Transcriptional reprogramming of the stress response and translational-secretory machinery enhances recombinant secreted protein production in Yarrowia lipolytica.
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.
Abstract
Protein synthesis and secretion processes are non-pathway traits. The genetic complexity makes them very challenging to engineer and genetically optimize. Therefore, we exploit transcriptional reprogramming to create a favorable cellular environment for a trait of interest. In this study, we investigate the effects of transcription factor (TF) engineering on secretory recombinant protein (rProt) production in Y. lipolytica. High-throughput genome-wide pre-screening guided the TFs selection. Here, we run high-throughput functional studies, followed by transcriptome analysis for selected TF-modified strains from semi-steady state. We contrasted the overexpression (OE) vs. deletion (KO) genotypes for their capacity to produce two reporter proteins (fluorescent model and glucoamylase). Despite a wide set of TF-modified strains analyzed here, only several displayed consistent, unbiased phenotypes. Collected data indicated that the known general stress response TFs, Msn4 and Hsf1, elicit their highly beneficial rProt-promoting function specifically towards a "challenging" rProt, while the levels of "simple" rProt remain unaffected. Deletion of the previously identified strong repressor of intracellular rProt synthesis, Azf1, significantly enhanced production of secretory rProt. At the transcriptomic level, deletion of Azf1 was associated with massive upregulation of ribosome biogenesis and cytoplasmic translation, which underwent alternative splicing (global differential exon usage analysis). The results of contrasting the Azf1- and Dep1-driven regulomes (the latter identified as a strong repressor of intracellular rProt synthesis, with no positive effect on the secretory rProt production) suggest that for efficient rProt secretion, upregulation in the vesicle-mediated transportation must co-occur, as in the case of Azf1-KO. In our experimental setting, OE of the general stress response TFs was beneficial for the production of "problematic", UPR-awakening rProt, and deletion of Azf1 for tuning a molecular background beneficial for secretory rProt.
Komagataella phaffii has emerged as an important eukaryotic microbial cell factory for the production of industrial enzymes, biopharmaceuticals, and food-related proteins owing to its rapid growth, capacity for eukaryotic post-translational modifications, and suitability for high-cell-density fermentation. However, efficient heterologous protein production remains constrained by limitations in transcriptional regulation, protein secretion, and endoplasmic reticulum (ER) protein-folding capacity. This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign (e.g., upstream activating sequence (UAS) duplication and core promoter mutagenesis), signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response (UPR). Rather than focusing on individual expression modules, this review highlights a systems-level engineering perspective that integrates regulatory elements, intracellular processing, and secretory pathway optimization, illustrating the ongoing transition from empirical modification of individual expression elements to systems-level engineering of the secretory pathway. Recent advances in synthetic biology and artificial intelligence (AI)-assisted approaches are further accelerating the development of predictive and precision engineering approaches for K. phaffii. Together, these advances provide a foundation for the rational design of next-generation K. phaffii cell factories for the sustainable production of structurally complex and high-value recombinant proteins.
Ruyue Han, Ruizheng Hu, Anran Liu et al.· Journal of Fungi· 0 citations
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.
Jiacheng Shi, Zhanqing Lv, Chaoyu Lu et al.· Microbial Biotechnology· 0 citations
The TCP (Teosinte branched 1/Cycloidea/Proliferating) gene family comprises plant-specific transcription factors essential for regulating growth, development, and environmental adaptation. Utilizing the high-quality ‘Rui Youpan 1’ (‘RYP1’) reference genome, we conducted a comprehensive genome-wide identification and characterization of the TCP family in peach (Prunus persica). We identified 20 PpTCP genes and systematically evaluated their physicochemical properties, chromosomal distribution, phylogeny, gene architecture, and promoter cis-regulatory elements. Notably, the enrichment of abscisic acid-responsive elements (ABREs) in their promoters suggests a significant role in abiotic stress signaling. Integrated RNA-seq and RT-qPCR analyses identified six putative candidates (PpTCP3, 5, 6, 11, 14, and 15) with pronounced differential expression under drought conditions. Subcellular localization confirmed that all tested PpTCPs function within the nucleus. Moreover, the results of STRING-based computer simulations predicting protein–protein interactions indicate that PpTCP3 and PpTCP5 interact with key hormone pathways and stress-related transcription factors (TFs), including auxin signaling and strigolactone signaling.
Yanfu Jing, Yang Yu, Zimin Xiao et al.· International Journal of Mol...· 0 citations
The yeast Komagataella phaffii (formerly Pichia pastoris) is a robust host for recombinant protein production, capable of growing at high cell densities, performing post-translational modifications, and efficiently secreting heterologous proteins. However, gene expression still relies mainly on the methanol-inducible AOX1 promoter (PAOX1) and the constitutive GAPDH promoter (PGAP), highlighting the need for alternative promoters with distinct regulatory properties. This study aimed to identify and characterize promoters from the most highly transcribed endogenous genes across diverse culture conditions as strong promoter candidates to expand the genetic toolbox for this yeast. Seven promoters were identified through RNA-seq analysis from genes showing transcript levels comparable to or higher than those of genes regulated by PAOX1 or PGAP under specific culture conditions. These promoters were further transcriptionally characterized by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) under different carbon sources, growth phases, and temperatures through the measurement of transcript levels of their corresponding genes, revealing distinct transcriptional profiles. Two major clusters were identified: one associated with genes upregulated during the stationary phase and the other with genes upregulated during the exponential phase. These transcriptional profiles provided a basis for the rational design of fed-batch strategies tailored to each promoter when driving heterologous gene expression. The 0208 and HSP12 promoters showed particular potential at low specific growth rates, favoring high recombinant protein titers with reduced biomass accumulation. In silico analyses suggested that 0208 encodes a mitochondrial membrane-associated protein with a possible functional relationship to Hsp12. Overall, this work provides well-characterized K. phaffii strong promoter candidates, supporting future biotechnological applications.
Karla B. Fernández-Cano, J. M. Viader-Salvadó, M. Guerrero-Olazarán· PeerJ· 0 citations
Yarrowia lipolytica (Y. lipolytica) is an attractive host for terpenoid biosynthesis due to its high acetyl-CoA availability and robust genetic tractability. However, how fermentation temperature influences intracellular metabolic states remains insufficiently characterized. Here, the engineered mevalonate-producing strain YL17 was employed as a platform to characterize temperature-dependent metabolic responses in Y. lipolytica. Fermentation experiments across a range of temperatures revealed a clear growth-production trade-off, whereby increasing temperature enhanced mevalonate synthesis at the expense of cellular growth. 13C metabolic flux analysis revealed that higher temperature promoted a significant redistribution of carbon flux toward glycolysis, increasing acetyl-CoA generation and precursor supply. Notably, transcriptomic analysis indicated a global downregulation of genes involved in glycolysis, indicating a decoupling between transcriptional levels and flux regulation. To explore the regulatory basis underlying this phenotype, differentially expressed transcription factors were systematically analyzed, leading to the identification of YALI1_A16891g as a key regulatory candidate associated with glycolytic flux redistribution. Further analysis suggested that YALI1_A16891g may regulate downstream glycolysis-related genes under elevated temperature conditions, thereby influencing glycolytic carbon allocation and mevalonate production specifically at high temperature in Y. lipolytica. These results demonstrate 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.
This study aimed to elucidate transcriptional adaptation mechanisms in Zygosaccharomyces mellis under high-glucose (50%) conditions and to identify cellular processes involved in osmotic stress tolerance. Transcriptomic analysis was performed to characterize global gene expression changes in cells cultured in yeast extract-malt medium containing 50% glucose (G50). Genes involved in glycolysis, including GAL7 and FBP1, were downregulated, whereas PYC2, associated with the tricarboxylic acid cycle, was upregulated, suggesting suppression of fermentative metabolism and maintenance of respiratory activity. Genes involved in membrane phospholipid synthesis (AGPAT and PLPP) were downregulated, while an acid sphingomyelinase-like gene (ASM) was upregulated, suggesting altered membrane lipid metabolism under osmotic stress. In addition, the cytokinesis-related gene MYO1 was upregulated, suggesting structural reinforcement under osmotic stress. Furthermore, the growth of Δmmr1 and Δhxt4 mutants in Saccharomyces cerevisiae was significantly impaired under G50 conditions, highlighting the importance of mitochondrial inheritance and regulated glucose uptake. These findings suggest that Z. mellis adapts to high-glucose stress by coordinating changes in gene expression associated with respiratory activity, lipid metabolism, and cell division processes.
T. Tamura· Letters in Applied Microbiol...· 0 citations