Impact of cell wall polysaccharide modifications on the performance of Pichia pastoris: novel mutants with enhanced fitness and functionality for bioproduction applications
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ABSTRACT:
SUBMITTER: Cheng B
PROVIDER: S-EPMC10874062 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production. RESULTS: Here we present five novel repressible Pichia pastoris promoters of different strength: PSER1, PMET3, PTHR1, PPIS1 and PTHI11. eGFP was expressed under the control of each of these promoters and its fluorescence could be successfully decreased in liquid culture by adding different supplements. We also expressed the essential genes with different native promoter strength, ERO1 and PDI1, under the control of two of the novel promoters. In our experiments, a clear down-regulation of both repressible promoters on transcriptional level could be achieved. Compared to the transcript levels of these two genes when expressed under the control of their native promoters, only ERO1 was significantly down-regulated. CONCLUSION: Our results show that all of the novel promoters can be used for repression of genes in liquid culture. We also came to the conclusion that the choice of the repressible promoter is of particular importance. For a successful repression experiment it is crucial that the native promoter of a gene and the repressible promoter in its non-repressed state are of similar strength.
| S-EPMC3599224 | biostudies-literature
Project description:Comparison of transcription profile of Pichia pastoris cells grown on Glucose medium with Pichia pastoris cells grown on Methanol/Glycerol medium, the fermentations were done in a chemostat.
2015-08-31 | GSE67690 | GEO
Project description:UnlabelledBackgroundRecent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification.ResultsFour pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH.ConclusionsP. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.
| S-EPMC3500269 | biostudies-literature
Project description:Proteins derived from the natural extracellular matrix like collagen or gelatin are common in clinical research, where they are prized for their biocompatibility and bioactivity. Cells are able to adhere, grow and remodel scaffolds based on these materials. Usually, collagen and gelatin are sourced from animal material, risking pathogenic transmission and inconsistent batch-to-batch product quality. A recombinant production in yeast circumvents these disadvantages by ensuring production with a reproducible quality in animal-component-free media. A gelatin mimetic protein, based on the alpha chain of human collagen I, was cloned in Pichia pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. A producing clone was selected and cultivated at the 30 L scale. The protein was secreted into the cultivation medium and the final yield was 3.4 g·L-1. Purification of the target was performed directly from the cell-free medium by size exclusion chromatography. The gelatin mimetic protein was tested in cell culture for biocompatibility and for promoting cell adhesion. It supported cell growth and its performance was indistinguishable from animal-derived gelatin. The gelatin-mimetic protein represents a swift strategy to produce recombinant and human-based extracellular matrix proteins for various biomedical applications.
| S-EPMC6787575 | biostudies-literature
Project description:Samples of PCH, LH73H and LH119H were cultivated according to the culture method of shake flask cultivation and then collected for protein extraction.
2024-08-10 | PXD048269 | Pride
Project description:ImportanceEngineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.
| S-EPMC10653784 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bonds via an oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 from Neurospora crassa (NcPMO-2) was heterologously expressed in Pichia pastoris to facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressing NcPMO-2 from a glycoengineered strain of P. pastoris and by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a large NcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.
| S-EPMC5297926 | biostudies-literature
Project description:Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris.
| S-EPMC5089815 | biostudies-literature
Project description:Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as Saccharomyces cerevisiae and Pichia pastoris have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for P. pastoris, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural AOX1 promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields.
| S-EPMC3964828 | biostudies-literature
Project description:Recombinant chimeric α-amylase (Ba-Gt-amy) has been produced extracellularly in Pichia pastoris under AOX promoter. Clones of P. pastoris with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in α-amylase titre as compared to a clone with a copy of the gene. The recombinant P. pastoris integrated eight copies of Ba-Gt-amy in the genome of P. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of Ba-Gt-amy was higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed in P. pastoris is a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60°C with T1/2 of 40 min at 70°C. The Kinetic parameters and end product analysis suggested that glycosylation has no effect on catalytic properties of Ba-Gt-amy. The enzyme saccharifies soluble as well as raw starches efficiently and generates maltose and maltooligosaccharides, thus, useful in baking and sugar syrup industries. The strategy for generating multi-copy clones is being reported for the first time, which could be useful in enhancing the production of other recombinant proteins.
| S-EPMC5360700 | biostudies-literature