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Proteins are essentially the ‘workhorses’ of cells. They are involved in almost all major functions within the cell, from replicating DNA, to breaking down energy sources, to providing a strong and consistent cellular structure.

Some of the major families of proteins that are often subjected to engineering in synthetic biology include: 

  • Enzymes – These are essentially biological catalysts. They have a specific 3D structure which enables them to increase the rate of biological reactions. For example, the enzyme amylase is required for the breakdown of long-chain carbohydrates like starch to occur at a rapid enough rate during digestion. Modifying enzymes can enable changes in the rate of these biological reactions to be achieved. 

  • Transcription factors – These are regulators of the rate of transcription. They bind to regulatory regions of DNA and change how quickly mRNA is produced from target genes. Transcription factors can either increase or decrease the rate of transcription through a variety of different mechanisms. Typically the final level of transcription from a specific gene will be due to the combined action of several transcription factors. 

Modifying proteins typically involves the selective change of specific amino acids in the peptide sequence of the protein being altered. These changes can have a structural effect on the protein, for example by changing the shape of the active site of an enzyme. Alternatively they can alter the chemical properties of the protein, such as by introducing or neutralising local charges, which in turn affects how it interacts with other components of the cell.

RuBisCO and improving photosynthesis

Spacefilling structure of RuBisCO
One protein which has been targeted for re-engineering is Ribulose-1,5-bisphosphate carboxylase/oxygenase - or RuBisCO for short. It is the enzyme used by plants to fix carbon from CO2 in the atmosphere. It is hugely abundant and arguably the most important protein in the world for its contribution to sustaining almost all forms of plant (and by extension animal) life. 

Despite its importance, RuBisCO is far from a perfect enzyme. It is the rate limiting step for photosynthesis and when compared to other enzymes, it is relatively slow and inefficient. Another inconvenient quirk for the enzyme is that RuBisCO also sometimes fixes oxygen instead of carbon dioxide (hence the word oxygenase in the enzyme name). This oxygenation activity can have a large negative effect on the photosynthetic ability of plants.

It is clear that engineering of an improved RuBisCO, with either a faster overall rate of activity or a reduced relative oxygenation:carboxylation ratio, would have the potential for a large effect on the growth of plants expressing it. Many researchers have investigated different avenues for re-engineering RuBisCO, with limited success. Due to the difficulty in developing viable, re-engineered versions of RuBisCO with improved efficiency, some have suggested that in fact the enzyme may already be at, or close to, its evolutionary optimum, in spite of the enzyme's obvious shortcomings.

Limitations of protein modification

Attempts to modify RuBisCO highlight an important feature of protein modification, namely that it is often very difficult. There are several steps involved in producing an organism expressing a protein modified to improve a certain trait, all of which present potential problems:

  1. Identifying how to improve the protein - This means finding specific sites within the protein relevant to the function being modified and then altering them in the right way. It is often very difficult to know how to change a protein e.g. which amino acids to change and to what, in order to produce the desired effect.
  2. Ensuring normal function is not disrupted - Because different parts of the protein often interact with each other due to folding effects, change at one site can have long-ranging effects. It is important to ensure that changing a protein does not interfere with other important parts of its function, such as its interaction with other proteins or localisation to a specific part of the cell.
  3. Expressing the protein - Actually producing the protein in the organism of interest produces yet another roadblock. The genome of an organism has to be altered specifically and accurately, with appropriate expression levels. Often expressing a modified protein in an organism has unexpected effects on its overall phenotype which are hard to explain. For example, expression of an 'improved' RuBisCO in a tobacco line produced mutants with improved CO2 fixation rates but a slower growth rate.

 

What do you think?

About the author

View full profile Jake Curtis from London

I am a student at Cambridge University who has just finished a BA in Natural Sciences, focusing on Genetics in my third year. I am now studying for an MSc in Systems Biology.

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