Technology

Engineered proteins are vitally important components of new diagnostics, vaccines, therapeutics, biopolymers and catalysts…but are expensive and difficult to obtain at large scale.

Plantcode exploits a novel plant system, Marchantia, to express proteins in chloroplasts for low-cost, large-scale, fast and GMO-contained bioproduction.

Plantcode work is fueled by a series of technical innovations:

  • The adoption of Marchantia polymorpha as a ‘bare-bones’ model plant that is extraordinarily facile to manipulate and analyse;

  • the speed of chloroplast genome transformation in this system;

  • the invention of novel DNA elements for driving very high levels of gene expression;

  • development of low-cost methods for rapid propagation of Marchantia plant material by indoor farming;

  • industrial tools for juicing and filtering vegetable material at large scale;

  • protein-based affinity chromatography and trimming for single-step production of native proteins;

  • development of yeast shuttle vectors for genome-scale DNA assembly and editing;

  • the prospect of constructing synthetic chloroplast genomes for transplantation, faster transformation and move to field crops.

Major benefits of working with Marchantia are:

  1. The plant is weed-like, small and grows rapidly in soil, artificial media, and hydroponic systems.

  2. The thalloid liverwort is a descendant of earliest land plants, with the haploid gametophyte form dominant in its lifecycle. This allows the exploitation of haploid genetic systems, like microbes.

  3. Sexual reproduction in male and female plants can be induced by far red light. The gamete bearing structures are easily identified and used for crosses. A single genetic cross results in the formation of millions of spores, which can be stored stably.

  4. Spores will germinate and grow through the entire process of plant development in an entirely open fashion, accessible to modern quantitative microscopy techniques.

  5. Marchantia plants also spontaneously produce vegetative propagules, called gemmae, which allow simple clonal propagation of lines.

  6. Gemma can be stored for 6 months as refrigerated stabs.

  7. Gemma are ideal tissues for microscopic analysis of early development, with defined simple morphology and ability to rapidly progress through key developmental and physiological stages after germination.

  8. The genome sequence has been determined. Marchantia has a highly streamlined genome architecture, with reduced gene families.

  9. Isolated Marchantia tissues spontaneously regenerate, allowing efficient plant nuclear and chloroplast transformation.

  10. CRISPR-Cas9 based approaches for genome editing are highly efficient.

Why Marchantia?

Marchantia polymorpha is the best characterised liverwort plant. It is a common weed, and can grow quickly and resiliently. The relative simplicity of genetic networks in Marchantia, combined with the growing set of genetic manipulation, culture and microscopy techniques, make this simple plant a major new system for analysis and engineering.

DNA code

In Cambridge, we have built an extensive library of standardised DNA parts for reprogramming the nuclear and chloroplast genomes in Marchantia. The sequences are formatted according to the Phytobricks standard. This allows use of liquid-handling robots for high throughput assembly of genetic circuits using our Loop assembly method.

We have assembled a toolkit of compatible DNA parts and vectors that include novel synthetic elements to drive high levels of gene expression in chloroplasts. (images from Pollak et al., Loop Assembly: a simple and open system for recursive fabrication of DNA circuits. 2018)

Chloroplast transformation

Chloroplasts provide a platform for low-cost, large-scale production of proteins. Chloroplasts have a small genome which is present in very high copy numbers in plant cells. There is no gene-silencing in the chloroplast, and the organelles are capable of expressing prodigious amounts of foreign protein at little cost to the plant. Despite this promise, use of chloroplast-based expression has been hindered by slow and difficult transformation procedures, a relatively small set of amenable plant hosts, and variability in expression levels. Plant has developed proprietary technologies for chloroplast transformation and bioproduction in Marchantia.

Hyperexpression of chloroplast encoded genes

Marchantia provides the fastest system for chloroplast transformation due to its explosive growth and extraordinary efficiency of regeneration after transformation. Plants spontaneously produce propagules in a short life cycle, grow rapidly in soil-free culture, and can be cultivated under GMO-containment conditions using low-cost soil-free indoor farming techniques.

Further, we have developed proprietary methods for ensuring high levels of protein accumulation in transformed Marchantia plants, with engineered proteins produced over 15% of total soluble protein. Plants contain high levels of expressed protein in sap that can be readily processed at industrial scales. (images from Frangedakis et al., Construction of DNA tools for hyperexpression in Marchantia chloroplasts. 2021)

Rapidly growing plants as bioreactors

Protein production does not require large capital expenditure. The protein-producing bioreactors are the plants themelves, self-assembled as they grow rapidly in a 24-day cycle on hybrid membranes, requiring only light and a simple hydroponic media. The plants are true-breeding, and can be continuously re-planted for bulk production.

One step protein purification

Chloroplast-based systems have exceptional capacity for low cost protein production. However, one of the main burdens for production of isolated proteins is the cost of purification. We are exploiting the capacity and programmability of the chloroplast system by genetically encoding functions for affinity purification and processing of native proteins. The functional elements are encoded within the genes expressed in the chloroplast, and the process can be scaled cheaply.

Scaling-up bioproduction

Controlled environment agriculture (CEA), also termed vertical farming is increasingly used to grow select crops. The associated costs are much lower than those required for animal cell culture or microbial fermentation, and compared to field cultivation, provides benefits of all-year production, high yields and in our case, GMO containment. The infrastructure required to set up a large indoor growth facility is relatively cheap and readily available. (images from www.sananbio.com)

Competing technologies

Companies have been using microbial, animal cell and plant systems to fulfill the growing demand for purified proteins in industry and research. Microbial and animal cell production is most commonly employed, but this appoach requires substantial capital expenditure to support culture in bioreactors, and need to feed these with sterile growth media. Producers are exploring the potential benefits of plant systems, which allow low cost growth, and are useful for unglycosylated proteins. Two methods are used to create high yields of expressed protein: (i) The HyperTrans system uses transient Agrobacterium-mediated gene delivery that provides high yields over a few days, but must be repeated for each production cycle. Medicago have implemented the approach for commercial vaccine production, but it does require expensive roboticised glasshouses and large scale microbial culture. (ii) Alternatively, chloroplast transformation allows high levels of stable expression (>10% total soluble protein), due to high gene copy and lack of silencing in the organelle genome. Chloroplast transformation is possible in only a few plant species, with tobacco being the leading platform. Field-grown tobaccos have been used for production of industrial enzymes, but the approach suffers from slow speed of transformation (>6 months) and generation times, and regulatory constaints due to environmental release of GMO plants.

In contrast, chloroplast transformation of Marchantia is fast, with homoplasmic plants available in 8 weeks, and very short and prolific life cycles. Plants can be propagated vegetatively through gemmae or simply cut tissues, or sexually via spores (a single cross will generate millions of spores). Transformed plant material can be propagated rapidly on low-cost soil-free media, using indoor farming techniques on a 24 day, all year production cycle.

Potential market size

Chloroplast-based expression can offer benefits for low-cost large-scale production of unmodified proteins such as:

(i) Protein cytokines. The global cytokine market is expected to grow from $204 billion in 2021 to $611 billion in 2028, growing at a CAGR of 17.0% (https://www.marketwatch.com)

(ii) Single chain antibody reagents. The global monoclonal antibody therapy market size is projected to grow from $178 billion in 2021 to $452 billion in 2028 at a CAGR of 14.1%. (https://www.fortunebusinessinsights.com/). The research antibodies market size was estimated at USD 3.6 billion in 2020 and is expected to expand at a compound annual growth rate (CAGR) of 6.4% from 2021 to 2028. (https://www.grandviewresearch.com)

(iii) Vaccine components. The global vaccines market is projected to grow from $61 billion in 2021 to $125 billion in 2028 at a CAGR of 10.8% in forecast period, 2021-2028. (https://www.fortunebusinessinsights.com)

(iv) Protein therapeutics for veterinary and human use. The global protein therapeutics market size was valued at $284 Billion in 2020, and is estimated to reach $567 Billion by 2030, growing at a CAGR of 7.1% from 2021 to 2030. (https://www.alliedmarketresearch.com/protein-therapeutics-market)

(v) Industrial enzymes. The industrial enzyme market was valued at over $6 billion in 2021, and projected to reach $9.1 billion by 2026, recording a CAGR of 6.6%. (https://www.marketsandmarkets.com)

(vi) Structural proteins and biopolymers. The global recombinant proteins market is projected to grow from $1 billion in 2021 to $1.7 billion by 2026, at a CAGR of 9.8% during the forecast period. (https://www.researchandmarkets.com)

The forecast demand for all of these products is high. Further, the growth of bio-based industries is likely to push demand for new protein-based products. Protein cytokines are a major component of production expenses, and provide a market opportunity for lower-cost proteins.

Opportunities

The lower cost of production of engineered proteins in Marchantia provides opportunities to undercut existing production costs, and develop a portfolio of products for direct B2B sales. Taking protein cytokines as an example, Plantcode would produce cytokines at large scale for producers of cultured meat and stem cell engineering applications. Further, Plantcode can provide nanobody proteins and derivatives to manufacturers of diagnostic devices. The potentially lower cost of the Plantcode proteins opens new opportunities for applications both at large-scale, and in new low-resource settings.

Construction of DNA Tools for Hyperexpression in Marchantia Chloroplasts
Eftychios Frangedakis, Fernando Guzman-Chavez, Marius Rebmann, Kasey Markel, Ying Yu, Artemis Perraki, Sze Wai Tse, Yang Liu, Jenna Rever, Susanna Sauret-Gueto, Bernard Goffinet, Harald Schneider, Jim Haseloff. ACS Synthetic Biology 16;10(7):1651-1666 (2021).

Rapid and Modular DNA Assembly for Transformation of Marchantia Chloroplasts
Eftychios Frangedakis, Kasey Markel, Susana Sauret-Gueto, Jim Haseloff. Methods Mol Biol. 2317:343-365 (2021).

Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha
Susanna Sauret-Gueto, Eftychios Frangedakis, Linda Silvestri, Marius Rebmann, Marta Tomaselli, Kasey Markel, Mihails Delmans, Anthony West, Nicola J Patron, Jim Haseloff. ACS Synthetic Biology 9, 4, 864–882 (2020).

Opening options for material transfer.
Linda Kahl, Jennifer Molloy, Nicola Patron, Colette Matthewman, Jim Haseloff, David Grewal, Richard Johnson, Drew Endy. Nature Biotechnology 36:923-927 (2018).

Loop Assembly: a simple and open system for recursive fabrication of DNA circuits.
Bernardo Pollak, Ariel Cerda, Mihails Delmans, Simón Álamos, Tomás Moyano, Anthony West, Rodrigo A Gutiérrez, Nicola Patron, Fernán Federici, Jim Haseloff. New Phytologist, (2018).

Analysis of Cambridge isolates of Marchantia polymorpha.
Bernardo Pollak, Mihails Delmans and Jim Haseloff, Supplementary material for Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome,Cell, 171:287–304 (2017).

Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.
John L. Bowman, Takayuki Kohchi, Katsuyuki T. Yamato, Jerry Jenkins, Shengqiang Shu, Kimitsune Ishizaki, Shohei Yamaoka, Ryuichi Nishihama, Yasukazu Nakamura, Frédéric Berger, Catherine Adam, Shiori Sugamata Aki, Felix Althoff, Takashi Araki, Mario A. Arteaga-Vazquez, Sureshkumar Balasubrmanian, Kerrie Barry, Diane Bauer, Christian R. Boehm, Liam Briginshaw, Juan Caballero-Perez, Bruno Catarino, Feng Chen, Shota Chiyoda, Mansi Chovatia, Kevin M. Davies, Mihails Delmans, Taku Demura, Tom Dierschke, Liam Dolan, Ana E. Dorantes-Acosta, D. Magnus Eklund, Stevie N. Florent, Eduardo Flores-Sandoval, Asao Fujiyama, Hideya Fukuzawa, Bence Galik, Daniel Grimanelli, Jane Grimwood, Ueli Grossniklaus, Takahiro Hamada, Jim Haseloff, Alexander J. Hetherington, Asuka Higo, Yuki Hirakawa, Hope N. Hundley, Yoko Ikeda, Keisuke Inoue, Shin-ichiro Inoue, Sakiko Ishida, Qidong Jia, Mitsuru Kakita, Takehiko Kanazawa, Yosuke Kawai, Tomokazu Kawashima, Megan Kennedy, Keita Kinose, Toshinori Kinoshita, Yuji Kohara, Eri Koide, Kenji Komatsu, Sarah Kopischke, Minoru Kubo, Junko Kyozuka, Ulf Lagercrantz, Shih-Shun Lin, Erika Lindquist, Anna M. Lipzen, Chia-Wei Lu, Efraín De Luna, Robert A. Martienssen, Naoki Minamino, Masaharu Mizutani, Miya Mizutani, Nobuyoshi Mochizuki, Isabel Monte, Rebecca Mosher, Hideki Nagasaki, Hirofumi Nakagami, Satoshi Naramoto, Kazuhiko Nishitani, Misato Ohtani, Takashi Okamoto, Masaki Okumura, Jeremy Phillips, Bernardo Pollak, Anke Reinders, Moritz Rövekamp, Ryosuke Sano, Shinichiro Sawa, Marc W. Schmid, Makoto Shirakawa, Roberto Solano, Alexander Spunde, Noriyuki Suetsugu, Sumio Sugano, Akifumi Sugiyama, Rui Sun, Yutaka Suzuki, Mizuki Takenaka, Daisuke Takezawa, Hirokazu Tomogane, Masayuki Tsuzuki, Takashi Ueda, Masaaki Umeda, John M. Ward, Yuichiro Watanabe, Kazufumi Yazaki, Ryusuke Yokoyama, Yoshihiro Yoshitake, Izumi Yotsui, Sabine Zachgo, Jeremy Schmutz, , Cell, 171:287–304 (2017).

Synthetic Botany.
Christian R. Boehm*, Bernardo Pollak*, Nuri Purswani, Nicola Patron and Jim Haseloff, Cold Spring Harbor Perspectives in Biology, doi: 10.1101/cshperspect.a023887, (2017).

MarpoDB: An Open Registry for Marchantia Polymorpha Genetic Parts.
Mihails Delmans*, Bernardo Pollak* and Jim Haseloff, Plant Cell Physiol. 58: e5(1–9) (2016).

A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha.
Boehm CR, Ueda M, Nishimura Y, Shikanai T, Haseloff J. Plant Cell Physiol. 57:291-9. (2016).

The Naming of Names: Guidelines for Gene Nomenclature in Marchantia.
Bowman JL, Araki T, Arteaga-Vazquez MA, Berger F, Dolan L, Haseloff J, Ishizaki K, Kyozuka J, Lin SS, Nagasaki H, Nakagami H, Nakajima K, Nakamura Y, Ohashi-Ito K, Sawa S, Shimamura M, Solano R, Tsukaya H, Ueda T, Watanabe Y, Yamato KT, Zachgo S, Kohchi T. Plant Cell Physiol. 57:257-61, (2016).

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.
Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, Raitskin O, Leveau A, Farré G, Rogers C, Smith A, Hibberd J, Webb AA, Locke J, Schornack S, Ajioka J, Baulcombe DC, Zipfel C, Kamoun S, Jones JD, Kuhn H, Robatzek S, Van Esse HP, Sanders D, Oldroyd G, Martin C, Field R, O'Connor S, Fox S, Wulff B, Miller B, Breakspear A, Radhakrishnan G, Delaux PM, Loqué D, Granell A, Tissier A, Shih P, Brutnell TP, Quick WP, Rischer H, Fraser PD, Aharoni A, Raines C, South PF, Ané JM, Hamberger BR, Langdale J, Stougaard J, Bouwmeester H, Udvardi M, Murray JA, Ntoukakis V, Schäfer P, Denby K, Edwards KJ, Osbourn A, Haseloff J. New Phytologist 208:13-9. (2015).

Integrated genetic and computation methods for in planta cytometry.
Federici F, Dupuy L, Laplaze L, Heisler M & Haseloff J. Nature Methods, 9:483-485 (2012).

High resolution, live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy.
Maizel A, von Wangenheim D, Federici F, Haseloff J, Stelzer EH. Plant Journal 68:377-385 (2011).

Coordination of plant cell division and expansion in a simple morphogenetic system.
Dupuy, L., Mackenzie, J. and Haseloff, J. Proc. Natl. Acad. Sci. USA 107:2711-6 (2010).

Synthetic biology: history, challenges and prospects.
Haseloff J and Ajioka J. Royal Society Interface 4:S389-91 (2009).

Published work