Abstract:Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive infl… Show more
“…The reduced oxidative stress could also be related to an improved supply of NADPH. Increased supply of NADPH via the pentose phosphate pathway (PPP) was shown beneficial for higher protein production in Chinese Hamster Ovary (CHO) cells and Pichia pastoris 47 , 48 . Although no increased expression was seen for PPP associated genes, we found reduced biomass yields of the mutant strains.…”
The biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. We confirm our findings by inverse engineering and physiological characterization and show that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion.
“…The reduced oxidative stress could also be related to an improved supply of NADPH. Increased supply of NADPH via the pentose phosphate pathway (PPP) was shown beneficial for higher protein production in Chinese Hamster Ovary (CHO) cells and Pichia pastoris 47 , 48 . Although no increased expression was seen for PPP associated genes, we found reduced biomass yields of the mutant strains.…”
The biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. We confirm our findings by inverse engineering and physiological characterization and show that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion.
“…This is partly explainable by error propagation, as measurement errors on the respective isotopologue fraction are propagated to the resulting tandem mass isotopomer fractions, since the obtained mass spectral ratio of a fragment on MS2 level is applied to the respective isotopologue fraction.Fig. 2Isotopologue distribution and tandem mass isotopomer distribution of P. Pastoris cell extract, fed with either (a,b) 50: 50 = 12 C 1 methanol: 13 C 1 methanol or (c,d) U 13 C glucose mixed with natural glucose (20: 80) [12, 17]. Data obtained from the PRM combined with data-dependent fragmentation LC-MS approach, indicated by “LC-QTOFMS AutoMSMS”, is shown in grey, whereas bars in blue depict the obtained fractions from the already published GC-CI-(Q)TOFMS approach.…”
A novel analytical approach based on liquid chromatography coupled to quadrupole time of flight mass spectrometry, employing data-dependent triggering for analysis of isotopologue and tandem mass isotopomer fractions of metabolites of the primary carbon metabolism was developed. The implemented QTOFMS method employs automated MS/MS triggering of higher abundant, biologically relevant isotopologues for generating positional information of the respective metabolite. Using this advanced isotopologue selective fragmentation approach enables the generation of significant tandem mass isotopomer data within a short cycle time without compromising sensitivity. Due to a lack of suitable reference material certified for isotopologue ratios, a Pichia pastoris cell extract with a defined 13C distribution as well as a cell extract from a 13C-based metabolic flux experiment were employed for proof of concept. Moreover, a method inter-comparison with an already established GC-CI-(Q)TOFMS approach was conducted. Both methods showed good agreement on isotopologue and tandem mass isotopomer distributions for the two different cell extracts.
Graphical abstractSchematic overview of data-dependent isotopologue fragmentation for acquisition of isotopologue and tandem mass isotopomer fractions
“…It will be also useful to study if co‐overexpression of the 6‐gluconolactonase ( SOL3 ), the second enzyme in the PPP would improve the secretion of RABV‐G, as demonstrated for intracellular expression by Nocon et al. ().…”
Section: Discussionmentioning
confidence: 99%
“…Our study suggests that overexpression of ZWF1 would also improve the expression level of heterologous proteins targeted to secretion in this yeast. It will be also useful to study if co-overexpression of the 6-gluconolactonase (SOL3), the second enzyme in the PPP would improve the secretion of RABV-G, as demonstrated for intracellular expression by Nocon et al (2016).…”
Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV‐G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (
AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV‐G in P. pastoris.
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