Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 13, Num. 3, 1996, pp. 209

CLONING OF THE Penicillium minioluteum GENE ENCODING DEXTRANASE AND ITS EXPRESSION IN Pichia pastoris

Hernan Roca, Bianca Garcia, Efrain Rodriguez, Jose Cremata, Rossana Garcia, Tirso Pons and Julio Delgado

Bioindustry Division, Center for Genetic Engineering and Biotechnology P.O. Box 6162, Havana, Cuba.

 
Code Number:BA96087 
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Introduction

Dextranase (EC 3.2.1.11, alpha-1,6 glucan-6 glucanohydrolase) hydrolyzes the alpha (1,6) glucosidic bond contained in dextran to release smaller isomaltosaccharides, commonly 3 to 5 glucose units long. Dextranases have been found in a wide variety of Mycomycetes species, Actinomycetes species and bacteria. In sugarcane processing this enzyme is used to degrade the dextran polymer to smaller molecules that do not create excessive viscosity, and there exists in the sugar industry a considerable demand for dextranase. The cloning and expression of a fungal dextranase gene in P. pastoris would make it possible to generate large amounts of enzyme with relative ease to be of potential utility in industrial fields.

Previously we have isolated by differential hybridization a cDNA encoding an extracellular dextranase from P. minioluteum (Garcia B, et al. Unpublished data). In this report we describe the cloning and characterization of the dextranase gene, as well as the expression of this gene in the methylotrophic yeast P. pastoris. We also compare the enzymatic properties of the dextranases from P. minioluteum and P. pastoris cells.

Materials and Methods

The strains used in this study were the P. minioluteum HI-4 (MUCL 38929) and P. pastoris (his3 mutant) MP36 (MUCL 38930). The dextranase protein was purified from the culture supernatant according Raices, et al. (1). The N-terminal sequencing of purified dextranase was performed by Edman degradation procedure. BamHI genomic library was constructed using the pUC19 vector. DNA sequencing was performed by dideoxynucleotide chain termination method using Sequenase (USB, Amersham). The integrating plasmid pPS7 (2) was used for dextranase expression in P. pastoris. MP36 strain was transformed using the eletroporation procedure.

Results and Discussion

The DEX gene encoding an extracellular dextranase was isolated from a partial genomic DNA library of P. minioluteum by hybridization using the dex-tranase cDNA as a probe. In contrast with the majority of the isolated filamentous fungal genes, sequencing data showed that the dextranase gene does not contains introns in its nucleotide sequence. The longest open reading frame (ORF) determined from the gene sequence encodes a protein composed of 608 aa with a predicted molecular weigth of 65942 Da. There are four potential initiation codons in the ORF, however only the second initiation codon has a nucleotide sequence, CACAAUG in its vicinity, which matches very well to the consensus sequence around reported initiation codons of filamentous fungi (3). According to the leaky scanning mechanism propossed by Kozac (4), it seems that in the dextranase mRNA the ribosomes initiate translation from the first and second AUG codons. Comparison of the aminoacid sequence of the N-terminus of the mature dextranase protein with the deduced protein sequence translated from the first AUG codon indicates the presence of a leader peptide of 34 aa. However the -1,-3 rule and the analysis by the S-factor method (5) predict the cleavage by signal peptidase occurs at least 13 aa preceding the N-terminus of mature dextranase protein. This analysis suggests that dextranase is synthesized as a preproenzyme which means that the protein undergoes another postranslational modification: the propeptide removal by proteolytic processing. The gene encoding dextranase was expressed in the methylotrophic yeast P. pastoris. The fusion of the coding region of mature dextranase protein to the yeast invertase (SUC2) signal under the control AOX1 promoter led to the secretion of high level (over 3.1 g/L) of active dextranase enzyme to the culture medium. The N-terminus of Pichia secreted dextranase was determined to be identical to the native product. This result implies the correct recognition and processing of the SUC2 signal peptide. The native (P. minioluteum) and the recombinant (P. pastoris) dextranases enzymes are N-glycosylated and the extend of N-glycosylation between the enzymes seems to be different, however having both the same specific activity (1 000 DU/mg protein).

References

1 Raices M, et al. Biotecnologia Aplicada 1991;8(2):248-255.

2 Yong V, et al. European Patent Application 438200 A1.

3 Ballance DJ Molecular Industrial Mycology. Marcel Dekker NY 1991;p.1-29.

4 Kozac M. NAR1984;12:3873-3893.

5 von Heijne G. NAR 1986;14(11): 4683-4690.

Copyright 1996 Elfos Scientiae