Does regulation hold the key to optimizing lipopeptide production in Pseudomonas for biotechnology?

Abstract

Lipopeptides (LPs) produced by Pseudomonas spp. are specialized metabolites with diverse structures and functions, including powerful biosurfactant and antimicrobial properties. Despite their enormous potential in environmental and industrial biotechnology, low yield and high production cost limit their practical use. While genome mining and functional genomics have identified a multitude of LP biosynthetic gene clusters, the regulatory mechanisms underlying their biosynthesis remain poorly understood. We propose that regulation holds the key to unlocking LP production in Pseudomonas for biotechnology. In this review, we summarize the structure and function of Pseudomonas-derived LPs and describe the molecular basis for their biosynthesis and regulation. We examine the global and specific regulator-driven mechanisms controlling LP synthesis including the influence of environmental signals. Understanding LP regulation is key to modulating production of these valuable compounds, both quantitatively and qualitatively, for industrial and environmental biotechnology.

Keywords: Pseudomonas; antibiotics; bioengineering; bioprocessing; biosurfactants; lipopeptides; regulation; specialized metabolites.

FIGURE 1

Graphical abstract showing strategies to exploit the regulatory pathways controlling lipopeptide production in Pseudomonas for applications in industrial and environmental biotechnology. Created with BioRender.com.

FIGURE 2

General overview of the organization of lipopeptide biosynthetic gene clusters in lipopeptide mono-producers.

FIGURE 3

Organization of the lipopeptide biosynthetic gene clusters in non-quorum sensing lipopeptide poly-producers. Representative strains are shown. See Supplementary Table S1 for strain and sequence information.

FIGURE 4

Organization of lipopeptide biosynthetic gene clusters in quorum sensing lipopeptide poly-producers. Representative strains are shown. See Supplementary Table S1 for strain and sequence information.

FIGURE 5

Regulation of lipopeptide production in the massetolide mono-producer P. lactis SS101. Massetolide production is under the control of the Gac/Rsm signal transduction pathway. The sensor kinase GacS is triggered by unknown environmental signals in the periplasm resulting in autophosphorylation. The phosphate group is transferred to the GacA response regulator via a phospho-relay system. Phosphorylated GacA binds to the GacA box in the promoter region of the rsmY and rsmZ genes encoding small RNAs. The resulting small RNAs RsmY and RsmZ bind to the repressor proteins RsmA and RsmE, relieving translational repression at the ribosomal binding site of the mRNAs of the luxR1 (massAR) gene. LuxR1 (MassAR) and LuxR2 (MassBCR) activate transcription of the massetolide biosynthetic gene cluster. The serine protease ClpP and its chaperone ClpA additionally regulate massetolide production via LuxR1, the heat shock proteins DnaK and DnaJ, and proteins involved in the citric acid cycle. DnaK and DnaJ may be required for proper folding of LuxR proteins or for assembly of the NRPS complex. Phgdh: D-3 phosphoglycerate dehydrogenase involved in the biosynthesis of the amino acid L-serine. Serine makes up two of the nine amino acids in massetolide. PrtR: antisigma factor which interacts with extra-cytoplasmic function sigma factors and affects the transcription of both luxR1 and luxR2 by an unknown mechanism. Secretion of massetolide occurs by the ABC transporter PleABC. Motifs: GacA-binding box see (Humair et al., 2010), Rsm box, see (Olorunleke et al., 2017). Created with BioRender.com.

FIGURE 6

Organization of lipopeptide biosynthetic gene clusters in lipopeptide mono-producers. See Supplementary Table S1 for strain and sequence information.

FIGURE 7

Phylogenetic tree of LuxR-type regulatory proteins in lipopeptide mono-producers. Neighbor-Joining phylogenetic tree (JTT model) constructed with MEGA 11 with MUSCLE alignment of LuxR amino acid sequences from LP mono-producers. Bootstrap values (percentage of 1,000 replicates) are shown in the figure. LuxR1 protein encoding genes (yellow) are located upstream of the lipopeptide NRPS genes, LuxR2 protein encoding genes (orange) are located downstream of the lipopeptide NRPS genes. See Supplementary Table S1 for strain and sequence information.

FIGURE 8

Regulation of lipopeptide production in Mycin and Peptin producers. Top: Non-quorum sensing regulation of lipopeptide production in the syringomycin and syringopeptin producers P. syringae pv. syringae B301D and B728a. Syringomycin and Syringopeptin production is under the control of the SalA regulon. SalA is activated by the Gac/Rsm signal transduction pathway in response to plant signals. SalA positively regulates its own transcription and activates the expression of both syrG and syrF. SyrG acts as a transcriptional activator of syrF. SyrF binds as a homodimer to a specific syr-syp box (indicated with a pin) in the promoter region of syringomycin and syringopeptin biosynthesis genes and transporters. Secretion of syringomycin and syringopeptin occurs by the ABC transporter PleABC, the cytoplasmic membrane protein SyrD and the RND transporter PseABC. Operons in the biosynthetic gene cluster are underlined. Bottom: Proposed model of quorum-sensing regulation of lipopeptide production in the nunamycin and nunapeptin producer P. nunensis In5. PcoI is an acyl-homoserine lactone (AHL) synthase encoding the autoinducer N-hexanoyl-L-homoserine lactone (C₆-AHL), NupR1 is a LuxR family protein lacking an N-AHL binding domain under the control of the Gac/Rsm regulon, NupR2 is a LuxR protein with an N-AHL binding domain. The promoter region of nunamycin and nunapeptin biosynthesis genes and transporters harbor a specific lux box (indicated with a pin) to which a NupR1-NupR2-C₆-AHL complex may bind (not experimentally proven). Production of nunamycin and nunapeptin is triggered by fungal signals that activate nunF. How NunF further activates lipopeptide synthesis and secretion is unknown. Nunamycin and nunapeptin secretion probably occurs by the ABC transporter PleABC, the cytoplasmic membrane protein NupD and the RND transporter PseABC. Motifs: GacA-binding box, see (Humair et al., 2010); Rsm-binding box, see (Olorunleke et al., 2017); syr-syp box, see (Wang et al., 2006a); lux-box, see (Whiteley and Greenberg, 2001). Created with BioRender.com.

FIGURE 9

Phylogenetic tree of LuxR-type regulatory proteins in lipopeptide mono- and poly-producers. Neighbor-Joining phylogenetic tree (JTT model) constructed with MEGA 11 with MUSCLE alignment of LuxR amino acid sequences from mono- and poly-producers. Bootstrap values (percentage of 1,000 replicates) are shown in the figure. Clade I: JesR2-type regulatory proteins with a helix-turn-helix (HTH, indicated in blue) and an autoinducer binding motif (indicated in green) produced by LP poly-producers. This clade also contains the CipR2 protein from P. cichorii (purple star). Clade II: LuxR2-type regulatory proteins with a HTH motif (indicated in blue) found in LP mono-producers downstream of the LP BGCs. This clade also contains the JesR1 type regulators found in poly-producers with a quorum sensing system (indicated in green) and the SalA (red star), CipR1 (purple star) and AspR4 (blue star) regulatory proteins of P. syringae pv. syringae, P. cichorii and P. fuscovaginae, respectively. Clade III: LuxR1-type regulatory proteins with a HTH motif (in blue) located upstream of the LP BGCs in mono- and poly-producers. This clade also contains the SyrF homologues (indicated in red in the tree) located downstream of the Mycin or Brabantamide BGCs in LP poly-producers. See Supplementary Table S1 for strain and sequence information.

FIGURE 10

Organisation of lipopeptide biosynthetic gene clusters in tolaasin/sessilin producers. See Supplementary Table S1 for strain and sequence information.