毕赤酵母液滴微流控高通量筛选方法的建立与应用

Development and application of a droplet-based microfluidic high-throughput screening of Pichia pastoris

巴斯德毕赤酵母是当前应用最为方便和广泛的外源蛋白表达系统之一,为了进一步提高其表达外源蛋白的能力,文中建立了基于液滴微流控的毕赤酵母高通量筛选方法,并以木聚糖酶融合荧光蛋白为例,筛选获得木聚糖酶表达和分泌能力提高的突变株。通过PCR扩增得到木聚糖酶xyn5基因和绿色荧光蛋白gfp基因融合片段,并克隆到毕赤酵母表达载体pPIC9K中构建出木聚糖酶融合绿色荧光蛋白的质粒pPIC9K-xyn5-gfp,电转化至毕赤酵母GS115中得到表达木聚糖酶和绿色荧光蛋白的毕赤酵母SG菌株。该菌株经过常压室温等离子体诱变后进行单细胞液滴包埋,液滴培养24h后进行微流控筛选,获得高表达木聚糖酶的突变菌株,进而用于下一轮的诱变突变库构建和筛选。以此类推,经过5轮液滴微流控筛选,获得一株高产菌株SG-m5,其木聚糖酶活为149.17U/mg,较出发菌株提升300%,分泌外源蛋白的能力较出发菌株提高160%。文中建立的毕赤酵母单细胞液滴微流控高通量筛选方法能达到每小时10万菌株的筛选通量,筛选百万级别的菌株库仅需10h,消耗荧光试剂体积100μL,对比传统的微孔板筛选方法降低试剂成本近百万倍,为高效、低成本筛选获得表达和分泌外源蛋白能力提高的毕赤酵母提供了一条新途径。

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 100μL 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.