LV10502B - Novel fungal strain and use thereof in preparation of antibiotics - Google Patents
Novel fungal strain and use thereof in preparation of antibiotics Download PDFInfo
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- LV10502B LV10502B LVP-93-115A LV930115A LV10502B LV 10502 B LV10502 B LV 10502B LV 930115 A LV930115 A LV 930115A LV 10502 B LV10502 B LV 10502B
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Description
-2- LV 10502
This invention relates to novel, genetically engineered fungal strains, and use thereof in preparation of antibiotics. More specifically, it relates to novel, genetically engineered strans of Aspergillus and their use in preparation of the drug lovastatin and analogs thereof.
Lovastatin is, chemically [lS-[loc(R*), 3α, 7β, 8β (2S*, 4S*), 8αβ]]-2-methylbutanoic acid 1, 2, 3, 7, 8, 8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)-ethyl]-l-naphthalenyl ester, and has the Chemical formula:
It is usefiil as an antihypercholesterolemic, being a potent inhibitor of HMG-CoA reductase, the rāte controlling enzyme in cholesterol biosynthesis. It is a fungal metabolite produced by fermentation processes using selected fungal strains.
Antibiotics such as lovastatin are metabolites which require sets of several enzymes for their synthesis. To permit their production by molecular cloning of anyibiotic-producing microorganisms requires the isolation, analysis and, perhaps modification of the corresponding genes for the several enzymes. Attempts to isolate such genes from such fungal species have so far yielded clones carrying either individual genes of the set, or only incomplete gene sets - see Malpartida and Hopwood, "Molecular Cloning of the Whole Biosynthetic Pathway of a Streptomvces Antibiotic and its Expression in a Heterologous Host", Nature (1984), 309 pp 462-464. -3-
Canadian Patent 1.129.794 Endo. describes the preparation of lovastatin by fermentation using a strain of the fungal species Monascus ruber.
Canadian Patent 1.161.380 Monaghan et ai., describes the preparation of lovastin by fermentation using strains of the fimgal species Aspereillus terreus.
In both of these prior art processes, lovastatin is produced along with other, very similar Chemical compounds, in substantial quantities, from which it must be separeted. In its production using Monascus ruber. lovastatin co-occurs with monacolin J. In its production using Aspergillus terreus. it co-occurs with dihydrolovastatin and with the hydroxy acid. Whilst the hydroxy acid can be readily lactonized to lovastatin, the dihydrolovastatin must be separated from it.
It is an object of the present invention to provide novel, genetically engineered mutant fungal strains capable of use in fermentation processes to make lovastatin.
It is a further object of the invention to provide novel processes for making lovastatin by fermentation using such strains.
According to one aspect of the present invention, novel mutant fungal strains of appropriate species of the gēnus Aspergillus are provided, which are capable of expressing and secreting lovastatin.These strains contain the genes of the lovastatin-producing enzyme set, in functioning relationship, derived from the DNA oflovastatin-producing Aspergillus terreus strains. They are produced by a process of protoplast transformation of the DNA from the lovastatin-producing Aspergillus terreus strain, also containing an appropriate selectable marker, with another , non-lovastatin producing Aspergillus species, selected from A. orvzae. A. fiimigatus. A. niger. A. nidulans and A. flavus, selečtion of the transformants so formed on the basis of the selectable marker, Identification and isolation of the lovastatin-producing transformants and sub-cloning thereof. -4- LV 10502
According to another aspect of the invention, there is privided a process of preparing lovastatin which comprises fermenting a nutrient medium with a transformant Aspergillus microorganism containing genes derived from Aspergillus terreus and coding for the set of lovastatin-producing enzymes, and recovering the lovastatin so formed.
The process of the present invention producēs lovastatin with significantly smaller amounts of contaminating dihydrolovastatin and hydroxy acid derivatives, as compared with processes using Aspergillus terreus.
In the process of making the transformants of the present invention, Standard techniques of preparing protoplasts from the selected, non-lovastatin producing Aspergillus strain, and Standard techniques of extracting the DNA from the lovastatin-producing Aspergillus terreus strain can be employed. These techniques are well known to those of skill in the art and do not need detailed discussion herein. Similary, the techniques and procedures of protoplast transformation useful herein are known and Standard.
The preferred choise of non-lovastatin producing Aspergillus strain is a strain of Aspergillus orvzae. but this is not critical to success in practising the invention. Other species of Aspergillus namely A. niger. A. nidulans. A, fumigatus and A, flavus. can be used. A, orvzae is chosen as the preferred species on account of its inertness, which renders it easy and safe to handle in the laboratory and in commercial scale fermentations.
In order to select transformant microorganisms from non-transformants after the protoplast transformation process, the DNA from the A. terreus should contain a selectable marker. There is a wide choice of selectable markers available to and selectable by the skilled worker, and the precise choice is not critical to success in vvorking the invention. Markers of antibiotic resistance such as ampicillin resistance, rifampicin resistance, streptomycin resistance etc. can be used, and the resulting mixture of transformants and non-transformants can be cultured in a medium containing the appropriate antibiotic, so that only the transformants, which contain the selectable marker, will survive for isolation. -5-
Particulary preferred as a selectable marker, on the basis of convenience, is cycloheximide resistance. Cycloheximide is an inhibitor of protein synthesis, so that the presence of a cycloheximide-resistant strain or transformant in a culture broth is readily detected. 5
After selection of the transformants on the basis of the selectable marker, they are screened for those which will express and secrete lovastatin. Only a relatively small number of the total transformants produced in the protoplast transformation process have this capability. They are recognized by separate culturing in Standard 10 culture broth, and analysis of the resulting medium, e.g. by HPLC, for the presence of lovastatin. These testing positive for the presence of lovaststin are sub-cultured and grown, to yeld colonies of novel, lovastatin-producing transformant fungal microorganisms of the Aspergillus gēnus and preferably of the Aspereillus orvzae species. 15
The invention is further described for illustrative purposes in the following experimental accounts.
In the accompanying dravvings: 20 FIGURĒ 1 is a graphical presentation of the HPLC analysis of the crude fungal extracts produced according to the Example 1 experiments described below; FIGURĒ 2 is the !H-NMR spectrum of the lovastatin compound odtained and 25 purified from the hybrid strain; FIGURĒ 3 is the mass spectrum of the same compound; FUGTJRE 4 is a depiction of the morphological characteristics of novel 30 transformant AoAt/NBJ-V. -6- LV 10502 EXAMPLE 1
MATERIALS AND METHODS FUNGAL CULTURE - fungal isolates Aspereillus terreus and A. orvzae used in the 5 present study were isolated from orchid soil samples collected from Agriculture Canada, Research Station, Beaverlodge, Alberta, Canada.
SELECTION OF CYCLOHEXIMIDE RESISTANT MUTANTS 10 Having grown both Aspergilli isolates on Czapek's dox media containing cycloheximide (conc. 0.1 - 5 mM) for 6 days at 28 ± 2°C, Spores (108) of each isolate A. terreus and A. orvzae were dispensed on 10 mM cycloheximide Czapek's dox broth (50 ml) and incubated for 30 min., centrifuged at 10,000 g for 10 min., and then re-suspended in sterile distilled water. They were washed three times by aqitation and 15 residimented by centrifugation. The spores were then suspended in sterile 1 % (v/V) Triton Χ-100 solution and their numbers determined in an aliquot on a haemocytometer. An appropriate dilution was dispensed on the 10 mM cycloheximide selection medium and resistant mutants were isolated after 10 days of incubation at 28 ± 2°C. Isolates were examined for lovastatin production. A. cycloheximide 20 resistant lovastatin producing isolate of A. terreus was selected for further transformation study.
PROTOPLAST AND DNA PREPARATION 25 Aspereillus orvzae and the cycloheximide-resistant lovastatin-producing A, terreus (herein designated JAG-4703) isolate were cultured separately in 50 ml of Czapek's dox broth. The cultures were incubated for 58 hours at 28 ± 2°C on a rotary shaker (New Brunswick Scientific Inc.) at 200 rpm, then harvested and washed with sterile distilled water by repeated centrifugation. -7-
Protoplasts from cultures of both species were obtained by using Novozym 234 (Novo-Nordisk, Novo Aile, DK 2880, Bagsvaerd, Denmark) to remove the celi walls during 10-12 hour digestion, generally follovving the method described by Dickinson & Isenberg, J. Gen. Microbiol. 128. p. 651-654 (1982). Aliquots of 5 protoplasts from each species regenerated viable, single celi walled spores at 28°C after 24 hours in regenerating medium (R) containing 0.1 g agar (Difco) 5 g sorbose and 0.35 g EDTA in 50 ml distilled water. On germination these spores developed ali the cultural characteristics of their parents. 10 The total DNA was isolated fforn Aspergillus terreus protoplasts according to the procedure described by Schlief & Wensink, "Practical Methods in Molecular Biology", 33, 21-29 (1981). The protoplasts were suspended in a solution consisting of 10 mg/ml SDS, 0.1 M NaCl and 0.1 M tris-HCl, pH 9.0. An equal volume of phenol saturated with the tris-HCl buffer was added. The mixture was centrifuged at 15 12,000 g for 10 min. in an Eppendorf Centrifuge tube (Brinkmann Instruments,
Canada Ltd., Rexdale, Ontario).The upper phase, containing the DNA, was removed, mixed with 95 % ethanol, stored at -20°C for 60 min., then centrifuged as before for 10 min. The peliet was resuspended in buffer, pH 7.0, and incubated with RNase (Sigma, St. Louis, MO, U.S.A.) treated with phenol and the DNA precipitated from 20 the aqueous layer. The isolated DNA was purified by adsorption and vvashing on DEAE-cellulose (DE-52, Whatman), presoaked in 10 mM tris-HCl buffer, pH 7.5 and 0.3 M NaCl and contained in a pasteur pipette. The DNA was eluted with 10 mM tris-HCl buffer, pH 7.5 containing 1.5 M NaCl. This solution was diluted to 0.2 M NaCl and the DNA was precipitated with two volumes of ethanol. The purity of the 25 DNA was estimated from the 200-320 nm spectra by determining the A260/A280 absorbance ratio. Only DNAs with ratios between 1.50 and 2.00 were used in the transformations. Six samples, each 0.1 mg of the isolated DNA were incubated in the regenerating medium to ensure the absence of viable protoplasts and none of them developed any colony. -8- LV 10502
PROTOPLAST -DNAINCUBATION
Approximately 5.2 χ 104 A. orvzae protoplasts, estimated by haemocytometer counts, were incubated with 10-100 ng of A. terreus DNA in 5 ml regenerating medium, the composition of which was as described above, and regenerated as described above. In order to study the possible Chemical effects of DNA on lovastatin expression, 10-100 μg calf-thymus DNA (Sigma Chemical Co., St. Louis, MO., U.S.A.), DNA (Bethesda Research Laboratories, Gaitherberg, MD, U.S.A.) and homologous A. orzvae DNA were incubated with similar lots of A. orvzae protoplasts. When DNA was included in the incubations, protoplasts regeneration was reduced to less than 10 % and comparable numbers of such treated protoplasts (105) failed to yield any cycloheximide resistance.
LOVASTATIN PRODUCTION AND ASSAY
The suspension of spores developed frorn A, orvzae protoplasts incubated with A. terreus DNA was inoculated, 1 ml per petriplate, onto Czapek'dox agar medium containing 10 mM cycloheximide. Afler incubation for 48 hours at 28 ± 2°C, cultures attained a diameter of 6-8 mm. Each developed separately from a single spore under these conditions. Those which grew on the cycloheximide medium were each inoculated separately onto Czapek'dox media broth (50 ml in 500 ml Erlenmeyer flask), incubated on rotary shaker (200 rpm) at 28 ±2°C for 12 days. Each growth media were examined for lovastatin production by the procedure described below and HPLC analysis.
EXTRACTION
Fermented broth (50 ml) was acidified to pH 4.0 with 17 N HC1 and then fractionated against ethylacetate (100 ml, 3x). Pooled ethylacetate fractions were dried in vacuo at 30°C, the residue was collected in acetonitrile (5 ml) and then subjected to HPLC analysis. -9-
HPLC ANALYSIS
The HPLC equipment (Beckman Modei 420) was supplied by Beckman Instruments, Toronto, Canada and consisted of an Altex pump (Modei 110 A) and 5 injection valve. The LC-UV detector was set at 237 nm. A hypersil C-18 ODS silica column (25 χ 0.46 cm, i.d.) and a solvent system of acetonitrile and water (55 : 45, V/V) at flow rāte of 1.0 ml/min. were used. Lovastatin produced was compared and qantitated ffom a Standard lovastatin curve construced.
10 EXAMPLE 1 - RESULTS
Seventy-nine cycloheximide-resistant isolates,including four lovastatin-producing isolates, were obtained ffom 5.2 χ 105 A orvzae protoplasts incubated with the DNA ffom the cycloheximide-resistant lovastatin-producing mutant of 15 Aterreus. The transformation experiments were performed six times.
The results are given in the following Table 1. -10- LV 10502 TABLE 1 Cycloheximide-resistant and lovastatin-expressing Aspereillus orvzae protoplasts incubated 05 35'3 u. (Uo, 05 c o C3 4-iI bQ g • HO3 XOΙΟ, G c5
05 cd>O
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Xa> 05 Π3Έ,oo tna
<u G<U P*5 o <s <n ο ο o §
bcG o o ιη o t~~ W"> 0-1 Ο O CS 1-1 co «J t-i<uGω bc<u VO O-l Ο (N O VO t> t"- iri o Ό l> 05 «3 d. O +-> O ε 3£ «Λ «n «r» 05 «O in l-i o O O O O o Ph cf 1-H i-H r-H i—l 1-H 1—H <5 .2 X X X X X X C/3 P r—4 T-H f- f-_ ir» *—; -< Ph m CS l—H 1—4
05 O o 05 O 05 O 05 o i—I r*4 T—1 1—' r-H X X X X X X <N <N o VO o‘ iri o-i X (N
:0l x 91 -11 -
One of the transformēti cultures AO-II, retained its original transformed characteristics for six months. Fig. 1 illustrates the HPLC analysis of the extracts obtained from the recipient and donor cultures of A. orvzae, and those treated with A, terreus DNA. The production of lovastatin by transformed isolates is shown below in Table 2. TABLE2 Lovastatin production bv transformed isolates of Aspereillus orvzae in Czapek'dox broth fpH 6.81 Isolate # Lovastatin Yield (mg/1) AO-I 15.36 ±1.78 ΑΟ-Π 26.89 ±3.76 AO-III 13.46 ±4.56 ΑΟ-ΙΥ 9.67 ± 0.75
Isolate no. AO-I was sub-cultured five times, to yield a transformant AoAt - NBJ/5 which has been deposited on February 25, 1992 as a viable, permanent culture thereof with American Type Culture Collection under reference number ATCC 74135.
The lovastatin is produced in association with the hydroxy acid analog thereof, which is readily convertible to lovastatin e.g. by refluxing in toluene, to accomplish lactonization and thereby to increase the lovastatin yield. The lactonization was not undertaken in these experiments.
Lovastatin produced by A. terreus parent culture in Czapek'dox was 166.72 ± 5.92 mg/1, and parent A, orvzae did not producē any lovastatin.
Figurē 2 of the accompaning dravvings is the 1H-NMP spectrum of the lovastatin compound purified by HPLC from the hybrid strain. By comparison with the known, Standard spectrum of lovastatin, this spectrum confirms of the priduct.
Figurē 3 of the accompaying drawings is the mass spectrum of the same product, and shows that lovastatin was obtained at a purity of 99 %. -12- LV 10502
Concentrations of DNA ranging from 5 - 20 ng per ml of medium affected neither the regeneration capacity of the A. orvzae protoplasts nor the recovery of cycloheximide resistant and lovastatin producing isolates from incubations with A. terreus DNA. The data indicate that these concentrations were not a limiting factor 5 in the production of lovastatin. Similary, DNA from calf-thymus, DNA and homologous A. orvzae DNA at contentrations from 5 ng to 5 μg per ml of medium did not affect the regeneration of A. orvzae protoplasts when compared with untreated Controls. However, DNA at 10 and 100 μg per ml of medium reduced . regeneration to maximum of 26 % and 1 % respectively. These DNA treatments did 10 not elicit lovastatin expression.
The regenerating medium yielded numbers of protoplasts comparable to those obtained by Acha et al. J. Gen. Microbiol., 45, 515-523 (1966), using a more complex mineral medium. Conidia and freshly germinated conidia produced better yields of 15 viable protoplasts and of DNA than the mycelia. The formation of cellular aggregates during protoplast regeneration described by Acha et al. (1966) was not observed in this system. The apparent transfer of factors responsible for the expression of cycloheximide resistance and lovastatin expression from A. terreus to A. orvzae indicates interspecies DNA transformation. The co-transformation of cycloheximide 20 resistance and lovastatin production was unexpectedly satisfactory and suggests a physical proximity and possible clusterting of the genes involved in the expression of these characteristics.
The morphology of the novel transformant AoAt-NBJ/5 is depicted in 25 Figurē 4 and may be characterized as follows: MORPHOLOGY OF AoAt-NBJ/5.
Conidial heads columnar, light brown. Conidiophores smooth, colourless. 30 Vesicles hemispherical, covered up to one-half or two-thirds by phialides arranged in two layers. Conidia globose or ellipsoidal, smooth colonies on Czapek agar growing very rapidly, either floccose or velvety cinnamon brown due to production of conidia. Orange exudate, reverse yellow to brown. - 13-
These experiments demonstrate that, in principle, genetically determined antibiotic producing characteristics of one fungal species can be expressed in another. EXAMPLE 2 - METHOD OF MANUFACTĪ JRE 5
Fungal culture: A transformed culture of Aspergillus oryzae (ATCC#74135) AoAt/NBJ-V was used to producē lovastatin.
FERMENTATION 10
Seed preparation: The fungal culture (lyophilized vial) was transferred aseptically onto the Potato-dextrose agar slant and allowed to grow at 25°C for 4 - 5 days. At the end of incubation, the conidial suspension was prepared by adding 10 ml of triton Χ-100 (0.01 %) solution, agitated vigorously and transferred the conidial 15 suspension onto sterile rice (50 g) contained in an Erlenmeyer flask (250 ml capacity). The flask was supplemented with an additional 10 ml sterile water, agitated vigorously and incubated at 28°C for 5 - 7 days. At the end of incubation period, sterile water (50 ml) was added.
The conidial suspension was passed through sterile muslin cloth and the filtrate 20 containing (1 χ 108 conidia/ml) was transferred onto seed fermenter (50 L capacity) with 30 L of seed media. The composition of seed media used was as foliows: ..
D-glucose 20 g/L
Malt extract 20 g/L
25 Neo-peptone 3 g/L
Tap water 1 L pH 6.8 (adjusted with 10 % - NaOH). (Media was sterilized at 121°C for 30 min (15 psi). The seed was allowed to grow for 17-20 hours at 28°C (aeration rāte 30 0.3 - 0.5 vvm) with 80 - 100 % dissolved oxygen (200 rpm). - 14- LV 10502
PRODUCTION OF LOVASTATIN
The seed (30 L) was transferred onto the production fermenter vessel (1,000 L, working volume) containing production media. The production media 5 composition was as follows:
Lactose 60 g/L Ardamine pH lOg/L Soy protein 2 g/L KCL 2 g/L kh2po4 0.8 g/L MnS044H20 0.03 g/L Betaine 0.6 g/L ^2000 2 ml Tap Water 1 L pH 6.8 (before sterilization, adjusted with 105 Na0H/H2S04) sterilization at 121°C for 1 hour at 15 - 20 psi. 20 After inoculation, the culture was allowed to grow for 7 - 8 days at 28°C with pH control at 6.0 - 6.2 (with 10 % H2S04).
The aeration rāte was maintained between 0.7 - 1.0 wm (% dissolved oxygen 80 - 100 %). -15-
2ND PRODUCTION STAGE
The 7 day old fermented broth (50 L) was transferred aseptically into a seed fermenter (2,000 L working volume) containing 1,500 L of seed media (composition given above). The seed was allowed to grow for 24 hours at 28°C (200 rpm) D.O. 80 - 100 %. The broth (200 L) was transferred aseptically to production fermenter (30,000 L) capacity with working volume 20,000 L containing 18,000 L of production media. The culture was allowed to grow further 7-9 days at 28°C with pH control at 6.0 - 6.2. After 60 hours of fermentation, pH was not controlled. The fermented broth was supplemented with feed media (rāte 10 L/hr) for 5 days. The feed media composition was as follovvs:
D-glucose 285.7 g/L
Ardamine pH 71.4 g/L
Soy protein 14.3 g/L
Tap Water 1 L pH 6.8 (before sterilization) - sterilized at 121°C for 30 - 45 min. At the end of fermentation, the broth was acidified to pH 4.0 with 10N-HCL and centrifuged. The fiingal cake as dried at 55°C and subjected to extraction.
EXTRACTION AND PURIFICATTION
The dried fiingal cake (20 kg) was homogenized with cold ethyl acetate (10 -15 min) and the homogenized material was refluxed at 80-85°C for 4-6 hours, allowed to cool and filtered. The filtrate was dried in vacuo to a black tarry material (2.5 kg - 3 kg). The black tarry materual was mixed with silica gel (2.5 kg) and CH2C12 (5 litre). The CH2C12 was evaporated in vacuo and the silica blended crude tarry material was poured onto a silica gel glass column (100 cm length χ 22.5 cm diam.) developed with EtOAc : Hexane (1 :2v.v.). Samples (2,000 jl, 12x) were collected. After 24 hours the eluting solvent mixture was changed to EtOAc : Hexane (1 : 1, v/v) and run for another 24 hours. Samoles containing the product were pooled, dried (280 g) snd crystallized with methanol. -16- LV 10502
CRYSTALLIZATION
The yellow dried product (280 g) was dissolved in 2.8 L of methanol and boiled for 40 - 60 minūtes at 60 - 65°C. The hot methanol mixture was filtered and the filtrate incubated for 4 - 6 hours at 4°C. The white ctystalline needles were separated from mother liquor and rinsed with cold methanol. The crystals were dried in vacuo, weighed (190 g) and ķept in dessicator at 4°C.
The process of the present invention can result in lovastatin yelds that are high enough to cause rupture of the celi walls of the novel Aspergillus orvzae transformants, so that in this way the step of lysing the celis prior to recovering the lovastatin can be eliminated. This substantially simplifies the dovvnstream recovery and purification process. As described above, the lovastatin according to the present process can be recovered by a solvent extraction process, without the need to form esters, salts etc, thereof during the course of the recovery. Solvent extraction is a much simpler and more economical process for recovery and purification than chomatography.
Whilst the invention has been described in detail by references of specific experiments, it will be understood that it is not restriced thereto. Its scope is defined in the appended claims.
Claims (6)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US83349692A | 1992-02-10 | 1992-02-10 | |
| CA002062023A CA2062023A1 (en) | 1992-02-10 | 1992-02-27 | Novel fungal strains and use thereof in antibiotic production |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| LV10502A LV10502A (en) | 1995-02-20 |
| LV10502B true LV10502B (en) | 1995-08-20 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| LVP-93-115A LV10502B (en) | 1992-02-10 | 1993-02-10 | Novel fungal strain and use thereof in preparation of antibiotics |
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|---|---|
| EP (1) | EP0556699A1 (en) |
| JP (1) | JPH0622780A (en) |
| CN (1) | CN1076965A (en) |
| AU (1) | AU667498B2 (en) |
| BG (1) | BG61180B1 (en) |
| BR (1) | BR9300467A (en) |
| CZ (1) | CZ17293A3 (en) |
| EE (1) | EE03048B1 (en) |
| FI (1) | FI930561A (en) |
| HR (1) | HRP930134A2 (en) |
| HU (1) | HUT67061A (en) |
| IL (1) | IL104481A0 (en) |
| LV (1) | LV10502B (en) |
| NO (1) | NO930446L (en) |
| NZ (1) | NZ245713A (en) |
| PL (1) | PL297691A1 (en) |
| SI (1) | SI9300068A (en) |
| SK (1) | SK5593A3 (en) |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6100054A (en) * | 1989-05-05 | 2000-08-08 | Baylor College Of Medicine | Production for recombinant lactoferrin and lactoferrin polypeptides using DNA sequences in various organisms |
| IL94183A (en) | 1989-05-05 | 2003-09-17 | Baylor College Medicine | cDNA SEQUENCE CODING FOR HUMAN LACTOFERRIN PROTEIN OR PORTION THEREOF AND LACTOFERRIN PROTEIN PRODUCED FROM SAID SEQUENCE |
| US5571691A (en) * | 1989-05-05 | 1996-11-05 | Baylor College Of Medicine | Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms |
| US5849881A (en) * | 1989-05-05 | 1998-12-15 | Baylor College Medicine | Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms |
| US5766939A (en) * | 1989-05-05 | 1998-06-16 | Baylor College Of Medicine | Production of recombinant lactoferrin and lactoferrin polypeptides using CDNA sequences in various organisms |
| SI9300303A (en) * | 1993-06-08 | 1994-12-31 | Krka Tovarna Zdravil | Process for isolation of hypolipemic effective substance |
| EP0726940A4 (en) * | 1993-11-02 | 2001-05-23 | Merck & Co Inc | Dna encoding triol polyketide synthase |
| US6221637B1 (en) | 1996-03-05 | 2001-04-24 | Takeda Chemical Industries, Ltd. | Xanthene derivatives, their production and use |
| US6111081A (en) * | 1996-05-31 | 2000-08-29 | Baylor College Of Medicine | Lactoferrin variants and uses thereof |
| CZ299290B6 (en) * | 1997-02-20 | 2008-06-11 | Dsm Ip Assets B.V. | Process for preparing beta-lactam compound, process for preparing and/or improving fibrous microbial strain and use of chemically defined fermentation medium |
| IL132822A0 (en) * | 1998-03-20 | 2001-03-19 | Biogal Gyogyszergyar | Metabolic controlled fermentation procedure for the manufacture of lovastatin hydroxy acid |
| US6391583B1 (en) * | 1998-12-18 | 2002-05-21 | Wisconsin Alumni Research Foundation | Method of producing antihypercholesterolemic agents |
| EP1176208A1 (en) * | 2000-07-28 | 2002-01-30 | Société des Produits Nestlé S.A. | Koji molds and use thereof for preparing cholesterol-lowering products |
| DE60218822T2 (en) * | 2001-02-09 | 2007-07-12 | Unilever N.V. | FOODS CONTAINING SOY PROTEIN AND STATIN |
| KR100423892B1 (en) * | 2001-12-03 | 2004-03-22 | 씨제이 주식회사 | A new process of lactonization in the preparation of statins |
| WO2006035295A1 (en) * | 2004-09-27 | 2006-04-06 | Ranbaxy Laboratories Limited | Process for the purification of lovastatin |
| CN102533893A (en) * | 2010-12-09 | 2012-07-04 | 浙江海正药业股份有限公司 | Method for preparing monacolin J |
| CN104277980B (en) * | 2013-07-09 | 2020-04-21 | 丰益(上海)生物技术研发中心有限公司 | Method for separating and purifying aspergillus oryzae transformant |
| CN105602856B (en) * | 2015-12-16 | 2019-04-16 | 浙江师范大学 | Aspergillus niger (Aspergillus niger) An-19 bacterial strain and its purposes and fermentation process of the production for Lovastatin |
| CN108118042B (en) * | 2016-11-30 | 2021-01-15 | 中国科学院青岛生物能源与过程研究所 | 2-methylbutyrate side chain hydrolase, Monacolin J-producing aspergillus strain, and construction method and application thereof |
| CN111117894B (en) * | 2019-11-26 | 2023-11-10 | 漳州大北农农牧科技有限公司 | Aspergillus oryzae strain and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU535944B2 (en) * | 1979-06-15 | 1984-04-12 | Merck & Co., Inc. | Hypocholestermic fermentation products from aspergillus |
| WO1987003907A1 (en) * | 1985-12-17 | 1987-07-02 | Lubrizol Genetics, Inc. | Isolation of genes for biosynthesis of polyketide antibiotics |
| FI104984B (en) * | 1988-08-11 | 2000-05-15 | Dsm Nv | Method for Identifying and Using Biosynthetic or Regulatory Genes to Improve Secondary Metabolite Production |
-
1993
- 1993-01-20 NZ NZ245713A patent/NZ245713A/en unknown
- 1993-01-22 IL IL104481A patent/IL104481A0/en unknown
- 1993-01-28 AU AU32121/93A patent/AU667498B2/en not_active Ceased
- 1993-02-02 SK SK55-93A patent/SK5593A3/en unknown
- 1993-02-03 BR BR9300467A patent/BR9300467A/en not_active Application Discontinuation
- 1993-02-08 HR HR930134A patent/HRP930134A2/en not_active Application Discontinuation
- 1993-02-09 EP EP93102016A patent/EP0556699A1/en not_active Withdrawn
- 1993-02-09 NO NO93930446A patent/NO930446L/en unknown
- 1993-02-09 FI FI930561A patent/FI930561A/en not_active Application Discontinuation
- 1993-02-09 BG BG97421A patent/BG61180B1/en unknown
- 1993-02-10 PL PL29769193A patent/PL297691A1/en unknown
- 1993-02-10 LV LVP-93-115A patent/LV10502B/en unknown
- 1993-02-10 JP JP5061492A patent/JPH0622780A/en active Pending
- 1993-02-10 SI SI19939300068A patent/SI9300068A/en unknown
- 1993-02-10 CN CN93102686A patent/CN1076965A/en active Pending
- 1993-02-10 HU HU9300330A patent/HUT67061A/en unknown
- 1993-02-10 CZ CZ93172A patent/CZ17293A3/en unknown
-
1994
- 1994-11-14 EE EE9400129A patent/EE03048B1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| FI930561A0 (en) | 1993-02-09 |
| CZ17293A3 (en) | 1993-12-15 |
| EE03048B1 (en) | 1997-10-15 |
| BR9300467A (en) | 1993-08-17 |
| BG97421A (en) | 1994-03-24 |
| CN1076965A (en) | 1993-10-06 |
| JPH0622780A (en) | 1994-02-01 |
| SI9300068A (en) | 1993-09-30 |
| HRP930134A2 (en) | 1995-10-31 |
| NZ245713A (en) | 1994-12-22 |
| EP0556699A1 (en) | 1993-08-25 |
| NO930446L (en) | 1993-08-11 |
| AU3212193A (en) | 1993-08-12 |
| HUT67061A (en) | 1995-01-30 |
| BG61180B1 (en) | 1997-02-28 |
| NO930446D0 (en) | 1993-02-09 |
| HU9300330D0 (en) | 1993-04-28 |
| LV10502A (en) | 1995-02-20 |
| IL104481A0 (en) | 1993-05-13 |
| SK5593A3 (en) | 1993-12-08 |
| PL297691A1 (en) | 1994-01-24 |
| AU667498B2 (en) | 1996-03-28 |
| FI930561A (en) | 1993-08-11 |
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