Auto-induction Screening Protocol for Ranking Clonal Libraries of Pichia pastoris MutS Strains

Wollborn, David; Müller, Rebecca Luise; Munkler, Lara Pauline; Horstmann, Rebekka; Germer, Andrea; Blank, Lars M.; Büchs, Jochen

doi:10.1007/s12257-022-0006-z

Cited by 5 publications

(4 citation statements)

References 51 publications

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“…The reference with 700 mg/L PO 4 3− is shown in black. The OTR increases exponentially over time, reaching a maximum of 24 mmol/L/h after 21 h. This marks the depletion of glucose in the medium (Wollborn et al, 2022). After the first OTR peak, two further peaks can be seen at 23 and 26 h, indicating the metabolization of some overflow metabolites (Anderlei & Büchs, 2001; Käppeli, 1986).…”

Section: Resultsmentioning

confidence: 99%

“…The concentrations, which show the first signs of limitation are 210 mg/L PO 4 3− and 200 mg/L NH 4 + , respectively. The use of glycerol eliminates overflow production by P. pastoris (Macauley‐Patrick et al, 2005; Wollborn et al, 2022). Similar to Figure 4a, in Figure 4c the effect of internal phosphate pools is obvious.…”

Section: Resultsmentioning

confidence: 99%

“…Fit parameters are given in Supporting Information S1: Table S3, line 1. Cultivation conditions: half concentrated Syn6-MES medium (100 mM MES), c Glucose = 5 g/L, OD 600 (0 h) = 0.2, 96 deep square well plate, V = 2.4 mL, V L = 600 μL, T = 30°C, 85% r. glucose in the medium (Wollborn et al, 2022). After the first OTR peak, two further peaks can be seen at 23 and 26 h, indicating the metabolization of some overflow metabolites (Anderlei & Büchs, 2001;Käppeli, 1986).…”

Section: Nutrient Limitation In Invitrogen Medium Due To Struvite Pre...mentioning

confidence: 99%

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Investigation into struvite precipitation: A commonly encountered problem during fermentations on chemically defined media

Steimann,

Wollborn,

Röck

et al. 2023

Biotech & Bioengineering

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Chemically defined mineral media are widely used in bioprocesses, as these show less batch to batch variation compared with complex media. Nonetheless, the recommended media formulations often lead to the formation of precipitants at elevated pH values. These precipitates are insoluble and reduce the availability of macronutrients to the cells, which can result in limiting growth rates and lower productivity. They can also damage equipment by clogging pipes, hoses, and spargers in stirred tank fermenters. In this study, the observed precipitate was analyzed via X‐ray fluorescence spectroscopy and identified as the magnesium ammonium phosphate salt struvite (MgNH4PO4 × 6H2O). The solubility of struvite crystals is known to be extremely low, causing the macronutrients magnesium, phosphate, and ammonium to be bound in the struvite crystals. Here, it was shown that struvite precipitates can be redissolved under common fermentation conditions. Furthermore, it was found that the struvite particle size distribution has a significant effect on the dissolution kinetics, which directly affects macronutrient availability. At a certain particle size, struvite crystals rapidly dissolved and provided unlimiting growth conditions. Therefore, struvite formation should be considered during media and bioprocess development, to ensure that the dissolution kinetics of struvite are faster than the growth kinetics.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Nutrient Limitation In Invitrogen Medium Due To Struvite Pre...mentioning

confidence: 99%

See 1 more Smart Citation

Investigation into struvite precipitation: A commonly encountered problem during fermentations on chemically defined media

Steimann,

Wollborn,

Röck

et al. 2023

Biotech & Bioengineering

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“…Notably, the target protein concentration shows a minimal increase prior to induction. A possible explanation is basal expression, already shown for recombinant protein expression under the P CAT promotor in K. phaffii under carbon limiting conditions prior to induction [ 23 ]. The P CAT promotor has even been suggested as a derepressed promotor enabling inducible, methanol-free processes [ 24 , 25 ].…”

Section: Resultsmentioning

confidence: 99%

Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production

Steimann,

Heite,

Germer

et al. 2024

Microb Cell Fact

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Background Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. Results This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. Conclusions This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.

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β-glucosidase production by recombinant Pichia pastoris strain Y1433 under optimal feed profiles of fed-batch cultivation

et al. 2022

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Auto-induction Screening Protocol for Ranking Clonal Libraries of Pichia pastoris MutS Strains

Cited by 5 publications

References 51 publications

Investigation into struvite precipitation: A commonly encountered problem during fermentations on chemically defined media

Investigation into struvite precipitation: A commonly encountered problem during fermentations on chemically defined media

Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production

β-glucosidase production by recombinant Pichia pastoris strain Y1433 under optimal feed profiles of fed-batch cultivation

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