Engineering of Bioenergy Crops: Dominant Genetic Approaches to Improve Polysaccharide Properties and Composition in Biomass.

Large-scale, sustainable production of lignocellulosic bioenergy from biomass will depend on a variety of dedicated bioenergy crops. Despite their great genetic diversity, prospective bioenergy crops share many similarities in the polysaccharide composition of their cell walls, and the changes needed to optimize them for conversion are largely universal. Therefore, biomass modification strategies that do not depend on genetic background or require mutant varieties are extremely valuable. Due to their preferential fermentation and conversion by microorganisms downstream, the ideal bioenergy crop should contain a high proportion of C6-sugars in polysaccharides like cellulose, callose, galactan, and mixed-linkage glucans. In addition, the biomass should be reduced in inhibitors of fermentation like pentoses and acetate. Finally, the overall complexity of the plant cell wall should be modified to reduce its recalcitrance to enzymatic deconstruction in ways that do no compromise plant health or come at a yield penalty. This review will focus on progress in the use of a variety of genetically dominant strategies to reach these ideals. Due to the breadth and volume of research in the field of lignin bioengineering, this review will instead focus on approaches to improve polysaccharide component plant biomass. Carbohydrate content can be dramatically increased by transgenic overexpression of enzymes involved in cell wall polysaccharide biosynthesis. Additionally, the recalcitrance of the cell wall can be reduced via the overexpression of native or non-native carbohydrate active enzymes like glycosyl hydrolases or carbohydrate esterases. Some research in this area has focused on engineering plants that accumulate cell wall-degrading enzymes that are sequestered to organelles or only active at very high temperatures. The rationale being that, in order to avoid potential negative effects of cell wall modification during plant growth, the enzymes could be activated post-harvest, and post-maturation of the cell wall. A potentially significant limitation of this approach is that at harvest, the cell wall is heavily lignified, making the substrates for these enzymes inaccessible and their activity ineffective. Therefore, this review will only include research employing enzymes that are at least partially active under the ambient conditions of plant growth and cell wall development

Plant Glycosyltransferases Beyond CAZy: A Perspective on DUF Families

The carbohydrate active enzyme (CAZy) database is an invaluable resource for glycobiology and currently contains 45 glycosyltransferase families that are represented in plants. Glycosyltransferases (GTs) have many functions in plants, but the majority are likely to be involved in biosynthesis of polysaccharides and glycoproteins in the plant cell wall. Bioinformatic approaches and structural modeling suggest that a number of protein families in plants include GTs that have not yet been identified as such and are therefore not included in CAZy. These families include proteins with domain of unknown function (DUF) DUF23, DUF246, and DUF266. The evidence for these proteins being GTs and their possible roles in cell wall biosynthesis is discussed

Synthesis of molecularly imprinted polymer for glucose binding

Molecularly imprinted polymer (MIP) is an attractive technique for the synthesis of highly selective polymeric receptors having artificial generated recognition sites. These materials were synthesized with polymerizable functional monomers and crosslinker that were surrounded around the template molecule. After polymerization, a template molecule was removed leaving in the polymer selective recognition sites with shape, size and functionalities complementary to the template. This study presents a synthesis of MIP selectively for glucose binding. Glucose phosphate salt (GPS) was used as a template molecule with poly(allylamine hydrochloride) (PAA.HCl) as a functional monomer. Three types of crosslinkers which are epichlorohydrin (EPI), ethylene glycol diglycidyl ether (EDGE) and glycerol diglycidyl ether (GDE) were studied during the MIP synthesis. MIP prepared using EPI as a crosslinking showed the highest glucose binding capacity around 0.84 mg glucose/mg dried gel. The binding capacity of MIP prepared using EGDE and GDE are 0.78 mg glucose/mg gel and 0.38 mg glucose/mg gel respectively. It is also found that the increase on GPS monomer concentration will contribute to increase in glucose bindin

A reversible <i>Renilla </i>luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta

Comparison of dilute acid and ionic liquid pretreatment of switchgrass: Biomass recalcitrance, delignification and enzymatic saccharification

The efficiency of two biomass pretreatment technologies, dilute acid hydrolysis and dissolution in an ionic liquid, are compared in terms of delignification, saccharification efficiency and saccharide yields with switchgrass serving as a model bioenergy crop. When subject to ionic liquid pretreatment (dissolution and precipitation of cellulose by anti-solvent) switchgrass exhibited reduced cellulose crystallinity, increased surface area, and decreased lignin content compared to dilute acid pretreatment. Pretreated material was characterized by powder X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy and chemistry methods. Ionic liquid pretreatment enabled a significant enhancement in the rate of enzyme hydrolysis of the cellulose component of switchgrass, with a rate increase of 16.7-fold, and a glucan yield of 96.0% obtained in 24 h. These results indicate that ionic liquid pretreatment may offer unique advantages when compared to the dilute acid pretreatment process for switchgrass. However, the cost of the ionic liquid process must also be taken into consideration

Three novel rice genes closely related to the <em>Arabidopsis</em> _{9<em>IRX</em>}, _{9<em>IRXL</em>}, and_{14<em> IRX</em>} genes and their roles in xylan biosynthesis

Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength

The elaborate route for UDP-arabinose delivery into the Golgi of plants

Indexación: Scopus.In plants, L-Arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDPAraf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transported back into the lumen. This step is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Thus, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgilocalized UDP-Araf transporters in Arabidopsis. The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo.http://www.pnas.org/content/114/16/4261.ful

Molecular Studies of the Protein Complexes Involving Cis-Prenyltransferase in Guayule (Parthenium argentatum), an Alternative Rubber-Producing Plant

Guayule (Parthenium argentatum) is a perennial shrub in the Asteraceae family and synthesizes a high quality, hypoallergenic cis-1,4-polyisoprene (or natural rubber; NR). Despite its potential to be an alternative NR supplier, the enzymes for cis-polyisoprene biosynthesis have not been comprehensively studied in guayule. Recently, implications of the protein complex involving cis-prenyltransferases (CPTs) and CPT-Binding Proteins (CBPs) in NR biosynthesis were shown in lettuce and dandelion, but such protein complexes have yet to be examined in guayule. Here, we identified four guayule genes – three PaCPTs (PaCPT1-3) and one PaCBP, whose protein products organize PaCPT/PaCBP complexes. Co-expression of both PaCBP and each of the PaCPTs could complemented the dolichol (a short cis-polyisoprene)-deficient yeast, whereas the individual expressions could not. Microsomes from the PaCPT/PaCBP-expressing yeast efficiently incorporated 14C-isopentenyl diphosphate into dehydrodolichyl diphosphates; however, NR with high molecular weight could not be synthesized in in vitro assays. Furthermore, co-immunoprecipitation and split-ubiquitin yeast 2-hybrid assays using PaCPTs and PaCBP confirmed the formation of protein complexes. Of the three PaCPTs, guayule transcriptomics analysis indicated that the PaCPT3 is predominantly expressed in stem and induced by cold-stress, suggesting its involvement in NR biosynthesis. The comprehensive analyses of these PaCPTs and PaCBP here provide the foundational knowledge to generate a high NR-yielding guayule

Industrial brewing yeast engineered for the production of primary flavor determinants in hopped beer

Production of aromatic monoterpene molecules in hop flowers is affected by genetic, environmental, and processing factors. Here, the authors engineer brewer’s yeast for the production of linalool and geraniol, and show pilot-scale beer produced by engineered strains reconstitutes some qualities of hop flavor

Field performance of switchgrass plants engineered for reduced recalcitrance

Switchgrass (Panicum virgatum L.) is a promising perennial bioenergy crop that achieves high yields with relatively low nutrient and energy inputs. Modification of cell wall composition for reduced recalcitrance can lower the costs of deconstructing biomass to fermentable sugars and other intermediates. We have engineered overexpression of OsAT10, encoding a rice BAHD acyltransferase and QsuB, encoding dehydroshikimate dehydratase from Corynebacterium glutamicum, to enhance saccharification efficiency in switchgrass. These engineering strategies demonstrated low lignin content, low ferulic acid esters, and increased saccharification yield during greenhouse studies in switchgrass and other plant species. In this work, transgenic switchgrass plants overexpressing either OsAT10 or QsuB were tested in the field in Davis, California, USA for three growing seasons. No significant differences in the content of lignin and cell wall-bound p-coumaric acid or ferulic acid were detected in transgenic OsAT10 lines compared with the untransformed Alamo control variety. However, the transgenic overexpressing QsuB lines had increased biomass yield and slightly increased biomass saccharification properties compared to the control plants. This work demonstrates good performance of engineered plants in the field, and also shows that the cell wall changes in the greenhouse were not replicated in the field, emphasizing the need to validate engineered plants under relevant field conditions