Cloning, Codon Optimization And Expression Of Rhizopus Oryzae Lipase Gene In Pichia Pastoris

Lipase from Rhizopus oryzae (ROL) with high specificity of 1,3-sn and stereoselectivity, is widely used in the synthesis of aromatic esters, biodiesel, chiral compounds and organic compounds, etc. To achieve an efficient industrial production of ROL, genetic engineering strategy such as genetic codes optimization and application of high efficient expression system were generally selected. In this study, we cloned the lipase gene ROL from R. oryzae CICC3005. Through point mutation and whole gene syntheis, the codon of ROL was optimized and secretorily expressed in Pichia pastoris. This laid a foundation for future genetic engineering and scale production of ROL. The main results are listed as follows:(1) The prosequence and mature lipase gene (pro-ROL and mature-ROL) were cloned from the total genomic DNA of R. oryzae CICC3005 by PCR, and achieved a secretory expression in P. pastoris. The measured activities of the lipases encoded by pro-ROL and m-ROL were 13.4 U/mL and 12.6 U/mL, respectively. The molecular mass of the recombinant lipases of pro-ROL and m-ROL determined by SDS-PAGE were 35 kDa and 30 kDa, respectively. The preliminary study on the characteristics of both recombinant lipases showed there was significant difference between them. The recombinant m-ROL lipase exhibited higher hydrolysis activity towards middle-chain (C10) than others, and got maximum activity at 30℃and pH 9.0,while recombinant pro-ROL lipase exhibited higher hydrolysis activity towards short-chain (C4) and showed high hydrolysis activity at 30℃and pH 8.0. Moreover, pro-ROL lipase possessed stronger tolerance to high temperature than m-ROL lipase. These differences were probably due to the existence of pro-sequences, for the previous study reported that pro-sequence could affect folding, modification, expression and secretion process of the lipase.(2) According to the codon usage frequency of P. pastoris, the codon of mature ROL gene was optimized through point mutation, and eight bases were substituted. After methnal inducible expression in P. postoris, the lipase activity of the optimal ROL gene reached 15.5 U/mL, 30 % higher than that of the native gene. This proved that codon optimization strategy could be an effective way to increase the expression quantity of the target protein.(3) Gene artificial construction through in vitro total gene synthesis is becoming more and more popular, and the ability to synthesize efficiently and accurately long DNA sequence at low cost is becoming increasingly important. In this study, taking lipase genes from R. oryzae CICC3005 as a starting gene, we designed a novel two-step gene synthesis strategy, which is defined as assembly PCR-enzyme ligation rections. The sequencing results indicated that the two-step gene synthesis method had many advantages, such as simple steps, low cost, efficiently enhanced successful ratio for full-length gene synthesis, exhibiting a potential prospect in the future.