Characterization and production of biosurfactants from Bacillus thuringiensis – Bt-Surf

This project aims to increase the production of a biosurfactant. To do that different technological approaches are implemented: molecular biology is used to genetically modify the bacteria in order to produce larger quantities of the molecule.
Microbial physiology studies are performed to characterize the influence of environmental factors on the production of this biosurfactant. A production process is then developed to produce sufficient quantities of the product in order to study its foaming and emulsifying properties.
The potential for exploitation in the phytosanitary field is tested.

Genetic modifications of a B. thuringiensis strain has increased the expression of genes involved in the synthesis of kurstakine and increased its export into the extracellular medium. Thus, we obtained for the first time a strain producing kurstakine in the culture supernatant. Our results show that kurstakine plays a role in biofilm formation and is an antifungal molecule active against plant pathogen fungi. These results concern industrial producers of lipopeptides, like the Lipofabrik company.

The study has allowed a better understanding of the factors limiting the synthesis of kurstakine.
Genetic changes have increased production of kurstakine in sporulation deficient bacteria.
The kurstakine has a role in the formation of biofilms and has antifungal properties against plant pathogen fungi.
Our study provides possibilities for a large-scale production of kurstakine and for developing a phytosanitary application.

Antifungal activity of kurstakine
A. Activity of the wt and ?krsC strains against Galactomyces geotrichum (INRA)
B. Activity of the purified kurstakine on Botrytis cinerea (University of Lille)

Surfactants are amphiphilic compounds which reduce the surface tension of water. They are used, among other applications, as detergents, emulsifyers, anti-foaming, and dispersing agents. Most of the surfactants currently used are chemically synthesized. However, interest in biosurfactants of microbial origin has significantly increased during the last decade. Biosurfactants are highly diverse and readily biodegradable. Moreover, the reduction of production costs through fermentation processes and the diversity of their potential applications make these molecules highly attractive in socio-economic terms. Among the microorganims which produce biosurfactants, the Bacillus species are the most known.
A new area of research is now emerging which focuses on the search for new biosurfactant molecules, especially lipopeptides, which can be used as phytosanitary products or adjuvants to improve both the ecological fitness and the efficacy of existing biopesticides. Among the set of known lipopeptides, kurstakins produced by Bacillus thuringiensis (Bt) is quite interesting because this is one of the sole cationic lipopeptide family. Bt is the most successful biopesticide used worldwide in agriculture, forest management, and mosquito control. This bacterium, which is recognized as a safe (GRAS) microorganism for environment, animals and humans, is easy to cultivate in large amounts with a low production cost and is fully accepted in organic farming. The capacity of the bacteria to multiply and sporulate in the insect cadaver increases the horizontal transmission of Bt on the leaf surface and might be a key factor for bacterial spreading and, in fine, for the success of the biopesticide treatment. We have recently shown that lipopeptides named kurstakins are essential to the saprophytic properties of Bt, permitting the bacteria to survive and to sporulate in the cadaver of an infected host (an insect larva). Moreover, the results show that these lipopeptides are essential for swarming and biofilm formation, two surface-associated traits requiring biosurfactants. These results indicate that kurstakins might be interesting enhancers of colonisation and persistence of Bacillus strains in the rhizosphere or the phyllosphere. However the main technological barriers for the production and extraction at industrial scale of these compounds are the low level of expression of kurstakin genes and and the resulting low production of the lipopeptide.
Our project aims at characterizing and overproducing, on a large scale, a Bt lipopeptide, and at confirming its interest to improve the efficacy of the biopesticide formulations. It is divided into four tasks handled by three teams with complementary competences. The selection of a kurstakin variant, with the most efficient biosurfactant activity and the lowest ecotoxicity, will be performed in task 1. A second task aims to construct a non-sporulating and overproducing strain. This construction will be based on various data including very recent results concerning the regulation of kurstakin expression in Bt obtained by Team 1. A task will focus on the properties and the use of kurstakins as an adjuvant for the formulation of biopesticides. Finally, an important task of this project will be the determination of an appropriate bioprocess for industrial production and purification of kurstakin based on an integrated bioprocess for biosurfactant production recently patented by Team 2. An additional task concerning the commercial development of this technology will be achieved by the INRA-Transfert team.