Molecular cloning of a lytic beta-1,3-glucanase gene from Oerskovia xanthineolytica LLG109. A beta-1,3-glucanase able to selectively permeabilize the yeast cell wall.
Molecular cloning of the beta gIII gene encoding for an endo-beta-1,3-glucanase (beta gl II) from Oerskovia xanthineolytica LLG109, a yeast-lytic gram-positive bacterium, has been conducted in order to elucidate its primary sequence and subsequently express it into B. subtilis. This endo-beta-1,3-glucanase exhibits low yeast-lytic activity toward viable S. cerevisiae cells, and it has shown ability to selectively permeabilize the yeast cell wall and release intracellular proteins produced by yeast. Highly degenerate oligonucleotides have been used to PCR-amplify a region of the beta-1,3-glucanase II encoding gene from O. xanthineolytica LLG109. The amplified fragment has been cloned and sequenced. The deduced amino acid sequence contains regions identical to the amino acid sequences previously determined by direct sequencing of the purified enzyme from O. xanthineolytica LLG109. By using the 180-bp PCR product as a homologous probe, we have been able to isolate four positive clones harboring plasmids pPF1A, pPF1B, pPF8A, and pPF9A, respectively, from a partial genomic library from O. xanthineolytica LLG109. All four plasmids contained a 2.7-kb BamHI insert that hybridized to the PCR probe under high stringency conditions. The 2.7-kb fragment seemed to be identical in all four cases regarding preliminary partial restriction mapping analysis done on the four plasmids. The 1.5-kb BamHI/KpnI restriction fragment from pPF8A and pPF9A hybridizing with the 180-bp PCR probe is presently being sequenced. The cloning of the lytic beta-1,3-glucanase from O. xanthineolytica LLG109 expands the number of yeast lytic beta-glucanases so far cloned. The availability of the nucleotide sequences of such a family of genes will allow further understanding of the role and mode of action of these enzymes in yeast cell wall degradation. In addition, a more extensive study on the structure and functional relationships of these enzymes will allow us to engineer "tailor-made" lytic beta-1,3-glucanases for use in new and improved large-scale selective cell permeabilization (SCP) and selective protein recovery (SPR) from yeast cells, not only from S. cerevisiae but also from alternative yeast expression systems such as Hansenula polymorpha, Pichia pastoris, and others, which are becoming of increasing importance in biotechnology.
Ferrer
P
P
Biochemical Engineering Laboratory, University of Reading, England.
Hedegaard
L
L
Halkier
T
T
Diers
I
I
Savva
D
D
Asenjo
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002463:Cell Membrane Permeability; D002473:Cell Wall / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli; D015698:Genomic Library; D043326:Glucan 1,3-beta-Glucosidase; D006094:Gram-Positive Bacteria / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase