Microparticles globally reprogram Streptomyces albus toward accelerated morphogenesis, streamlined carbon core metabolism, and enhanced production of the antituberculosis polyketide pamamycin

Abstract

Streptomyces spp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline the performance of in these microbes. Here, we studied the production of pamamycins, macrodiolide homologs with a high activity against multiresistant pathogenic microbes, using recombinant Streptomyces albus J1074/R2. Talc particles (hydrous magnesium silicate, 3MgO•4SiO 2 •H 2 O) of micrometer size, added to submerged cultures of the recombinant strain, tripled pamamycin production up to 50 mg/L. Furthermore, they strongly affected morphology, reduced the size of cell pellets formed by the filamentous microbe during the process up to sixfold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and precursor (CoA thioester) supply of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3,341 genes (56% of all genes), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to 1,024-fold). Furthermore, the microparticles perturbed genes encoding for CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased fourfold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. The novel insights into the behavior

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

“…Taken together, fine‐tuned re‐balancing of the expression of individual cluster genes obviously mediated the improved biosynthesis. This picture differs largely from that obtained for talc‐enhanced pamamycin production in S. albus , where the entire gene cluster was activated, up to 1000‐fold (Kuhl et al, 2020). Likely, the difference originated from the different expression control strategies: the expression control of the bottromycin cluster in S. lividans was of synthetic nature (Horbal et al, 2018), whereas the pamamycin cluster was expressed under its native control (Kuhl et al, 2020).…”

“…To investigate the effect of the microparticles in detail, a bottromycin‐producing mutant of S. lividans was studied in liquid culture with 10 g L −1 talc (Figure 2), an amount recently found optimal for the related strain S. albus J1074/R2 (Kuhl et al, 2020). A culture without talc served as control.…”

“…For appropriate mixing and oxygen transfer, the microtiter plates contained flower‐shaped wells, optimized in geometry for the applied 3 mm shaking diameter of the instrument (Funke et al, 2009). To validate the microliter scale against conventional shake flasks, a first round of experiments was conducted for S. lividans TK24 DG2‐Km‐P41hyg + (Figure 2) and S. albus J1074/R2 (Kuhl et al, 2020), for which the stimulating microparticle effects had been already proven in shake flasks. Both strains were grown in microtiter plates over 5 days at different levels of talc (0–50 g L −1 ) on their preferred medium (Table 1), followed by culture harvesting, natural product extraction and HPLC‐ESI‐MS analysis.…”

“…2) are available from GEO (GSE168044). For statistical analysis, Student's t ‐test was carried out and the data were filtered for genes with a log 2 ‐fold change ≥ 1 ( p ≤ 0.05) (Kuhl et al, 2020). RNA extraction and sequencing were conducted as biological triplicates for each strain.…”

“…The 5 µl culture broth was transferred onto a glass for bright-field microscopy (Olympus IX70 microscope). The software ImageJ 1.52 (Schneider et al, 2012) was used to automatically determine the size T A B L E 1 Strains and natural products investigated in this study and corresponding culture conditions of pellets formed during growth (Kuhl et al, 2020). At least 150…”