Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.

Deletion of rapQONML from the rapamycin gene cluster of Streptomyces hygroscopicus gives production of the 16-O-desmethyl-27-desmethoxy analog.

Five contiguous genes in the rapamycin gene cluster, rapQONML, of Streptomyces hygroscopicus ATCC29253 were replaced with a neomycin resistance marker by double homologous recombination. The resulting strain, if fed pipecolate, produced the analog 16-O-desmethyl-27-desmethoxyrapamycin instead of rapamycin. This indicates that the P450 hydroxylase encoded by rapN is specific for C-27, and that the O-methyltransferases encoded by rapQ and rapM methylate the hydroxyl groups on C-16 and C-27. By inference, the remaining P450 hydroxylase and methyltransferase genes (rapI and rapJ) are responsible for hydroxylation of C-9 and methylation of the C-39 hydroxyl, consistent with their homology to fkbD and fkbM, respectively, in the FK506 cluster. The relatively high level of 16-O-desmethyl-27-desmethoxyrapamycin produced indicates that the reactions at C-9 and C-39 do not require previous modification of the macrolactone precursor at either C-16 or C-27.

The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units.

FK520 (ascomycin) is a macrolide produced by Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) that has immunosuppressive, neurotrophic and antifungal activities. To further elucidate the biosynthesis of this and related macrolides, we cloned and sequenced an 80kb region encompassing the FK520 gene cluster. Genes encoding the three polyketide synthase (PKS) subunits (fkbB, fkbC and fkbA), the peptide synthetase (fkbP), the 31-O-methyltransferase (fkbM), the C-9 hydroxylase (fkbD) and the 9-hydroxyl oxidase (fkbO) had the same organization as the genes reported in the FK506 gene cluster of Streptomyces sp. MA6548 (Motamedi, H., Shafiee, A., 1998. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 256, 528-534). Disruption of a PKS gene in the cluster using the φC31 phage vector, KC515, led to antibiotic non-producing strains, proving the identity of the cluster. Previous labeling data have indicated that FK520 biosynthesis uses novel polyketide extender units (Byrne, K.M., Shafiee, A., Nielson, J., Arison, B., Monaghan, R.L., Kaplan, L., 1993. The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var, ascomyceticus. Dev. Ind. Microbiol. 32, 29-45). Genes in the flanking regions of the FK520 cluster were identified that appear to be involved in synthesis of these extender units. All but two of these genes were homologous to genes with known function. In addition to a crotonyl-CoA reductase gene (fkbS), at least two other genes are proposed to be involved in biosynthesis of the atypical PKS extender unit ethylmalonyl-CoA, which accounts for the ethyl side chain on C-21 of FK520. A set of five contiguous genes (fkbGHIJK) is proposed to be involved in biosynthesis of an unusual PKS extender unit bearing an oxygen on the alpha-carbon, and leading to the 13- and 15-methoxy side chains. These putative precursor synthesis genes in the flanking regions of the FK520 cluster are not found in the flanking regions of the rapamycin cluster (Molnár, I., Aparicio, J.F., Haydock, S.F., Khaw, L.E., Schwecke, T., König, A., Staunton, J., Leadlay, P.F., 1996. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene 169, 1-7), consistent with labeling data showing that rapamycin biosynthesis uses only malonyl and methylmalonyl extender units.

Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene.

BACKGROUND: Lovastatin, an HMG-CoA reductase inhibitor produced by the fungus Aspergillus terreus, is composed of two polyketide chains. One is a nonaketide that undergoes cyclization to a hexahydronaphthalene ring system and the other is a simple diketide, 2-methylbutyrate. Fungal polyketide synthase (PKS) systems are of great interest and their genetic manipulation should lead to novel compounds. RESULTS: An A. terreus mutant (BX102) was isolated that could not synthesize the nonaketide portion of lovastatin and was missing a approximately 250 kDa polypeptide normally present under conditions of lovastatin production. Other mutants produced lovastatin intermediates without the methylbutyryl sidechain and were missing a polypeptide of approximately 220 kDa. The PKS inhibitor cerulenin reacted covalently with both polypeptides. Antiserum raised against the approximately 250 kDa polypeptide was used to isolate the corresponding gene, which complemented the BX102 mutation. The gene encodes a polypeptide of 269 kDa containing catalytic domains typical of vertebrate fatty acid and fungal PKSs, plus two additional domains not previously seen in PKSs: a centrally located methyltransferase domain and a peptide synthetase elongation domain at the carboxyl terminus. CONCLUSIONS: The results show that the nonaketide and diketide portions of lovastatin are synthesized by separate large multifunctional PKSs. Elucidation of the primary structure of the PKS that forms the lovastatin nonaketide, as well as characterization of blocked mutants, provides new details of lovastatin biosynthesis.

Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity.

We demonstrate a novel and efficient bioprocess for production of the cephalosporin intermediates, 7-aminocephalosporanic acid (7-ACA) or 7-amino deacetoxycephalosporanic acid (7-ADCA). The Streptomyces clavuligerus expandase gene or the Cephalosporium acremonium expandase-hydroxylase gene, with and without the acetyltransferase gene, were expressed in a penicillin production strain of Penicillium chrysogenum. Growth of these transformants in media containing adipic acid as the side chain precursor resulted in efficient production of cephalosporins having an adipyl side chain, proving that adipyl-6-APA is a substrate for either enzyme in vivo. Strains expressing expandase produced adipyl-7-ADCA, whereas strains expressing expandase-hydroxylase produced both adipyl-7-ADCA and adipyl-7-ADAC (aminodeacetylcephalosporanic acid). Strains expressing expandase-hydroxylase and acetyltransferase produced adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. The adipyl side chain of these cephalosporins was easily removed with a Pseudomonas-derived amidase to yield the cephalosporin intermediates.

Epidermoid cyst of the spleen presenting as a generalized peritonitis.

In this report we reviewed 159 cases of epidermoid cyst of the spleen reported since 1929 and we added one case of our own with a unique clinical presentation. In these cases, the patients' age at presentation ranged from newborn to 51 years, with a mean age of 17.7 years. Female-to-male ratio was 2.0 to 1.0. Patients with this lesion usually present with asymptomatic abdominal mass and/or abdominal pain. Only in rare reports has there been infection (4 cases) or rupture (4 cases) of the cyst. In our case, the patient presented with an acute surgical abdomen and diffuse peritonitis. As in three of the previously reported cases associated with infection, Salmonella group organisms were cultured from the cyst abscess. Splenectomy is the surgical treatment of choice and the initial antibiotic regime should include Salmonella coverage.

Analyses of alpha/beta-type gliadin genes from diploid and hexaploid wheats.

The alpha/beta-gliadin genes isolated from both hexaploid wheat (cv. Yamhill) and the diploid A genome progenitor Triticum urartu had remarkably similar sequences and differ by only a few point mutations. Primer extension analysis indicated that the transcriptional start points for individual genes in the family cluster within a few nucleotides. Comparison of the promoter region of several alpha/beta-gliadin and B-hordein genes reveals two conserved regions at about -130 and -250 bp. DNA from the hexaploid cultivars, Cheyenne and Chinese Spring, and the diploid progenitors T. urartu and Aegilops squarrosa was analysed by Southern blotting. Restriction fragment lengths of the alpha/beta-gliadin genes varied only slightly between the various wheats, although the overall copy number varied significantly. A region between approx. -1700 and -700 bp upstream from the TATA box was highly repeated in all three wheat genomes. For the hexaploid-derived gene, over 1700 bp of sequence upstream from the TATA box was determined, revealing an additional open reading frame between approx. -1550 and -1250 bp relative to the gliadin TATA box. Northern blot analysis indicated that RNA homologous to this repeated sequence family was present only in developing seed and accumulated to a maximum at late stages of maturation.

Gene Expression in Developing Wheat Endosperm : Accumulation of Gliadin and ADPglucose Pyrophosphorylase Messenger RNAs and Polypeptides.

The developmental accumulation pattern of messenger RNA transcripts and polypeptides for wheat gliadins and ADPglucose pyrophosphorylase was determined using cDNA and antibody probes. Gliadin mRNA was detected on Northern and RNA dot blots at 3 days after flowering, it increased 100-fold by 10 days and decreased subsequent to 14 days. The abundant mRNAs encoding alpha/beta- and gamma-type gliadins and mRNA for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, accumulated coordinately. Despite the coordinate accumulation of their mRNA transcripts, the accumulation of gliadin and ADPglucose pyrophosphorylase polypeptides, as determined by Western blot, differed significantly. The time at which gliadin and ADPglucose pyrophosphorylase mRNAs began accumulating was also the time when the overall pattern of gene expression, as seen by two-dimensional gel electrophoresis of in vitro translation products, changed most significantly. However, the accumulation of a number of other mRNAs or polypeptides having unknown function occurred at other times during endosperm development. The pattern of expression in the earliest stages of development was strikingly similar to that of coleoptile, another rapidly growing, nonphotosynthetic tissue. Thus, the pattern of gene expression reflects the program of development observed cytologically.

ADPglucose Pyrophosphorylase Is Encoded by Different mRNA Transcripts in Leaf and Endosperm of Cereals.

Western blots of soluble protein from wheat, rice, and corn showed that ADPglucose pyrophosphorylase subunits have a size of 50 kilodaltons from endosperm tissue and 43 and 46 kilodaltons from leaf. Antisera to ADPglucose pyrophosphorylase precipitated in vitro translation products of 73 and 76 kilodaltons when leaf poly(A)(+) RNA was used, whereas endosperm mRNA directed the synthesis of 50 and 56 kilodalton polypeptides. To further study the nature of these mRNA species, an ADPglucose pyrophosphorylase cDNA clone from rice endosperm polyadenylated RNA was obtained and used as a hybridization probe. Northern blots showed that ADPglucose pyrophosphorylase mRNA was slightly larger in leaf (2100 bases) than in endosperm tissue (1900 bases). These studies indicated that in cereals there are at least two tissue specific forms of ADPglucose pyrophosphorylase that are encoded by distinct mRNA transcripts. Analysis of genomic DNA by Southern blotting suggested that ADPglucose pyrophosphorylase is encoded by a small gene family.

Evolution and heterogeneity of the alpha-/beta-type and gamma-type gliadin DNA sequences.

Near full length cDNA clones for both alpha-/beta- and gamma-type gliadins were isolated and studied for sequence diversity. Based on restriction site polymorphism and cross-hybridization studies, alpha-/beta- and gamma-type clones could be divided into five and three homology classes, respectively. Clones representing each of the different classes were sequenced and compared. Sequence divergence between the classes was due to single-base substitutions and to duplications or deletions within or near direct repeats. Thus, through numerous duplications and subsequent divergence, the gliadin multigene family encodes a polymorphic set of polypeptides differing in both isoelectric point and molecular size. Southern blot analysis of wheat DNA suggested that the number of genes encoding the alpha-/beta-type gliadins was extremely large (greater than 100 copies/haploid genome). Inasmuch as hybridization patterns were the same using DNA isolated from seeds or leaves, amplification or rearrangement of DNA does not occur during development. The complete coding sequence of a gamma-gliadin was similar to that observed for the alpha-/beta-gliadins, but with several notable differences. Comparison of gamma-type gliadin cDNA sequences showed that, unlike the conserved dodecamer repeat common to all the alpha-/beta-gliadins, the tandem repeat unit differed among gamma-gliadin clones.

Role of silicon in diatom metabolism. Messenger RNA and polypeptide accumulation patterns in synchronized cultures of Cylindrotheca fusiformis.

Patterns of in vitro translation products from isolated mRNA and in vivo polypeptide accumulation in synchronized cultures of Cylindrotheca fusiformis were analysed by two-dimensional gel electrophoresis. The way in which the availability of silicon, the specific cell cycle stage, or the illumination conditions affected the pattern of gene expression was distinguished by comparing the timing of polypeptide and mRNA accumulation in cultures synchronized by two different methods. A rapid and dramatic shift in the relative abundance of in vitro translation products from mRNA followed either the removal or the readdition of silicate to the media as well as the transition from dark to light. Eleven mRNAs appeared to be expressed specifically between mid-S phase and cell separation, as their increase was observed at this stage in both synchronies. In addition, three mildly acidic polypeptides from the soluble protein fraction of C. fusiformis, each representing about 0.05% of the total protein, increased several-fold between mid-S and cell separation. Thus, silicon appears to affect gene expression both directly and, due to its effect on cell cycle progression, indirectly. Both effects are primarily at a level before translation.

Role of Silicon in Diatom Metabolism : Cyclic Nucleotide Levels, Nucleotide Cyclase, and Phosphodiesterase Activities during Synchronized Growth of Cylindrotheca fusiformis.

Adenylate cyclase, guanylate cyclase, and the cyclic nucleotide phosphodiesterases of Cylindrotheca fusiformis were characterized in crude and partially purified preparations. Both cyclases were membrane-bound and required Mn(2+) for activity, though Mg(2+) gave 50% activity with adenylate cyclase. Properties of adenylate cyclase were similar to those of higher eukaryotic cyclases in some respects, and in other respects were like lower eukaryotic cyclases. Guanylate cyclase was typical of other lower eukaryotic enzymes.Two phosphodiesterase activities were found, one selective for cyclic AMP, the other for cyclic GMP. The 5'-nucleoside monophosphate was the major product of both activities and each of the enzymes had distinctive divalent cation requirements, pH optima, and kinetic parameters. Both phosphodiesterases were similar to those of other lower eukaryotes with one notable difference: the cyclic AMP enzyme was inhibited by calcium.Changes in the cyclic nucleotide levels were quantitated in light-dark and silicon-starvation synchronized cultures using a more sensitive radioimmunoassay than used in a previously published study (Borowitzka and Volcani 1977 Arch Microbiol 112: 147-152). Contrary to the previous report, the cyclic GMP level did not change significantly in either synchrony. The cyclic AMP level increased dramatically very early in the period of DNA replication with the peak cyclic AMP accumulation substantially preceding that of DNA synthesis in both synchronies. There was no significant change in the activity of either cyclase or either phosphodiesterase during either synchrony. Thus, the mechanism for the rise in cAMP level remains unclear.

Control of breathing in Sherpas at low and high altitude.

Sherpas are well known for their physical performance at extreme altitudes, yet they are reported to have blunted ventilatory responses to acute hypoxia and relative hypoventilation in chronic hypoxia. To examine this paradox, we studied ventilatory control in Sherpas in comparison to that in Westerners at both low and high altitude. At low altitude, 25 Sherpas had higher minute ventilation, higher respiratory frequency, and lower end-tidal carbon dioxide tension than 25 Westerners. The hypoxic ventilatory response of Sherpas was found to be similar to that in Westerners, even though long altitude exposure had blunted the responses of some Sherpas. At high altitude, Sherpas again had higher minute ventilation and a tendency toward higher arterial oxygen saturation than Westerners. Oxygen administration increased ventilation further in Sherpas but decreased ventilation in Westerners. We conclude that Sherpas differ from other high-altitude natives; their hypoxic ventilatory response is not blunted, and they exhibit relative hyperventilation.

Posttraumatic ischemic stenosis of the small bowel.

In three patients, signs and symptoms developed of chronic, incomplete small-bowel obstruction three or more weeks after blunt abdominal trauma. At surgery, a stenotic segment of small bowel was found adjacent to a mesenteric rent that apparently devascularized the small bowel, leading to stenosis and mucosal ulceration. Resection of the stenotic small-bowel segment effected a cure in each patient. Posttraumatic strictures of the small bowel should be suspected in patients on whom and abdominal laparotomy was not performed at the time of injury and in whom signs and symptoms of incomplete small-bowel obstruction develop three of more weeks after blunt abdominal trauma.

Differential diagnosis of hypercalcemia by the chloride/phosphate ratio.

The serum chloride and phosphate levels were measured and the chloride/phosphate ratios calculated in a group of eighty-four hypercalcemic patients. Although patients with hyperparathyroidism frequently had phosphate levels in the low normal range (less than 3 mg/100 ml) and chloride levels in the nigh normal range (greater than 102 mEq/L), they were nevertheless significantly different from the groups of patients with nonparathyroid hypercalcemia in whom phosphate levels were usually higher (greater than 3 mg/100 ml) and chloride levels usually lower (less than 102 mEq/L). The chloride/phosphate ratio was higher than 33 in 94 per cent of hyperparathyroid patients and lower than 33 in 96 per cent of other hypercalcemic patients. Thus, the measurements of serum phosphate and chloride levels and the calculation of the chloride/phosphate ratios were useful diagnostic screening tests that discriminated between patients with hypercalcemia of parathyroid and nonparathyroid origin with an accuracy of 95 per cent.