Selection and mutant properties of Pichia guilliermondii yeasts with derepressed GTP cyclohydrolase, the enzyme of the 1st step in flavinogenesis

A positive method is proposed for selecting Pichia guilliermondii mutants with derepressed GTP cyclohydrolase. Mutants with the incompletely blocked gene RIB2 were used as parent strains; these can grow in a medium without riboflavin (RF) only if the enzyme is derepressed as the result of iron deficiency in cells. Strains growing in a medium without RF at the optimal supply of cells with iron were selected as regulatory mutants. The mutants accumulated 6,7-dimethylpterin in high concentrations and a small amount of RF in the medium and in the cells. The activity of GTP cyclohydrolase rather than that of RF synthase increased in the mutants; the activity of RF kinase and FAD pyrophosphorylase was not elevated. Hybrids produced by crossing the regulatory mutants with wild type strains did not accumulate 6,7-dimethylpterin in the medium and the activity of the GTP cyclohydrolase did not increase; this is indicative of the negative regulation for the expression of the structural gene for GTP cyclohydrolase. The authors propose a model for the regulation of GTP cyclohydrolase and RF synthase at the gene level involving iron ions as a corepressor.     

Localization of the genes coding for GTP cyclohydrolase II and riboflavin synthase on the chromosome of Escherichia coli K-12

A conjugation analysis of riboflavin-dependent mutants of Escherichia coli K-12 has been made by means of various F'-factors. It is shown that the GTP cyclohydrolase II gene is localized in the chromosome map site between 27 and 30 min; the riboflavin synthase gene--in the site between 56 and 59 min; and the gene in which mutation causes accumulation of 2,6-dioxy-5-amino-4-ribitylamino-pyrimidine--in the site limited by 61-65 min. So it may be concluded that riboflavin biosynthesis genes in the E. coli chromosome are not clustered.     

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes

Improved strains for the production of riboflavin (vitamin B₂) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l⁻¹ for 72 h in a 5-l jar fermentor without any end product inhibition. Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999     

Biosynthesis of riboflavin in plants. The ribA gene of Arabidopsis thaliana specifies a bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase

A cDNA segment from Arabidopsis thaliana with similarity to the ribA gene of Bacillus subtilis was sequenced. A similar gene was cloned from tomato. The open reading frame of A. thaliana was fused to the malE gene of Escherichia coli and was expressed in a recombinant E. coli strain. The recombinant fusion protein was purified and shown to have GTP cyclohydrolase II activity as well as 3,4-dihydroxy-2-butanone 4-phosphate synthase activity. The cognate gene was amplified by polymerase chain reaction from chromosomal Arabidopsis DNA and was shown to contain six introns. Intron 4 is located in the region connecting the GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase domain of the putative domains catalyzing the two reaction steps. By comparison with the bacterial ribA gene, the Arabidopsis gene contains an additional 5' element specifying about 120 amino acid residues. This segment contains numerous serine and threonine residues and does not show similarity with other known sequences. The N-terminal segment is not required for catalytic activity and is likely to serve as signal sequence for import into chloroplasts.     

Production of sepiapterin in Escherichia coli by coexpression of cyanobacterial GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase

Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.     

The red mutations impair the regulation of flavinogenesis and metal homeostas in yeast Pichia guilliermondii

A method of positive selection of mutants with impaired regulation of flavinogenesis and metal homeostasis in yeast Pichia guilliermondii was developed. This positive selection system was based on the isolation of pseudo-wild-type revertants (the Rib+ phenotype) in riboflavin-dependent rib1-86 mutant (the Rib- phenotype) of yeast P. guilliermondii. Mutation rib1-86 blocks activity of the GTP cyclohydrolase II catalyzing the first step in riboflavin (RF) biosynthesis. Study of a collection of spontaneous Rib+ revertants allowed the identification of a considerably large number of genetic loci responsible for the suppression of rib1-86, which include both previously identified three loci (rib80, rib81, and hit1) and six new loci designated red1-red6 (reduction). A comparative analysis of the wild-type strain and red mutants revealed that these mutants had higher activity levels of GTP cyclohydrolase and RF-synthase, elevated levels of RF biosynthesis, enhanced Fe/Cu reductase activity and higher total iron content in cells and that they are characterized by enhanced sensitivity to transition metals (Fe(III), Cu(II), Cd(II), Co(II), Zn(II), Ag(I), and to H2O2. The metal hypersensitivity of mutant cells can be prevented by an increased amount of extracellular iron ions. Mutations red1 and red6 synergistically interact with the locus rib81 in the course of RF biosynthesis. Obviously, each RED gene plays an important role in the regulation of both flavinogenesis and metal homeostasis in P. guilliermondii cells.     

RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors

During translation termination, class II release factor RF3 binds to the ribosome to promote rapid dissociation of a class I release factor (RF) in a GTP-dependent manner. We present the crystal structure of E. coli RF3*GDP, which has a three-domain architecture strikingly similar to the structure of EF-Tu*GTP. Biochemical data on RF3 mutants show that a surface region involving domains II and III is important for distinct steps in the action cycle of RF3. Furthermore, we present a cryo-electron microscopy (cryo-EM) structure of the posttermination ribosome bound with RF3 in the GTP form. Our data show that RF3*GTP binding induces large conformational changes in the ribosome, which break the interactions of the class I RF with both the decoding center and the GTPase-associated center of the ribosome, apparently leading to the release of the class I RF.     

Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli.

An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E. coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A. W., and Brown, G. M. (1968) J. Biol. Chem. 243, 2349-2358). Some of the properties of GTP cyclohydrolase II are: (a) divalent cations are required for activity (Mg2+ is most effective); (b) its molecular weight, estimated by filtration on Sephadex G-200, is 44,000; (c) the K-m for GTP is 41 mum; (d) its pH optimum is 8.5; and (e) its activity is inhibited by inorganic pyrophosphate, one of the products of the reaction. Compounds not used as substrate are: GDP, GMP, guanosine, dGTP, ATP, ITP, and XTP. Properties a, b, c, and e (above), as well as the nature of the products, distinguish this enzyme from GTP cyclohydrolase I. Since GTP cyclohydrolase II apparently is not concerned with the biosynthesis of folic acid, the possible physiological role of this enzyme in the biosynthesis of riboflavin is considered in the light of the present investigations and the previously published work on riboflavin biosynthesis by other investigators.     

Complementation of the fol2 deletion in Saccharomyces cerevisiae by human and Escherichia coli genes encoding GTP cyclohydrolase I

Saccharomyces cerevisiae is so far the only organism where a knock-out mutant in the gene encoding GTP cyclohydrolase I (FOL2) has been obtained. GTP cyclohydrolase I controls the de novo biosynthetic pathway of tetrahydrobiopterin and folic acid. Since deletion of yeast FOL2 leads to a recessive auxotrophy for folinic acid, we used a yeast fol2Delta mutant for an in vivo functional assay of heterologous GTP cyclohydrolases I. We show that the GTP cyclohydrolase I, encoded either by the E. coli folE gene or by the human cDNA, complements the yeast fol2Delta mutation by restoring folate prototrophy. Furthermore the folE-3x allele of the E. coli gene, carrying three base substitutions, failed to complement the yeast fol2Delta defect. This allele behaved as a negative semidominant to the wild type folE and, when overexpressed, completely abolished complementation of fol2Delta by folE. Thus, the yeast fol2 null mutant is a suitable system to characterize mutations in genes encoding GTP cyclohydrolase I.     

Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli.

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.     

Heterologous expression of the acyl–acyl carrier protein thioesterase gene from the plant Umbellularia californica mediates polyhydroxyalkanoate biosynthesis in recombinant Escherichia coli

The acyl-acyl carrier protein (ACP) thioesterase cDNA from the plant Umbellularia californica was functionally expressed in various recombinant Escherichia coli strains in order to establish a new metabolic route toward medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis from non-related carbon sources. Coexpression of the PHA synthase genes from Ralstonia eutropha and Pseudomonas aeruginosa, or only the PHA synthase gene from P. aeruginosa, respectively, showed PHA(MCL) accumulation when the type II PHA synthase from P. aeruginosa was produced. Both wild-type E. coli and various fad mutants were investigated; and only when the beta-oxidation pathway was impaired PHA(MCL) accumulation from gluconate was observed, contributing to about 6% of cellular dry weight. Thus coexpression of type II PHA synthase gene with cDNA encoding the medium-chain acyl-ACP thioesterase from U. californica established a new PHA(MCL) biosynthesis pathway, connecting fatty acid de novo biosynthesis with fatty acid beta-oxidation, using a non-related carbon source.     

Biosynthesis and metabolism of pterins in peripheral blood mononuclear cells and leukemia lines of man and mouse

The cellular origin and the control of neopterin release associated with immune stimulation was studied in cell cultures. Using purified human mononuclear cells, the intracellular change in concentrations of GTP and pterins was measured under various kinds of stimulation. Three enzymes involved in tetrahydrobiopterin biosynthesis, i.e. GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase, were also determined. Human macrophages stimulated with culture supernatant from activated T-lymphocytes were the main producers of neopterin. In these cells, GTP cyclohydrolase I activity was elevated due to high GTP levels and therefore neopterin accumulated. Human macrophages lack 6-pyruvoyl tetrahydropterin synthase activity. Exogenous tetrahydrobiopterin added to the culture medium of stimulated T cells and macrophages suppressed the elevation of GTP cyclohydrolase I activity and neopterin concentration, but not the elevation of intracellular GTP. Stimulation of macrophages with recombinant human interferon-gamma and neutralization of the effect of T cell supernatants by addition of a monoclonal antibody specific for human interferon-gamma showed that immune interferon induced the alterations in GTP cyclohydrolase I activity and neopterin concentration. In the human macrophage line U-937 and in the leukemia line HL-60, no GTP cyclohydrolase I activity or intracellular pterins were detected, but high levels of GTP. In mouse mononuclear cells, no neopterin was detected, but biopterin and pterin. After stimulation, biopterin was elevated in the same way as neopterin in human mononuclear cells. This is explained by the different regulation of the rate-limiting steps of tetrahydrobiopterin biosynthesis in man and in mouse. These results suggest that neopterin is an unspecific marker for the activation of the cellular immune system.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.     

GTP cyclohydrolase II structure and mechanism

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-beta-ribosyl-4(3H)-pyrimidinone 5'-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase II catalyzes a distinctive overall reaction from GTP cyclohydrolase I; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-A resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II.GMPCPP complex structure shows Arg(128) interacting with the alpha-phosphonate, and thus in the case of GTP, Arg(128) is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr(105) is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step.     

Expression of the Saccharomyces cerevisiae PIS gene and synthesis of phosphatidylinositol in Escherichia coli.

Expression of the Saccharomyces cerevisiae PIS gene encoding phosphatidylinositol synthase in Escherichia coli was achieved by inserting its coding sequence into lacZ on pUC8. The fused gene encoded a phosphatidylinositol synthase whose amino-terminal three amino acids had been replaced by the amino-terminal five amino acids of E. coli beta-galactosidase. E. coli cells bearing this recombinant plasmid produced a significant level of phosphatidylinositol synthase in the presence of a lacZ inducer, isopropylthio-beta-D-galactopyranoside. When the culture medium was supplemented with myo-inositol and isopropylthio-beta-D-galactopyranoside, the cells accumulated a substantial amount of phosphatidylinositol in their membranes. When a saturating level of myo-inositol was added, phosphatidylinositol constituted about 4% of the total phospholipids. Phosphatidylinositol accumulation occurred at the expense of phosphatidylglycerol. The ratio of phosphatidylethanolamine to total acidic phospholipids remained constant. The growth rate of phosphatidylinositol-containing E. coli cells did not differ significantly from that of cells with the normal phospholipid composition.     

Mapping of New Escherichia coli K and 15 Restriction Sites on Specific Fragments of Bacteriophage φX174 DNA

We have isolated several new phiX174 mutants which contain sites sensitive to restriction by Escherichia coli. One contains an E. coli 15 restriction site and three are double mutants containing an E. coli K site as well as the E. coli 15 site. The replicative form (RF) DNA of one of the mutants containing a K site has been shown to be restricted in spheroplasts of a K-12 strain. The infectivity of this RF, but not wild-type RF, has also been shown to be inactivated by an E. coli K extract and by purified K restriction enzyme in vitro. The product of the RF treated with purified K restriction enzyme in vitro is a full length linear molecule. The mutant sites have also been localized to specific regions of the phiX174 genome by a fragment mapping technique, making use of specific fragments of phiX174 RF DNA obtained by digestion with a specific endonuclease.     

Osmotic adaptation of Escherichia coli with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone.

It has been reported that Escherichia coli is able to grow in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) when ATP is produced by glycolysis (N. Kinoshita et al., J. Bacteriol. 160:1074-1077, 1984). We investigated the effect of CCCP on the osmotic adaptation of E. coli growing with glucose. When E. coli growing in rich medium containing CCCP was transferred to medium containing sucrose, its growth stopped for a while and then started again. This lag time was negligible in the absence of CCCP. The same results were obtained when the osmolarity was increased by N-methylglucamine-maleic acid. In addition to adapting itself to the hyperosmotic rich medium, E. coli adapted itself to hyperosmolarity in a minimal medium containing CCCP, again with a lag time. Hyperosmotic shock decreased the internal level of potassium ion rather than causing the accumulation of external potassium ion in the presence of CCCP. The internal amount of glutamic acid increased in cells growing in hyperosmotic medium in the presence and absence of CCCP. Large elevations in levels of other amino acids were not observed in the cells adapted to hyperosmolarity. Trehalose was detected only in hyperosmosis-stressed cells in the presence and absence of CCCP. These results suggest that E. coli can adapt to changes in the environmental osmolarity with a negligible accumulation of osmolytes from the external milieu but that the accumulation may promote the adaptation.