By Simon Hadlington

Ready access to complex compounds will allow pharmacological studies of
potential painkillers.
Yeast cells have been turned into biological factories that manufacture a
range of alkaloids - naturally occurring chemical compounds such as morphine
that contain nitrogen atoms and that often have useful pharmaceutical
properties. The work opens the way to commercially producing previously
unobtainable and potentially valuable alkaloids.

Thousands of different alkaloids are known to exist, but only a handful of
them can be obtained in useful quantities, usually by extracting them from
plants such as the opium poppy. Alkaloids are synthesised by sequences of
biochemical reactions involving many enzymes and sophisticated regulatory
mechanisms.

Intermediate molecules that could have interesting properties are produced
in these pathways, but the complexity of these chemicals and the fact that
they they occur in tiny amounts means that extracting or synthesizing them
is difficult and expensive.

Painkiller pathway

?The obvious approach to getting more of these compounds would be to
genetically engineer plants to stop production along the pathway, so that a
particular intermediate would accumulate,? says Christina Smolke, a chemist
at the California Institute of Technology in Pasadena. ?People have tried
this but with limited success ? if you knock out one enzyme you end up
knocking out a large part of the pathway.?

Other scientists have already used yeast to produce useful compounds such as
hydrocortisone1 and the antimalarial drug precursor artemisinic acid2. Now,
Smolke and her co-worker Kristy Hawkins have successfully reconstructed ?
within a yeast cell ? many of the key elements of the elaborate pathways for
synthesising alkaloids. Their research is published in Nature Chemical
Biology3.

Hawkins and Smolke focused on the benzylisoquinoline alkaloids (BIAs), which
include the painkillers morphine and codeine. They inserted into yeast cells
genes from three plants: the opium poppy, Papaver somniferum, the common
meadow rue, Thalictrum flavum and thale cress, Arabidopsis thaliana. These
genes make enzymes that help to produce the BIAs from simpler chemical
building blocks. They also added the gene for a human enzyme, P450, which is
known to act on a range of alkaloid molecules.

High yields

By mixing and matching different enzyme combinations, the researchers were
able to create substantial amounts of seven different BIAs. ?Now that we
have access to intermediates that were not previously available, people will
want to do careful studies on their pharmacological activity,? Smolke says.
?And we were getting yields of 100 to 200 milligrams per litre, which is
respectable for potentially valuable molecules. With relatively simple
optimization of the fermentation you could obtain 10 or 100 times more than
this.?

Hawkins and Smolke also devised a way to tune the system so that the yeast
produced the optimum amount of each enzyme to synthesise whichever alkaloid
they wanted, and did not waste energy making an excess of any given enzyme.

Sarah O?Connor, an expert on the biosynthesis of natural products at the
Massachusetts Institute of Technology, Cambridge, is impressed by the work.
?It?s very exciting that plant alkaloid pathways are starting to be
reconstituted in microbes. Very importantly, Smolke has also shown how this
strain can be used to discover new enzymes that catalyse biosynthetic
transformations.?

Smolke says, ?We are now hoping to extend the pathway both ways ? to get a
broader range of intermediates downstream, including the end products, and
to be able to start with simpler substrates upstream.?

?The system will also allow us to start producing non-natural alkaloids by
using enzymes from different sources and in combinations that do not occur
in nature.?

References
1. Szczebara, F. M. et al. Nature Biotechnol. 21, 143-149 (2003).
2. Ro, D. K. et al. Nature 440, 940-943 (2006).
3. Hawkins K. M. and Smolke C.D. Nature Chem. Biol. doi:
10.1038/nchembio.105 (2008).

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