Tricksters, Toolsters, Tinkerers, and Traders
A roundup of PlantSynbio19 by Steven Burgess
Aug 7-9, San Jose, California – the American Society for Plant Biology (ASPB) showcased the latest research in Plant Synthetic Biology. With topics ranging from modifying plants for desalination to reimagining carbon fixation.
Plants provide food, fuel and medicinal compounds for society. Cultivation and extraction processes are well established but are under strain from environmental changes and population growth. In a series of visionary presentations leading members of the plant synbio community presented ways of teaching plants new tricks to address societal challenges.
Prof June Medford, from Colorado State University, introduced salt tolerance into the model plant Arabidopsis as part of a learn-by-building approach that is currently under review. 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 and was able to generate plants which survived saline treatment. Expanding on this work, her lab went on to develop a ‘new-to-nature’ system for desalination, where plants grown on brine exude pure water through their leaves of a similar quality to bottled ‘Fuji water’.
Prof. Sean Cutler, from the University of California Riverside, talked about his group’s efforts to produce new tools from maximizing crop productivity. Normally there is a trade off between crop yield and water use efficiency: the more water efficient you make a plant the slower it grows as you restrict its ability to photosynthesize. 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. This technology is being explored as a means of accelerating citrus breeding as part of attempts to improve resistance to emerging pathogens.
The Plant Synthetic Biology community has seen progress in the development of tools to manipulate signalling pathways and gene expression. However, the number and variety of tools available to plant researchers currently lags behind those working in microbes. The need for new systems is being tackled by researchers at the forefront of tool development.
Give Me Some Mo’ Tools
Plant parts can be prone to a number of issues, including silencing, cross-talk with endogenous systems and failure when transferring plant or viral-derived promoters and terminators between species. A potential way of overcoming these problems is to use synthetic promoter and repressor libraries. Therefore the lab of Patrick Shi, Director of plant biosystem design at JBEI and assistant professor at UC Davis, has made a library of synthetic parts using cis-elements from eukaryotes, and Jennifer Brophy, a postdoc in Jose Dinneny’s lab, presented libraries with parts dervied from bacteria. Shi is using his tools in a ‘plug-and-play’ approach to design new metabolic pathways and has been able to produce a new antifungal compound that works with a similar efficacy to current agrochemicals. Whereas Brophy has combined her parts to create Boolean NOR and NIMPLY gates that can be used to manipulate root system architecture to improve agronomic performance.
Faster, Better, Phenotyping
As well as the need for tools, plant synthetic biology suffers from a slow test phase in the ‘design-build-test-learn’ cycle. Compared to microbes, current plant growth and transformation timescales are long and low throughput. It can take in the order of 6-12 months to generate stable transgenic lines of commonly used crops compared to the hour to day timescale for microbes. However, there is hope that classic methods used in plant research can be modified to facilitate high-throughput phenotyping. Three strategies are being pursued to address this problem.
The first is to modify plant tissues so they can be handled like microbes. In this approach, tissues are broken down into individual cells known as protoplasts by enzymatic digestion. Protoplasts can then be transformed with construct libraries followed by phenotyping. However there are no commonly used protocols for high-throughput handling and phenotyping of protoplasts. Ben Pouvreau, a postdoctoral researcher the CSIRO Future Science Platform, presented a FACS screen of construct libraries in protoplasts coupled to single cell sequencing to identify the best performing plasmids, and Naomi Nakayama’s lab has been developing a microfludic platform for analysis of protoplasts subjected to mechanical forces, potentially allowing researchers to test the performance of tens of thousands of designs at a time.
Protoplasts require preparation and are fragile making them tricky to handle. Prof. Tobias Erb, from the Max Planck Institute for Terrestrial Microbiology, Germany, presented an alternative method which involves building minimal systems in vitro which can be optimized before transferring into higher organisms. Using microfludics Erb’s lab analyzes the performance of combinations of enzymes as is commonly done elsewhere. But the ultimate aim is to power these reactions using the energy from sunlight. Therefore his lab has isolated and encapsulated photosynthetic membranes, known as thylakoids in lipid droplets. Erb demonstrated the thylakoids can produce ATP and NADPH when exposed to light, and run simple reactions, providing a model system for manipulating chloroplasts.
Some applications, much as synthetic morphology to influence agronomic traits, require maintenance of plant 3D structure which precludes the use of single cells or in vitro systems. Arjun Khakhar, a postdoc in Dan Voytas lab at the University of Minnesota, presented a system for rapid transcriptional reprogramming in plants called VipariNama (from the Sanskrit “to change”). However, a number of limitations of viral vectors, such as a limit on the size of cargo that can be delivered has restricted their widespread use. Therefore Khakhar has introduced several improvements including incorporating movement enhancer sequences (FT protein from Arabidopsis) to try and overcome patchiness of expression, co-delivering synthetic satellite viruses to reduced suppression of constructs and using an ensemble of vectors instead of a single large construct, to alleviate the need for generation of new stable lines to test each effector.
Several speakers talked about introducing alternatives to the Calvin-Benson-Bassham cycle into plants to improve the efficiency of carbon-fixation. 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. When rubisco accepts O2 rather than CO2 it produces a toxic byproduct that is recycled through a set of reactions known as photorespiration. Reducing photorespiratory energy losses of only 5% could result in a 500 million dollar annual savings in US agriculture by ‘expanding the pie’ of carbon available to crops, boosting yields.
Tobias Erb, presented a synthetic pathway that requires fewer molecules of ATP to fix CO2 than the Calvin-Benson-Bassham Cycle (4 compared to 7). Starting from first principles, the Erb lab used the process of retrosynthesis, which starts with the end product before working out which reactions are required to synthesize it. They 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.
A big challenge moving forward will be to translate plant synbio research from the lab into the field. It is widely understood that findings from small scale experiments under controlled environments cannot always be replicated when plants are exposed to environmental fluctuations. Paul South, assistant professor at Louisiana State University, has been addressing this by testing an analogous approach to reduce photorespiration in the model plant Tobacco. South tested three alternative photorespiratory (AP) bypasses, one of which (AP3) resulted in a ~20% increase in dry weight biomass in greenhouse trials. AP3 was taken forwarded for field experiments and showed a similar increase in dry weight biomass compared to control plants. South went on to show preliminary data where he has been translating his alternative bypass into potato, early indications suggest suppressing photorespiration may translate into increased tuber number and work is ongoing to verify these findings in field trials.
As well as increasing the amount of carbon fixed, researchers are focusing on how to use this more efficiently. Prof, Andrew Hanson, from the University of Florida, presented a discussion on improving the efficiency of carbon utilization in plants based on a review of this topic. Respiration consumes about 50% of carbon fixed by plants and a significant fraction of this is the result of energy expended from protein turnover. Most enzyme turnover is the result of wear and tear and they can be reimagined as non-robust parts that can be improved.
Hanson outlined his vision to build and install more efficient enzymes by rational redesign or directed evolution giving the example of THI4, a suicide enzyme, the turnover of which is responsible about 2-12% of respiration.
Plants as production factories
There is also interest in using plants to produce bioproducts either directly or as co-products to make processes economically viable. In addition to tools, Shi presented his group’s work on manipulating metabolism to accumulate co-products in plants, with the aim of making biomass competitive with petroleum based fuel. The DOE has set a target price of $2.50 per gallon of ethanol produced from plant biomass. But current production processes are still far too expensive. Therefore efforts have turned to simultaneously producing valuable compounds that can be used to make the process economically viable. Shi presented a model to help guide engineering efforts, produced with researchers at JBEI, which provides target concentration for any bioproduct to be produced along with biomass in order to reach economic viability.
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.
Producing plant derived compounds in microbes
Sometimes producing and extracting compounds of interest from plants can be difficult: often target molecules are produced by slow growing species, for which there is no genetic transformation protocol, and in low amounts. An alternative approach is to move biosynthetic pathways out of plants and in to yeast or bacteria to boost yields and manipulate compounds to produce a wide range of analogs. As a result, a number of talks merged classic plant biochemistry with conventional synthetic biology.
Matt Mattozzi from Conagen provided an industry perspective on the production of food additives (for more see his interview Plantae). Reliably sourcing natural flavors can be a big challenge as the result of batch effects, difficulties in extraction and global events such as droughts and flooding. But by starting from raw ingredients it can be possible to synthesize additives in yeast. Elaborating on the production of RebM BESTEVIA, one of thirty products made by Conagen, Matt described the process of bioprospecting, high-throughput cloning, automation and screening, to identify enzymes for the creation of new sweeteners with improved taste profiles.
Another area of active research is the production of medicinal compounds. Vincent Martin, Co-director of the Centre for Synthetic Biology at Concordia University, presented work to create a platform yeast strain to create (S)-reticuline, a key intermediate in opioid synthesis pathway. His work 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. The problem now is to create these compounds in large amounts. Martin 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. Following this process Martin’s group has managed to increase production by ~38,000 fold relative to their first generation strain.
One of the fastest growing areas of industrial interest is the production of biologicals (microbes) for use in agriculture. By focusing on microbes rather than plants, researchers are able to rapidly engineer strains with the potential to quickly reach the market. Today the majority of corn grown in the US Midwest comes with a seed treatment that includes performance inducing microbes. However, current biologicals tend to be sourced using conventional means restricting their use. John Margolis, CSO at JoynBio talked about using highly characterized plant colonizing microbes as chassis to create a new generation of biologicals focusing on crop nutrition, protection and quality. Margolis touched upon active programs at Joyn, focusing on nitrogen fixation and crop protection, involving bioprospecting for new enzymes which are then further improved by protein engineering in a genome foundry before testing on plants.
Continuing with the theme, Karsten Temme, CEO at Pivot Bio, presented a vision for the sustainable fertilization of crops (an extended interview can be seen here). Leaching of fertilizer during torrential downpours can result in pollution of waterways and a loss in yield due to nutrient deprivation. Pivot Bio seeks to address this issue by reawakening the ability of root microbes to fix nitrogen in the presence of fertilizer and provide a consistent supply of nitrogen throughout the growing season. By applying ‘shovel-omics’ Temme estimates Pivot Bio’s microbes can produce anything from 25-35% of crop needs, reducing the amount of fertilizer needed and helping to reduce the carbon footprint of farming. 2019 is the first year of Pivot Bio PROVEN is being used across the Midwest and the company now plans to scale up production and tap into new markets.
Steven Burgess is a postdoctoral researcher at the University of Illinois at Urbana-Champaign. He works for the Realizing Increased Photosynthetic Efficiency (RIPE) project and is coordinating the Plant Synthetic Biology Network on the American Society for Plant Biology website Plantae. You can follow him on twitter @sjb015
Banner Image: San Jose skyline at night. Photo by Ben Loomis (CC BY 2.0)
The ASPB Synthetic Biology was really great, lots of excellent discussion about the prospects of implementing syn bio strategies in crops.