Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase.
An important industrial enzyme, Candida rugosa lipase (CRL) possesses several different isoforms encoded by the lip gene family (lip1-lip7), in which the recombinant LIP1 is the major form of the CRL multigene family. Previously, 19 of the nonuniversal serine codons (CTG) of the lip1 gene hav been successfully converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP1 in the yeast Pichia pastoris. To improve the expression efficiency of recombinant LIP1 in P. pastoris, a regional synthetic gene fragment of lip1 near the 5' end of a transcript has been constructed to match P. pastoris-preferred codon usage for simple scale-up fermentation. The present results show that the production level (152 mg/L) of coLIP1 (codon-optimized LIP1) has an overall improvement of 4.6-fold relative to that (33 mg/L) of non-codon-optimized LIP1 with only half the cultivation time of P. pastoris. This finding demonstrates that the regional codon optimization the lip1 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP1 in the P. pastoris system. More distinct biochemical properties of the purified recombinant LIP1 for further industrial applications are also determined and discussed in detail.
Chang
Shu-Wei
SW
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 202, Taiwan.
Lee
Guan-Chiun
GC
Shaw
Jei-Fu
JF
D001483:Base Sequence; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D003062:Codon; D004271:DNA, Fungal / Q000737:chemistry; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics; D008049:Lipase / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013696:Temperature
D003062:Codon; D004271:DNA, Fungal; D007527:Isoenzymes; D011994:Recombinant Proteins; D008049:Lipase