Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2-year comparative analysis of field-grown lines modified for target gene or genetic element expression.

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4-KD, miRNA156-OE, MYB4-OE, COMT-KD and FPGS-KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second- versus the first-year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second-year growth of transgenics targeted for wall modification, GAUT4-KD, MYB4-OE, COMT-KD and FPGS-KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next-generation bio-feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Phenotypic characterization and comparative transcriptomics of evolved Saccharomyces cerevisiae strains with improved tolerance to lignocellulosic derived inhibitors.

BACKGROUND: Lignocellulosic biomass continues to be investigated as a viable source for bioethanol production. However, the pretreatment process generates inhibitory compounds that impair the growth and fermentation performance of microorganisms such as Saccharomyces cerevisiae. Pinewood specifically has been shown to be challenging in obtaining industrially relevant ethanol titers. An industrial S. cerevisiae strain was subjected to directed evolution and adaptation in pretreated pine biomass and resultant strains, GHP1 and GHP4, exhibited improved growth and fermentative ability on pretreated pine in the presence of related inhibitory compounds. A comparative transcriptomic approach was applied to identify and characterize differences in phenotypic stability of evolved strains. RESULTS: Evolved strains displayed different fermentative capabilities with pretreated pine that appear to be influenced by the addition or absence of 13 inhibitory compounds during pre-culturing. GHP4 performance was consistent independent of culturing conditions, while GHP1 performance was dependent on culturing with inhibitors. Comparative transcriptomics revealed 52 genes potentially associated with stress responses to multiple inhibitors simultaneously. Fluorescence microscopy revealed improved cellular integrity of both strains with mitochondria exhibiting resistance to the damaging effects of inhibitors in contrast to the parent. CONCLUSIONS: Multiple potentially novel genetic targets have been discovered for understanding stress tolerance through the characterization of our evolved strains. This study specifically examines the synergistic effects of multiple inhibitors and identified targets will guide future studies in remediating effects of inhibitors and further development of robust yeast strains for multiple industrial applications.

Development of Agave as a dedicated biomass source: production of biofuels from whole plants.

BACKGROUND: Agave species can grow well in semi-arid marginal agricultural lands around the world. Selected Agave species are used largely for alcoholic beverage production in Mexico. There are expanding research efforts to use the plentiful residues (bagasse) for ethanol production as the beverage manufacturing process only uses the juice from the central core of mature plants. Here, we investigate the potential of over a dozen Agave species, including three from cold semi-arid regions of the United States, to produce biofuels using the whole plant. RESULTS: Ethanol was readily produced by Saccharomyces cerevisiae from hydrolysate of ten whole Agaves with the use of a proper blend of biomass degrading enzymes including inulinase that overcomes inhibition of most of the species tested. As an example, US grown Agave neomexicana produced 119 ± 11 mg ethanol/g biomass. Unlike yeast fermentations, Clostridium beijerinckii produced n-butanol plus acetone from all species tested. Butyric acid, a precursor of n-butanol, was also present due to incomplete conversion during the screening process. Since Agave contains high levels of free and polyfructose which are readily destroyed by acidic pretreatment, a two-step procedure was developed to depolymerize polyfructose while maintaining its fermentability. The hydrolysate from before and after dilute acid processing was used in C. beijerinckii fermentations with selected Agave species with A. neomexicana producing 144 ± 4 mg fermentation products/g biomass. CONCLUSIONS: Results showed Agave's potential to be a source of fermentable sugars beyond the existing beverage species to now include many species previously unfermentable by yeast, including cold-tolerant lines. This development should stimulate development of Agave as a dedicated feedstock for biofuels in semi-arid regions throughout the globe.

Consolidated bioprocessing of transgenic switchgrass by an engineered and evolved Clostridium thermocellum strain.

BACKGROUND: Switchgrass is an abundant and dedicated bioenergy feedstock, however its inherent recalcitrance is one of the economic hurdles for producing biofuels. The downregulation of the caffeic acid O-methyl transferase (COMT) gene in the lignin pathway of switchgrass reduced lignin content and S/G ratio, and the transgenic lines showed improved fermentation yield with Saccharomyces cerevisiae and wild-type Clostridium thermocellum (ATCC 27405) in comparison to the wild-type switchgrass. RESULTS: Here we examine the conversion and yield of the COMT transgenic and wild-type switchgrass lines with an engineered and evolved C. thermocellum (M1570) strain. The fermentation of the transgenic switchgrass by M1570 had superior conversion relative to the wild-type control switchgrass line with an increase in conversion of approximately 20% and ethanol being the primary product accounting for 90% of the total metabolites measured by HPLC analysis. CONCLUSIONS: The engineered and evolved C. thermocellum M1570 was found to respond to the apparent reduced recalcitrance of the COMT switchgrass with no substrate inhibition, producing more ethanol on the transgenic feedstock than the wild-type substrate. Since ethanol was the main fermentation metabolite produced by an engineered and evolved C. thermocellum strain, its ethanol yield on a transgenic switchgrass substrate (gram/gram (g/g) glucan liberated) is the highest produced thus far. This result indicates that the advantages of a modified feedstock can be combined with a modified consolidated bioprocessing microorganism as anticipated.

Patterns of cell division, cell differentiation and cell elongation in epidermis and cortex of Arabidopsis pedicels in the wild type and in erecta.

Plant organ shape and size are established during growth by a predictable, controlled sequence of cell proliferation, differentiation, and elongation. To understand the regulation and coordination of these processes, we studied the temporal behavior of epidermal and cortex cells in Arabidopsis pedicels and used computational modeling to analyze cell behavior in tissues. Pedicels offer multiple advantages for such a study, as their growth is determinate, mostly one dimensional, and epidermis differentiation is uniform along the proximodistal axis. Three developmental stages were distinguished during pedicel growth: a proliferative stage, a stomata differentiation stage, and a cell elongation stage. Throughout the first two stages pedicel growth is exponential, while during the final stage growth becomes linear and depends on flower fertilization. During the first stage, the average cell cycle duration in the cortex and during symmetric divisions of epidermal cells was constant and cells divided at a fairly specific size. We also examined the mutant of ERECTA, a gene with strong influence on pedicel growth. We demonstrate that during the first two stages of pedicel development ERECTA is important for the rate of cell growth along the proximodistal axis and for cell cycle duration in epidermis and cortex. The second function of ERECTA is to prolong the proliferative phase and inhibit premature cell differentiation in the epidermis. Comparison of epidermis development in the wild type and erecta suggests that differentiation is a synchronized event in which the stomata differentiation and the transition of pavement cells from proliferation to expansion are intimately connected.