[Show abstract][Hide abstract] ABSTRACT: The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.
Proceedings of the National Academy of Sciences 06/2015; 112(28). DOI:10.1073/pnas.1424031112 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Photosynthetic hydrocarbon production bypasses the traditional biomass hydrolysis process and represents the most direct conversion of sunlight energy into the next-generation biofuels. As a major class of biologically derived hydrocarbons with diverse structures, terpenes are also valuable in producing a variety of fungible bioproducts in addition to the advanced ‘drop-in’ biofuels. However, it is highly challenging to achieve the efficient redirection of photosynthetic carbon and reductant into terpene biosynthesis. In this review, we discuss four major scientific and technical barriers for photosynthetic terpene production and recent advances to address these constraints. Collectively, photosynthetic terpene production needs to be optimized in a systematic fashion, in which the photosynthesis improvement, the optimization of terpene biosynthesis pathway, the improvement of key enzymes and the enhancement of sink effect through terpene storage or secretion are all important. New advances in synthetic biology also offer a suite of potential tools to design and engineer photosynthetic terpene platforms. The systemic integration of these solutions may lead to ‘disruptive’ technologies to enable biofuels and bioproducts with high efficiency, yield and infrastructure compatibility.
[Show abstract][Hide abstract] ABSTRACT: Lignin utilization during biomass conversion has been a major challenge for lignocellulosic biofuel. In particular, the conversion of lignin along with carbohydrate for fungible fuels and chemicals will both improve the overall carbon efficiency and reduce the need for chemical pretreatments. However, few biomass-converting microorganisms have the capacity to degrade all cell wall components including lignin, cellulose, and hemicellulose. We hereby evaluated a unique oleaginous fungus strain Cunninghamella echinulata FR3 for its capacity to degrade lignin during biomass conversion to lipid, and the potential to carry out consolidated fermentation without chemical pretreatment, especially when combined with sorghum (Sorghum bicolor) bmr mutants with reduced lignin content. The study clearly showed that lignin was consumed together with carbohydrate during biomass conversion for all sorghum samples, which indicates this organism has the potential for biomass conversion without chemical pretreatment. Even though dilute acid pretreatment of biomass resulted in more weight loss during fungal fermentation than untreated biomass, the lipid yields were comparable for untreated bmr6/bmr12 double mutant and dilute acid-pretreated wild-type biomass samples. The mechanisms for lignin degradation in oleaginous fungi were further elucidated through transcriptomics and chemical analysis. The studies showed that in C. echinulata FR3, Fenton Reaction may play an important role in lignin degradation. This discovery is among the first to show that a mechanism for lignin degradation similar to ones found in white and brown rot basidiomycetous fungi exists in an oleaginous fungus. This study suggests that oleaginous fungus such as C. echinulata FR3 can be employed for complete biomass utilization in a consolidated platform without chemical pretreatment, or can be used to convert lignin waste into lipids.
Green Chemistry 12/2014; 17(3). DOI:10.1039/C4GC01529K · 6.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Efficient degradation and utilization of lignocellulosic biomass remains a challenge for sustainable and affordable biofuels. Various natural biomass utilization systems (NBUS) evolved the capacity to combat the recalcitrance of plant cell walls. The study of these NBUS could enable the development of efficient and cost-effective biocatalysts, microorganisms, and bioprocesses for biofuels and bioproducts. Here, we reviewed the recent research progresses for several NBUS, ranging from single cell microorganisms to consortiums such as cattle rumen and insect guts. These studies aided the discovery of biomass-degrading enzymes and the elucidation of the evolutionary and functional relevance in these systems. In particular, advances in the next generation 'omics' technologies offered new opportunities to explore NBUS in a high-throughput manner. Systems biology helped to facilitate the rapid biocatalyst discovery and detailed mechanism analysis, which could in turn guide the reverse design of engineered microorganisms and bioprocesses for cost-effective and efficient biomass conversion.
Current Opinion in Biotechnology 03/2014; 27C:195-203. DOI:10.1016/j.copbio.2014.02.007 · 8.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Basic research has provided a much better understanding of the genetic networks and regulatory hierarchies in plants. To meet the challenges of agriculture, we must be able to rapidly translate this knowledge into generating improved plants. Therefore, in this Review, we discuss advanced tools that are currently available for use in plant biotechnology to produce new products in plants and to generate plants with new functions. These tools include synthetic promoters, 'tunable' transcription factors, genome-editing tools and site-specific recombinases. We also review some tools with the potential to enable crop improvement, such as methods for the assembly and synthesis of large DNA molecules, plant transformation with linked multigenes and plant artificial chromosomes. These genetic technologies should be integrated to realize their potential for applications to pressing agricultural and environmental problems.
[Show abstract][Hide abstract] ABSTRACT: Genetically engineered (GE) ringspot virus-resistant papayas cultivars 'Rainbow' and 'SunUp' have been grown in Hawai'i for over 10 years. In Hawai'i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities.
We assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/mul of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%.
This method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai'i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.
[Show abstract][Hide abstract] ABSTRACT: High abundance proteins like Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. In order to address this challenge, we developed and evaluated Polyethyleneimine (PEI) Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery.
[Show abstract][Hide abstract] ABSTRACT: Metagenome analysis of the gut symbionts of three different insects was conducted as a means of comparing taxonomic and metabolic diversity of gut microbiomes to diet and life history of the insect hosts. A second goal was the discovery of novel biocatalysts for biorefinery applications. Grasshopper and cutworm gut symbionts were sequenced and compared with the previously identified metagenome of termite gut microbiota. These insect hosts represent three different insect orders and specialize on different food types. The comparative analysis revealed dramatic differences among the three insect species in the abundance and taxonomic composition of the symbiont populations present in the gut. The composition and abundance of symbionts was correlated with their previously identified capacity to degrade and utilize the different types of food consumed by their hosts. The metabolic reconstruction revealed that the gut metabolome of cutworms and grasshoppers was more enriched for genes involved in carbohydrate metabolism and transport than wood-feeding termite, whereas the termite gut metabolome was enriched for glycosyl hydrolase (GH) enzymes relevant to lignocellulosic biomass degradation. Moreover, termite gut metabolome was more enriched with nitrogen fixation genes than those of grasshopper and cutworm gut, presumably due to the termite's adaptation to the high fiber and less nutritious food types. In order to evaluate and exploit the insect symbionts for biotechnology applications, we cloned and further characterized four biomass-degrading enzymes including one endoglucanase and one xylanase from both the grasshopper and cutworm gut symbionts. The results indicated that the grasshopper symbiont enzymes were generally more efficient in biomass degradation than the homologous enzymes from cutworm symbionts. Together, these results demonstrated a correlation between the composition and putative metabolic functionality of the gut microbiome and host diet, and suggested that this relationship could be exploited for the discovery of symbionts and biocatalysts useful for biorefinery applications.
[Show abstract][Hide abstract] ABSTRACT: To overcome the daunting technical barriers of algae biofuels and photosynthetic biorefineries, a novel cultivation technology has been developed to concentrate, harvest, and enhance microalgae-based biofuels and bioproducts through pelletization. The technology involves the co-cultivation of microalgae with fungi to achieve optimized pelletization with a 2-to-10-mm diameter. This pelletization enables the complete removal of single algal cells from the liquid medium to allow their extraction and harvest by simple filtration. In addition, the pelletization process results in significantly increased biomass, lipid, and bioproduct yields. If successfully scaled up, this technology has the potential to improve the sustainability and economic viability of the production of algal biofuels.
Algal Research 01/2013; 2(1):28–33. DOI:10.1016/j.algal.2012.11.004 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multidimensional protein identification technology (MudPIT)-based shot-gun proteomics has been proven to be an effective platform for functional proteomics. In particular, the various sample preparation methods and bioinformatics tools can be integrated to improve the proteomics platform for applications like target organelle proteomics. We have recently integrated a rapid sample preparation method and bioinformatics classification system for comparative analysis of plant responses to two plant hormones, zeatin and brassinosteroid (BR). These hormones belong to two distinct classes of plant growth regulators, yet both can promote cell elongation and growth. An understanding of the differences and the cross-talk between the two types of hormone responses will allow us to better understand the molecular mechanisms and to identify new candidate genes for plant engineering.
As compared to traditional organelle proteomics, the organelle-enrichment method both simplifies the sample preparation and increases the number of proteins identified in the targeted organelle as well as the entire sample. Both zeatin and BR induce dramatic changes in signaling and metabolism. Their shared-regulated protein components indicate that both hormones may down-regulate some key components in auxin responses. However, they have shown distinct induction and suppression of metabolic pathways in mitochondria and chloroplast. For zeatin, the metabolic pathways in sucrose and starch biosynthesis and utilization were significantly changed, yet the lipid biosynthesis remained unchanged. For BR, lipid biosynthesis and β-oxidation were both down-regulated, yet the changes in sucrose and starch metabolism were minor.
We present a rapid sample preparation method and bioinformatics classification for effective proteomics analysis of plant hormone responses. The study highlighted the largely differing response to zeatin and brassinosteroid by the metabolic pathways in chloroplast and mitochondria.
[Show abstract][Hide abstract] ABSTRACT: NAC domain transcription factors are important transcriptional regulators involved in plant growth, development and stress responses. Recent studies have revealed several classes of NAC transcriptional factors crucial for controlling secondary cell wall biosynthesis. These transcriptional factors mainly include three classes, SND, NST and VND. Despite progress, most current analysis is carried out in the model plant Arabidopsis. Moreover, many downstream genes regulated by these transcriptional factors are still not clear.
In order to identify the key homologue genes across species and discover the network controlling cell wall biosynthesis, we carried out comparative genome analysis of NST, VND and SND genes across 19 higher plant species along with computational modelling of genes regulated or co-regulated with these transcriptional factors.
The comparative genome analysis revealed that evolutionarily the secondary-wall-associated NAC domain transcription factors first appeared in Selaginella moellendorffii. In fact, among the three groups, only VND genes appeared in S. moellendorffii, which is evolutionarily earlier than the other two groups. The Arabidopsis and rice gene expression analysis showed specific patterns of the secondary cell wall-associated NAC genes (SND, NST and VND). Most of them were preferentially expressed in the stem, especially the second internodes. Furthermore, comprehensive co-regulatory network analysis revealed that the SND and MYB genes were co-regulated, which indicated the coordinative function of these transcriptional factors in modulating cell wall biosynthesis. In addition, the co-regulatory network analysis revealed many novel genes and pathways that could be involved in cell wall biosynthesis and its regulation. The gene ontology analysis also indicated that processes like carbohydrate synthesis, transport and stress response, are coordinately regulated toward cell wall biosynthesis.
Overall, we provided a new insight into the evolution and the gene regulatory network of a subgroup of the NAC gene family controlling cell wall composition through bioinformatics data mining and bench validation. Our work might benefit to elucidate the possible molecular mechanism underlying the regulation network of secondary cell wall biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: Background: Insect gut symbionts are essential for biomass degradation, nutrient synthesis and toxin detoxification. We carried out comparative analysis of gut symbionts from four insect species to investigate the adaptation to different food types and to evaluate the potential for biofuel applications. Results: The study revealed significant variation of insect gut symbiont composition as a function to adapt to different food types. The composition–function relationship has been discussed from different perspectives, including biomass utilization and nutrient biosynthesis. The existence of species such as Klebsiella oxytoca and the capacity for these species to produce unique biomass degradation enzymes indicated that grasshopper and woodborer gut systems may be enriched resources for biofuel applications. For functional verification and concept validation, we have cloned and characterized a β-glucosidase from K. oxytoca in the grasshopper gut system. Conclusion: The research suggested that different insect gut systems can be good resources for biocatalyst and microbial strain discovery for bioenergy applications.
[Show abstract][Hide abstract] ABSTRACT: After herbivore damage, many plants increase their emission of volatile compounds, with terpenes usually comprising the major group of induced volatiles. Populus trichocarpa is the first woody species with a fully sequenced genome, enabling rapid molecular approaches towards characterization of volatile terpene biosynthesis in this and other poplar species. We identified and characterized four terpene synthases (PtTPS1-4) from P. trichocarpa which form major terpene compounds of the volatile blend induced by gypsy moth (Lymantria dispar) feeding. The enzymes were heterologously expressed and assayed with potential prenyl diphosphate substrates. PtTPS1 and PtTPS2 accepted only farnesyl diphosphate and produced (-)-germacrene D and (E,E)-α-farnesene as their major products, respectively. In contrast, PtTPS3 and PtTPS4 showed both mono- and sesquiterpene synthase activity. They produce the acyclic terpene alcohols linalool and nerolidol but exhibited opposite stereospecificity. qRT-PCR analysis revealed that the expression of the respective terpene synthase genes was induced after feeding of gypsy moth caterpillars. The TPS enzyme products may play important roles in indirect defense of poplar to herbivores and in mediating intra- and inter-plant signaling.
[Show abstract][Hide abstract] ABSTRACT: Insect guts represent unique natural biocatalyst systems for biocatalyst discovery and biomass deconstruction mechanism studies.
In order to guide the further research for enzyme discovery and biodiversity analysis, we carried out comprehensive xylanase
and cellulase activity assays for the gut contents of three insect species representing different orders and food sources.
The three insect species are grasshopper (Acrididae sp.), woodborer (Cerambycidae spp.), and silkworm (Bombyx mori) to represent the wood-consuming, grass-consuming, and leaf-consuming insects from Orthoptera, Coleoptera, and Lepidoptera
orders, respectively. Generally speaking, the enzyme activity assays have shown that the cellulase and xylanase activities
for grasshopper and woodborer guts are significantly higher than those of silkworm under various conditions. In addition,
both pH and temperature have a significant impact on the enzyme activities in the gut contents. For the grasshopper gut, the
means of xylanase and cellulase activities at pH7 were 3,397 and 404μM mg−1 min−1, which are significantly higher than the activities at pH4 and 10 (P < 0.05). However, woodborer guts have shown the highest cellulase activity at pH10. The results suggested that systems similar
to woodborer guts could be good resources for discovering alkaline-tolerant enzymes. Moreover, the enzyme activities in response
to different substrate concentrations were also analyzed, which indicated that grasshopper gut had particularly high cellulase
activity. The enzyme activities in response to the reaction time were also examined, and we found that the enzyme activities
(micromolar per milligram per minute) of different insect gut juices in response to the increase of incubation time fit well
to the power function equation (E
c = K ⋅ t
) with high coefficients (r
2 > 0.99). The newly developed model serves well to compare the characteristics of the enzyme mixtures among different insect
species, which can be applied to other studies of natural biocatalyst systems for the future. Overall, the data indicated
that grasshopper and woodborer guts are valuable resources for discovering the novel biocatalysts for various biorefinery
KeywordsInsect gut-Cellulase-Xylanase-Natural biocatalyst system
BioEnergy Research 03/2011; 4(1):1-10. DOI:10.1007/s12155-010-9096-0 · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
HDX mass spectrometry is a powerful platform to probe protein structure dynamics during ligand binding, protein folding, enzyme catalysis, and such. HDX mass spectrometry analysis derives the protein structure dynamics based on the mass increase of a protein of which the backbone protons exchanged with solvent deuterium. Coupled with enzyme digestion and MS/MS analysis, HDX mass spectrometry can be used to study the regional dynamics of protein based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX mass spectrometry data. Despite the progresses, proper and explicit statistical treatment is still lacking in most of the current HDX mass spectrometry software. In order to address this issue, we have developed the HDXanalyzer for the statistical analysis of HDX mass spectrometry data using R, Python, and RPY2.
Implementation and results
HDXanalyzer package contains three major modules, the data processing module, the statistical analysis module, and the user interface. RPY2 is employed to enable the connection of these three components, where the data processing module is implemented using Python and the statistical analysis module is implemented with R. RPY2 creates a low-level interface for R and allows the effective integration of statistical module for data processing. The data processing module generates the centroid for the peptides in form of m/z value, and the differences of centroids between the peptides derived from apo and ligand-bound protein allow us to evaluate whether the regions have significant changes in structure dynamics or not. Another option of the software is to calculate the deuterium incorporation rate for the comparison. The two types of statistical analyses are Paired Student’s t-test and the linear combination of the intercept for multiple regression and ANCOVA model. The user interface is implemented with wxpython to facilitate the data visualization in graphs and the statistical analysis output presentation. In order to evaluate the software, a previously published xylanase HDX mass spectrometry analysis dataset is processed and presented. The results from the different statistical analysis methods are compared and shown to be similar. The statistical analysis results are overlaid with the three dimensional structure of the protein to highlight the regional structure dynamics changes in the xylanase enzyme.
Statistical analysis provides crucial evaluation of whether a protein region is significantly protected or unprotected during the HDX mass spectrometry studies. Although there are several other available software programs to process HDX experimental data, HDXanalyzer is the first software program to offer multiple statistical methods to evaluate the changes in protein structure dynamics based on HDX mass spectrometry analysis. Moreover, the statistical analysis can be carried out for both m/z value and deuterium incorporation rate. In addition, the software package can be used for the data generated from a wide range of mass spectrometry instruments.