Yuhui Sun

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Verified

Yuhui verified their affiliation via an institutional email.

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• Professor
• Principal Investigator at Huazhong University of Science and Technology

The PKS genes for biosynthesis of the polyether nanchangmycin are organized to encode two sets of proteins (six and seven ORFs, respectively), but are separated by independent ORFs that encode an epimerase, epoxidase, and epoxide hydrolase, and, notably, an independent ACP. One of the PKS modules lacks a corresponding ACP. We propose that the proce...

The complete DNA sequence of plasmid pHZ1358, a widely used vector for targeted gene disruption and replacement experiments in many Streptomyces hosts, has been determined. This has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pIJ101 of Streptomyces lividans 1326...

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in...

Gentamicin C complex from Micromonospora echinospora remains a globally important antibiotic, and there is revived interest in the semisynthesis of analogs that might show improved therapeutic properties. The complex consists of five components differing in their methylation pattern at one or more sites in the molecule. We show here, using specific...

Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER1/2 ins...

Base editors are effective tools for introducing base conversions without double‐strand breaks, showing broad applications in biotechnological and clinical areas. However, their non‐negligible bystander mutations and off‐target effects have raised extensive safety concerns. To address these issues, a novel method is developed by inserting specific...

Non‐ribosomal peptide synthetases (NRPSs) are key enzymes in pharmaceutical synthesis, with condensation (C) domains catalyzing amide bond formation between aminoacyl substrates. However, recent research has elucidated that the catalytic capabilities of C domains extend beyond the traditional formation of peptide bonds. In this study, we elucidate...

Branched aromatic α-amino acids are valuable building blocks in natural products and pharmaceutically active compounds. However, their chemical or enzymatic synthesis is challenging due to the presence of two stereocenters. We design phenylalanine ammonia lyases (PAL) variants for the direct asymmetric synthesis of β-branched aromatic α-amino acids...

Aminoglycosides are essential antibiotics used to treat severe infections caused mainly by Gram-negative bacteria. Gentamicin is an aminoglycoside and, despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation...

Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSC...

Aminoglycosides (AGs) are a class of antibiotics with a broad spectrum of activity. However, their use is limited by safety concerns associated with nephrotoxicity and ototoxicity, as well as drug resistance. To address these issues, semi-synthetic approaches for modifying natural AGs have generated new generations of AGs, however, with limited typ...

Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we proposed that a set of genes co-evolved with BGCs to support biosynthetic profici...

Base editors, which enable targeted locus nucleotide conversion in genomic DNA without double‐stranded breaks, have been engineered as powerful tools for biotechnological and clinical applications. However, the application of base editors is limited by their off‐target effects. Continuously expressed deaminases used for gene editing may lead to unw...

About two-thirds of small molecule drugs contain methyl group and it plays a very important role in the drug development. So, methyltransferases catalyzing the methylation have always attracted great attention. Hangtaimycin (HTM) is a potent hepatoprotective agent. Previous study showed that its biosynthetic gene cluster contained three methyltrans...

The soil-dwelling filamentous bacteria, Streptomyces, is widely known for its ability to produce numerous bioactive natural products. Despite many efforts toward their overproduction and reconstitution, our limited understanding of the relationship between the host's chromosome three dimension (3D) structure and the yield of the natural products es...

An improved synthesis for tryptophan-dehydrobutyrine diketopiperazine (TDD), a co-metabolite of the hybrid polyketide/non-ribosomal peptide hangtaimycin, starting from ʟ-tryptophan is presented. Comparison to TDD isolated from the hangtaimycin producer Streptomyces spectabilis confirmed its S configuration. The X-ray structure of the racemate shows...

Two new ansamycin derivatives, damavaricin H (1) and protostreptovaricin VI (2) were isolated from the Streptomyces spectabilis CCTCC M2017417 derived mutants of ΔstvP5 and ΔstvA2, respectively. The structures of 1 and 2 were established by analysis of the HRESIMS as well as 1D and 2D NMR datasets. The minimum inhibitory concentration (MIC) results...

Endowing mesophilic microorganisms with high-temperature resistance is highly desirable for industrial microbial fermentation. Here, we report a cold-shock protein (CspL) that is an RNA chaperone protein from a lactate producing thermophile strain ( Bacillus coagulans 2–6), which is able to recombinantly confer strong high-temperature resistance to...

Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathways of gentamicin have been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that th...

A bioassay‐guided fractionation led to the isolation of hangtaimycin (HTM) from Streptomyces spectabilis CCTCC M2017417 and the discovery of its hepatoprotective properties. Structure elucidation by NMR suggested the need for a structural revision. A putative HTM degradation product was also isolated and its structure was confirmed by total synthes...

Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin struc...

A new streptovaricin analogue, namely 3-desmethyl protostreptovaricin I (1), was isolated from the culture of the genetically engineered strain ΔstvM2 derived from Streptomyces spectabilis CCTCC M2017417. Its structure was elucidated on the basis of extensive spectroscopic analyses, including 1D and 2D NMR tests, and high resolution mass spectromet...

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P...

The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end‐product, has become a cornerstone of knowledge‐based genome mining. Here we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synth...

Two new spirotetronate natural products, lobophorin L (1) and lobophorin M (2), together with three known lobophorin-like spirotetronate antibiotics (3–5) and two known ansamycins (6–7), were isolated from the marine-derived Streptomyces sp. 4506. The structures of 1 and 2 were established on the basis of HRESIMS as well as 1D and 2D NMR datasets....

Background: The alarming spread of antimicrobial resistance requires the development of novel anti-infective drugs. Despite the recent research focus on the human microbiome and its likely value to understand and exploit inter-bacterial inhibitory phenomena as a source for antimicrobial strategies, the human microbiota has barely been investigated...

A new ring‐fused streptovaricin analogue, named ansavaricin J ( 1 ), together with the known compound ansavaricin E, were unprecedently isolated from the culture of the genetically modified strains ΔstvP5 and ΔstvP4 which derived from Streptomyces spectabilis CCTCC M2017417, respectively. Their structures were elucidated via comprehensive spectrosc...

Hygromycin B is an aminoglycoside antibiotic widely used in industry and biological research. However, most of its biosynthetic pathway has not been completely identified due to the immense difficulty in genetic manipulation of the producing strain. To address this problem, we developed an efficient system that combines clustered regularly interspa...

Enzymes that catalyse remarkable Diels–Alder-like [4+2] cyclizations have been previously implicated in the biosynthesis of spirotetronate and spirotetramate antibiotics. Biosynthesis of the polyether antibiotic tetronasin is not expected to require such steps, yet the tetronasin gene cluster encodes enzymes Tsn11 and Tsn15, which are homologous to...

Conventional CRISPR/Cas genetic manipulation has been profitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desired recombinants. We have adapted for Streptomyces recently-introduced cytidine base editor...

The readthrough of premature termination codons (PTCs) is a promising strategy for curing PTC‐causing diseases. In cancers, the p53 anti‐tumor activity is often disabled by forming premature terminated p53 protein (p53PTC). Currently, there are lack of p53PTC‐rescuing drugs. Herein we designed a feasible strategy for identifying p53PTC‐rescuing com...

Microbial natural products (NPs) especially of the Streptomyces genus have been regarded as an unparalleled resource for pharmaceutical drugs discovery. Moreover, recent progress in sequencing technologies and computational resources further reinforces to identify numerous NP biosynthetic gene clusters (BGCs) from the genomes of Streptomyces. Howev...

Hygromycin B is an aminoglycoside antibiotic with a structurally distinctive orthoester linkage. Despite its long history of use in industry and in the laboratory, its biosynthesis remains poorly understood. We show here, by in-frame gene deletion in vivo and detailed enzyme characterization in vitro, that formation of the unique orthoester moiety...

Polyketides are a large family of pharmaceutically important natural products, and the structural modification of their scaffolds is significant for drug development. Herein, we report one apo- and two complex structures (with a pentynylmalonyl and benzylmalonyl, respectively) of the broadly selective acyltransferase (AT) from the splenocin polyket...

Polyketides are a large family of pharmaceutically important natural products, and the structural modification of their scaffolds is significant for drug development. Herein, we report one apo- and two complex structures (with a pentynylmalonyl and benzylmalonyl, respectively) of the broadly selective acyltransferase (AT) from the splenocin polyket...

Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterised as an S-adenosyl-L-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3''-N-methylation of 3''-dehydro-3''-amino-gentamicin A2, an es...

Supplementary

Detailed analysis of the modular Type Ip olyketide synthase (PKS) involved in the biosynthesis of the margino-lactone azalomycin Fi nm angrove Streptomyces sp.2 11726 has shown that only nineteen extension modules are required to accomplish twenty cycles of polyketide chain elongation. Analysis of the products of aP KS mutant specifically inactivat...

Following the in vivo investigation of thiotetronate assembly in Lentzea sp. and in S. thiolactonus NRRL 15439 (Havemann et al., Chem. Commun., 2017, DOI: 10.1039/c6cc09933e), the minimal set of genes required for thiolactomycin production was determined through heterologous expression and the mechanism for polyketide assembly was established in vi...

Chemical 'chain termination' probes were utilised for the investigation of thiotetronate antibiotic biosynthesis in the filamentous bacteria Lentzea sp. and Streptomyces thiolactonus NRRL 15439. The use of these tools led to the capture of biosynthetic intermediates involved in the thiotetronate polyketide backbone assembly, providing first insight...

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an...

Details of all molecular biological materials and procedures, growth conditions and analytical data.

Polyoxin, produced by Streptomcyes cacaoi var. asoensis and Streptomyces aureochromogenes, contains two non-proteinogenic amino acids, carbamoylpolyoxamic acid (CPOAA) and polyoximic acid. Although the CPOAA moiety is highly unusual, its biosynthetic logic has remained enigmatic for decades. Here, we address CPOAA biosynthesis by reconstitution of...

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein, Cas9, is a RNA-guided endonuclease that uses RNA–DNA base pairing to recognize and cleave double-stranded DNA (dsDNA) with a protospacer adjacent motif (PAM). It is widely accepted that the most commonly used Streptococcus pyogenes Cas9 (SpyCas9) protei...

This is a detail protocol for highly efficient editing in Streptomyces by CRISPR/Cas9-CodA system

This is a detail protocol for in vitro CRISPR/Cas9 editing (ICE) system

Thiolactomycin (TLM) is a thiotetronate antibiotic that selectively targets bacterial fatty acid biosynthesis through inhibition of the b-ketoacyl-acyl carrier protein synthases (KASI/II) that catalyse chain elongation on the type II (dissociated) fatty acid synthase. It has proved effective in in vivo infection models of Mycobacterium tuberculosis...

The current diminishing returns in finding useful antibiotics and the occurrence of drug-resistance bacteria call for the need to find new antibiotics. Moreover, the whole genome sequencing revealed that the biosynthetic potential of Streptomyces, which has produced the highest numbers of approved and clinical-trial drugs, has been greatly underest...

Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamic...

Gentamicin C complex is a mixture of aminoglycoside antibiotics used to treat severe Gram-negative bacterial infections. We report here key features of the late-stage biosynthesis of gentamicins. We show that the intermediate gentamicin X2, a known substrate for C-methylation at C-6' to form G418 catalyzed by the radical SAM-dependent enzyme GenK,...

Carbonates are known to exist in natural products, but their biosynthesis has not been linked to any enzymes. An unusual fungal Baeyer-Villiger monooxygenase CcsB is now revealed to catalyze an in-line carbonate formation via a two-step mechanism of oxygen atom insertions.

A novel nitrilase superfamily amidase gene, designated azl13, was cloned from Streptomyces sp. 211726. Bioinformatic and biochemical analysis indicated that Azl13 belongs to a new subfamily in branch 13 of the nitrilase superfamily. His6-Azl13 was expressed in Escherichia coli BL21(DE3) and had the expected molecular mass of 31 kDa, and the enzymat...

Gentamicin C complex is a mixture of aminoglycoside antibiotics used to treat severe Gram-negative bacterial infections. We report here key features of the late-stage biosynthesis of gentamicins. We show that the intermediate gentamicin X2, a known substrate for C-methylation at C-6' to form G418 catalyzed by the radical SAM-dependent enzyme GenK,...

Tetronate antibiotics, a growing family of natural products featuring a characteristic tetronic acid moiety, are of importance and of particular interest for their typical structures, especially the spirotetronate structure, and corresponding versatile biological activities. Considerable efforts have persistently performed since the first tetronate...

The identity and reactivity of the intermediates in agglomerin biosynthesis were established and the respective roles of the acetyltransferase Agg4 and the eliminating enzyme Agg5 identified. It is proposed that enzymes homologous to Agg4 and Agg5 carry out the dehydration steps in all spirotetronate biosynthetic pathways. If this proves correct, i...

Say “when”: Analysis of the biosynthetic gene cluster for the polyether antibiotic and anticancer agent salinomycin (1) shows that its core structure is synthesised by a nine-multienzyme modular polyketide synthase. Deletion of the salC gene, which is required for oxidative cyclisation, has led to the detection of a novel metabolite whose structure...

Lankacidin biosynthesis is processed by an unusual NRPS/PKS. A previously unrecognised dehydratase domain was shown in knockout experiments to act together with the discrete dehydratase LkcB in lankacidin biosynthesis and may control the switch from the iterative to the modular mode of operation that is implied in this system.

The protein phosphatase inhibitor RK-682 is one of a number of potentially valuable tetronate polyketide natural products. Understanding how the tetronate ring is formed has been frustrated by the inaccessibility of the putative substrates. We report the heterologous expression of rk genes in Saccharopolyspora erythraea and reconstitution of the RK...

The protein phosphatase inhibitor RK-682 is one of a number of potentially valuable tetronate polyketide natural products. Understanding how the tetronate ring is formed has been frustrated by the inaccessibility of the putative substrates. We report the heterologous expression of rk genes in Saccharopolyspora erythraea and reconstitution of the RK...

Five meilingmycins, A to E, with A as the major component, were isolated from Streptomyces nanchangensis NS3226. Through nuclear magnetic resonance (NMR) characterization, meilingmycins A to E proved to be identical to reported milbemycins alpha11, alpha13, alpha14, beta1, and beta9, respectively. Sequencing of a previously cloned 103-kb region ide...

When an enoylreductase enzyme of a modular polyketide synthase reduces a propionate extender unit that has been newly added to the growing polyketide chain, the resulting methyl branch may have either S or R configuration. We have uncovered a correlation between the presence or absence of a unique tyrosine residue in the ER active site and the chir...

The biosynthetic gene cluster for tetronomycin (TMN), a polyether ionophoric antibiotic that contains four different types of ring, including the distinctive tetronic acid moiety, has been cloned from Streptomyces sp. NRRL11266. The sequenced tmn locus (113 234 bp) contains six modular polyketide synthase (PKS) genes and a further 27 open-reading f...

The biosynthetic pathway to the unusual tetronate ring of certain polyketide natural products, including the antibiotics abyssomicin and tetronomycin (TMN) and the antitumour compound chlorothricin (CHL), is presently unknown. The gene clusters governing chlorothricin and tetronomycin biosynthesis both contain a gene encoding an atypical member of...

Nigericin was among the first polyether ionophores to be discovered, but its biosynthesis remains obscure. The biosynthetic gene cluster for nigericin has been serendipitously cloned from Streptomyces sp. DSM4137, and deletion of this gene cluster abolished the production of both nigericin and the closely related metabolite abierixin. Detailed comp...

Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-p...

The polyketide synthase (PKS) for the biosynthesis of the polyether nanchangmycin lacks an apparent thioesterase comparable to the type I thioesterase domains of the modular PKSs responsible for macrolide biosynthesis. Three candidate polyether chain-releasing factors were examined. Both the putative CR domain and the NanE protein appeared to be ge...

A cluster encoding genes for the biosynthesis of meilingmycin, a macrolide antibiotic structurally similar to avermectin and milbemycin alpha11, was identified among seven uncharacterized polyketide synthase gene clusters isolated from Streptomyces nanchangensis NS3226 by hybridization with PCR products using primers derived from the sequences of a...

The chromosomal DNA of Streptomyces hygroscopicus 10-22, a derivative of strain 5102-6, was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestions with AseI gave 11 fragments with a total length of ca. 7.36 Mb. The AseI sites were mapped by analysis of overlapping chromosomal deletions in...

Streptomyces hygroscopicus 10-22 harbors a conjugative, autonomously replicating linear plasmid pHZ6 of ca. 70 kb, which shows no obvious homology with chromosomal DNA and is temperature-sensitive for replication, being stable in the host at 28 degrees C but easily lost at 37 degrees C. On a lawn of the wild-type S. hygroscopicus 10-22 cured of pHZ...

Several independent gene clusters containing varying lengths of type I polyketide synthase genes were isolated from 'Streptomyces nanchangensis' NS3226, a producer of nanchangmycin and meilingmycin. The former is a polyether compound similar to dianemycin and the latter is a macrolide compound similar to milbemycin, which shares the same macrolide...