Poppies without opiates

Addictive drugs have been a scourge of civilizations for millenia. When drugs become available to citizens they face a seemingly easy choice: work hard or get high. The economy slows down, social systems fall apart and the whole society collapses because nobody is working. Governments have to step in and take action or their state fails. This has happened countless times throughout history, yet the problem remains. This issue has been attacked from many angles, from production and distribution of drugs to discouragement of their use through legislation, treatment of addicts to stop abuse, and even improvement of the lives of those who might fall victim to their allure.

Poppies are plants that produce opiates, which are natural, very addictive drugs. Opiates are used to make heroin and other narcotics. These plants are grown in troubled regions around the world due to their high profit potential, usually by criminal organizations that exploit local populations. However, imagine if a breed of poppy plants was available that did not produce opiates. Seeds from this hybrid could then be spread throughout the world, eventually replacing current opiate producing varieties. This can be accomplished with technology available today, similar to how herbicide resistant crop plants were developed.

One of the goals of Apimba is to use the tools of molecular biology to help deal with social problems, therefore the overall purpose of this project is to develop a variety of poppy plant that looks exactly like the wild type plant, but does not produce any opiates. Of course opiates could still be synthesized, but that would require large facilities and resources, which are easier for government level authorities to control.

Development of a new breed of poppy that does not produce opiates can be accomplished in a variety of ways using molecular biology:

  1. Stopping one of the early steps of opiate biosynthesis.
  2. Inhibiting one of the early steps of opiate biosynthesis.
  3. Diverting one of the early steps of opiate biosynthesis.
  4. Randomly interfering with opiate production in poppy plants.
  5. Modifying the biosynthetic pathway to produce a different outcome.

Each of these methods will be outlined on separate pages for this project, but a detailed proposal will only be presented for the first method since it will provide the highest chance of producing a viable hybrid that meets the requirements of this project.

The first stage in a molecular biology project like this is to choose an activity assay, which is an analytical method that can be utilized to show that whatever change has been made to an organism has resulted in an appropriate change to the desired activity. In this case the 'activity' is production of opiates, so the assay needs to be able to detect the presence of opiates and their precursors in cell extracts of poppy plants. Even at the beginning stages involving genetic work, the activity of the enzymes under study will be production of an opiate, so an analytical technique that detects opiates and their precursors can be used.

The best assays are easy, fast, inexpensive, and capable of testing many samples at once. An LC/MS method could work as an assay for this project, and there are many such available for opiates.

The second stage in a molecular biology project is a review of the biosynthetic pathway, in other words, how the poppy plant produces opiates. The scientific name for poppy is Papaver somniferum, and it is an annual herb with 52 botanical varieties. Opiates are secondary metabolites in poppy plants, which means that they provide a useful but non-essential function for the plant, in this case defense against pathogens. As secondary metabolites these pathways can usually be altered without affecting the plant itself.

The ultimate starting material for opiate biosynthesis is the amino acid tyrosine. There is a long pathway involving many enzymes to reach opiates, but all the steps are well-studied. A quick summary of the pathway, with intermediates noted by abbreviations, will provide an overall look at the system:

Tyrosine->4HPP->4HPA->Nor->Coc->NMC->3HM->SRet->DHR->RRet->Sale->Salo->Theb->two branches to get to->morphine

Complications that can occur with a pathway this long are:

  1. Stopping anything after the first step will lead to a buildup of intermediates in the organism, which could be detrimental to plant health.
  2. Other pathways could intersect with this one, so if an early step in this one is blocked, other paths will take up the slack and continue to produce opiates.
  3. Some of these intermediates could be used in other useful pathways for the plant.
  4. The enzyme that is blocked in the project could be used in other pathways for the organism, which could be detrimental to plant health.

It is difficult to predict which of these problems will end up being an issue for this project due to the complexity of eukaryotic metabolism, but these outcomes need to be kept in mind as the project advances.

Since the method of choice for this project is to block an early step in the biosynthesis of opiates, the initial target choice is the conversion of tyrosine to 4HPP. A review of this step is thus in order.

The first step in the conversion of tyrosine to opiates in Papaver somniferum is catalyzed by the enzyme Tyrosine Aminotransferase (TyrAT)(Plant Phys 2011 Nov 157(3):1067-1078 or here). This plant physiology paper provides much of the technical information that will be needed for this project, including gene sequences and bioassays. The reaction according to the Plant Phys paper is: Tyrosine + pyridoxal 5' phosphate (PLP) + alpha-keto glutarate (AKG) + ethylene diamine tetraacetic acid (EDTA) -> 4HPP.

Modification of genes in plants is particularly difficult due to the sequestration of genomic DNA in the nucleus, so the best way to change the DNA of a plant is to utilize the plant's own DNA repair pathways to fix a break that we make. Because of how and where the break is made, the 'repair' will actually result in a change in the sequence, which will change what is produced by that sequence (such as an enzyme), which, when the plant produces seeds, will result in a 'new' plant with new functions. In other words, domestication of plants that doesn't take a thousand years.

A number of ways have been to developed to get to the gene of interest to make a break that the plant itself will then fix. The whole process is referred to as knockout. One of these methods, called CRISPR will be chosen to block the TyrAT reaction.