毕赤酵母
发起人
合成生物学
生物
计算生物学
基因
调节顺序
遗传学
基因表达调控
转录因子
代谢工程
基因表达
表情盒
重组DNA
载体(分子生物学)
作者
Jing Lai,Lingang Song,Yuyu Zhou,Hong Zong,Bin Zhuge,Xiaoyun Lu
标识
DOI:10.1021/acssynbio.3c00534
摘要
As a desirable microbial cell factory, Pichia pastoris has garnered extensive utilization in metabolic engineering. Nevertheless, the lack of fine-tuned gene expression components has significantly constrained the potential scope of applications. Here, a gradient strength promoter library was constructed by random hybridization and high-throughput screening. The hybrid promoter, phy47, performed best with 2.93-fold higher GFP expression levels than GAP. The broad applicability of the novel hybrid promoter variants in biotechnological production was further validated in the biosynthesis of pinene and rHuPH20 with higher titers. The upstream regulatory sequences (UASE and URSD) were identified and applied to promoters GAP and ENO1, resulting in a 34 and 43% increase and an 18 and 37% decrease in the expression level, respectively. Yeast one-hybrid analysis showed that transcription factor HAP2 activates the hybrid promoter through a direct interaction with the crucial regulatory region UASH. Furthermore, a short segment of tunable activation sequence (20 bp) was also screened, and artificial promoters were constructed in tandem with the addition of regulatory sequence, resulting in a 61% expansion of the expression range. This study provides a molecular tool and regulatory elements for further synthetic biology research in P. pastoris.
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