Enzymatic fluorination in Streptomyces cattleya. Setting a framework for biotechnological development.

Streptomyces cattleya is a bacterium which has the unusual ability to generate fluorometabolites. These are the toxic compound fluoroacetate and the fluorinated amino acid 4-fluorothreonine (an antibiotic). In recent years we have been able to identify the enzyme responsible for the initial fluorination event in S. cattleya. This enzyme converts S-adenosyl-L-methionine (SAM) and fluoride ion to 5'-fluoro-5'-deoxy-adenosine (5'-FDA) and L-methionine. This is an important discovery because organo-fluorine compounds as a class, have made a huge impact in the fine chemicals, pharmaceuticals and agrochemicals industries and they continue to have a growing impact in the discovery and development of new pharmaceutical products. All fluorinated compounds to date are made by chemical methods thus the identification of this enzyme, which can take inorganic fluoride and convert it to organic fluorine, and the genes associated with this enzyme, offer new prospects for the biotechnological, rather than the chemical, production of organo-fluorine compounds. We envisage that in the near future we will be able to move the fluorinase gene and the related biosynthetic and management genes (eg. fluoroacetate resistance gene, fluoride ion up take genes) into host organisms and 'kick start' fluorometabolite production in the hosts. This is novel and exciting in the context of organo-fluorine chemistry. To do this we need to identify as many of the relevant genes as possible and establish their roles. This will be done by gene knockout experiments and exploring the consequences. Recent progress has identified the fluorinase gene as well as a cluster of about 10 genes flanking this gene. In this cluster some, but not all, of the biosynthetic pathway genes to fluoroacetate and 4-fluorothreonine are present. A major focus of this proposal is to identify the remaining genes, explore the gene products (enzymes/proteins) and characterise these. We will also use these genes as the focal point for further gene walking to explore flanking genes. The overall focus of this research is to characterise fluorometabolite biosynthesis at the genetic level and assess the protein products of relevant genes and the function and mechanisms of relevant enzymes. This information will be used to develop biotechnological tools for the production of novel organofluorine compounds by fermentation methods.

The research will aim to identify all of the genes and enzymes involved in the biosynthesis of the fluorinated secondary metabolites of Streptomyces cattleya and identify to what extend that they are clustered. The products of the biosynthetic genes will be expressed and characterised (mechanism, structure etc). This knowledge will be important for engineering fluorometabolite production into host organisms. Several genes will be targeted (isomerse, aldolase, PLP transaldolase, Na+/H+antiporter) and where relevant, gene knockouts will be carried out to establish their biosynthesis role. The programme will have a heavy technical focus on Streptomyces molecular biology. We know that the fluorinase and the next enzyme (a PNP), lie adjacent on the genome among a cluster of management genes. Also there is a mutase enzyme. These three genes have been characterised and their products over-expressed. In very recent work we have begun to sequence the gene for a fourth enzyme (PLP transaldolase) on the pathway. The project will move towards a complete genetic understanding of this pathway and the role (mechanism/structure) of some management genes (eg Na+/H+ antiporter) will be studied. Thus in overview the project aims to establish a deeper understanding of the requirements for fluoride uptake and fluorometabolite biosynthesis with the longer term goal of being able to technically transfer fluorination genes into a host and generate fluorination ability within the host. Subsequent metabolism should develop novel fluorination products by fermentation in the host organism.