Transcriptomic analysis-driven identification and transcriptional characterization of Komagataella phaffii promoters from highly transcribed endogenous genes across diverse culture conditions
This work provides well-characterized K. phaffii strong promoter candidates, supporting future biotechnological applications and identifying 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.
Abstract
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.
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
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
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.
Results identify P SD13 as a phage-derived promoter exhibiting strong activity in both Streptomyces and E. coli, suggesting its potential as a useful genetic element for Streptomyces engineering and heterologous gene expression.
Nana Lu· Applied Microbiology and Bio...· 0 citations
Transient plant expression, which relies on Agrobacterium carrying T-DNA with target genes infiltrated into leaves for transient nuclear expression, is crucial for gene function research and the rapid production of pharmaceutical proteins. Commonly used laboratory Agrobacterium strains differ in their genetic backgrounds and modified helper Ti plasmids, which may induce varying degrees and durations of host immune responses and transcriptional reprogramming. However, the differential responses of N. benthamiana, the most commonly used host, to these strains remain unclear. In this study, we for the first time systematically investigated the transcriptional defense responses of N. benthamiana to six common laboratory Agrobacterium strains (Agrobacterium tumefaciens C58C1, GV3101, LBA4404, AGL-1, EHA105, and the Rhizobium rhizogene strain ArA4). Transcriptome profiling of tobacco leaves was conducted at 3 and 7 dpi to clarify the conserved and divergent regulation of tobacco immune pathways and stress responses by different strains. Additionally, we selected genes significantly upregulated in all strain-infiltrated groups and verified three novel defence-related genes, PAR1, WRKY81, and DMR6, that negatively regulate recombinant protein expression efficiency. We also analysed the expression patterns of previously reported immune-related genes that regulate recombinant protein yields, revealing the differential regulatory characteristics of tobacco in response to infiltration by different Agrobacterium strains. The results show that different strains have divergent activation patterns of immune genes and may adopt distinct colonization modes. We provide an important basis for selecting Agrobacterium strains for transient expression in N. benthamiana, optimizing tobacco transient expression systems, and constructing high-efficiency plant bioreactors.
Tairu Wu, Weisong Pan, Waichin Li et al.· Frontiers in Plant Science· 0 citations
ABSTRACT Leishmania displays an unconventional mode of transcription, with long clusters of genes being transcribed polycistronically from Transcription Start Regions (TSRs), being processed into monocistronic units prior to translation. It has long been believed that transcription is constitutive: failure to identify consensus sequences across TSRs (except a GT-rich motif supporting transcription in Trypanosoma brucei) and absence of canonical eukaryotic transcription factors led to the conclusion that regulation is primarily post-transcriptional, with epigenetics playing a role in triggering transcription initiation. This study stems from our previous findings identifying a few genes to be activated in a cell cycle-dependent manner. Using nuclear run-on assays to analyze nascent transcripts of two chromosomes, chromosomes 2 and 14, we find that while most genes are constitutively transcribed, a subset of genes gets activated at specific cell cycle stages. Reporter assays reveal that this transcriptional activation is driven by the regions immediately upstream of the genes. Sequence analyses of these TSRs lying in polycistronic intergenic regions (PIRs) uncovered a 10-mer GT-rich motif, in synchrony with earlier findings in T. brucei identifying a GT-rich motif at bidirectional TSRs. We also identify a second 25-mer motif at these TSRs, and deletion analyses find this motif to be critical for regulating gene expression. The findings of this study reveal that transcriptional events in these unicellular parasites are more complex than believed thus far: not all transcriptional events are constitutive, polycistronic transcription is not the only mode of transcription, and cis-acting sequence elements regulate at least some transcriptional events in these parasites. IMPORTANCE Endemic to 90 countries, Leishmania parasites cause a spectrum of diseases called Leishmaniases. No vaccines for human use are available to date, and the drugs currently used to treat the disease are expensive, have toxic side effects, and have complex administration regimens, with emerging drug resistance compounding problems. Researchers continue to investigate Leishmania cellular processes, with the hope of uncovering new therapeutic target sites. Gene regulation in these parasites is unusual, being modulated by various mechanisms, including epigenetic modifications, gene dosage, and post-transcriptional processing. Transcription is typically polycistronic and constitutive, initiating from Transcription Start Regions (TSRs) lying upstream of the first gene in the polycistronic transcription unit (PTU). The work presented here reveals that a subset of genes is transcribed monocistronically in a cell cycle-dependent manner from Transcription Start Regions lying in the PTU-intergenic regions (PIRs), underscoring the complexities of gene regulation in these parasites. Endemic to 90 countries, Leishmania parasites cause a spectrum of diseases called Leishmaniases. No vaccines for human use are available to date, and the drugs currently used to treat the disease are expensive, have toxic side effects, and have complex administration regimens, with emerging drug resistance compounding problems. Researchers continue to investigate Leishmania cellular processes, with the hope of uncovering new therapeutic target sites. Gene regulation in these parasites is unusual, being modulated by various mechanisms, including epigenetic modifications, gene dosage, and post-transcriptional processing. Transcription is typically polycistronic and constitutive, initiating from Transcription Start Regions (TSRs) lying upstream of the first gene in the polycistronic transcription unit (PTU). The work presented here reveals that a subset of genes is transcribed monocistronically in a cell cycle-dependent manner from Transcription Start Regions lying in the PTU-intergenic regions (PIRs), underscoring the complexities of gene regulation in these parasites.
A. Singh, Vishal Dashora, Arushi Khanna et al.· Microbiology spectrum· 0 citations