PCB Biosynthesis in Yeast
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What is PCB and where do you get it? One of the most technically difficult and time consuming aspects of this project was extracting phycocyanobilin from Spirulina algae. Functional Phytochrome B is not just a peptide, it requires the covalent attachment of a prosthetic group. Normally, plants and algae produce this prosthetic group, phycocyanobilin, or PCB, which attaches to the PhyB auto-catalytically. However, since yeast cells don’t normally express Phytochrome B, they also do not have the enzymes required to create PCB. Thus PCB had to be added to cultures for PhyB to function.
PCB is light sensitive, so its extraction required hours of reflux with heat in the dark in a hood, a condition very difficult to achieve—in our facilities there are no darkrooms with chemical fume hoods. Hence, we had to construct a darkroom, lit only with a green safelight that would not induce the breakdown of the PCB. In addition, extraction of the PCB required the use of numerous toxic chemicals including mercuric chloride, which requires special disposal and is highly toxic. In short, the PCB extraction protocol was inefficient and environmentally unfriendly.
Why pursue PCB biosynthesis? Because of the difficulty of extraction, we decided to insert into yeast the genes used by Spirulina to make PCB to allow the yeast to produce PCB themselves. This PCB biosynthesis by yeast would not only be more efficient, removing the need to do any further PCB extractions, but also be more environmentally friendly as it would not necessitate the use of any more mercuric chloride. PCB is a derivative of heme, a prosthetic group made in almost all cells. Heme is most commonly known as being the prosthetic group bound to hemoglobin which allows it to bind oxygen. There are only two enzymes in the PCB biosynthesis pathway from heme, hence making this pathway an ideal candidate for insertion in to yeast. Such reconstitution of biosynthetic pathways from one species in another is one of the major goals of synthetic biology, as it would theoretically allow for easy production of valuable biological small molecules.
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PCB Plasmid for insertion into yeast. To allow yeast to express the PCB biosynthesis genes, we inserted the two genes, HO1 and PcyA, into a yeast dual expression vector, both under the same promoter. This would allow us to express both genes from the same plasmid, and in equimolar amounts. This plasmid remains to be tested.
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