MOESM1 of Integrated foam fractionation for heterologous rhamnolipid production with recombinant Pseudomonas putida in a bioreactor

Additional file 1: Table S1: Sequence of the synthetic oligonucleotide Term-lov-Term

TREX: A Universal Tool for the Transfer and Expression of Biosynthetic Pathways in Bacteria

Secondary metabolites represent a virtually inexhaustible source of natural molecules exhibiting a high potential as pharmaceuticals or chemical building blocks. To gain broad access to these compounds, sophisticated expression systems are needed that facilitate the transfer and expression of large chromosomal regions, whose genes encode complex metabolic pathways. Here, we report on the development of the novel system for the <u>tr</u>ansfer and <u>ex</u>pression of biosynthetic pathways (TREX), which comprises all functional elements necessary for the delivery and concerted expression of clustered pathway genes in different bacteria. TREX employs (i) conjugation for DNA transfer, (ii) randomized transposition for its chromosomal insertion, and (iii) T7 RNA polymerase for unimpeded bidirectional gene expression. The applicability of the TREX system was demonstrated by establishing the biosynthetic pathways of two pigmented secondary metabolites, zeaxanthin and prodigiosin, in bacteria with different metabolic capacities. Thus, TREX represents a valuable tool for accessing natural products by allowing comparative expression studies with clustered genes

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Hemagglutination of rabbit red blood cells after incubation with <i>P. aeruginosa</i>.

<p>Erythrocytes were treated with papain and L-cysteine and then incubated with either PBS buffer in the absence and presence of 20 mM fucose or PBS-buffer containing <i>P. aeruginosa</i> PAO1 wild-type and mutants Δ<i>lecB</i> and Δ<i>oprF</i>. The positive control additionally contained purified LecB protein (concentration: 10 µg ml⁻¹). Non-agglutinated red blood cells form a distinct concentric pellet in the middle of the well whereas agglutinated red blood cells form a more extended and plaque-like structure.</p

Identification of LecB interaction partners in <i>P. aeruginosa</i>.

<p><b>A.</b> Far-Western blot analysis of membrane fractions from <i>P. aeruginosa</i> PAO1 using purified LecB and a LecB-specific antiserum. <b>B.</b> SDS-PAGE analysis of putative LecB ligands isolated from cell lysates of <i>P. aeruginosa</i> PAO1. Proteins were purified by affinity chromatography on mannose agarose, the column washed with 100 mM Tris-HCl (pH 8) and proteins eluted with 20 mM mannose in 100 mM Tris-HCl (pH 8) The <i>lecB</i> deficient mutant <i>P. aeruginosa</i> PATI2 served as a negative control. <b>C</b>.2D electrophoretic separation of protein bands shown in Fig. 2B; spots were subsequently analysed by MALDI-TOF mass spectrometry and identified as OprF and PA1337.</p

Release of LecB from the cell surface of <i>P. aeruginosa</i>.

<p>Biofilm cells were incubated with 20 mM L-fucose for 1 h at 4°C. Cell pellets (P) were separated from supernatants (S) by centrifugation and LecB was detected in both fractions by immunoblotting. Cells treated with 20 mM D-galactose served as a negative control. Additionally, blots were incubated with an antiserum against the periplasmic protein DsbA to monitor putative cell lysis during L-fucose treatment.</p

MOESM1 of Designer rhamnolipids by reduction of congener diversity: production and characterization

Additional file 1. Compilation of extra information for better comprehension of the contents of the study. The file contains a more detailed description of some methods, as well as some additional figures for the presented results

Subcellular localization of LecB in biofilm cells of the <i>oprF</i>-deficient mutant <i>P. aeruginosa</i> H636 grown for 48 h at 37°C. A.

<p>The same amount of culture supernatant, periplasm, cytoplasm and total membrane were subjected to SDS-PAGE analysis followed by immunoblotting using LecB antiserum. Fractions obtained from <i>P. aeruginosa</i> wild-type (wt) served as a positive control. Fractionation controls: <b>B.</b> Cellular fractions were analyzed using antisera directed against EstA (an outer membrane protein) and DsbA-(a periplasmic protein) and <b>C.</b> by determination of relative glucose-6-phosphate dehydrogenase (cytoplasmic protein) activities. The percentages of relative enzyme activities present in the cytoplasm (CP), the periplasm (PP), the membrane fraction (MF) and the culture supernatant (SN) are shown.</p