Synthesis And Expression Of Optimized Designed Aspergillus Niger Lipase Gene In Yeast

Lipase (Triacylgycerol acylhydrolase, Lipase, EC3.1.1.3) is a kind of enzymes widely present in plant seeds, animal tissue and microbes. It can catalyze the hydrolysis of long chain polyglyceryl fatty esters to long chain fatty acids and glycerol. Aspergillus niger lipases are important biocatalysis widely used in industries for food processing and pharmaceutical preparation. High-level expression and large scale production recombinants can lead to cost effective lipase.The secreted expression of Pichia pastoris can increase the amount of the target protein. In this paper, the Aspergillus niger lipase gene was optimized designed, synthesised and expressed in Pichia pastoris.The paper content about the following studies:1. The 99 amino acid of the Aspergillus niger lipase gene in 5’terminal were mutanted according to the Pichia pastoris codon bias and synthesised the mutated primer. After the mutation gene was coloned by PCR, the recombinant expression vector pPIC9K-lipm and pPIC9K-lipm-27 were constructed and transformed into Pichia pastoris GS115 strains by electroporation. This two proteins secreted expression in Pichia pastoris GS115 were confirmed by SDS-PAGE analysis, but a lower activity was detected.2. The biology informations of Aspergillus niger lipase gene from Genbank (GenBank Accession No. DQ647700) were analized.The result showed that the lipA gene was 1044bp in length, encoding a 348-amino acid polypeptide; while the mature peptide was 894bp in length, encoding 298-amino acid mature peptide. The (G+C)% content was 55.59% in the lipA gene, and the (G+C)% content of the third site of codon was 66.1%. According to P.pastoris preferred codon, the analysis of lipA gene showed that the number of condons whose relative synonymous condon usage(RSCU) value less than 0.3, was 190 codon, accounting for 63.7% of the whole codons. The mRNA structure of lipA gene analysis showed that the folding mRNA of minimum free energy (△G)=-293.74 kcal/mol. The lipAm gene was optimized designed and synthesized firstly after the optimized scheme was established. The nucleic acid sequence of the synthesis lipAm gene was desinged from the amino acid sequences of Aspergillus niger lipase based on the Pichia pastoris preferred condons. In order to insert the gene into pPIC9K to construct the recombinant plasmid, the synthetic gene was edsigned with EcoR I and Not I restriction enzyme sites at the 5’and 3’ terminal, respectively.3. The Aspergillus niger lipase gene expression vector pPIC9K-lipAm and pPIC9K-lipAm were constructed successfully after artificial synthesis and sequencing. The expression vectors (pPIC9K-lipAm and pPIC9K-lipAm) linearized by Sal I were transformed into Pichia pastoris GS115 by electroporation. After selection by MD and MM plate screening, colony-PCR testing the positive recombinant strains. After shake-flask expression by methanol induction, the lipAm was secreted expression in the Pichia pastori. The results showed that the activity of recombinant Pichia pastoris activity of the optimized gene lipAm is 5 times than lipm.