疏棉状嗜热丝孢菌脂肪酶基因在毕赤酵母工程菌中的遗传稳定性

【背景】重组工程菌的传代稳定性是保证外源蛋白高效稳定表达的前提,是决定工业化生产能力的关键因素之一。【目的】对异源的疏棉状嗜热丝孢菌脂肪酶基因在毕赤酵母重组工程菌中的遗传稳定性进行研究。【方法】将工程菌连续传代15次,取第1、5、10、15代作为受检代次,结合重组菌的菌落及菌体形态、水解酶活、目的基因片段、外源基因拷贝数等指标综合评价其遗传稳定性。【结果】传代过程中重组菌的菌落和菌体形态、目的蛋白分子量、目的基因序列均保持一致。目的基因的整合拷贝数经5次传代后发生一定损失,但随后稳定为7左右,而脂肪酶的相对酶活则提高至90%以上。【结论】适量的整合拷贝数更有利于该脂肪酶基因在毕赤酵母重组菌中的表达,经综合评价此工程菌的遗传稳定性良好,应用于工业化大规模生产是可行的。

Hereditary stability of Thermomyces lanuginosus lipase gene in recombinant Pichia pastoris strain

[Background] hereditary stability of recombinant strain is the prerequisite for high-level and stable expressing, which determines the success of industrial applications. [Objective] hereditary stability of Pichia pastoris recombinant strain containing heterologous Thermomyces lanuginosus lipase gene was studied. [Methods] recombinant strain was subcultured for 15 generations and its stability judged from the following aspects: colony morphology, cell morphology, hydrolytic activity of lipase, DNA sequence and integrated copy number of the target gene. [Results] colony and cell morphology of the 1st, 5th, 10th and 15th generations of strains remained the same, as well as the molecular weight of the target protein and the target gene sequence. It was also found that after pass-generation culture for 5 times, the integrated copy number of lipase gene was greatly reduced and then stabilized at about 7 in the 10th and 15th generations, while the relative hydrolytic activity of lipase reached up to 90%. [Conclusion] high-level expression of the lipase is achieved by moderate gene copy number. this recombinant strain has a good hereditary stability and is feasible for large-scale production.