Molecular Duckweed and  The Duckweed Genome Project

Genome Project   and   Cloning Duckweed Genes
Duckweed Chromosomes:     Under the Microscope  |   Counting Chromosomes   |   DNA Content
Duckweed Genetic Engineering:  GM Duckweed
Pharmaceutical Proteins from Duckweed

Duckweed Genetics  and  Duckweed Mutants

Glossary

The Duckweed Genome Project

Several years ago The Charms of Duckweed, with tongue-in-cheek, announced the formation of a very small genome project, devoted to the DNA sequences of the duckweeds. Only, now it's really happening.  The project is led by Joachim Messing at Rutgers University and focuses on Spirodela polyrhiza.  Overall funding is from the US Department of Energy.


Left:  An interview with Joachim Messing on the Duckweed Genome Project and the future of duckweed applications.


Spirobase Logo

Timeline:

2003:  Studies by G. Salazar et al. and cited by Soltis indicated that the Lemna has a very small genome size (1C = 0.60 pg, where C = the one chromosome set). Compare this for example to to a primitive diploid wheat, Triticum monococcum (1C = 6.23 pg). The small genome size of Lemna compares to that of early flowering plants.(1)

2008:  Andrey Mardanov and colleagues at the Bioengineering Center of the Russian Academy of Sciences in Moscow announced the sequencing of the Lemna minor chloroplast Genome.(2)

2008: US Department of Energy (DOE) Joint Genome Institute announced the Community Sequencing Program will fund the sequencing the genome of the giant duckweed, Spirodela polyrhiza, which has a genome size similar to that of Arabidopsis (150 MB). This Duckweed Genome Project was a priority project for DOE in 2009.  The research intended to facilitate new biomass and bioenergy programs.(3)

2011:  W. Wang and Joachim Messing at Rutgers published a DNA sequence comparison of three duckweed chloroplast genomes Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana.(4)

2102: Wang, Wu and Messing published the mitochondrial genome of Spirodela.(5)

2014:  An international team led by Messing publishes the complete an overall study of the Spirodela genome and interprets their findings to explain important anatomical and physiological attributes of the duckweeds.(6)

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(1) Soltis, D.E. et al. (2003) American Journal of Botany. 90:1596-1603. http://www.amjbot.org/cgi/content/full/90/11/1596#F1 

(2) Mardanov, A.V. et al. (2008) Complete Sequence of the Duckweed ( Lemna minor ) Chloroplast Genome: Structural Organization and Phylogenetic Relationships to Other Angiosperms. J. Molec. Evol. 66(6):555-564. http://www.springerlink.com/content/8042060xp665646x/

(3) Duckweed genome sequencing has global implications. E! Science News. http://esciencenews.com/articles/2008/07/08/duckweed.genome.sequencing.has.global.implications

(4) Wang W, Messing J. High-throughput sequencing of three Lemnoideae (duckweeds) chloroplast genomes from total DNA. PLoS One. 2011;6(9):e24670. doi: 10.1371/journal.pone.0024670. Epub 2011 Sep 9. PubMed PMID: 21931804; PubMed Central PMCID: PMC3170387.

(5) Wang, W, Wu Y, Messing J.  2012.  The mitochondrial genome of an aquatic plant, Spirodela polyrhiza. PLoS One. 2012;7(10):e46747. doi: 10.1371/journal.pone.0046747. Epub 2012 Oct 4. PubMed PMID: 23056432; PubMed Central PMCID: PMC3464924. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0046747

(6) Wang, W et al. The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle.   Nature Communications  5, Article number:
    3311, doi:10.1038/ncomms4311 Published 19 February 2014 . http://www.nature.com/ncomms/2014/140219/ncomms4311/full/ncomms4311.html

DNA double helix 


Cloning Duckweed Genes

Duckweed genes are similar to those of other plants.  Many laboratories have cloned (isolated) genes from duckweeds for research in several areas, particularly photosynthesis and phytochrome regulation of plant development. Elaine Tobin's lab (UCLA) has studied the regulation of chloroplast development by phytochrome.  Cheryl Smart (ETH, Zurich) has cloned a plant ABC transporter regulated by Abscisic Acid (ABA) from Spirodela .  ABC transporters protect against toxic substances by pumping them out of the cell at the expense of ATP.

Bibliography of duckweed population genetics, molecular biology and tissue culture.


Duckweed Genetic Engineering

The desirable characteristics of duckweeds in agriculture and the environment spurred scientists to think of more and more novel uses for these plants.   Many of these new applications may require new capabilities for duckweed metabolism, growth or nutritional composition.  Genetic modifications will be required to give duckweeds these new characteristics, and the new technology of genetic engineering is a powerful technique that can help bring many of these dreams to reality.  This technology is being used to modify the activity of existing duckweed genes, or to introduce new genes into duckweeds. 

Pharmaceutical Proteins from Duckweed.

Some of the most exciting prospects in duckweed technology are aimed at using this plant as a factory for biopharmaceuticals.  The advantages of this technology are many: 

 The total absence of potentially hazardous animal proteins, cells and viruses means that many of the challenges of producing biopharmaceuticals are vastly simplified and the cost of production reduced accordingly.  At first these advantages were merely theoretical, but now this technology is making rapid strides towards practical commercialization.

 Anne-Marie StompA North Carolina State University scientist, Anne-Marie Stomp, developed the first procedure to genetically engineer duckweed, a common aquatic weed, to produce therapeutic proteins.  Stomp's spin-off company, now owned by Synthon, has achieved promising levels of  recombinant protein expression in duckweed.

Stomp estimates the market for pharmaceutical proteins like insulin is worth about $10 billion yearly.  
Review article:  Stomp AM.  "The duckweeds: A valuable plant for biomanufacturing." Biotechnol Annu Rev. 2005;11:69-99.

[ Read Dr. Stomp's Patent ]  

Dr. Stomp made this advance with the aid of an EPA research grant, Genetic Improvement of Duckweed (Lemna gibba) for Wastewater Treatment.  The final report of this grant is still available on line.


Research paper:  Genetic transformation of duckweed.

Yamamoto Y.T.*; Rajbhandari N.; Lin X.; Bergmann B.A.; Nishimura Y.; Stomp A.M. (2001)  "Genetic transformation of duckweed Lemna gibba and Lemna minor."   In Vitro Cellular and Development Biology - Plant,   37(3):349-353.

"We developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer.  Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing -glucuronidase and nptII expression cassettes.  Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin.  Transgenic duckweed fronds were regenerated from selected nodules.  We demonstrated that transgenic duckweed could be regenerated within 3 months after Agrobacterium-mediated transformation of nodules.  Furthermore, we developed a method for transforming L. minor 8627 in 6 weeks. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins."
 

Duckweed tissue culture research from this lab.

H. K. Moon and A. M. Stomp (1996) Effects of Medium Components and Light on Callus Induction, Growth, and Frond Regeneration in Lemna gibba (Duckweed). In Vitro Plant Cellular & Developmental Biology 33(1):20-25.Tamara Lynn Tatroe. (1998)

Investigation of growth parameters for a plant-based gene expression system using duckweed.  M.S. Thesis, Biological and Agricultural Engineering, N.C. State University. 

 

Identification of superior clones for bioremedation:

Bergmann, B.A., Cheng, J., Classen, J., Stomp, A.M. (2000) In vitro selection of duckweed geographical isolates for potential use in swine lagoon effluent renovation. Bioresource technology. 73 (1):13-20.

"The objective of this study was to select superior duckweed (Lemnaceae) genotypes for the utilization of nutrients in animal wastes. A two-step protocol was used to select promising duckweed geographic isolates to be grown on swine lagoon effluent. Forty-one geographic  isolates from the worldwide germplasm collection were used in an in vitro screening test, because they were noted to be fast-growing genotypes during routine collection maintenance. In vitro screening was accomplished by growing geographic isolates on a synthetic medium that approximated swine lagoon effluent in terms of nutrient profile, total ionic strength, pH, and buffering capacity. 

"Large differences among geographic isolates were observed for wet weight gain during the 11-day growing period, percent dry weight, and percent protein in dry biomass. Total protein production per culture jar differed 28-fold between the most disparate of the 41 geographic isolates and was the variable used for selection of superior geographic isolates. The challenge of eight of the 41 geographic isolates with full-strength swine lagoon effluent in the greenhouse led to the selection of three that are promising as genotypes to be grown on lagoon effluent."

Protein Glycosylation

Cox KM et al.  Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor.   Nat Biotechnol. 2006 Dec;24(12):1591-7.

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.”

 

 

Synthon's SYNLEX Recombinant Protein Technology

SYNLEX™ is a proprietary therapeutic protein expression technology that uses cultures of Lemna minor (duckweed) to enable development of pharmaceutical proteins. According to the company, this expression platform is capable of generating glycan-engineered monoclonal antibodies, hard-to-make proteins, follow-on biologics, vaccines and veterinary medicines.

Synthon's Synlex recombinant protein technology

The technology was first developed by Biolex a start-up company created expressly to manufacture high-quality pharmaceutical proteins in duckweed.  It was spun-off from the technology developed by Prof. Anne-Marie Stomp at North Carolina State University.  In July 2012 Biolex's Lemna-based technology was sold to Synthon, a Netherlands-based pharmaceutical company with a presence in Research Triangle Park, North Carolina.

Much of the information below comes from the former Biolex corporate website, which is no longer on-line:

"Biolex, Inc. is a biotechnology company based in Pittsboro, North Carolina, just outside of Research Triangle Park. Biolex is focused on developing the next generation of patented, recombinant protein production technologies in a plant based system.  The foundation of the Biolex technology is its patented Lemna System™. This system couples the natural characteristics of the green plant, Lemna, with advanced genetic engineering and protein recovery methods to create a recombinant protein production platform."

"The Lemna System™ has unique, innate characteristics that provide enormous value for recombinant protein production"  The stated advantages include:  versatility, efficient genetic engineering, fast and flexible operation, low capital costs for facilities, low operating costs, high protein recovery and simple purification, product safety, and environmental safety."

Biolex has claimed to have successfully expressed at least twelve recombinant proteins in duckweeds, including small peptides, Fab fragments (Fabs), monoclonal antibodies (mAbs), and large multimeric enzymes. These include interferon alpha 2b (IFN), human growth hormone (hGH), a Fab, and a mAb.

A Biolex publication stated that IFN and hGH was successfully isolated from Lemna growth medium,

"At least 50% of IFN and hGH was secreted into asceptic growth media with pre-purification titers as high as 609 mg/L. Both proteins were found to be biologically active with IFN shown to be at least equal to the commercial source, Intron A. Efficient secretion of these recombinant proteins into an inorganic media with no need for viral inactivation offers substantial cost advantages in downstream purification."

It should be noted that Biolex defines "pre-purification titer" to include a 50x concentration step, so that their actual titer in Lemna medium would be 609/50 =  12 mg/L.  Biolex indicated that it was further optimizing protein secretion from duckweed cultures.

Another key advantage of recombinant Lemna cultures is that, unlike mammalian tissue culture systems, animal viruses cannot multiply.  Therefore, this system should provide an increased level of pathogen safety in the final product.  Currently many recombinant proteins are produced in mammalian cell cultures, where processes and tests to assure pathogen safety are major costs of production.

References:  

John R. Gasdaska, David Spencer and Lynn Dickey "Advantages of Therapeutic Protein Production in the Aquatic Plant Lemna" BioProcessing Journal, Mar/Apr 2003.

Katharina Schoebi, "One weed you do not want to get rid of" Checkbiotech April 15, 2005.

 
Duckweeds Engineered for Bioremediation.

USDA scientists in Peoria, Illinois are developing ways to convert renewable agricultural resources into value-added products.  Their research may also show how genetically modified duckweeds can improve waste-water processing systems.  One object of this research is to develop a duckweed which produces cellulase, the enzyme that converts cellulose into simple sugars.  By converting cellulose into sugars, agricultural wastes containing cellulose could be used to make many valuable products, including lactic acid, ethanol, and glycerol.

Patents for GM Duckweed Applications.

Much of the technology for genetic modification of duckweeds can be found in patent literature.  These patents can be read on-line and have considerable information about this technology.


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Background Information on Plant Genetic Engineering

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Revised:  February 22, 2014