Codon Optimization And Synthesis Of Nattokinase Gene And Its High-Level Expression In Pichia Pastoris

Nattokinase(NK) is a strong fibrinolytic enzyme from Bacillus subtilis natto, which could obviously dissolve fibrin,blood clot,enhance fibrinolytic systerm's function of animal and human thrombus model.In vivo,the natto-saline extract on normal human volunteers by oral administration revealed a significant elevation of the fibrinogen degradation products and euglobulin fibrinolysis,and an increase of t-PA antigen in the plasma,indicating release of endogenous plasminogen activator was probably from endothelial cell.NK also acts as a plasmin-like proteinase and also cleaves and inactivates plasminogen activator inhibitor-1(PAI-1).But until now,their application is limited due to the short supply and high price.In fact,to make this enzyme available for more people,a convenient and widely used delivery vehicle is needed.The purpose of this research is to achieve a high level expression of nattokinase in Pichia pastoris through the technique of gene engineering and protein engineering with the property right reserved and offer theoretical basis for exploitation and application of nattokinase.The research includes the items as follows:1.The mature peptide gene of nattokinase(NK) was obtained by PCR amplification from Bacillus natto mutant strain DU115 which was protoplast mutagenesis with UV and wild-type strain BN10 genomic DNA,and sub-cloned into pPICZα-A. Sequencing of the amplified fragment revealed a synonymous mutation C396T and a nonsynonymous mutation A107G leading to amino-acid substitution D36G (Asp→Gly).The recombinants pPICZα-A-nkD and pPICZα-A-nkB were expressed in Pichia pastoris X-33.The comparison experiments of purified mutant enzymes DNK with natural BNK showed that its thermostability was increased by 20%,when it was exposed for 15min at 65℃and specific activity was increased by 16.6%.We conclude that D36G mutation was positive for the thermostability and activity after a comparison of their structure.All things considered,the heat stability of nattokinase from strain DU115 was better and was used for further research.2.When we employed the yeast Pichia pastoris to express nattokinase derived from Bacillus subtilis var.natto DU115,the yield did not exceed 20 mg/L in shake flask cultures.As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and production,we designed the full-length nattokinase mature-peptide gene synkD by choosing the P.pastoris most-preferred codons,the second-preferred codons were alternatively used acoding to native gene while eliminating of stable secondary structures.The optimized nattokinase gene 848 bp in length was synthesized and cloned into the pMD18-T vector.Sequencing analysis demonstrated that the pMD18- synkD carried synkD gene correctly.3.The synthetic nattokinase gene synkD was cloned into the plasmid pPICZα-A to construct the recombinant expression vector pPICZα-A-synkD,and transformed into yeast host strain X-33.The yield of the nattokinase expressed by P.pastoris with optimized gene synkD was 3.5 times higher than that of the wild type gene nkD. Investigation of culture conditions revealed that nattokinase expression were achieved at the density of the seed yeast amounted to OD₆₀₀ value 5-6,at pH7.0 of the culturing BMMY medium,with 1.5%methanol after 84 h induction.It was found that the expression level of the recombinant synkD was increased to 112μg/mL,512 IU/mL fibrinolytic activity and transformants showed quite good genetic stability.The P.pastoris strain expressed the optimized nattokinase gene showing great potential as a commercial nattokinase production system.