Summary Plant‐based co‐production of polyhydroxyalkanoates (PHAs) and seed oil has the potential to create a viable domestic source of feedstocks for renewable fuels and plastics. PHAs, a class of biodegradable polyesters, can replace conventional plastics in many applications while providing full degradation in all biologically active environments. Here we report the production of the PHA poly[( R )‐3‐hydroxybutyrate] (PHB) in the seed cytosol of the emerging bioenergy crop Camelina sativa engineered with a bacterial PHB biosynthetic pathway. Two approaches were used: cytosolic localization of all three enzymes of the PHB pathway in the seed, or localization of the first two enzymes of the pathway in the cytosol and anchoring of the third enzyme required for polymerization to the cytosolic face of the endoplasmic reticulum (ER). The ER‐targeted approach was found to provide more stable polymer production with PHB levels up to 10.2% of the mature seed weight achieved in seeds with good viability. These results mark a significant step forward towards engineering lines for commercial use. Plant‐based PHA production would enable a direct link between low‐cost large‐scale agricultural production of biodegradable polymers and seed oil with the global plastics and renewable fuels markets.

Plant genetic engineering is an important tool used in current efforts in crop improvement, pharmaceutical product biosynthesis and sustainable agriculture. However, conventional genetic engineering techniques target the nuclear genome, prompting concerns about the proliferation of foreign genes to weedy relatives. Chloroplast transformation does not have this limitation, since the plastid genome is maternally inherited in most plants, motivating the need for organelle-specific and selective nanocarriers. Here, we rationally designed chitosan-complexed single-walled carbon nanotubes, utilizing the lipid exchange envelope penetration mechanism. The single-walled carbon nanotubes selectively deliver plasmid DNA to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and transient expression in mature Eruca sativa, Nasturtium officinale , Nicotiana tabacum and Spinacia oleracea plants and in isolated Arabidopsis thaliana mesophyll protoplasts. This nanoparticle-mediated chloroplast transgene delivery tool provides practical advantages over current delivery techniques as a potential transformation method for mature plants to benefit plant bioengineering and biological studies.

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.

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.

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 company's 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.

L'invention concerne une plante terrestre transgenique. La plante terrestre transgenique comprend une proteine de transporteur putatif de bicarbonate d'une algue eucaryote comestible. La proteine de transporteur putatif de bicarbonate de l'algue eucaryote comestible est heterologue en ce qui concerne la plante terrestre transgenique. La proteine de transporteur putatif de bicarbonate est un orthologue de CCPl de Chlamydomonas reinhardtii de SEQ ID NO : 1. La proteine de transporteur putatif de bicarbonate est localisee dans des mitochondries de la plante terrestre transgenique sur la base d'un signal de ciblage mitochondrial intrinseque a la proteine de transporteur putatif de bicarbonate.

•Plant production of novel polymers increases materials available from agriculture.•Synthesis of PHB illustrates challenges of plant-based production of novel materials.•PHB can be produced at high levels in biomass and oil seed crops.•Competition for substrate from endogenous pathways limits PHB and/or plant yield.•Future efforts should focus on increasing substrate for PHB synthesis and plant growth.

Crotonic acid has the potential to be produced from renewable resources at low cost but currently has a limited market. We are investigating catalytic routes to exploit the functionalities of crotonic acid to produce a range of established industrial chemicals. Here we report our work on converting crotonates to acrylates, where a cost-competitive bio-based alternative can provide a market advantage. Our optimized reaction conditions for the cross-metathesis between crotonates and ethylene resulted in an increase in catalyst turnover numbers by 2 orders of magnitude compared with literature values. Control experiments showed the cross-metathesis with ethylene to be an equilibrium reaction. The turnover-number-limiting factor was found to be the stability of the metathesis catalyst.

BACKGROUND:Switchgrass (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.RESULTS:A 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.CONCLUSION:The 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.

Poly-3-hydroxybutyrate (PHB) production in plastids of Camelina sativa seeds was investigated by comparing levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed-specific promoters for expression of transgenes. Genes encoding PHB biosynthetic enzymes were modified at the N-terminus to encode a plastid targeting signal. PHB levels of up to 15% of the mature seed weight were measured in single sacrificed T-1 seeds with a genetic construct containing the oleosin and glycinin promoters. Amore detailed analysis of the PHB production potential of two of the best performing binary vectors in a Camelina line bred for larger seed size yielded lines containing up to 15% polymer in mature T-2 seeds. Transmission electron microscopy showed the presence of distinct granules of PHB in the seeds. PHB production had varying effects on germination, emergence and survival of seedlings. Once true leaves formed, plants grew normally and were able to set seeds. PHB synthesis lowered the total oil but not the protein content of engineered seeds. A change in the oil fatty acid profile was also observed. High molecular weight polymer was produced with weight-averaged molecular weights varying between 600000 and 1500000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T-4 seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed-based platform for PHB production.

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing β-ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the β-ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10(6)  Da. These results are a major step forward in engineering a high biomass C4 grass for the commercial production of PHB.

BACKGROUND:Polyhydroxyalkanoates are linear biodegradable polyesters produced by bacteria as a carbon store and used to produce a range of bioplastics. Widespread polyhydroxyalkanoate production in C4 crops would decrease petroleum dependency by producing a renewable supply of biodegradable plastics along with residual biomass that could be converted into biofuels or energy. Increasing yields to commercial levels in biomass crops however remains a challenge. Previously, lower accumulation levels of the short side chain polyhydroxyalkanoate, polyhydroxybutyrate (PHB), were observed in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells in transgenic maize (Zea mays), sugarcane (Saccharum sp.), and switchgrass (Panicum virgatum L.) leading to a significant decrease in the theoretical yield potential. Here we explore various factors which might affect polymer accumulation in mesophyll cells, including targeting of the PHB pathway enzymes to the mesophyll plastid and their access to substrate.RESULTS:The small subunit of Rubisco from pea effectively targeted the PHB biosynthesis enzymes to both M and BS chloroplasts of sugarcane and switchgrass. PHB enzyme activity was retained following targeting to M plastids and was equivalent to that found in the BS plastids. Leaf total fatty acid content was not affected by PHB production. However, when fatty acid synthesis was chemically inhibited, polymer accumulated in M cells.CONCLUSIONS:In this study, we provide evidence that access to substrate and neither poor targeting nor insufficient activity of the PHB biosynthetic enzymes may be the limiting factor for polymer production in mesophyll chloroplasts of C4 plants.

The mild pyrolysis of switchgrass/poly-3-hydroxybutyrate (P3HB) blends that mimic P3HB-producing switchgrass lines was studied in a pilot scale fluidized bed reactor with the goal of simultaneously producing crotonic acid, a switchgrass-based bio-oil, and bio-char. Factors such as pyrolysis temperature, reactor residence time, flow rate and particle size of the P3HB were studied to determine their effects on the recovery of crotonic acid as a component of the pyrolysis oil produced from the mixture. Crotonic acid yields were maximized at 45 wt% of the input P3HB by using small P3HB particles and a pyrolysis temperature of 375 degrees C. The remaining components of the liquid product were similar to those produced via fast pyrolysis of switchgrass alone. Fractional collection within the condensation system of the pyrolysis process development unit (PDU) did not significantly fractionate crotonic acid more than the total liquids collected. Concentrations of 6 to 10 wt% crotonic acid in the liquids were found in all fractions and crotonic acid was effectively collected by both condensation and electrostatic precipitation suggesting that pyrolysis of P3HB produces crotonic acid in both gas and aerosol phases. Published by Elsevier B.V.

Using metabolic engineering approaches, the biopolymer poly-3-hydroxybutyrate (P3HB) can be overproduced in organisms such as bacteria and plants. Thermolysis of P3HB, either in isolated form or within biomass, yields crotonic acid, a potential bioderived platform chemical. Reduction of crotonic acid provides n-butanol, which has value as a fuel and as a commodity chemical. Herein, we report optimization work on the hydrogenation of the n-butyl ester of crotonic acid to n-butanol and the potential of this chemistry to be incorporated into the production of bio-n-butanol from P3HB containing biomass.

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.