The shikimic acid pathway and polyketide biosynthesis

The shikimic acid pathway, ubiquitous in microorganisms and plants, provides precursors for the biosynthesis of primary metabolites such as the aromatic amino acids and folic acid. Several branchpoints from the primary metabolic pathway also provide aromatic and, in some unusual cases, nonaromatic precursors for the biosynthesis of secondary metabolites. We report herein recent progress in the analysis of two unusual branches of the shikimic acid pathway in streptomycetes; the formation of the cyclohexanecarboxylic acid (CHC)-derived moiety of the antifungal agent ansatrienin and the dihydroxycyclohexanecarboxylic acid (DHCHC) starter unit for the biosynthesis of the immunosuppressant ascomycin. A gene for 1-cyclohexenylcarbonyl-CoA reductase, chcA, which plays a role in catalyzing three of the reductive steps leading from shikimic acid to CHC has been characterized from Streptomyces collinus. A cluster of six open reading frames (ORFs) has been identified by sequencing in both directions from chcA and the putative role of these in CHC biosynthesis is discussed. The individual steps involved in the biosynthesis of DHCHC from shikimic acid in Streptomyces hygroscopicus var ascomyceticus has been delineated and shown to be stereochemically and enzymatically distinct from the CHC pathway. A dehydroquinate dehydratase gene (dhq) likely involved in providing shikimic acid for both DHCHC biosynthesis and primary metabolism has been cloned, sequenced and characterized. Received 17 February 1998/ Accepted in revised form 26 April 1998     

Genetic manipulation of the biosynthetic process leading to phoslactomycins, potent protein phosphatase 2A inhibitors

Phoslactomycins (PLMs) represent an unusual structural class of natural products secreted by various streptomycetes, containing an α,β-unsaturated δ-lactone, an amino group, phosphate ester, conjugated diene and a cyclohexane ring. Phosphazomycins, phospholines and leustroducsins contain the same structural moieties, varying only in the acyl substituent at the C-18 hydroxyl position. These compounds possess either antifungal or antitumor activities or both. The antitumor activity of the PLM class of compounds has been attributed to a potent and selective inhibition of protein phosphatase 2A (PP2A). The cysteine-269 residue of PP2Ac-subunit has been shown to be the site of covalent modification by PLMs. In this article, we review previous work on the isolation, structure elucidation and biological activities of PLMs and related compounds and current status of our work on both PLM stability and genetic manipulation of the biosynthetic process. Our work has shown that PLM B is surprisingly stable in solution, with a pH optimum of 6. Preliminary biosynthetic studies utilizing isotopically labeled shikimic acid and cyclohexanecarboxylic acid (CHC) suggested PLM B to be a polyketide-type antibiotic synthesized using CHC as a starter unit. Using a gene (chcA) from a set of CHC-CoA biosynthesis genes from Streptomyces collinus as a probe, a 75 kb region of 29 ORFs encoding PLM biosynthesis was located in the genome of Streptomyces sp. strain HK803. Analysis and subsequent manipulation of plmS ₂ and plmR ₂ in the gene cluster has allowed for rational engineering of a strain that produces only one PLM analog, PLM B, at ninefold higher titers than the wild type strain. A strain producing PLM G (the penultimate intermediate in PLMs biosynthesis) has also been generated. Current work is aimed at selective in vitro acylation of PLM G with various carboxylic acids and a precursor-directed biosynthesis in a chcA deletion mutant with the aim of generating novel PLM analogs.     

Draft Genome Sequence of Streptomyces sp. Strain SS,Which Produces a Series of Uridyl Peptide Antibiotic Sansanmycins

Streptomyces sp. SS produces a series of uridyl peptide antibiotic sansanmycins. Here, we present a draft genome sequence of Streptomyces sp. SS containing the biosynthetic gene cluster for the antibiotics. The identification of the biosynthetic gene cluster of sansanmycins may provide further insight into biosynthetic mechanisms for uridyl peptide antibiotics.     

Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis: IDENTIFICATION AND CHARACTERIZATION OF THE GE81112 BIOSYNTHETIC GENE CLUSTER*

The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.     

Molecular Interaction of Novel Compound 2-Methylheptyl Isonicotinate Produced by Streptomyces sp. 201 with Dihydrodipicolinate Synthase (DHDPS) Enzyme of Mycobacterium tuberculosis for its Antibacterial Activity

Antibiotic resistance is a growing problem in multi-drug-resistant tuberculosis which is caused by Mycobacterium tuberculosis (MTB). Hence there is an urgent need for designing or developing a novel or potent anti-tubercular agent. The Lysine/DAP biosynthetic pathway is a promising target because of its role in cell wall and amino acid biosynthesis. In our study we performed a molecular docking analysis of a novel antibacterial isolated from Streptomyces sp. 201 at three different binding site of dihydrodipicolinate synthase (DHDPS) enzyme of MTB. The molecular docking studies suggest that the novel molecule shows favourable interaction at the three different binding sites as compared to five experimentally known inhibitors of DHDPS.     

Cloning and identification of saprolmycin biosynthetic gene cluster from Streptomyces sp. TK08046

Saprolmycins A–E are anti-Saprolegnia parasitica antibiotics. To identify the gene cluster for saprolmycin biosynthesis in Streptomyces sp. TK08046, polymerase chain reaction using aromatase and cyclase gene-specific primers was performed; the spr gene cluster, which codes for angucycline biosynthesis, was obtained from the strain. The cluster consists of 36 open reading frames, including minimal polyketide synthase, ketoreductase, aromatase, cyclase, oxygenase, and deoxy sugar biosynthetic genes, as defined by homology to the corresponding genes of the urdamycin, Sch-47554, and grincamycin biosynthetic gene clusters in Streptomyces fradiae, Streptomyces sp. SCC-2136, and Streptomyces lusitanus, respectively. To establish the function of the gene cluster, an expression cosmid vector containing all 36 open reading frames was introduced into Streptomyces lividans TK23. The transformant was confirmed to express the biosynthetic genes and produce saprolmycins by liquid chromatography–mass spectrometry analysis of the extract.     

Structures and biosynthesis of 12-membered macrocyclic depsipeptides from Streptomyces sp. ML55

Two novel depsipeptides (1–2) were isolated from Streptomyces sp. ML55 together with two known analogues (3–4). Their structures were elucidated using a combination of NMR experiments, as well as detailed MS/MS experiments. The biosynthetic pathway of isolated compounds was dissected by genome sequencing data analysis for a hybrid nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) assembly line.     

Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces

A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin.     

Identification and catalytic characterization of a nonribosomal peptide synthetase-like (NRPS-like) enzyme involved in the biosynthesis of echosides from Streptomyces sp. LZ35

Echosides, isolated from Streptomyces sp. LZ35, represent a class of para-terphenyl natural products that display DNA topoisomerase I and IIα inhibitory activities. By analyzing the genome draft of strain LZ35, the ech gene cluster was identified to be responsible for the biosynthesis of echosides, which was further confirmed by gene disruption and HPLC analysis. Meanwhile, the biosynthetic pathway for echosides was proposed. Furthermore, the echA-gene, encoding a tri-domain nonribosomal peptide synthetase (NRPS)-like enzyme, was identified as a polyporic acid synthetase and biochemically characterized in vitro. This is the first study to our knowledge on the biochemical characterization of an Actinobacteria quinone synthetase, which accepts phenylpyruvic acid as a native substrate. Therefore, our results may help investigate the function of other NRPS-like enzymes in Actinobacteria.     

Biochemical detection of a metallo-β-lactamase in carbapenem resistant strain ofStreptomyces sp. CN229 isolated from soil

Streptomyces sp. CN229 was isolated from Tunisia soil. This strain displayed antimicrobial activity against Gram positive and Gram negative bacteria. In addition it is resistant to most β-lactam antibiotics including imipenem and meropenem (MIC imipenem >70 μg/ml). Metallo-β-lactamase (MβL) production was confirmed by either imipenem MIC decrease in the presence of ethylene diamine tetraactic acid (EDTA) or the inhibition zone enhancement around EDTA-impregnated imipenem, or meropenem discs. Isolectric focusing analysis demonstrated the production of β-lactamase with pI of 5.8 that is inhibited by EDTA.Streptomyces sp. CN229 was screened for the imipenem resistance genes,bla _VIM andbla _IMP previously identified inPseudomonas aeruginosa. The presence of these genes was not confirmed by specific PCR analysis. We concluded that carbapenem resistance inStreptomyces sp. CN229 strain is mainly due to production of a novel carbapenemase. Our data show for the first time that MβL is produced byStreptomyces sp. MβL-mediated imipenem and meropenem resistance inStreptomyces is a cause for concern in the study of resistance evolution and antibiotic cluster biosynthetic genes.     

Biosynthesis of an engineered tautomycetin analogue via disruption of tmcK-encoding terminal decarboxylase in Streptomyces CK4412

Tautomycetin (TMC), produced by Streptomyces sp. CK4412, is an antifungal secondary metabolite with an unusual ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Recently, TMC was identified to possess additional biological functions including T cell-specific immunosuppressive and anti-cancer activities through differential inhibition of protein phosphatases, such as PP1, PP2A, and SHP2. These findings led us to isolate and characterize its entire biosynthetic and regulatory pathway gene cluster. In silico database comparisons revealed that the deduced products of two translationally coupled genes, a 666-bp tmcJ and a 1458-bp tmcK located on the 3′-terminus of the polyketide synthase gene, were found to have amino acid sequence homologies with putative bacterial decarboxylase genes. Targeted gene disruption of tmcK, but not tmcJ, from the Streptomyces sp. CK4412 chromosome resulted in production of a 5-deoxy-3″-carboxylic TMC. The tmcK mutant strain was functionally complemented using an integrative plasmid carrying tmcK and/or tmcJ–tmcK in order to restore TMC biosynthesis, a result suggesting that only TmcK is a functional TMC terminal decarboxylase. Unlike an authentic TMC, this engineered 5-deoxy-3″-carboxylic TMC analogue failed to show PP1 selectivity over PP2A, and it showed significantly reduced cytotoxicity against a human lung cancer cell line. These results imply that regio-specific modifications of TMC polyketide moiety, such as C3″-terminal carboxylation and/or C5-deketonization, could differentiate multiple biological activities in TMC produced from Streptomyces sp. CK4412.     

The cyclohexane moiety of rapamycin is derived from shikimic acid inStreptomyces hygroscopicus

Summary Although the addition of shikimic acid to the medium had no effect on the level of production of rapamycin byStreptomyces hygroscopicus,¹⁴C-shikimic acid was incorporated into rapamycin to a very high degree.¹³C-Shikimic acid was successfully prepared from 1-[¹³C]-glucose using a mutant ofKlebsiella pneumoniae, and used to label rapamycin. It was found that¹³C-shikimic acid was incorporated into the cyclohexane moiety of rapamycin, thereby establishing the shikimic acid pathway origin of the seven-carbon starter unit.     

Discovery of nosokophic acid,a predicted intermediate of moenomycins,from nosokomycin-producing Streptomyces sp. K04-0144

A new actinomycete metabolite designated nosokophic acid was isolated from the culture broth of nosokomycin-producing Streptomyces sp. K04-0144, and the structure was elucidated by various NMR experiments. Nosokophic acid was found to be 3-phosphoglycosyl-2-sesquiterpenyl dihydroxypropionic acid, a predicted biosynthetic intermediate of nosokomycin-related moenomycins. The compound showed no activity against MRSA, but potentiated imipenem activity against MRSA by 512-fold.     

Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces tsukubaensis’

‘Streptomyces tsukubaensis’ was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain ‘S. tsukubaensis’ NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in ‘S. tsukubaensis’ necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to ‘S. tsukubaensis’ have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non‐defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in ‘S. tsukubaensis’.     

Production of chromopyrrolic acid by coexpression of inkOD in a heterologous host Streptomyces albus

Two ink genes, inkO and inkD, responsible for the earliest steps of K252a biosynthetic pathway, from Nonomurea longicantena JCM 11136 were heterologously coexpressed in Streptomyces albus J1074. The resultant strain accumulated compound that was purified by HPLC and studied by NMR. Coexpression of inkOD yielded chromopyrrolic acid, the key intermediate in an indolocarbazole biosynthesis.     

Characterization of the bagremycin biosynthetic gene cluster in Streptomyces sp. Tü 4128

Bagremycin A and bagremycin B isolated from Streptomyces sp. Tü 4128 have activities against Gram-positive bacteria, fungi and also have a weak antitumor activity, which make them have great potential for development of novel antibiotics. Here, we report a draft genome 8,424,112 bp in length of S. sp. Tü 4128 by Illumina Hiseq2000, and identify the bagremycins biosynthetic gene cluster (BGC) by bioinformatics analysis. The putative bagremycins BGC includes 16 open reading frames (ORFs) with the functions of biosynthesis, resistance and regulation. Disruptions of relative genes and HPLC analysis of bagremycins production demonstrated that not all the genes within the BGC are responsible for the biosynthesis of bagremycins. In addition, the biosynthetic pathways of bagremycins are proposed for deeper inquiries into their intriguing biosynthetic mechanism.     

Novel desosaminyl derivatives of dihydrochalcomycin from a genetically engineered strain of <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

Dihydrochalcomycin from Streptomyces sp. KCTC 0041BP is a 16-membered macrolide antibiotic containing two deoxysugars (d-chalcose and d-mycinose) that are O-glycosylated at the C-5 and C-20 positions, respectively. The desosamine sugar cassette was constructed from pikromycin-deoxysugar biosynthetic genes and transformed into Streptomyces sp. GerSM1, which was engineered for deletion of the genes related to TDP-d-chalcose biosynthesis (gerB, gerN and gerMI). Novel 16-membered macrolides (5-O-desosaminyl derivatives of dihydrochalcomycin) were detected by ESI-MS, LC/MS, and MS/MS thereby demonstrating combinatorial biosynthesis of the deoxysugar in 16-membered macrolide antibiotics.     

免疫抑制剂他克莫司生物合成的研究进展

他克莫司(FK506)是一种具有免疫抑制活性的23元大环内酯类化合物,临床上广泛用于防止器官移植术后的免疫排斥反应。生物合成法是他克莫司制备方法的研究热点,但他克莫司生物合成的研究还存在一定生产技术上的瓶颈。基于此,本文主要从他克莫司代谢途径改造和发酵过程控制等方面对他克莫司生物合成进展进行综述,以期为今后突破他克莫司生物合成的技术瓶颈提供参考,进而利用代谢工程、发酵工程等技术提升他克莫司的生物合成水平。