Pantoea sp. P37 as a novel nonpathogenic host for the heterologous production of rhamnolipids

Abstract

Microbially derived surfactants, so-called biosurfactants, have attracted significant attention as an environmentally friendly alternative to their chemically synthesized counterparts. Particularly, rhamnolipids offer a large potential with their outstanding surfactant properties such as complete biodegradability, low toxicity, and stability. Rhamnolipids are naturally synthesized by the opportunistic human pathogen Pseudomonas aeruginosa under the tight regulation of a highly complex quorum-sensing system. The heterologous production of mono-rhamnolipids by a newly isolated nonpathogenic strain of the genus Pantoea was investigated. Analysis of the genome obtained by a chimeric assembly of Nanopore long reads and high-quality Illumina reads suggested that the strain has evolved to an epiphytic rather than a pathogenic lifestyle. Functional heterologous expression of the mono-rhamnolipid operon rhlAB derived from a P. aeruginosa strain was established and confirmed by HPLC analysis. Transcriptome analysis indicated destabilizing effects of the produced rhamnolipids on the cell envelope of the host resulting in the induction of molecular stress responses. After integration of the rmlBCDA operon, extracellular rhamnolipids in amounts up to 0.4 g/L could be detected and were identified as a mono-rhamnolipid Rha-C₁₀ -C₁₀ by MALDI-TOF mass spectrometry.

Keywords: Pantoea; biosurfactants; genome sequencing; heterologous expression; rhamnolipids.

Figure 1

Genome map of the main chromosome of Pantoea sp. P37. The outer ring displays specific biosynthetic clusters (blue), MFS transporter, multidrug efflux pumps and transporter (red), hypothetical proteins (purple), and phage‐associated genes (yellow). The second outer ring displays genomic islands (red). The third outer ring represents the GC content. The fourth ring represents the GC skew

Figure 2

Genome map of Pantoea sp. P37 plasmid 1. The outer ring displays specific biosynthetic clusters (blue), MFS transporter, multidrug efflux pumps and transporter (red), hypothetical proteins (purple), and phage‐associated genes (yellow). The second outer ring displays genomic islands (red). The third outer ring shows the GC content. The fourth ring displays the GC skew

Figure 3

MALDI‐TOF‐MS spectra of extracts from culture supernatants of recombinant Pantoea sp. P37 isolate (pET3110), Pantoea sp. P37 wild type (WT) and P. aeruginosa isolate TGR2A

Figure 4

Differential expression analysis of genes expressed in the recombinant Pantoea sp. P37 (pETrhlAB8) and its wild type. Blue dots: |log2(FoldChange)| < −2; Red dots: |log2(FoldChange)|> 2; Orange dots: 1.5 <|log2(FoldChange)| < 2. p‐Value < .005. tgt: Queuine tRNA‐ribosyltransferase; Siroheme synthase 2; groS: 10 kDa chaperonin GroES; leuC1: 3‐isopropylmalate dehydratase large subunit 1; cydB2: cytochrome bd‐I ubiquinol oxidase subunit 2; atpA: ATP synthase subunit alpha; rplV: 50S ribosomal protein L22; kdpA: potassium‐transporting ATPase potassium‐binding subunit; ybaY: lipoprotein YbaY; rpoB: DNA‐directed RNA polymerase subunit beta; pstS2: phosphate‐binding protein PstS 2; rpsE: 30S ribosomal protein S5; rpoC: DNA‐directed RNA polymerase subunit beta; groL: 60 kDa chaperonin GroEL; cyoB: cytochrome o ubiquinol oxidase subunit I; lpp: major outer membrane prolipoprotein Lpp; rplF: 50S ribosomal protein L6; rplO: 50S ribosomal protein L15

Figure A1

Maximum likelihood tree showing the phylogenetic relationship between Pantoea sp. P37 and selected Pantoea strains based on 16S rRNA, gyrB and rpoB. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Red colored dots indicate a BSL2 classification according to the German Federal Institute for Occupational Safety and Health (2015)

Figure A2

Genome map of the Pantoea sp. P37 plasmids 2, 3 and 4. The outer ring displays all known CDS (blue), MFS transporter, multidrug efflux pumps and transporter (red), hypothetical proteins (purple), and phage‐associated genes (yellow). The second outer ring displays genomic islands (red). The third outer ring shows the GC content. The fourth ring displays the GC skew