Can we build better systems? Can we go from the Wright Brothers to a Boeing 747?
Day 1 Plant Synthetic Biology 2019
Aug 7, San Jose, California - the American Society for Plant Biology (ASPB) showcased the latest research into engineering plants to address grand societal challenges on the first day of the inaugural Plant Synthetic Biology meeting. With topics ranging from modifying plants for desalination to reimagining carbon fixation, speakers outlined visions which suggest we are limited only by our imagination.
Physicist Markita Landry opened proceedings with her group’s work to improve transformation of plants. The Landry lab has recently published several high impact papers investigating the use of nano-materials to deliver DNA and RNA into plant cells, in a process that is non-pathogenic and non-integrating.
Not all plant species are currently amenable to genetic transformation and typically making transgenic plants is a costly process requiring the use of modified plant pathogens or specialist equipment. Transformation is often limited to amenable cultivars and researchers have little control over the frequency or location of integration of DNA.
The major barrier to plant transformation is the cell wall, which has a size exclusion limit of 5-20nm, as a result the Landry lab is investigating the use of both single carbon nanotubes (~1 nm in diameter) and DNA nanostructures to deliver cargos into both the nucleus and chloroplast. By tuning the strength of nucleic acid–carrier interactions, size, shape and tensile strength of delivery materials they have been able to optimize protocols to facilitate transformation of previously recalcitrant species. As the nucleic acid delivered by carbon nanotubes does not integrate into the host genome, the technique is particularly useful for high throughput transient assays, RNAi and transgene free gene editing using CRISPR technologies.
Prof June Medford, from Colorado State University, introduced the need for digital gene expression systems to deal with environmental fluctuations stressing the importance of simple mathematical modelling when creating designs.
“Plants are really, really good at calculations” - Prof. June Medford
Medford went on to present soon to be published work that aims to understand salt tolerance by building a system from scratch. Looking at mangrove trees, specially adapted to grow in brackish water, her lab characterized salt tolerance as requiring three components: (1) filters (2) transporters (3) and the ability to retain pure water. Taking a “Jurassic Park approach” June’s lab filled in gaps in the mangrove genome with information from sequenced halophytes, and managed to introduce salt tolerance into the model plant Arabidopsis.
Following on from their success, the Medford lab went on to develop a fully artificial system for desalination where plants obtain water from the sea and exude pure water that could be collected by human uses. Following growth in salt water, engineered Arabidopsis plants exuded highly purified water of a similar quality to bottled ‘Fuji water’. Medford left the audience with the challenge “Can we build better systems? Can we go from the Wright Brothers to a Boeing 747?” ending “we are better than we think”.
Prof. Sean Cutler, from the University of California Riverside, talked about his group’s efforts to produce new tools from maximizing crop productivity. The Cutler lab is pioneering efforts to manipulate water use efficiency of crops with ABA receptors and synthetic analogs to reduce crop losses from drought.
Normally there is a trade off between crop yield and water use efficiency. The Cutler lab addresses this issue by designing and discovery of molecules to modulate ABA receptors in order for farmers to manipulate water use dynamically.
Cutler first presented a decade of research into creation of ABA antagonists such as Quinabactin then went on to talk about groundbreaking work to engineer ABA receptors to be responsive to a commonly used agrochemical, opening up the possibility of manipulating water use within current agronomic practices. Looking to the future, his lab has further redesigned the receptor to decouple it from ABA signaling to create an orthogonal receptor that can be connected to any synthetic signaling pathway to be manipulated at will. The Cutler lab is currently exploring one application of this technology - accelerating tree breeding by simulating inducible flowering in citrus as part of attempts to improve resistance to emerging pathogens.
Tobias Erb, from the Max Planck Institute for Terrestrial Microbiology, Germany, presented work where his lab designed a pathway that requires fewer molecules of ATP to fix CO2 than the Calvin-Benson-Bassham Cycle (4 compared to 7).
Prof. Erb spoke about redesigning carbon fixation in plants from first principles using the process of retrosynthesis, where you start with the end product and design the synthesis around it. All designs had to be fast and pass the test of being thermodynamically feasible, work as a cycle, include a reductive carboxylation reaction, and have an output module, so carbon can be taken directly into metabolism central metabolism such as pyruvate.
The problem with most plants is the enzyme rubisco, which suffers from competitive inhibition of carboxylation by oxygen, and is often reported to be slow. The Erb lab decided to use a new carboxylase to catalyse CO2 fixation - enoyl-CoA carboxylase, which has the advantage of no side reactions with oxygen, being highly active and 4-10x more efficient than rubisco. Using this enzyme and 16 others taken from a variety of organisms they created the crotonyl–coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle.
However, when tested in vitro the first cycle was very slow, the issue turned out to be putting together enzymes that have not coevolved. In order to address this the Erb lab went on to include metabolite recycling, proofreading and enzyme redesign to dramatically improve the system and it is now reaching rates of biological relevance.
Future work includes automating and miniaturizing the system for testing new variations of the CETCH cycle using microfluidics, as well as coupling carbon fixation to photosynthesis. To date, the Erb lab has demonstrated encapulsation of thylakoids in droplets, which can produce ATP and NADPH when exposed to light, and run simple reactions, providing a model system for playing around with chloroplasts. These steps open up the way for assembly and testing of new cycles.
Erb closed by emphasizing we can use synthetic biology to come up with radical new solutions to nature’s problems. We can build minimal systems in vitro then optimize them before transferring into higher organisms.
Prof Poul Erik Jensen, from the University of Copenhagen, Denmark, presented his vision for using solar energy to drive chemical synthesis in plant chloroplasts using reducing equivalents from photosystem I. Working with a class of enzymes known as cytochrome P450s, Jensen presented a proof of concept where his group produced a plant defense compound dhurrin, which is usually synthesized in the ER using NADPH, in the chloroplast with electrons derived from photosynthetic electron transport chain.
However, competition with native processes that also use reducing equivalents, such as sulphur reduction limited enzyme activity. To overcome this challenge, his lab improved electron transport by fusing P450 directly to an electron carrier.
Vincent Martin, Co-director of the Centre for Synthetic Biology at Concordia University, presented optimizing the synthesis of medicinal compounds from plants. The work initially started with PhytoMetaSyn which aimed to identify biosynthetic genes and pathways in plants that produce interesting natural products and move them into more tractable heterologous systems such as yeast, allowing brewing which can fit into industrial production systems.
Martin focused on creating a platform yeast strain to create central intermediate to many of these compounds (S)-reticuline. This coincided with a paper published by Christina Smolke’s lab in 2015, which reported the complete synthesis of opioids in yeast – but at very low yields. Martin states the problem now is to create these compounds in large amounts. He went on to show a series of painstaking experiments described as metabolic engineering “whack-a-mole’ to lower competing side reactions, highlighting the importance of the order in which genes are deleted, to eventually increase production by up to ~38,000 fold relative their first generation strain.
In addition, a series of talks presented new tools for use in plant synthetic biology. Naomi Nakayama spoke about a variety of tools including Mobius Assembly, a golden-gate cloning system compatible with the Plant MoClo standard, a high throughput, microfluidic protoplast gene expression system for characterizing different parts, double and triple step inducible systems and cell factory lines.
Alexander Jones, a group leader at the Sainsbury Laboratory in Cambridge, UK, spoke about making new optogenetic tools to answer basic biology questions. Optogentics can provide exquisite control of gene expression and is widely used in mammalian systems, but cross-talk with endogenous systems can occur in plants. To overcome this issue, using serial redesign, his group has created a modified CcaS-CcaR ratiometric photosensor “highlighter system” to work in plants.
Jennifer Brophy, a postdoc in Jose Dinneny’s lab presented work on engineering size and shape of root systems for agronomic performance using genetic circuits. Brophy has created a new library of well characterized synthetic repressors and activators using parts from bacteria. These parts have been compiled to create Boolean NOR and NIMPLY gates opening up the possibility for complex computations that can be used to manipulate root system architecture.
Finally, Oliver Windram, a NERC research fellow at Imperial College London, presented work to transcriptionally rewire stress responses. He showed work utilizing AI to quantitatively measure subjective phenotypes, system biology approaches to pick targets for rewiring based on transcription factor network topology and synthetic biology to improve tolerance to pathogens in Arabdiopsis and is expanding this approach to crops such as wheat.