Researchers from Australia recently reported a novel way to study a key enzyme in photosynthesis and carbon fixation. Using directed evolution, they opened the way to greater crop productivity.
Ribulose bisphosphate carboxylase/oxygenase (Rubisco) catalyses the addition of CO2 to ribulose-1-5-bisphosphate, which acts as the entry point for the majority of carbon entering the biosphere. Rubisco is also widely reported to be inefficient, which has led it to be a long standing target for modification. But despite decades of research, progress has been slow.
Rob Wilson, a postdoctoral researcher at the Max Planck Institute, explains, “we know from efforts to survey the catalytic diversity of Rubisco (kudos to Douglas Orr of Lancaster University) that variation exists out there which could be of benefit in certain photosynthetic contexts.” However, “rational design, even assisted by computers, is extremely difficult for Rubisco” says Rob. “There is a complicated network of epistasis that underpins Rubisco evolution and the complex interactions of many essential chaperones further complicates strategies which look at the final structure or primary amino acid sequence. One mutation can upset the apple cart and all of a sudden the enzyme will be totally insoluble.”
To address this challenge, Rob’s aim is to ”mutate, mass purify, and characterise Rubisco from Eukarya” using directed evolution. Until recently this would have been impossible. Mutating Rubisco in plants and algae requires chloroplast transformation, which is technically challenging and is only widely performed in Tobacco. So instead of using plant systems, Rob decided to switch to Eschericheia coli: “[it] is probably the easiest host for manipulating proteins of interest. [Chloroplast transformation is] total hell in comparison.”
Using a bacterium rather than a plant has advantages beyond speed and ease of transformation: as E. coli doesn’t have the assembly factors required for Rubisco synthesis, the process can be manipulated without interference. “Abstracting Rubisco from its photosynthetic environment allows greater control over the plasticity of evolution” says Rob, “E. coli lets us work on our understanding on how to manufacture Rubisco in the best way possible so we can put everything into a photosynthetic host, hopefully, without any unexpected chaos.”
Working with Spencer Whitney at ANU in Canberra, Australia, Rob used E. coli to evolve a better form of the Rubisco from the cyanobacterium Thermosynechococcus elongatus. In doing so they managed to identify several amino acid changes that could improve the specificity, efficiency and rate of the enzyme, disproving the claims of many critics that the enzyme must have been optimized by evolution already.
However, future engineering efforts will likely need to focus on Rubisco from higher plants. This is tricky - although cyanobacterial enzymes can be produced in plants, there are concerns over compatibility. “The key feature about eukaryotic Rubisco expression is a strict dependence on molecular chaperones for folding and assembly,” says Rob. “If you want to fiddle about with Rubisco in plants you have to bring along the factory to produce it as well.”
Therefore, even if you make a better Rubisco, it is not a straightforward process to translate these findings into crops. “You can't just make a Maserati in a BMW factory” says Rob, “even if you imported the materials and crew, no-one can understand each other, it's a mess - just as in a plant, if you have the endogenous machinery present it's not an ideal situation”.
The breakthrough came in 2017 in work co-authored by Rob that demonstrated the ability to assemble higher plant Rubiscos in E. coli. They overcame previous issues by co-expressing the enzyme with 6 chaperones that had been identified through over 20 years of careful genetic analysis. It was a leap of faith - nobody knew what the minimum number of genes were required to synthesize a higher plant Rubisco as up until that point most studies had been done in planta and the existence of additional unidentified factors couldn’t be ruled out.
Now this limitation has been surpassed, Rob is optimistic about the future, “recombinant expression of plant Rubisco in E. coli really opens the door to a whole new world of investigation which I hope will fast-track improvements to photosynthesis.”
Evolution and selective breeding has not fully optimized our crops for maximum growth, but with work like this, along with other advances in photosynthesis research, scientists are starting to take matters into their own hands.
Disclaimer: Steven is employed on the same project as Douglas Orr who was mentioned in the post and researchers who produce the publication hyperlinked in the final paragraph
Editor’s note: This article is cross-posted on the PLOS Synthetic Biology Community