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PubMed2003

Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: prevalence of CO(2)-dependent auxotrophs and characterization of deficient arg genes in L. plantarum.

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO(2). We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO(2) requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.

Arginine↗

PubMed2001

Substrate specificity of the sialic acid biosynthetic pathway.

Unnatural analogues of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogues bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogues with ketone-containing N-acyl groups that varied in the length or steric bulk was chemically synthesized and tested for metabolic conversion to cell surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

Carbon↗

PubMed2004

DpgC is a metal- and cofactor-free 3,5-dihydroxyphenylacetyl-CoA 1,2-dioxygenase in the vancomycin biosynthetic pathway.

3,5-Dihydroxyphenylglycine is a crucial amino acid monomer in the nonribosomal glycopeptide antibiotic vancomycin. This nonproteinogenic amino acid is constructed from malonyl-CoA by a set of four enzymes, DpgA-D, in the biosynthetic cluster. DpgC is an unusual metal-free, cofactor-free enzyme that consumes O(2) during the conversion of 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) to the penultimate intermediate 3,5-dihydroxyphenylglyoxylate (DPGx). We show that in anaerobic incubations, DpgC catalyzes the exchange of the C(2)-methylene hydrogens of DPA-CoA at unequal rates, consistent with enzyme-mediated formation of the substrate-derived C(2)-carbanion as an early intermediate. Incubations with (18)O(2) reveal that DpgC transfers both atoms of an O(2) molecule to DPGx product. This establishes DpgC as a 1,2-dioxygenase that mediates thioester cleavage by the oxygen transfer process. These results are consistent with a DPA-CoA C(2)-peroxy intermediate, followed by enzyme-directed alpha-peroxylactone formation and collapse by O-O bond cleavage.

Anaerobiosis↗

PubMed1995

Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species.

Polyketides are an extensive class of secondary metabolites with diverse molecular structures and biological activities. A plasmid-based multicomponent polyketide synthase expression cassette was constructed using a subset of actinorhodin (act) biosynthetic genes (actI-orf1, actI-orf2, actI-orf3, actIII, actVII, and actIV) from Streptomyces coelicolor which specify the construction of the anthraquinone product aloesaponarin II, a molecule derived from acetyl coenzyme A and 7 malonyl coenzyme A extender units. This system was designed as an indicator pathway in Streptomyces parvulus to quantify polyketide product formation and to examine the functional significance of specific polyketide synthase components, including the act beta-ketoacyl synthase (beta-KS; encoded by actI-orf1 and actI-orf2) and the act cyclase/dehydrase (encoded by actVII-orf4). Site-directed mutagenesis of the putative active site Cys (to a Gln) in the actI-orf1 beta-KS product completely abrogated aloesaponarin II production. Changing the putative acyltransferase active-site Ser (to a Leu) located in the actI-orf1 beta-KS product led to significantly reduced but continued production of aloesaponarin II. Replacement of the expression cassette with one containing a mutant form of actI-orf2 gave no production of aloesaponarin II or any other detectable polyketide products. However, an expression cassette containing a mutant form of actVII-orf4 gave primarily mutactin with low-level production of aloesaponarin II.

Anthraquinones↗

PubMed2000

Metabolism of chiral ionylideneacetic acids on the abscisic acid biosynthetic pathway in Cercospora.

A Chiralcel OJ column was used to determine the absolute configuration of naturally occurring alpha-ionylideneacetic acid from Cercospora rosicola and gamma-ionylideneacetic acid from C. cruenta as (R) enantiomers in accordance with their biosynthetic product, (S)-ABA. Both enantiomers of [1, 2-(13)C2]-alpha and gamma-ionylideneacetic acids were prepared and fed to C. rosicola and C. cruenta. Six combinations of feeding experiments comparatively and unequivocally demonstrated stereoselectivity in the biosynthetic conversions, including stepwise hydroxylation at C-1' and 4'. Enzymatic isomerization from the gamma to alpha-intermediate was suggested not to be involved in ABA biosynthesis in C. rosicola.

Abscisic Acid↗

PubMed2005

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp.

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.

Amino Acid Sequence↗

PubMed1975

Pyrimidine biosynthetic pathway of Baccillus subtilis.

Biochemical and genetic data were obtained from a series of 51 Pyr- strains of Bacillus subtilis. The observed enzymatic deficiencies allowed the mutants to be placed into 12 clases, some of which represent defects in more than one of the six known pyrimidine biosynthetic enzymes. Mapping analysis by transformation has shown that all the Pyr- mutations are located in a single small area of the B. subtilis genome. A correlation of the biochemical defects and the genetic data has been made. Those mutations conferring similar enzymatic deficiencies were found to be contiguous on the B. subtilis map. Regulatory aspects of the pyrimidine pathway have also been investigated and are compared to previously reported results from other organisms. Evidence is presented which bears upon the possible physical association of the first three enzymes and the association of at least some of the enzymes of this pathway with particulate elements of the cell. A model for the organization of the enzymes is presented with dihydroorotate dehydrogenase as the central enzyme in a proposed aggregate.

Aspartate Carbamoyltransferase↗

PubMed2004

Total synthesis of macquarimicins using an intramolecular Diels-Alder approach inspired by a biosynthetic pathway.

A total synthesis of the macquarimicins A-C (1-3), novel natural products with intriguing tetra- or pentacyclic frameworks, has been achieved. The synthesis features an extensive investigation of the biosynthesis-based intramolecular Diels-Alder (IMDA) reactions of (E,Z,E)-1,6,8-nonatrienes. Considering possible biosynthetic sequences, four types of substrates were synthesized, and their IMDA reactions were examined. From one of the four substrates, the total synthesis was achieved via a transannular Diels-Alder reaction, which led to the stereoselective construction of the unique molecular framework. The convergent and efficient synthetic pathway afforded (+)-1 in 27 linear steps with 4.3% and 9.9% overall yields from readily available ethyl (2E,4S)-4,5-(isopropylidene)dioxy-2-pentenoate (22) and (R)-epichlorohydrin (30), respectively. Furthermore, efficient syntheses of 2, 3, and the 9-epi-cochleamycins A (57) and B (58) were accomplished. Additionally, the present work established the absolute stereochemistry of macquarimicins and revised the C(2)--C(3) geometry of 1.

Biomimetics↗

PubMed1992

The evolution of aminoacyl-tRNA synthetases, the biosynthetic pathways of amino acids and the genetic code.

In this paper the partition metric is used to compare binary trees deriving from (i) the study of the evolutionary relationships between aminoacyl-tRNA synthetases, (ii) the physicochemical properties of amino acids and (iii) the biosynthetic relationships between amino acids. If the tree defining the evolutionary relationships between aminoacyl-tRNA synthetases is assumed to be a manifestation of the mechanism that originated the organization of the genetic code, then the results appear to indicate the following: the hypothesis that regards the genetic code as a map of the biosynthetic relationships between amino acids seems to explain the organization of the genetic code, at least as plausibly as the hypotheses that consider the physicochemical properties of amino acids as the main adaptive theme that lead to the structuring of the code.

Amino Acids↗

PubMed2000

Generation of hybrid elloramycin analogs by combinatorial biosynthesis using genes from anthracycline-type and macrolide biosynthetic pathways.

Elloramycin and oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tü2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6-dideoxyhexose attached to the macrolide antibiotic oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandrose and L-rhamnose were coexpressed with cos16F4 in S. albus. Three new hybrid elloramycin analogs were produced by these recombinant strains through combinatorial biosynthesis, containing elloramycinone or 12a-demethyl-elloramycinone (= 8-demethyl-tetracenomycin C) as aglycone moiety encoded by S. olivaceus genes and different sugar moieties, coded by the S. antibioticus genes. Among them is L-olivose, which is here described for the first time as a sugar moiety of a natural product.

Anthracyclines↗

PubMed1982

The biosynthetic pathway of ecdysone: studies with vitellogenic ovaries of Locusta migratoria (Orthoptera).

Ovaries of adult females of Locusta migratoria synthesize impressive amounts of the steroid hormone ecdysone (and related ecdysteroids) during the late phases of vitellogenesis. The present study, aimed at elucidating the sequence of the biosynthetic steps that lead from cholesterol to ecdysone, has taken benefit of this remarkable biological model by using a double approach: (1) isolation and physico-chemical identification of endogenous biogenetic intermediates; (2) metabolic study of labelled putative precursor molecules. The data presented in this paper lead us to propose the following sequence of events: conversion of cholesterol to 3 beta-hydroxy-5 beta-cholest-7-en-6-one (via several intermediates not identified in this study) followed by 14 beta-hydroxylation to 3 beta, 14 alpha-dihydroxy-5 beta-cholest-7-en-6-one; hydroxylation on the side-chain at C-25 and C-22 (in this order) to 2-deoxyecdysone; hydroxylation at C-2 to ecdysone.

Animals↗

PubMed2005

Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2-2: designing new cytotoxic compounds.

Safracin is an antibiotic with anti-tumour activity produced by Pseudomonas fluorescens A2-2. The entire safracin synthetic gene cluster spanning 17.5 kb has been identified, cloned and sequenced. The safracin cluster comprises 10 open reading frames (ORFs) encoding proteins for three non-ribosomal peptide synthetases (NRPS), three safracin precursor biosynthetic enzymes, two safracin tailoring enzymes, a safracin resistance protein and a small hypothetical protein of unknown function. These genes are organized in two divergent operons of eight and two genes respectively. This pathway exhibits unusual features when compared with other NRPS systems. We have demonstrated by heterologous expression of the cluster that it is able to direct the synthesis of safracin in other strains. Cross-feeding experiments have confirmed that 3-hydroxy-5-methyl-O-methyltyrosine is the precursor of two amino acids of the molecule. Genetic analyses have allowed us to demonstrate that the bicistronic operon encodes the hydroxylation and N-methylation activities of the pathway. The cloning and expression of the safracin cluster has settled the basis for the in vivo and in vitro production of a wide variety of compounds, such as the promising ecteinascidins anti-cancer compounds.

Anti-Bacterial Agents↗

PubMed1989

afsB stimulates transcription of the actinorhodin biosynthetic pathway in Streptomyces coelicolor A3(2) and Streptomyces lividans.

The pleiotropic regulatory gene, afsB, from Streptomyces coelicolor A3(2), possibly encoding a DNA-binding protein, is required for actinorhodin production in this organism. Northern blot hybridization using a DNA fragment covering part of the set of cloned actinorhodin biosynthetic gene cluster (act) as the probe showed lack of the act transcripts in an afsB-negative mutant of S. coelicolor A3(2); the transcripts were restored on introduction of a cloned afsB gene. Introduction of the cloned afsB gene into Streptomyces lividans stimulated transcription of the act genes under conditions in which they are normally silent in this strain, leading to production of actinorhodin in large quantity. These data show that afsB exerts its positive regulatory effect by means of transcriptional stimulation of its target genes.

Anthraquinones↗

PubMed1993

Evidence for a gene cluster involving trichothecene-pathway biosynthetic genes in Fusarium sporotrichioides.

Two overlapping cosmid clones (Cos1-1 and Cos9-1) carrying the Tox5 gene were isolated from a library of F. sporotrichioides strain NRRL 3299 genomic DNA. These cosmids were used to transform three T-2 toxin-deficient mutants that are blocked at different steps in the trichothecene pathway. Both cosmids restored T-2 toxin production to Tox3-1- or Tox4-1- mutants but neither restored T-2 toxin production to a Tox1-2- mutant. The production of T-2 toxin by the complemented Tox3-1- and Tox4-1- mutants, as well as the production of diacetoxycirpenol by the cosmid-transformed Tox1-2- mutant, were 2- to 10-fold higher than in strain NRRL 3299. In addition, those transformants carrying Cos9-1 produced significantly higher levels of trichothecenes than transformants carrying Cos1-1. Two different DNA fragments (FSC13-9 and FSC14-5), representing the region of overlap between the cosmid clones, were isolated. These fragments specifically complemented either the Tox3-1- mutant (FSC14-5) or the Tox4-1- mutant (FSC13-9). The trichothecene-production phenotype of these transformants was similar to NRRL 3299. These results suggest that two or more genes involved in the biosynthesis of trichothecenes are closely linked to Tox5.

Cloning, Molecular↗

PubMed2005

A new family of glucose-1-phosphate/glucosamine-1-phosphate nucleotidylyltransferase in the biosynthetic pathways for antibiotics.

Aminoglycoside antibiotics are composed of aminosugars and a unique aminocyclitol aglycon including 2-deoxystreptamine (DOS), streptidine, actinamine, etc., and nucleotidylyltransferases, sugar modifying enzymes, and glycosyltransferases appear to be essential for their biosynthesis. However, the genes encoding those enzymes were unable to be identified by a standard homology search in the butirosin biosynthetic btr gene cluster, except that the btrM gene appeared to be a glycosyltransfease. Disruption studies of the btrD gene indicated that BtrD was involved in the supply of a glycosyl donor immediately prior to the glycosylation of DOS giving paromamine. As anticipated, BtrD expressed in Escherichia coli was able to catalyze UDP-D-glucosamine formation from D-glucosamine-1-phosphate and UTP. Both dTTP and UTP were good NTP substrates, and D-glucose-1-phosphate and D-glucosamine-1-phosphate were good sugar phosphates for the enzyme reaction. This finding is the first to identify an enzyme which activates a sugar donor in the DOS-containing antibiotics. Interestingly, BtrD homologues have been reported as functionally unknown open reading frames (ORFs) in the biosynthetic gene clusters for several antibiotics including teicoplanin, balhimycin, chloroeremomycin, and mitomycin C. It appears therefore that gene clusters for antibiotic biosynthesis provide their own nucleotidylyltransferases, and the BtrD homologues are among the secondary metabolism specific enzymes.

Amino Acid Sequence↗

PubMed2005

A novel actinomycete strain de-replication approach based on the diversity of polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways.

The actinomycetes traditionally represent one of the most important sources for the discovery of new metabolites with biological activity; and many of these are described as being produced by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We present a strain characterization system based on the metabolic potential of microbial strains by targeting these biosynthetic genes. After an initial evaluation of the existing bias derived from the PCR detection in a well defined biosynthetic systems, we developed a new fingerprinting approach based on the restriction analysis of these PKS and NRPS amplified sequences. This method was applied to study the distribution of PKS and NRPS biosynthetic systems in a collection of wild-type actinomycetes isolated from tropical soil samples that were evaluated for the production of antimicrobial activities. We discuss the application of this tool as an alternative characterization approach for actinomycetes and we comment on the relationship observed between the presence of PKS-I, PKS-II and NRPS sequences and the antimicrobial activities observed in some of the microbial groups tested.

Actinobacteria↗

PubMed2003

Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous.

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

Basidiomycota↗

PubMed2002

Engineering deoxysugar biosynthetic pathways from antibiotic-producing microorganisms. A tool to produce novel glycosylated bioactive compounds.

A plasmid (pLN2) was generated in which genes involved in the biosynthesis of L-oleandrose in the oleandomycin producer Streptomyces antibioticus ATCC11891 were cloned. pLN2 was used to direct the biosynthesis of different deoxysugars by exchanging and/or adding genes from other antibiotic biosynthetic clusters. Transfer of the synthesized deoxysugars to the tetracenomycin C aglycon, 8-demethyl-tetracenomycin C, through the use of the "sugar flexible" glycosyltransferase ElmGT, validated the system. Several pLN2 derivatives were constructed by replacement of the oleU 4-ketoreductase gene by different 4-ketoreductase genes. Some of them, such as EryBIV and UrdR, reduced the keto group of the 4-keto intermediates, generating L-olivosyl and D-olivosyl derivatives, respectively. The system was also used to generate an L-rhamnosyl derivative (through a two-gene deletion) and an L-rhodinosyl derivative (through a combination of a gene replacement and a gene addition).

Anti-Bacterial Agents↗