Pichia pastoris (Komagataella sp.) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER). To restore their proper folding, the cell triggers the unfolded protein response (UPR); however, if the proteins cannot be repaired, they are degraded, which impairs process productivity. Moreover, a non-producing/non-secreting subpopulation of cells might occur, which also decreases overall productivity. Therefore, an in depth understanding of intracellular protein fluxes and population heterogeneity is needed to improve productivity. Under industrially relevant cultivation conditions in bioreactors, we cultured P. pastoris strains producing three different recombinant proteins: penicillin G acylase from Escherichia coli (EcPGA), lipase B from Candida antarctica (CaLB) and xylanase A from Thermomyces lanuginosus (TlXynA). Extracellular and intracellular product concentrations were determined, along with flow cytometry-based single-cell measurements of cell viability and the up-regulation of UPR. The cell population was distributed into four clusters, two of which were viable cells with no UPR up-regulation, differing in cell size and complexity. The other two clusters were cells with impaired viability, and cells with up-regulated UPR. Over the time course of cultivation, the distribution of the population into these four clusters changed. After 30 h of production, 60% of the cells producing EcPGA, which accumulated in the cells (50-70% of the product), had up-regulated UPR, but only 13% of the cells had impaired viability. A higher proportion of cells with decreased viability was observed in strains producing CaLB (20%) and TlXynA (27%). The proportion of cells with up-regulated UPR in CaLB-producing (35%) and TlXynA-producing (30%) strains was lower in comparison to the EcPGA-producing strain, and a smaller proportion of CaLB and TlXynA (<10%) accumulated in the cells. These data provide an insight into the development of heterogeneity in a recombinant P. pastoris population during a biotechnological process. A deeper understanding of the relationship between protein production/secretion and the regulation of the UPR might be utilized in bioprocess control and optimization with respect to secretion and population heterogeneity.
- Publikační typ
- časopisecké články MeSH
Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts. Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.
- MeSH
- bodová mutace MeSH
- chromozomy hub MeSH
- CRISPR-Cas systémy * MeSH
- editace genu metody MeSH
- geneticky modifikované mikroorganismy MeSH
- guide RNA, Kinetoplastida MeSH
- klonování DNA MeSH
- kvasinky genetika MeSH
- metabolické inženýrství MeSH
- Pichia genetika MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae genetika MeSH
- technologie gene drive MeSH
- Yarrowia genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (q p) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l(-1)) and at a growth rate close to μ max (0.15 h(-1)). Between 0.017 and 0.115 h(-1) in glycerol-limited fedbatch cultures, basal levels of q p > 0.4 mg g(-1) h(-1) CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h(-1), an elevated q p occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the q p(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h(-1) and vice versa. Changes in q p were related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.
- MeSH
- fermentace MeSH
- fungální proteiny genetika metabolismus MeSH
- glycerol metabolismus farmakologie MeSH
- kinetika MeSH
- lipasa genetika metabolismus MeSH
- methanol analýza MeSH
- Pichia genetika růst a vývoj metabolismus MeSH
- promotorové oblasti (genetika) * MeSH
- rekombinantní proteiny metabolismus MeSH
- techniky vsádkové kultivace MeSH
- Publikační typ
- časopisecké články MeSH
Pure methanol, which is required as an inducer of the AOX1 promoter and a carbon/energy source in processes for recombinant protein production by Pichia pastoris, is impracticable and therefore generally undesirable. As an alternative, a procedure using double carbon substrate was examined (11.7g(carbon)l(-1), 60%/40% carbon from glucose/methanol). The effects on methanol metabolism, extracellular formation of porcine trypsinogen, biomass growth and cell viability were analyzed. In contrast to batch cultures, where the glucose and methanol were utilized sequentially, in carbon/energy-limited continuous cultures (operated between dilution rates 0.03 and 0.20h(-1)) the repressive effect of glucose on methanol utilization was eliminated up to 0.15h(-1) (ca. 130% of μ(max) with methanol). With the mixture, the yield of biomass (1.54±0.12) g(CDW)g(carbon)(-1) was found to be 1.4 times larger than the yield with methanol alone. Despite the current widespread view that glucose has a repressive effect on the AOX1 promoter, the product was synthesized over the entire range of dilution rates, with maximum productivities of (0.70±0.12)mgg(CDW)(-1) h(-1) at 0.07h(-1). Thus, glucose was shown to be a feasible partial substitute for methanol in recombinant protein production by P. pastoris Mut(+) strain while enhancing process productivity.
- MeSH
- biomasa MeSH
- bioreaktory MeSH
- glukosa metabolismus MeSH
- kinetika MeSH
- kultivační média MeSH
- methanol metabolismus MeSH
- Pichia genetika metabolismus MeSH
- prasata MeSH
- rekombinantní proteiny biosyntéza MeSH
- trypsinogen biosyntéza MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH