Synthetic Biology Pathways to Isoquinoline Alkaloids

For thousands of years mankind has been using plants and medicinal compounds from plants for treating pain, diseases, infections and also for recreational purposes. Many of these plant derived compounds have also found their way into modern drug use for example morphine and codeine for pain relief, atropine to increase heart rate and for irritable bowel syndrome, vinblastine to treat cancers and quinine for malaria. There are over 14,000 alkaloid chemical structures known and these represent a wealth of potential biological activity. However many of these alkaloids occur in small amounts or in plants that are rare or only grow in parts of the world that are difficult to access. The pathways that plants use to synthesise alkaloids are often long with many biosynthetic steps. There are some key intermediate compounds that are common to many plant alkaloid pathways and the complexity of alkaloid structures comes from the different later steps in the pathways that different plants have. It would be of great benefit to be able to make some of these key intermediate alkaloid compounds in a more controlled way and to be able to modify these alkaloid structures using enzymes or chemistry. In this way novel biologically active drugs could be found. The pathways for the synthesis of several alkaloids have begun to be determined but many of the steps for some of the more complex alkaloids are still unknown. In the last few years researchers have begun to isolate the enzymes (and their genes) for some of the key steps in the synthesis of some alkaloids. Some of these enzymes carry out very important steps such as the joining together of rather simple compounds derived from simple amino acids. We intend to use the techniques of whole gene synthesis, synthetic biology and pathway design to make some of these key alkaloid synthesising enzymes and put them into a laboratory bacterium. In this way we can combine different plant based and bacterial enzymes and build synthetic pathways for the creation of new chemical compounds that could be screened for useful medicinal activities. By having the pathways in bacteria we would be able to generate them in a reproducible way using a renewable resource and not be dependant on the growth of difficult or rare plants. In addition, extensive chemical transformations using methods with high environmental impact would not be required which are currently used to synthesise such isoquinolines.

We aim to construct recombinant Escherichia coli carrying synthetic engineered pathways for the production of alkaloids of the isoquinolines class. To do this we will use synthetic genes for the main coupling enzyme from the benzylisoquinoline (BIA) pathway, the norcoclaurine synthase (NCS). There are two types of NCS and these genes will be made synthetically and expressed in E. coli. The NCS joins dopamine to 4-hydroxyphenylacetaldehyde to make (S)-norcoclaurine. The substrate specificity of these two classes of wild type enzyme will be determined towards a range of aromatic amines such as substituted dopamines and tryptamines. The specificity towards a range of aldehydes including modified phenylacetaldehyes (4-OH, 3-OH, 3,4-diOH, halogen, amino etc), indoleacetaldehyde and aliphatic or cyclic aldehydes will be established. The NCS enzymes will be tested and used as stand alone enzymes for the coupling of the amine and aldehyde. Mutant NCS enzymes will be made with the ability to accept different and non-natural amines and aldehydes. The structures of the compounds formed by the native and mutant NCS enzymes will be determined using mass spectrometry and NMR spectroscopy. We will also construct the pathways which will enable E. coli to synthesise the dopamine and 4-hydroxyphenylacetaldehyde for the NCS and to combine these pathways to make strains which will make (S)-norcoclaurine de novo. Using synthetic genes for many of these feeder pathway components will allow different aromatic aldehydes to be made in vivo. E. coli expressing only one or the other pathway for the NCS substrates will allow non-natural amines or aldehydes to be fed to strains which carry pathways for the other NCS substrate and the NCS. Cytochrome P450 enzymes systems which have previously been constructed will be expressed together with NCS enzymes and the feeder pathways plus NCS. In this way we aim to build strains which can make diverse alkaloids of the isoquinoline class.