Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase.
The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site-directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pastoris cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lipl of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.
Brocca
S
S
Institut für Technische Biochemie, Universität Stuttgart, Germany.
Schmidt-Dannert
C
C
Lotti
M
M
Alberghina
L
L
Schmid
R D
RD
D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007221:Industry; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D007527:Isoenzymes; D011994:Recombinant Proteins; D008049:Lipase