A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.

Aims:  To analyse the effects of plipastatin operon disruption and constitutive expression of surfactin operon in Bacillus subtilis 168 on surfactin productivity, in vitro invasive growth and antagonism against fungi. Methods and Results:  The srfA native promoter was replaced by the constitutive promoter PrepU in B. subtilis 168 after integration of a functional sfp gene. Moreover, the plipastatin synthesis was further disrupted in the B. subtilis 168 derivatives. In liquid media, an earlier and higher expression of PrepU, than that found with PsrfA, led to a specific surfactin production fivefold higher after 6 h of culture. On solid media, not only the invasive growth and the haemolytic activity but also the antifungal activity of the constitutive strains were improved when compared to the parental strain BBG111. As expected, the disruption of the plipastatin operon strongly reduced in vitro antifungal properties but, interestingly, enhanced specific surfactin production (1·47 g g−1 of biomass), spreading behaviour and haemolytic activity of the strains. Conclusions:  This work demonstrates for the first time the interdependency of surfactin and plipastatin regarding their biosynthesis as well as their influence on the biological activities of the producing strain. Significance and Impact of the Study:  The constitutive overproduction of surfactin enhances the invasive growth and the in vitro antagonistic activity of the mutant strain. Both properties are known to play an important role in the biocontrol of plant diseases. Plipastatin operon disruption increases the surfactin productivity of mutant strains. These mutants are interesting for use in continuous bioprocesses for surfactin production or in bioremediation.

Lipopeoptides are amphiphilic compounds combining interesting physicochemical properties and biological activities. Due to their high foaming capacity in aerated bioreactor, the development of scalable bioprocesses for their production is a major bottleneck. In addition, the genes involved in the biosynthesis of these lipopeptides are mainly regulated by the quorum sensing, a global regulatory mechanism depending on cell density and known to be activated in biofilms. Several approaches have thus been considered in literature taking into account two criteria, on one side, to favor, control or avoid foam formation and on the other side, to use planktonic or immobilized (biofilm) cells. These different bioprocesses are discussed in the present review along with the purification strategies proposed for extracting and concentrating these biosurfactants.