Codon optimization, expression and enzymatic comparison of Rhizopus oryzae lipases pro-ROL and m-ROL in Pichia pastoris / 生物工程学报

Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.

Two-step synthesis of the full length Aspergillus niger lipase gene lipA leads to high-level expression in Pichia pastoris / 生物工程学报

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.