Codon Optimization Of Candida Antarctica Lipase B Gene In Pichia Pastoris By Full Gene Synthesis Strategy

For its broad spectrum of substrate acceptance and enantioselectivity, Candida antarctica lipase B (CALB) has been widely used in the fields of food, chemicals, drug precursors synthesis and bio-energy. In order to realize the industrial production of this enzyme, it is important that using genetic engineering achieves high expression of the lipase in the heterologus host.In this study, a codon optimization and gene synthesis strategy were used to improve the heterogenous expression level of CalB (GenBank: Z30645.1) in Pichia pastoris.Initialy, condons of original CalB gene were re-designed and optimized by bioinformatics measures, and then genes of the original gene CalB and the codon-optimized gene CalBM were both synthesized by PCR-based accurate synthesis (PAS) method. Then, they were integrated into the constitutive and inductive expression vectors of P. pastoris expression system. The recombinant vectors were respectively transformed into P. pastoris GS115, SMD1168 and X-33. The functional expression of these recombinants in all hosts have been realized and the overexpression genetic engineering strains were obtained. The properties of the recombinant CALB were also characterized. The main works and the results were listed as follows:1. In order to improve the expression level of CalB in Pichia pastoris, the codons of several amino acids, such as Leu, Thr, Gly, Val, Ala, Arg, Ser, Pro, Lys, Ile, Gln, Tyr, Asn, Phe, Cys and Asp, which have a low codon usage frequency in P. pastoris, were optimized with the assistance of software DNA2.0 according to codon usage frequency in P. pastoris. A total of 172 positions were replaced to the most, second and third frequency codon usage. The final content of G+C was 53.99% (while the original gene was 61.89%), and the removals of rich GC and AT regions successfully decreased the complexity of the RNA secondary structure.2. Four different primary structure forms of original CalB gene, CalBSP,CalBP,CalB,CalB His-tag were synthesized by full gene synthesis strategy of PAS protocol.3. In this reaserch, recombinant expression vectors pPIC9K-CalB, pPIC9KM-CalB, pGAPZα-CalB, pPICZα-CalB, pPIC9K-CalBM, pPIC9KM-CalBM, pGAPZα-CalBM, pPICZα-CalBM, pPIC9K-CalB-His and pPIC9K-CalBM-His were constructed, which used original gene CalB and optimized gene CalBM as foreign genes, pPIC9K, pPIC9KM and pPICZαas inductive expression vectors, pGAPZαas constitutive expression vector. Then, they were transformed into P. pastoris GS115, SMD1168 and X-33, respectively. Functional expressions of these recombinants were realized in all genetic engineering strains and the high-level expression strains were obtained. As a result, the optimized gene CalBM expressed in P. pastoris showed a higher hydrolysis activity and protein expression level, enhanced about 40%. And the transformants used pPICZαA as expression vector and X-33 as a host showed the highest expression level with the measured highest hydrolysis activity of ethyl octanonate and protein expression level was 246.3 U/mL and 179.1 mg/L respectively.4. CALB was purified through Ni-NTA affinity chromatography and ultrafiltration, and was then analyzed by SDS-PAGE. The result showed that the CALB protein expressed by P. pastoris was about 35 kDa, which might contain the glycosylated brach chain. After Endo H digestion, the molecular weight of CALB became 33 kDa, silimar to the nature CALB. Thus, it was demonstrated that CALB had glycosylation in protein processing.5. The enzymatic characteristics of the recombinant CALB were also determined. The results showed that it had a maximal hydrolysis activity for medium chain ester (C8), the optimal temperature and pH of the CALB were 40℃and pH8.0, respectively. The enzyme activity was strongly stimulated in the presence of Ca²⁺, Mg²⁺, K+ and Tween 80, while inhibited by Cu²⁺, TrionX-100 and SDS.