Lü Tong, Tu Ran, Yuan Huiling, Liu Hao, Wang Qinhong
College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China.
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Sheng Wu Gong Cheng Xue Bao. 2019 Jul 25;35(7):1317-1325. doi: 10.13345/j.cjb.190058.
Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.
毕赤酵母是最便捷且应用广泛的异源蛋白表达系统之一。为进一步提高其表达异源蛋白的能力，我们开发了一种基于微滴微流控的高通量毕赤酵母筛选方法，并通过筛选获得木聚糖酶表达和分泌能力增强的突变体来验证该方法。我们使用聚合酶链反应（PCR）扩增获得木聚糖酶xyn5基因与绿色荧光蛋白gfp基因的融合片段，并将该片段克隆到毕赤酵母表达载体pPIC9K中，构建重组木聚糖酶和绿色荧光蛋白DNA片段的质粒pPIC9K-xyn5-gfp。该质粒通过电穿孔进入毕赤酵母GS115后，获得了能够表达木聚糖酶和绿色荧光蛋白的毕赤酵母SG菌株。然后，上述菌株通过常压室温等离子体诱变，随后封装形成单细胞液滴。液滴培养24小时后，进行微流控筛选以获得木聚糖酶高表达的突变菌株，用于进一步构建和筛选下一个诱变文库。经过五轮微滴微流控筛选，获得了高产菌株毕赤酵母SG-m5。所表达木聚糖酶的活性为149.17 U/mg，比原始菌株SG表达的木聚糖酶活性高300%。该菌株分泌异源蛋白的能力比原始菌株高160%。本研究开发的基于单细胞微滴微流控的高通量毕赤酵母筛选系统，筛选通量为每小时100000个菌株，仅需10小时就能筛选百万菌株文库，且仅消耗100 μL荧光试剂，与传统微孔板筛选相比，试剂成本降低了数百万倍，更重要的是，为通过高效低成本筛选获得具有高表达和分泌异源蛋白能力的毕赤酵母提供了一种新方法。
