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

Dominika Gorniaková,Tomáš Zelenka,Roman Grabic,Kateřina Petříčková,David Kahoun,Alica Chroňáková,Miroslav Petříček

doi:10.3390/microorganisms9030559

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.

Highlights

IntroductionGrowing knowledge on the genetics and biochemistry of bacterial secondary metabolism together with the availability of a huge number of bacterial genomic sequences has opened a new space for screening projects to get novel compounds and novel producers via genetic screening and genome mining [1]
Like many other secondary metabolites, manumycins possess numerous bioactivities based mostly on the enzyme inhibition mechanism. They have mostly been studied for their cancerostatic activity, with over 350 studies published on the topic so far
Based on our previous work [15], it seems that the upper polyketide chain represents a key structure of the molecules for determining whether pro-apoptotic, cancerostatic activities prevail over the immunosuppressive ones, which is the case for the commercially available manumycins A and B

Summary

Introduction

Growing knowledge on the genetics and biochemistry of bacterial secondary metabolism together with the availability of a huge number of bacterial genomic sequences has opened a new space for screening projects to get novel compounds and novel producers via genetic screening and genome mining [1]. This, has raised a new challenge for researchers: the identified biosynthetic gene clusters (BGCs) are quite often cryptic and sometimes difficult to activate. Various techniques have been applied to solve the problem, including heterologous expression, ribosome engineering, chemical elicitors application, co-culture approaches, promoter engineering or exchange, and others, reviewed recently by, e.g., Nguyen [2] and Kang [3].

Objectives

Methods

Results

Conclusion