In the present study, green fluorescence protein (GFP) was used as a candidate protein to test and optimize a robust chemical transformation procedure in P. pastoris. Towards this, it was adjudged that pretreatment of P. pastoris cells with lithium chloride (LiCl) and its optimal concentration is critical for efficient transformation. Using three different methods (M1: 100 mM LiCl, 10 min, M2: 1 M LiCl, 10 min and M3: 1 M LiCl, 1 h), it was found that concentration and incubation time for LiCl treatment significantly affects the transformation efficiency. The transformation efficiency (transformants/μg DNA) was observed to be 1.01 × 10², 5.07 × 10³ and 6.52 × 10³ using methods M1, M2 and M3, respectively, indicating the superiority of M3. Moreover, presence of the GFP gene in the positive transformants was confirmed using a novel colony PCR method where the colonies were treated with LiCl prior to GFP specific amplification. Also, it was established using fluorescence microscopy and western blot analysis that increasing zeocin concentration as a post transformational vector amplification (PTVA) strategy increased the fluorescence and gene expression, respectively. Further, RT-qPCR revealed that the gene copy number using methods M1, M2 and M3 were 2.97, 5.26 and 7.19, respectively, when 500 μg/ml zecocin was used for selection, thus corroborating western blot results. In conclusion, we demonstrate a cheap and robust chemical method for achieving higher transformation efficiency in P. pastoris and a simple procedure for colony-PCR based-diagnosis alleviating the need for enzymatic treatment.

在本研究中，绿色荧光蛋白（GFP）被用作候选蛋白来测试和优化巴斯德毕赤酵母的稳健化学转化程序。为此，判定使用氯化锂（LiCl）预处理巴斯德毕赤酵母细胞及其最佳浓度对于有效转化至关重要。使用三种不同的方法（M1：100 mM LiCl，10分钟； M2：1 M LiCl，10分钟； M3：1 M LiCl，1小时），发现LiCl处理的浓度和孵育时间会显着影响转化效率。转化效率（转化子/μgDNA）被观察到为1.01×10 ²，5.07×10 ³和6.52×10 ³分别使用方法M1，M2和M3表示M3的优越性。此外，使用新型菌落PCR方法确认了阳性转化体中GFP基因的存在，其中在GFP特异性扩增之前用LiCl处理了菌落。此外，使用荧光显微镜和蛋白质印迹分析已确定增加zeocin浓度作为转化后载体扩增（PTVA）策略分别增加了荧光和基因表达。此外，RT-qPCR显示，当使用500μg/ ml zecocin进行选择时，使用方法M1，M2和M3的基因拷贝数分别为2.97、5.26和7.19，从而证实了蛋白质印迹结果。总之，我们证明了一种便宜而稳健的化学方法可实现更高的转化效率。巴斯德毕赤酵母和基于菌落-PCR的诊断的简单程序减轻了对酶处理的需求。