal and similar to that of the recipient, and the transformants were highly resistant to glyphosate at the germination and seedling stages.
CONCLUSION
The fusion Bt protein was accumulated on cell walls and endowed the rice with high resistance to lepidopteran pests without unintended effects in agronomic traits. © 2023 Society of Chemical Industry.
X-ray Fluorescence Microscopy (XFM), also known as Synchrotron Radiation based X-Ray Fluorescence (SRXRF) or Microprobe Synchrotron X-ray fluorescence (mSXRF), is a powerful and versatile technique for the investigation of elemental content in biological samples. Whole cells can be imaged with resolutions better than 100 nm and their elemental content 3D reconstructed despite a cell thickness of 10 microns or more; with some compromises in the spatial resolution even samples as thick as 100 s of microns can also be imaged in 3D. The resultant elemental map is quantitative – concentrations of the different elements are detected simultaneously pixel by pixel, as the fluorescence radiation emitted is proportional to the atom’s concentration within the sample. Detection limits as low as 0.1–5 ppm can be achieved for metals. With new technical developments such as “fourth generation” synchrotrons, faster detectors and even “X-ray focusing” optics, it is likely that XFM will continue to develop toward ever higher resolution and speed of data acquisition. While XFM can be used for detection of radionuclides in biological material, due to extremely low background for such elements in samples collected in non-contaminated areas, radionuclide quantities are generally low and imaging them is difficult. Moreover, radioactive decay and resultant elemental transitions further decrease numbers of atoms of interest that are available for detection. With the increase in brightness, new generations of synchrotrons and their further updates can be expected to improve sensitivity of radionuclide detection.
