Glucoamylase CDNA

A method of producing a DNA sequence coding for fungal glucoamylase protein, wherein said DNA sequence is free of introns, is capable of hybridizing to Aspergillus awamori or Aspergillus niger mRNA, and is capable, when correctly combined with a cleaved expression vector, of expressing a non-native protein which has glucoamylase enzyme activity and can degrade starch in an aqueous slurry of starch, or starch hydrolysate, to glucose molecules upon transformation of a host by an expression vector correctly combined with said sequence, which method comprises the steps of:

• (a) selecting a fungal source consisting of Aspergillus awamori or Aspergillus niger for genomic DNA;
• (b) preparing digest fragments of the selected fungal genomic DNA;
• (c) providing a glucoamylase probe;
• (d) using the glucoamylase probe to identify genomic digest fragments containing glucoamylase genomic gene regions;
• (e) molecularly cloning the identified genomic digest fragments;
• (f) molecularly cloning a cDNA or partial cDNAs;
• (g) sequencing the genomic and cDNA clones;
• (h) comparing the sequenced glucoamylase gene regions with all or a portion of the amino acid sequence of the mature glucoamylase enzyme to determine the existence and location of all the exons and introns in the genomic clones;
• and
• (i) constructing a gene whose nucleotide sequence is identical to that of the glucoamylase genomic gene than about 80% of the sequences comprising the introns are deleted.

A recombinant DNA comprising a recombinant DNA sequence of the following sequence in a 5' to 3' direction:

• __________________________________________________________________________ GCG ACC TTG GAT TCA TGG TTG AGC AAC GAA GCG ACC GTG GCT CGT ACT GCC ATC CTG AAT AAC ATC GGG GCG GAC GGT GCT TGG GTG TCG GGC GCG GAC TCT GGC ATT GTC GTT GCT AGT CCC AGC ACG GAT AAC CCG GAC TAC TTC TAC ACC TGG ACT CGC GAC TCT GGT CTC GTC CTC AAG ACC CTC GTC GAT CTC TTC CGA AAT GGA GAT ACC AGT CTC CTC TCC ACC ATT GAG AAC TAC ATC TCC GCC CAG GCA ATT GTC CAG GGT ATC AGT AAC CCC TCT GGT GAT CTG TCC AGC GGC GCT GGT CTC GGT GAA CCC AAG TTC AAT GTC GAT GAG ACT GCC TAC ACT GGT TCT TGG GGA CGG CCG CAG CGA GAT GGT CCG GCT CTG AGA GCA ACT GCT ATG ATC GGC TTC GGG CAA TGG CTG CTT GAC AAT GGC TAC ACC AGC ACC GCA ACG GAC ATT GTT TGG CCC CTC GTT AGG AAC GAC CTG TCG TAT GTG GCT CAA TAC TGG AAC CAG ACA GGA TAT GAT CTC TGG GAA GAA GTC AAT GGC TCG TCT TTC TTT ACG ATT GCT GTG CAA CAC CGC GCC CTT GTC GAA GGT AGT GCC TTC GCG ACG GCC GTC GGC TCG TCC TGC TCC TGG TGT GAT TCT CAG GCA CCC GAA ATT CTC TGC TAC CTG CAG TCC TTC TGG ACC GGC AGC TTC ATT CTG GCC AAC TTC GAT AGC AGC CGT TCC GGC AAG GAC GCA AAC ACC CTC CTG GGA AGC ATC CAC ACC TTT GAT CCT GAG GCC GCA TGC GAC GAC TCC ACC TTC CAG CCC TGC TCC CCG CGC GCG CTC GCC AAC CAC AAG GAG GTT GTA GAC TCT TTC CGC TCA ATC TAT ACC CTC AAC GAT GGT CTC AGT GAC AGC GAG GCT GTT GCG GTG GGT CGG TAC CCT GAG GAC ACG TAC TAC AAC GGC AAC CCG TGG TTC CTG TGC ACC TTG GCT GCC GCA GAG CAG TTG TAC GAT GCT CTA TAC CAG TGG GAC AAG CAG GGG TCG TTG GAG GTC ACA GAT GTG TCG CTG GAC TTC TTC AAG GCA CTG TAC AGC GAT GCT GCT ACT GGC ACC TAC TCT TCG TCC AGT TCG ACT TAT AGT AGC ATT GTA GAT GCC GTG AAG ACT TTC GCC GAT GGC TTC GTC TCT ATT GTG GAA ACT CAC GCC GCA AGC AAC GGC TCC ATG TCC GAG CAA TAC GAC AAG TCT GAT GGC GAG CAG CTT TCC GCT CGC GAC CTG ACC TGG TCT TAT GCT GCT CTG CTG ACC GCC AAC AAC CGT CGT AAC GTC GTG CCT TCC GCT TCT TGG GGC GAG ACC TCT GCC AGC AGC GTG CCC GGC ACC TGT GCG GCC ACA TCT GCC ATT GGT ACC TAC AGC AGT GTG ACT GTC ACC TCG TGG CCG AGT ATC GTG GCT ACT GGC GGC ACC ACT ACG ACG GCT ACC CCC ACT GGA TCC GGC AGC GTG ACC TCG ACC AGC AAG ACC ACC GCG ACT GCT AGC AAG ACC AGC ACC AGT ACG TCA TCA AOC TCC TGT ACC ACT CCC ACC GCC GTG GCT GTG ACT TTC GAT CTG ACA GCT ACC ACC ACC TAC GGC GAG AAC ATC TAC CTG GTC GGA TCG ATC TCT CAG CTG GGT GAC TGG GAA ACC AGC GAC GGC ATA GCT CTG AGT GCT GAC AAG TAC ACT TCC AGC GAC CCG CTC TGG TAT GTC ACT GTG ACT CTG CCG GCT GGT GAG TCG TTT GAG TAC AAG TTT ATC CGC ATT GAG AGC GAT GAC TCC GTG GAG TGG GAG AGT GAT CCC AAC CGA GAA TAC ACC GTT CCT CAG GCG TGC GGA ACG TCG ACC GCG ACG GTG ACT GAC ACC TGG CGG __________________________________________________________________________
• and further comprising a region adjoining the 5' end of said sequence which encodes a signal sequence.