Plant Metabolic Engineering Using Synthetic Enzymes Targets Increased Crop Yields

In a recent study published in the journal PNAS, by Devin Trudeau, PhD, and colleagues from the Max Planck Institute of Molecular Plant Physiology and Terrestrial Microbiology and the Weizmann Institute of Science, a synthetic plant metabolic pathway was constructed to increase carbon fixation efficiency. The group used enzyme and metabolic engineering to establish synthetic routes that bypass the natural and inefficient metabolic pathway of photorespiration. Twist gene fragments were featured in the  construction and testing of novel enzymes in vitro as a proof-of-concept for the potential use of a new-to-nature metabolic pathway in plants.

In order to feed the world of the future, we need better crop yields. Researchers are thinking out-of-the-box and testing out synthetic biology techniques to improve a plant’s light-converting abilities. At an average conversion rate of 3 to 6%, photosynthesis is actually an extremely inefficient process for converting sunlight into biomass. One reason for this inefficiency is a wasteful metabolic pathway called photorespiration.

The process of photorespiration occurs in 85% of plant species including rice, soybeans, wheat, and all trees. It’s estimated that this wasteful pathway decreases U.S. soybean and wheat yields by 36% and 20%, respectively. If photorespiration was eliminated in these plants, crop yields could dramatically improve.

Photorespiration begins in the second half of photosynthesis, when the carbon-fixing enzyme RuBisCO binds to an O2 molecule rather than CO2. Carbon fixation is the process by which inorganic carbon is added to an organic molecule. Photorespiration happens about 25% of the time, as RuBisCO has a higher affinity for CO2, but will depend on the concentrations of CO2 and O2 in the plant. RuBisCO is then oxygenated by O2and the product, glycolate-2P, must go through a long line of chemical reactions to be able to enter the Calvin Cycle, where additional chemical reactions take place, and complete carbon fixation.  

Simplified photorespiration and Calvin Cycle: When RuBisCO picks up an O2 molecule instead of CO2, it is oxygenated into the form glycolate-2P (denoted in the figure as 2-PGlycolate), which must go through photorespiration, losing CO2 and energy, to be converted back into RuBisCO and enter the Calvin Cycle. (Source: Wikicommons. Image licensed under CC BY-SA 3.0)

Not only does it take a lot of energy to achieve photorespiration, but it also releases CO2 in the process, making the entire carbon fixation reaction less efficient. In contrast, if RuBisCO had picked up CO2 instead, the molecules would immediately enter into the Calvin Cycle, avoiding energy dissipating reactions, and result in a much more efficient pathway to carbon fixation.

RuBisCO naturally has a low affinity for O2 and engineering the enzyme is a highly complex challenge. Instead, researchers are trying to create alternative chemical pathways for the oxygenation products of RuBisCO to enter the Calvin Cycle without going through photorespiration.

In a recent study published in the journal PNAS, by Devin Trudeau, PhD, and colleagues from the Max Planck Institute of Molecular Plant Physiology and Terrestrial Microbiology and the Weizmann Institute of Science, entitled, “Design and in vitro realization of carbon-conserving photorespiration,” a novel synthetic pathway to bypass photorespiration was developed and tested using synthetic gene fragments from Twist Bioscience.

Several photorespiration bypasses have been suggested in the past, but were limited to existing natural enzymes and pathways that still released CO2. The researchers took a novel approach to the challenge of establishing synthetic routes for bypassing photorespiration using the power of enzyme and metabolic engineering. They first used computational tools, thermodynamic analyses, and a comprehensive literature review to try and determine a novel photorespiration bypass pathway.

The group was able to identify several potential carbon-neutral bypass pathways using this method, and found that all of them relied on the reduction of an intermediate molecule called glycolate to glycolaldehyde. The conversion of glycolate to glycolaldehyde is not a reaction found in nature, and required two reactions using novel enzymes. The first reaction converts glycolate to glycol-CoA, and the second converts glycol-CoA to glycolaldehyde:

The researchers first set out to engineer a novel enzyme - glycol-CoA synthetase - for glycolate conversion to glycol-CoA. They started with an enzyme naturally found in E. coli called acetyl-CoA synthetase (EcACS), which converts a molecule similar to glycolate, acetate, to acetyl-CoA and has the potential to also accept glycolate as a substrate. The researchers tested this in vitro, and found that EcACS was in fact able to convert glycolate to glycol-CoA, although at a very low efficiency. To achieve a higher efficiency, the researchers used enzyme engineering methods to predict novel enzymes, starting with several computational designs.

Natural enzymes are inherently unstable. Additionally, when mutations are made in an enzyme sequence, the majority will have a destabilizing effect. Combining these two factors causes significant loss of diversity in the enzyme screening process, as many of the engineered enzymes will fall apart at functional temperatures. To circumvent this issue, the researchers first applied Protein Repair One-Stop Shop (PROSS), a protein stability design algorithm, to acetyl-CoA synthase in order to establish a similar, but more stable enzyme for further engineering. This allowed the researchers’ target enzyme to potentially accommodate significantly more potentially useful mutations than would be possible with natural enzymes and more functional diversity could be screened.

Using PROSS, a variant of EcACS with 57 mutations that the researchers called ACSsta, it was determined that ACSstab was, as predicted, significantly more stable than natural acetyl-CoA synthases. Starting with  ACSstab, the researchers then used another computational algorithm to find variants predicted to have high catalytic activity for converting glycolate to glycol-CoA. ACS19, a variant with 2 amino acid mutations in the substrate binding pocket and 3 adjacent mutations was discovered and shown to have a 16-fold higher activity of glycolate over acetate as a substrate as compared to EcACS.

With the success of establishing an effective glycol-CoA synthetase, the researchers moved on to engineer a novel glycol-CoA reductase, which would convert glycol-CoA into glycolaldehyde. Using a similar approach to the engineering of glycol-CoA synthetase, they started with the natural bacterial enzyme propanediol utilization protein (PduP), which reduces propionyl-CoA to propionaldehyde and has low activity for reducing acetyl-CoA and glycol-CoA. Instead of using computational methods to predict the activity of PduP variants, the researchers manually created mutational libraries, only changing amino acids within the substrate binding site and testing the resulting enzyme variants on their acetyl-CoA and glycol-CoA reducing activity in vitro. One variant, called GCR, exhibited a ∼12-fold improved selectivity for glycolyl-CoA over acetyl-CoA compared with natural PduP and had preferential use of the co-factor NADPH over NADH, an important distinction within this pathway.

The next step was to test the combination of the newly engineered glycol-CoA synthetase (ACS19) and glycol-CoA reductase (GCR) to see if the two enzymes could complete the desired reaction of converting glycolate to glycolaldehyde. The researchers tested this combination, as well as combinations with the non-engineered versions of the enzymes for comparison. Indeed, the combination of ACS19 and GCR with NADPH as a co-factor successfully and selectively converted glycolate to glycolaldehyde, in vitro, establishing a completely novel glycolate reduction pathway.

Finally, the researchers were able to use their engineered enzymes ACS19 and GCR within a complete photorespiration bypass reaction with the addition of several natural enzymes from various bacterium. The complete reaction for the bypass starts with the oxygenation product of RuBisCO, glycolate-2P, and converts it back into RuBisCO without losing CO2. Using a total of 7 enzymes, 2 of which were the products of enzyme engineering, they were able to show that a new-to-nature bypass to photorespiration was possible. In vitro, the researchers successfully converted glycolate-2P to RuBisCO without the loss of CO2, a proof-of-concept that in practice, could offer improvements in some of the ways we grow our food. Due to recent developments like this, the promotion of sustainable agriculture is creating an exciting path toward in being able to not only meet society’s food needs in the future, but also doing so in conscientious and environmentally friendly way.

Featured image: Soybeans (Adobe Stock)