High-throughput optimization of medium components and culture conditions for the efficient production of a lipopeptide pseudofactin by Pseudomonas fluorescens BD5

Abstract

BackgroundLipopeptides are a promising group of surface-active compounds of microbial origin (biosurfactants). These diverse molecules are produced mainly by Bacillus and Pseudomonas strains. Because of their attractive physiochemical and biological properties, biosurfactants are considered to be “green and versatile molecules of the future”. The main obstacles in widespread use of biosurfactants are mainly their low yields and high production costs. Pseudofactin (PF) is a lipopeptide produced by Pseudomonas fluorescens BD5. Recently, we identified two analogues, PF1 (C16-Val) and PF2 (C16-Leu), and reported that PF2 has good emulsification and foaming activities, as well as antibacterial, antifungal, anticancer, and antiadhesive properties. Reported production of PF in a mineral salt medium was approximately 10 mg/L.ResultsHere, we report successful high-throughput optimization of culture medium and conditions for efficient PF production using P. fluorescens BD5. Compared with production in minimal medium, PF yield increased almost 120-fold, up to 1187 ± 13.0 mg/L. Using Plackett–Burman and central composite design methodologies we identified critical factors that are important for efficient PF production, mainly high glycerol concentration, supplementation with amino acids (leucine or valine) and complex additives (e.g. tryptone), as well as high culture aeration. We also detected the shift in a ratio of produced PF analogues in response to supplementation with different amino acids. Leucine strongly induces PF2 production, while valine addition supports PF1 production. We also reported the identification of two new PF analogues: PF3 (C18-Val) and PF4 (C18-Leu).ConclusionsIdentification of critical culture parameters that are important for lipopeptide production and their high yields can result in reduction of the production costs of these molecules. This may lead to the industrial-scale production of biosurfactants and their widespread use. Moreover, we produced new lipopeptide pure analogues that can be used to investigate the relationship between the structure and biological activity of lipopeptides.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0968-x) contains supplementary material, which is available to authorized users.

Genome and transcriptome analysis of Bacillus velezensis BS‐37, an efficient surfactin producer from glycerol, in response to d‐/l‐leucine

“…Many previously characterized bacteria, such as B. subtilis 168 and Bacillus amyloliquefaciens MT45, can utilize sucrose to produce surfactin with high efficiency, but few of these can efficiently use glycerol (Amani, Haghighi, & Keshtkar, ; Biniarz, Coutte, Gancel, & Lukaszewicz, ; Zhu, Zhang, Chen, Cai, & Lin, ). It is therefore notable that BS‐37 shows the opposite substrate preference.…”

Generation of Xylose-inducible promoter tools forPseudomonasspecies and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

“…Pseudomonas species have been suggested for industrial production and bioremediation of molecules including, but not limited to, β-peptides (25), polyhdroxyalkanoates (26), phenazine-1-carboxyamide (27), proteases (28, 29), pseudofactin (30), rhamnolipids (31–33), silver nanoparticles (34), and toluene (35). The ability of P xut to activate expression of gene expression in the majority of cells and its relative strength in P. fluorescens compared to P BAD suggest that it may be useful for larger scale gene synthetic applications.…”

Development of a Bioprocess for the Production of Cyclic Lipopeptides Pseudofactins With Efficient Purification From Collected Foam

“…Pseudofactins (PFs) are a family of CLPs produced by the Arctic isolate Pseudomonas fluorescens BD5 (Janek et al, 2010). Four PF structural analogs were previously identified (PF1 -PF4), with PF2 being the most abundant in P. fluorescens BD5 cultures (Janek et al, 2010;Biniarz et al, 2018). PFs are probably synthesized by a single NRPS complex, with one of its modules lacking substrate specificity for leucine and valine (data not shown).…”

“…In our previous work we identified critical parameters essential for PFs production, mainly high glycerol and tryptone concentration, high culture aeration, and the presence of amino acids: leucine (Leu), valine (Val), or isoleucine (Ile). These experiments allowed us to develop a laboratory-scale optimized culture conditions and to achieve two goals: (1) increase PFs production ∼120-fold to meet the demand for large amounts of pure PFs needed for further experiments, and (2) separate individual PF variants, for investigating structure-properties relationship of CLPs (Biniarz et al, 2018). Simultaneously, we established a protocol for the precise quantification of PFs, together with their structural analysis by LC-MS/MS (Biniarz and Łukaszewicz, 2017).…”

“…The aim of this work was to develop a bioprocess for the increased production and purification of PFs in laboratory-scale bioreactors and then to transfer this process to technical scale. To this end, we established an efficient culturing of P. fluorescens BD5 in bioreactors, initially in 2.5 L and next in 30 L of working volumes, using optimized media and conditions (Biniarz et al, 2018). We also demonstrated an efficient process of raw PFs purification from the foam collected from cultures, together with the purification and separation of PF structural analogs with semi-preparative RP-HPLC.…”