Total RNA was isolated from Aspergillus niger B 1 in the following manner: Aspergillus niger BI was grown in liquid culture in mineral media (NH4)2S04 3H20 (0.5 gil), KH2P04 (0.2 gil), MgS04 (0.2 gil), CaCI2 H20 (0.1 gil), FeS04 6 H20 (0.001 gil), ZnS04 7 H20 (0.001 gil), and 2 mM citric acid, at pH 3.5 with 1% wlv bran as carbon source. The me- dium was autoclaved, cooled, inoculated with A. niger B 1 (106 spores/ml). Baffled flasks
ENHANCEMENT OF AROMA IN GRAPES AND WINES 235 were used with shaking at 200 RPM at 3ic. The appearance of beta-glucosidase activity was monitored by placing 5 ~l of growth medium on 1% agar plates containing 0.5 mM MUGIc. Activity was detected after 15 h. of shaking. The mycelium was harvested after 24 h. and medium was removed by filtering through GF A glass micro fibre (Whatman Inter.
Ltd., Maidstone, England). The mycelium was then frozen with liquid nitrogen and ground to a fme powder with mortar and pestle. Total RNA was then produced from this powder by the Guanidine thiocyanate (TriReagentTM) method (Molecular Research Center, Inc.).
cDNA was produced in the following manner: Reverse transcriptase reaction using the total RNA from the extraction described above was carried out with the Stratagene RT- PCR kit (Stratagene, La Jolla, CA). The reaction volume was 50 III and contained: 10 Ilg total RNA, 11lg Oligo dT)s, 20 units RNase Block Ribonuclease Inhibitor, 1 x buffer (50 mM Tris-HCl, pH8.3, 75 mM KCl, 10 mM DTT, 3 mM MgCI2) 500 11M of each dNTP and 300 units reverse transcriptase. The RNA was denatured at 70°C, cooled slowly at R.T.
to allow the annealing of primers before it was added to the reaction mixture. The reaction mixture was incubated at 3ic for 1 h and then heated at 95°C for an additional 5 minutes.
The cDNA from the reaction was kept at -70 ° C and used for a PCR reaction with degener- ate primers. Degenerate primers were synthesized based on part of the N-terminal sequence and an internal sequence determined by Edman degradation.
Amino acid sequence 1: SPPYYP
Primer 1: 5' -C(G)A(CGT)CCA(CGT)CCA(CGT)TAC(T)TAC(T)CC-3' Amino acid sequence 2: QPILP AGG
Primer 2: 5'-TCCIGCT(G/C/A)GGT(G/C/A)AG(A)T(G/A)ATT (G/C/A) GGT(C)TG-3'.
DNA amplification was carried out in a 25 III reaction volume containing 1 III Reverse Transcriptase reaction product from above, 2.5 III 10 x PCR buffer (Promega Corp., Madi- son, WI), 250 roM each dNTP, 2.0 mM MgClz, 250 pmol of each of the degenerate prim- ers, 3 units pfo DNA polymerase (Strategene, La Jolla, CA) and 25 f.l1 mineral oil. The reaction was performed in an automated heating block (Programmable thermal controller MJ Research, Inc.). The resulting 2.2 kb cDNA gene fragment recovered was purified on a 1.2% (w/v) agarose/TBE gel isolated using the Gel Extraction Kit (QIAGEN, Hilden, Germany) and cloned into pGEM-T cloning vector (promega Corp., Madison, WI).
Preparation of genomic DNA plasmid library: An A. niger B 1 genomic library was constructed in the pYEAUra3 yeastlE.coli shuttle vector (Clontech Lab. Inc. Palo Alto, CA). A. niger B1 was grown in liquid culture as described above for total RNA isolation except that the mycelium was harvested after 48 h .. The mycelium ground with liquid nitrogen was used to produce genomic DNA by the CT AB method of Murray and Thompson (1980). The library was constructed from partially digested Sau3A genomic DNA cloned into the BamHI site of the pYEUra3 yeast shuttle vector (Clontech Lab.
Inc. Palo Alto, CA). pYEAUra3 yeastlE.coli shuttle vector was digested with BamHI and dephosphorylated with CIP to prevent self ligation. The partially digested genomic DNA was then cloned into the shuttle vector with T4 ligase and used to transform TOPIO E.coli electro-competent cells which were then plated on LB-agar (ampicillin 50 uglml).
The 2.2 kb partial cDNA was digested with Pst I to produce a 1.2 kb fragment DNA
236 O. SHOSEYOV and B. BRA VDO
probe. A total of 4 X 104 colonies were grown on LB-agar (50ug/ml ampicillin) plates and then blotted to Hybond™-N membranes. The colonies were screened using the 1.2 kb fragment. A sample (25 ng) of the probe was labeled with [32p] dCTP by using the random sequence nanonucleotide rediprime DNA labelling system (Amersham Phar- macia Biotech AB, Buckinghamshire, England). Positive clones were subcloned in pUC18 and nucleotide sequences were determined at the Weizmann Institute's Depart- ment of Biological Services, Rehovot, Israel. The bgll cDNA and the genomic gene were successfully cloned and sequenced (accession #:AJ132386). The cDNA sequence perfectly matched the DNA sequence of the combined exons. The open reading frame was found to encode a polypeptide with a predicted molecular weight of 92 kDa. The genomic gene consisted of 7 exons intercepted by 6 introns. Sequence analysis of the DNA sequence upstream to the sequence encoding for the mature protein revealed a pu- tative leader sequence intercepted by an 82bp intron.
9. EXPRESSION OF BGLI eDNA IN SACCHAROMYCES CEREVISIAE AND PICHIA PASTORIS
The pYES2 vector (Invitrogen Inc, San Diego, CA) was used to_successfully clone the bgll cDNA gene into the pYES2-bgll plasmid using the HindIIIIBamHI sites, and trans- form Saccharomyces cerevisiae using the lithium acetate method (Ito et al., 1983). The BGLl was expressed by inducing the Gall promoter according to the manufacturer's protocol. Saccharomyces cerevisiae strain INVSc2 (MATa, his3-D200, ura3-J67) was used as the host. Pichia pastor is strain GSI15 (his4 mutant) was used as the host for shuttle and expression vector plasmid pHIL-SI (Invitrogen Inc, San Diego, CA). The bgl1 cDNA was cloned into the EcoRIIBamHI sites of pHIL-S I, yielding the pHIL-S 1- bgll expression and secretion vector. Expression in P. pastoris was carried out according to the manufacturer's protocol. Screening of B-glucosidase-expressing clones was facili- tated by top-agar containing 50 mg X-Glc, 30 ml methanol and I % agar per liter. Blue color indicated a colony producing active beta-glucosidase.
Recombinant BGLI was successfully expressed both in S. cerevisiae and P. pastoris.
In S. cerevisiae a relatively low level of expression was found. The recombinant protein was detected by a Western blot analysis (Fig. 9.2A). The total protein extract of S. cere- visiae expressing bgll cDNA had a beta-glucosidase activity of 1.9 units/mg protein. No beta-glucosidase activity was detected in control S. cerevisiae transformed with the vec- tor only under the same assay conditions. However, no protein band corresponding to rBGLl could be detected by coomassie blue staining. P. pastoris transformed with bgll secreted relatively high levels of rBGL I to the medium (about 0.5 gIL) appearing as al- most pure protein in the culture supernatant (Fig. 9.2B). This recombinant enzyme was very active (124 units/mg protein) and even without any purification, its specific activity was very close to that of the pure native enzyme. The amount of BGL I per liter of fer- mented broth of the transgenic P. pastoris, was 100 folds higher than that obtained from
ENHANCEMENT OF AROMA IN GRAPES AND WINES 237
B
1 2 3 kDa 1 2 3
205- 116.
97 - 66 -
Figure 9.2. A) Western blot analysis of rBGLl expressed in S. cerevisiae. I. Native BGLl (posi- tive control). 2. Total protein extract of S. cerevisiae expressing rBGLl. 3. Total protein extract of S. cerevisiae without the vector (negative control). B) SDS-PAGE analysis and commassei of rBGLl secreted from P. pastor/ãs. I. M.W. marker. 2. Medium supernatant of P. pastoris express- ing rBGLl. 3. Medium supernatant of P. pastoris host without the vector (negative control).
A. niger. In comparison, recombinant Aspergillus nidulans producing Candida molischi- ana beta-glucosidase have been constructed (Sanchez et al., 1998). Depending on the growing conditions, the recombinant strain produce up to 4 times more beta-glucosidase, than the wild type strain.
The characterisation of A. niger beta-glucosidase gene and the current biotechnologi- cal methods, pave the way for future manipulation of must and wine aroma via trans- genic yeast and grape vines.
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