Abstract

Hypersolutes, compatible solutes from hyperthermophiles, are superior to their mesophilic counterparts in the preservation of the performance of a variety of proteins. A major bottleneck for industrial applications of hypersolutes is the large-scale production at a competitive cost. The major goal of this project is the development of cell systems optimised for the production of mannosylglycerate, the most widely distributed hypersolute.

Two strategies will be pursued:

• Engineering of expression and secretion systems in suitable hosts.
• Optimisation of fermentation of natural producers.

Trials will be conducted that will widen the range of application of mannosylglycerate. These trials will focus on the utilisation of hypersolutes for the pharmaceutical and cosmetics industry and their application in drug delivery and proteomics. The involvement of industrial partners ensures that the results will reach end-users.

Objectives

• The main objective of this project is the development of microbial systems suitable for the industrial production of the hypersolute, mannosylglycerate to be used for the protection and stabilisation of labile biomaterials against stress caused by heat, drying, freezing, UV-radiation and operational conditions.
• A second objective is to assess the protecting properties of mannosylglycerate (and mannosylglyceramide) in a variety of biological systems, namely proteins, membranes, viruses, mammalian cells and artificial tissues to uncover new fields of application for hypersolutes.

Description of the work

To reach the objectives of the project three main lines of research will be followed:

• Establishment of specific fermentation conditions that will induce high production of mannosylglycerate in selected natural producers.
• Modification by genetic engineering of thermophilic candidates to production hosts.
• Generation of a mannosylglycerate synthase functioning at ambient temperatures to enable the use of the easily genetically manipulated organism, E. coli, as a producer of mannosylglycerate.

To decrease costs from down-stream processing, prospective industrial producers must secrete efficiently the intracellular solutes upon hypo-osmotic shock and be amenable to its reuse by a process designated "bacterial milking". To accomplish this goal, mechanosensitive systems responsible for solute efflux will be characterised in natural producers, improved by genetic alteration, and expressed in high producing strains.

The workplan is divided in four major interlinked research areas according to the type of methodology used and objectives envisaged:

• Characterisation of metabolic pathways, key enzymes and efflux channels in natural producers.
• Genetically-engineered strains for the production of hypersolutes.
• Assessment of the effectiveness of hypersolutes as protecting agents of labile biomaterials.
• Optimisation of fermentation conditions for maximal production and secretion of mannosylglycerate in natural and recombinant strains.

• Pilot-scale production of mannosylglycerate.
• Key-enzymes for the synthesis of mannosylglycerate and expression in E. coli.
• Engineered thermophilic strains for overproduction.
• Optimised fermentation of recombinant and natural producing strains.
• Engineered optimal-efflux systems.
• Assessment of the effectiveness of mannosylglycerate to stabilise a variety of biomaterials.

Contacts

Coordinator

• Helena SANTOS / Instituto de Tecnologia quimica e Biologica PORTUGAL

Participant

• Thomas SCHWARZ / Bitop Gesellschaft für Biotechnische Optimierung mbH GERMANY
• Dr Neil RAVEN / Centre for Applied Microbiology & Research / UNITED KINGDOM
• De Montfort University TEAM Research Group / UNITED KINGDOM
• Simonetta BARTOLUCCI / Dipartimento di Chimica organica e Biologica ITALY
• Orfeu FLORES / STAB - Tratamento de Aguas e Biotecnologia PORTUGAL
• Garabed ANTRANIKIAN / TU Hamburg-Harburg Biotechnologie/Technische Mikrobiologi / GERMANY
• Milton COSTA / Universidade de Coimbra Centro de Neurociêncas de Coimbra / PORTUGAL
• Winfried BOOS / Universität Konstanz Department of Biology / GERMANY