Activation of a Cryptic Manumycin-Type Biosynthetic Gene Cluster of Saccharothrix espanaensis DSM44229 by Series of Genetic Manipulations

Abstract

(1) Background: Manumycins are small actinomycete polyketides with prominent cancerostatic and immunosuppressive activities via inhibition of various eukaryotic enzymes. Their overall activity towards human cells depends on the structural variability of both their polyketide chains, mainly the upper one. In our genetic screening project to find novel producers of anti-inflammatory manumycins, the strain Saccharothrix espanaensis DSM44229 was identified as containing a novel manumycin-type biosynthetic gene cluster (BGC). (2) Methods: The biosynthetic genes appeared to be silent under all assayed laboratory conditions. Several techniques were used to activate the BGC, including: (i) heterologous expression in various hosts, (ii) overexpression of putative pathway-specific regulatory genes, and (iii) overexpression of a bottleneck cyclizing aminolevulinate synthase gene in both natural and heterologous producers. (3) Results: Multiple novel manumycin-type compounds were produced at various levels by genetically-modified strains, sharing a tetraene lower chain structure with a colabomycin subgroup of manumycins, but possessing much shorter and saturated upper chains. (4) Conclusions: A cryptic manumycin-type BGC was successfully activated by genetic means to gain production of novel manumycin-type compounds for future comparative activity assays. Heterologously produced compounds were identical to those found after final activation of the BGC in the original strain, proving the intactness of the cloned BGC.

Keywords: Saccharothrix; actinomycetes; cancerostatics; colabomycin; cryptic BGC activation; immunomodulators; manumycin; secondary metabolites.

Figure 2

Manumycin-type gene cluster of Saccharothrix espanaensis DSM44229. The genes were assigned by homology to the genes of the asukamycin biosynthetic gene cluster (BGC) in Streptomyces nodosus ssp. asukaensis ATCC 29757, GenBank: GQ926890.1 [28]. The gene functions and homology to asukamycin biosynthetic genes are listed in the table below. 1T2 and 2T2 cosmid clone inserts extents are indicated.

Figure 1

Manumycin-type polyketides. (a) General structure. Lower polyketide chains are colored in blue, upper chains in green. (b) Examples of variability in both polyketide chains. The lower chain contains 3,4-AHBA as a starter unit prolonged with linear, usually polyene, 4- to 8-carbon chains, but may be saturated in rare cases (U-62,162). The upper chain shows the highest variability affecting length, saturation, branching pattern, and starter unit selection.

Figure 3

Activation of the manumycin-type BGC by the heterologous expression, UHPLC record. Three novel metabolites were produced in K4-114 × 1T2 and detected at λ = 350 nm: A: ES-: 507.2173, C₂₈H₃₂N₂O₇, B: ES-: 521.2343, C₂₉H₃₄N₂O₇, C: ES-: 533.2308, C₃₀H₃₄N₂O₇.

Figure 4

Comparison of ESI⁻ LC-MS/MS spectra of tetraene colabomycin E (a), triene asukamycin A (b), and compound A (c) The corresponding fragment structures (d) were predicted by Mass Frontier 7.1 software (Thermo Scientific) based on the exact molecular mass. Typical fragments shared by all manumycins containing the C₅N unit are shown (I, II, III) together with trienic (V) and tetraenic (IV) lower chain-specific fragments.

Figure 5

LC-HRMS analysis of the production of manumycin-type compounds encoded by the DSM44229 BGC in various producer strains (ESI⁻). The list of strains is provided in the left column, the total amounts of manumycin-type compounds produced (relative amounts reflected by peak areas) and relative production levels calculated as percentages of that in DSM44229 × pMS17 are shown in the right columns. The best producer strain is shown in bold. Composition and ratio of the compounds produced are indicated for each strain in color: compound A in three isomers sharing the same MS/MS fragmentation pattern, but showing different retention times (507a: 7.57 min, 507b: 7.03 min, 507c: 5.72 min); type II form of compound A (A+2H)—two isomers double peak (509: 7.01–7.21 min); compound B in three isomers (521a: 7.94 min, 521b: 7.38 min, 521c: 6.04 min); type II form of compound B (B + 2H)—two isomers double peak (523: 7.36–7.55 min), and the minor congener compound C (533: 8.10 min). (a) Saccharothrix espanaensis DSM44229-derived strains, (b) Streptomyces lividans K4-114-derived strains, (c) Streptomyces coelicolor M512-derived strains. Amounts of the extract loaded were equal to 4 mL of the original post-fermentation culture volumes.

Figure 6

Pathway intermediate structure predicted by NMR. Measured for ¹H and ¹³C at 700.13 MHz for ¹H and at 176.05 MHz for ¹³C in CDCl₃ at 20 °C. (a) NMR data summary, (b) structure predicted.