Weixin Tao

Unusual Acetylation–Elimination in the Formation of Tetronate Antibiotics

Tetronate antibiotics comprise an important and growing family of polyketide natural products possessing a characteristic tetronate (4-hydroxy-[5H]furan-2-one) ring system. They have been isolated from both terrestrial and marine bacteria, and show a diverse range of biological activities. They include the tetronate polyethers tetronomycin and tetronasin, the fatty acyltetronate antibiotic agglomerin, and the structurally closely related protein phosphatase inhibitor RK682 (Figure 1). Of particular interest are the structurally intriguing spirotetronates (Figure 1a), including the antibacterial compounds chlorothricin and abyssomicin, the antiviral compound quartromicin, and the antitumour compounds tetrocarcin and kijanimicin. These compounds appear to arise through an enzyme-catalyzed Diels– Alder reaction after specific dehydration of an initially formed tetronate precursor, as shown for atrop-abyssomicin C in Figure 1b. Analysis of the biosynthetic gene clusters for several of these natural products 5–10] has highlighted the presence of a set of highly conserved genes unique to tetronate biosynthesis, whose predicted products include candidate enzymes that might catalyze formation of the tetronate ring C C and C O bonds. Reconstitution of RK-682 biosynthesis in vitro has been used to show that RkE is a glyceryl-S-acyl carrier protein (ACP) synthase, and that the ketoacyl-S-ACP synthase FabH-like RkD is necessary and sufficient to catalyze formation of the tetronate ring in vitro starting from a 3-ketoacyl thioester and glyceryl-S-ACP. Similar results were recently obtained for the FabH-like QmnD5 in quartromicin biosynthesis and it seems highly likely that most (if not all) tetronates follow an analogous biosynthetic pathway. Until now, the course of the dehydration step in spirotetronate biosynthesis, which provides the dienophile for the ensuing Diels–Alder-like reaction, has remained obscure. We show here, by cloning and analysis of the gene cluster for biosynthesis of agglomerins A–D in Pantoea agglomerans (formerly Enterobacter agglomerans) PB-6042, its heterologous expression in Escherichia coli, and the total reconstitution of agglomerin biosynthesis in vitro, that the mechanism of dehydration actually involves two steps after formation of the tetronate ring: O-acetylation catalyzed by Agg4, followed by elimination of acetic acid to form the exocyclic double bond catalyzed by Agg5. We propose that the biosynthesis of spirotetronates involves the same two-step reaction sequence, catalyzed by enzymes homologous to Agg4 and Agg5. The agglomerin biosynthetic pathway of P. agglomerans provided an attractive system in which to study these key steps, given the relative simplicity of the structures of agglomerin A and its congeners agglomerins B–D, which differ from each other only in the nature of the fatty acyl sidechain (Figure 1). Cosmid and whole-genome sequencing of P. agglomerans PB-6042 was used to reveal a circular chromosome of approximately 4.3 Mbp (bp = base pairs), within which the agglomerin cluster was readily identified through its similarity to that of RK-682 (Supporting Information, Figure S1). A 12 kbp DNA sequence encodes seven ORFs that could be plausibly assigned to the cluster. As well as the expected high homology between several genes in the agg and rk clusters, key differences in enzymology could also be inferred from the comparison of these clusters: whereas RK-682 obtains its linear precursor from palmitic acid, which is activated and then elongated on a modular polyketide synthase (PKS) to give 3-oxo-stearoyl-S-ACP, no counterpart of the rkC PKS could be found anywhere on the P. agglomerans chromosome. The precursors for agglomerins A–D appear to be taken directly from primary metabolism, probably as the corresponding 3-oxoacyl-CoA thioesters. In support of this, when the seven genes of the putative [*] C. Kanchanabanca, Dr. H. Hong, Dr. M. Samborskyy, Prof. Dr. P. F. Leadlay Department of Biochemistry, University of Cambridge 80 Tennis Court Road, Cambridge CB2 1GA (UK) E-mail: pfl10@cam.ac.uk Dr. W. Tao, Y. Liu, Prof. Dr. Z. Deng, Prof. Dr. Y. Sun Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and School of Pharmaceutical Sciences, Wuhan University 185 East Lake Road, Wuchan 430071 (P.R.China) E-mail: yhsun@whu.edu.cn

Inactivation of Chloramphenicol and Florfenicol by a Novel Chloramphenicol Hydrolase

ABSTRACT Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.

Isolation and characterization of a family VII esterase derived from alluvial soil metagenomic library

A novel esterase gene, estDL30, was isolated from an alluvial metagenomic library using function-driven screening. estDL30 consisted of 1,524 nucleotides and encoded a 507-amino acid protein. Sequence analysis revealed that EstDL30 is similar to many type B carboxylesterases, containing a G-E-S-A-G pentapeptide with a catalytic Ser residue. Phylogenetic analysis suggested that EstDL30 belongs to the family VII lipases, together with esterases from Bacillus subtilis (P37967), Streptomyces coelicolor A3(2) (CAA22794), and Arthrobacter oxydans (Q01470). Purified EstDL30 showed its highest catalytic efficiency toward p-nitrophenyl butyrate, with a kcat of 2293 s−1 and kcat/Km of 176.4 s−1mM−1; however, little activity was detected when the acyl chain length exceeded C8. Biochemical characterization of EstDL30 revealed that it is an alkaline esterase that possesses maximal activity at pH 8 and 40° C. The effects of denaturants and divalent cations were also investigated. EstDL30 tolerated well the presence of methanol and Tween 20. Its activity was strongly inhibited by 1 mM Cu2+ and Zn2+, but stimulated by Fe2+. The unique properties of EstDL30, its high activity under alkaline conditions and stability in the presence of organic solvents, may render it applicable to organic synthesis.

Biosynthesis of tetronate antibiotics：A growing family of natural products with broad biological activities

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 was isolated, to elucidate the biosynthesis of natural tetronate products, by isotope-labeled feeding experiments, genetical characterization of biosynthetic gene clusters, and biochemical reconstitution of key enzymatic catalyzed reactions. Accordingly, the biosynthesis of spirotetronates has been gradually determined, including biosynthesis of a polyketide-derived backbone for spirotetronate aglycone, incorporation of a glycerol-derived three-carbon unit into tetronic acid moiety, formation of mature aglycone via Diels-Alder-like reaction, and decorations of aglycone with various deoxysugar moieties. In this paper, the biosynthetic investigations of natural tetronates are well documented and a common biosynthetic route for this group of natural products is summarized accordingly.

In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing

ABSTRACT 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 vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters. IMPORTANCE Recent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways. Recent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.

Soil metagenome-derived 3-hydroxypalmitic acid methyl ester hydrolases suppress extracellular polysaccharide production in Ralstonia solanacearum

Autoinducers are indispensable for bacterial cell-cell communication. However, due to the reliance on culture-based techniques, few autoinducer-hydrolyzing enzymes are known. In this study, we characterized soil metagenome-derived unique enzymes capable of hydrolyzing 3-hydroxypalmitic acid methyl ester (3-OH PAME), an autoinducer of the plant pathogenic bacterium Ralstonia solanacearum. Among 146 candidate lipolytic clones from a soil metagenome library, 4 unique enzymes capable of hydrolyzing the autoinducer 3-OH PAME, termed ELP86, ELP96, ELP104, and EstDL33, were selected and characterized. Phylogenetic analysis revealed that metagenomic enzymes were novel esterase/lipase candidates as they clustered as novel subfamilies of family I, V, X, and family XI. The purified enzymes displayed various levels of hydrolytic activities towards 3-OH PAME with optimum activity at 40-50 °C and pH 7-10. Interestingly, ELP104 also displayed N-(3-oxohexanoyl)-L-homoserine lactone hydrolysis activity. Heterologous expression of the gene encoding 3-OH PAME hydrolase in R. solanacearum significantly decreased exopolysaccharide production without affecting bacterial growth. mRNA transcription analysis revealed that genes regulated by quorum-sensing, such as phcA and xpsR, were significantly down-regulated in the stationary growth phase of R. solanacearum. Therefore, metagenomic enzymes are capable of quorum-quenching by hydrolyzing the autoinducer 3-OH PAME, which could be used as a biocontrol strategy against bacterial wilt.

Functional Analysis of Cytochrome P450s Involved in Streptovaricin Biosynthesis and Generation of Anti-MRSA Analogues

The streptovaricins, chemically related to the rifamycins, are highly effective antibacterial agents, particularly against mycobacteria. Herein, a bioassay-guided investigation of Streptomyces spectabilis CCTCC M2017417 has led to the characterization of streptovaricins as potent compounds against methicillin-resistant Staphylococcus aureus (MRSA). We identified the streptovaricin biosynthetic gene cluster from S. spectabilis CCTCC M2017417 based on genomic sequencing and bioinformatic analysis. Targeted in-frame deletion of five cytochrome P450 genes (stvP1-P5) resulted in the identification of four new streptovaricin analogues and revealed the functions of these genes as follows: stvP1, stvP4, and stvP5 are responsible for the hydroxylation of C-20, Me-24, and C-28, respectively. stvP2 is possibly involved in formation of the methylenedioxy bridge, and stvP3, a conserved gene found in the biosynthetic cluster for naphthalenic ansamycins, might be related to the formation of a naphthalene ring. Biochemical verification of the hydroxylase activity of StvP1, StvP4, and StvP5 was performed, and StvP1 showed unexpected biocatalytic specificity and promiscuity. More importantly, anti-MRSA studies of streptovaricins and derivatives revealed significant structure-activity relationships (SARs): The hydroxyl group at C-28 plays a vital role in antibacterial activity. The hydroxyl group at C-20 substantially enhances activity in the absence of the methoxycarbonyl side chain at C-24, which can increase the activity regardless of the presence of a hydroxyl group at C-20. The inner lactone ring between C-21 and C-24 shows a positive effect on activity. This work provides meaningful information on the SARs of streptovaricins and demonstrates the utility of the engineering of streptovaricins to yield novel anti-MRSA molecules.

CRISPR/Cas9-Based Editing of Streptomyces for Discovery, Characterization, and Production of Natural Products

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. However, the majority of these BGCs are silent or poorly expressed in native strains and remain to be activated and investigated, which relies heavily on efficient genome editing approaches. Accordingly, numerous strategies are developed, especially, the most recently developed, namely, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) system reveals remarkable higher accuracy and efficiency for genome editing in various model organisms including the Streptomyces. In this mini review, we highlight the application of CRISPR/Cas9-based approaches in Streptomyces, focus on the editing of BGCs either in vivo or in vitro, as well as target cloning of large-sized BGCs and heterologous expression in a genetically manipulatable host, for discovery, characterization, reengineering, and production of potential pharmaceutical drugs.