Understanding and Engineering Alkaloid Biosynthesis

Lead Research Organisation: John Innes Centre
Department Name: Contracts Office

Abstract

My group’s work encompasses the area of natural product biosynthesis, with an emphasis on the chemistry, enzymology and metabolic engineering of plant natural product pathways.

We are actively working to identify uncharacterized alkaloid biosynthetic enzymes in the medicianl plant Catharanthus roseus. Transcripts that encode putative biosynthetic enzymes displaying the expected sequence homology, and/or having an expression profile consistent with the metabolite production levels, will be cloned, subjected to heterologous expression and evaluated by in vitro biochemical assay. Alternatively, we will use Viral Induced Gene Silencing (VIGS) to silence the gene candidate in planta and then assess the resulting natural product profile in silenced C. roseus seedlings. We will subject and newly discovered enzymes to in depth mechanistic analysis. Furthermore, we also note that a number of alkaloid biosynthetic enzymes show homology to enzymes from primary metabolism; for example, the N-methyl transferase described in Section 1 shows sequence homology to a C-methyl transferase involved in tocopherol biosynthesis. We propose to compare the sequences of these enzymes and use site directed mutagenesis to explore how nature may have potentially evolved an enzymatic function from a preexisting one.

Finally, we have also developed strategies to enable C. roseus to produce “new-to-nature” products. By silencing tryptophan decarboxylase, which makes the alkaloid precursor tryptamine, we have generated a C. roseus culture that lacks all alkaloids. Thus, adding unnatural tryptamine analogs to the culture medium of the “silenced” system results in the formation alkaloids derived exclusively from the unnatural starting substrates. We are in the process of initiating screening efforts to determine the medicinal activity of these compounds.

Publications

10 25 50
 
Description This is my BBSRC-funded "core account" at the John Innes centre. It encompasses 8,000 pounds per year, so the aim of this grant is to provide some modest support for more robustly funded projects. On its own, this grant is too small to have had any meaningful impact. However, in combination with my BBSRC response mode grants and my ERC funding, we have used this grant to discover new enzymes involved in biosynthesis of high value plant compounds, structural characterisation of these enzymes to understand how they work, so that we can use them more widely and development of a Nicotiania and yeast expression system to produce these corresponding compounds. Examples of recent gene discovery include the last remaining enzymes of vinblastine biosynthesis.
Exploitation Route for improved production of high value plant products
Sectors Agriculture, Food and Drink,Chemicals

 
Description To assemble production platforms for high value plant products., namely anticancer alkaloids. This has led to numerous academic collaborations and a collaboration with Conagen.
First Year Of Impact 2015
Sector Agriculture, Food and Drink,Chemicals
Impact Types Economic

 
Description Buell Group Bioinformatics 
Organisation Michigan State University
Country United States 
Sector Academic/University 
PI Contribution The Buell group has provided sequencing and bioinformatics support for our plants. We do the functional characterisation and biochemistry.
Collaborator Contribution The Buell group has seqeunced plants for us, assembled genomes/transcriptomes/annotated them, provided extensive bioinformatics support.
Impact Numerous papers (see publications); an NSF grant from the USA; an extensive database of plant sequence data; more papers underway
Start Year 2009
 
Description Collaboration with Vincent Courdavault 
Organisation François Rabelais University or University of Tours
Country France 
Sector Academic/University 
PI Contribution Courdavaults group has done microscopy to show localization of these enzymes. Our role in this project has been to discover the enzymes
Collaborator Contribution Our partners have provided localization data for the enzymes that we have discovered.
Impact See co-authored papers in publications
 
Description Transporter Collaboration 
Organisation University of Copenhagen
Department Department of Plant and Environmental Sciences
Country Denmark 
Sector Academic/University 
PI Contribution Collaboration with University of Copenhagen PIs Barbara Halkier and Hassam Nour Eldin. They perform biochemical assays with plant transporters that we have identified.
Collaborator Contribution They performed assay of transporters in oocytes.
Impact Publication in Nature Plants in 2017
Start Year 2016
 
Description Talk at Kew Gardens 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A Kew organized event called "State of the World's Plants"
Year(s) Of Engagement Activity 2017