Functional Gene-Guided Discovery of Type II Polyketides from Culturable Actinomycetes Associated with Soft Coral Scleronephthya sp

Compared with the actinomycetes in stone corals, the phylogenetic diversity of soft coral-associated culturable actinomycetes is essentially unexplored. Meanwhile, the knowledge of the natural products from coral-associated actinomycetes is very limited. In this study, thirty-two strains were isolated from the tissue of the soft coral Scleronephthya sp. in the East China Sea, which were grouped into eight genera by 16S rDNA phylogenetic analysis: Micromonospora, Gordonia, Mycobacterium, Nocardioides, Streptomyces, Cellulomonas, Dietzia and Rhodococcus. 6 Micromonospora strains and 4 Streptomyces strains were found to be with the potential for producing aromatic polyketides based on the analysis of KS(α) (ketoacyl-synthase) gene in the PKS II (type II polyketides synthase) gene cluster. Among the 6 Micromonospora strains, angucycline cyclase gene was amplified in 2 strains (A5-1 and A6-2), suggesting their potential in synthesizing angucyclines e.g. jadomycin. Under the guidance of functional gene prediction, one jadomycin B analogue (7b, 13-dihydro-7-O-methyl jadomycin B) was detected in the fermentation broth of Micromonospora sp. strain A5-1. This study highlights the phylogenetically diverse culturable actinomycetes associated with the tissue of soft coral Scleronephthya sp. and the potential of coral-derived actinomycetes especially Micromonospora in producing aromatic polyketides.     

芳香聚酮后修饰氧化酶

氧化酶在芳香聚酮生物合成后修饰中普遍存在并对终产物的结构产生关键影响。本文简要总结了芳香聚酮后修饰氧化酶中几类最常见的氧化酶的结构和功能,并以杰多霉素生物合成途径中的后修饰氧化酶为例,阐明这些氧化酶在后修饰反应中发生作用的方式。并对后修饰氧化酶在组合生物学中的应用做了展望。     

A method to type the potential angucycline producers in actinomycetes isolated from marine sponges

Angucyclines are aromatic polyketides with antimicrobial, antitumor, antiviral and enzyme inhibition activities. In this study, a new pair of degenerate primers targeting the cyclase genes that are involved in the aromatization of the first and/or second ring of angucycline, were designed and evaluated in a PCR protocol targeting the jadomycin cyclase gene of Streptomyces venezuelae ISP5230. The identity of the target amplicon was confirmed by sequencing. After validation, the primers were used to screen 49 actinomycete isolates from three different marine sponges to identify putative angucycline producers. Seven isolates were positively identified using this method. Sequence analysis of the positive amplicons confirmed their identity as putative angucycline cyclases with sequence highly similar to known angucycline cyclases. Phylogenetic analysis clustered these positives into the angucycline group of cyclases. Furthermore, amplifications of the seven isolates using ketosynthase-specific primers were positive, backing the results using the cyclase primers. Together these results provided strong support for the presence of angucycline biosynthetic genes in these isolates. The specific primer set targeting the cyclase can be used to identify putative angucycline producers among marine actinobacteria, and aid in the discovery of novel angucyclines.     

非典型角蒽环聚酮氧化开环酶的功能与进化

非典型角蒽环聚酮化合物是一类经过氧化重排反应形成的具有独特骨架结构的芳香聚酮类化合物。近年来的研究表明,尽管此类化合物具有多种多样的骨架结构,它们都是由共同的生物合成中间体Dehydrorabelomycin生成的。一个独特的加氧酶家族(称为非典型角蒽环氧化开环酶)催化了Dehydrorabelomycin的氧化碳-碳键断裂与重排反应。尽管这些酶属于同一个蛋白质家族,催化相同的底物发生氧化开环反应,但是通过不同的重排方式形成了对应于各自生物合成终产物的骨架结构,对这类化合物最终结构的形成起到了关键作用。对这一家族的加氧酶进行深入的催化功能与反应机理研究,不仅有助于对已知芳香聚酮的结构改造与新颖骨架结构芳香聚酮的发现,也有助于加深对于蛋白质序列进化与功能演化的认识。     

Biosynthesis of plant-specific flavones and flavonols in Streptomyces venezuelae

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3beta-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.     

鉴别杰多霉素生物合成后修饰氧化酶JadH中参与底物结合或催化的关键残基

JadH是羟化脱水双功能酶,参与杰多霉素生物合成中的聚酮后修饰反应,将2,3-dehydro-UWM6催化为dehydrorabelomycin。为了分析杰多霉素生物合成途径中后修饰氧化酶JadH结合、催化底物的关键氨基酸,构建了JadH与底物复合物的三维结构模型。利用该模型并结合JadH同源蛋白氨基酸序列比对分析,推测出JadH活性中心中可能参与底物结合或催化的关键氨基酸(R50、G51、L52、G53、F100、R221、I223、P295和G298)。通过定点突变及体外酶学实验对这些位点的突变体的催化活性进行评价,结果显示这些突变株活性均显著低于野生型,表明这9个氨基酸是JadH参与底物结合或催化的关键氨基酸。     

A repressor-response regulator gene pair controlling jadomycin B production in Streptomyces venezuelae ISP5230.

A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.     

Regulation of jadomycin B production in Streptomyces venezuelae ISP5230: involvement of a repressor gene, jadR2.

The nucleotide sequence of a region upstream of the type II polyketide synthase genes in the cluster for biosynthesis of the polyketide antibiotic jadomycin B in Streptomyces venezuelae contained an open reading frame encoding a sequence of 196 amino acids that resembeled sequences deduced for a group of repressor proteins. The strongest similarity was to EnvR of Escherichia coli, but the sequence also resembled MtrR, AcrR, TetC, and TcmR, all of which are involved in regulating resistance to antibiotics or toxic hydrophobic substances in the environment. Disruption of the nucleotide sequence of this putative S. venezuelae repressor gene (jadR2), by insertion of an apramycin resistance gene at an internal MluI site, and replacement of the chromosomal gene generated mutants that produced jadomycin B without the stress treatments (exposure to heat shock or to toxic concentrations of ethanol) required for jadomycin B production by the wild type. When cultures of the disruption mutants were ethanol stressed, they overproduced the antibiotic. From these results it was concluded that expression of the jadomycin B biosynthesis genes are negatively regulated by jadR2.     

Flavoprotein hydroxylase PgaE catalyzes two consecutive oxygen-dependent tailoring reactions in angucycline biosynthesis

A simplified model system composed of a NADPH-dependent flavoprotein hydroxylase PgaE and a short-chain alcohol dehydrogenase/reductase (SDR) CabV was used to dissect a multistep angucycline modification redox cascade into several subreactions in vitro. We demonstrate that the two enzymes are sufficient for the conversion of angucycline substrate 2,3-dehydro-UWM6 to gaudimycin C. The flavoenzyme PgaE is shown to be responsible for two consecutive NADPH- and O(2)-dependent reactions, consistent with the enzyme-catalyzed incorporation of oxygen atoms at C-12 and C-12b in gaudimycin C. The two reactions do not significantly overlap, and the second catalytic cycle is initiated only after the original substrate 2,3-dehydro-UWM6 is nearly depleted. This allowed us to isolate the product of the first reaction at limiting NADPH concentrations and allowed the study of the qualitative and kinetic properties of the separated reactions. Dissection of the reaction cascade also allowed us to establish that the SDR reductase CabV catalyzes the final biosynthetic step, which is closely coupled to the second PgaE reaction. In the absence of CabV, the complete PgaE reaction leads invariably to product degradation, whereas in its presence, the reaction yields the final product, gaudimycin C. The result implies that the C-6 ketoreduction step catalyzed by CabV is required for stabilization of a reactive intermediate. The close relationship between PgaE and CabV would explain previous in vivo observations: why the absence of a reductase gene may result in the lack of C-12b-oxygenated species and, vice versa, why all C-12b-oxygenated angucyclines appear to have undergone reduction at position C-6.     

Novel jadomycins: incorporation of non-natural and natural amino acids

Electrospray ionization mass spectrometry of extracts from Streptomyces venezuelae ISP5230 cultures grown on chemically synthesized non-natural L-amino acids, D-amino acids or any of the 20 natural amino acids demonstrated incorporation of the amino acid into a jadomycin B analogue.     

The role of erythromycin C-12 hydroxylase, EryK, as a substitute for PikC hydroxylase in pikromycin biosynthesis

The substrate flexibility of the erythromycin C-12 hydroxylase from Saccharopolyspora erythraea, EryK, was investigated to test its potential for the generation of novel polyketide structures. We have shown that EryK can accept the substrates of PikC from Streptomyces venezuelae which is responsible for the hydroxylation of YC-17 and narbomycin. In a S. venezuelae pikC deletion mutant, EryK could catalyze the hydroxylation of YC-17 and narbomycin to generate methymycin/neomethymycin and pikromycin, respectively. Molecular modeling of the enzyme-substrate complex suggested the possible interaction of EryK with alternative substrates. The results indicate that EryK is flexible toward some alternative polyketides and can be useful for structural diversification of macrolides by post-polyketide synthase hydroxylation.     

Biochemistry and genetics of chloramphenicol production

In Streptomyces venezuelae, chloramphenicol is derived by an unusual diversion of chorismate, the branchpoint intermediate of the pathway involved in the biosynthesis of aromatic amino acids. In the chloramphenicol-producing organism, the DAHP synthetase was neither feedback inhibited nor repressed. Chorismate mutase was not repressed or inhibited by the intermediates or end-products of the shikimate-chorismate pathway. However, anthranilate synthetase and prephenate dehydratase are feedback inhibited by tryptophan and phenylalanine, respectively. During growth, when primary metabolism is not perfectly coordinated, decreasing demand for aromatic amino acids results in shunting of chorismate towards chloramphenicol biosynthesis.The endogenous synthesis of chloramphenicol produced by Streptomyces venezuelae is inhibited by the increasing concentration of chloramphenicol in the medium. Arylamine synthetase, the first enzyme involved in chloramphenicol biosynthesis, is repressed by the secreted chloramphenicol, by dl-p-aminophenylalanine and l-threo-p-aminophenylserinol. The excess intracellular chorismate pool is diverted to other aromatic shunt metabolites if biosynthesis of chloramphenicol is inhibited. There appears to be a glutamine binding protein subunit which is shared by several enzymes involved in amination of the aromatic ring of chorismate.Chloramphenicol producing organism also inactivated intracellular chloramphenicol. However, the resistance of the streptomycetes is due to inducible impermeability of the organism to chloramphenicol during antibiotic production. Streptomyces venezuelae is sensitive to chloramphenicol when it is not engaged in antibiotic production. The resistance to and production of chloramphenicol are induced simultaneously.A linkage map for 17 marker loci using Streptomyces venezuelae has been constructed. Restriction enzyme map of a plasmid from the chloramphenicol-producing streptomycetes has also been developed. The role of the plasmid in chloramphenicol biosynthesis and the life-cycle of the Streptomyces venezuelae is not yet understood.     

芳香聚酮早期后修饰环化酶的结构分类和功能分析

聚酮是一类结构和生物活性多样的天然产物,根据结构特点可以分为芳香聚酮和复合聚酮两大类。芳香聚酮环化酶是芳香聚酮生物合成过程中一种非常重要的早期后修饰酶,是决定芳香聚酮骨架结构的主要影响因素。根据序列和结构的相似性,芳香聚酮环化酶可以分为不同的种类。本文主要对其中3类芳香聚酮环化酶结构和功能进行了简要总结,从晶体结构、催化反应和催化机制等方面对它们进行了分类描述和功能分析,并结合自己实验室工作介绍了杰多霉素B环化酶催化机制的研究方法。     

The Streptomyces peucetius dpsY and dnrX Genes Govern Early and Late Steps of Daunorubicin and Doxorubicin Biosynthesis

The Streptomyces peucetius dpsY and dnrX genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of dpsY and dnrX. Replacement of dpsY with a mutant form disrupted by insertion of the aphII neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of dnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that dnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.     

Purification, properties and immunological detection of a bromoperoxidase-catalase from Streptomyces venezuelae and from a chloramphenicol-nonproducing mutant

A new bromoperoxidase-catalase was purified from the chloramphenicol-producing actinomycete Streptomyces venezuelae ISP 5230. The homogeneous enzyme showed brominating activity, catalase activity and a very low peroxidase activity. The spectral properties and pH dependence of the catalase activity showed similarities to conventional catalases. In contrast to other haem-bromoperoxidases, the bromoperoxidase-catalase was stable when treated with an ethanol/chloroform mixture. Gel filtration gave an estimated Mr of 127,000-136,000. SDS-PAGE showed a single band corresponding in mobility to a species with an Mr of 61,000. The pI was estimated to be 4.5. The bromoperoxidase-catalase was not present in active form in a mutant of S. venezuelae ISP 5230, blocked in the chlorination step of chloramphenicol biosynthesis. However, an inactive species of the enzyme was detected in crude extracts of the mutant by using antibodies. From these results it is concluded that this bromoperoxidase participates in the chlorination step during chloramphenicol biosynthesis.     

Functional analysis of desVIII homologues involved in glycosylation of macrolide antibiotics by interspecies complementation

The DesVIII is an auxiliary protein which enhances the transfer of TDP-d-desosamine catalyzed by DesVII glycosyltransferase in the biosynthesis of macrolide antibiotics, neomethymycin, methymycin and pikromycin, in Streptomyces venezuelae ATCC 15439. Homologues of the desVIII gene are present in a number of aminosugar-containing antibiotic biosynthetic gene clusters including eryCII from the erythromycin producer Saccharopolyspora erythraea, oleP1 from the oleandomycin producer Streptomyces antibioticus, dnrQ from the doxorubicin producer Streptomyces peucetius, and tylMIII from the tylosin producer Streptomyces fradiae. In order to gain further insight into the function of these DesVIII homologues, interspecies complementation experiments were carried out by expressing each gene in a desVIII deletion mutant strain of S. venezuelae. Complementation by expressing EryCII, OleP1, and DnrQ in this mutant strain restored the production of glycosylated macrolides to an approximate level of 66%, 26% and 26%, respectively, compared to self-complementation by DesVIII. However, expression of TylMIII did not restore the antibiotic production. These results suggest that the DesVIII homologues (except for TylMIII) can functionally replace the native DesVIII for glycosylation to proceed in vivo and their functions are similar in acting as glycosyltransferase auxiliary proteins. The requirement of glycosyltransferase auxiliary protein seems to be more widespread in polyketide biosynthetic pathways than previously known.     

Structural basis for macrolactonization by the pikromycin thioesterase

Polyketides are a class of biologically active microbial and plant-derived metabolites that possess a high degree of structural and functional diversity and include many human therapeutics, among them anti-infective and anti-cancer drugs, growth promoters and anti-parasitic agents. The macrolide antibiotics, characterized by a glycoside-linked macrolactone, constitute an important class of polyketides, including erythromycin and the natural ketolide anti-infective agent pikromycin. Here we describe new mechanistic details of macrolactone ring formation catalyzed by the pikromycin polyketide synthase thioesterase domain from Streptomyces venezuelae. A pentaketide phosphonate mimic of the final pikromycin linear chain-elongation intermediate was synthesized and shown to be an active site affinity label. The crystal structures of the affinity-labeled enzyme and of a 12-membered-ring macrolactone product complex suggest a mechanism for cyclization in which a hydrophilic barrier in the enzyme and structural restraints of the substrate induce a curled conformation to direct macrolactone ring formation.     

Biosynthesis of glycosylated derivatives of tylosin in Streptomyces venezuelae

Streptomyces venezuelae YJ028, bearing a deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes, was used as a bioconversion system for combinatorial biosynthesis of glycosylated derivatives of tylosin. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of TDP-3-O-demethyl-D-chalcose or TDP-Lrhamnose in conjunction with the glycosyltransferaseauxiliary protein pair DesVII/DesVIII were expressed in a S. venezuelae YJ028 mutant strain. Supplementation of each mutant strain capable of producing TDP-3-O-demethyl- D-chalcose or TDP-L-rhamnose with tylosin aglycone tylactone resulted in the production of the 3-O-demethyl- D-chalcose, D-quinovose, or L-rhamnose-glycosylated tylactone.