为了克服巴斯德毕赤酵母高密度发酵过程中的溶氧限制，提高外源蛋白的表达量，本文以启动子YPT介导透明颤菌血红蛋白（VHb）在培养过程中组成型表达。同时以启动子AOX1介导的南极假丝酵母脂肪酶B（CALB）基因作为报告基因，使CALB在甲醇诱导阶段表达。另外，还将绿色荧光蛋白在过氧化物表面表达，将红色荧光蛋白在过氧化物基质中表达以检测信号肽能否定位在相应的位置。最后，将VHb分别在过氧化物酶体表面和基质两个位置表达，以观察表达位置的不同所产生的影响。通过SDS-PAGE和Western Blot分析表明，CALB和VHb在重组菌中成功实现了共表达。在限氧条件下，重组菌株GS115/ExVHb、GS115/VHbSKL的外源蛋白CALB的表达量比对照菌株GS115/CALB分别提高了27.57%和20.52%。这说明VHb与外源基因共表达可以改善酵母在发酵过程中的摄氧情况，提高外源蛋白的表达量。

To alleviate the dissolved oxygen (DO) limitation and improve Candida antarctica lipase B (CALB) production during high-cell density fermentation, the CALB gene and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the AOX1 and YPT promoter control, respectively. In addition, to detect the effect of signal peptide, GFP was expressed on the surface of peroxide, while RFP was expressed in the peroxide matrix. Finally, GFP or RFP was replaced by VHb protein to find out the impaction of the different positions. SDS-PAGE and Western Blot analysis results indicated that VHb and CALB were successfully co-expressed in recombinant strains. Compared with the control strain, expression of CALB in GS115/ExVHb and GS115/VHbSKL under oxygen limitation was improved by 27.57% and 20.52%, respectively. Therefore, expression of VHb in P. pastoris is an effective strategy to improve CALB production.

国家自然科学青年基金(31000027)