Aug 8, San Jose California – day two of the Plant Synthetic Biology Meeting invited speakers presented work with an emphasis on metabolic engineering and the necessary tool development.
Tools
Patrick Shi, Director of plant biosystem design at JBEI and assistant professor at UC Davis, 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. However, current production processes are still far too expensive, so 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.
In addition to choosing the right molecule, commercial production will require well-characterized parts to predictably modulate metabolism. Shi presented jSTACK hierarchal assembly, that his lab has been using for the construction of multigene constructs, and synthetic promoter and repressor libraries he has created using cis-elements from eukaryotes to provide precise control and avoid issues of silencing that can be encountered with native plant parts.
Shi went on to talk about using these tools in a ‘plug-and-play’ approach to designing new metabolic pathways. As a proof of principle, he has started with a class of plant defense compounds related to Brassinin which is produced by Brassicas, where he was able to produce a new antifungal compound that works with similar efficacy to current agrochemicals.
Ben Pouvreau, a postdoctoral researcher on the CSIRO Future Science Platform, presented a method for high-throughput that could potentially test thousands of constructs at a time. The approach involves isolating protoplasts and transforming with a library of elements, selecting the best performing cells with FACS, then using single cell sequencing to identify which were the best constructs. He presented a proof of principle experiment to increase lipid production in leaves and is looking for collaborators to utilize this technology.
Radin Sadre, a Senior Research Associate at Michigan State University, on using synthetic lipid droplets for production and accumulation of high-value compounds in leaves and Sally Anne Buck, a PhD student at ANU, Canberra, who has been investigating the use of bacterial chaperones to overcome current problems with folding and expand the range of heterologous Rubisco isoforms that can be functionally produced in chloroplasts to improve photosynthesis.
Metabolic Engineering
Paul South, assistant professor at Louisiana State University, presented work on improving photosynthesis under field conditions. Previous work has suggested a reduction of photorespiratory energy losses of only 5% could result in a 500 million dollar saving in US agriculture. South tested three alternative photorespiratory (AP) bypasses, in Tobacco, one of which (AP3) resulted in an ~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 increase tuber number and work is ongoing to verify these findings in field trials.
Continuing the theme of reducing photorespiration, Nathan Wlison, from the Sederoff lab at NC State University, presented work to engineer a reverse TCA cycle in Camelina as an alternate carbon fixation pathway. Marc Sven-Roell, from the Weber Lab, HHU, Dusseldorf, Germany, talked about engineering CO2 neutral photorespiratuion by introducing a new pathway to generate 1C intermediates using formate. Formate is also the starting molecule for many alternate photorespiratory pathways, but has the potential to produce formaldehyde as a toxic side product. Jenelle Patterson, presented work to address this, where she has identified an enzyme, known as pepP, involved in formaldehyde tolerance in microbes through bioprospecting.
Looking towards producing bioproducts, Mattheos Koffas, Professor at Rensselaer Polytechnic Institute, talked about development of an E. coliplatform for production of flavonoids, derived from plants. Koffas uses E. coli to produce anthocyanins as replacements for artificial food colors, anthocyanins are target molecules because they are water soluble and can give a wide range of colors. Koffas has been using stoichiometric-based modeling to predict fluxes within the cell to identify targets for genetic modification with the OptForce algorithm, and his group has been able to boost anthocyanin yields through overexpression and gene knockout.
In addition, Stacie Kim, from the Sattely Lab at Stanford, presented work to develop N. benthamiana as a screening platform for investigation of lignin synthesis, compounds derived from phenylalanine and can have nutritional and medicinal benefits, and Iris Ngo, from the University of Calgary, talked about reconstituting the pathway for synthesis Salvinorin A from plants in microbes. Current methods of extracting this compound from plants and chemical synthesis are difficult so a yeast based system could be used to boost yields and manipulate the compound to produce a wide range of analogs.
Finally, 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 ware 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. Hanson showed parts prospecting has identified versions which are non-suicidal, from organisms that grow in anaerobic or low oxygen environments. Looking to the future he envisions using directed evolution, with systems such as EvolvR in E. coli could to create versions which function in oxygen and could be used in plants.