Title of Invention	GENETICALLY MODIFIED ASPERGILLUS ORYZAE AND A PLASMID TO USE IN ASPERGILLUS FOR PRODUCTION OF ALPHA-AMYLASE AND PROCESSES THEREOF.
Abstract	Genetically modified A.oryzae (MTCC 5177) for better production of amylase having sequence as herein described from locally available GRAS fungus A.oryzae E212 from the plasmid pAAC3 (9) obtained from cloning of 2.8213Kbpamylase fragment having a blunt end BamH1 side and a staggered end EcoR1 at both the ends derived from plasmid pSPK14 (1) and the 5Kbf"acetamidase gene of A. nidulans derived from p3SR2 (4) having a EcoR1 blunt end and Sal1 staggered end at both the ends with linearized plasmid pGEM 3Z having Sal1 and EcoR1 sides at both the ends and process thereof.

Title of Invention

GENETICALLY MODIFIED ASPERGILLUS ORYZAE AND A PLASMID TO USE IN ASPERGILLUS FOR PRODUCTION OF ALPHA-AMYLASE AND PROCESSES THEREOF.

Abstract

Genetically modified A.oryzae (MTCC 5177) for better production of amylase having sequence as herein described from locally available GRAS fungus A.oryzae E212 from the plasmid pAAC3 (9) obtained from cloning of 2.8213Kbpamylase fragment having a blunt end BamH1 side and a staggered end EcoR1 at both the ends derived from plasmid pSPK14 (1) and the 5Kbf"acetamidase gene of A. nidulans derived from p3SR2 (4) having a EcoR1 blunt end and Sal1 staggered end at both the ends with linearized plasmid pGEM 3Z having Sal1 and EcoR1 sides at both the ends and process thereof.

Full Text	FIELD OF THE INVENTION :- This invention relates to genetically modified Aspergillus oryzae (EIPW16, MTCC 5177) and a Plasmid to use in Aspergillus for production of alpha-amylase and processes for preparation of the same. More particularly Genetically modified filamentous fungus Aspergillus oryzae for hyper-production (4 to 5 times of original amylase producing Strain Aspergillus oryzae EIPW 212) and preparation of amylase from the said fungus by solid-state fermentation using agrowastes particularly wheat bran as the substrate, for use in degradation of starch. The invention further discloses the process for protein expression in solid state fermentation by using a genetically modified GRAS fungus. It encompasses a process of transformation with the recombinant plasmid containing the DNA-sequence and a suitable marker for selection of transformants and the selection of a stable transformants of GRAS fungus Aspergillus oryzae having multiple integrations of the DNA-sequence encoding the desired protein product at various chromosomal sites facilitating higher amount of expression of the protein coded by the DNA-sequence in solid state fermentation. The process permits the industrial production of secretory proteins that are expressed in the host GRAS fungus which are economic and easy to implement in an industrial process for doing the same. BACKGROUND OF THE INVENTION :- Aspergillus oryzae is a filamentous fungus is being used from prehistoric dates in production of fermented food in Asian countries. A. oryzae has been designated as generally regarded as safe (GRAS) organism by the US Food and Drug Administration. It has earned considerable interest in the biotechnology and enzyme producing industries due to its ability to produce and secrete hydrolyzing enzymes. A local isolate of filamentous fungus of GRAS origin Aspergillus oryzae(EIPW 212), has been studied for its capacity to secrete hydrolyzing enzymes. Aspergillus oryzae is a significant filamentous fungus used in the fermentation industry It is being used for its capability to secrete the important enzyme Alpha-Amylases Taka-amylase A (TAA) [EC 3.2.1.1, a - 1, 4- giucan-4-glucanohydrolase] which constitute a group of enzymes catalyzing hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides to yield dextrin during the fermentation processes. TAA is a glycoprotein consisting of a single polypeptide chair of 478 amino acids residues of which the sequence except for the signal peptide has been determined. Employing the synthetic oligonucleotides (26 oligomer each) as DNA probes corresponding to the N-terminal end and C-temninal end, the TAA gene was cloned frorr the genomic library of A.oryzae. The gene was located in a 3.7Kbp EcoR1 fragment anc A.oryzae transformants containing the EcoR1 fragment showed 2 to 5 fold enhancement in TAA activity. The complete nucleotide sequence of the gene was determined and it was found that the gene consisted of 2040 bp, with eight introns anc twenty-one amino acids comprising of a signal peptide. The deduced amino acid sequence contains one insertion, one deletion and ten substitutions of amino acids compared to that of the sequence reported earlier. a-Amylase enzyme and its coding multiple gene(s)/family members has been isolated and characterized from various sources of bacterial and eukaryotic systems. EP 0 238 023 A2 teaches a process for expression of a protein product in Aspergillus oryzae and is disclosed. The process comprises transforming Aspergillus oryzae with a vector system comprising DNA-sequences encoding functions facilitating gene expression, a suitable marker for selection of transformants, and a DNA-sequence encoding the desired protein product. The process enables industrial production of many different polypeptides and proteins in A. oryzae. Examples of such products are chymosin or prochymosin and other rennets, proteases, lipases and amylases. Also disclosed is an effective promoter for expression of a protein in Aspergillus. A preferred promoter is the TAKA-amylase promoter or functional parts thereof. EP 0 439 997 A1 states further a variant of a parent Fungamyl-like fungal alpha-amylase, which exhibits improved thermal stability at acidic pH suitable for, e.g., starch processes. US Patent 7,163,816 further discloses a method of constructing a variant of a parent Termamyl-like alpha-amy/ase, which variant has alpha-amy/ase activity and at least one altered property as compared to the parent alpha-amy/ase, comprising (a) analyzing the structure of the parent Termamyl-like alpha-amy/ase to identify at least one amino acid residue or at least one structural part of the Termamyl-like alpha-amy/ase structure, which amino acid residue or structural part is believed to be of relevance for altering the property of the parent Termamyl-like alpha-amy/ase (as evaluated on the basis of structural or functional considerations), (b) constructing a Termamyl-like alpha-amy/ase variant, which as compared to the parent Termamyl-like alpha-amy/ase, has been modified in the amino acid residue or structural part identified in (a) so as to alter the property, and (c) testing the resulting Termamyl-like alpha-amy/ase variant for the property in question. US Patent 6,664,095 describes an improved solid state fermentation device that combines all of the operations of microorganism cultivation (sterilization, inoculation, cultivation, extraction, and post extraction treatment). This solid state fermentation device is modular in nature and operates in a contained manner so that the live microorganisms from the reactor cannot come into contact with the environment and pollute the environment and also so that the environment inside the bioreactor is aseptic. Another aspect of this invention allows fermentation of microorganisms without inhibiting the growth of the microorganism. Specifically, the bioreactor is designed to remove heat that accumulates inside the bioreactor duing fermentation by conduction. Additionally, there is a mechanism to add fluid to the interior of the bioreactor that permits equal distribution and precise control of a variety of environmental parameters. For example, the bioreactor of the present invention provides a means to add nutritive media to the microorganisms at any time during the fermentation process without disturbing the fermenting microorganisms. Furthermore, the bioreactor of the present invention provides a mechanism to mix the contents of the bioreactor at any time and for any duration during the fermentation process. Finally, the present bioreactor provides a means of extracting desired microbial products from the bioreactor without opening the bioreactor. No prior art describes as such the requirements of production of alpha-amylase in high yield through genetically modified Aspergillus oryzae at a cheaper industrial process. Accordingly, there remains a need for a method of genetically modified Aspergillus oryzae and a Plasmid to use in Aspergillus for production of alpha-amylase and process for preparation of the same. Cloning of TAA and hyper-expression TAA activity to the extent of 2 to 5 folds by transformants of A. oryzae with TAA gene was reported. In this report the enhanced TAA activity has been demonstrated by growing the TAA-transformant of A. oryzae in liquid Czapek-Dox medium supplemented with 1% starch with shaking for 5 days. Accordingly, an attempt has been made to identify the local strain Aspergillus oryzae (EIPW212) and used here has been demonstrated to yield enhanced TAA activity to the extend of 4 to 5 fold upon solid state fermentation for more or less 48 hours using the agro waste wheat bran and like substances. The production of TAA employing the said strain is much cheaper and it produces TAA by employing the general method of solid state fermentation, which is considered to produce higher amount of TAA activity compared to liquid submerged fermentation culture. OBJECT OF THE INVENTION :- The object of the invention is to prepare genetically modified Aspergillus oryzae (EIPW16, MTCC 5177) for production of alpha-amylase using agrowastes e.g. wheat bran as substrate for degradation of starch. The further object of the invention is to produce a Plasmid to use in Aspergillus for production of alpha-amylase. The further object of the invention is to develop a solid-state fermentation process for preparation of alpha-amylase from hyper-producing (4 to 5 times of original amylase producing Strain Aspergillus oryzae EIPW 212), genetically modified Aspergillus oryzae (EIPW 16, MTCC 5177) for degradation of starch. STATEMENT OF INVENTION Genetically modified A.oryzae (MTCC) for better production of amylase having sequence as herein described from locally available GRAS fungus A.oryzae E212 from the plasmid pAAC3 (9) obtained from cloning of 2.826 Kbp amylase fragment having a blunt end BamH1 side and a staggered end EcoR1 at both the ends derived from plasmid pSPK14 (1) and the 5Kbp acetamidase gene of A. nidulans derived from p3SR2 (4) having a EcoR1 blunt end and Sail staggered end at both the ends with linearized plasmid pGEM 3Z having Sail and EcoR1 sides at both the ends and process thereof. DESCRIPTION OF THE INVENTION WITH ACCOMPANYING DRAWINGS: FIG. 1 Schematic Diagram of Preparation for the Construct having the amylase gene and the acetamidase gene biomarker in a recombinant plasmid pAAC3 (9) FIG. 2 Pair of Primer Sequences of ASP1 and ASP4. FIG. 3 DNA SEQUENCE of amylase gene (Sequence ID No. 1) derived from A. oryzae EIPW 212, cloned in pSPK14. According to the invention, the genetically modified Aspergillus oryzae (EIPW16 having MTCC No. 5177) has been prepared comprising of the following steps. i. Cloning (path lii, fig.1) of appropriate genomic DNA fragment from the host A. oryzae capable of expressing and secreting out of amylase enzyme in the medium supporting the growth of the fungus. The DNA fragment should code for the functional regions (including the preregion upstream activating sequences and promoter) to code and express for the enzyme amylase capable to perform the amylolytic activity. The DNA fragment should also code for the essential region to confer the functions of Promoter activity or the functional parts thereof optionally preceded by upstream activating sequences. It can also have additional DNA sequences related to the expressions. The coding region of the DNA should also have the essential leader sequences leading to expression of the pre-protein to facilitate the transport of expressed protein for producing the active and mature-amylase enzyme. It is preferred that the expressed protein should be secreted out of the cell without accumulating inside. Proteins that are secreted out of the cells are preferred for isolation as the cells are not required to be disrupted resulting less deterioration of the proteins due to the processes. Thus the presence of a preregion which is a leader peptide or a synthetic sequence or a naturally occurring signal or functional parts thereof ensures the effective direction of the expressed product into the secretory pathway of the cell. The preregion results the secretion and is generally cleaved from the desired product during secretion leaving the mature product ready for isolation from the culture medium. The DNA sequence should also have the transcription termination and polyadenylation signal essential to terminate the expression of the protein. ii. Genomic DNA was isolated from the GRAS Aspergillus oryzae E1PW 212 a local isolate of filamentous fungus. Restriction endonucleases mediated digestions of the isolated chromosomal DNA and fractionation of the digested chromosomal DNA fragments is performed followed by the southern hybridization employing a probe DNA fragment capable of specific hybridization to the genomic DNA fragment that codes for the amylolytic activity or a part thereof of the host A. oryzae EIPW 212. The DNA fragment that codes for the amylase activity or a part thereof was fished out from the chromosomal DNA isolated from A. oryzae EIPW 212 using a specific probe that was designed from the genomic gene sequence of Aspergillus oryzae (Norihiro, T. et al, Gene, vol. 84, 1989, pages 319 - 327). Genomic clones of amylase enzyme have been studied to find the most conserved region. A conserved region in all these clones was identified. The probe has been designed to the corresponding location of the amylase enzyme having the conserved region of the enzymatic activity encompassing the active sites of the amylase enzyme. A pair of primers ASP1 and ASP4 (fig.2) was synthesized corresponding to the two_ sites in or adjacent to the conservative region of amylase enzyme. Therefore it was suggested that the unknown genomic clone of A. oryzae EIPW 212 might also have partial or a full homology to the conserved region. iii. Chromosomal DNA isolated from A. oryzae EIPW 212 was amplified by PCR employing the pair of primers ASP1 and ASP4 corresponding to the two specific sites in the conservative region of amylase enzyme. A PCR product of 1.589 Kbp DNA was obtained using very stringent parameters of PCR amplification. iv. The PCR amplified 1.589 KbPDNA fragment was radiolabeled to be used as a probe in the DNA hybridization to identify the homologous chromosomal DNA fragments from BamH1 and EcoR1 restriction enzymes digested A. oryzae EIPW 212 in a southern blotting experiment. This specific homologous DNA fragments complementary to the probe has been purified and cloned in. appropriate site of a suitable plasmid pGEM4 to construct the recombinant plasmid pSPK14 (1, fig.1) and transformed in the E. coli using the standard technique of gene cloning. v. The transformed E. coli cells were detected by colony hybridization employing the PCR amplified 1.589 Kbp radiolabeled DNA fragment probe. Bacterial colonies harboring the A. oryzae EIPW 212 DNA fragments coding for amylase gene or a part thereof was detected by the specific colony hybridization experiment. The bacterial colony was further purified and the plasmid DNA pSPK14 containing the DNA fragment of fungal origin was isolated. DNA sequencing of the plasmid pSPK14 containing the 2.826 KbpDNA fragment derived from the fungal origin was done and the amylase gene of A. oryzae EIPW 212 has been detected in the sequence. vi. Recombinant plasmid pAAC3 (9, fig. 1) was constructed harboring the amylase gene of fungus A.oryzae EIPW 212 and a suitable marker from plasmid p3SR2 (4, fig. 1) to permit selection of transformants of the host A oryzae (ATCC 66222) funqal strain. The successful transformants was screened out by its ability to utilize the biomarker. In this case the nitrogen utilization gene of amdS from A. nidulans was employed for the selection of transformants by its ability to grow in acetamide containing growth plates (Kelly, J.M. and Hynes, M.J., EMBO Journal vol. 4, 1985, pages 475 - 479). vii. From all the transformants the fungal transformants having efficient and homologous hyper expression of the amylase enzyme in appropriate media of solid state fermentation using wheat bran as the substrate was screened out. Aspergillus oryzae ATCC 66222 strain was chosen as the host for homologous expression of the amylase gene due to its ability to produce amylase enzyme and its inability to grow stongly on acetamide as the sole nitrogen source. When A. nidulans amdS gene (Tilburn, J. G. et al Gene 26 1983, pages 1470-1474) is used for the genetic transformation of the host A. oryzae ATCC 662225the transformed cell can be screened on the acetamide as the transformed fungus are able to utilize nitrogen utilizing amdS (biomarker) gene. Chromosomal DNA isolated from A. oryzae EIPW 212 were digested with restriction enzymes BamH1 and subjected to southern hybridization using the radiolabeled 1.589Kb DNA probe corresponding to the conserved region of amylase enzyme. Autoradiography revealed the hybridized bands at positions of 7.3KbP6.4Kbpand 3.8Kbp When BamH1 and EcoR1 double digested chromosomal DNA of A. oryzae EIPW 212 was utilized for the southern hybridization employing the same DNA probe it revealed the hybridized bands at the positions of 5.3 Kbpand 2.9 Kbp The intensity of the band located at the region of 2.9 Kbpwas found to be higher than the intensity of the hybridized band located at 5.3 Kbpband. BamH1 and EcoR1 double digested chromosomal DNA of A. oryzae ATCC 66222 strain was utilized for southern hybridization employing the radiolabeled 1.589KbpPCR fragment and hybridized bands at the positions of 5.8Kbp4.7Kbjand 3.7Kbpwere located to indicate the presence of the amylase gene or a part thereof in these fragments of hybridized bands. The DNA fragments corresponding to the 2.9 Kbphybridized DNA fragment derived from BamH1 and EcoR1 digested A. oryzae EIPW 212 chromosomal DNA and the acetamide utilizing amdS from A nidulans were cloned in a plasmid pGEM3Z (7, fig. 1) to construct a recombinant plasmid pAAC3 (9, fig. 1) harboring the fungal amylase gene located in a BamH1 and EcoR1 2.9KbpDNA fragment of A. oryzae EIPW 212 origin. The protoplasts of Aspergillus oryzae have been transformed to the amdS+ phenotype with the recombinant plasmid pAAC3 (9). The particular transformants for the hyper producing phenomenon of amylase production was picked up after a thorough screening. Amylase hyper producing recombinant strain was further tested by isolation of the chromosomal DNA from the recombinant amylase hyper producing A. oryzae (EIPW16, MTCC 5177). The chromosomal DNA was digested with BamH1 and EcoR1 restriction enzymes and southern blotting was performed using the radiolabeled 1.589KbpDNA fragment and also with the amdS gene. Autoradiography revealed that both the radiolabeled 1.589 KbpDNA fragment and the acetamide utilizing amdS gene has been inserted in the chromosome of A. oryzae 66222 in sites as revealed by the multiple hybridization bands with a very predominant higher intensity in a one band using both the above mentioned probes in separate experiments. The chromosomal DNA of local Aspergillus oryzae (EIPW 212), a filamentous fungus capable of producing the enzyme amylase was isolated (exemplified in example 1) after harvesting the fungal mass grown in M-15 starch media for 72 hours at 32°C. The purified chromosomal DNA was doubly digested with BamH1 and EcoR1 restriction enzymes and resolved in 0.8% agarose gel, followed by Southern blotting to Nytran membrane following the standard procedures. From the genomic amylase gene sequence of Aspergillus oryzae (Sequence Ref: Norihiro, T. et al, Gene, vol. 84, 1989 pages 319-327, 1989) oligo nucleotide primers within the conserved active sites of the enzyme amylase were chosen. The locations of primer ASP1 (24 mer) is within 967 5' 3: 990 bp and ASP4 (24 mer) is within 2556 5-3 2533 bp in the 2935 bp sequence of amylase gene. ASP1 and ASP4 (FIG. 2) primers are employed to PCR amplify the corresponding segment (967-2556 bp) of amylase gene of A. oryzae, using the isolated local A. oryzae EIPW 212 chromosomal DNA as the template. A PCR amplified product of 1.589 kbpwas obtained (exemplified in example 2) and purified. 1.589 kbcsegment of the amylase gene was radiolabelled by random primer labeling system using the radionucleotide dATP- =c P32 and was used as the probe for hybridization experiment with the above Nytran membrane already southern blotted with BamH1 and EcoR1 double digested chromosomal DNA from A. oryzae EIPW 212. Autoradiography revealed the hybridized bands at the positions of 5.3 Kbpand 2.9 Kbp However, the intensity of 5.3 Kbfband was found to be less than 2.9 Kbfband. The DNA fragments corresponding to the 2.9 Kbpsize, was purified and cloned in EcoR1- BamH1 sites of pGEM4 plasmid employing E. coli DH 5 Screening for the fungal amylase clone in the mini-genomic library was performed employing the colony hybridization technique, using the same radiolabelled probe of 1.589 KbpPCR amplified segment of amylase gene. About nine thousand colonies were screened. Five transformed colonies lighted up consistently after repeated screening. The recombinant plasmids namely pSPK 7, pSPK 14, pSPK 15, pSPK 16 and pSPK 18 were isolated from these five clones. PCR experiments and Southern hybridization experiments using the radiolabelled 1.589 Kbp probe revealed that these five recombinant plasmids harbor the amylase gene of A. oryzae EIPW 212 or the part of it. Subsequent restriction enzyme mapping and sequencing experiments were carried out using the di-deoxy sequencing procedure of nucleic acid employing T7 and SP6 RNA polymerase promoter primers. It was found that all these five clones contain the same sequence of the amylase gene cloned in EcoR1 - BamH1 sites of pGEM4 plasmid. Later the entire sequence of the amylase genomic gene was derived by DNA sequencing of the entire EcoR1 and BamH1 bound insert of 2.826KbpDNA fragment (exemplified in example 3). The entire sequence data (fig.3) confirmed the presence of the genomic gene as of amylase gene of A. oryzae. In order to enhance the production capability of amylase enzyme from the GRAS fungus Aspergillus oryzae in solid state fermentation using wheat bran as the substrate the 2.826Kbp amylase gene was cloned in a suitable host strain Aspergillus oryzae (ATCC 66222 ), herein described as Aspergillus, oryzae 222 (exemplified in example 4). The amylase gene was introduced into the host Aspergillus oryzae 222 by transformation technique. This transformation system in this filamentous fungus has been standardized. A. oryzae 222 grows poorly on acetamide as a nitrogen or carbon source and lacks sequences homologous to the amdS gene encoding the acetamidase of Aspergillus nidulans (exemplified in example 5). We have taken advantage of these observations to standardize the transformation system for Aspergillus oryzae using the amdS gene as a selection marker for selecting transformants on the basis of acetamide utilization. For this purpose protoplasts of Aspergillus oryzae 222 have been prepared and subsequently transformed to the amdS+ phenotype with a recombinant plasmid (exemplified in example 6). The recombinant plasmid was constructed by the introduction of 2.826Kbp amylase gene from Aspergillus oryzae (EIPW 212) and acetamidase (amdS) gene of Aspergillus nidulans obtained from the plasmid p3SR2 into the pGEM 3Z vector. With this constructed recombinant plasmid the protoplasts of Aspergillus oryzae 222 were transformed to the amdS+ phenotype. Pure culture of the amdS+ transformants were further screened and isolated. Each of the transformants was subjected to solid state fermentation using wheat bran, for amylase production (exemplified in example 7). We have found that 7 out of 50 amdS+ transformants have been producing higher amount of amylase than the wild-type strain. Mitotic stability of the clone has been determined periodically by first streaking for single colonies on non-selective medium and then testing a sample of 20 individual colonies, using selected medium for the transformed phenotype. Among the colonies tested, none was found to have a wild type phenotype. Several cycles of repeated growth has been done to check the stability of the transformants in both selective and non-selective conditions. Analysis of DNA from amdS+ transformants showed that transformation had occurred by integration of vector DNA sequences including the 2.826Kbp amylase gene from Aspergillus oryzae (EIPW 212) and acetamidase (amdS) gene in to the genome and confirmed increased copy number of the amylase gene in the transformants. This specific integration of DNA at the particular site of integration resulted in producing higher amount of amylase (exemplified in example 7). The amylase hyper producing clone has been designated as Aspergillus oryzae EIPW 16. Thus Aspergillus oryzae (EIPW 16) was derived by genetic manipulation with an amylase gene of its own origin for enhancing the yield of the product. This strain was further deposited with international Depository Authority (IDA) at Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh after obtaining the necessary permission from the appropriate authority and was assigned the MTCC number MTCC 5177. The solid-state fermentation process for production of alpha-amylase using Aspergillus oryzae (MTCC 5177) has been developed to obtain alpha-amylase in high yield from the processes as herein described. Process for the expression of proteins in fungus has been described. The expression ol proteins can be of homologous or heterologous in origin. As used in here "homologous proteins" means proteins produced by A. oryzae itself whereas heterologous proteins' means proteins not produced by A. oryzae. More specifically the processes to express homologous protein in A. oryzae as reported in the document comprises of following examples. EXAMPLE 1 Isolation of fungal Chromosomal DNA. (Ref. PNAS (1984), Vol. 81, 1470-74 and Methods of enzymology, Vol65, pages 404-411; Vol. 152, pages 181-183.) The culture of the fungus A. oryzae was grown in 50ml M15 starch medium in 37°C shaker (180 RPM or more) incubator for 48 to 72 hours. The culture was chilled and the mycelium was collected by filtration through a suitable cloth. The collected mycelium was washed in water at 4°C and immediately frozen at liquid nitrogen. The frozen mycelium was crushed to powder in the constant presence of liquid nitrogen by pressing in a porcelain mortar and pestle. The crashed frozen mycelium powder was added in small portions in 20 ml TEN9 buffer pH 9.0 (50mM Tris-HCL pH 9.0, 100mM EDTA, 200nM Nad) containing 10mg ProteinaseK. SDS was added to a final concentration of 1% to get a viscous turbid solution. Further 200 pi Pronase (from a stock 20mg/ml in buffer containing 10mM Tris pH 7.5 and 10mM Nad) was added. It is incubated at 37°C in a slow rocking (30 RPM) platform for overnight. After overnight incubation, a very gentle and careful crushing with the help of a sterile spatula crushed any presence of small lumps of cells. It should be reasonably clear viscous solution. Phenol and chloroform extraction was performed and the aqueous phase containing the chromosomal DNA was collected by centrifugation at room temperature to avoid the precipitation of SDS, NaOAc (pH 6.5) was added to a final concentration of 0.3M followed by the addition of 0.8 volume of isopropanol. Filament of the chromosomal DNA appeared and was collected by swirling around a bent tip Pasteur pipette. The filamentous chromosomal DNA was washed in 70% ethanol and dried. The DNA precipitate was dissolved in 2ml TE (10mM Tris pH 7.8 and 1mM EDTA) at 4°C. It is further treated with 20 pi RNAse A (lOmg/ml) at 37°C. Further phenol and chloroform extraction was done followed by DNA precipitation by the addition of NaOAc and ethanol. The filamentous chromosomal DNA precipitate was washed with 70% ethanol, dried and was dissolved in buffer TE at a concentration of 1.5ug per ul. EXAMPLE 2 PCR amplification of 1.589 KbpDNA for identification of A oryzae amylase gene or a part thereof. Synthetic DNA oligomer ASP1 and ASP4, fig (2) was employed in a standard PCR reaction set up using 10ng of fungal chromosomal DNA as the template. PCR reaction for the specific amplification of the desired DNA fragment was optimized by performing the first denaturation of the chromosomal DNA at 94°C for 40 seconds and then repetition of 29 PCR cycles each comprises of steps a) 94°C for 40 seconds followed by b) 70nC for 60 seconds. The PCR was completed by the final polymerization reaction at 70°C for 4 minutes. The optimized PCR produced a single DNA band of 1,589Kbpwhich was subsequently radiolabeled by primer extension method to generate the radioactive probe to selectively fish out genomic gene of amylase of A. oryzae origin. EXAMPLE 3 Construction of plasmid pSPK14 (1, fig. 1) The PCR product of 1.589KbpDNA fragment was made radioactive by primer extension method and employed in the southern hybridization experiments to hybridize BamH1 and EcoR1 digested chromosomal DNA derived from A. oryzae EIPW 212. Autoradiography revealed the hybridized bands at 5.3kbpand 2.9kbp, The intensity of autoradiograph at 2.9Kbpband was higher. The BamH1 and EcoR1 digested DNA fragments were eluted from the position of the 2.9Kbp autoradiograph band in a preparative gel that was identically done to match the conditions to reproduce the same autoradiograph as described. The purified 2.9KbPONA fragments were cloned in BamH1 and EcoR1 sites of the plasmid pGEM4 and transformed in competent E. coli DH5a cells to form the mini-genomic library in growth plates using ampicillin as the selection marker. Subsequent screening of about nine thousand colonies from this mini-genomic library was done by colony hybridization method using the same radio-labeled 1.589Kb DNA probe. Five transformed colonies lighted up consistently after repeated screening and the recombinant plasmids were isolated namely pSPK7, pSPK14, pSPK15, pSPK16 and pSPK 18. All these plasmids digested with BamH1 and EcoR1 produced the insert of 2.826 KbpDNA fragments which co-migrates with the liberalized vector pGEM4 and also hybridize with the 1.589Kb?DNA probe in a southern hybridization experiment. Subsequent restriction enzymes mapping showed the same identity in all of the inserts. Further, sequencing experiments were carried out using the di-deoxy sequencing procedure of nucleic acid employing T7 and SP6 RNA polymerase promoter primers. It was found that all these five clones contain the same terminal sequences of the amylase gene. The clone pSPK14 was sequenced to find the entire sequence of the amylase genomic gene. It is found to have 2.826KbpDNA fragment to code for the complete amylase gene of A. oryzae. EXAMPLE 4 Construction of plasmid pAAC3 (9, fig. 1) Path I: The plasmid pSPK14 (1, fig. 1) was digested with BamH1 and treated with klenow polymerase to blunt end the site (2, fig.1). The plasmid was digested with EcoR1 and the released 2.826KhpDNA fragment (3, fig.1) bound with BamM blunt end in one side and EcoR1 staggered end in the other side was purified (path 1. fig. 1) Path II: The plasmid p3SR2 (4, fig.1) was digested with EcoR1 and treated with klenow polymerase to blunt end the site (5, fig.1). The linearized plasmid was further digested with restriction enzyme Sal 1 to generate 5KbfDNA fragment (6, fig.1) containing the acetamidase gene bound with EcoR1 blunt side in one end and Sail staggered end. This fragment was purified (path II, fig. 1). Path III: The plasmid pGEM3Z (7, fig.1) was digested with EcoR1 and Sail and the linearized plasmid DNA (8, fig. 1) bound with staggered ends of EcoR1 and Sail sites was purified (path III, fig.1). Three piece DNA ligations with T4DNA ligase was carried out with the DNA fragments that were generated and purified as described in the above mentioned Path I, Path II and Path III. The ligated DNA was transformed in competent E. coli DH5a and the desired recombinant plasmid pAAC3 (9, fig.1) was constructed. EXAMPLE 5 Utilization of acetamide for screening the transformants. The growth of A. oryzae ATCC 66222 is very poor on nitrogen less minimal growth (Cove, D. J. 1966 Biochim. Biophys. Acta, vol. 113 page 51 - 56) agar plates containing 10mM acetamide as the only nitrogen source. The chromosomal DNA isolated from the strain lacks the acetamidase gene as revealed by the no detection of hybridized band in the southern hybridization experiment employing the radiolabeled 5Kbpacetamidase gene as the probe to hybridize restriction enzyme digested chromosomal DNA from this fungal strain, The transformed A. oryzae was selected in the nitrogen less minimal growth agar plates due to its ability to utilize the acetamidase gene in the presence of 10mM acetamide as the only nitrogen source. EXAMPLE 6 TRANSFORMATION IN A oryiae Spores of A. oryzae(ATCC 65222) were collected from a fresh slants (grown at 25CC in Czapekdox broth containing Sucrose 3%, Sodium nitrate 0.3%, Dipotassium hydrogen phosphate 0.1%, Magnesium Sulphate 7H20 0.05%, Potassium chloride 0.05% and Ferrous sulphate 7H20 0.001%. pH 7.3) and suspended in 10 ml H20 containing 0.005% Tween 80 followed by sonication. The spore (up to 107 spore per ml) suspension is inoculated in Czapekdox broth and incubated at 25CC for 16 to 20 hours with shaking to achieve germination of the spores. All the following operations were at 4-6°C or at the temperature as described herein. The germinated mycelium was collected by suitable sterile cloth filtration and resuspended in 5ml osmotic media containing 1.2 M Magnesium Sulphate and 10mM Disodium hydrogen phosphate pH 7. Either 15mg or 7mg Novozyme had been added followed by the addition of 250 pi BSA from a stock of 12 mg BSA per 1ml of osmotic medium. The viscous mycelia suspension was incubated at 30°C at an initial shaking rate of 200 RPM which was lowered gradually as the viscosity decreased and the incubation was continued for a period of 1.5 to 3 hours until a large number of protoplasts wem visible,,under the microscope in representative samples during the incubation. It was centrifuged using HB-6 rotor at 4000 RPM for 10 minutes to collect the precipitate of treated mycelia and was further washed with osmotic medium. Finally the washed mycelia was resuspended in 3 ml osmotic medium and was over layered with the same volume of trapping buffer (100mM Tris-HCl pH 7 and 600mM Sorbitol) to form two phases and kept in ice for 5 to 10 minutes followed by centrifugation as earlier. At the junction of two phases a denser band appears which was carefully collected and monitored under the microscope to observe protoplasts. The collected spherical protoplasts were mixed with equal volume of STC buffer (1X) containing 10mM Tris-HCl pH 7.5, 1.2 M Sorbitol and 10mM CaCI2 and centrifuged as described earlier. The precipitated protoplasts were suspended in STC buffer and were reprecipitated. Finally the protoplasts were suspended in 0.2 to 1ml 3TC buffer. Plasmids were prepared as pyrogen free preparations either by the CsCb density gradient ultracentrifugation method of plasmid preparation or by using the appropriate commercially available EndoFree plasmid preparation kits. Plasmids were finally dissolved in a concentration of 10ug to 20ug of plasmid per 10 ul of STC buffer (2X). Aliquots of 100ul protoplasts suspension was mixed with 10 pi of appropriate plasmid DNA and 200 pi 60 % PEG 4000 (dissolved in 10mMCaCI2 and 10 mM Tns-HCI pH 7.5). It was mixed by carefully rolling the tube and was incubated for 15 minutes. Finally 1ml of same 60% PEG solution had been added to each tube and was mixed carefully. The mixture was left at the room temperature for 15 minutes. 5ml of STC buffer (1X) was added and the protoplasts were precipitated by centrifugation as described earlier and was resuspended in 300 pi of STC buffer (1X). 100 pi of protoplasts, thus transformed with the appropriate plasmid were plated on nitrogen less minimal growth (Cove, D. J. 1966 Biochim. Biophys. Acta, vol. 113 page 51 - 56.) agar plates containing 10mM acetamide as the only nitrogen source. The growth plates were incubated at 37°C for 4 to 5 days for the growth of the resistant A. oryzae colonies. The transformed protoplasts mediated by the plasmid pAAC3 and the control plasmid p3SR2 produced resistant colonies in the above minimal growth plates containing the selection marker acetamide due to its ability to utilize acetamide as the sole nitrogen source. The protoplasts that have not received either of these plasmids failed to grow on the acetamide containing plates. EXAMPLE 7 Genetic stability of the transformants and amylase production. Spores from acetarnide resistant colonies, transformec with the plasmid pAAC3 or the plasmid p3SR2 were collected and suspended in sterile water and further spread on acetarnide containing minimal growth agar plates. This process of growth was repeated twice. Finally the transformed resistant A. oryzae colonies were grown on Czapekdox growth agar plates without any selection pressure, in the absence of acetarnide. The colonies appeared in the growth plates containing no acetarnide were again spread on the minimal growth agar plates containing 10 mM acetarnide and incubated for the growth. A good growth on the acetarnide containing plates indicated the mitotic stability of the permanently transformed A. oryzae to show the acetarnide utilizing phenotype and the transformant is hereby termed as A. oryzae EIPW16 Further to characterize the A. oryzae (EIPW16, MTCC 5177) chromosomal DNA had been isolated from the strain. Chromosomal DNA from this transformed strain and the original host A. oryzae ATCC 66222 were digested with restriction enzymes BamH1 and EcoR1 and southern hybridization experiments with the PCR amplified radiolabeled 1.589 Kbp DNA probe revealed the specific integration of amylase gene in the transformed A. oryzae (EIPW16, MTCC 5177) chromosomal DNA. Southern hybridization had also been performed employing the radiolabeled 5KbpDNA fragment (6, fig. 1) containing the acetamidase gene (derived from the p3SR2 plasmid by restriction enzymes digestion EcoR1 and Sail; path 2, fig 1). It also revealed the specific integration of the acetamidase containing DNA fragment in the A. oryzae (EIPW16, MTCC 5177) Further A. oryzae EIPW 16, the host strain A. oryzae ATCC 66222 and the donor strain A. oryzae EIPW 212 were plated on M15 starch media containing Starch 2%, Ammonium nitrate 0.3%, Potassium dihydrogen phosphate 0.1%, Magnesium sulphate 7H20 0.1%, Ferrous sulphate trace amount, Agar 1.8 % and incubated at 25°C for 72 hours. These amylase secreting strains produce starch depleting spherical zones on the above growth plates and the comparative analysis of starch hydrolyzing potential were compared. A.oryzae EIPW16 produces the highest hydrolyzed zone indicating the highest potential of this strain to produce amylase. Further the acetamide utilizing A. oryzae had been checked for their ability to produce amylase enzyme in the solid state fermentation using wheat bran as the substrate and a trend to hyper produce amylase enzyme had been observed an all the transformed A. oryzae isolates. To access the starch hydrolyzing ability of the transformed A. oryzae strains spores from different slants of transformed strains and the host A. oryzae 66222 were resuspended in aqueous solution containing 0.1% Triton X. The numbers of spores were measured by hemocytometer and appropriate dilutions were spread on M15 Starch Agar plates to get not more than 15 colonies on a 90mm growth plate. The plates were incubated at 25°C for 72 hours and 0.02M iodine was poured on Petri plates to visualize zone of clearance due to the starch hydrolyzing activity of the amylase produced by the individual colonies. Diameter of zone of clearance (Dc) and diameter of zone of fungal mass (Df) were measured for each of the colonies of the transformed and the host strains. The ratio (Dc/Df = R) was calculated for each colony. (Industrial Biotechnology; Oxford & IBH Publication 1992; editors Vedpal S.Malik & Padma Sridhar; p-425). The host strain showed an average R value of 1.9 and the higher starch hydrolyzing transformed strains showed R values in the range of 2.7 to 3.2. The permanently transformed A oryzae strains showing comparatively higher starch-hydrolyzing potential in the M15 starch growth plates were subjected to laboratory scale solid state fermentation using wheat bran as the substrate (Ramdas, M. et "aT, World Journal of Microbiology & Biotechnology, vol. 12, 1996, pages 267-271. Pandey, A. Process Biochemistry, vol. 27, 1992, pages 109-116. Madhavan N. K. & Pandey, A. Biotechnology Letters, vol. 18, no. 2, 1996, pages 199-204). These selected transformed A oryzae strains were grown in Czapekdox agar media for 5 days at 25°C. Tablel. Production of amylase enzyme in the Solid State Fermentation using wheat bran from Aspergillus oryzae and some of its genetically transformed varieties. The spore suspension from each of these selected cultures were collected and used as the inoculums for solid state fermentation for 48 hours at 25°C using sterile wheat bran as the substrate. After 48 hours the moulded bran were harvested and extracted with sterile water. The clear supernatant containing the enzyme extracts were subjected to amylase assay following the IP method of amylase assay (IP 2007, volume 2, page 734 - 735). The amylolytic activities are compared in the table 1. After a thorough screening the transformed A. oryzae AAC3 #16 (EIPW16, MTCC 5177) strain has been identified for its ability to hyper produce the amylase enzyme in the solid state fermentation using wheat bran. Different embodiments of the invention are possible to achieve the best method of performance and to obtain the product as stated earlier. It will be understood that skilled persons with many modifications, variations and adaptations may carry out the invention into practice without departing from its sprit in describing the invention for the purpose of illustration. We claim 1. Genetically modified A.oryzae (MTCC 5177) for production of amylase having sequence ID No. 1 from locally available GRAS (generally regarded as safe) fungus A.oryzae E212. 2. A recombinant plasmid pSPK14 (1) constructed to harbor the genomic clone of amylase gene derived from a locally isolated GRAS fungus A. oryzae E212. 3. A plasmid pACC3 (9) constructed to use in A. oryzae for production of fungal alpha amylase through cloning of a DNA fragment harboring the amylase gene from pSPK14 (1) and a biomarker amdS gene from a plasmid p3SR2 (4) in an E. coli plasmid pGEM3Z (7). 4. A process for producing a plasmid pSPK14 (1) where PCR parameters have been optimized for specific amplification of a DNA probe to produce an unique 1.589Kbp PCR fragment to specifically identify fungal amylase gene from A.oryzae fungal genome. 5. A process for producing a genetically modified A. oryzae (MTCC 5177) comprises of the following steps i. Providing a recombinant plasmid pACC3 (9) as here in described. ii. Transforming A.oryzae ATCC 66222 host with the constructed recombinant plasmid pACC3 (9) to produce stable genetically modified hyper producing amylase fungal strain having amdS+ phenotype. iii. Culturing the transformed A. oryzae (MTCC 5177) in a suitable culture medium. 6. A process has claimed in claim 5 wherein the plasmid pAAC3 (9) construction comprises of the following steps. i. Providing the 2.826Kbp amylase fragment having a blunt end BamH1 side and a staggered end EcoR1 at both the ends, from plasmid pSPK14 (1). ii. Providing the 5Kbp acetamidase gene of A. nidulans derived from p3SR2 (4) having EcoR1 blunt end and Sail staggered end. iii. Providing linearized plasmid pGEM 3Z having Sal 1 and EcoR1 sides at both the ends. iv. Cloning of the products obtained from steps i, ii and iii to construct the recombinant plasmid pAAC3 (9). 7. A genetically modified A. oryzae (MTCC 5177) substantially herein described. 8. A plasmid pAAC3 (9) substantially herein described. 9. A process for producing A. oryzae (MTCC 5177) substantially herein described. Genetically modified A.oryzae (MTCC 5177) for better production of amylase having sequence as herein described from locally available GRAS fungus A.oryzae E212 from the plasmid pAAC3 (9) obtained from cloning of 2.8213Kbpamylase fragment having a blunt end BamH1 side and a staggered end EcoR1 at both the ends derived from plasmid pSPK14 (1) and the 5Kbf"acetamidase gene of A. nidulans derived from p3SR2 (4) having a EcoR1 blunt end and Sal1 staggered end at both the ends with linearized plasmid pGEM 3Z having Sal1 and EcoR1 sides at both the ends and process thereof.

Full Text

FIELD OF THE INVENTION :-
This invention relates to genetically modified Aspergillus oryzae (EIPW16, MTCC 5177) and a Plasmid to use in Aspergillus for production of alpha-amylase and processes for preparation of the same. More particularly Genetically modified filamentous fungus Aspergillus oryzae for hyper-production (4 to 5 times of original amylase producing Strain Aspergillus oryzae EIPW 212) and preparation of amylase from the said fungus by solid-state fermentation using agrowastes particularly wheat bran as the substrate, for use in degradation of starch.
The invention further discloses the process for protein expression in solid state fermentation by using a genetically modified GRAS fungus. It encompasses a process of transformation with the recombinant plasmid containing the DNA-sequence and a suitable marker for selection of transformants and the selection of a stable transformants of GRAS fungus Aspergillus oryzae having multiple integrations of the DNA-sequence encoding the desired protein product at various chromosomal sites facilitating higher amount of expression of the protein coded by the DNA-sequence in solid state fermentation. The process permits the industrial production of secretory proteins that are expressed in the host GRAS fungus which are economic and easy to implement in an industrial process for doing the same.
BACKGROUND OF THE INVENTION :-
Aspergillus oryzae is a filamentous fungus is being used from prehistoric dates in production of fermented food in Asian countries. A. oryzae has been designated as
generally regarded as safe (GRAS) organism by the US Food and Drug Administration.

It has earned considerable interest in the biotechnology and enzyme producing
industries due to its ability to produce and secrete hydrolyzing enzymes. A local isolate
of filamentous fungus of GRAS origin Aspergillus oryzae(EIPW 212), has been studied
for its capacity to secrete hydrolyzing enzymes.
Aspergillus oryzae is a significant filamentous fungus used in the fermentation industry It is being used for its capability to secrete the important enzyme Alpha-Amylases Taka-amylase A (TAA) [EC 3.2.1.1, a - 1, 4- giucan-4-glucanohydrolase] which constitute a group of enzymes catalyzing hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides to yield dextrin during the fermentation processes. TAA is a glycoprotein consisting of a single polypeptide chair of 478 amino acids residues of which the sequence except for the signal peptide has been determined.
Employing the synthetic oligonucleotides (26 oligomer each) as DNA probes corresponding to the N-terminal end and C-temninal end, the TAA gene was cloned frorr the genomic library of A.oryzae. The gene was located in a 3.7Kbp EcoR1 fragment anc A.oryzae transformants containing the EcoR1 fragment showed 2 to 5 fold enhancement in TAA activity. The complete nucleotide sequence of the gene was determined and it was found that the gene consisted of 2040 bp, with eight introns anc twenty-one amino acids comprising of a signal peptide. The deduced amino acid sequence contains one insertion, one deletion and ten substitutions of amino acids compared to that of the sequence reported earlier. a-Amylase enzyme and its coding multiple gene(s)/family members has been isolated and characterized from various sources of bacterial and eukaryotic systems.
EP 0 238 023 A2 teaches a process for expression of a protein product in Aspergillus oryzae and is disclosed. The process comprises transforming Aspergillus oryzae with a vector system comprising DNA-sequences encoding functions facilitating gene expression, a suitable marker for selection of transformants, and a DNA-sequence encoding the desired protein product. The process enables industrial production of many different polypeptides and proteins in A. oryzae. Examples of such products are chymosin or prochymosin and other rennets, proteases, lipases and amylases. Also disclosed is an effective promoter for expression of a protein in Aspergillus. A preferred promoter is the TAKA-amylase promoter or functional parts thereof.
EP 0 439 997 A1 states further a variant of a parent Fungamyl-like fungal alpha-amylase, which exhibits improved thermal stability at acidic pH suitable for, e.g., starch processes.
US Patent 7,163,816 further discloses a method of constructing a variant of a parent Termamyl-like alpha-amy/ase, which variant has alpha-amy/ase activity and at least one altered property as compared to the parent alpha-amy/ase, comprising (a) analyzing the structure of the parent Termamyl-like alpha-amy/ase to identify at least one amino acid residue or at least one structural part of the Termamyl-like alpha-amy/ase structure, which amino acid residue or structural part is believed to be of relevance for altering the property of the parent Termamyl-like alpha-amy/ase (as evaluated on the basis of structural or functional considerations), (b) constructing a Termamyl-like alpha-amy/ase variant, which as compared to the parent Termamyl-like alpha-amy/ase, has been modified in the amino acid residue or structural part identified in (a) so as to alter the property, and (c) testing the resulting Termamyl-like alpha-amy/ase variant for the property in question.
US Patent 6,664,095 describes an improved solid state fermentation device that combines all of the operations of microorganism cultivation (sterilization, inoculation, cultivation, extraction, and post extraction treatment). This solid state fermentation device is modular in nature and operates in a contained manner so that the live microorganisms from the reactor cannot come into contact with the environment and pollute the environment and also so that the environment inside the bioreactor is aseptic. Another aspect of this invention allows fermentation of microorganisms without inhibiting the growth of the microorganism. Specifically, the bioreactor is designed to remove heat that accumulates inside the bioreactor duing fermentation by conduction. Additionally, there is a mechanism to add fluid to the interior of the bioreactor that permits equal distribution and precise control of a variety of environmental parameters. For example, the bioreactor of the present invention provides a means to add nutritive media to the microorganisms at any time during the fermentation process without
disturbing the fermenting microorganisms. Furthermore, the bioreactor of the present invention provides a mechanism to mix the contents of the bioreactor at any time and for any duration during the fermentation process. Finally, the present bioreactor provides a means of extracting desired microbial products from the bioreactor without opening the bioreactor.
No prior art describes as such the requirements of production of alpha-amylase in high yield through genetically modified Aspergillus oryzae at a cheaper industrial process. Accordingly, there remains a need for a method of genetically modified Aspergillus oryzae and a Plasmid to use in Aspergillus for production of alpha-amylase and process for preparation of the same.
Cloning of TAA and hyper-expression TAA activity to the extent of 2 to 5 folds by transformants of A. oryzae with TAA gene was reported. In this report the enhanced TAA activity has been demonstrated by growing the TAA-transformant of A. oryzae in liquid Czapek-Dox medium supplemented with 1% starch with shaking for 5 days.
Accordingly, an attempt has been made to identify the local strain Aspergillus oryzae (EIPW212) and used here has been demonstrated to yield enhanced TAA activity to the extend of 4 to 5 fold upon solid state fermentation for more or less 48 hours using the agro waste wheat bran and like substances. The production of TAA employing the said strain is much cheaper and it produces TAA by employing the general method of solid state fermentation, which is considered to produce higher amount of TAA activity compared to liquid submerged fermentation culture.
OBJECT OF THE INVENTION :-
The object of the invention is to prepare genetically modified Aspergillus oryzae (EIPW16, MTCC 5177) for production of alpha-amylase using agrowastes e.g. wheat bran as substrate for degradation of starch.
The further object of the invention is to produce a Plasmid to use in Aspergillus for production of alpha-amylase.
The further object of the invention is to develop a solid-state fermentation process for preparation of alpha-amylase from hyper-producing (4 to 5 times of original amylase producing Strain Aspergillus oryzae EIPW 212), genetically modified Aspergillus oryzae (EIPW 16, MTCC 5177) for degradation of starch.
STATEMENT OF INVENTION
Genetically modified A.oryzae (MTCC) for better production of amylase having sequence as herein described from locally available GRAS fungus A.oryzae E212 from the plasmid pAAC3 (9) obtained from cloning of 2.826 Kbp amylase fragment having a blunt end BamH1 side and a staggered end EcoR1 at both the ends derived from plasmid pSPK14 (1) and the 5Kbp acetamidase gene of A. nidulans derived from p3SR2 (4) having a EcoR1 blunt end and Sail staggered end at both the ends with linearized plasmid pGEM 3Z having Sail and EcoR1 sides at both the ends and process thereof.
DESCRIPTION OF THE INVENTION WITH ACCOMPANYING DRAWINGS:
FIG. 1 Schematic Diagram of Preparation for the Construct having the amylase gene and the acetamidase gene biomarker in a recombinant plasmid pAAC3 (9)
FIG. 2 Pair of Primer Sequences of ASP1 and ASP4.
FIG. 3 DNA SEQUENCE of amylase gene (Sequence ID No. 1) derived from A. oryzae EIPW 212, cloned in pSPK14.
According to the invention, the genetically modified Aspergillus oryzae (EIPW16 having MTCC No. 5177) has been prepared comprising of the following steps.
i. Cloning (path lii, fig.1) of appropriate genomic DNA fragment from the host A. oryzae capable of expressing and secreting out of amylase enzyme in the medium supporting the growth of the fungus.
The DNA fragment should code for the functional regions (including the preregion upstream activating sequences and promoter) to code and express for the enzyme amylase capable to perform the amylolytic activity.
The DNA fragment should also code for the essential region to confer the functions of Promoter activity or the functional parts thereof optionally preceded by upstream activating sequences.
It can also have additional DNA sequences related to the expressions.
The coding region of the DNA should also have the essential leader sequences leading to expression of the pre-protein to facilitate the transport of expressed protein for producing the active and mature-amylase enzyme. It is preferred that the expressed protein should be secreted out of the cell without accumulating inside. Proteins that are secreted out of the cells are preferred for isolation as the cells are not required to be disrupted resulting less deterioration of the proteins due to the processes. Thus the presence of a preregion which is a leader peptide or a synthetic sequence or a naturally occurring signal or functional parts thereof ensures the effective direction of the expressed product into the secretory pathway of the cell. The preregion results the secretion and is generally cleaved from the desired product during secretion leaving the mature product ready for isolation from the culture medium.
The DNA sequence should also have the transcription termination and polyadenylation signal essential to terminate the expression of the protein.
ii. Genomic DNA was isolated from the GRAS Aspergillus oryzae E1PW 212 a local isolate of filamentous fungus. Restriction endonucleases mediated digestions of the isolated chromosomal DNA and fractionation of the digested chromosomal DNA fragments is performed followed by the southern hybridization employing a probe DNA fragment capable of specific hybridization to the genomic DNA fragment that codes for the amylolytic activity or a part thereof of the host A. oryzae EIPW 212.
The DNA fragment that codes for the amylase activity or a part thereof was fished out from the chromosomal DNA isolated from A. oryzae EIPW 212 using a specific probe that was designed from the genomic gene sequence of Aspergillus oryzae (Norihiro, T. et al, Gene, vol. 84, 1989, pages 319 - 327).
Genomic clones of amylase enzyme have been studied to find the most conserved region. A conserved region in all these clones was identified.
The probe has been designed to the corresponding location of the amylase enzyme having the conserved region of the enzymatic activity encompassing the active sites of the amylase enzyme. A pair of primers ASP1 and ASP4 (fig.2) was synthesized corresponding to the two_ sites in or adjacent to the conservative region of amylase enzyme.
Therefore it was suggested that the unknown genomic clone of A. oryzae EIPW 212 might also have partial or a full homology to the conserved region.
iii. Chromosomal DNA isolated from A. oryzae EIPW 212 was amplified by PCR employing the pair of primers ASP1 and ASP4 corresponding to the two specific sites in the conservative region of amylase enzyme. A PCR product of 1.589 Kbp DNA was obtained using very stringent parameters of PCR amplification.
iv. The PCR amplified 1.589 KbPDNA fragment was radiolabeled to be used as a probe in the DNA hybridization to identify the homologous chromosomal DNA fragments from BamH1 and EcoR1 restriction enzymes digested A. oryzae EIPW 212 in a southern blotting experiment. This specific homologous DNA fragments complementary to the probe has been purified and cloned in. appropriate site of a suitable plasmid pGEM4 to construct the recombinant plasmid pSPK14 (1, fig.1) and transformed in the E. coli using the standard technique of gene cloning.
v. The transformed E. coli cells were detected by colony hybridization employing the PCR amplified 1.589 Kbp radiolabeled DNA fragment probe. Bacterial colonies harboring the A. oryzae EIPW 212 DNA fragments coding for amylase gene or a part thereof was detected by the specific colony hybridization experiment. The bacterial colony was further purified and the plasmid DNA pSPK14 containing the DNA fragment of fungal origin was isolated. DNA sequencing of the plasmid pSPK14 containing the 2.826 KbpDNA fragment derived from the fungal origin was done and the amylase gene of A. oryzae EIPW 212 has been detected in the sequence.
vi. Recombinant plasmid pAAC3 (9, fig. 1) was constructed harboring the amylase gene of fungus A.oryzae EIPW 212 and a suitable marker from plasmid p3SR2 (4, fig. 1) to permit selection of transformants of the host A oryzae (ATCC 66222) funqal strain. The successful transformants was screened out by its ability to utilize the biomarker. In this case the nitrogen utilization gene of amdS from A. nidulans was employed for the selection of transformants by its ability to grow in acetamide containing growth plates (Kelly, J.M. and Hynes, M.J., EMBO Journal vol. 4, 1985, pages 475 - 479).
vii. From all the transformants the fungal transformants having efficient and homologous hyper expression of the amylase enzyme in appropriate media of solid state fermentation using wheat bran as the substrate was screened out.
Aspergillus oryzae ATCC 66222 strain was chosen as the host for homologous expression of the amylase gene due to its ability to produce amylase enzyme and its inability to grow stongly on acetamide as the sole nitrogen source. When A. nidulans amdS gene (Tilburn, J. G. et al Gene 26 1983, pages 1470-1474) is used for the genetic transformation of the host A. oryzae ATCC 662225the transformed cell can be screened on the acetamide as the transformed fungus are able to utilize nitrogen utilizing amdS (biomarker) gene.
Chromosomal DNA isolated from A. oryzae EIPW 212 were digested with restriction enzymes BamH1 and subjected to southern hybridization using the radiolabeled 1.589Kb DNA probe corresponding to the conserved region of amylase enzyme. Autoradiography revealed the hybridized bands at positions of 7.3KbP6.4Kbpand 3.8Kbp When BamH1 and EcoR1 double digested chromosomal DNA of A. oryzae EIPW 212 was utilized for the southern hybridization employing the same DNA probe it revealed the hybridized bands at the positions of 5.3 Kbpand 2.9 Kbp The intensity of the band located at the region of 2.9 Kbpwas found to be higher than the intensity of the hybridized band located at 5.3 Kbpband.
BamH1 and EcoR1 double digested chromosomal DNA of A. oryzae ATCC 66222 strain was utilized for southern hybridization employing the radiolabeled 1.589KbpPCR fragment and hybridized bands at the positions of 5.8Kbp4.7Kbjand 3.7Kbpwere located to indicate the presence of the amylase gene or a part thereof in these fragments of hybridized bands.
The DNA fragments corresponding to the 2.9 Kbphybridized DNA fragment derived from BamH1 and EcoR1 digested A. oryzae EIPW 212 chromosomal DNA and the acetamide utilizing amdS from A nidulans were cloned in a plasmid pGEM3Z (7, fig. 1) to construct a recombinant plasmid pAAC3 (9, fig. 1) harboring the fungal amylase gene located in a BamH1 and EcoR1 2.9KbpDNA fragment of A. oryzae EIPW 212 origin.
The protoplasts of Aspergillus oryzae have been transformed to the amdS+ phenotype with the recombinant plasmid pAAC3 (9). The particular transformants for the hyper producing phenomenon of amylase production was picked up after a thorough screening.
Amylase hyper producing recombinant strain was further tested by isolation of the chromosomal DNA from the recombinant amylase hyper producing A. oryzae (EIPW16, MTCC 5177). The chromosomal DNA was digested with BamH1 and EcoR1 restriction enzymes and southern blotting was performed using the radiolabeled 1.589KbpDNA fragment and also with the amdS gene.
Autoradiography revealed that both the radiolabeled 1.589 KbpDNA fragment and the acetamide utilizing amdS gene has been inserted in the chromosome of A. oryzae 66222 in sites as revealed by the multiple hybridization bands with a very predominant higher intensity in a one band using both the above mentioned probes in separate experiments.
The chromosomal DNA of local Aspergillus oryzae (EIPW 212), a filamentous fungus capable of producing the enzyme amylase was isolated (exemplified in example 1) after harvesting the fungal mass grown in M-15 starch media for 72 hours at 32°C. The purified chromosomal DNA was doubly digested with BamH1 and EcoR1 restriction enzymes and resolved in 0.8% agarose gel, followed by Southern blotting to Nytran membrane following the standard procedures.
From the genomic amylase gene sequence of Aspergillus oryzae (Sequence Ref: Norihiro, T. et al, Gene, vol. 84, 1989 pages 319-327, 1989) oligo nucleotide primers within the conserved active sites of the enzyme amylase were chosen. The locations of primer ASP1 (24 mer) is within 967 5' 3: 990 bp and ASP4 (24 mer) is within 2556 5-3 2533 bp in the 2935 bp sequence of amylase gene. ASP1 and ASP4 (FIG. 2) primers are employed to PCR amplify the corresponding segment (967-2556 bp) of amylase
gene of A. oryzae, using the isolated local A. oryzae EIPW 212 chromosomal DNA as the template. A PCR amplified product of 1.589 kbpwas obtained (exemplified in example 2) and purified. 1.589 kbcsegment of the amylase gene was radiolabelled by random primer labeling system using the radionucleotide dATP- =c P32 and was used as the probe for hybridization experiment with the above Nytran membrane already southern blotted with BamH1 and EcoR1 double digested chromosomal DNA from A. oryzae EIPW 212. Autoradiography revealed the hybridized bands at the positions of 5.3 Kbpand 2.9 Kbp However, the intensity of 5.3 Kbfband was found to be less than 2.9 Kbfband. The DNA fragments corresponding to the 2.9 Kbpsize, was purified and cloned in EcoR1- BamH1 sites of pGEM4 plasmid employing E. coli DH 5 Screening for the fungal amylase clone in the mini-genomic library was performed employing the colony hybridization technique, using the same radiolabelled probe of 1.589 KbpPCR amplified segment of amylase gene. About nine thousand colonies were screened. Five transformed colonies lighted up consistently after repeated screening. The recombinant plasmids namely pSPK 7, pSPK 14, pSPK 15, pSPK 16 and pSPK 18 were isolated from these five clones. PCR experiments and Southern hybridization experiments using the radiolabelled 1.589 Kbp probe revealed that these five recombinant plasmids harbor the amylase gene of A. oryzae EIPW 212 or the part of it. Subsequent restriction enzyme mapping and sequencing experiments were carried out using the di-deoxy sequencing procedure of nucleic acid employing T7 and SP6 RNA polymerase promoter primers. It was found that all these five clones contain the same sequence of the amylase gene cloned in EcoR1 - BamH1 sites of pGEM4 plasmid. Later the entire sequence of the amylase genomic gene was derived by DNA sequencing of the entire EcoR1 and BamH1 bound insert of 2.826KbpDNA fragment (exemplified in example 3). The entire sequence data (fig.3) confirmed the presence of the genomic gene as of amylase gene of A. oryzae.
In order to enhance the production capability of amylase enzyme from the GRAS fungus Aspergillus oryzae in solid state fermentation using wheat bran as the substrate the 2.826Kbp amylase gene was cloned in a suitable host strain Aspergillus oryzae (ATCC 66222 ), herein described as Aspergillus, oryzae 222 (exemplified in example 4).
The amylase gene was introduced into the host Aspergillus oryzae 222 by transformation technique. This transformation system in this filamentous fungus has been standardized. A. oryzae 222 grows poorly on acetamide as a nitrogen or carbon source and lacks sequences homologous to the amdS gene encoding the acetamidase of Aspergillus nidulans (exemplified in example 5). We have taken advantage of these observations to standardize the transformation system for Aspergillus oryzae using the amdS gene as a selection marker for selecting transformants on the basis of acetamide utilization. For this purpose protoplasts of Aspergillus oryzae 222 have been prepared and subsequently transformed to the amdS+ phenotype with a recombinant plasmid (exemplified in example 6).
The recombinant plasmid was constructed by the introduction of 2.826Kbp amylase gene from Aspergillus oryzae (EIPW 212) and acetamidase (amdS) gene of Aspergillus nidulans obtained from the plasmid p3SR2 into the pGEM 3Z vector. With this constructed recombinant plasmid the protoplasts of Aspergillus oryzae 222 were transformed to the amdS+ phenotype. Pure culture of the amdS+ transformants were further screened and isolated. Each of the transformants was subjected to solid state fermentation using wheat bran, for amylase production (exemplified in example 7). We have found that 7 out of 50 amdS+ transformants have been producing higher amount of amylase than the wild-type strain. Mitotic stability of the clone has been determined periodically by first streaking for single colonies on non-selective medium and then testing a sample of 20 individual colonies, using selected medium for the transformed phenotype. Among the colonies tested, none was found to have a wild type phenotype. Several cycles of repeated growth has been done to check the stability of the transformants in both selective and non-selective conditions.
Analysis of DNA from amdS+ transformants showed that transformation had occurred by integration of vector DNA sequences including the 2.826Kbp amylase gene from Aspergillus oryzae (EIPW 212) and acetamidase (amdS) gene in to the genome and confirmed increased copy number of the amylase gene in the transformants. This specific integration of DNA at the particular site of integration resulted in producing higher amount of amylase (exemplified in example 7). The amylase hyper producing clone has been designated as Aspergillus oryzae EIPW 16.
Thus Aspergillus oryzae (EIPW 16) was derived by genetic manipulation with an amylase gene of its own origin for enhancing the yield of the product. This strain was further deposited with international Depository Authority (IDA) at Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh after obtaining the necessary permission from the appropriate authority and was assigned the MTCC number MTCC 5177.
The solid-state fermentation process for production of alpha-amylase using Aspergillus oryzae (MTCC 5177) has been developed to obtain alpha-amylase in high yield from the processes as herein described.
Process for the expression of proteins in fungus has been described. The expression ol proteins can be of homologous or heterologous in origin. As used in here "homologous proteins" means proteins produced by A. oryzae itself whereas heterologous proteins' means proteins not produced by A. oryzae.
More specifically the processes to express homologous protein in A. oryzae as reported in the document comprises of following examples.
EXAMPLE 1
Isolation of fungal Chromosomal DNA.
(Ref. PNAS (1984), Vol. 81, 1470-74 and Methods of enzymology, Vol65, pages 404-411; Vol. 152, pages 181-183.)
The culture of the fungus A. oryzae was grown in 50ml M15 starch medium in 37°C shaker (180 RPM or more) incubator for 48 to 72 hours. The culture was chilled and the mycelium was collected by filtration through a suitable cloth. The collected mycelium was washed in water at 4°C and immediately frozen at liquid nitrogen. The frozen mycelium was crushed to powder in the constant presence of liquid nitrogen by pressing in a porcelain mortar and pestle. The crashed frozen mycelium powder was added in small portions in 20 ml TEN9 buffer pH 9.0 (50mM Tris-HCL pH 9.0, 100mM EDTA, 200nM Nad) containing 10mg ProteinaseK. SDS was added to a final concentration of 1% to get a viscous turbid solution. Further 200 pi Pronase (from a stock 20mg/ml in buffer containing 10mM Tris pH 7.5 and 10mM Nad) was added. It is incubated at 37°C in a slow rocking (30 RPM) platform for overnight. After overnight incubation, a very gentle and careful crushing with the help of a sterile spatula crushed any presence of small lumps of cells. It should be reasonably clear viscous solution. Phenol and chloroform extraction was performed and the aqueous phase containing the chromosomal DNA was collected by centrifugation at room temperature to avoid the precipitation of SDS, NaOAc (pH 6.5) was added to a final concentration of 0.3M followed by the addition of 0.8 volume of isopropanol. Filament of the chromosomal DNA appeared and was collected by swirling around a bent tip Pasteur pipette. The filamentous chromosomal DNA was washed in 70% ethanol and dried. The DNA precipitate was dissolved in 2ml TE (10mM Tris pH 7.8 and 1mM EDTA) at 4°C. It is further treated with 20 pi RNAse A (lOmg/ml) at 37°C. Further phenol and chloroform extraction was done followed by DNA precipitation by the addition of NaOAc and
ethanol. The filamentous chromosomal DNA precipitate was washed with 70% ethanol, dried and was dissolved in buffer TE at a concentration of 1.5ug per ul.
EXAMPLE 2
PCR amplification of 1.589 KbpDNA for identification of A oryzae amylase gene or a part thereof.
Synthetic DNA oligomer ASP1 and ASP4, fig (2) was employed in a standard PCR reaction set up using 10ng of fungal chromosomal DNA as the template. PCR reaction for the specific amplification of the desired DNA fragment was optimized by performing the first denaturation of the chromosomal DNA at 94°C for 40 seconds and then repetition of 29 PCR cycles each comprises of steps a) 94°C for 40 seconds followed by b) 70nC for 60 seconds. The PCR was completed by the final polymerization reaction at 70°C for 4 minutes.
The optimized PCR produced a single DNA band of 1,589Kbpwhich was subsequently radiolabeled by primer extension method to generate the radioactive probe to selectively fish out genomic gene of amylase of A. oryzae origin.
EXAMPLE 3
Construction of plasmid pSPK14 (1, fig. 1)
The PCR product of 1.589KbpDNA fragment was made radioactive by primer extension method and employed in the southern hybridization experiments to hybridize BamH1 and EcoR1 digested chromosomal DNA derived from A. oryzae EIPW 212. Autoradiography revealed the hybridized bands at 5.3kbpand 2.9kbp, The intensity of autoradiograph at 2.9Kbpband was higher. The BamH1 and EcoR1 digested DNA fragments were eluted from the position of the 2.9Kbp autoradiograph band in a
preparative gel that was identically done to match the conditions to reproduce the same autoradiograph as described. The purified 2.9KbPONA fragments were cloned in BamH1 and EcoR1 sites of the plasmid pGEM4 and transformed in competent E. coli DH5a cells to form the mini-genomic library in growth plates using ampicillin as the selection marker. Subsequent screening of about nine thousand colonies from this mini-genomic library was done by colony hybridization method using the same radio-labeled 1.589Kb DNA probe. Five transformed colonies lighted up consistently after repeated screening and the recombinant plasmids were isolated namely pSPK7, pSPK14, pSPK15, pSPK16 and pSPK 18. All these plasmids digested with BamH1 and EcoR1 produced the insert of 2.826 KbpDNA fragments which co-migrates with the liberalized vector pGEM4 and also hybridize with the 1.589Kb?DNA probe in a southern hybridization experiment. Subsequent restriction enzymes mapping showed the same identity in all of the inserts. Further, sequencing experiments were carried out using the di-deoxy sequencing procedure of nucleic acid employing T7 and SP6 RNA polymerase promoter primers. It was found that all these five clones contain the same terminal sequences of the amylase gene. The clone pSPK14 was sequenced to find the entire sequence of the amylase genomic gene. It is found to have 2.826KbpDNA fragment to code for the complete amylase gene of A. oryzae.
EXAMPLE 4
Construction of plasmid pAAC3 (9, fig. 1)
Path I: The plasmid pSPK14 (1, fig. 1) was digested with BamH1 and treated with klenow polymerase to blunt end the site (2, fig.1). The plasmid was digested with EcoR1 and the released 2.826KhpDNA fragment (3, fig.1) bound with BamM blunt end in one side and EcoR1 staggered end in the other side was purified (path 1. fig. 1)
Path II: The plasmid p3SR2 (4, fig.1) was digested with EcoR1 and treated with klenow polymerase to blunt end the site (5, fig.1). The linearized plasmid was further digested
with restriction enzyme Sal 1 to generate 5KbfDNA fragment (6, fig.1) containing the acetamidase gene bound with EcoR1 blunt side in one end and Sail staggered end. This fragment was purified (path II, fig. 1).
Path III: The plasmid pGEM3Z (7, fig.1) was digested with EcoR1 and Sail and the linearized plasmid DNA (8, fig. 1) bound with staggered ends of EcoR1 and Sail sites was purified (path III, fig.1).
Three piece DNA ligations with T4DNA ligase was carried out with the DNA fragments that were generated and purified as described in the above mentioned Path I, Path II and Path III. The ligated DNA was transformed in competent E. coli DH5a and the desired recombinant plasmid pAAC3 (9, fig.1) was constructed.
EXAMPLE 5
Utilization of acetamide for screening the transformants.
The growth of A. oryzae ATCC 66222 is very poor on nitrogen less minimal growth (Cove, D. J. 1966 Biochim. Biophys. Acta, vol. 113 page 51 - 56) agar plates containing 10mM acetamide as the only nitrogen source.
The chromosomal DNA isolated from the strain lacks the acetamidase gene as revealed by the no detection of hybridized band in the southern hybridization experiment employing the radiolabeled 5Kbpacetamidase gene as the probe to hybridize restriction enzyme digested chromosomal DNA from this fungal strain,
The transformed A. oryzae was selected in the nitrogen less minimal growth agar plates due to its ability to utilize the acetamidase gene in the presence of 10mM acetamide as the only nitrogen source.
EXAMPLE 6
TRANSFORMATION IN A oryiae
Spores of A. oryzae(ATCC 65222) were collected from a fresh slants (grown at 25CC in Czapekdox broth containing Sucrose 3%, Sodium nitrate 0.3%, Dipotassium hydrogen phosphate 0.1%, Magnesium Sulphate 7H20 0.05%, Potassium chloride 0.05% and Ferrous sulphate 7H20 0.001%. pH 7.3) and suspended in 10 ml H20 containing 0.005% Tween 80 followed by sonication. The spore (up to 107 spore per ml) suspension is inoculated in Czapekdox broth and incubated at 25CC for 16 to 20 hours with shaking to achieve germination of the spores. All the following operations were at 4-6°C or at the temperature as described herein. The germinated mycelium was collected by suitable sterile cloth filtration and resuspended in 5ml osmotic media containing 1.2 M Magnesium Sulphate and 10mM Disodium hydrogen phosphate pH 7. Either 15mg or 7mg Novozyme had been added followed by the addition of 250 pi BSA from a stock of 12 mg BSA per 1ml of osmotic medium. The viscous mycelia suspension was incubated at 30°C at an initial shaking rate of 200 RPM which was lowered gradually as the viscosity decreased and the incubation was continued for a period of 1.5 to 3 hours until a large number of protoplasts wem visible,,under the microscope in representative samples during the incubation. It was centrifuged using HB-6 rotor at 4000 RPM for 10 minutes to collect the precipitate of treated mycelia and was further washed with osmotic medium. Finally the washed mycelia was resuspended in 3 ml osmotic medium and was over layered with the same volume of trapping buffer (100mM Tris-HCl pH 7 and 600mM Sorbitol) to form two phases and kept in ice for 5 to 10 minutes followed by centrifugation as earlier. At the junction of two phases a denser band appears which was carefully collected and monitored under the microscope to observe protoplasts. The collected spherical protoplasts were mixed with equal volume of STC buffer (1X) containing 10mM Tris-HCl pH 7.5, 1.2 M Sorbitol and 10mM CaCI2 and centrifuged as described earlier. The precipitated protoplasts were suspended in
STC buffer and were reprecipitated. Finally the protoplasts were suspended in 0.2 to 1ml 3TC buffer.
Plasmids were prepared as pyrogen free preparations either by the CsCb density gradient ultracentrifugation method of plasmid preparation or by using the appropriate commercially available EndoFree plasmid preparation kits. Plasmids were finally dissolved in a concentration of 10ug to 20ug of plasmid per 10 ul of STC buffer (2X).
Aliquots of 100ul protoplasts suspension was mixed with 10 pi of appropriate plasmid DNA and 200 pi 60 % PEG 4000 (dissolved in 10mMCaCI2 and 10 mM Tns-HCI pH 7.5). It was mixed by carefully rolling the tube and was incubated for 15 minutes. Finally 1ml of same 60% PEG solution had been added to each tube and was mixed carefully. The mixture was left at the room temperature for 15 minutes. 5ml of STC buffer (1X) was added and the protoplasts were precipitated by centrifugation as described earlier and was resuspended in 300 pi of STC buffer (1X).
100 pi of protoplasts, thus transformed with the appropriate plasmid were plated on nitrogen less minimal growth (Cove, D. J. 1966 Biochim. Biophys. Acta, vol. 113 page 51 - 56.) agar plates containing 10mM acetamide as the only nitrogen source. The growth plates were incubated at 37°C for 4 to 5 days for the growth of the resistant A. oryzae colonies. The transformed protoplasts mediated by the plasmid pAAC3 and the control plasmid p3SR2 produced resistant colonies in the above minimal growth plates containing the selection marker acetamide due to its ability to utilize acetamide as the sole nitrogen source. The protoplasts that have not received either of these plasmids failed to grow on the acetamide containing plates.
EXAMPLE 7
Genetic stability of the transformants and amylase production.
Spores from acetarnide resistant colonies, transformec with the plasmid pAAC3 or the plasmid p3SR2 were collected and suspended in sterile water and further spread on acetarnide containing minimal growth agar plates. This process of growth was repeated twice. Finally the transformed resistant A. oryzae colonies were grown on Czapekdox growth agar plates without any selection pressure, in the absence of acetarnide. The colonies appeared in the growth plates containing no acetarnide were again spread on the minimal growth agar plates containing 10 mM acetarnide and incubated for the growth. A good growth on the acetarnide containing plates indicated the mitotic stability of the permanently transformed A. oryzae to show the acetarnide utilizing phenotype and the transformant is hereby termed as A. oryzae EIPW16
Further to characterize the A. oryzae (EIPW16, MTCC 5177) chromosomal DNA had been isolated from the strain. Chromosomal DNA from this transformed strain and the original host A. oryzae ATCC 66222 were digested with restriction enzymes BamH1 and EcoR1 and southern hybridization experiments with the PCR amplified radiolabeled 1.589 Kbp DNA probe revealed the specific integration of amylase gene in the transformed A. oryzae (EIPW16, MTCC 5177) chromosomal DNA. Southern hybridization had also been performed employing the radiolabeled 5KbpDNA fragment (6, fig. 1) containing the acetamidase gene (derived from the p3SR2 plasmid by restriction enzymes digestion EcoR1 and Sail; path 2, fig 1). It also revealed the specific integration of the acetamidase containing DNA fragment in the A. oryzae (EIPW16, MTCC 5177)
Further A. oryzae EIPW 16, the host strain A. oryzae ATCC 66222 and the donor strain A. oryzae EIPW 212 were plated on M15 starch media containing Starch 2%, Ammonium nitrate 0.3%, Potassium dihydrogen phosphate 0.1%, Magnesium sulphate
7H20 0.1%, Ferrous sulphate trace amount, Agar 1.8 % and incubated at 25°C for 72 hours. These amylase secreting strains produce starch depleting spherical zones on the above growth plates and the comparative analysis of starch hydrolyzing potential were compared. A.oryzae EIPW16 produces the highest hydrolyzed zone indicating the highest potential of this strain to produce amylase.
Further the acetamide utilizing A. oryzae had been checked for their ability to produce amylase enzyme in the solid state fermentation using wheat bran as the substrate and a trend to hyper produce amylase enzyme had been observed an all the transformed A. oryzae isolates.
To access the starch hydrolyzing ability of the transformed A. oryzae strains spores from different slants of transformed strains and the host A. oryzae 66222 were resuspended in aqueous solution containing 0.1% Triton X. The numbers of spores were measured by hemocytometer and appropriate dilutions were spread on M15 Starch Agar plates to get not more than 15 colonies on a 90mm growth plate. The plates were incubated at 25°C for 72 hours and 0.02M iodine was poured on Petri plates to visualize zone of clearance due to the starch hydrolyzing activity of the amylase produced by the individual colonies. Diameter of zone of clearance (Dc) and diameter of zone of fungal mass (Df) were measured for each of the colonies of the transformed and the host strains. The ratio (Dc/Df = R) was calculated for each colony. (Industrial Biotechnology; Oxford & IBH Publication 1992; editors Vedpal S.Malik & Padma Sridhar; p-425). The host strain showed an average R value of 1.9 and the higher starch hydrolyzing transformed strains showed R values in the range of 2.7 to 3.2.
The permanently transformed A oryzae strains showing comparatively higher starch-hydrolyzing potential in the M15 starch growth plates were subjected to laboratory scale solid state fermentation using wheat bran as the substrate (Ramdas, M. et "aT, World Journal of Microbiology & Biotechnology, vol. 12, 1996, pages 267-271. Pandey, A.
Process Biochemistry, vol. 27, 1992, pages 109-116. Madhavan N. K. & Pandey, A. Biotechnology Letters, vol. 18, no. 2, 1996, pages 199-204). These selected transformed A oryzae strains were grown in Czapekdox agar media for 5 days at 25°C.
Tablel. Production of amylase enzyme in the Solid State Fermentation using wheat bran from Aspergillus oryzae and some of its genetically transformed varieties.

The spore suspension from each of these selected cultures were collected and used as the inoculums for solid state fermentation for 48 hours at 25°C using sterile wheat bran as the substrate. After 48 hours the moulded bran were harvested and extracted with sterile water. The clear supernatant containing the enzyme extracts were subjected to amylase assay following the IP method of amylase assay (IP 2007, volume 2, page 734 - 735). The amylolytic activities are compared in the table 1.
After a thorough screening the transformed A. oryzae AAC3 #16 (EIPW16, MTCC 5177) strain has been identified for its ability to hyper produce the amylase enzyme in the solid state fermentation using wheat bran.
Different embodiments of the invention are possible to achieve the best method of performance and to obtain the product as stated earlier. It will be understood that skilled persons with many modifications, variations and adaptations may carry out the invention into practice without departing from its sprit in describing the invention for the purpose of illustration.
We claim
1. Genetically modified A.oryzae (MTCC 5177) for production of amylase having sequence ID No. 1 from locally available GRAS (generally regarded as safe) fungus A.oryzae E212.
2. A recombinant plasmid pSPK14 (1) constructed to harbor the genomic clone of amylase gene derived from a locally isolated GRAS fungus A. oryzae E212.
3. A plasmid pACC3 (9) constructed to use in A. oryzae for production of fungal alpha amylase through cloning of a DNA fragment harboring the amylase gene from pSPK14 (1) and a biomarker amdS gene from a plasmid p3SR2 (4) in an E. coli plasmid pGEM3Z (7).
4. A process for producing a plasmid pSPK14 (1) where PCR parameters have been optimized for specific amplification of a DNA probe to produce an unique 1.589Kbp PCR fragment to specifically identify fungal amylase gene from A.oryzae fungal genome.
5. A process for producing a genetically modified A. oryzae (MTCC 5177) comprises of the following steps
i. Providing a recombinant plasmid pACC3 (9) as here in described.
ii. Transforming A.oryzae ATCC 66222 host with the constructed recombinant plasmid pACC3 (9) to produce stable genetically modified hyper producing amylase fungal strain having amdS+ phenotype.
iii. Culturing the transformed A. oryzae (MTCC 5177) in a suitable culture medium.
6. A process has claimed in claim 5 wherein the plasmid pAAC3 (9) construction comprises of the following steps.
i. Providing the 2.826Kbp amylase fragment having a blunt end BamH1 side
and a staggered end EcoR1 at both the ends, from plasmid pSPK14 (1).
ii. Providing the 5Kbp acetamidase gene of A. nidulans derived from p3SR2 (4) having EcoR1 blunt end and Sail staggered end.
iii. Providing linearized plasmid pGEM 3Z having Sal 1 and EcoR1 sides at both the ends.
iv. Cloning of the products obtained from steps i, ii and iii to construct the recombinant plasmid pAAC3 (9).
7. A genetically modified A. oryzae (MTCC 5177) substantially herein described.
8. A plasmid pAAC3 (9) substantially herein described.
9. A process for producing A. oryzae (MTCC 5177) substantially herein described.

Genetically modified A.oryzae (MTCC 5177) for better production of amylase having
sequence as herein described from locally available GRAS fungus A.oryzae E212 from
the plasmid pAAC3 (9) obtained from cloning of 2.8213Kbpamylase fragment having a
blunt end BamH1 side and a staggered end EcoR1 at both the ends derived from
plasmid pSPK14 (1) and the 5Kbf"acetamidase gene of A. nidulans derived from p3SR2
(4) having a EcoR1 blunt end and Sal1 staggered end at both the ends with linearized
plasmid pGEM 3Z having Sal1 and EcoR1 sides at both the ends and process thereof.

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Patent Number

243393

Indian Patent Application Number

482/KOL/2007

PG Journal Number

42/2010

Publication Date

15-Oct-2010

Grant Date

12-Oct-2010

Date of Filing

27-Mar-2007

Name of Patentee

EAST INDIA PHARMACEUTICAL WORKS LIMITED

Applicant Address

6, LITTLE RUSSELL STREET, KOLKATA

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. SUBRATA K. GHOSH	IA-301, FLAT NO. 7, SALT LAKE, KOLKATA 700 097.
2	DR. AMBIKA C. BANERJEE	FLAT-1A, P-171 LAKE TOWN, BLOCK-A, KOLKATA 700 089.
3	MR. SAIKAT MUKHERJEE	MERLIN LAUREL GARDEN, FLAT-3F, PEARL BLOCK, 71, NRISINGHA DUTTA ROAD, KOLKATA 700 008.
4	DR. BARUN K. BHATTACHARYYA	A-11/557, KALYANI, P.O.: KALYANI, DIST.: NADIA, PIN 741 235.
5	DR. SOUMITRA DEB	84A, GOPI MOHAN DUTTA LANE, KOLKATA 700 003.
6	MR. DIPANKAR DUTTAGUPTA	28/1 A, GARIAHAT ROAD, BLOCK-2, FLAT 15, KOLKATA 700 029.
7	DR. AMITABHA KUNDU	BC-137, SALT LAKE, KOLKATA 700 064.

PCT International Classification Number

C12P7/10

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA