title: 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 C10fatty acids.
  
  
  CONCLUSIONS
  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.
authors: Department of Systems Biology, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.
authors: Vinoth
authors: V
authors: Wigneswaran
authors: Department of Systems Biology, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.
authors: Kristian Fog
authors: KF
authors: Nielsen
authors: Department of Systems Biology, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.
authors: Claus
authors: C
authors: Sternberg
authors: National Food Institute, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark.
authors: Peter Ruhdal
authors: PR
authors: Jensen
authors: National Veterinary Institute, Technical University of Denmark, 1870, Frederiksberg C, Denmark.
authors: Anders
authors: A
authors: Folkesson
authors: Department of Systems Biology, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark. lj@bio.dtu.dk.
authors: Lars
authors: http://orcid.org/0000-0002-5759-9769
authors: L
authors: Jelsbak
mesh_terms: D018441:Biofilms; D006017:Glycolipids / Q000096:biosynthesis*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D006017:Glycolipids; C418382:rhamnolipid
keywords: Biofilm; Biosurfactants; Heterologous biosynthesis; Pseudomonas putida KT2440; Rhamnolipids; Synthetic promoter library; UHPLC-HRMS; UHPLC-MS/HRMS