Gene cloning, codon optimization and functional expression of Yarrawia lipolytica lipase Lip

Abstract:

Abstract: [Objective] To implement inducible and constitutive over-expression of Yarrowia lipolytica lipase gene lip1 in Pichia pastoris using codon optimization. [Methods] We cloned Y. lipolytica lipase gene lip1 according to codon bias of P. pastoris, and optimized lip1 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 Lip1 was p-nitrophenyl butyrate (C4), the optimum temperature and pH was 45℃and 8.5, respectively. [Conclusion] 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.

Key words:Keywords: Yarrawia lipolytica lipase gene lip1; clone; codon optimization; GAP promoter; functional expression