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In the course of photosynthesis, an enzyme named rubisco facilitates a crucial reaction — the combination of carbon dioxide into organic substances to generate sugars. Nonetheless, rubisco, which is thought to be the most prevalent enzyme on our planet, is quite inefficient when compared to the other enzymes engaged in the photosynthetic process.
Chemists at MIT have demonstrated that they can significantly enhance a variant of rubisco present in bacteria from a low-oxygen habitat. Through a method known as directed evolution, they discovered mutations that could elevate rubisco’s catalytic efficiency by as much as 25 percent.
The scientists now intend to implement their method on various forms of rubisco that could be utilized in plants, aiming to enhance their photosynthesis rates, potentially leading to improved crop productivity.
“This is, in my opinion, a compelling illustration of the successful enhancement of rubisco’s enzymatic characteristics, offering great promise for engineering different variations of rubisco,” states Matthew Shoulders, the Class of 1942 Chemistry Professor at MIT.
Shoulders and Robert Wilson, a research scientist in the Chemistry Department, serve as the senior authors of the recent study, which is published this week in the Proceedings of the National Academy of Sciences. MIT graduate student Julie McDonald is the principal author of the paper.
Advancement of efficacy
When plants or photosynthetic bacteria harness energy from the sun, they initially transform it into energy-retaining molecules such as ATP. In the subsequent stage of photosynthesis, cells utilize that energy to convert a molecule known as ribulose bisphosphate into glucose, which necessitates several additional reactions. Rubisco facilitates the first of those reactions, termed carboxylation. During this reaction, carbon from CO2 is added to ribulose bisphosphate.
In comparison to the other enzymes involved in photosynthesis, rubisco operates at a slow pace, catalyzing merely one to ten reactions per second. Furthermore, rubisco can engage with oxygen, leading to a competing reaction that incorporates oxygen instead of carbon — a process that squanders some of the energy captured from sunlight.
“For protein engineers, that presents a fascinating set of challenges because those traits seem likely to be improved by modifying the enzyme’s amino acid sequence,” mentions McDonald.
Earlier studies have resulted in enhancements in rubisco’s stability and solubility, leading to minor gains in enzyme efficiency. The majority of those investigations employed directed evolution — a method where a naturally occurring protein is randomly mutated and then screened for the emergence of new, advantageous characteristics.
This process is generally achieved using error-prone PCR, a technique that initially creates mutations in vitro (outside of living organisms), typically introducing just one or two mutations in the target gene. In prior investigations on rubisco, this library of mutations was subsequently introduced into bacteria that grow at a rate correlating with rubisco activity. Restrictions in error-prone PCR and in the efficiency of introducing new genes limit the total number of mutations that can be generated and evaluated using this method. Manual mutagenesis and selection processes also contribute additional time to the procedure through multiple rounds of evolution.
The MIT team, however, employed a more recent mutagenesis method that the Shoulders Lab had previously developed, called MutaT7. This approach allows the researchers to conduct both mutagenesis and screening within living cells, significantly expediting the process. Their method also permits them to mutate the target gene at an increased rate.
“Our continuous directed evolution technique enables you to examine many more mutations in the enzyme than has been previously possible,” says McDonald.
Enhanced rubisco
For this study, the researchers initiated their work with a variant of rubisco, extracted from a group of semi-anaerobic bacteria known as Gallionellaceae, which is one of the fastest rubisco variants found in nature. During the directed evolution experiments conducted in E. coli, the researchers maintained the microbes in an environment with normal oxygen levels, creating evolutionary pressure to adapt to oxygen.
After six iterations of directed evolution, the researchers pinpointed three distinct mutations that enhanced rubisco’s resistance to oxygen. Each of these mutations is situated near the enzyme’s active site (where it performs carboxylation or oxygenation). The researchers are confident that these mutations enhance the enzyme’s ability to preferentially interact with carbon dioxide over oxygen, resulting in an overall rise in carboxylation efficiency.
“The fundamental question here is: Can you modify and enhance the kinetic properties of rubisco to function better in the environments where optimal performance is desired?” Shoulders queries. “What changed through the directed evolution process was that rubisco became less inclined to react with oxygen. This adaptation allows rubisco to perform effectively in an oxygen-rich context, where it would typically be distracted and react with oxygen, which is undesirable.”
In their ongoing research, the scientists are applying this methodology to other forms of rubisco, including those derived from plants. It is thought that plants lose approximately 30 percent of the solar energy they capture through a phenomenon called photorespiration, which occurs when rubisco interacts with oxygen instead of carbon dioxide.
“This genuinely paves the way for numerous exciting new inquiries, representing a step beyond the engineering approaches that have previously dominated rubisco engineering,” indicates Wilson. “There are clear advantages to agricultural productivity that could be harnessed through a more effective rubisco.”
The study received funding, in part, from the National Science Foundation, the National Institutes of Health, an Abdul Latif Jameel Water and Food Systems Lab Grand Challenge grant, and a Martin Family Society Fellowship for Sustainability.
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