18O and mass spectrometry in chlorophyll research: Derivation and loss of oxygen atoms at the periphery of the chlorophyll macrocycle during biosynthesis,degradation and adaptation

Chlorophylls, magnesium-containing tetrapyrrolic pigments of photosynthesis, are widely-distributed in Nature and participate in both light harvesting and in the transduction of light energy to chemical energy for the photosynthetic fixation of carbon dioxide. We briefly discuss the extensive role of various isotopic labelling techniques in elucidating the pathway of tetrapyrrole-pigment biosynthesis and we acknowledge the classic and meticulous research of David Shemin who, approximately 50 years ago, introduced isotopic tracer techniques with ¹⁵N and ¹⁴C isotopes to study the biosynthesis of the carbon/nitrogen macrocycle of haem, an iron tetrapyrrole. The main focus of this review is the application of mass spectrometry and ¹⁸O labelling to the study of the incorporation of oxygen atoms from molecular oxygen or water into the periphery of the chlorophyll macrocycle during biosynthesis and their loss during degradation and light acclimation. In particular, we review the mechanism of formation of the isocyclic ring of chlorophylls, in higher plants, green algae and various photosynthetic bacteria, which concomitantly incurs formation of the 13¹-oxo group that is present in all photosynthetically-active chlorophylls. In addition we discuss the formation of the ubiquitous 13³- and 17³-carboxyl groups and also the formation of the 7-formyl group of chlorophyll b and the 3-acetyl group of bacteriochlorophyll a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biosynthesis of vitamin B12: Isobacteriochlorins,the link between corrins and sulphite reductases

The beautiful structure of vitamin B₁₂ has attracted great interest in its biosynthesis. This review briefly outlines the background and then covers very recent developments which have shown the importance of isobacteriochlorins in relation to the B₁₂ biosynthetic pathway. These isobacteriochlorin systems are also key materials for sulphite and nitrite reductase enzymes, and thus they act as a bridge, presumably an evolutionary bridge, between these enzymes and vitamin B₁₂. Determination of the structures of the isobacteriochlorins opens up the central part of the biosynthetic pathway to the corrin macrocycle.     

Natural and directed biosynthesis of communesin alkaloids

A role for tryptophan, acetate, mevalonate and methionine in the biosynthesis of communesins A and B, novel structurally-related and biologically-active Penicillium metabolites, has been established by isotopic labelling techniques. The incorporation of (14)C-tryptamine has also been demonstrated. dl-2-(13)C-tryptophan specifically enriched two carbon atoms in the (13)C NMR spectrum, thereby defining the intra-molecular arrangement of the two tryptophan-derived moieties. Feeding differentially labelled precursors during communesin production showed that tryptophan and methionine are involved early in the biosynthesis and that mevalonate provides an isoprene which is added later. A biosynthetic pathway involving an early precursor based on tryptophan is proposed. Indole-N-((13)C-methyl) tryptophan was not incorporated into communesins implying that N-methylation of tryptophan is not the first step of the communesin biosynthetic pathway. During deamination of indole-N-((13)C-methyl) tryptophan to 1-(13)C-methylindole-3-carboxylic acid communesin biosynthesis was inhibited. Of several halogenated indoles tested for directed biosynthesis, only dl-6-fluoro-tryptophan and 6-fluoro-tryptamine caused accumulation of the corresponding monofluoro-analogues of communesins A and B.     

Vitamin B1 de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine

Vitamin B1 (thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase (ThiD) and thiamine phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K(m)app values of 68 microM for THZ and 12 microM for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B1 biosynthesis, which depends on external provision of thiamine precursors.     

Identification and characterization of a novel vitamin B12 (cobalamin) biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing flavin, heme, and Fe-S cofactors

One of the most intriguing steps during cobalamin (vitamin B12) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework. The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ. However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663. To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B12 pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA. Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction. HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG. Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B12 itself is a modified tetrapyrrole. A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed.     

Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes

Using comparative analysis of genes, operons, and regulatory elements, we describe the cobalamin (vitamin B12) biosynthetic pathway in available prokaryotic genomes. Here we found a highly conserved RNA secondary structure, the regulatory B12 element, which is widely distributed in the upstream regions of cobalamin biosynthetic/transport genes in eubacteria. In addition, the binding signal (CBL-box) for a hypothetical B12 regulator was identified in some archaea. A search for B12 elements and CBL-boxes and positional analysis identified a large number of new candidate B12-regulated genes in various prokaryotes. Among newly assigned functions associated with the cobalamin biosynthesis, there are several new types of cobalt transporters, ChlI and ChlD subunits of the CobN-dependent cobaltochelatase complex, cobalt reductase BluB, adenosyltransferase PduO, several new proteins linked to the lower ligand assembly pathway, l-threonine kinase PduX, and a large number of other hypothetical proteins. Most missing genes detected within the cobalamin biosynthetic pathways of various bacteria were identified as nonorthologous substitutes. The variable parts of the cobalamin metabolism appear to be the cobalt transport and insertion, the CobG/CbiG- and CobF/CbiD-catalyzed reactions, and the lower ligand synthesis pathway. The most interesting result of analysis of B12 elements is that B12-independent isozymes of the methionine synthase and ribonucleotide reductase are regulated by B12 elements in bacteria that have both B12-dependent and B12-independent isozymes. Moreover, B12 regulons of various bacteria are thought to include enzymes from known B12-dependent or alternative pathways.     

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.     

Secondary metabolite biosynthesis: the first century

An account of work on the biosynthesis of secondary metabolites up to 1965 is presented. The earliest suggestions for three of the four major pathways were speculative; for the isoprene rule, hypotheses date to 1877, for the polyketide rule to 1907, and for a role for amino acids in alkaloid biosynthesis to 1910. The fourth major pathway based on intermediates of the shikimic acid pathway has a much shorter history because shikimic acid itself was only identified as a primary metabolite in 1951. In addition to speculation, biomimetic syntheses were carried out in which chemists attempted to duplicate possible biosynthetic pathways in vitro. The classic example was Robinson's synthesis of tropinone in 1917. Direct examination of secondary metabolite biosynthesis was possible with the use of the isotopic tracer technique. This methodology, applied extensively to primary metabolism beginning in 1935 and to secondary metabolism from about 1950, was facilitated by the increasing availability of the 14C isotope. With the use of isotopes as tracers, the broad outlines of secondary metabolite biosynthesis, reviewed here, were established in the period 1950 to 1965.     

A Salmonella typhimurium cobalamin-deficient mutant blocked in 1-amino-2-propanol synthesis.

Salmonella typhimurium synthesizes cobalamin (vitamin B12) when grown under anaerobic conditions. All but one of the biosynthetic genes (cob) are located in a single operon which includes genes required for the production of cobinamide and dimethylbenzimidazole, as well as the genes needed to form cobalamin from these precursors. We isolated strains carrying mutations (cobD) which are unlinked to any of the previously described B12 biosynthetic genes. Mutations in cobD are recessive and map at minute 14 of the linkage map, far from the major cluster of B12 genes at minute 41. The cobD mutants appear to be defective in the synthesis of 1-amino-2-propanol, because they can synthesize B12 when this compound is provided exogenously. Labeling studies in other organisms have shown that aminopropanol, derived from threonine, is the precursor of the chain linking dimethylbenzimidazole to the corrinoid ring of B12. Previously, a three-step pathway has been proposed for the synthesis of aminopropanol from threonine, including two enzymatic steps and a spontaneous nonenzymatic decarboxylation. We assayed the two enzymatic steps of the hypothetical pathway; cobD mutants are not defective in either. Furthermore, mutants blocked in one step of the proposed pathway continue to make B12. We conclude that the aminopropanol for B12 synthesis is not made by this pathway. Expression of a lac operon fused to the cobD promoter is unaffected by vitamin B12 or oxygen, both of which are known to repress the main cob operon, suggesting that the cobD gene is not regulated.     

Metabolic engineering provides insight into the regulation of thiamin biosynthesis in plants

Thiamin (or thiamine) is a water-soluble B-vitamin (B1), which is required, in the form of thiamin pyrophosphate, as an essential cofactor in crucial carbon metabolism reactions in all forms of life. To ensure adequate metabolic functioning, humans rely on a sufficient dietary supply of thiamin. Increasing thiamin levels in plants via metabolic engineering is a powerful strategy to alleviate vitamin B1 malnutrition and thus improve global human health. These engineering strategies rely on comprehensive knowledge of plant thiamin metabolism and its regulation. Here, multiple metabolic engineering strategies were examined in the model plant Arabidopsis thaliana. This was achieved by constitutive overexpression of the three biosynthesis genes responsible for B1 synthesis, HMP-P synthase (THIC), HET-P synthase (THI1), and HMP-P kinase/TMP pyrophosphorylase (TH1), either separate or in combination. By monitoring the levels of thiamin, its phosphorylated entities, and its biosynthetic intermediates, we gained insight into the effect of either strategy on thiamin biosynthesis. Moreover, expression analysis of thiamin biosynthesis genes showed the plant’s intriguing ability to respond to alterations in the pathway. Overall, we revealed the necessity to balance the pyrimidine and thiazole branches of thiamin biosynthesis and assessed its biosynthetic intermediates. Furthermore, the accumulation of nonphosphorylated intermediates demonstrated the inefficiency of endogenous thiamin salvage mechanisms. These results serve as guidelines in the development of novel thiamin metabolic engineering strategies.     

Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants

Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin B12 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway.     

茄科蔬菜花青素苷分子调控研究进展

花青素苷是一种分布广泛的水溶性色素,不仅赋予了果实多彩的外表,还是天然食用色素的重要来源。近年来有关茄科蔬菜花青素苷的研究逐渐增多,文中从花青素苷结构及其生物合成途径、茄科蔬菜中花青素苷合成代谢的结构基因和调节基因、影响合成的环境因素等方面进行回顾和总结,为进一步阐明茄科蔬菜花青素苷的合成及调控机理、更好利用花青素苷进行果色品质育种的创新提供一些参考。     

Variation in oxygen isotope fractionation during cellulose synthesis: intramolecular and biosynthetic effects

The oxygen isotopic composition of plant cellulose is commonly used for the interpretations of climate, ecophysiology and dendrochronology in both modern and palaeoenvironments. Further applications of this analytical tool depends on our in-depth knowledge of the isotopic fractionations associated with the biochemical pathways leading to cellulose. Here, we test two important assumptions regarding isotopic effects resulting from the location of oxygen in the carbohydrate moiety and the biosynthetic pathway towards cellulose synthesis. We show that the oxygen isotopic fractionation of the oxygen attached to carbon 2 of the glucose moieties differs from the average fractionation of the oxygens attached to carbons 3-6 from cellulose by at least 9%, for cellulose synthesized within seedlings of two different species (Triticum aestivum L. and Ricinus communis L.). The fractionation for a given oxygen in cellulose synthesized by the Triticum seedlings, which have starch as their primary carbon source, is different than the corresponding fractionation in Ricinus seedlings, within which lipids are the primary carbon source. This observation shows that the biosynthetic pathway towards cellulose affects oxygen isotope partitioning, a fact heretofore undemonstrated. Our findings may explain the species-dependent variability in the overall oxygen isotope fractionation during cellulose synthesis, and may provide much-needed insight for palaeoclimate reconstruction using fossil cellulose.     

Effect of porphyrins and their derivatives on biosynthesis of vitamin B 12 and the development of Propionibacterium shermanii]

The effect of uroporphyrin, coproporphyrin and their cobalt-containing derivatives on the biosynthesis of vitamin B12 and development of propionibacterium shermanii was studied. The compounds under study stimulated the vitamin synthesis by growing cultures and resting suspensions of these bacteria. Cobalt porphyrins as the sole source of cobalt were used in the vitamin B12 biosynthesis. An addition of cobalt porphyrins to the growing culture of propionic bacteria increased in accumulation of their biomass. Possible mechanisms of porphyrin involvement in the biosynthesis of vitamin B12 and the specific role of cobalt porphyrins in the bacterial activity are discussed.     

Multiple pathways for isoleucine biosynthesis in the spirochete Leptospira

Spirochetes of the genus Leptospira have previously been shown to use an unusual pathway to synthesize isoleucine. For reasons of convenience, we assume that only one unusual pathway is found in the genus, and we refer to it as the pyruvate pathway. We determined the distribution of this pyruvate pathway in representatives of the seven Leptospira DNA hybridization groups. Our method included labeling the representative strains with radioactive carbon dioxide and other radioactive precursors, fractionating the cells, and determining the specific activities (counts detected per nanomole) of the amino acids found in the protein fractions. On the basis of isoleucine biosynthesis, we found that the genus can be classified as follows: class I primarily, if not exclusively, uses the well-known threonine pathway; class II uses mostly the pyruvate pathway, with a minor amount of isoleucine being synthesized via the threonine pathway; and class III uses the pyruvate pathway exclusively. No relationship appears to exist between the degree of DNA hybridization and the classes of isoleucine biosynthesis. Although the precise intermediates on the pyruvate pathway are unknown, the origin of the carbon skeleton of isoleucine synthesized by this pathway is consistent with a borrowing of the leucine biosynthetic enzymes. However, we found that the pyruvate pathway is not controlled by leucine and that the two isoleucine pathways are independently regulated. Finding major and highly evolved multiple biosynthetic pathways of a specific amino acid within one genus is unique, and, conceivably, represents phylogenetic diversity within Leptospira.     

Molecular and carbon isotopic composition of leaf wax in vegetation and aerosols in a northern prairie ecosystem

We measured the molecular and carbon isotopic composition of major leaf wax compound classes in northern mixed mesic prairie species (Agropyron smithii, Stipa viridula, Bouteloua gracilis, Tragopogon dubius) and in selected crops (Triticum aestivum, Brassica napus, Hordeum vulgare, Medicago sativa) of southern Alberta and also in aerosols collected 4 m above the prairie canopy. Our aims were to better constrain the wax biosynthetic carbon isotopic fractionation relative to the plant's carbon isotopic discrimination and to quantitatively assess the correspondence between wax composition in vegetation and in boundary layer aerosols. Wax molecular composition of the C(3)prairie species and bulked vegetation was characterized by high abundance of C(28) n-alkanol and C(31) n-alkane compounds whereas the C(4) species B. gracilis had several co-dominant n-alkanol and n-alkane compounds. Wax molecular composition of crop species differed significantly from that of prairie vegetation and was often dominated by a single compound. Results indicate that leaf wax isotopic composition is quantitatively related to the plant's carbon isotopic discrimination. Although species variations were evident, n-alcohol, n-acid and n-alkane wax compounds were on average depleted in (13)C by approximately 6.0+/-1 per thousand relative to total plant carbon. The magnitude of the depletion in wax delta(13)C was unaffected by environmental factors which altered photosynthetic carbon isotopic discrimination. No consistent difference in the magnitude of wax biosynthetic fractionation was observed between C(3) and C(4) species, indicating that photosynthetic pathway has little influence on the isotopic fractionation of wax during biosynthesis. The isotopic composition of ablated waxes in aerosols collected above the canopy was similar to that of the grassland vegetation but the molecular composition differed significantly and indicated that the source "footprint" of the ablated leaf wax particles we sampled in boundary layer air masses was of a regional or larger spatial scale.     

Regulation of biosynthesis of secondary metabolites

The biosynthetic activity of mutants with altered morphological and biochemical characteristics, obtained from two strains ofStreptomyces aureofaciens by means of physical and chemical mutagens, was studied. The majority of mutants formed pigments different from pigments of the parent strains, which did not usually give fluorescence in UV-light and, in addition, differed as to the solubility in organic solvents. The production of further secondary metabolites was investigated chromatographically in the extracts from mycelium and in the fermentation fluid of submerged cultures. According to the results of chromatographical analysis, the obtained mutants can be divided into 12 metabolic types. In most of them the production of substances was found which are different both mutually and from the metabolites of parent strains in physical and chemical properties. A direct correlation was observed between the character of colonies and biosynthetic properties of mutants.     

Evidence for a new, oxygen-regulated biosynthetic pathway for the pyrimidine moiety of thiamine in Salmonella typhimurium.

The synthesis of the pyrimidine moiety of thiamine (vitamin B1) shares five reactions with the de novo purine biosynthetic pathway. Aminoimidazole ribotide (AIR) is the last common intermediate before the two pathways diverge. Evidence for the existence of a new pathway to the pyrimidine which bypasses the de novo purine biosynthetic pathway is reported here. This pathway is only expressed under anaerobic growth conditions and is denoted alternative pyrimidine biosynthesis or APB. Labeling studies are consistent with pantothenate being a precursor to the pyrimidine moiety of thiamine that is synthesized by the APB pathway. The APB pathway is independent of the alternative purF function which was proposed previously (D. M. Downs and J. R. Roth, J. Bacteriol. 173:6597-6604, 1991). The alternative purF function is shown here to be affected by temperature and exogenous pantothenate. Although the evidence suggests that the APB pathway is separate from the alternative purF function, the relationship between this function and the APB pathway is not yet clear.     

Biosynthesis of porphyrins and corrins.

Haem, chlorophyll and vitamin B12 are all derived ultimately from four molecules of the pyrrole porphobilinogen (PBG) and the initial enzyme catalysed condensation of PBG leads to the unsymmetrical type III isomer of uroporphyrinogen. On the basis of straightforward chemical considerations the type I isomer should be formed and so the porphyrinogen-forming enzymes of all living systems must catalyse a highly specific rearrangement process. The nature and chemical mechanism of this rearrangement poses one of the most fascinating problems in the porphyrin field and so it is not surprising that over 20 hypothetical schemes have been proposed to account for it. Analysis of the problem suggested that the incorporation of doubly 13C-labelled precursors into the rearranged macrocyclic rings would give valuable new information on the nature of the rearrangement process. In this approach the meso=bridge atoms are of crucial importance, and several unambiguous syntheses of 13C-labelled pyrroles and porphyrins were developed to allow rigorous n.m.r. assignments to be made, and also to provide substrates for enzymic experiments. Studies carried out with enzymes from both avian blood and from Euglena gracilis have revealed the precise nature of the assembly of four PBG molecules into the type-III macrocycle: it is the same in both systems despite their vastly different evolutionary development. Complementary studies are in progress in order to determine the intermediates involved in the conversion of PBG into uroporphyrinogen III. The synthesis of amino methyl pyrromethanes and their interaction in the presence of PBG with the appropriate enzyme systems are described. It is important for the work to be able to separate not only isomeric pyrromethanes but also the four isomeric coproporphyrins. Powerful methods are described which make use of high pressure liquid chromatography for both types of separation process. Once uroporhyrinogen III has been built enzymically, there is a stepwise enzymic decarboxylation of the four acetic acid residues. A heptacarboxylic porphyrin shown to be a type-III porphyrin is isolated from the action of avian blood enzymes on porphobilinogen. Spectroscopic studies with 13C-labelling limit the possible structures to two and total synthesis of these substances shows that the natural product carries its methyl group on ring D. An isomeric heptacarboxylic porphyrin having its methyl group on ring C is of particular interest in relation to the biosynthesis of vitamin B12. This substance is synthesized together with uroporphyrin III, 14C-labelled specifically in ring C. This latter product is used to settle one of the key questions concerning nature's route to vitamin B12 - that is, does the corrin macrocycle arise from uroporphyrinogen III? Incorporation studies and specific degradations prove specific incorporation of uroporphyrinogen III into cobyrinic acid, which is the known precursor of vitamin B12.     

Total biosynthesis of hydrocortisone from a simple carbon source in yeast

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.