STARLAB Contractor Programmes

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Cell Engineering of Lactococcus lactis

Co-ordinators:
Dr. Gerald Fitzgerald, University College, Cork, Ireland.
Douwe van Sinderen , University College, Cork, Ireland.
Partners:

OBJECTIVES

This project aims to optimise the performance of Lactococcus lactis as a cell factory for the production of existing and novel industrially relevant compounds. The project will use a multidisiplinary approach that combines physiology, biochemistry, biophysics and molecular genetics to understand, improve, model and manipulate metabolic flux and the biosynthesis of polysaccharides. Improvement of strain performance will be sought through the analysis of stress and environmental response and will be coupled to the metabolic engineering aspects of the project. A significant part of the research programme will be devoted to metabolic engineering of the glycolytic flux and will focus on pyruvate as the gateway to a variety of metabolic routes. The potential to develop Lactococcus lactis as a cell factory for the production of novel metabolites of industrial value will be explored using alanine biosynthesis as a model system.

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Control of Bacteriophage Development in Lactic Acid Bacteria
Towards a rational solution to a major problem of food fermentation

Co-ordinator: Dr. Marie-Christine Chopin, INRA, France.

Partners:

OBJECTIVES

To construct highly resistant lactic acid bacteria strains for a long-term efficient control of lactic fermentation. This will be achieved by an increase in knowledge of phage gene functions and on global regulatory circuits to develop new phage defence strategies and to characterize natural phage defence mechanisms.

SCIENTIFIC PROGRAMME

Design of new phage defence strategies:
A better knowledge of phage biology would allow the abolition of phage development either by preventing some key steps of phage growth, or by using phage regulatory circuits to control expression of toxic genes and to induce premature death of infected cells. Gene functions essential to phage development will be characterized and global regulatory circuits will be studied. Lactococcal phages of two main species of industrial concern will be studied. Defence mechanisms preventing phage replication or inducing cell death will be designed. The background knowledge acquired on lactococcal phages will be adapted to phage species of Streptococcus thermophilus, Lactobacillus casei and Lactobacillus helveticus.

Increase of the efficiency of natural phage defence mechanisms:
To protect strains efficiently against a large phage spectrum, it will be necessary to increase the effectiveness of such mechanisms and to associate those acting on different steps of the phage growth and/or on different phage species. Therefore, additional mechanisms, present in industrial strains, will be characterized and the efficiency of two mechanisms which have been shown to have a very large spectrum will be improved.
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The Molecular Biology and Genetics of Thermophilic Lactic Acid Bacteria

Co-ordinator: Maurilio de Felice, Universita di Napoli Federico II, Italy.

Partners:

OBJECTIVES

The goal of this project is to improve the knowledge of relevant metabolic traits and the response to environmental stress in thermophilc lactic acid bacteria. The project is expected to generate new multi-functional starter strains able to enhance flavour and texturing properties in yoghurt and cheese making.

SCIENTIFIC PROGRAMME

The biology of thermophilic LAB is poorly known, although some of them, and principally Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus helveticus, are widely used in the manufacturing of many French, Italian and Swiss cheeses as well as yoghurt and acidified milk products.
The objective of this project is to fill the gap existing between thermophilic LAB and other industrially important microorganisms and relies on the confidence that genetic tools made recently available from studies with other organisms may apply successfully to thermophilic LAB. Focusses of the research will be:

Metabolic Engineering:
Thermophilic LAB often rely on symbiotic partners for proteolytic activity and nutritional factors. Furthermore: (i) their lactose utilization and acidification rate differ from what is known for Lactococcus lactis, and (ii) polysaccharide secretion, an important texturing property, is an unstable character in LAB. These features, which are a serious impediment to the development of new dairy processes, will be deeply studied.

Environmental and stress response:
Little is known about how thermophilic LAB adapt to changes of pH, temperature, nutrient and salt concentration, which are common in food fermentations. A better knowledge of the adaptative mechanisms will be pursued in order to allow the development of LAB starter strains for improved technological processes.
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Lactic Acid Bacteria with Modified Proteolytic Properties in Milk Fermentation

Coordinator: Prof. Wil N. Konings, University of Groningen, The Netherlands.

Partners:

OBJECTIVES

The aim of this project is to construct strains of Lactococcus lactis in which the proteolytic pathway has been engineered, to use these strains as starters in cheese fermentation, and to analyse in pilot studies their influence on the rate of cheese ripening and the development of organoleptic properties. The outcome of these experiments will be used to design new starter cultures which may lead to improved or newly developed milk fermentation products.

SCIENTIFIC PROGRAMME

Fermentation of milk by lactic acid bacteria is essential for the production of cheese, yoghurt and other dairy products. The degradation of milk proteins during milk fermentations is catalyzed by many different enzymes which together form the proteolytic system. The activity of this proteolytic system determines the rate and extent of the fermentation process and plays a crucial role in the development of the rheological and organoleptic properties of the final product. In this project various complementary approaches will be used to genetically modify the proteolytic properties of Lactococcus lactis. Strains will be constructed in which:

  1. The activity, specificity and/or stability of the proteinase PrtP has been engineered;
  2. The peptide transport activity and specificity has been engineered;
  3. The activity of one or multiple peptidases has been affected either by gene deletion or overexpression. The genes encoding 8 unique peptidases of Lactococcus lactis and at least 5 other unique enzymes from Lactobacillus species are available for these studies;
  4. A combination of these enzymes are affected.

Food-grade cloning techniques, based on self-cloning, will be used to genetically engineer Lactococcus lactis, which will allow the evaluation of the modified strains in cheese trials. The breakdown of caseins by these modified strains will be followed in detail by on-line HPLC-ion mass spectrometry measurements, which will reveal bottlenecks in the proteolytic pathway and identify the relative importance of specific peptides as nitrogen sources for the organism and/or sources of flavour compounds. Following the construction of the first generation of modified Lactococcus lactis strains and their biochemical characterisation, cheese slurry experiments and small scale cheese trials will be carried out with single and mixed starter cultures. The proteolysis in the cheeses will be evaluated by official cheese examiners. Based on the outcome of these studies, new (second generation) strains will be constructed and selected strains (with desirable properties) will be tested further in large scale cheese trials.

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Lactic Acid Bacteria as Cell Factories for the Production and Delivery of Mucosal Immunogens

Co-ordinator: Dr. Annick Mercenier, Institut Pasteur de Lille

Partners:

OBJECTIVES:

The use of live microorganisms as antigen delivery systems has received much attention in the development of new vaccines. Most of the bacterial or viral vectors currently investigated are derived from pathogenic invasive microorganisms for which attenuated variants have been isolated. The lactic acid bacteria (LAB) could represent original alternative live vaccine vectors since they are safe organisms that are well known for their use in food fermentation processes and as probiotics. Some LAB species are commensals of the gastrointestinal and urogenital tracts of healthy individuals (humans and animals) where they participate in the maintenance of an equilibrated microflora. Consequently, the LAB should be especially suitable vehicles to deliver protective antigens at mucosal surfaces. A distinct advantage of the mucosal route of immunization is that it is able to elicit local antibody responses at the portals of entry for most pathogens. Moreover, there is still a need to develop low-cost vaccines which could be administered easily (e.g. orally) to a large number of individuals including infants, elderly and immunocompromised people. In this scope, LAB offer a number of advantages (ease of oral or local administration and long-lasting experience in producing and preserving LAB strains at industrial scale) which render them attractive as live antigen carriers.

SCIENTIFIC PROGRAMME

The ultimate goal of the research program is to design original and efficient local vaccines targeting mucosal pathogens. Specifically, the work is focused on the construction and immunological evaluation of recombinant LAB strains expressing protective antigens from pathogenic organisms. Three bacterial host systems are being evaluated in a mouse model: Lactococcus lactis (non-colonizing microorganism), Streptococcus gordonii and Lactobacillus (colonizing organisms). The project comprises three interconnected areas:

Suitable LAB strains are also selected for future application in humans, which should improve our understanding of the health beneficial traits exhibited by certain LAB strains.

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Carbon Catabolite Control in Food Grade Lactobacilli to Provide the Tools for Strain Improvement

Co-ordinator: Dr. Josef Deutscher, Institute of Biology and Chemistry of Proteins, France.

Partners:

OBJECTIVES:

This project relates to studies to improve the properties of industrial Lactobacillus strains for the production of high- and low-molecular weight compounds and their use in milk an soy fermentation.
Bacteria impose regulatory mechanisms on metabolic processes in order to ensure that the needs of the cell are met but not exceeded. These regulatory mechanisms interfere with the desired exhaustive industrial production of low- and high-molecular weight compounds by bacteria and their use in the food industry. The most important of these regulatory mechanisms is catabolite repression (CR) which controls carbon and nitrogen metabolism and carbon fluxes under conditions where rapidly metabolizable carbon sources are abundant. It is the aim of this project to exploit the knowledge gained in the previous BIOTECH programme for the construction of novel strains of industrially important food grade lactobacilli which are insensitive to CR and deregulated at the enzyme level. Lactobacillus has been chosen because it plays an important role in enzyme production, fermentation processes and food production.

SCIENTIFIC PROGRAMME:

The two main goals of this proposal are;

The knowledge gained about the mechanisms of CR and carbon flux regulation in lactobacilli will specifically be used to improve storage properties of fermented milk with Lb. casei of DANONE (postacidification), to improve the production of alpha-amylase by Lb. pentosus, to optimize the fermentation of pentose sugars by lactobacilli for soy production as well as to improve the nutritive value of feed and to construct Lb. casei strains of DANONE in which carbon fluxes important to milk fermentation and flavour production can be controlled.

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For further information about the STARLAB project or to make contributions to these pages please contact Dr. Jerry Wells.
This page was created by C. Coward 1997. Please send comments or bug reports by email to cc122@mole.bio.cam.ac.uk or use the form.