BioMatNet Item: QLK3-2002-02086 - Exploiting the secretion machinery of Pseudomonas for the nanotechnological production of pharmaceuticals (NANOFOLDEX)

[BioMatNet Database - FP5 Quality of Life Programme]

QLK3-2002-02086
Exploiting the secretion machinery of Pseudomonas for the nanotechnological production of pharmaceuticals (NANOFOLDEX)

Contacts

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Nanotechnology : Quality of Life - 3. The Cell Factory

Type of Project	Shared Cost Research
Contract No	QLK3-2002-02086
Total Cost
EC Contribution	1,828,658 EUR
Start Date	01-01-2003
Duration	36 Months

Abstract

The use of enzymes as biocatalysts is a rapidly developing field of immense academic and industrial interest. Several biocatalysts such as lipases and penicillin acylases proved to be optimally suited for the nanotechnological production of therapeutic compounds including enantiopure pharmaceuticals. At present, however, these biocatalysts cannot be produced in sufficient amounts and at a reasonable price because traditional production hosts like Bacillus and Aspergillus do not support their correct folding and secretion. This proposal aims at providing a solution to this problem by construction of a Gram-negative secretion host strain with its periplasm engineered into a compartment optimised for the assembly and folding of complex biocatalysts. The barrier in the secretion pathway imposed by the bacterial outer membrane will be overcome by relaxing the selectivity and specificity of the secretion machinery using nano-biotechnological strategies.

Objectives

Many biocatalysts cannot be produced efficiently in traditional industrial host strains, usually because of protein-folding problems. A Gram-negative bacterium could be an ideal host for the production of these enzymes, because of the presence of the periplasm, which is an excellent folding compartment. Traditionally, Gram-negatives are not considered as industrial production hosts for secretory proteins because of the barrier in the secretion process imposed by the outer membrane. Pseudomonas strains may offer a solution to this problem since they possess a nano-machine for the secretion of folded proteins. The main objective of this proposal is the design of a potent Pseudomonas secretion host by engineering protein folding and secretion as well as global gene expression and carbon flow. This secretion host will be used for the production of therapeutic peptides and enantioselective biocatalysts enabling the nano-technological production of new and improved pharmaceuticals.

Activities

Pseudomonas aeruginosa will be used as the model strain because a wealth of knowledge is available on its biochemistry and molecular genetics including the genome sequence. The knowledge gained will be transferred to the related industrial strain, P. alcaligenes. To optimise periplasmic protein folding, periplasmic functions involved in protein folding will be identified by genomic and proteomic approaches. The mechanism of action of selected chaperones will be investigated. Deleterious protease activity will be eliminated, while maintaining the putative chaperone function of these enzymes. To manipulate the efficiency and specificity of the secretion machinery the interaction of exo-proteins with the outer membrane secretion machinery will be studied, thereby focusing on the identification of the secretion signal. This knowledge will be used to develop vectors for the secretion of non-secretory proteins. To optimise gene expression and carbon flow for exoprotein production, new global regulatory systems, including a post-transcriptional regulation system probably also involved in carbon flow, will be studied. Strains will be constructed with an optimal co-ordination between exo-protein synthesis and secretion, and in which metabolic resources are optimally used to drive secretion.

All available tools derived from this explorative work will be combined to construct a versatile secretion host, which is able to secrete selected high-value added biocatalysts. Beta-lactam acylases and enantioselective lipases will be engineered for secretion. Furthermore, an efficient lipase-secretion process will be employed for the secretion of therapeutic peptides.

Deliverables

The following deliverables are anticipated:

a potent Gram-negative secretion host.
an optimised periplasmic folding compartment for the production of valuable biocatalysts, including enantioselective lipases and complex multimeric penicillin acylases.
vectors for the efficient secretion of these biocatalysts by the optimised secretion host.
a cost-effective system for the production of therapeutic peptides.

Contacts

Coordinator

Jan TOMMASSEN / Universiteit Utrecht Leerstoelgroep Microbiologie / NETHERLANDS

Participant