Biofilm as a production platform for heterologous production of rhamnolipids by the non-pathogenic strain Pseudomonas putida KT2440

Abstract

BackgroundAlthough a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used.ResultsIn the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C10 fatty acids.ConclusionThis study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0581-9) contains supplementary material, which is available to authorized users.

Fly ash based robust biocatalyst generation: a sustainable strategy towards enhanced green biosurfactant production and waste utilization

“…35 The intercellular communications within a biolm further stimulated the up-and-down regulation of gene expression, enabling temporal adaptations, such as phenotypic variation and the ability to survive in nutrient-decient conditions, 36 thereby promoting the biosurfactant production. This study demonstrated the successful application of FA as a platform to stimulate the biosynthesis of lipopeptide through biolm-encased cells and the nding in this study was comparable to that generated by Wigneswaran et al 37 This result further proved the enhanced production mechanism described in Fig. 1(a).…”

Rheological Behavior of High Cell Density Pseudomonas putida LS46 Cultures during Production of Medium Chain Length Polyhydroxyalkanoate (PHA) Polymers

“…Other extracellular secretions associated with Pseudomonas spp. include alginate [67], DNA [68][69][70], gellan [71], proteins [72], glycolipids and lipopolysaccharides [64,73,74], organic acids, [30,75], as well as acetylated sugars and uronic acids [65,66].…”

Effects of sodium citrate on the structure and microbial community composition of an early-stage multispecies biofilm model

“…Sodium citrate and sodium acetate are the most commonly used carbon sources for supplementing natural or artificial low-nutrient media to accelerate the formation of monospecies or multispecies biofilm in laboratorybased biofilm experiments [19][20][21][22][23][24][25][26] . However, the potential biased effects caused by supplementation with such carbon sources on the structural attributes, microbial community composition, and microbial interaction networks of biofilms remain unexplored.…”

“…One strategy commonly used to accelerate biofilm formation in nutrient-poor media is the addition of an organic carbon source. In biofilm experiments using nutrient poor media, sodium citrate or sodium acetate is occasionally supplemented as the sole carbon source for accelerating biofilm development in monospecies biofilm models [19][20][21][22] or multispecies consortia [23][24][25][26] . It is through carbon source supplementation that such studies were able to accelerate biofilm development processes needed for conducting timely quantitative and qualitative biofilm analyses, thereby providing an attractive means for experimental productivity.…”