Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Engineering Poplar for Production of Co-Products Muconic Acid and 2-Pyrone-4,6-Dicarboxylic Acid

Authors:

Nidhi Dwivedi1,2* (ndwivedi@bnl.gov), Yunjun Zhao1,  Shuncang Zhang1, Henrik Scheller2, Aymerick Eudes2, Chang-Jun Liu1,2, Jay Keasling2

Institutions:

1Biology Department, Brookhaven National Laboratory; 2Feedstock Division, Joint Bioenergy Institute

Goals

Designing and testing metabolic engineering strategies for production of the value-added co-products muconic acid (MA) and 2-pyrone-4,6-dicarboxylic acid (PDC) in woody biomass of bioenergy crop poplar.

  1. Identifying key enzymes that can be manipulated to divert metabolic flux towards the production of MA and PDC.
  2. Establishing expression cassettes with stacking of multiple genes involved in MA or PDC biosynthesis and transforming them into poplar.
  3. Analyzing transgenic plants for production of the desired MA and
  4. Observing the effects of pathway engineering on plant growth and development, and woody biomass processability.

Abstract

Increasing fossil fuel consumption, and concerns about climate change and energy security have been driving the worldwide revolution towards more sustainable and renewable energy sources derived from biomass, agricultural crops, and waste materials. However, the high processing cost of biofuel production hampers its application. Production of high-value co-products in biomass has been regarded as one of the solutions to reduce the overall cost of biofuel in biorefinery (Yang et al 2020). In this project, researchers tested the possibility in redirecting the shikimate pathway metabolic flux towards the production of valuable chemicals Muonic acid (MA) and 2-Pyrone-4,6-dicarboxylic acid (PDC) through systematic metabolic engineering in the bioenergy crop poplar. MA services as a platform chemical for the manufacture of functional resins, bio-plastics, food additives, agrochemicals, and pharmaceuticals, and for generation of bulk chemicals like adipic acid, terephthalic acid and trimellitic acid (Eudes et al 2018). PDC is utilized for manufacturing the performance-advantaged biodegradable polymers with strong adhesive properties, high elasticity, and rigidity (Lin et al 2021). For MA production, the salicylic acid pool derived from chorismate via the shikimate pathway is converted to catechol and MA by the sequential activities of bacterial salicylate hydroxylase (NahG) and catechol 1, 2-dioxygenase (CatA). Multiple genes (AroG*, Irp9, NahG, and CatA), driven by the xylem preferential

expression promoters, were stacked and transformed into Populus tremula x P. alba via agrobacterium-mediated gene transformation. Transgenic lines grew similar to the vector control plants and did not show detrimental growth defects. LC-MS analysis of stem samples showed a three-times higher accumulation level of catechol in transgenic lines than in the control plants, and up  to  9  ug/g  dry  weight  MA  production.  For  PDC  production,  five  genes AroG, QsuB, PmdA, PmdB, and PmdC were stacked on an expression cassette directed into plastid and transformed in hybrid poplar. The transgenic plants exhibited strong growth defects, compared to the wild type plants under normal conditions. LC-MS analysis of soluble phenolics in the leaves of transgenic plants revealed a substantial increase in the accumulation level of protocatechuate (PCA), an intermediate for production of PDC, showing 40 to 100 times higher levels compared to the wild-type plants. These results demonstrate the potential of engineering the production of value-added chemicals in poplar, highlighting the importance of genetic manipulation and optimization of metabolic pathways for biomass-based biorefinery applications. These findings also pave the way for further research and development in utilizing plant-based production of sustainable and renewable alternatives to petroleum-derived chemicals.

References

Eudes, A., et al. 2018. “Production of Muconic Acid in Plants,” Metabolic Engineering 46, 13–19.

Lin, A., 2021. “In-Planta Production of the Biodegradable Polyester Precursor 2-Pyrone-4,6-Dicarboxylic Acid (PDC): Stacking Reduced Biomass Recalcitrance with Value-Added Co-Product,” Metabolic Engineering 66, 148–56.

Yang, M., et al. 2020. “Accumulation of High-Value Bioproducts in Planta Can Improve the Economics of Advanced Biofuels,” The Proceedings of the National Academy of Sciences 117(15) 8639–48.

Funding Information

This work was supported by the Joint BioEnergy Institute, one of the Bioenergy Research Centers of the U.S. DOE, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. DOE.