[Gene cloning, codon optimization and functional expression of Yarrawia lipolytica lipase Lip1].
OBJECTIVE
To implement inducible and constitutive over-expression of Yarrowia lipolytica lipase gene lipl in Pichia pastoris using codon optimization.


METHODS
We cloned Y. lipolytica lipase gene lip1 according to codon bias of P. pastoris, and optimized lipl using overlap extension PCR synthesis. Then, we cloned the original and optimized genes into the induced vector pPIC9K and newly built constitutive carrier pGAP9K, and electrotransformated the resultant expression plasmids into P. pastoris GS115. Through G418 resistance screening, high copy transformatants were selected and fermented in shake flasks. P-nitrophenyl palmitate (pNPP) was used as substrates for assay the activities of the expressed lipase, and the characteristics of the lipase were further examined.


RESULTS
We successfully cloned lipase gene lip1 from Y. lipolytica, nucleotide sequence revealed that the open reading frame (ORF) had 1461 nucleotides, encoding 486 amino acids, without any intron or any signal peptide. SDS-PAGE analysis and fermentation result showed that the optimized gene had a higher expression level than the original one, and the constitutive expression was superior to the inducible expression. Preliminary analysis showed that the optimal substrate of Lipl was p-nitrophenyl butyrate (C4), the optimum temperature and pH was 45 degrees C and 8.5, respectively.


CONCLUSIONS
Y. lipolytica lipase gene lip1 can be over-expressed through both inducible and constitutive expressions using codon optimization, which lays a solid foundation to further study Y. lipolytica lipase family, and also provides an important prerequisite for scale production and industrial application of the lipase.
Zhang
Li
L
Key Laboratory of Molecular Bio-physics, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. ymw537@163.com
Zhao
Heyun
H
Xu
Li
L
Liu
Yun
Y
Yan
Yunjun
Y
D003001:Cloning, Molecular; D003062:Codon; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D025062:Yarrowia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D003062:Codon; D005656:Fungal Proteins; D008049:Lipase