Improvement of compactin (ML-236B) production by genetic engineering in compactin high-producing Penicillium citrinum.

An increase in compactin (ML-236B) production was achieved by introducing a whole compactin biosynthetic gene cluster or the regulatory gene mlcR into compactin high-producing Penicillium citrinum. In the previous report, we introduced mlcR encoding the positive regulator of compactin biosynthetic genes into compactin high-producing strain no. 41520, and most of the transformants produced higher amounts of compactin. Here, we characterize one of the resulting high producers (strain TIR-35, which produced 50% more compactin) and reveal that TIR-35 contained five copies of mlcR and that early, enhanced expression of mlcR caused compactin overproduction. Similarly, the introduction of mlcR into strain T48.19, which was created previously from strain no. 41520 by introducing a partial compactin biosynthetic gene cluster, enhanced compactin production further. Our results indicated that genetic engineering is an effective tool to improve compactin production, even in compactin high producers.

Identification of the specific sequence recognized by Penicillium citrinum MlcR, a GAL4-type transcriptional activator of ML-236B (compactin) biosynthetic genes.

MlcR is a pathway-specific transcriptional activator of the ML-236B biosynthetic genes in Penicillium citrinum. The MlcR-binding sequences were identified by an in vitro gel-shift assay and an in vivo reporter assay for the region between mlcA and mlcC as a model. The gel-shift assay showed that recombinant MlcR bound to the DNA sequence 5'-ACGGCGTTATTCGG-3' and most of the bases in this motif were required for the interaction between MlcR and DNA. In the reporter assay using beta-glucuronidase (GUS), substitution of the bases in this binding sequence resulted in the drastic reduction of GUS activities. These data clearly indicate that this MlcR-binding sequence is essential for the transcriptional activation of mlcA and mlcC in P. citrinum. Similar motifs were found in other loci of the ML-236B biosynthetic gene cluster and the consensus-binding motif for MlcR was predicted to be a direct repeat, 5'-WCGG-N(6)-TCGG-3'.

Targeted disruption of the genes, mlcR and ariB, which encode GAL4-type proteins in Penicillium citrinum.

The role of two genes, mlcR and ariB, was investigated by gene disruption experiments. The mlcR gene in the ML-236B biosynthetic gene cluster of Penicillium citrinum encodes a putative 50.2-kDa protein with a Zn (II) 2Cys6 DNA-binding domain, and has similarity to most of the GAL4-type regulatory proteins. The mlcR disruptant did not produce ML-236B or its intermediates, suggesting that mlcR is involved in ML-236B biosynthesis. Transcriptional analysis of the mlcR disruptant by Northern hybridization and RT-PCR indicated that MlcR activates the transcription of mlcA, B, C,D, F, G and H in a pathway-specific manner. On the other hand, MlcR did not affect the transcription of mlcE and the genes outside the ML-236B cluster. The ariB gene, next to mlcR, encodes another GAL4-type protein. Transcriptional analysis of the ariB disruptant indicated that it is a transcriptional activator of the genes outside the ML-236B cluster, and is not related to ML-236B biosynthesis.

Functional analysis of mlcR, a regulatory gene for ML-236B (compactin) biosynthesis in Penicillium citrinum.

The mlcR gene encodes a putative 50.2-kDa protein with a Zn(II)(2)Cys(6) DNA-binding domain, which may be involved in the regulation of ML-236B biosynthesis in Penicillium citrinum. The induction of ML-236B production appears to correlate with the expression of mlcR, and the ML-236B biosynthetic genes mlcA- mlcH, and occurs mostly during the stationary phase. The present study was designed to examine the effects of alterations in mlcR expression on ML-236B biosynthesis. We first set out to increase the mlcR copy number in the chromosome of P. citrinum. Transformants with additional copies of native mlcR showed increased transcription of mlcR and produced larger amounts of ML-236B than the parent strain. Altered mlcR expression was also achieved by introducing a construct, designated pgkA(P)::mlcR, that contained the mlcR coding region fused to the (constitutively active) promoter and terminator sequences of the Aspergillus nidulans 3-phospho-glycerate kinase (pgkA) gene. Transformants carrying the pgkA(P)::mlcR construct expressed mlcR constitutively, and produced ML-236B during the exponential growth phase, suggesting that the pgkA(P)::mlcR construct does affect the regulation of ML-236B biosynthesis. Comparative expression analysis by RT-PCR showed that altering the expression profile of mlcR influenced the expression of some of the ML-236B biosynthetic genes. The evidence suggests that mlcR may indeed be involved in the transcriptional activation of some of the pathway-specific genes required for ML-236B biosynthesis.

Effect of increased dosage of the ML-236B (compactin) biosynthetic gene cluster on ML-236B production in Penicillium citrinum.

The gene cluster responsible for ML-236B (compactin) biosynthesis has recently been characterized from P. citrinum No. 41520. Here, we describe how the ML-236B-producing strain was improved using a cosmid-mediated recombination technique. The introduction of the cosmid pML48, which contains seven of the nine ML-236B biosynthetic genes, into P. citrinum No. 41520 resulted in transformants which produced increased amounts of ML-236B. Southern analysis showed that pML48 had been incorporated by a homologous recombination event, and all high producers possessed two copies of each of the seven genes, mlcA- mlcF and mlcR, suggesting that increased dosage of the biosynthetic gene cluster was responsible for the enhanced production of ML-236B. On the other hand, various kinds of mutants with decreased titers of ML-236B were also obtained. Characterization of one such mutant, designated as T48.28, which was more sensitive to ML-236B than the parental strain, suggested that one of the ML-236B biosynthetic genes, mlcD, which encodes a putative HMG-CoA reductase, was involved in conferring resistance to ML-236B.

Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum.

Cloning of genes encoding polyketide synthases (PKSs) has allowed us to identify a gene cluster for ML-236B biosynthesis in Penicillium citrinum. Like lovastatin, which is produced by Aspergillus terreus, ML-236B (compactin) inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Genomic sequencing and Northern analysis showed that nine predicted genes for ML-236B biosynthesis were located within a 38-kb region and were transcribed when ML-236B was produced. The predicted amino acid sequences encoded by these nine genes, designated mlcA- mlcH and mlcR, were similar to those encoded by the genes for lovastatin synthesis, and were therefore assumed to be involved either directly or indirectly in ML-236B biosynthesis. Targeted disruption experiments provided evidence that two PKS genes in the cluster, mlcA and mlcB, are required for the biosynthesis of the nonaketide and the diketide moieties, respectively, of ML-236B, suggesting that the gene cluster as a whole is responsible for ML-236B biosynthesis in P. citrinum. Bioconversion of some of the predicted intermediates by an mlcA-disrupted mutant was also investigated in order to analyze the ML-236B biosynthetic pathway. The molecular organization of the gene cluster and proposed functions for the ML-236B biosynthetic genes in P. citrinum are described.

Maternal beta-catenin establishes a 'dorsal signal' in early Xenopus embryos.

In previous work, we demonstrated that maternally encoded beta-catenin, the vertebrate homolog of armadillo, is required for formation of dorsal axial structures in early Xenopus embryos (Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., Kintner, C., Yoshida-Noro, C. and Wylie, C. (1994). Cell 79, 791-803). Here we investigated, firstly, the role(s) of beta-catenin in spatial terms, in different regions of the embryo, by injecting beta-catenin mRNA into individual blastomeres of beta-catenin-depleted embryos at the 32 cell stage. The results indicate that beta-catenin can rescue the dorsal axial structures in a non-cell-autonomous way and without changing the fates of the injected cells. This suggests that cells overexpressing beta-catenin send a 'dorsal signal' to other cells. This was confirmed by showing that beta-catenin overexpressing animal caps did not cause wild-type caps to form mesoderm, but did cause isolated beta-catenin-deficient marginal zones to form dorsal mesoderm. Furthermore beta-catenin-deficient vegetal masses treated with overexpressing caps regained their ability to act as Nieuwkoop Centers. Secondly, we studied the temporal activity of beta-catenin. We showed that zygotic transcription of beta-catenin starts after the midblastula transition (MBT), but does not rescue dorsal axial structures. We further demonstrated that the vegetal mass does not release a dorsal signal until after the onset of transcription, at the midblastula stage, suggesting that maternal beta-catenin protein is required at or before this time. Thirdly we investigated where, in relationship to other gene products known to be active in axis formation, beta-catenin is placed. We find that BVg1, bFGF, tBR (the truncated form of BMP2/4R), siamois and noggin activities are all downstream of beta-catenin, as shown by the fact that injection of their mRNAs rescues the effect of depleting maternally encoded beta-catenin. Interference with the action of glycogen synthase kinase (GSK), a vertebrate homolog of the Drosophila gene product, zeste white 3 kinase, does not rescue the effect, suggesting that it is upstream.

COPI- and COPII-coated vesicles bud directly from the endoplasmic reticulum in yeast.

The cytosolic yeast proteins Sec13p-Sec31p, Sec23p-Sec24p, and the small GTP-binding protein Sar1p generate protein transport vesicles by forming the membrane coat termed COPII. We demonstrate by thin section and immunoelectron microscopy that purified COPII components form transport vesicles directly from the outer membrane of isolated yeast nuclei. Another set of yeast cytosolic proteins, coatomer and Arf1p (COPI), also form coated buds and vesicles from the nuclear envelope. Formation of COPI-coated, but not COPII-coated, buds and vesicles on the nuclear envelope is inhibited by the fungal metabolite brefeldin A. The two vesicle populations are distinct. However, both vesicle types are devoid of endoplasmic reticulum (ER) resident proteins, and each contains targeting proteins necessary for docking at the Golgi complex. Our data suggest that COPI and COPII mediate separate vesicular transport pathways from the ER.

Yeast beta- and beta'-coat proteins (COP). Two coatomer subunits essential for endoplasmic reticulum-to-Golgi protein traffic.

To understand better the role of non-clathrin coat proteins in membrane traffic, we have cloned and characterized two essential genes encoding subunits of the yeast coatomer, SEC26 and SEC27. Sec26p is a 109-kDa protein that shares 43% sequence identity with mammalian beta-coat protein (beta-COP). Sec26p-depleted cells accumulate endoplasmic reticulum (ER) forms of secretory precursor proteins, and growth ceases after a dramatic accumulation of ER membranes. Sec26p overproduction partially suppresses sec27-1, a new mutant that shows a temperature-sensitive defect in ER-to-Golgi transport. The SEC27 gene was cloned, and the sequence predicts a 99.4-kDa protein with 45% sequence identity to mammalian beta'-COP. Our sequence data support a two-domain model for the SEC27 protein: a conserved amino-terminal domain, composed of five WD-40 repeats similar to those found in beta-subunits of trimeric G proteins, and a less conserved carboxyl-terminal domain. Genetic interactions connect sec27-1 and sec21-1 (coatomer gamma subunit) with the ARF1 and ARF2 genes and with the SEC22, BET1, and BOS1 genes, which encode membrane proteins involved in ER-to-Golgi transport.

COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum.

In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.

Application of computer to monitoring and control of fermentation process: Microbial conversion of ML-236B Na to pravastatin.

An automatic feeding process for microbial hydroxylation of ML236B sodium salt (ML-236B Na; compactin) by Streptomyces carbophilus SANK 62585 was developed. The hydroxylated product, pravastatin sodium salt (pravastatin; trade name Mevalotin), is an inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A reductase (HMG-CoA reductase) used as cholesterol-lowering drug. The hydroxylation activity of S. carbophilus was induced by the addition of ML236B Na to culture broth but inhibited by high concentration of ML236B Na. In order to obtain high conversion yield, it was necessary to maintain optimum ML236B Na concentration throughout the fermentation by continuous feeding. For this purpose, we developed an on-line monitoring method, which mainly consisted of a cross-flow filtration module, high-performance liquid chromatography (HPLC) analyzer, feed pump, and microcomputer for regulation of ML236B Na concentration. An algorithm for control of ML236B Na feed rate based on feedback and feed-forward control where conversion rate after Deltat was estimated by using regression analysis of the five latest values of conversion rate. In a fed-batch culture employing this system, the concentration of ML236B Na was maintained at optimum level during the fermentation and the productivity of pravastatin was increased threefold over that obtained in manual control culture.

SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer.

Non-clathrin coated vesicles have been implicated in early steps of intercompartmental transport. A distinct set of coat proteins are peripherally associated with the exterior of purified mammalian intra-Golgi transport vesicles. The 'coatomer', a cytosolic complex containing a similar subunit composition to and sharing at least one subunit (beta-COP) with the coat found on vesicles, has been postulated to be the precursor of this non-clathrin coat. Here we describe the characterization of SEC21, an essential gene required for protein transport from the endoplasmic reticulum to the Golgi in the yeast Saccharomyces cerevisiae. The 105K product of this gene, Sec21p, participates in a cytosolic complex that we show to be a yeast homologue of the mammalian coatomer. These observations demonstrate that a non-clathrin coat protein plays an essential role in intercompartmental transport.

Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

Purification and characterization of cytochrome P-450sca from Streptomyces carbophilus. ML-236B (compactin) induces a cytochrome P-450sca in Streptomyces carbophilus that hydroxylates ML-236B to pravastatin sodium (CS-514), a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase.

Pravastatin sodium (CS-514) is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in cholesterol biosynthesis. This compound is obtained by microbial hydroxylation of sodium ML-236B (compactin) carboxylate. The soluble cytochrome P-450 was induced by sodium ML-236B carboxylate in Streptomyces carbophilus of Actinomycetes as detected in its cell-free extract. This cytochrome P-450 was designated as cytochrome P-450sca after its origin. Cytochrome P-450sca was purified by successive chromatography on anion-exchange, gel filtration and hydroxyapatite columns. On hydroxyapatite cytochrome P-450sca was further separated into minor and major peaks, designated cytochrome P-450sca-1 and cytochrome P-450sca-2, respectively. Each peak yielded a single band on sodium dodecyl sulfate/polyacrylamide gels with molecular masses of 46 +/- 1 kDa. The activity hydroxylating sodium ML-236B carboxylate to pravastatin sodium was reconstituted in the presence of an electron transport system, an NADPH-generating system and oxygen. The Ks values of the cytochromes P-450sca-1 and P-450sca-2 for sodium ML-236B carboxylate were 179 microM and 229 microM, respectively. The CO versus reduced difference spectra of both cytochromes P-450 showed an absorption maximum at 448.5 nm. Their substrate difference spectra with sodium ML-236B carboxylate showed an absorption maximum at 386 nm. Amino acid analysis indicated that cytochrome P-450sca-1 and P-450sca-2 contained 46% and 47% hydrophobic residues, respectively. On Western blotting, cytochromes P-450sca-1 and P-450sca-2 were immunologically identical.

Effects of transforming growth factor beta on growth of human mammary epithelial cells in culture.

Normal human mammary epithelial cells (HMEC) from different individual reduction mammoplasty specimens were all growth inhibited, and showed a flattened, elongated morphology in response to human recombinant transforming growth factor beta 1 (TGF beta). The degree of growth inhibition varied among specimens, but none of the normal HMEC maintained growth in the continued presence of TGF beta. The degree of growth inhibition also varied with cell age in vitro, cells closer to senescence being more sensitive. TGF beta sensitivity was additionally assayed in two established cell lines derived from one of the reduction mammoplasty specimens after exposure to benzo(a)pyrene. Although varying degrees of growth inhibition and morphologic changes were observed in the cell lines, both lines contained populations that maintained active growth in the presence of TGF beta. Subclones of these lines demonstrated a great plasticity in their growth response to TGF beta, with individual clones ranging from strongly growth inhibited to nearly unaffected. These results suggest that multiple factors influence the extent of TGF beta-induced growth effects on both normal and transformed mammary epithelial cells, and that some of these factors may act through epigenetic mechanisms.

Cellulase production by a solid state culture system.

Production of cellulase using solid culture systems of Trichoderma reesei QM9414 and Sporotrichum cellulophilum on wheat bran was studied. By using moisture-controlled solid culture equipment, the effect of water content of wheat bran on cell growth and cellulase production was investigated. Cellular biomass grown on solid substrate was estimated by measuring oxygen consumption rate and glucosamine content of the cells. These parameters were shown to have a good linear correlation with the specific growth rate. This reliable method of estimating the cell growth rate enabled us to simulate the enzyme production in a solid culture system by means of multiple linear regression analysis which takes into account of the water content, cell mass, and the oxygen consumption rate as variables. The cell growth and cellulase production were maximized at different water content of the medium. A high water content, 57% for T. reesei and 70% for S. cellulophilum, favored mycelial growth, while the maximum cellulase activity was obtained at a lower water content such as 50% for both fungi. It was observed that cellulase production by T. reesei depended on the culture conditions that support the optimal growth rate for the maximum enzyme production.