Fine-Tuned Gene Expression Elements from Hybrid Promoter Libraries in Pichia pastoris.

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.

Analysis of acid-tolerance mechanism based on membrane microdomains in Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae has been used in the biosynthesis of acid products such as organic acids owing to its acid tolerance. Improving the acid tolerance of S. cerevisiae is beneficial for expanding its application range. Our previous study isolated the TAMC strain that was tolerant to a pH 2.3 through adaptive laboratory evolution; however, its mechanism underlying tolerance to low pH environment remains unclear. RESULTS: In this study, through visual observation and order analysis of plasma membrane and membrane microdomains, we revealed that the membrane microdomains of TAMC strain play an indispensable role in acid tolerance. Transcriptomic analysis showed an increase in the expression of genes related to key components of membrane microdomains in TAMC strain. Furthermore, an obvious reduction was observed in the acid tolerance of the strain with sterol C-24 methyltransferase encoding gene ERG6 knockout for inhibiting membrane microdomain formation. Finally, colocalization analysis of H+-ATPase PMA1 and plasma membrane protein PMP1 showed that disruption of membrane microdomains could inhibit the formation of the H+-ATPase complex. CONCLUSIONS: Membrane microdomains could provide a platform for forming H+-ATPase complexes to facilitate intracellular H+ homeostasis, and thereby improve cell acid resistance. This study proposed a novel acid tolerance mechanism, providing a new direction for the rational engineering of acid-tolerant strains.