Post-crotonylation oxidation by a monooxygenase promotes acetyl-CoA synthetase degradation in Streptomyces roseosporus.

Protein post-translational modifications (PTMs) with various acyl groups play central roles in Streptomyces. But whether these acyl groups can be further modified, and the influences of these potential modifications on bacterial physiology have not been addressed. Here in Streptomyces roseosporus with rich crotonylation, a luciferase monooxygenase LimB is identified to elaborately regulate the crotonylation level, morphological development and antibiotic production by oxidation on the crotonyl groups of an acetyl-CoA synthetase Acs. This chemical modification on crotonylation leads to Acs degradation via the protease ClpP1/2 pathway and lowered intracellular crotonyl-CoA pool. Thus, we show that acyl groups after PTMs can be further modified, herein named post-PTM modification (PPM), and LimB is a PTM modifier to control the substrate protein turnover for cell development of Streptomyces. These findings expand our understanding of the complexity of chemical modifications on proteins for physiological regulation, and also suggest that PPM would be widespread.

Degradation mechanism of AtrA mediated by ClpXP and its application in daptomycin production in Streptomyces roseosporus.

The efficiency of drug biosynthesis depends on different transcriptional regulatory pathways in Streptomyces, and the protein degradation system adds another layer of complexity to the regulatory processes. AtrA, a transcriptional regulator in the A-factor regulatory cascade, stimulates the production of daptomycin by binding to the dptE promoter in Streptomyces roseosporus. Using pull-down assays, bacterial two-hybrid system and knockout verification, we demonstrated that AtrA is a substrate for ClpP protease. Furthermore, we showed that ClpX is necessary for AtrA recognition and subsequent degradation. Bioinformatics analysis, truncating mutation, and overexpression proved that the AAA motifs of AtrA were essential for initial recognition in the degradation process. Finally, overexpression of mutated atrA (AAA-QQQ) in S. roseosporus increased the yield of daptomycin by 225% in shake flask and by 164% in the 15 L bioreactor. Thus, improving the stability of key regulators is an effective method to promote the ability of antibiotic synthesis.

Comprehensive investigation of long non-coding RNAs in an endophytic fungus Calcarisporium arbuscula NRRL 3705.

Long non-coding RNAs (lncRNAs) play an important role in eukaryotic cells. However, there is no report of lncRNAs in endophytic fungi Calcarisporium arbuscula. Here, in Calcarisporium arbuscula NRRL 3705, an endophytic fungus predominantly producing mycotoxins aurovertins, the genome-wide identification of lncRNAs was carried out based on RNA-Seq. Totally, 1332 lncRNAs were identified, including 1082 long intergenic noncoding RNAs, 64 long intronic noncoding RNAs and 186 long noncoding natural antisense transcripts. The average length of lncRNA and mRNA were 254 and 1102 bp, respectively. LncRNAs were shorter, with fewer exons and lower expression levels. Moreover, there were 39 up-regulated lncRNAs and 10 down-regulated lncRNAs in the ΔaurA mutant, which lacks the aurovertin biosynthetic enzyme AurA. Interestingly, expression of genes related to the metabolism of linoleic acid and methane were significantly down regulated in the ΔaurA mutant. This study enriches the endophytic fungal lncRNA database and provide a basis for further research.

A novel strategy of gene screen based on multi-omics in Streptomyces roseosporus.

Daptomycin is a new lipopeptide antibiotic for treatment of severe infection caused by multi-drug-resistant bacteria, but its production cost remains high currently. Thus, it is very important to improve the fermentation ability of the daptomycin producer Streptomyces roseosporus. Here, we found that the deletion of proteasome in S. roseosporus would result in the loss of ability to produce daptomycin. Therefore, transcriptome and 4D label-free proteome analyses of the proteasome mutant (Δprc) and wild type were carried out, showing 457 differential genes. Further, five genes were screened by integrated crotonylation omics analysis. Among them, two genes (orf04750/orf05959) could significantly promote the daptomycin synthesis by overexpression, and the fermentation yield in shake flask increased by 54% and 76.7%, respectively. By enhancing the crotonylation modification via lysine site mutation (K-Q), the daptomycin production in shake flask was finally increased by 98.8% and 206.3%, respectively. This result proved that the crotonylation modification of appropriate proteins could effectively modulate daptomycin biosynthesis. In summary, we established a novel strategy of gene screen for antibiotic biosynthesis process, which is more convenient than the previous screening method based on pathway-specific regulators. KEY POINTS: • Δprc strain has lost the ability of daptomycin production • Five genes were screened by multi-omics analysis • Two genes (orf04750/orf05959) could promote the daptomycin synthesis by overexpression.

Discovery of a Potential Liver Fibrosis Inhibitor from a Mushroom Endophytic Fungus by Genome Mining of a Silent Biosynthetic Gene Cluster.

Liver fibrosis has accounted for liver diseases and overall mortality, but no relevant drug has been developed. Filamentous fungi are important resources of natural products for pharmaceutical development. Calcarisporium arbuscula is a mushroom endophytic fungus, which primarily produces aurovertins. Here, in an aurovertin null-production mutant, one silent gene cluster (mca17) was activated by overexpression of a pathway-specific zinc finger transcriptional regulator, and a tetramic acid-type compound (1, MCA17-1) was identified. Along with detailed structural characterization, its biosynthesis was proposed to be produced from the core PKS-NRPS hybrid enzyme. Moreover, 1 suppressed the activation of LX-2 upon transforming growth factor-ß (TGF-ß) challenge and had stronger bioactivity than the positive control obeticholic acid (OCA) against liver fibrosis. Our work suggested that this engineered fungus could be a producer of 1 for promising pharmaceutical development, and alternatively, it would be developed as a mushroom ingredient in dietary therapy to prevent liver fibrosis.

A Cell Factory of a Fungicolous Fungus Calcarisporiumarbuscula for Efficient Production of Natural Products.

Fungal natural products are rich sources of clinical drugs. Particularly, the fungicolous fungi have a large number of biosynthetic gene clusters (BGCs) to produce numerous bioactive natural products, but most BGCs are silent in the laboratory. We have shown that a fungicolous fungus Calcarisporiumarbuscula NRRL 3705 predominantly produces the highly reduced polyketide-type mycotoxins aurovertins. Here after evaluation of the aurovertin-null mutant ΔaurA as an efficient host, we further screened two strong promoters aurBp and A07068p based on RNA-Seq, and successfully activated an endogenous gene cluster from C. arbuscula as well as three additional exogenous BGCs from other fungi to produce polyketide-type natural products. Thus, we showed an efficient expression system from the fungicolous fungus C. arbuscula, which will be highly beneficial and complementary to the conventional Aspergillus and Penicillium fungal cell factories, and provides a useful toolkit for genome-wide mining of bioactive natural products from fungicolous fungi.

Regulation of Protein Post-Translational Modifications on Metabolism of Actinomycetes.

Protein post-translational modification (PTM) is a reversible process, which can dynamically regulate the metabolic state of cells through regulation of protein structure, activity, localization or protein-protein interactions. Actinomycetes are present in the soil, air and water, and their life cycle is strongly determined by environmental conditions. The complexity of variable environments urges Actinomycetes to respond quickly to external stimuli. In recent years, advances in identification and quantification of PTMs have led researchers to deepen their understanding of the functions of PTMs in physiology and metabolism, including vegetative growth, sporulation, metabolite synthesis and infectivity. On the other hand, most donor groups for PTMs come from various metabolites, suggesting a complex association network between metabolic states, PTMs and signaling pathways. Here, we review the mechanisms and functions of PTMs identified in Actinomycetes, focusing on phosphorylation, acylation and protein degradation in an attempt to summarize the recent progress of research on PTMs and their important role in bacterial cellular processes.

Crotonylation of key metabolic enzymes regulates carbon catabolite repression in Streptomyces roseosporus.

Due to the plethora natural products made by Streptomyces, the regulation of its metabolism are of great interest, whereas there is a lack of detailed understanding of the role of posttranslational modifications (PTM) beyond traditional transcriptional regulation. Herein with Streptomyces roseosporus as a model, we showed that crotonylation is widespread on key enzymes for various metabolic pathways, and sufficient crotonylation in primary metabolism and timely elimination in secondary metabolism are required for proper Streptomyces metabolism. Particularly, the glucose kinase Glk, a keyplayer of carbon catabolite repression (CCR) regulating bacterial metabolism, is identified reversibly crotonylated by the decrotonylase CobB and the crotonyl-transferase Kct1 to negatively control its activity. Furthermore, crotonylation positively regulates CCR for Streptomyces metabolism through modulation of the ratio of glucose uptake/Glk activity and utilization of carbon sources. Thus, our results revealed a regulatory mechanism that crotonylation globally regulates Streptomyces metabolism at least through positive modulation of CCR.

Multi-Layer Controls of Cas9 Activity Coupled With ATP Synthase Over-Expression for Efficient Genome Editing in Streptomyces.

Efficient genome editing is a prerequisite of genetic engineering in synthetic biology, which has been recently achieved by the powerful CRISPR/Cas9 system. However, the toxicity of Cas9, due to its abundant intracellular expression, has impeded its extensive applications. Here we constructed a genetic cassette with triple controls of Cas9 activities at transcriptional, translational and protein levels, together with over-expression of the ATP synthase ß-subunit AtpD, for the efficient genome editing in Streptomyces. By deletion of actII-ORF4 in Streptomyces coelicolor as a model, we found that constitutive expression of cas9 had about 90% editing efficiency but dramatically reduced transformation efficiency by 900-fold. However, triple controls of Cas9 under non-induction conditions to reduce its activity increased transformation efficiency over 250-fold, and had about 10% editing efficiency if combined with atpD overexpression. Overall, our strategy accounts for about 30-fold increased possibility for successful genome editing under the non-induction condition. In addition, about 80% editing efficiency was observed at the actII-ORF4 locus after simultaneous induction with thiostrepton, theophylline and blue light for Cas9 activity reconstitution. This improved straightforward efficient genome editing was also confirmed in another locus redD. Thus, we developed a new strategy for efficient genome editing, and it could be readily and widely adaptable to other Streptomyces species to improve genetic manipulation for rapid strain engineering in Streptomyces synthetic biology, due to the highly conserved genetic cassettes in this genus.

Dual regulation between the two-component system PhoRP and AdpA regulates antibiotic production in Streptomyces.

Antibiotic production during secondary metabolism in Streptomyces spp. is elaborately controlled by multiple environmental signals and intracellular cascades. These include the two-component system PhoRP responding to phosphate starvation and a conserved signaling pathway mediated by the pleiotropic regulator AdpA. However, little information exists about how these two pathways work together for secondary metabolite production of Streptomyces. Herein, we report the dual regulation from the phosphate starvation-responsive regulator PhoP and AdpA on atrA promoter (atrAp) for the production of daptomycin, an antibiotic produced by Streptomyces roseosporus. We found that PhoP directly binds to atrAp, positively regulates atrA expression and thus daptomycin production. We also observed positive auto-regulation of phoRP expression during fermentation for daptomycin production. Moreover, partial overlap between PhoP- and AdpA-binding sites on atrAp was observed, which results in partial competitive binding between these two regulators. This partial overlapping and competition between PhoP and AdpA was further confirmed by mutations and binding assays. In summary, our findings have revealed dual regulation of PhoP and AdpA on the same promoter for antibiotic production in Streptomyces. This mechanism would be beneficial to further environment-responsive fermentation optimization for antibiotic production.