Abstract

The project aims at a robust bioprocess to produce heterologous proteins of biopharmaceutical relevance based on the exploitation of Streptomyces lividans protein secretion. Delivery of the key scientific and technical objectives will be secured by implementing the following elements of a Project plan:

• Investigation and modification where required, of the specificity of the interacting target proteins and secretory machineries to improve overall secretion yield.
• Routing of proteins of interest to the most efficient secretion pathway, including the recently discovered twin-arginine translocation pathway.
• Investigation and optimisation of the fermentation conditions for recombinant proteins of interest.
• Interrogation of the physiological basis of growth and productivity of heterologous proteins within fermentation systems by DNA arrays.

Objectives

Amelioration of the Cell Factory capacity of Streptomyces to secrete bioactive heterologous proteins by using a comprehensive multidisciplinary and highly focussed set of scientific tools and by intertwining fundamental with industrial research and its applications. This will be realised through:

• Better insight into the molecular determinants for substrate recognition by the different secretion pathways including the newly discovered twin-arginine translocation pathway, permitting rational engineering of the relevant components to tailor-made Streptomyces strains.
• Insight into the molecular physiology of Streptomyces by interrogating the differential expression of genes involved with the pioneering use of DNA array analysis for Streptomyces.
• Novel rationally-optimised large-scale fermentation processes for Streptomyces protein secretion.

Description of the work

By taking advantages of the recent progress made in the elucidation of the protein secretion pathway of Streptomyces concerning the identification and characterisation of the genes involved, and by benefiting from the nearly completed S. coelicolor genome project, it is now feasible to detect eventual bottlenecks that hamper the secretion of specific proteins, to look for ways to overcome these and to select classes of heterologous proteins for which S. lividans can be an ideal host.

This will be accomplished through an improved knowledge of the different secretion pathways and of the substrate recognition specificity of the interacting proteins including the signal peptidases, chaperones, SecA and other Sec proteins and foldases. Deliverables will be obtained using a combination of mutations, biochemical and biophysical tools and will allow the construction of tailor-made secretion strains for a number of relevant biopharmaceutical proteins. Development of the necessary detection systems will allow monitoring of the quantity and quality of the proteins of interest expressed and secreted by engineered strains, first at lab-scale, and later in larger fermentors. Substrate kinetics will be studied and special attention will be given to the determination of the most favourable nutrient composition in order to maximise the suppression of protease activity. Based on the known genome sequence of S. coelicolor, DNA arrays will be developed to investigate what sets of metabolic genes or genes involved in secretion are turned on/off during such growth conditions. The resultant information will be used for a more rational design of media and of appropriate expression/secretion host strains.

Deliverables

Novel pilot-scale robust bioprocessing tools for the production of biopharmaceutically relevant proteins by exploiting the Streptomyces cell factory, made possible through:

• A better insight into the molecular determinants' substrate recognition by the different secretion pathways, including the Tat pathway.
• A better knowledge of molecular physiology of Streptomyces by DNA array analysis.
• By novel optimised large-scale processes for Streptomyces secretion biotechnology.