Combining culture optimization and synthetic biology to improve production and detection of secondary metabolites in Myxococcus xanthus: application to myxoprincomide

Abstract

Microbial secondary metabolites play crucial ecological roles in governing species interactions and contributing to their defense strategies. Their unique structures and potent bioactivities have been key in discovering antibiotics and anticancer drugs. Genome sequencing has undoubtedly revealed that myxobacteria constitute a huge reservoir of secondary metabolites as the well-known producers, actinomycetes. However, because most secondary metabolites are not produced in the laboratory context, the natural products from myxobacteria characterized to date represent only the tip of the iceberg. By combining the engineering of a dedicated Myxococcus xanthus DZ2 chassis strain with a two-step growth medium protocol, we provide a new approach called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) for the production of non-ribosomal peptides synthetases (NRPS)/polyketides synthases (PKS) secondary metabolites from myxobacteria. We further show that the 2PRIM-BOOST strategy will facilitate the screening of secondary metabolites for biological activities of medical interest. As proof of concept, using a constitutive strong promoter, the myxoprincomide from M. xanthus DZ2 has been efficiently produced and its biosynthesis has been enhanced using the 2PRIM-BOOST approach, allowing the identification of new features of myxoprincomide. This strategy should allow the chances to produce and discover new NRPS, PKS, and mixed NRPS/PKS hybrid natural metabolites that are currently considered as cryptic and are the most represented in myxobacteria.IMPORTANCEMicrobial secondary metabolites are important in species interactions and are also a prolific source of drugs. Myxobacteria are ubiquitous soil-dwelling bacteria constituting a huge reservoir of secondary metabolites. However, because most of these molecules are not produced in the laboratory context, one can estimate that only one-tenth have been characterized to date. Here, we developed a new strategy called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) that combines the engineering of a dedicated Myxococcus xanthus chassis strain together with growth medium optimization. By combining these strategies with the insertion of a constitutive promoter upstream the biosynthetic gene cluster (BGC), the production of myxoprincomide, a characterized low-produced secondary metabolite, was successfully and significantly increased. The 2PRIM-BOOST enriches the toolbox used to produce previously cryptic metabolites, unveil their ecological role, and provide new molecules of medical interest.

Keywords: bioactive compounds; biosynthetic gene cluster; chassis strain; myxobacteria; natural products; secondary metabolites; synthetic biology.

Fig 1

Comparison of the WT metabolomes using the standard and the 2PRIM protocols. (A) Description of the standard and the 2PRIM protocols. The protocols are divided into two steps: first, a growth step to produce biomass, and the second, to enrich the culture with metabolites with the addition of 2% of Amberlite XAD16 resin. For the standard protocol (blue oval), the CTT rich medium is used for the two steps. In the 2PRIM protocol (red oval), the biomass production step is performed in CYE rich medium, and cells were transferred in CF minimal medium for the enrichment step. (B) Prepared crude extracts of M. xanthus DZ2 strain were analyzed by Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HR-MS²), followed by untargeted and targeted analysis using MZmine, MetaboAnalyst, GNPS, and SIRIUS. (C–F) Volcano plot of data matrix 476FT showing the fold change difference of a given metabolite from the 2PRIM and the standard protocols (2PRIM/standard ratio). (C) Merged volcano of D, E, and F. Features with a positive log₂ fold change value (UP in 2PRIME) or with a negative log₂ fold change value (UP in standard protocol) above the thresholds (P value below 0.05 and fold change above 2) are represented. Some features are highlighted; FT318 corresponds to S-acetyl-pantetheine, FT329 to myxoprincomide c-506, FT445 to cittilin A, FT587 to DKxanthene 534, and FT738 to myxalamid A. The dotted circle surrounds the 41 MS features that are the most produced in 2PRIM relative to the standard protocol. Total volcano plot is decomposed to highlight the amino acids and derivates (purple dots) (D), the fatty acids and lipids (yellow dots) (E), and unknown metabolites (gray dots) (F). Metabolites from the blank medium of each protocol were subtracted. Extracts from each protocol (n = 4) were normalized at 5 mg/mL in MeOH.

Fig 2

M. xanthus accumulates derivates of pantetheine and the myxoprincomide SM in the 2PRIM protocol. (A) Analysis of the spectral family 54, identified as thioester derivates of pantetheine, in extracts obtained using the standard and the 2PRIM protocols. Each feature is identified by its RT and m/z. The pie chart represents the relative concentration of features in blue for the standard protocol and in red for the 2PRIM protocol. (B) MS2 spectrum annotation of FT318 identified as S-acetyl-pantetheine. (C) EIC of myxoprincomide c-506 m/z: 506.2715 [M + 2H]²⁺. Shown in red is the myxoprincomide EIC from the 2PRIM protocol, and in blue, the one from the standard protocol.

Fig 3

The deletion of the main secondary metabolites does not affect the growth of the BOOST strain. (A) Scheme of the genotype of the BOOST chassis strain in which the four BGC encoding the pathways to produce the SM myxalamids, myxochelins, DKxanthenes, and myxovirescin were deleted. (B) Genomic structure of BGC encoding myxochelins, myxovirescin, DKxanthenes, and myxalamids. Genes in red were deleted by double recombination. (C) Colony morphology and motility of M. xanthus DZ2 WT and mutant strains. Pictures of the colonies were taken after 48 h of growth on CYE plates with 1.5% agar. MXC-, myxochelins null strain; DKx-, DKxanthenes null strain; MXV-, myxovirescin null strain; and MXA-, myxalamids null strain.

Fig 4

Comparison of the metabolome composition between the BOOST and the WT strains. (A) Upper part: comparison of the base peak chromatogram (BPC) of the WT (in blue) and BOOST (in red) strains. Lower part: the EICs of the main SM of M. xanthus are highlighted. EIC from the WT strain (in blue) and the BOOST strain (in red). Cittilin A EIC is highlighted in gray for both strains because they have the same intensity. (B) Volcano plot showing the fold change difference of an FT between the BOOST and the WT strains following the 2PRIM protocol (BOOST/WT ratio). Features with a positive log₂ fold change value are more produced in the BOOST strain, and features with a negative log₂ fold change value are more produced in the WT strain (the threshold was set with a P value below 0.05 and a fold change above 2). The FT present in the same proportion for both strains are indicated in gray. DKX, DKxanthene; MXV, myxovirescin; MXA, myxalamid; MXC, myxochelin; CTL, cittilin. Some other FT defined by their cluster index (FTXXX) are also highlighted and described in Table S4. Metabolites from the blank medium of each protocol were subtracted. Extracts from each protocol (n = 4) were normalized at 5 mg/mL in MeOH.

Fig 5

The BOOST strain exhibits a significant loss of biological activities. (A) The table sums up results of the inhibition zone of S. aureus and B. subtilis on agar plates. Size of the inhibition zone is expressed in millimeters and is measured from the border of the well to the limit of the inhibition zone as shown by the black line in panel B. For agar test, 2 mg of dimethyl sulfoxide (DMSO) extracts from the standard protocol and 1 mg of DMSO extracts from the 2PRIM protocol were used. The blank was performed with DMSO only. Standard errors were calculated from two biological replicates. (B) The inhibition zones of M. xanthus DMSO extracts on LB agar plates against S. aureus with the standard protocol extracts and against B. subtilis with the 2PRIM extracts. (C) The 50% inhibitory concentrations (IC₅₀) on the proliferation of human lung cancer epithelial cells (A549 cells) were calculated after exposure to DMSO extracts of M. xanthus strains prepared following the standard or 2PRIM protocols. Errors bars show the standard error of three technical replicates, and statistical analysis was performed with a Tukey test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).

Fig 6

The combination of 2PRIM-BOOST and a constitutive strong promoter highly enhances the production of myxoprincomide. (A) Scheme of the 2PRIM-BOOST concept to produce cryptic or weakly produce SM by combining the 2PRIM protocol to the BOOST strain with the insertion of a constitutive strong promoter to induce the expression of the desired BGC and the corresponding SM. (B) Scheme of the construction to express myxoprincomide BGC (mxan_3779) with the synthetic strong promoter BBA_J23104 and the RBS BBa_B0034 inserted into the locus by a single step of homologous recombination. (C) The production of myxoprincomide c-506 is normalized to the production in the WT strain calibrated to 1 following the standard protocol. The myxoprincomide c-506 amount following the 2PRIM protocol was calculated by comparison of the integration of each EIC from WT, BOOST, and BOOST_MXP strain extracts. The proportional correlation of the integration of the EIC of myxoprincomide c-506 was verified by making a standard curve from diluted extracts of the BOOST_MXP strain. Error bars show the standard error of four biological replicates.