毕赤酵母截短PGK1启动子与不同终止子组合调控外源基因表达

Truncated PGK1 promoter is paired with varied terminators to regulate heterologous gene expression in Pichia pastoris

摘要:

【目的】调控多基因表达对于优化代谢途径和合成生物学应用至关重要，构建不同的启动子和终止子组合，可作为毕赤酵母代谢途径改造和优化外源基因表达的有力分子调控工具。【方法】首先，将毕赤酵母组成型磷酸甘油酸激酶基因的启动子P_PGK1进行截短，构建截短启动子分别调控报告基因(绿色荧光蛋白基因egfp和β-半乳糖苷酶基因lacZ)表达的毕赤酵母重组菌。检测重组菌的报告基团转录水平、荧光强度和β-半乳糖苷酶产量。然后，构建了不同强度启动子和终止子组合(共27种组合)调控egfp表达的重组菌。最后，选取能调控基因高、中、低表达的6个启动子-终止子组合，调控β-呋喃果糖苷酶基因表达，构建β-呋喃果糖苷酶分泌表达的重组菌。【结果】构建的截短启动子(P_PP、P_PE、P_PG和P_PD)的强度是野生型启动子P_PGK1的70%–190%，最强的启动子为P_PD。分别与9个终止子组合时，P_PG、P_PE和P_PD启动子驱动egfp基因表达的强度最高的和最低的相比分别达到4倍、7倍和10倍。6个启动子-终止子组合调控β-呋喃果糖苷酶分泌表达的重组菌胞外酶产量最高的和最低的相比可达6倍。【结论】构建了不同的启动子-终止子组合，调控基因表达水平最高的和最低的相比达到10倍，可为优化毕赤酵母代谢工程和合成生物学应用中控制不同外源基因的表达量提供有力的分子工具。

关键词:毕赤酵母;终止子;代谢工程;合成生物学;β-呋喃果糖苷酶

Abstract:

Objective] Pichia pastoris (syn. Komagataella phaffii) has been extensively used as a versatile cell factory for the production of industrial enzymes and chemicals. However, well-tuned co-expression of multiple genes is a common challenge for P.pastoris in metabolic engineering and synthetic biology. Therefore, in this work, we constructed a set of terminators and paired them with varied promoters to tune the protein levels in P.pastoris.[Methods] We constructed the P.pastoris strains expressing reporter genes (egfp and lacZ) under the control of truncated constitutive 3-phosphoglycerate kinase (PGK1) promoters, and then measured the transcript levels of reporter genes, yEGFP fluorescence intensity and β-galactosidase activity of these strains. Next, we created a total of 27 promoter-terminator pairs to regulate the transcription of egfp, and used 6 promoter- terminator pairs to alter the secretory expression of β-fructofuranosidase (β-Ffase). [Results] The promoter activities of the truncated P_PGK1 variants (P_PP, P_PE, P_PG and P_PD) relative to that of the native P_PGK1 ranged from 70% to 190%. Furthermore, when paired with the weak promoter P_PG, moderate promoter P_PE, and strong promoter P_PD, the terminators had the tuning ranges of 4, 7 and 10 folds (comparing between the strongest and weakest terminator), respectively. Finally, we demonstrated the utility of the promoter-terminator pairs for tuning the expression of the industrial enzyme β-Ffase, which showed an overall tuning range of 6 folds. [Conclusion] The promoter-terminator pairs constructed not only provide valuable information for understanding the modulatory roles of terminator regions in gene expression but also serve as a useful toolbox enabling the metabolic engineering of P.pastoris and the application of P.pastoris in synthetic biology.

Key words:Pichia pastoris;terminator;metabolic engineering;synthetic biology;β-fructofuranosidase