Microparticles enhance the formation of seven major classes of natural products in native and metabolically engineered actinobacteria through accelerated morphological development.

Martin Kuhl,Jörn Kalinowski,Lars Gläser,Christoph Wittmann,Selma Beganovic,Andriy Luzhetskyy,Christian Rückert

doi:10.1002/bit.27818

Martin Kuhl, Jörn Kalinowski + Show 5 more

Open Access

https://doi.org/10.1002/bit.27818

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Abstract

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.

Highlights

Natural products are chemically diverse molecules that are synthetized by living organisms (Harvey, 2000)
The genus Streptomyces provides more than two‐third of all known antibiotics of microbial origin (Bibb, 2013) and more than half of the FDA‐approved antibacterial natural products (Patridge et al, 2015) and related actinobacteria such as Micromonospora, Actinoplanes, and Amycolatopsis (Barka et al, 2016) have emerged as producers of potent bioactive molecules
A promising line of research seems to exploit the link between natural product formation and the unique morphological life cycle of actinobacteria (Chater, 1984), which differentiates them from most other bacteria (Barka et al, 2016)

Summary

| INTRODUCTION

Natural products are chemically diverse molecules that are synthetized by living organisms (Harvey, 2000). General strategies that allow to enhance natural product formation in actinobacteria are highly desired At this point, a promising line of research seems to exploit the link between natural product formation and the unique morphological life cycle of actinobacteria (Chater, 1984), which differentiates them from most other bacteria (Barka et al, 2016). Reprogrammed recombinant Streptomyces albus J1074/R2 to live a life of accelerated morphogenesis, which enabled a three‐fold enhanced production of the antituberculosis polyketide pamamycin (Kuhl et al, 2020). The microparticle approach was scaled down to the microtiter plate scale to investigate its potential in overproducing twelve natural compounds of commercial interest from ten major structural classes in native and metabolically engineered actinobacteria, including various Streptomyces and other genera and families (Figure 1)

| MATERIALS AND METHODS

| RESULTS

| DISCUSSION

Findings

| CONCLUSIONS