Information

7.15B: Genomics and Biofuels - Biology


Learning Objectives

  • Explain the process of creating new biofuels by using microbial genomics

Knowledge of the genomics of microorganisms is being used to find better ways to harness biofuels from algae and cyanobacteria. The primary sources of fuel today are coal, oil, wood, and other plant products, such as ethanol. Although plants are renewable resources, there is still a need to find more alternative renewable sources of energy to meet our population ‘s energy demands. The microbial world is one of the largest resources for genes that encode new enzymes and produce new organic compounds, and it remains largely untapped.

For microbial biomass breakdown, many candidates have already been identified. These include Clostridia species for their ability to degrade cellulose, and fungi that express genes associated with the decomposition of the most recalcitrant features of the plant cell wall, lignin, the phenolic “glue” that imbues the plant with structural integrity and pest resistance. The white rot fungus Phanerochaete chrysosporium produces unique extracellular oxidative enzymes that effectively degrade lignin by gaining access through the protective matrix surrounding the cellulose microfibrils of plant cell walls.

Another fungus, the yeast Pichia stipitis, ferments the five-carbon “wood sugar” xylose abundant in hardwoods and agricultural harvest residue. Pichia‘s recently-sequenced genome has revealed insights into the metabolic pathways responsible for this process, guiding efforts to optimize this capability in commercial production strains. Pathway engineering promises to produce a wider variety of organisms able to ferment the full repertoire of sugars derived from cellulose and hemicellulose and tolerate higher ethanol concentrations to optimize fuel yields. For instance, the hindgut contents of nature’s own bioreactor, the termite, has yielded more than 500 genes related to the enzymatic deconstruction of cellulose and hemicellulose.

Key Points

  • Microorganisms can encode new enzymes and produce new organic compounds that can be used as biofuels.
  • Genomic analysis of the fungus Pichia will allow optimization of its use in fermenting ethanol fuels.
  • Analysis of the microbes in the hindgut of termites have found 500 genes that may be useful in enzymatic destruction of cellulose.
  • Genetic markers have been used in forensic analysis, like in 2001 when the FBI used microbial genomics to determine a specific strain of anthrax that was found in several pieces of mail.
  • Genomics is used in agriculture to develop plants with more desirable traits, such as drought and disease resistance.

Key Terms

  • renewable resource: a natural resource such that it is replenished by natural processes at a rate comparable to its rate of consumption by humans or other users
  • biofuel: any fuel that is obtained from a renewable biological resource

17.4 Applying Genomics

By the end of this section, you will be able to do the following:

Introducing DNA sequencing and whole genome sequencing projects, particularly the Human Genome project, has expanded the applicability of DNA sequence information. Many fields, such as metagenomics, pharmacogenomics, and mitochondrial genomics are using genomics. Understanding and finding cures for diseases is the most common application of genomics.

Predicting Disease Risk at the Individual Level

Predicting disease risk involves screening currently healthy individuals by genome analysis at the individual level. Health care professionals can recommend intervention with lifestyle changes and drugs before disease onset. However, this approach is most applicable when the problem resides within a single gene defect. Such defects only account for approximately 5 percent of diseases in developed countries. Most of the common diseases, such as heart disease, are multi-factored or polygenic , which is a phenotypic characteristic that involves two or more genes, and also involve environmental factors such as diet. In April 2010, scientists at Stanford University published the genome analysis of a healthy individual (Stephen Quake, a scientist at Stanford University, who had his genome sequenced. The analysis predicted his propensity to acquire various diseases. The medical team performed a risk assessment to analyze Quake’s percentage of risk for 55 different medical conditions. The team found a rare genetic mutation, which showed him to be at risk for sudden heart attack. The results also predicted that Quake had a 23 percent risk of developing prostate cancer and a 1.4 percent risk of developing Alzheimer’s. The scientists used databases and several publications to analyze the genomic data. Even though genomic sequencing is becoming more affordable and analytical tools are becoming more reliable, researchers still must address ethical issues surrounding genomic analysis at a population level.

Visual Connection

In 2011, the United States Preventative Services Task Force recommended against using the PSA test to screen healthy men for prostate cancer. Their recommendation is based on evidence that screening does not reduce the risk of death from prostate cancer. Prostate cancer often develops very slowly and does not cause problems, while the cancer treatment can have severe side effects. The PCA3 test is more accurate, but screening may still result in men who would not have been harmed by the cancer itself suffering side effects from treatment. What do you think? Should all healthy men receive prostate cancer screenings using the PCA3 or PSA test? Should people in general receive screenings to find out if they have a genetic risk for cancer or other diseases?

Pharmacogenomics and Toxicogenomics

Pharmacogenomics , or toxicogenomics, involves evaluating drug effectiveness and safety on the basis of information from an individual's genomic sequence. We can study genomic responses to drugs using experimental animals (such as laboratory rats or mice) or live cells in the laboratory before embarking on studies with humans. Studying changes in gene expression could provide information about the transcription profile in the drug's presence, which we can use as an early indicator of the potential for toxic effects. For example, genes involved in cellular growth and controlled cell death, when disturbed, could lead to cancerous cell growth. Genome-wide studies can also help to find new genes involved in drug toxicity. Medical professionals can use personal genome sequence information to prescribe medications that will be most effective and least toxic on the basis of the individual patient’s genotype. The gene signatures may not be completely accurate, but medical professionals can test them further before pathologic symptoms arise.

Microbial Genomics: Metagenomics

Traditionally, scholars have taught microbiology with the view that it is best to study microorganisms under pure culture conditions. This involves isolating a single cell type and culturing it in the laboratory. Because microorganisms can go through several generations in a matter of hours, their gene expression profiles adapt to the new laboratory environment very quickly. In addition, the vast majority of bacterial species resist culturing in isolation. Most microorganisms do not live as isolated entities, but in microbial communities or biofilms. For all of these reasons, pure culture is not always the best way to study microorganisms. Metagenomics is the study of the collective genomes of multiple species that grow and interact in an environmental niche. Metagenomics can be used to identify new species more rapidly and to analyze the effect of pollutants on the environment (Figure 17.16).

Microbial Genomics: Creation of New Biofuels

Knowledge of the genomics of microorganisms is being used to find better ways to harness biofuels from algae and cyanobacteria. The primary sources of fuel today are coal, oil, wood, and other plant products, such as ethanol. Although plants are renewable resources, there is still a need to find more alternative renewable sources of energy to meet our population’s energy demands. The microbial world is one of the largest resources for genes that encode new enzymes and produce new organic compounds, and it remains largely untapped. Microorganisms are used to create products, such as enzymes that are used in research, antibiotics, and other antimicrobial mechanisms. Microbial genomics is helping to develop diagnostic tools, improved vaccines, new disease treatments, and advanced environmental cleanup techniques.

Mitochondrial Genomics

Mitochondria are intracellular organelles that contain their own DNA. Mitochondrial DNA mutates at a rapid rate and scientists often use it to study evolutionary relationships. Another feature that makes studying the mitochondrial genome interesting is that the mitochondrial DNA in most multicellular organisms passes from the mother during the fertilization process. For this reason, scientists often use mitochondrial genomics to trace genealogy.

Experts have used information and clues from DNA samples at crime scenes as evidence in court cases, and they have used genetic markers in forensic analysis. Genomic analysis has also become useful in this field. The first publication showcasing the first use of genomics in forensics came out in 2001. It was a collaborative attempt between academic research institutions and the FBI to solve the mysterious cases of anthrax communicated via the US Postal Service. Using microbial genomics, researchers determined that the culprit used a specific anthrax strain in all the mailings.

Genomics in Agriculture

Genomics can reduce the trials and failures involved in scientific research to a certain extent, which could improve agricultural crop yield quality and quantity. Linking traits to genes or gene signatures helps improve crop breeding to generate hybrids with the most desirable qualities. Scientists use genomic data to identify desirable traits, and then transfer those traits to a different organism. Researchers are discovering how genomics can improve agricultural production's quality and quantity. For example, scientists could use desirable traits to create a useful product or enhance an existing product, such as making a drought-sensitive crop more tolerant of the dry season.


Synthetic genomics and synthetic biology applications between hopes and concerns

New organisms and biological systems designed to satisfy human needs are among the aims of synthetic genomics and synthetic biology. Synthetic biology seeks to model and construct biological components, functions and organisms that do not exist in nature or to redesign existing biological systems to perform new functions. Synthetic genomics, on the other hand, encompasses technologies for the generation of chemically-synthesized whole genomes or larger parts of genomes, allowing to simultaneously engineer a myriad of changes to the genetic material of organisms. Engineering complex functions or new organisms in synthetic biology are thus progressively becoming dependent on and converging with synthetic genomics. While applications from both areas have been predicted to offer great benefits by making possible new drugs, renewable chemicals or clean energy, they have also given rise to concerns about new safety, environmental and socio-economic risks - stirring an increasingly polarizing debate. Here we intend to provide an overview on recent progress in biomedical and biotechnological applications of synthetic genomics and synthetic biology as well as on arguments and evidence related to their possible benefits, risks and governance implications.

Keywords: Applications Benefits Biofuels Biomedicine Environment Risks Synthetic biology. Synthetic genomics.

Figures

Synthetic genomics and synthetic biology…

Synthetic genomics and synthetic biology applications in biomedicine and health. A. “Prosthetic” cell…

Synthetic biology approaches to environmental…

Synthetic biology approaches to environmental applications. A. Whole-cell biosensor array that is frequency-modulated…

Different generations of biofuels and…

Different generations of biofuels and their carbon cycles.

Approaches involving synthetic biology concepts…

Approaches involving synthetic biology concepts to generate biofuels. A . A synthetic pathway…


Biotechnological tools for genetic improvement

Tissue culture

Efficient switchgrass cell and tissue culture is required for the production of transgenic plants as well as vegetative propagation. Prior to 1991, little switchgrass tissue culture research had been conducted. The initiation of US Bioenergy Feedstock Development Program enhanced opportunities for the long-term improvement of switchgrass [11]. Thus, in the 1990s, this program spurred research exploring explant types, tissue culture and regeneration of switchgrass with the ultimate goal of increasing the resource-base for developing transgenic lines. Switchgrass is amenable to regeneration after somatic embryogenesis and organogenesis.

Embryogenic callus

Somatic embryogenesis was used by Denchev and Conger [24] who reported high frequency plantlet regeneration. They used mature caryopses (seeds) and young leaf segments of the lowland cultivar ‘Alamo’ as explants to produce embryogenic callus on solidified Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The ease of handling and callus induction from mature caryopses made these valuable explants. When leaves were used as explants, there was a response gradient with regards to tissue age for callus initiation young tissue is better than old tissue. Although somatic embryogenesis could be induced from embryogenic calli, regeneration of somatic embryos directly from the cells of the explants was not observed [24]. Somatic embryogenesis has also been reported from young infloresences of ‘Alamo’ [25, 26]. The cyclic production of plants from embryogenic callus renders this technique a viable option for rapid clonal propagation of switchgrass. However, compared with seed production, clonal propagation would be quite expensive and probably only used for the most valuable lines.

One disadvantage to the use of embryogenic callus- and seed-derived callus systems is that they generally have limited lifespans (months) of usefulness before they cease to be regenerable. Whereas the longevity of embryo viability can be only two months, the recently described switchgrass medium, LP9, increased the viability of callus and the ability to maintain it for a duration of over six months, making it more efficient for use in a transformation pipeline [27]. LP9 combined N6 macroelements and B5 microelements for the production and maintenance of switchgrass callus and its regeneration [27]. Also, the callus obtained was categorized as type II callus, which is more effective in grass transformation and regeneration [27] than type I callus obtained from previously described tissue culture systems [25, 26].

Cell suspension cultures

Cells divide faster in liquid suspension cultures compared with callus cells grown on solidified medium [28]. For large scale propagation, mutant selection, gene transfer and protoplast isolation, development of embryogenic cell suspension cultures would be advantageous. Cell suspension cultures were first obtained by Bob Conger’s group that used young inflorescences of ‘Alamo’ as explants, which could directly yield embryogenic callus, which could be regenerated into plants [25]. This same group [26] showed that the utilization of osmotic pretreatment had a positive effect on the initiation and induction of somatic embryogenesis from suspension cultures derived from in vitro-cultured inflorescences of ‘Alamo.’ It was also observed that younger cultures gave a higher embryogenic response as compared with older cultures [26]. The HR8 line that was developed from a recurrent tissue culture selection of ‘Alamo’ had a higher seed germination capacity, and germinating seeds gave rise to higher percentage of somatic embryogenic callus [29]. Although this HR8 line, and indeed all improved Conger materials other than ‘Alamo2’ have been lost, the improved germplasm demonstrated very rapid propagation. These sorts of materials would have great use in breeding programs [11].

Cell suspensions are also excellent starting materials for the isolation of protoplasts. Protoplasts are useful in a wide range of applications including cell fusion and genetic manipulation [30]. Recently, Mazarei et al. reported protoplast isolation from switchgrass cell suspension cultures established from embryogenic callus [31]. They demonstrated that protoplast isolation efficiency was highly dependent on the type of cell suspension. Currently, our and other research groups are using cell suspension cultures for a variety of biotechnology-to-synthetic biology applications including deciphering the cell wall biology for improvments and high throughput multi-target genetic engineering and screening.

Organogenesis

Organogenesis illustrates a significant capability of plants to adapt to their altering environment this process allows organ genesis from undifferentiated cells [32–34]. Switchgrass regeneration from organogenesis has been accomplished [24, 35]. Explants include mature caryopses, young leaf segments and young seedling explants and MS medium supplemented with auxins (2,4-D or picloram) and BAP is effective [24, 35]. The combination of 2,4-D and BAP induced a high regeneration frequency in both nonembryogenic and embryogenic calli derived from mature caryopses, while induction of shoots from young seedling explants was more effective when picloram was used in combination with BAP [35]. Protocols for high-throughput callus induction by plating whole dehusked caryopses and plant regeneration from new, higher yielding switchgrass cvs. ‘NSL’ and ‘SL93’ have been optimized [36]. Seed pretreatments, such as dehusking with sulfuric acid, chilling for two weeks at 4°C prior to plating, and sterilizing with sodium hypochlorite and ethanol, were found to have significant effect on callus induction and subsequent plant regeneration.

Micropropagation

As mentioned earlier, vegetative/micropropagation using tissue culture might be useful for valuable germplasm and also for research. Advanced regeneration techniques have been developed for switchgrass. For the efficient multiplication of switchgrass genotypes, micropropagation has been established using nodal explants especially the nodes below the top node [37]. Regardless of their position on the culm, all nodes exhibited shoot induction at a similar rate. It was also reported that 500 plantlets could be regenerated from a single parent plant in 12 weeks [37]. Clonal propagation can be used for scaling up the number of plants obtained from selected cultivars, for controlled pollination studies for use in breeding programs, in genetic transformation experiments, and also as an important explant source for additional in vitro culture initiation.

In switchgrass, the regeneration capacity is highly genotype-dependent [38, 39]. The recalcitrance of upland cultivars warranted the development of new efficient regeneration systems. Intact seedlings of both lowland (‘Alamo’) and upland (‘Trailblazer’ and ‘Blackwell’) cultivars exhibited multiple shoot regeneration on MS medium supplemented with various combinations of 2,4-D and thidiazuron (TDZ) [38]. This technique of inducing multiple shoots from intact seedlings was less labor intensive and more rapid, efficient and consistent across genotypes, and the shoots appeared to originate from enlarged shoot apice [38]. Since each caryopsis vary for genotype, owing to self-incompatibility and natural outcrossing that is inherent to switchgrass, this system did not have utility for clonal propagation.

Immature inflorescences are a significant resource for in vitro culture establishment. Young inflorescences of switchgrass have been utilized for callus induction and plant regeneration [40]. To reduce the damage caused by harvesting, endogenous or exogenous fungal and bacterial contamination, and toxicity of sterilization solutions on inflorescences, growth establishment in axenic cultures might be beneficial. A protocol for in vitro production of inflorescences from node cultures derived from greenhouse grown tillers of ‘Alamo’ has been reported [41]. These inflorescences, with completely developed spikelets and terminal florets, were used as axenic explants for callus induction and plant regeneration. This highly efficient procedure for the development of organ-specific differentiating tissues provides a vehicle for genetic transformation using microprojectile bombardment in switchgrass. In vitro- grown mature florets also provide an aseptic source of anthers for the production of haploids, and open up the possibilities for in vitro fertilization techniques to enhance breeding experiments between ecotypes that are naturally difficult to cross.

Genetic engineering

Genetic transformation is useful for gene discovery and characterization in plant biology. The commercial use of transformation is to introduce traits into plants that would not be possible by conventional breeding alone and also to increase trait development rate [42]. The main trait targets to address using genetic engineering in switchgrass include domestication, plant architecture, and especially reduced recalcitrance for cell wall conversion into biofuel and valuable bioproducts [6, 43]. The recent focus on the use of switchgrass as a biofuel crop has led to its large-scale production and genetic engineering (Table 1 Figure 3) for incorporating traits by overexpressing exotic genes and knocking down the expression of endogenous genes [44]. These genes may be for increasing the saccharification efficiency, modifying the cell wall structure and/or composition, enhancing biomass yields or affecting the growth and development of switchgrass plants [6, 9, 44, 45].

Flow chart of transgenic production in switchgrass [Photo credits: Wegi A. Wuddineh and M Nageswara-Rao].

The first transgenic switchgrass was obtained through bombardment of immature inflorescence-derived embryogenic calluses of ‘Alamo’ using a dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein GFP) driven by the rice actin (Act1) promoter and the selectable bar gene (conferring tolerance to the herbicide Basta) driven by the maize ubiquitin (Ubi1) promoter [46]. The leaf tissues and pollen of transgenic plants exhibited GFP and were also tolerant to Basta. T1 seedlings from crosses between transgenic and non-transgenic control plants that inherited the bar transgene were also tolerant to Basta [46]. Agrobacterium tumefaciens-mediated transformation has been accomplished in switchgrass, and appears to be the most common method for switchgrass transformation. The hypervirulent A. tumefaciens strain AGL1 carrying the binary vector pDM805 containing the bar gene under the control of the Ubi1 promoter and the uidA gene driven by Act1 promoter was used for transforming four different explant types of which somatic embryos gave the highest transformation frequency [47]. This opened up new opportunities for genetic manipulation of switchgrass as Agrobacterium-mediated transformation is often the preferred method since it favors the integration of a low copy number of transgenes. Somleva et al. [48] was able to influence the transformation efficiency of switchgrass by manipulating explant type and genotype, pre-culture treatment of the explant, wounding of explants preceding infection, addition of acetosyringone during inoculation and cocultivation, and selection. These experiments have been valuable in making switchgrass transformation more routine.

Embryogenic calli derived from caryopses or inflorescences of ‘Alamo’ were transformed using A. tumefaciens strain EHA105 in combination with the binary vectors pCAMBIA 1301 (carrying a gusA from E. coli) and pCAMBIA 1305.2 (carrying a GUSPlus from Staphylococcus spp.) [49]. Since both binary vectors carried the hygromycin phosphotransferase gene (hpt) as a selectable marker, the transgenic plants were selected on medium supplemented with hygromycin. T1 plants from crosses between transgenic and non-transgenic control plants that had multiple copies exhibited transgene silencing, whereas lines harboring only one insert expressed the transgene [49]. One of the largest sources, if not the largest source of efficiency improvement, has come from genotype. Highly regenerable and transformation-competent embryogenic calli developed from seeds of ‘Alamo’, ‘Performer’ and ‘Colony’ were used for genetic transformation using A. tumefaciens strain EHA105 containing the binary vectors pTOK47 (carrying a 20 kb KpnI fragment of Ti plasmid from pTiBo542, which contains virB, virC and virG virulence genes) and pJLU13 (a derivative of pCAMBIA 1301 containing hpt and sgfp genes) [50]. It appears that lines of ‘Performer’ are probably the best switchgrass for tissue culture and transformation. Application of vacuum during infection and dehydration at co-cultivation also enhanced the transformation efficiency, as did resting after infection and before culturing onto the selection medium [50]. Transformation efficiency can be improved by the optimization of the gene delivery system, and the appropriate selection and regeneration of transformed cells. Transformation efficiency was enhanced by utilizing the basal parts of ‘Alamo’ seedlings that had higher regeneration potential [51]. Genetic transformation of the type II callus derived from the inflorescences of switchgrass on LP9 medium [27] exhibited transformation efficiency of as high as 34% and also decreased the time taken for transgenic production by one month [52].

Though a number of procedures are well established for switchgrass plant transformation, evaluation of the transgene expression may take several weeks. To reduce this time required for testing gene constructs, transient transgene expression could be a rapid screen [53]. Inoculation of germinating ‘Alamo’ seedlings using an Agrobacterium-mediated transient gene expression system (agroinfiltration) was optimized using AGL1, C58, EHA105, and GV3101 strains, of which AGL1 showed the highest efficiency in gene delivery [54]. In another study, it was reported that EHA105 was more effective in gene delivery than LBA4404 or GV3101 [51]. To study the effects of agroinfiltration conditions such as mechanical wounding (bead beating, sonication or vortexing), concentration of the surfactant (Break-Thru S 240, Silwet L77 or Li700), and application of vacuum on transient β-glucuronidase expression, experiments were performed using harvested switchgrass leaves or seedlings [53, 54]. Though bead beating wounded the leaf surface, it did not have any effect on the transient β-glucuronidase expression [53]. On the other hand, utilization of sonication and vortexing with carborundum had a positive effect on the transient expression [54]. Use of ‘Break-Thru S 240’ under low vacuum application improved the transient expression [53] while Silwet L77 or Li700 had a negative effect [54]. Transient expression was also enhanced by increasing the vacuum application when surfactant concentration was low [53]. Incorporation of chemicals (L-cysteine and dithiothreitol), heat stress and separation by centrifugation also influenced transient transgene expression [54]. Agroinfiltration might provide a quick assay for overexpression studies in switchgrass.

Mazarei et al. [55] developed a protoplast system using leaves and roots of ‘Alamo’ and the ‘Alamo2’ clone followed by transient expression of polyethylene glycol (PEG) mediated DNA uptake in protoplasts [55]. GUS driven by either the CaMV 35S promoter or the maize ubi1 promoter was utilized as the reporter gene. To develop a transformation system for upland cultivars, calli were induced from seedling segments of the upland octoploid cultivar ‘Cave-in-Rock.’ However, the callus was not amenable for regeneration and produced only roots [51]. Since the tissue culture and transformation systems have been developed for ‘Alamo’ or its derivatives, for a wide applicability across the species, there is a need to create more genotype-independent methodologies for switchgrass. It is also highly crucial to select the right candidate gene(s) for genetic transformation, and develop appropriate protocols for evaluation of transgenics with the non-transgenics [56]. Given the strong germplasm effects observed, this might be a difficult task. In addition, ‘Alamo’ and ‘Performer’ are both agronomically viable lowland cultivars.

A wide variety of promoters have been used for monocot transformation [57–59], but only a few of these have been utilized in switchgrass [46, 47, 50]. Thus, attention has been given toward promoter testing and discovery for switchgrass genetic engineering [60, 61]. Two novel switchgrass ubiquitin gene (PvUbi1 and PvUbi2) promoters have been tested [60]. Particle bombardment of callus using these two promoters exhibited expression patterns comparable to the maize Ubi1 promoter and much higher than that using the 35S promoter [60].

To rapidly screen transgenes in switchgrass, monocot-effective plant expression vectors are required. One such new vector set is pANIC, which uses a Gateway-compatible cassette for over-expression or RNAi of the target gene [62]. The set contains selectable marker and visible marker cassettes for Agrobacterium-mediated transformation as well as biolistic bombardment [62]. These vectors were designed especially for switchgrass and are being routinely used in several switchgrass transformation labs.

Production of bioproducts in transgenic switchgrass

Somleva et al. [63] demonstrated the amenability of transgenic switchgrass to synthesize polyhydroxybutyrate (PHB), a biodegradable polyhydroxyalkanoate biobased plastic, in which the pathway was engineered into switchgrass. PHB was accumulated to 3.72% and 1.23% (dry weight) in the leaves and whole tillers respectively. PHB production was stable in the next plant generation too. This study has shown the incorporation of a complex trait in switchgrass is possible for biomanufacturing.

Cell wall modification

Genetically modified feedstocks play an important role in scenarios for next-generation biofuel production [64]. Reducing lignin biosynthesis can lead to lower recalcitrance and higher saccharification efficiency, making lignin composition and amount an obvious target to change in lignocellulosic feedstocks [6]. Recalcitrance of cell walls conversion to biofuels is perhaps the greatest hurdle in realizing the economic potential of switchgrass and other lignocellulosic biofuel feedstocks [64, 65]. Currently, to enable efficient enzymatic degradation of cellulose, harsh physical or chemical pretreatment is required for the modification of the cell wall structures, removal of lignin and degradation of the hemicelluloses [66]. For augmenting the biofuel production from lignocellulosic feedstocks, changing lignin composition and amount are being performed [67, 68].

Fu et al. reported a reduction in lignin content, and increase (38%) in ethanol yield from transgenic switchgrass in which the endogenous caffeic acid O-methyltransferase (COMT) gene was down-regulated [69]. The syringyl:guaiacyl monolignol ratio was decreased and the transgenic plants required less pretreatment and enzymes to yield the same levels of ethanol using simultaneous saccharification and fermentation. As a result, there was also enhanced forage quality in the COMT down-regulated lines.

The last step in the biosynthesis of lignins is catalyzed by cinnamyl alcohol dehydrogenase (CAD) [70]. CAD deficiency modifies the lignin structure, reduces the lignin content, and augments the saccharification efficiency in grasses [71, 72]. Agrobacterium-mediated transformation was utilized for RNAi of CAD in switchgrass [73, 74]. These two studies reported a reduction in lignin content and increased saccharification efficiency in the transgenic lines. Another important enzyme involved in the biosynthesis of lignin is 4-coumarate:coenzyme A ligase (4CL). Xu et al. carried out phylogenetic analysis and gene expression studies, and suggested the involvement of Pv4CL1 in the biosynthesis of lignins [68]. Pv4CL1 down-regulated transgenic switchgrass plants, obtained by Agrobacterium-mediated transformation, had normal biomass yields with reduced lignin content and increased saccharification efficiency [68].

In contrast to the above-mentioned approach in which endogenous lignin biosynthesis genes were down-regulated, Hui Shen and colleagues targeted the overexpression of a key transcription factor affecting the expression of many lignin biosynthesis genes [75]. A decrease in recalcitrance in transgenic switchgrass was observed when the repressor, PvMYB4 was overexpressed [75]. The transgenic lines exhibited a drastic reduction in lignin, but no change in the S:G ratio. The plants were also morphologically affected, having more tillers and reduced height. The transgenics had increased cellulose and pectin contents, significantly reduced wall recalcitrance and phenolic fermentation inhibitors, and produced approximately 1.8-fold more ethanol using yeast based simultaneous saccharification and fermentation without pretreatment (Shen et al., in review).

These efforts have highlighted the usefulness of lignin biosynthesis or lignin repressor gene targets for down-regulation, and these genetically engineered plants for reduced lignin may contain higher levels of free monolignols and other phenylpropanoids. The accessibility of cell wall carbohydrates for the production of biofuels is negatively correlated with the amount of lignin present [76, 77]. Decrease in lignin content or alteration in its composition alleviated the digestibility of the cellulose and hemicelluloses. This led to enhanced saccharification efficiency, reduction in the severity of the pretreatment, decrease in enzyme requirements and increase in the energy available to microorganisms for breaking down the carbohydrates [69, 76, 78]. To change the lignin content of the biomass, dwarfing might also be of use as it shifts the biomass allocation from the stem to the leaves [44]. Reduced lignin content during the vegetative phase in switchgrass might also delay flowering, which could also increase vegetative biomass [44, 79].

In is unclear whether the lignin biosynthetic pathway is perfectly conserved between widely-studied model species and switchgrass. There might be many more genes and transcription factors that have not been discovered in switchgrass and be manipulated for improved biofuel production. Other cell wall targets include cellulose, reducing the crystallinity of cellulose, hemicellulose, pectin, and their interactions with lignin. Research on the expression of cellulases, in planta, under extreme conditions and its thermal stability also needs to be carried out. The cost of lignocellulosic ethanol production may also be reduced by genetically modifying switchgrass to produce the enzymes that are required during fermentation. Devising strategies for recycling these enzymes will also lead to reduction in biofuel production cost.

Altering switchgrass development: microRNAs and other targets

Improvement in the rate of saccharification efficiency, which is inhibited by the complex structure of the plant cell wall, is an important objective in developing a competent and lucrative biofuel industry [80, 81]. Biomass yield could be enhanced by manipulating microRNAs (miRNAs) that regulate transcription factors controlling growth and development in plants [69, 81–84]. The maize Corngrass1 (Cg1) gene, which produces a miR156, targets the SQUAMOSA PROMOTER BINDING LIKE (SPL) family and reduces lignification while promoting juvenile characteristics in plants [85, 86]. To study how juvenile characters improve the biofuel potential of switchgrass, the Cg1 gene was constitutively overexpressed in ‘Alamo’ [81]. A second miR156 study overexpressed the switchgrass PvmiR156 in switchgrass, [82]. In both studies, the transgenic plants had delayed flowering, variant morphology, and improved sugar release. Transgene expression levels were sufficient to allow three morphology categories to be observed. Low expressers resembled non-transgenic switchgrass. Moderate expression levels rendered plants that were shorter and with more tillers. The plants had delayed flowering, which could be useful in bioconfinement of transgenes. High levels of miR156 accumulation induced severe dwarfism and reduced biomass accumulation [81, 82]. Thus, targeted overexpression of miR156 could not only make biofuel production more efficient but allow the production of switchgrass that is more suitable for production. These studies highlight the potential utility of this approach for the domestication of new switchgrass cultivars, and the lack or delay in flowering will have important implications for the limitation or prevention of transgene flow into native/wild relatives or non-transgenic agronomic plantings of switchgrass. Recently, it was demonstrated that the expression levels of miR156 and miR162 could be changed under drought conditions in switchgrass [87].

Genetic engineering can also be used to increase the biomass by modifying the plant growth regulators such as increasing the biosynthesis of gibberellins [88] to improve the growth and increase the biomass in switchgrass. Thus, early transgenic research in switchgrass has revealed that multiple targets for improvement have been reached. It appears that there could be a tradeoff between sugar release and plant growth, but results are promising with regards to increasing liters per hectare. To date, there has been no transgene stacking in switchgrass, which should be pursued. For example, it makes sense to hybridize miRI56 plants with those with greatly reduced lignin, such as MYB4 overexpressers. In addition, tissue-specific and inducible expression of transgenes will also be valuable in decreasing off-target effects. Targeted expression is particularly needed for genes, such as those that are master regulators, to diminish or better control pleiotropic effects. The transgenic studies to date with switchgrass show the power of the technology, which is becoming increasingly routine.


Advanced Biology

Advanced Biology is a peer-reviewed, interdisciplinary and international journal that published its first issue in 2017 (under the former name of the journal, Advanced Biosystems). The journal has been mainly focusing on applied research and technologies that enhance and harness biological systems. To further develop the journal we have decided to expand the scope to cover all aspects of biology. Biology is understood in its broadest sense as life sciences research from multiple disciplines including but not limited to biology, chemistry, physics, medical science and computer science applied to biologically relevant systems at all scales ranging from molecular to whole organism level and beyond. The journal provides a forum for significant novel findings of wide biological relevance from both basic as well as applied research with a particular focus on – but not limited to – the following areas:

  • Cell and molecular biology, including aging, transcription, translation, DNA replication, biochemistry, circadian biology, metabolic pathways, host-pathogen interactions
  • Systems biology, including “omics” studies (genomics, proteomics, metabolomics) and bioinformatics
  • Synthetic biology, including artificial organisms, molecular machines, genetic engineering
  • Tissue engineering and regenerative medicine, including stem cells, artificial organs, organoids, immunoengineering
  • Biotechnology and bioengineering, including DNA and protein engineering, metabolic engineering, industrial microorganisms, biocatalysis and biofuels
  • Immunology, including adaptive and innate immunity, molecular immunology and signaling, allergy and inflammation
  • Microbiology, covering both prokaryotic (bacteria, archaea) and eukaryotic (fungi, protists, microalgae) microorganisms as well as viruses and prions
  • Structural biology, covering the molecular structure of biological macromolecules (proteins, DNA, RNA, lipids)
  • Neuroscience, including cell and molecular neuroscience, neuroengineering, developmental neuroscience, neurophysiology
  • Cancer, including metastasis, molecular aspects of cancer biology, biomarkers
  • Developmental biology, including regeneration and asexual reproduction
  • Genetics and epigenetics
  • Ecosystems and evolutionary biology

Currently 12 issues per year.

Readership

Life scientists working in all areas of biology from both academia and industry.

Keywords

artificial organisms, molecular machines, viral engineering, DNA synthesis, bioinspired/biomimetic systems, biophysics, bioelectronics, nano and microfluidics, DNA and protein engineering, metabolic engineering, industrial microorganisms, biocatalysis, biofuels, genetically modified organisms, genetics, biochemistry, cellular engineering, cellular programming, stem cells, artificial organs, neuroengineering, pharmaceuticals, vaccines, gene therapy, biomarkers, personalized medicine, drug delivery, immunology, diagnostics, microbiomics, genomics, proteomics, metabolomics, bioinformatics


Just how ‘green’ are biofuels? Why turning crops into energy might not be a worthwhile climate change solution

Credit: M Photography/Daryl Marshke

First-generation biofuels have been around for a long time. In fact, in the US, ethanol has been used as a fuel without interruption since 1933. In 1900, Rudolf Diesel (inventor of the Diesel Engine) demonstrated a model engine that ran entirely on peanut oil. Biofuels have received increased attention over the last two decades, as societies around the world strive to diversify their energy portfolios and address environmental degradation concerns.

There is a clear interest in the production of biofuels in many countries around the world. But what exactly are ethanol and biodiesel? What are the implications of their production and consumption? And where does Canada stand with its biofuel policies?

Ethanol

Ethanol is an alcohol that can be produced from feedstock in several ways, the most common being fermentation. Typically, ethanol is blended with gasoline to produce a fuel that has environmental advantages (burns cleaner) compared to gasoline alone. However, ethanol does have a lower energy density than gasoline. Specifically, the energy per unit volume of ethanol is 34% lower than that of gasoline. To get a better idea of this, you would need 1.5 gallons of ethanol to produce the same amount of energy from 1 gallon of gasoline. Despite this, ethanol does have a higher octane level than gasoline. This means that as the energy contained in ethanol is converted into mechanical energy (the energy used to move a vehicle by combustion), less energy from ethanol is wasted compared to gasoline.

In Canada, most gasoline-powered vehicles can run on a blend consisting of gasoline and up to 10% ethanol. According to the Renewable Fuels Regulations, fuel imported or produced in Canada must have an average renewable fuel content of at least 5% based on the volume of gasoline produced or imported, and at least 2% based on the volume of diesel fuel and heating distillate oil produced or imported.

According to the Renewable Fuels Association, the US is the world leader in the production, consumption, and export of ethanol (54% of total global production), and Brazil and Canada are the top two destinations of US ethanol exports. The top 10 world ethanol producers are presented in Figure 1.

Figure 1. Global Fuel Ethanol Production by Country in 2019
Source: 2020 Ethanol Industry Outlook p. 7

Biodiesel

Biodiesel is a diesel fuel substitute that can be used in diesel engines. It is typically made from renewable materials that are transformed into biodiesel through a process called transesterification, a chemical process that converts fats and oils into fatty acid methyl esters. The energy density of biodiesel is comparable to that of gasoline and petrodiesel. The most common biodiesel-petrodiesel blends are:

  • B2: 2% biodiesel mixed with 98% diesel
  • B5: 5% biodiesel mixed with 95% diesel
  • B10: 10% biodiesel mixed with 90% diesel
  • B20: 20% biodiesel mixed with 80% diesel
  • B100: 100% biodiesel with no diesel content

B20 is the best blend rate because it burns cleaner than petrodiesel and has better flow properties at low temperatures than pure biodiesel, which would clump up in the cold. While B100 is not a commonly used fuel, its energy output is 8% less than pure diesel, whereas the energy difference is virtually unnoticeable with B5 or lower.

Implications of biofuel production

Biofuel production and consumption are not without their critics, practically all of its supposed benefits have been called into question. To understand why, it’s necessary to look at how biofuels are produced. The logical starting point is with the world’s largest producer.

In the US, an average of 38 million acres of land a year (15.4 million ha), an area larger than the state of Illinois (¼ the size of Saskatchewan), are cultivated with corn to produce ethanol. The Renewable Fuel Standard (RFS), an energy policy intended to diversify energy sources and reduce carbon emissions, has the goal that by 2022, 35 billion gallons (160 billion liters) of ethanol-equivalent biofuels and 1 billion gallons of biomass-based diesel must be consumed in the US. This target is unlikely to be met. Even if that target was met, it is unlikely to reduce greenhouse gas emissions because reductions depend on how biofuels are produced.

A large amount of fossil fuel is required to produce, grow, harvest, transport, and process a gallon of ethanol, eating up much of the difference in carbon emissions between ethanol and gasoline alone. A 2009 study by the Congressional Budget Office found that “the demand for corn for ethanol production, along with other factors, exerted upward pressure on corn prices, which rose by more than 50 percent between April 2007 and April 2008. Rising demand for corn also increased the demand for cropland and the price of animal feed.” In the US, producing ethanol has had unintended effects on feed price ( and therefore also on meat price for consumers) and sends signals to farmers to produce corn (competing with the production of other crops). These issues persist today.

What is the biofuel picture in Canada?

The Canadian government has proposed rules for its Clean Fuel Standard (CFS), which are now in a 75-day comment period. If adopted they would come into force in 2022. The objective of the CFS “is to achieve 30 million tons of annual reductions in greenhouse gas emissions by 2030 … reducing national emissions by 30% below 2005 levels by 2030.” In 2005, Canada emitted 729 megatons of carbon dioxide equivalent (Mt CO2 eq).

The table below provides an overview of existing biofuel mandates in Canada.

Jurisdiction Ethanol Mandate (Year enacted) Biodiesel Mandate (Year enacted)
Canada 5.0% (2010) 2.0% (2011)
Ontario 5.0% (2007) 4.0% (2017)
Manitoba 8.5% (2008) 2.0% (2009)
Saskatchewan 7.5% (2007) 2.0% (2012)
Alberta 5.0% (2011) 2.0% (2011)
British Columbia 5.0% (2010) 2.0% (2010)
Source: CEC, 2016 p.10

With the pursuit of biofuel policies, it is thought they will provide a practical opportunity to achieve multiple objectives: reduce GHG emissions, increase economic opportunities for rural communities, improve air quality, and accelerate the development of next-generation biofuels.


Mechanisms of Salinity Tolerance

Rana Munns and Mark Tester
Vol. 59, 2008

Abstract

The physiological and molecular mechanisms of tolerance to osmotic and ionic components of salinity stress are reviewed at the cellular, organ, and whole-plant level. Plant growth responds to salinity in two phases: a rapid, osmotic phase that inhibits . Read More

Figure 1: Diversity in the salt tolerance of various species, shown as increases in shoot dry matter after growth in solution or sand culture containing NaCl for at least 3 weeks, relative to plant gr.

Figure 2: The growth response to salinity stress occurs in two phases: a rapid response to the increase in external osmotic pressure (the osmotic phase), and a slower response due to the accumulation .

Figure 3: The thermodynamics and mechanisms of Na+ and Cl− transport at the soil-root and stelar cell–xylem vessel interfaces in roots. Indicative cytosolic pH, ion concentrations, and voltages are de.

Figure 4: Differences in vacuolar concentrations of Na+ across roots of transpiring wheat plants growing in 150 mM NaCl. Concentrations were measured by quantitative and calibrated X-ray microanalysis.

Figure 5: Hypothetical relationships between salinity tolerance and leaf Na+ concentration for three different species, denoted by a, b, and c for rice, durum wheat, and barley. Within most species, t.

Figure 6: Relationships measured between salinity tolerance (biomass in salt as a % of biomass in control conditions) and leaf Na+ concentration in different wheat species. (a) Negative relationship f.


Plant and Microbial Biosciences Program

Faculty and students in the Plant and Microbial Biosciences (PMB) Program use a variety of experimental organisms to address fundamental and applied biological problems. Research on plant and microbial systems contributes to our understanding of the natural world and drives innovation in biomedicine, agriculture, and energy production.

The PMB Program trains world-class biologists who employ plants and microbes as their model systems and use state-of-the-art techniques and approaches across disciplines. The success of PMB graduates in obtaining postdoctoral fellowships, professorships at leading academic institutions, and leadership positions in the private sector attests to the inherent strengths of this philosophy.

PMB is fully integrated with other graduate programs in the Division of Biology and Biomedical Sciences (DBBS). PMB faculty maintain close ties with the Molecular Microbiology and Pathogenesis, Genetics and Genomics, Biochemistry, and Molecular Cell Biology programs. Students in PMB receive training not only in plant and microbial systems, but also in imaging, biochemistry, cell biology, genetics, systems biology, geochemistry, molecular evolution, and ecology, from leaders in the field.


Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production

Economic feasibility of biosynthetic fuel and chemical production hinges upon harnessing metabolism to achieve high titre and yield. Here we report a thorough genotypic and phenotypic optimization of an oleaginous organism to create a strain with significant lipogenesis capability. Specifically, we rewire Yarrowia lipolytica's native metabolism for superior de novo lipogenesis by coupling combinatorial multiplexing of lipogenesis targets with phenotypic induction. We further complete direct conversion of lipid content into biodiesel. Tri-level metabolic control results in saturated cells containing upwards of 90% lipid content and titres exceeding 25 g l(-1) lipids, which represents a 60-fold improvement over parental strain and conditions. Through this rewiring effort, we advance fundamental understanding of lipogenesis, demonstrate non-canonical environmental and intracellular stimuli and uncouple lipogenesis from nitrogen starvation. The high titres and carbon-source independent nature of this lipogenesis in Y. lipolytica highlight the potential of this organism as a platform for efficient oleochemical production.


Tracks in the Graduate Program

Students in the Plant Biology Program may choose from four research and curriculum tracks:

  1. Molecular and Cellular Biology/Genomics
  2. Plant Breeding and Genomics
  3. Horticulture and Plant Technology
  4. Plant Pathology
  5. Natural Products and Human Health

Specific curricular requirements for each student are developed within the general program requirements by his or her committee, with approval by the track coordinator and program director. The tracks are interwoven in that members of the graduate faculty may be members of more than one track and students are encouraged to take courses in more than one track area.

Students in the molecular and cellular biology/genomics track may specialize in biocontrol of pests and pathogens, biofuels and bioenergy, biotechnology and crop improvements, biotic and abiotic stress/interactions with the environment, circadian control, genetic control of plant development, metabolomics/primary and secondary metabolism, natural products and human health, programmed cell death/senescence and fruit ripening, plant diversity/anatomy/evolution and biogeography, interactiosn with pathogenic and symbiotic microbes, plastid molecular genetics, structural/functional and computational genomics, transcriptional and post-trnacsriptional gene regulation.

Students in the plant breeding and genomics track have the opportunity to study a broad range of topics related to plant breeding from cultivar development, tissue culture, gene mapping, biochemical mechanisms to the latest discoveries in plant genomics and bioinformatics. The plant breeding faculty conduct research on an array of differenct traits, including but not limited to, higher quality and greater yield of fruit, fiber and other plant consituents, resistance to biotic stresses caused by disease and insect pests, resistance to abiotic stresses such as heat and drought, and specialty products such as novel fatty acids, proteins and other plant metabolites.Students will gain experience on a diverse range of vegetable, horticultural and pomological crops including specialty crops like cranberry, blueberry, hazlenuts, dogwoods, hollies, turgrasses, peaches, apples, tomatoes and biofuels. Students will gain experience with both traditional and DNA-based marker-facilitated selection schemes. Studens will gain credentials that are desired to directo or lead plant breeding research at private companies or public institutions. Work experience on the various plant breeding projects often is available for students majoring in plant breeding.

The horticulture and plant technology track focuses on agricultural biotechnology, genetics, plant physiology, weed science, plant systematic, plant-microbe interactions, turfgrass science, crop science in agronomic crops and biofuels, horticultural science in fruits, vegetables, flowers and tree crops.

Among the issues that students in the plant pathology track may address are host/pathogen interactions, epidemiology and control of plant disease, plant virology, bacteriology, mycoplasmology, mycology, molecular biology of plant pathogenic or endophytic microorganisms, and biotechnology.

In the Natural Products and Human Health track, students are to study basics mechanisms of Natural products, mode of action and toxicity. Discover, identify, and characterize bioactive from plant and fungi through multidisciplinary approaches. Develop novel uses for natural products as medicines, foods, cosmetics, dietary supplements and crop protection agent.

The list below categorizes each faculty according to their respective track(s). Names in bold indicate track coordinators.