Gene coding for the E1 endoglucanase

• US 5,536,655 A
• Filed: 07/15/1994
• Issued: 07/16/1996
• Est. Priority Date: 09/26/1989
• Status: Expired due to Term

• Current Assignee: Alliance For Sustainable Energy LLC
• Original Assignee: Midwest Research Institute
• Inventors: Laymon, Robert A., Thomas, Steven R., Himmel, Michael E.
• Primary Examiner(s): Patterson, Jr., Charles L.
• Assistant Examiner(s): KIM, HYO
• Application Number:
  US08/276,213
• Time in Patent Office: 732 Days
• Field of Search: 435/69.1, 435/209, 435/252.3, 435/252.31, 435/252.33, 435/253.5, 435/254.21, 435/320.1, 536/22.1, 536/23.1, 536/23.2, 536/23.7
• US Class Current: 435/209
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The gene encoding Acidothermus cellulolyticus E1 endoglucanase is cloned and expressed in heterologous microorganisms. A new modified E1 endoglucanase enzyme is produced along with variants of the gene and enzyme. The E1 endoglucanase is useful for hydrolyzing cellulose to sugars for simultaneous or later fermentation into alcohol.

Claims

1. A DNA comprising a DNA encoding the following amino acid sequence:

AGGGYWHTSGREILDANNVPVRIAGINWFGFETCNYVVHGLWSRDYRSMLDQIKSLGYNTIR LPYSDDILKPGTMPNSINFYQMNQDLQGLTSLQVMDKIVAYAGQIGLRIILDRHRPDCSGQS ALWYTSSVSEATWISDLQALAQRYKGNPTVVGFDLHNEPHDPACWGCGDPSIDWRLAAERAG NAVLSVNPNLLIFVEGVQSYNGDSYWWGGNLQGAGQYPVVLNVPNRLVYSAHDYATSVYPQT WFSDPTFPNNMPGIWNKNWGYLFNQNIAPVWLGEFGTTLQSTTDQTWLKTLVQYLRPTAQYG ADSFQWTFWSWNPDSGDTGGILKDDWQTVDTVKDGYLAPIKSSIFDPVGASASPSSQPSPSV SPSPSPSPSASRTPTPTPTPTASPTPTLTPTATPTPTASPTPSPTAASGARCTASYQVNSDW GNGFTVTVAVTNSGSVATKTWTVSWTFGGNQTITNSWNAAVTQNGQSVTARNMSYNNVIQPG QNTTFGFQASYTGSNAAPTVACAAS (SEQ ID NO;

3).

2. The DNA according to claim 1 further comprising the following sequence attached to an amino terminal end:

MLRVGVVVAVLALVAALANLAVPRPARA, (SEQ ID NO;

4).

3. The DNA according to claim 2 further comprising the following sequence attached to an amino terminal end:

VPRALRRVPGSRV, SEQ ID NO;

5.

4. The DNA according to claim 1 comprising the following sequence:

space="preserve" listing-type="tabular">__________________________________________________________________________GGATCCACGT TGTACAAGGT CACCTGTCCG TCGTTCTGGT AGAGCGGCGG 50 GATGGTCACC CGCACGATCT CTCCTTTGTT GATGTCGACG GTCACGTGGT 100 TACGGTTTGC CTCGGCCGCG ATTTTCGCGC TCGGGCTTGC TCCGGCTGTC 150 GGGTTCGGTT TGGCGTGGTG TGCGGAGCAC GCCGAGGCGA TCCCAATGAG 200 GGCAAGGGCA AGAGCGGAGC CGATGGCACG TCGGGTGGCC GATGGGGTAC 250 GCCGATGGGG CGTGGCGTCC CCGCCGCGGA CAGAACCGGA TGCGGAATAG 300 GTCACGGTGC GACATGTTGC CGTACCGCGG ACCCGGATGA CAAGGGTGGG 350 TGCGCGGGTC GCCTGTGAGC TGCCGGCTGG CGTCTGGATC ATGGGAACGA 400 TCCCACCATT CCCCGCAATC GACGCGATCG GGAGCAGGGC GGCGCGAGCC 450 GGACCGTGTG GTCGAGCCGG ACGATTCGCC CATACGGTGC TGCAATGCCC 500 AGCGCCATGT TGTCAATCCG CCAAATGCAG CAATGCACAC ATGGACAGGG 550 ATTGTGACTC TGAGTAATGA TTGGATTGCC TTCTTGCCGC CTACGCGTTA 600 CGCAGAGTAG GCGACTGTAT GCGGTAGGTT GGCGCTCCAG CCGTGGGCTG 650 GACATGCCTG CTGCGAACTC TTGACACGTC TGGTTGAACG CGCAATACTC 700 CCAACACCGA TGGGATCGTT CCCATAAGTT TCCGTCTCAC AACAGAATCG 750 GTGCGCCCTC ATGATCAACG TGAAAGGAGT ACGGGGGAGA ACAGACGGGG 800 GAGAAACCAA CGGGGGATTG GCGGTGCCGC GCGCATTGCG GCGAGTGCCT 850 GGCTCGCGGG TGATGCTGCG GGTCGGCGTC GTCGTCGCGG TGCTGGCATT 900 GGTTGCCGCA CTCGCCAACC TAGCCGTGCC GCGGCCGGCT CGCGCCGCGG 950 GCGGCGGCTA TTGGCACACG AGCGGCCGGG AGATCCTGGA CGCGAACAAC 1000 GTGCCGGTAC GGATCGCCGG CATCAACTGG TTTGGGTTCG AAACCTGCAA 1050 TTACGTCGTG CACGGTCTCT GGTCACGCGA CTACCGCAGC ATGCTCGACC 1100 AGATAAAGTC GCTCGGCTAC AACACAATCC GGCTGCCGTA CTCTGACGAC 1150 ATTCTCAAGC CGGGCACCAT GCCGAACAGC ATCAATTTTT ACCAGATGAA 1200 TCAGGACCTG CAGGGTCTGA CGTCCTTGCA GGTCATGGAC AAAATCGTCG 1250 CGTACGCCGG TCAGATCGGC CTGCGCATCA TTCTTGACCG CCACCGACCG 1300 GATTGCAGCG GGCAGTCGGC GCTGTGGTAC ACGAGCAGCG TCTCGGAGGC 1350 TACGTGGATT TCCGACCTGC AAGCGCTGGC GCAGCGCTAC AAGGGAAACC 1400 CGACGGTCGT CGGCTTTGAC TTGCACAACG AGCCGCATGA CCCGGCCTGC 1450 TGGGGCTGCG GCGATCCGAG CATCGACTGG CGATTGGCCG CCGAGCGGGC 1500 CGGAAACGCC GTGCTCTCGG TGAATCCGAA CCTGCTCATT TTCGTCGAAG 1550 GTGTGCAGAG CTACAACGGA GACTCCTACT GGTGGGGCGG CAACCTGCAA 1600 GGAGCCGGCC AGTACCCGGT CGTGCTGAAC GTGCCGAACC GCCTGGTGTA 1650 CTCGGCGCAC GACTACGCGA CGAGCGTCTA CCCGCAGACG TGGTTCAGCG 1700 ATCCGACCTT CCCCAACAAC ATGCCCGGCA TCTGGAACAA GAACTGGGGA 1750 TACCTCTTCA ATCAGAACAT TGCACCGGTA TGGCTGGGCG AATTCGGTAC 1800 GACACTGCAA TCCACGACCG ACCAGACGTG GCTGAAGACG CTCGTCCAGT 1850 ACCTACGGCC GACCGCGCAA TACGGTGCGG ACAGCTTCCA GTGGACCTTC 1900 TGGTCCTGGA ACCCCGATTC CGGCGACACA GGAGGAATTC TCAAGGATGA 1950 CTGGCAGACG GTCGACACAG TAAAAGACGG CTATCTCGCG CCGATCAAGT 2000 CGTCGATTTT CGATCCTGTC GGCGCGTCTG CATCGCCTAG CAGTCAACCG 2050 TCCCCGTCGG TGTCGCCGTC TCCGTCGCCG AGCCCGTCGG CGAGTCGGAC 2100 GCCGACGCCT ACTCCGACGC CGACAGCCAG CCCGACGCCA ACGCTGACCC 2150 CTACTGCTAC GCCCACGCCC ACGGCAAGCC CGACGCCGTC ACCGACGGCA 2200 GCCTCCGGAG CCCGCTGCAC CGCGAGTTAC CAGGTCAACA GCGATTGGGG 2250 CAATGGCTTC ACGGTAACGG TGGCCGTGAC AAATTCCGGA TCCGTCGCGA 2300 CCAAGACATG GACGGTCAGT TGGACATTCG GCGGAAATCA GACGATTACC 2350 AATTCGTGGA ATGCAGCGGT CACGCAGAAC GGTCAGTCGG TAACGGCTCG 2400 GAATATGAGT TATAACAACG TGATTCAGCC TGGTCAGAAC ACCACGTTCG 2450 GATTCCAGGC GAGCTATACC GGAAGCAACG CGGCACCGAC AGTCGCCTGC 2500 GCAGCAAGTT AATACGTCGG GGAGCCGACG GGAGGGTCCG GACCGTCGGT 2550 TCCCCGGCTT CCACCTATGG AGCGAACCCA ACAATCCGGA CGGAACTGCA 2600 GGTACCAGAG AGGAACGACA CGAATGCCCG CCATCTCAAA ACGGCTGCGA 2650 GCCGGCGTCC TCGCCGGGGC GGTGAGCATC GCAGCCTCCA TCGTGCCGCT 2700 GGCGATGCAG CATCCTGCCA TCGCCGCGAC GCACGTCGAC AATCCCTATG 2750 CGGGAGCGAC CTTCTTCGTC AACCCGTACT GGGCGCAAGA AGTACAGAGC 2800 GAACGGCGAA CCAGACCAAT GCCACTCTCG CAGCGAAAAT GCGCGTCGTT 2850 TCCACATATT CGACGGCCGT CTGGATGGAC CGCATCGCTG CGATCAACGG 2900 CGTCAACGGC GGACCCGGCT TGACGACATA TCTGGACGCC GCCCTCTCCC 2950 AGCAGCAGGG AACCACCCCT GAAGTCATTG AGATTGTCAT CTACGATCTG 3000 CCGG 3004 SEQ ID NO;

6. __________________________________________________________________________

5. A vector comprising the DNA according to claim 1 and a vector sequence encoding either an origin of replication or an integration site for a host genome.

6. A vector according to claim 5 further comprising DNA encoding a signal sequence operably linked thereto.

7. A vector according to claim 5 further comprising exogenous regulatory sequences capable of causing expression of said DNA in a suitable host.

8. A recombinant microorganism containing the vector according to claim 5.

9. A recombinant microorganism containing the vector according to claim 6.

10. A recombinant microorganism containing the vector according to claim 7.

11. A recombinant microorganism according to claim 5 wherein a genus of said microorganism is selected from the group consisting of Saccharomyces, Streptomyces, Bacillus, Zymomonas and Escherichia.

12. A method for producing an endoglucanase comprising culturing the recombinant microorganism according to claim 8 in a vessel under culture conditions sufficient to express said DNA and recovering said endoglucanase therefrom.

13. The method according to claim 12, further comprising separating the recombinant microorganism from microbial medium and recovering said endoglucanase from the medium.

14. A method for producing an endoglucanase according to claim 12, further comprising effectively increasing the permeability of a membrane of the recombinant microorganism to permit release of said endoglucanase.

15. A DNA comprising at least one domain but not all of the domains of the Acidothermus cellulolyticus E1 endoglucanase.

16. The DNA according to claim 15 further comprising at least one domain from a cellulase gene other than E1 endoglucanase.

17. The DNA according to claim 16 wherein the DNA encodes a protein having a cellulase activity.

18. The DNA according to claim 17 wherein the cellulase activity is an endoglucanase activity.