不同构建策略对D-氨基酸氧化酶表达的影响及发酵调节

Effect on expression of D-amino acid oxidase constructed by different construction strategies and fermentation regulation

目的构建不含编码β-内酰胺酶编码基因的D-氨基酸氧化酶(D-amino acid oxidase,DAAO)工程菌,对不同构建策略构建的重组菌进行发酵调节研究。方法采用分子生物学手段,利用不同的策略、不同的毕赤酵母宿主菌,构建DAAO工程菌;利用微生物发酵和调节手段,对工程菌的发酵条件进行优化。结果获得了表型不同的3株重组菌(Muts的PDK13和PDGA10,Mut+的PDG27),它们的最佳生长条件基本相似,诱导表达条件却不相同,在Basalsalts培养基内,当生长时间为36h,诱导培养基起始pH为6.0、甲醇浓度为0.5%时,最有利于DAAO表达。在菌体密度相同的条件下,Mut+表型(PDG27)甲醇代谢快,诱导24h,DAAO活力达到最高;Muts型(PDK13和PDGA10)甲醇代谢慢,诱导36h,DAAO活力达最高;通气量一定时,菌体密度增加,DAAO表达量增加,菌体增加到一定程度表达量反而下降,表明通气量应随菌体密度的增大而增加。结论Muts型重组菌对通气量要求低且更有利于高密度发酵。

Objective Recombinant P. pastoris producing D-amino acid oxidase （DAAO） was constructed,in which the AMP assistant gene was cut. The fermentation regulation of three recombinant strains constructed by different construction strategies was studied. Methods By molecular biology method recombinant P. pastoris strains （PDG PDK PDGA-） were constructed via different strategies with different host strains. The ferment conditions were optimized by microbiology technology. Results Three kinds of recombinant strains with two kinds of phenotype （Mut^＋ and Mut^s） were obtained. Their best growth conditions were similar. In basal salts medium the three recombinant strains grew for 36h with the cells benefited for expression under 0.5 % methanol induction. But the induction conditions were different between Mut^s and Mut^＋ ..for Mut^＋ ,PDG27 was induced for 24 h and needed higher aeration;for Mut^s, PDK13 and PDGA10, the DAAO activity reached the highest after induced for 36h under lower aeration. The aeration should be regulated with cell density. Conclusions The result showed that Mut^s was more suitble for high density fermentation.