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

Abstract

Background: Although 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.

Results: In 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.

Conclusion: This 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.

Keywords: Biofilm; Biosurfactants; Heterologous biosynthesis; Pseudomonas putida KT2440; Rhamnolipids; Synthetic promoter library; UHPLC-HRMS; UHPLC-MS/HRMS.

Fig. 1

Construction and utilization of a synthetic promoter library in P. putida. a Outline of the plasmid construction strategy. The genes gfp, rhlA and rhlB indicate the green fluorescence protein, rhamnosyltransferase chain A and rhamnosyltransferase chain B, respectively. SPL indicate synthetic promoter library. The X in pVW12-SPLX represents any of the 120 synthetic promoters constructed in this study (see “Methods” section). b Gfp fluorescence measurement in a series of control strains without SPL. The control strains are wild type strain (P. putida) and strains containing an empty vector (pVW10), plasmid containing rhlAB operon (pVW13) with no promoter and the rhlAB operon with the native promoter from PAO1 (pVW14). The asterisks represent a significant difference (P < 0.05). c The different promoter strengths in the constructed SPL determined as Gfp intensities. The controls strains depicted in b are shown in grey. The columns represent mean value and the error bars indicate standard deviation. The Gfp intensities have been replicated 1–3 times. An enlarged picture of c can be found in Additional file 1: Figure S1 in which the name of the constructed promoters is shown

Fig. 2

Outline of the different rhamnolipid congeners. The grey part of the structure is the varying part of the molecule. The numbers indicate the length of the fatty acid side chain. The most predominant congener produced by the recombinant P. putida is Rha-C₁₀-C₁₀. The different rhamnolipid congeners were determined based on their elemental composition

Fig. 3

Correlation between Rha-C₁₀-C₁₀ concentration and gfp expression. The linear correlation between gfp and Rha-C₁₀-C₁₀ enable fluorescence to be used as an indirect measure of rhamnolipid biosynthesis

Fig. 4

Correlation between rhamnolipid production and growth rate. Biosynthesis of rhamnolipids has an impact on planktonic growth rate. The cells grow slower as they produce more rhamnolipids

Fig. 5

Biofilm development in microtiter plate assays. The amount of biofilm was quantified for the reference strain (black), a medium rhamnolipid producer (red) and a high rhamnolipid producer (green) at the specified time points. Each data point represents the mean and the error bars indicate standard deviations of eight replicates

Fig. 6

Effect of rhamnolipid and the surfactant Tween 20 on swarming motility. The top row is the reference strain (KT2440/pVW10) and the bottom row is a rhamnolipid producer (KT2440/pVW12-SPL4). The right column has been added 0.0005% Tween 20 and the left column have not. The scale bars indicate the size of 1 cm on the pictured strain. The presence of Tween 20 increased the swarming motility of both strains but in particular for the reference strain

Fig. 7

Biofilm development of a high rhamnolipid producing P. putida (KT2440/pVW12-SPL115) and the reference strain (KT2440/pVW10). Depicted are representative pictures of biofilm from the specified days. The scale bar indicates the size of 50 µm

Fig. 8

Biofilm biomass quantification. The biomass was quantified based on the obtained CSLM pictures. a The reference strain (KT2440/pVW10), b the medium rhamnolipid producer (KT2440/pVW12-SPL4), and c the high rhamnolipid producer (KT2440/pVW12-SPL115). In d strains are combined and only the mean values are depicted for clarity. The symbols represent the mean biomass and the error bars the standard deviation based on two biological replicates each composed of 7–11 biofilm pictures

Fig. 9

Continuous rhamnolipid production in a biofilm system. The reference strain (KT2440/pVW10), a medium (KT2440/pVW12-SPL4) and a high rhamnolipid (KT2440/pVW12-SPL115) producer were cultivated for 7 days and the rhamnolipid production quantified. The symbols represent mean concentration and the error bars the standard deviation of two biological replicates