An engineered UPR‐adaptive chassis (UPR‐Ad+) was constructed by over‐expressing these three key regulators and enhanced the production of high‐copy secretory proteins by 30%–46%.
Abstract
Komagataella phaffii is one of the most widely used eukaryotic protein expression systems. Increasing the copy number of exogenous genes is a common method to enhance expression efficiency. However, at extremely high copy numbers, the resulting endoplasmic reticulum stress (ERS) can hinder further improvement in the expression efficiency of secreted proteins. And elucidating the underlying molecular mechanisms serves as the foundation for engineering and constructing stress‐adapted strains. High‐copy secretory expression was first confirmed to adversely affect yeast growth fitness, a phenotype consistent with DTT‐induced unfolded protein response (UPR). Screening of a kinase knockout library established that UPR signalling in K. phaffii was modulated by the PpHog1‐mediated MAPK pathway. PpHog1, the K. phaffii ortholog of the well‐characterized Saccharomyces cerevisiae Hog1 (67% sequence identity), is traditionally known for its roles in osmotic stress and cell wall integrity. Notably, while ScHog1 has also been implicated in ERS regulation, the downstream pathway remains obscure. Further comparison of the PpHog1 interactome with versus without DTT treatment, combined with follow‐up genetic knockout/knockdown screens, leads to the identification of two critical UPR regulators: PpLRR‐0498 and PpPINT‐0120. Subsequently, an engineered UPR‐adaptive chassis (UPR‐Ad+) was constructed by over‐expressing these three key regulators. Using human serum albumin (HSA), human calcium‐regulated actin binding protein LCP1, and α‐amylase as reporters, UPR‐Ad+ enhanced the production of high‐copy secretory proteins by 30%–46%. Collectively, this work advances the understanding of UPR regulation in K. phaffii and provides a strategic basis for designing high‐yield strains.
This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign, signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response.
Ru-Yue Han, Ruizheng Hu, An-Ran Liu et al.· Journal of Fungi· 0 citations
The SNF1‐related protein kinase 2 (SnRK2) pathway is a central regulator of abiotic stress signaling in land plants, yet its evolutionary origins and functional conservation across the green lineage remain poorly understood. To determine whether this signaling module predates the streptophyte–chlorophyte divergence, we investigated the chlorophyte alga Chlamydomonas reinhardtii, which diverged from the streptophyte lineage over 1 billion years ago. Using a combination of reverse genetics and protein–protein interaction assays, we identified and characterized a bona fide SnRK2 signaling module in C. reinhardtii. We demonstrate that the isoform SnRK2.7 is essential for osmotic stress tolerance and general cellular viability. SnRK2.7 localizes to the contractile vacuole, an osmoregulatory organelle lost during streptophyte evolution, revealing a lineage‐specific functional adaptation. Together with MAPKKK3, a B1/B3‐RAF kinase, and the clade A protein phosphatase PP2C3, these components constitute a MAPKKK3‐SnRK2.7‐PP2C3 regulatory module in C. reinhardtii. Our findings demonstrate the presence of a functional SnRK2 pathway in a chlorophyte alga, suggesting that core components were established early in the green lineage. This work provides a foundation for comparative studies across green plants and underscores the need for broader taxonomic sampling to reconstruct the ancestral signaling networks underlying stress adaptation.
Wenqing Yu, Kangning Guo, Yanyou Jiang et al.· New Phytologist· 0 citations
The results of contrasting the Azf1- and Dep1-driven regulomes suggest that for efficient rProt secretion, upregulation in the vesicle-mediated transportation must co-occur, as in the case of Azf1-KO.
Maria Gorczyca, P. Kubiak, E. Celińska· Microbial Cell Factories· 0 citations
Begonia semperflorens is an important ornamental plant worldwide, but its practical application is severely limited by low temperature sensitivity. However, the molecular regulatory mechanisms underlying low temperature-induced anthocyanin biosynthesis remain unclear. In this study, we assembled a high-quality chromosome-level B. semperflorens genome. Through genomic and MYB gene family analysis, we identified a key transcription factor BsTT2, which directly binds to the BsDFR promoter and enhances its activity, thereby driving anthocyanin accumulation in B. semperflorens. Furthermore, using BsTT2 as bait, we identified its interacting protein BsAlfin2. Under low temperature-induced reactive oxygen species (ROS) signaling, BsAlfin2 undergoes nuclear translocation and forms a complex with BsTT2, synergistically enhancing the activation of the BsDFR promoter and significantly improving anthocyanin production efficiency. Based on these results, we propose a previously uncharacterized BsAlfin2/BsTT2-BsDFR regulatory module, which reveals a molecular links low-temperature ROS signaling to anthocyanin biosynthesis in B. semperflorens. In summary, this study not only provides chromosome-level genomic resources for B. semperflorens research but also elucidates a key molecular module in low temperature-induced anthocyanin biosynthesis regulatory pathway, laying a theoretical and data foundation for future studies on leaf color improvement and stress resistance breeding in Begonia.
It is demonstrated that temperature can effectively reshape metabolic flux distribution and highlight transcription factor-mediated regulation as a practical target for improving precursor supply in engineered Y. lipolytica.
First comprehensive characterization of the RLCK gene family in sugarcane is presented, elucidating its evolutionary features, expression dynamics and functional roles, and providing compelling evidence that ScRLCK53 modulates salt tolerance through activation of the JA signaling pathway.
Shichao Wang, Pingping Lin, Deng Wu et al.· Plant physiology and biochem...· 0 citations