Fixing CO2 fixation

How biochemistry students can become multi-millionaires by making plants more efficient. Has someone finally succeeded?

Living organisms need carbon to grow and divide. Many get their carbon atoms from organic molecules such as glucose or acetate that have been synthesized in other species.

Most organisms can fix carbon directly from carbon dioxide by a variety of different reactions but this isn't necessarily the primary source of carbon atoms. (We can fix carbon using pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and phosphoenolpyruvate carboxykinase (PEPCK) among others.)

Some organism can get most of their carbon from inorganic carbon dioxide. As you might imagine, this is quite common for single-cell organisms living in the ocean where complex organic molecules aren't very abundant. One of the most common pathways is called the Calvin Cycle after Melvin Calvin (1911-1997) who won the Nobel Prize in Chemistry in 1961 "for his research on the carbon dioxide assimilation in plants."

The Calvin Cycle is quite complicated but that doesn't concern us here [The Calvin Cycle] [The Calvin Cycle: Regeneration]. It's the preferred pathway for fixing CO₂ in photosynthetic species because it requires a great deal of energy. The pathway is also used in non-photosynthetic species [Carbon Dioxide Fixation in the Dark Ocean].

(Incidentally, photosynthesis is often defined as a carbon dioxide fixation process but that's not correct [The Photosynthesis Song and a Pet Peeve].)

The most important enzyme in the Cavlin Cycle pathway is the very first one. It's called ribulose 1,5-bisphosphate carboxylase-oxygenase. That's quite a mouthful so it's usually shortened to "Rubisco." The enzyme takes a five-carbon sugar (ribulose 1,5-bisphosphate) and attaches carbon dioxide. It then splits the six-carbon product into two three-carbon compounds that can be used in a variety of metabolic reactions.

The reaction is complicated and so is the enzyme [Fixing Carbon: the Rubisco Reaction ] [Fixing Carbon: the Structure of Rubisco]. Here's a somewhat more detailed diagram of the reaction. (Yes, this will be on the exam! ¹)

Rubisco is a really bad enzyme. This is a difficult concept for most people because they are used to thinking that everything in the cell has evolved to perfection. Stop thinking like that! [Better Biochemistry: The Perfect Enzyme] That's not how evolution works.

Rubisco is terribly inefficient at doing its basic reaction— it only catalyzes about three reactions per second. It also gets easily confused. About 25% of the time it fixes oxygen instead of carbon dioxide in a reaction called "oxygenation" (hence, the name of the enzyme). It seems strange that after two billion years of evolution no species has evolved a more efficient enzyme but none has been found. This is why organisms contain huge amounts of Rubisco. It makes up about 50% of the soluble protein in plant leaves making it the most abundant enzyme on the planet.

It's abundant because it's a bad enzyme and you need a lot of it to do the job.

You could make bundles of money by being an intelligent designer and building a better Rubisco [Fixing Carbon: Building a Better Rubisco ]. You can bet your life that many companies have tried. All of them have failed so far.²

Photosynthetic organisms may have given up on trying to make a better enzyme but they've come up with a number of other solutions to improve efficiency. One of the solutions is to sequester Rubisco in cells that aren't exposed to the air (and oxygen) then transfer CO₂ specifically to the interior cells where Rubisco is active. This is called the C₄ pathway because the intermediates are four-carbon compounds. Corn (maize), sorghum, sugarcane, and many weeds use the C₄ pathway.

Another solution is employed by succulent plants like cactus that only fix carbon at night. They also have ways of concentrating CO₂ via four-carbon intermediates. The process is called "Crassulacean acid metabolism" (CAM).³

The strategy that concerns us today is one employed by cyanobacteria. They have special internal compartments called carboxysomes where Rubisco is concentrated along with carbonic anhydrase. There's a protein coat around the carboxysome that's impermeable to oxygen so the enzyme doesn't get confused. The coat also blocks carbon dioxide gas but dissolved carbon dioxide in the form of bicarbonate (HCO₃^⊖) can enter. Carbonic anhydrase converts bicarbonate to CO₂ so Rubisco is active in an environment that's rich in CO₂ and deficient in O₂. Pretty clever, eh?

Lin et al. (2014) know their biochemistry so they figured they could use this knowledge to improve crop plants. They picked tobacco—but not for the reasons you think. It's just easier to manipulate tobacco. Here's the abstract of their paper. It says all you need to know to appreciate what they have done.

In photosynthetic organisms, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO₂ into the biosphere1. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO₂-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco’s assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins.

There's still a little work to be done—plant "a" in the figure is wild-type and plants "b" and "c" are the transgenic plants all grown for 6 weeks.

I wonder if it will work in some flowering herbs, like Cannabis sativa?

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1. Just kidding. I would never ask students to memorize useless information.

2. The gods couldn't do it either!

3. Not to be confuse with Complementary and Alternative Medicine [Faculty of Medicine at the University of Toronto supports quackery].

Lin, M.T., Occhialini, A., Andralojc, P.J., Parry, M.A.J., and Hanson, M.R. (2014) A faster Rubisco with potential to increase photosynthesis in crops. Nature 513:547-550. [doi: 10.1038/nature13776]