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.
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 β-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.
The semipinacol rearrangement (SPR) is highly useful in the asymmetric synthesis of complex compounds. In biological systems, only a few semi-pinacolases belonging to a few families have been identified to catalyze the SPR on alkaloids. Here, based on the biosynthesis of a fungal mycotoxin asteltoxin (1), two semi-pinacolases AstD/MrvD were identified from the epoxide hydrolase family to catalyze type III SPR on the polyketide backbone. They were proposed to catalyze efficient regio-selective hydrolysis on the bis-epoxide and 2,3-migration on the epoxide alcohol for the rearrangement. Based on the comprehensive mutations and chemical calculations, a critical Asp residue was identified as an acid for the coupled catalysis of selective epoxide collapse and subsequent SPR, while other critical residues facilitated efficient hydrolysis and protected carbocation for SPR. Thus, this study expanded the SPR biocatalyst family and provided an understanding of the catalytic mechanisms of these bifunctional semi-pinacolases.
Nanozymes
have shown great promise in reactive oxygen species (ROS)-mediated
tumor therapy with mitigated side effects but are normally limited
by the complex tumor microenvironment (TME). Herein, to overcome the
adverse effects of TME, such as tumor hypoxia and high endogenous
glutathione (GSH), an aptamer-functionalized Pd@MoO3–x
nano-hydrangea (A-Pd@MoO3–x
NH) is constructed for high-efficiency cancer therapy. Utilizing
the irregular shape characteristics of nano Pd, the A-Pd@MoO3–x
NH nanozyme simultaneously exposes catalase-like
Pd(111) and oxidase-like Pd(100) surface facets as dual active centers.
This can catalyze cascade enzymatic reactions to overcome the negative
effects of tumor hypoxia caused by the accumulation of cytotoxic superoxide
(O2
•–) radicals in TME without
any external stimuli. In addition, the nanozyme can effectively degrade
the overexpressed glutathione (GSH) through the redox reaction to
avoid nontherapeutic consumption of O2
•– radicals. More significantly, as a reversible electron station,
MoO3–x
can extract electrons from
H2O2 decomposing on Pd(111) or GSH degradation
and transfer them back to Pd(100) through oxygen bridges or few Mo–Pd
bonds. This can synergistically enhance enzyme-like activities of
dual active centers and the GSH-degrading ability to enrich O2
•– radicals. In this way, the A-Pd@MoO3–x
NH nanozyme can selectively and
remarkably kill tumor cells while keeping the normal cell line unharmed.
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