Kristi D. Snell

Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop.

SUMMARY Polyhydroxyalkanoate bio-based plastics made from renewable resources can reduce petroleum consumption and decrease plastic waste disposal issues as they are inherently biodegradable in soil, compost and marine environments. In this paper, the successful engineering of the biomass crop switchgrass (Panicum virgatum L.) for the synthesis of polyhydroxybutyrate (PHB) is reported. Polymer production was monitored in more than 400 primary transformants grown under in vitro and glasshouse conditions. Plants containing up to 3.72% dry weight of PHB in leaf tissues and 1.23% dry weight of PHB in whole tillers were obtained. Results from the analysis of the polymer distribution at the cellular and whole plant levels are presented, and target areas for the improvement of PHB production are highlighted. Polymer accumulation was also analysed in the T(1) generation obtained from controlled crosses of transgenic plants. This study presents the first successful expression of a functional multigene pathway in switchgrass, and demonstrates that this high-yielding biomass crop is amenable to the complex metabolic engineering strategies necessary to produce high-value biomaterials with lignocellulose-derived biofuels.

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5′ end by the host plant’s psbA coding sequence and at the 3′ end by the host plant’s 3′ psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.

PHA Bioplastics, Biochemicals, and Energy from Crops

Large scale production of polyhydroxyalkanoates (PHAs) in plants can provide a sustainable supply of bioplastics, biochemicals, and energy from sunlight and atmospheric CO(2). PHAs are a class of polymers with various chain lengths that are naturally produced by some microorganisms as storage materials. The properties of these polyesters make them functionally equivalent to many of the petroleum-based plastics that are currently in the market place. However, unlike most petroleum-derived plastics, PHAs can be produced from renewable feedstocks and easily degrade in most biologically active environments. This review highlights research efforts over the last 20 years to engineer the production of PHAs in plants with a focus on polyhydroxybutryrate (PHB) production in bioenergy crops with C(4) photosynthesis. PHB has the potential to be a high volume commercial product with uses not only in the plastics and materials markets, but also in renewable chemicals and feed. The major challenges of improving product yield and plant fitness in high biomass yielding C(4) crops are discussed in detail.

Enhanced polyhydroxybutyrate production in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.

Production of novel biopolymers in plants: recent technological advances and future prospects.

The production of novel biopolymers in plants has the potential to provide renewable sources of industrial materials through agriculture. In this review we will highlight recent progress with plant-based production of polyhydroxyalkanoates (PHAs), silk, elastin, collagen, and cyanophycin with an emphasis on the synthesis of poly[(R)-3-hydroxybutyrate] (PHB), a renewable biodegradable PHA polymer with potential commercial applications in plastics, chemicals, and feed markets. Improved production of PHB has required manipulation of promoters driving expression of transgenes, reduction in activity of endogenous enzymes in competing metabolic pathways, insertion of genes to increase carbon flow to polymer, and basic plant biochemistry to understand metabolic limitations. These experiments have increased our understanding of carbon availability and partitioning in different plant organelles, cell types, and organs, information that is useful for the production of other novel molecules in plants.

Increased poly-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli

A second promoter (P1) was inserted to the PHA (poly-β-hydroxyalkanoate) operon (pSP2) of Esherichia coli DH5α with an optimal E. coli ribosome binding site and a trc strong promoter (pSJS1) to obtain poly-β-hydroxybutyrate (PHB) with long chain length. When the inducer, IPTG was added to the culture at 0.4 mM, the average molecular weight was 1.1 × 106 Da. However, an even greater increase of the PHB average molecular weight to 2.5 × 107 Da was observed without IPTG being added.

Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase.

BackgroundSwitchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated.ResultsA novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used.ConclusionThe results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.

Metabolic engineering to increase crop yield: From concept to execution

Although the return on investment over the last 20 years for mass screening of individual plant genes to improve crop performance has been low, the investment in these activities was essential to establish the infrastructure and tools of modern plant genomics. Complex traits such as crop yield are likely multigenic, and the exhaustive screening of random gene combinations to achieve yield gains is not realistic. Clearly, smart approaches must be developed. In silico analyses of plant metabolism and gene networks can move a trait discovery program beyond trial-and-error approaches and towards rational design strategies. Metabolic models employing flux-balance analysis are useful to determine the contribution of individual genes to a trait, or to compare, optimize, or even design metabolic pathways. Regulatory association networks provide a transcriptome-based view of the plant and can lead to the identification of transcription factors that control expression of multiple genes affecting a trait. In this review, the use of these models from the perspective of an Ag innovation companys trait discovery and development program will be discussed. Important decisions that can have significant impacts on the cost and timeline to develop a commercial trait will also be presented.

Novel transcription factors PvBMY1 and PvBMY3 increase biomass yield in greenhouse-grown switchgrass (Panicum virgatum L.)

Increasing crop yield requires the coordination of multiple metabolic pathways spanning photosynthetic carbon fixation, central carbon metabolism, and finally targeted carbon deposition to end product. In this study, we used a transcriptome-based gene regulatory association network to search for transcription factor genes that could play a role in increasing carbon flow through pathways associated with these processes to increase biomass yield in switchgrass. Two novel switchgrass transcription factors, PvBMY1 (BioMass Yield 1, belonging to the APETALA2/Ethylene Response Factor family of transcription factors) and PvBMY3 (BioMass Yield 3, a member of the Nuclear-Factor Y family of transcription factors), with predicted roles in the regulation of photosynthesis and related metabolism were identified. These genes were overexpressed in switchgrass to determine their impact on biomass yield. A significant increase in both aboveground and root biomass was observed in transgenic greenhouse grown plants compared to wild-type control plants with the best line producing 160% more aboveground biomass than controls. Transgenic lines with elevated electron transport rate of photosystems I and II as well as increased levels of starch and soluble sugars were identified.

Camelina sativa, an oilseed at the nexus between model system and commercial crop

The rapid assessment of metabolic engineering strategies in plants is aided by crops that provide simple, high throughput transformation systems, a sequenced genome, and the ability to evaluate the resulting plants in field trials. Camelina sativa provides all of these attributes in a robust oilseed platform. The ability to perform field evaluation of Camelina is a useful, and in some studies essential benefit that allows researchers to evaluate how traits perform outside the strictly controlled conditions of a greenhouse. In the field the plants are subjected to higher light intensities, seasonal diurnal variations in temperature and light, competition for nutrients, and watering regimes dictated by natural weather patterns, all which may affect trait performance. There are difficulties associated with the use of Camelina. The current genetic resources available for Camelina pale in comparison to those developed for the model plant Arabidopsis thaliana; however, the sequence similarity of the Arabidopsis and Camelina genomes often allows the use of Arabidopsis as a reference when additional information is needed. Camelina’s genome, an allohexaploid, is more complex than other model crops, but the diploid inheritance of its three subgenomes is straightforward. The need to navigate three copies of each gene in genome editing or mutagenesis experiments adds some complexity but also provides advantages for gene dosage experiments. The ability to quickly engineer Camelina with novel traits, advance generations, and bulk up homozygous lines for small-scale field tests in less than a year, in our opinion, far outweighs the complexities associated with the crop.