Performing Department
(N/A)
Non Technical Summary
The use of lignocellulosic biomass to produce biofuels and other chemicals has been impeded by the resistance of plant cell walls to enzymatic deconstruction, primarily due to the highly heterogenic polymer, lignin. Various pretreatment strategies have been developed to reduce this recalcitrance. While encouraging results have been achieved, the methods suffer from techno[1]economic challenges. Advances in molecular genetics have offered tremendous opportunities for producing enzymes in planta that can modify cell wall composition to overcome biomass recalcitrance. However, none of these recombinant enzymes have been applied in the biofuel industry on a commercial scale thus far. This approach also has its own set of drawbacks including the metabolic load associated with plants producing large quantities of enzymes, hence requiring more fertilizer inputs, and the risk of undesirable effects on normal plant development. Our recent study revealed that heterologous expression of a bacterial lignin-degrading peroxidase depolymerizes lignin and reduces biomass recalcitrance in transgenic tobacco (Nicotiana benthamiana). This project will explore the potential of highly efficient bacterial and fungal lignin-degrading peroxidases to improve biomass digestibility. Specific objectives include: 1) generating expression cassettes of a fungal Pleurotus eryngii (VPL2) and bacterial Amycolatopsis sp. 75iv2 DyP2 ligninases, a construct containing both genes for co-expression, and a chimeric VPL2::DyP2 construct obtained by swapping N- and C-terminal domains of the two proteins; 2) developing and characterizing transgenic switchgrass lines expressing one or two ligninases using molecular and biochemical tools; 3) determining lignin modification, saccharification, and fermentation efficiency of bioengineered biomass; 4) providing training opportunities for undergraduate students and junior level scientists. We intend to develop a strategy that will address the long-standing challenge of biomass recalcitrance. The platform established using switchgrass will serve as a model to engineer other bioenergy grasses and trees. The project will also provide unique training opportunities for underrepresented minority students in biotechnology and biofuels and prepare them for the scientific workforce. It will also enhance faculty skills to develop grant proposals to seek funding from federal agencies and private foundations. Furthermore, it will strengthen collaboration between Delaware State University with institutions equipped with advanced research facilities.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Goals / Objectives
The commercialization oflignocellulosic biomass as feedstock to efficiently produce biofuels and other economically important chemicals has been tracking behind industrial and Energy Independence and Security Act of 2007 (EISA) targets due to the resistance of plant cell walls to enzymatic deconstruction, primarily due to presence of the highly heterogenic polymer, lignin. Despite significant progress in developingeffective pretreatment strategies to reducesuch ligninrecalcitrance, current technologies are stillproven to be uneconomical.Advances in molecular genetics, genetic engineering, enzyme technology, discovery of novel lignin degrading enzymes offered tremendous opportunities for producing biofuel enzymesto cell wall compartmentin situto overcome biomass recalcitrance. However, application of recombinant lignin enzymes in the biofuel industry on a commercial scale is lacking. Drawbacks to this approach include the metabolic load associated with plants producing large quantities of enzymes, hence requiring more fertilizer inputs, and the risk of undesirable effects on normal plant development. Our recent study revealed that heterologous expression of a bacterial lignin-degrading peroxidase depolymerizeslignin in intact biomassin situand reduces biomass recalcitrance in transgenic tobacco (Nicotiana benthamiana).as demonstrated in saccharification data obtained from bioengineered biomass. Therefore, theoverall goal of this project is to reduce or eliminate lignocellulosic biomass recalcitrance by leveraging and deploy the power of genetic modificationtechnologies todeveloping novel designer biomass with enhanced cell wall degradability and reduce lignin recalcitrance. Hence, we hypothesize that targeted transgenic accumulation andin situactivation of lignin-degrading bacterial and fungal peroxidases in commercial dedicated feedstock such as switchgrass could lead to the development of novel designer switch grassendowed with novel molecular and biochemical capabilities to drive the sustainable production of both biofuels and products of high commercial value chemicals.The current project will explore the potential of highly efficient bacterial and fungal lignin-degrading peroxidases to improve biomass digestibility. Specifically, we will 1) generate expression cassettes using fungal Pleurotus eryngii (VPL2) and bacterial Amycolatopsis sp. (DyP2) ligninases. These constructs will contain both genes for co-expression, and a chimeric VPL2::DyP2 construct obtained by swapping N- and C-terminal domains of the two proteins based on computational analysis by Phyre2 software54; 2) developing and characterizing transgenic switch grass lines expressing one and two ligninases using routine molecular and biochemical tools; 3) determining lignin modification, saccharification, and fermentation efficiency of transgenic biomass; 4) providing training opportunities for undergraduate minority students. Overall, our teamintend to develop a strategy that will address the long-standing challenge of biomass recalcitrance. The novel bioengineering platform will serve as a model to engineer other economically bioenergy grasses and trees for reducing or elimination of lignin recalcitrance.?
Project Methods
Although native sequences encoding bacterial and fungal ligninolytic enzymes have been successfully express in plants, we intend to codon-optimize the sequence of both Amycolatopsis sp. 75iv2 Dyp2 and Pleurotus eryngii (VPL2) for switchgrass at GenScript (www.genescript.com). Since ER-targeting has several advantages including the presence of a suitable environment for enhanced protein expression, correct protein folding, and disulfide bridge formation by ER-chaperones, the proteins will be targeted to the ER using an N-terminal ER-signal peptide based on Arabidopsis endochitinase (ChiB) gene and the C-terminal retention signal for higher level protein accumulation. To confirm intracellular localization of the protein, the genes will be fused to green fluorescent protein (GFP) for transient expression in tobacco (N. benthamiana) leaves and detection using Confocal Laser Scanning Microscopy. Expression cassettes will be generated in pSAT-modular vector system in-frame with protein epitope tags, which will ease purification and detection of recombinant proteins using standardized methods. Gene expression will be under the control of the switchgrass PvUbi1 or PvUbi2 ubiquitin promoters and the rice actin promoter (OsAct), and a translation enhancer (TEV-TL). Single or double expression cassettes will be inserted into pPZP-RCS2 binary vector which has the neomycin phosphotransferase (NPTII) for transgenic tissue selection using aminoglycoside antibiotics such as kanamycin and paramomycin. We have successfully generated transgenic grasses (switchgrass and Brachypodium) using pPZP-RCS2 binary vector previously. We also intend to generate expression cassette of chimeric bacterial and fungal peroxidases by swapping protein domains which may lead to a superior ligninase variant. Fungal ligninolytic enzymes have higher catalytic efficiency in degrading lignin than bacterial enzymes, while bacterial enzymes usually express well in heterologous expression systems as compared to fungal proteins due to complexity of fungal genetics. Therefore, we anticipate that swapping protein domains in these constructs may lead to chimeric enzymes with improved enzyme production and catalytic activity. However, prior to heterologous expression in switchgrass, the chimeric genes will be inserted into pET24a under the control of strong bacteriophage T7 transcription and translation signals for expression in E. coli BL21(DE3) (Novagen) and the chimeric enzyme will be purified and tested on model compounds in vitro. Recombinant protein production in E. coli, purification and assay of recombinant enzyme activity is routinely in our laboratory. Expression cassettes of two chimeric ligninases (N-DyP2:C-VPL2, N[1]VPL2::C-DyP2; will be generated by swapping the N-terminal domain of VPL2 possessing a predicted substrate binding motif and most of the heme-binding motif, and the C-terminal domain of DyP2, which has putative catalytic residues. Motif analysis was performed in collaboration with Dr. David Schneider who is a computational biologist at USDA-ARS (Cornell University). The chimeric constructs will also be generated in pSAT shuttle vectors and assembled into the pPZP-RCS2 binary vector. Correctness of the expression cassettes will be confirmed by restriction enzyme digestion and sequencing prior to introduction into Agrobacterium and transformation of switchgrass calli. For Agrobacterium-mediated transformation, highly proliferating and regenerating embryogenic calli will be developed from two lowland switchgrass cultivars (Timber, which can produce high aboveground biomass yield60, and Performer that showed high Agrobacterium-mediated transformability of mature seeds[1]derived calli) based on established. The tissues will be transformed using Agrobacterium strain EHA105 harboring the binary vectors with expression cassettes of the ligninolytic peroxidases and selectable marker genes. Tissues will also be transformed with a binary vector containing the GUS reporter gene62 for visual monitoring of transformation efficiency. Transgene insertion will be confirmed by PCR using genomic DNA as a template. Transgenic seeds from regenerated T0 plants will be germinated to obtained T1 plants for characterization. Transgenic lines will be characterized phenotypically for growth parameters such as plant height, canopy area and biomass accumulation. Transgenic plants will be monitored by qPCR and western blots using gene-specific antibodies, and microscopic imaging. Recombinant enzymes will be purified from total protein extracted fromleaves using kits suitable for each epitope, His-select Ni-affinity purification kit (Sigma) for His-tagged protein and Pierce HA[1]Tag IP/Co-IP Kit (Thermo Scientific) for HA-tagged protein. The activity of recombinant enzymes will be tested using both standard peroxidase substrates and lignin model compounds. The catalytic activity of recombinant DyP2 on standard peroxidase substrates such as 2, 4-dichlorophenol and ABTS, and lignin model dimers, and MnP and LiP activities of VPL2 will be determined will be performed according to published procedures. Recombinant enzymes extracted from DyP2 and VPL2 co-expressing lines will be purified using HA and His-tags, respectively, and their activity will also be assayed. Chimeric recombinant enzymes will also be purified and assayed for DyP, LiP, or MnP activity initially, and since the chimeric enzymes may exhibit intermediate or unique properties, assay condition will be optimized as needed. To study the effect of peroxidase accumulation in the biomass on lignin polymerization, in situ lignin modification will be studied using microscopy techniques in collaboration with Dr. Charles Anderson, who has developed biochemical labeling and fluorescent staining techniques to analyze lignin polymerization and accumulation in plant tissues according to published methods. To assay lignin accumulation and localization, control and transgenic stems will be cryosectioned and stained with Basic Fuchsin, a dye that allows imaging[1]based relative quantification of lignin in different tissues, and imaged by spinning disk confocal microscopy using a Zeiss Cell Observer SD microscope at Dr. Charles Anderson's laboratory in Pennsylvania State University. This approach will also be used to determine lignin modification after activation of the recombinant enzyme directly in the biomass prior to saccharification. Optimal conditions for enzyme activation in the biomass will be determined based on the conditions previously used to analyze the activity of recombinant DypB in vitro at Delaware State University. We will also investigate cell wall ultrastructure before and after peroxidase activation prior to saccharification using Scanning Electron Microscopy on a Zeiss Sigma FESEM microscope also at Pennsylvania State University Microscopy and Cytometry Facility. The biomass modified by the peroxidase enzymes will be subjected to saccharification and fermentation in collaboration with Dr. Nancy Nichols.Dr. Nancy Nichols'slaboratory in ARS, in IL, Peoria usDionex ion chromatography for analysis of biomass composition and saccharification products, sugar fermentation and quantification of fermentation product such as bioethanol. Analysis of lignin degradation by the peroxidases including aromatic products will be performed at the Complex Carbohydrate Research Center, GA in collaboration with Dr. Parastoo Azadi.