Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a

Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the β-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g⁻¹ of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.     

Elucidation of the 2-aminoethylphosphonate biosynthetic pathway in Tetrahymena pyriformis

The biosynthetic reaction pathway leading to the natural product, 2-aminoethylphosphonate in Tetrahymena pyriformis has been elucidated. Incubation of [32P]PEP and [14C]PEP with T.pyriformis cellular homogenate fortified with Mg2+ and alanine/pyridoxal phosphate, yielded 2-aminoethylphosphonate as the minor reaction product (2-5% yield) and phosphoglycerate and pyruvate plus orthophosphate as the major products. Inclusion of thiamine pyrophosphate in the reaction mixture increased the yield of 2-aminoethylphosphonate by a factor of 10. Incubation of phosphonoacetaldehyde or phosphonopyruvate in the cellular homogenate also provided 2-aminoethylphosphonate. The cellular homogenate catalyzed the transformation of phosphonoacetaldehyde to 2-aminoethylphosphonate in an ca. 80% yield. However, the maximum yield of 2-aminoethylphosphonic acid obtained by use of phosphonopyruvate was only 15%. The major reaction pathways induced by treatment of phosphonopyruvate with the cellular extract involved its competitive conversion to PEP and pyruvate plus orthophosphate.     

Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway

Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.4 μg g⁻¹ parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding a costunolide and parthenolide 3β-hydroxylase was identified opening up further options to improve the water solubility of parthenolide and therefore its potential as a drug.     

Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis

Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron‐chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non‐ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC‐MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl‐coenzyme A (CoA)‐hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)‐CoA synthase Hcs1 in U. maydis that HMG‐CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.     

Elucidation of a carboxylate O-methyltransferase NcmP in nocamycin biosynthetic pathway

Nocamycins belong to the tetramic acid family natural products and show potent antimicrobial activity. Recently, the biosynthetic gene cluster of nocamycin was identified from the rare actinomycete Saccharothrix syringae and an S-adenosylmethionine (SAM) dependent methyltransferase gene NcmP was found to be located within the gene cluster. In this report, the methyltransferase gene NcmP was disrupted and a new nocamycin intermediate nocamycin E was isolated from the mutant strain. Meanwhile, NcmP was heterologously expressed in Escherichia coli BL21 (DE3) and biochemically characterized as a carboxylate O-methyltransferase in nocamycin biosynthetic pathway. Compared to nocamycin I, nocamycin E showed inferior antibacterial activity, indicating the methyl group is essential to antibacterial activity.     

Elucidation of the biosynthetic pathway of the allelochemical sorgoleone using retrobiosynthetic NMR analysis

NMR analyses of the labeling pattern obtained using various 13C-labeled precursors indicated that both the lipid tail and the quinone head of sorgoleone, the main allelopathic component of the oily root exudate of Sorghum bicolor, were derived from acetate units, but that the two moieties were synthesized in different subcellular compartments. The 16:3 fatty acid precursor of the tail is synthesized by the combined action of fatty-acid synthase and desaturases most likely in the plastids. It is then exported out of the plastids and converted to 5-pentadecatriene resorcinol by a polyketide synthase. This resorcinol intermediate was identified in root hair extracts. The lipid resorcinol intermediate is then methylated by a S-adenosylmethionine-dependent O-methyltransferase and subsequently dihydroxylated by a P450 monooxygenase to yield the reduced form of sorgoleone.     

Elucidation of biosynthetic pathway of a plant hormone abscisic acid in phytopathogenic fungi

ABSTRACT

Abscisic acid (ABA) is one of the plant hormones that regulates physiological functions in various organisms, including plants, sponges, and humans. The biosynthetic machinery in plants is firmly established, while that in fungi is still unclear. Here, we elucidated the functions of the four biosynthetic genes, bcABA1-bcABA4, found in Botrytis cinerea by performing biotransformation experiments and in vitro enzymatic reactions with putative biosynthetic intermediates. The first-committed step is the cyclization of farnesyl diphosphate to give α-ionylideneethane catalyzed by a novel sesquiterpene synthase, BcABA3, which exhibits low amino acid sequence identities with sesquiterpene synthases. Subsequently, two cytochrome P450s, BcABA1 and BcABA2, mediate oxidative modifications of the cyclized product to afford 1?,4?-trans-dihydroxy-α-ionylideneacetic acid, which undergoes alcohol oxidation to furnish ABA. Our results demonstrated that production of ABA does not depend on the nucleotide sequence of bcABA genes. The present study set the stage to investigate the role of ABA in infections.     

Evidence for the presence of a novel biosynthetic pathway for norspermidine in Vibrio

Enzymatic studies of the cell extracts of Vibrio alginolyticus and V. parahaemolyticus provided evidence that there exists a novel biosynthetic pathway for norspermidine (NH2(CH2)3NH(CH2)3NH2), a major polyamine species. In this pathway, the Schiff base formed between aspartic beta-semialdehyde and 1,3-diaminopropane is first reduced by a NADPH-dependent enzyme to yield "carboxynorspermidine" (NH2(CH2)3NH(CH2)2CH(NH2)COOH), which is in turn decarboxylated by a pyridoxal phosphate dependent enzyme to form norspermidine. The end product and its intermediate were identified by gas chromatography - mass spectrometry. Experiments with L-[U-14C]aspartic acid resulted in appreciable incorporation of the label into norspermidine. Putrescine could replace 1,3-diaminopropane as a substrate to produce spermidine, but at a reduced rate. The enzyme activity was greatly enhanced by dithiothreitol. Since the activity of an aminopropyltransferase that utilizes decarboxylated S-adenosylmethionine as an aminopropyl group donor could not be detected in any of the cell extracts by our assay method, it was concluded that this novel pathway is primarily responsible for producing norspermidine and spermidine in these species.     

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.     

Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae

Genes of the post-squalene ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been overexpressed in a systematic approach with the aim to construct yeast strains that produce high amounts of sterols from a squalene-accumulating strain. This strain had previously been deregulated by overexpressing a truncated HMG-CoA reductase (tHMG1) in the main bottleneck of the early ergosterol pathway. The overexpression of the gene ERG1 (squalene epoxidase) induced a significant decrease of the direct substrate squalene, a high increase of lanosterol, and a small increase of later sterols. The overexpression of the ERG11 gene encoding the sterol-14alpha-demethylase resulted in a decrease of lanosterol and an increase of downstream sterols. When these two genes were simultaneously overexpressed, later sterols from zymosterol to ergosterol accumulated and the content of squalene was decreased about three-fold, indicating that these steps had limited the transformation of squalene into sterols. The total sterol content in this strain was three-fold higher than in a wild-type strain.     

Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli.

The most important function of carotenoid pigments, especially beta-carotene in higher plants, is to protect organisms against photooxidative damage (G. Britton, in T. W. Goodwin, ed., Plant Pigments--1988, 1988; N. I. Krinsky, in O. Isler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). beta-Carotene also functions as a precursor of vitamin A in mammals (G. A. J. Pitt, in I. Osler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). The enzymes and genes which mediate the biosynthesis of cyclic carotenoids such as beta-carotene are virtually unknown. We have elucidated for the first time the pathway for biosynthesis of these carotenoids at the level of enzyme-catalyzed reactions, using bacterial carotenoid biosynthesis genes. These genes were cloned from a phytopathogenic bacterium, Erwinia uredovora 20D3 (ATCC 19321), in Escherichia coli and located on a 6,918-bp fragment whose nucleotide sequence was determined. Six open reading frames were found and designated the crtE, crtX, crtY, crtI, crtB, and crtZ genes in reference to the carotenoid biosynthesis genes of a photosynthetic bacterium, Rhodobacter capsulatus; only crtZ had the opposite orientation from the others. The carotenoid biosynthetic pathway in Erwinia uredovora was clarified by analyzing carotenoids accumulated in E. coli transformants in which some of these six genes were expressed, as follows: geranylgeranyl PPiCrtB----prephytoene PPiCrtE----phytoeneCrtI---- lycopeneCrtY----beta-caroteneCrtZ----zeaxanthinCrtX--- -zeaxanthin-beta- diglucoside. The carotenoids in this pathway appear to be close to those in higher plants rather than to those in bacteria. Also significant is that only one gene product (CrtI) for the conversion of phytoene to lycopene is required, a conversion in which four sequential desaturations should occur via the intermediates phytofluene, zeta-carotene, and neurosporene.     

Localization of two enzymes of the tetrahydrobiopterin biosynthetic pathway in embryonic chick retina.

Tetrahydrobiopterin (BH4) is an essential co-factor for the biosynthesis of catecholamine-type neurotransmitters and of nitric oxide (NO). The expression of the enzymes catalyzing the first two steps of the BH4 biosynthetic pathway was studied in the developing chicken retina by in situ hybridization and immunocytochemistry. GTP-cyclohydrolase-I (GTP-CH-I) and 6-pyruvoyl-tetrahydropterin synthase (PTPS) were already expressed in the undifferentiated and proliferating retina of E7. At stage E11 both enzymes were expressed in photoreceptors, amacrine cells, displaced amacrine cells, and ganglion cells, and in the plexiform layers in which synaptic connections take place. At stage E18 the labeling was comparable to E11 but appeared to be more concentrated in photoreceptors and ganglion cells.     

The cyanobacterium Anabaena sp. PCC 7120 has two distinct beta-carotene ketolases: CrtO for echinenone and CrtW for ketomyxol synthesis

Two beta-carotene ketolases, CrtW and CrtO, are widely distributed in bacteria, although they show no significant sequence homology with each other. The cyanobacterium Anabaena sp. PCC 7120 was found to have two homologous genes. In the crtW deleted mutant, myxol 2'-fucoside was present, but ketomyxol 2'-fucoside was absent. In the crtO deleted mutant, beta-carotene was accumulated, and the amount of echinenone was decreased. Therefore, CrtW catalyzed myxol 2'-fucoside to ketomyxol 2'-fucoside, and CrtO catalyzed beta-carotene to echinenone. This cyanobacterium was the first species found to have both enzymes, which functioned in two distinct biosynthetic pathways.     

Reconstruction of the violacein biosynthetic pathway from Duganella sp. B2 in different heterologous hosts

Violacein is a bacteria-originated indolocarbazole pigment with potential applications due to its various bioactivities such as anti-tumor, antiviral, and antifungal activities. However, stable mass production of this pigment is difficult due to its low productivities and the instability of wild-type violacein-producing strains. In order to establish a stable and efficient production system for violacein, the violacein synthesis pathway from a new species of Duganella sp. B2 was reconstructed in different bacterial strains including Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes by using different vectors. The gene cluster that encodes five enzymes involved in the violacein biosynthetic pathway was first isolated from Duganella sp. B2, and three recombinant expression vectors were constructed using the T7 promoter or the alkane-responsive promoter PalkB. Our results showed that violacein could be stably synthesized in E. coli, C. freundii, and E. aerogenes. Interestingly, we found that there were great differences between the different recombinant strains, not only in the protein expression profiles pertaining to violacein biosynthesis but also in the productivity and composition of crude violacein. Among the host strains tested, the crude violacein production by the recombinant C. freundii strain reached 1.68 g L⁻¹ in shake flask cultures, which was 4-fold higher than the highest production previously reported in flask culture by other groups. To the best of our knowledge, this is the first report on the efficient production of violacein by genetically engineered strains.     

PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16

AIMS: To assess the antibiotic biosynthetic potential of Amycolatopsis sp. strain UM16 and eight other Amycolatopsis species. METHODS AND RESULTS: Amycolatopsis genomic DNA was screened by PCR for the glycopeptide, Type-II (aromatic) polyketide and ansamycin biosynthetic gene clusters. Amycolatopsis sp. strain UM16, which exhibits weak antitubercular activity, was shown to have the glycopeptide oxyB gene and the Type-II (aromatic) polyketide-synthase KSalpha-KSbeta tandem gene pair, but not the AHBA synthase gene. The ristocetin (glycopeptide) producer, Amycolatopsis lurida NRRL 2430(T), was shown to have the oxyB gene and the Type-II polyketide-synthase KSalpha-KSbeta tandem gene pair. Amycolatopsis alba NRRL 18532(T) was shown to have the glycopeptide oxyB gene and the AHBA synthase gene. Phylogenetic analyses using Amycolatopsis oxyB and KSalpha-KSbeta gene sequences were conducted. CONCLUSIONS: Amycolatopsis sp. strain UM16 appears to have the biosynthetic potential to produce glycopeptide and Type-II polyketide antibiotics, but not ansamycins. The potential to synthesize aromatic polyketides may be more widely distributed in Amycolatopsis than is currently recognized. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening is a very useful tool for rapidly identifying the biosynthetic potential of an antibiotic-producing actinomycete isolate. Advanced knowledge of the type of antibiotic(s) produced will allow appropriate methods to be selected for antibiotic purification.     

The structure of two N-methyltransferases from the caffeine biosynthetic pathway

Caffeine (1,3,7-trimethylxanthine) is a secondary metabolite produced by certain plant species and an important component of coffee (Coffea arabica and Coffea canephora) and tea (Camellia sinensis). Here we describe the structures of two S-adenosyl-l-methionine-dependent N-methyltransferases that mediate caffeine biosynthesis in C. canephora 'robusta', xanthosine (XR) methyltransferase (XMT), and 1,7-dimethylxanthine methyltransferase (DXMT). Both were cocrystallized with the demethylated cofactor, S-adenosyl-L-cysteine, and substrate, either xanthosine or theobromine. Our structures reveal several elements that appear critical for substrate selectivity. Serine-316 in XMT appears central to the recognition of XR. Likewise, a change from glutamine-161 in XMT to histidine-160 in DXMT is likely to have catalytic consequences. A phenylalanine-266 to isoleucine-266 change in DXMT is also likely to be crucial for the discrimination between mono and dimethyl transferases in coffee. These key residues are probably functionally important and will guide future studies with implications for the biosynthesis of caffeine and its derivatives in plants.     

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway.

The poly-beta-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, beta-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.     

Thermocryptoxanthins: novel intermediates in the carotenoid biosynthetic pathway of Thermus thermophilus

Various thermozeaxanthins are the end products of the carotenoid biosynthetic pathway of the thermophilic eubacterium Thermus thermophilus. These compounds are zeaxanthin glucoside esters. Carotenoid analysis and inhibitory studies led to the identification of most of the intermediates of the pathway: β-carotene, β-cryptoxanthin, zeaxanthin, and several new carotenoids. The intermediates, identified by various spectroscopic methods as β-cryptoxanthin glucoside esters carrying fatty acid moieties of different chain lengths, were designated as thermocryptoxanthins. The use of the inhibitors diphenylamine and 2-(4-chlorophenylthio)-triethylamine-HCl resulted in the accumulation of the intermediates phytoene, lycopene, and γ-carotene derivatives, which normally are present in amounts below the detection limit. The levels of non-esterified glycosides were extremely low. The results presented were used to establish the complete carotenoid biosynthetic pathway of T. thermophilus. Received: 9 September 1995 / Accepted: 14 February 1996