Two-step synthesis of the full length Aspergillus niger lipase gene lipA leads to high-level expression in Pichia pastoris.
- Author:
Jiangke YANG
1
;
Xiangxiang YAN
;
Zhengping ZHANG
;
Xueqing JIANG
;
Yunjun YAN
Author Information
1. Key Laboratory of Molecular Bio-physics of Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China. jiangke_yang@hotmail.com
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Carboxylic Ester Hydrolases;
biosynthesis;
genetics;
Cloning, Molecular;
Genes, Synthetic;
Genetic Engineering;
methods;
Genetic Vectors;
genetics;
Molecular Sequence Data;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2009;25(3):381-387
- CountryChina
- Language:Chinese
-
Abstract:
Aspergillus niger lipases are important biocatalysis widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Full length gene synthesis is an efficient measure to enhance the expression level of the gene. In order to reduce the non-specific binding between oligonucleotides and bases mutation caused by the complicate secondary structure of DNA and excessive PCR amplification, a frequently phenomenon in one-step gene synthesis, we used a two-step method including assembly PCR (A-PCR) and digestion-ligation step to synthesis Aspergillus niger lipase gene lipA. Assisted by DNA2.0 and Gene2Oliga software, we optimized the codon usage and secondary structure of RNA and induced enzyme sites Cla I (237 site) and Pst I (475 site) into the gene. In the first step, fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) were separately synthesized by assembly PCR. In the second step, fragments F1, F2 and F3 were separately digested by Cla I and Pst I, and then ligated into a full length lipA gene. Two-step method efficiently enhanced successful ratio for full-length gene synthesis and dispersed the risk for gene redesign. The synthesized gene was cloned into pPIC9K vector and transferred into Pichia pastoris. After methanol inducement, the expression level of the codon optimized lipA-syn gene reached 176.0 U/mL, 10.8-fold of the original lipA gene (16.3 U/mL) in Pichia pastoris GS1115. The recombinant offers the possibility for lipase large-scale production.
