Characterization of benzoyl coenzyme A biosynthesis genes in the enterocin-producing bacterium "Streptomyces maritimus"

The novel benzoyl coenzyme A (benzoyl-CoA) biosynthesis pathway in "Streptomyces maritimus" was investigated through a series of target-directed mutations. Genes involved in benzoyl-CoA formation were disrupted through single-crossover homologous recombination, and the resulting mutants were analyzed for their ability to biosynthesize the benzoyl-CoA-primed polyketide antibiotic enterocin. Inactivation of the unique phenylalanine ammonia-lyase-encoding gene encP was previously shown to be absolutely required for benzoyl-CoA formation in "S. maritimus". The fatty acid beta-oxidation-related genes encH, -I, and -J, on the other hand, are necessary but not required. In each case, the yield of benzoyl-CoA-primed enterocin dropped below wild-type levels. We attribute the reduced benzoyl-CoA formation in these specific mutants to functional substitution and cross-talk between the products of genes encH, -I, and -J and the enzyme homologues of primary metabolism. Disruption of the benzoate-CoA ligase encN gene did not perturb enterocin production, however, demonstrating that encN is extraneous and that benzoic acid is not a pathway intermediate. EncN rather serves as a substitute pathway for utilizing exogenous benzoic acid. These experiments provide further support that benzoyl-CoA is formed in a novel bacterial pathway that resembles the eukaryotic assembly of benzoyl-CoA from phenylalanine via a beta-oxidative path.     

Biochemical characterization of a prokaryotic phenylalanine ammonia lyase

The committed biosynthetic reaction to benzoyl-coenzyme A in the marine bacterium "Streptomyces maritimus" is carried out by the novel prokaryotic phenylalanine ammonia lyase (PAL) EncP, which converts the primary amino acid L-phenylalanine to trans-cinnamic acid. Recombinant EncP is specific for L-phenylalanine and shares many biochemical features with eukaryotic PALs, which are substantially larger proteins by approximately 200 amino acid residues.     

Heterologous production of flavanones in<Emphasis Type="Italic"> Escherichia coli</Emphasis>: potential for combinatorial biosynthesis of flavonoids in bacteria

Chalcones, the central precursor of flavonoids, are synthesized exclusively in plants from tyrosine and phenylalanine via the sequential reaction of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL) and chalcone synthase (CHS). Chalcones are converted into the corresponding flavanones by the action of chalcone isomerase (CHI), or non-enzymatically under alkaline conditions. PAL from the yeast Rhodotorula rubra, 4CL from an actinomycete Streptomyces coelicolor A3(2), and CHS from a licorice plant Glycyrrhiza echinata, assembled as artificial gene clusters in different organizations, were used for fermentation production of flavanones in Escherichia coli. Because the bacterial 4CL enzyme attaches CoA to both cinnamic acid and 4-coumaric acid, the designed biosynthetic pathway bypassed the C4H step. E. coli carrying one of the designed gene clusters produced about 450 μg naringenin/l from tyrosine and 750 μg pinocembrin/l from phenylalanine. The successful production of plant-specific flavanones in bacteria demonstrates the usefulness of combinatorial biosynthesis approaches not only for the production of various compounds of plant and animal origin but also for the construction of libraries of "unnatural" natural compounds. Dedicated to Professor Sir David Hopwood.     

Enzymatic total synthesis of enterocin polyketides

Polyketides are clinically important natural products that often require elaborate organic syntheses owing to their complex chemical structures. Here we report the multienzyme total synthesis of the Streptomyces maritimus enterocin and wailupemycin bacteriostatic agents in a single reaction vessel from simple benzoate and malonate substrates. To our knowledge, our results represent the first in vitro assembly of a complete type II polyketide synthase enzymatic pathway to natural products.     

Expression of bacterial tyrosine ammonia-lyase creates a novel p-coumaric acid pathway in the biosynthesis of phenylpropanoids in Arabidopsis

Some flavonoids are considered as beneficial compounds because they exhibit anticancer or antioxidant activity. In higher plants, flavonoids are secondary metabolites that are derived from phenylpropanoid biosynthetic pathway. A large number of phenylpropanoids are generated from p-coumaric acid, which is a derivative of the primary metabolite, phenylalanine. The first two steps in the phenylpropanoid biosynthetic pathway are catalyzed by phenylalanine ammonia-lyase and cinnamate 4-hydroxylase, and the coupling of these two enzymes forms a rate-limiting step in the pathway. For the generation of p-coumaric acid, the conversion from phenylalanine to p-coumaric acid that is catalyzed by two enzymes can be theoretically performed by a single enzyme, tyrosine ammonia-lyase (TAL) that catalyzes the conversion of tyrosine to p-coumaric acid in certain bacteria. To modify the p-coumaric acid pathway in plants, we isolated a gene encoding TAL from a photosynthetic bacterium, Rhodobacter sphaeroides, and introduced the gene (RsTAL) in Arabidopsis thaliana. Analysis of metabolites revealed that the ectopic over-expression of RsTAL leads to higher accumulation of anthocyanins in transgenic 5-day-old seedlings. On the other hand, 21-day-old seedlings of plants expressing RsTAL showed accumulation of higher amount of quercetin glycosides, sinapoyl and p-coumaroyl derivatives than control. These results indicate that ectopic expression of the RsTAL gene in Arabidopsis enhanced the metabolic flux into the phenylpropanoid pathway and resulted in increased accumulation of flavonoids and phenylpropanoids.     

Purification of histidase from Streptomyces griseus and nucleotide sequence of the hutH structural gene.

Histidine ammonia-lyase (histidase) was purified to homogeneity from vegetative mycelia of Streptomyces griseus. The enzyme was specific for L-histidine and showed no activity against the substrate analog, D-histidine. Histidinol phosphate was a potent competitive inhibitor. Histidase displayed saturation kinetics with no detectable sigmoidal response. Neither thiol reagents nor a variety of divalent cations had any effect on the activity of the purified enzyme. High concentrations of potassium cyanide inactivated histidase in the absence of its substrate or histidinol phosphate, suggesting that, as in other histidases, dehydroalanine plays an important role in catalysis. The N-terminal amino acid sequence of histidase was used to construct a mixed oligonucleotide probe to identify and clone the histidase structural gene, hutH, from genomic DNA of the wild-type strain of S. griseus. The cloned DNA restored the ability of a histidase structural gene mutant to grow on L-histidine as the sole nitrogen source. The deduced amino acid sequence of hutH shows significant relatedness with histidase from bacteria and a mammal as well as phenylalanine ammonia-lyase from plants and fungi.     

Cloning, analysis, and heterologous expression of a polyketide synthase gene cluster of Streptomyces curacoi.

Streptomyces curacoi produces curamycin, an antibiotic based on a modified orsellinic acid skeleton that is synthesized by the polyketide pathway. We have cloned, characterized, and partly sequenced a polyketide synthase gene cluster of S. curacoi. The sequence data reveal an organization of open reading frames that is similar to those of other polyketide synthetic clusters, although the biosynthetic products differ considerably in size and structure. We propose that one of the predicted open reading frames (curA) encodes polykeptide synthase, on the basis of its homology with other enzymes with similar functions. Expression of the cloned chromosomal fragment in the heterologous host S. lividans leads to the production of a brown pigment in large quantities. The analysis and expression of the cur genes for detailed molecular studies of the mechanism of polyketide biosynthesis is discussed.     

Genes and enzymes involved in caffeic acid biosynthesis in the actinomycete Saccharothrix espanaensis

The saccharomicins A and B, produced by the actinomycete Saccharothrix espanaensis, are oligosaccharide antibiotics. They consist of 17 monosaccharide units and the unique aglycon N-(m,p-dihydroxycinnamoyl)taurine. To investigate candidate genes responsible for the formation of trans-m,p-dihydroxycinnamic acid (caffeic acid) as part of the saccharomicin aglycon, gene expression experiments were carried out in Streptomyces fradiae XKS. It is shown that the biosynthetic pathway for trans-caffeic acid proceeds from L-tyrosine via trans-p-coumaric acid directly to trans-caffeic acid, since heterologous expression of sam8, encoding a tyrosine ammonia-lyase, led to the production of trans-p-hydroxycinnamic acid (coumaric acid), and coexpression of sam8 and sam5, the latter encoding a 4-coumarate 3-hydroxylase, led to the production of trans-m,p-dihydroxycinnamic acid. This is not in accordance with the general phenylpropanoid pathway in plants, where trans-p-coumaric acid is first activated before the 3-hydroxylation of its ring takes place.     

Effects of competitive inhibitors of phenylalanine ammonia-lyase on the levels of phenylpropanoid enzymes in Solatium tuberosum

Abstract The accumulation of chlorogenic acid in illuminated discs of Solanum tuberosum tuber tissue is accompanied by rapid but transient increases in the activity levels of the biosynthetic enzymes phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl-CoA : quinate hydroxycinna-moyl transferase. Exogenous D-phenylalanine and L-α-aminooxy-β-phenylpropionic acid, competitive inhibitors of phenylalanine ammonia-lyase, inhibit the accumulation of chlorogenic acid and presumably reduce the endogenous pools of pathway intermediates such as cinnamic acid. These treatments prolong the phase of increase in phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase activities and indicate that product feedback modulation is important in maintaining the interrelationship between the levels of these two enzymes during the later stages of induction. In contrast,L-α-aminooxy-β-phenylpropionic acid inhibits the development of hydroxycinnamoyl transferase in illuminated discs supporting the idea that the light-stimulated increase in phenylalanine ammonia-lyase activity causes an increase in cinnamic acid production which mediates the light-stimulated increase in hydroxycinnamoyl transferase activity.     

Cloning and characterization of a polyketide synthase gene from Streptomyces fradiae Tü2717, which carries the genes for biosynthesis of the angucycline antibiotic urdamycin A and a gene probably involved in its oxygenation.

A DNA fragment was cloned as cosmid purd8, which encodes a polyketide synthase involved in the production of the angucycline antibiotic urdamycin from Streptomyces fradiae Tü2717. Deletion of the polyketide synthase genes from the chromosome abolished urdamycin production. In addition, purd8 conferred urdamycin resistance on introduction into Streptomyces lividans TK24. Sequence analysis of 5.7 kb of purd8 revealed six open reading frames transcribed in the same direction. The deduced amino acid sequences of the six open reading frames strongly resemble proteins from known type II polyketide synthase gene clusters: a ketoacyl synthase, a chain length factor, an acyl carrier protein, a ketoreductase, a cyclase, and an oxygenase. Heterologous expression of the urdamycin genes encoding a ketoacyl synthase and a chain length factor in Streptomyces glaucescens tetracenomycin C-nonproducing mutants impaired in either the TcmK ketoacyl synthase or TcmL chain length factor resulted in the production of tetracenomycin C. Heterologous expression of a putative oxygenase gene from the urdamycin gene cluster in S. glaucescens GLA.O caused production of the hybrid antibiotic 6-hydroxy tetracenomycin C.     

Conversion of phenylalanine into tyrosine by portulaca callus

The incorporation of [(14)C]phenylalanine and [1,6-(14)C]shikimic acid into tyrosine was investigated in the callus of Portulaca grandiflora, var. JR (L.). By inhibiting phenylalanine with 1-alpha-aminooxy-beta-phenyl-propionic acid and tyrosinase with 1-cysteine-HCl and hydrazine-hydrate, the possible synthesis of tyrosine from phenylalanine was demonstrated. Tetrahydropterine sulfate was an effective activator of this pathway and tyrosine accumulation via 4-hydroxy-prephenic acid is regulated by feedback inhibition. l-alpha-Amminooxy-beta-phenylpropionic acid inhibits both phenylalanine ammonia-lyase and the production of phenylalanine from prephenic acid.     

Biosynthesis of epsilon-rhodomycinone from glucose by Streptomyces C5 and comparison with intermediary metabolism of other polyketide-producing streptomycetes

The catabolism of glucose by Streptomyces C5, a producer of anthracycline antibiotics, was investigated to determine the pathways that supply precursors for anthracycline biosynthesis. Carbons for the biosynthesis of epsilon-rhodomycinone, an anthracycline aglycone, from radiolabelled glucose were derived primarily from the Embden-Meyerhof-Parnas pathway, with a minor contribution from the pentose phosphate pathway. Furthermore, the anthracycline-producing strain, Streptomyces C5, as well as Streptomyces aureofaciens and Streptomyces lividans, strains that produce nonanthracycline polyketide antibiotics, displayed enzyme activities indicative of the Embden-Meyerhof-Parnas and pentose phosphate glycolytic pathways. As determined from labelling patterns, Streptomyces C5 apparently has a complete tricarboxylic acid cycle, but does not have a glyoxylate bypass pathway.     

The Suitability of a Quantitative Spectrophotometric Assay for Phenylalanine Ammonia-lyase Activity in Barley, Buckwheat, and Pea Seedlings

It has been suggested by others that the spectrophotometric assay of phenylalanine ammonia-lyase based on changes in absorbance at 290 nm may be complicated by a side reaction involving transamination from phenylalanine onto α-keto acids. This would lead to the production of phenylpyruvate which would spontaneously tautomerize and form an enol borate complex absorbing at this wavelength. We find that the inclusion of 1 ml of either 60 μm α-ketoglutarate or 500 μm phenylpyruvate in our 3-ml reaction mixtures has no significant effect on the spectrophotometric assay of phenylalanine ammonia-lyase in shoots from young seedlings of barley (Hordeum vulgare), buckwheat (Fagopyrum esculentum), or pea (Pisum sativum). Although these side reactions may be involved in preparations with very low enzyme activity, the spectrophotometric determination of phenylalanine ammonia-lyase based on changes in absorbance at 290 nm appears to be a reliable and sensitive technique in these seedlings.     

Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase.

Mutations in the tcmII-tcmIV region of the Streptomyces glaucescens chromosome block the C-3 and C-8 O-methylations of the polyketide antibiotic tetracenomycin C (Tcm C). The nucleotide sequence of this region reveals the presence of two genes, tcmN and tcmO, whose deduced protein products display similarity to the hydroxyindole O-methyl transferase of the bovine pineal gland, an enzyme that catalyzes a phenolic O-methylation analogous to those required for the biosynthesis of Tcm C. The deduced product of the tcmN gene also has an N-terminal domain that shows similarity to the putative ActVII and WhiE ORFVI proteins of Streptomyces coelicolor. The tcmN N-terminal domain can be separated from the remainder of the tcmN gene product, and when coupled on a plasmid with the Tcm C polyketide synthase genes (tcmKLM), this domain enables high-level production of an early, partially cyclized intermediate of Tcm C in a Tcm C- null mutant or in a heterologous host (Streptomyces lividans). By analogy to fatty acid biosynthesis, the tcmKLM polyketide synthase gene products are probably sufficient to produce the linear decaketide precursor of Tcm C; thus, the tcmN N-terminal domain is most likely responsible for one or more of the early cyclizations and, perhaps, the attendant dehydrations that lead to the partially cyclized intermediate. The tcmN gene therefore appears to encode a multifunctional cyclase-dehydratase-3-O-methyl transferase. The tcmO gene encodes the 8-O-methyl transferase.     

Modulation of L-phenylalanine ammonia-lyase by pathway intermediates in cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.)

The increase in extractable phenylalanine ammonia-lyase (PAL;EC 4.3.1.5.) activity induced in French bean cell suspension cultures in response to treatment with autoclaved ribonuclease A was inhibited by addition of the phenylpropanoid pathway intermediates cinnamic acid, 4-coumaric acid or ferulic acid. The effectiveness of inhibition was in the order cinnamic acid>4-coumaric acid>ferulic acid. Cinnamic acid also inhibited the PAL activity increase induced by dilution of the suspensions into an excess of fresh culture medium. Addition of low concentrations (<10^-5M) of the pathway intermediates to cultures at the time of application of ribonuclease gave variable responses ranging from inhibition to 30–40% stimulation of the PAL activity measured at 8 h. Following addition of pathway intermediates to cultures 4–5 h after ribonuclease treatment, rapid increases followed by equally rapid declines in PAL activity were observed. The cinnamic acid-stimulated increase in enzyme activity was unaffected by treatment with cycloheximide at a concentration which gave complete inhibition of the ribonuclease-induced response. However, cycloheximide completely abolished the subsequent decline in enzyme activity. Treatment of induced cultures with -aminooxy--phenylpropionic acid (AOPPA) resulted in increased but delayed rates of enzyme appearance when compared to controls not treated with the phenylalanine analogue. The results are discussed in relation to current views on the regulation of enzyme levels in higher plants.Abbreviations AOPPA -aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - AOA -aminooxyacetic acid     

Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-β-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates.