Pharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogen-the inactive precursor of trypsin-is advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. The experiments were performed with two different pH values (5.0 and 5.9) and two constant specific growth rates (0.02 and 0.04 1/h), maintained using exponential addition of methanol. All the productivity data presented rely on an active determination of trypsin obtained by proteolysis of the trypsinogen produced. The pH of the medium did not affect cell growth, but significantly influenced specific production of trypsinogen: A 1.7-fold higher concentration of trypsinogen was achieved at pH 5.9 (64 mg/L at 0.02 1/h) compared to pH 5.0. EGFP was primarily used to facilitate detection of intracellular protein over the biosynthetic time course. Using flow cytometry with fluorescence detection, cell disruption was avoided, and protein extraction and purification prior to analysis were unnecessary. However, Western blot and SDS-PAGE showed that cleavage of EGFP-trypsinogen fusion protein occurred, probably caused by Pichia-endogenous proteases. The fluorescence analysis did therefore not accurately represent the actual trypsinogen concentration. However, we gained new experimentally-relevant insights, which can be used to avoid misinterpretation of tracking and quantifying as well as online-monitoring of proteins with the frequently used fluorescent tags.

• MeSH
• exprese genu MeSH
• koncentrace vodíkových iontů MeSH
• kultivační média chemie   metabolismus   MeSH
• Pichia genetika   růst a vývoj   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• prasata MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• trypsinogen genetika   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• enhanced green fluorescent protein MeSH Prohlížeč
• kultivační média MeSH
• rekombinantní fúzní proteiny MeSH
• trypsinogen MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: Biobutanol production by clostridia via the acetone-butanol-ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol. RESULTS: Dual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained-viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation. CONCLUSION: We have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.

• Klíčová slova
• ABE fermentation, Butanol, Clostridium, Cytometry, Fluorescence staining, Sporulation, Stress, Viability,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Candida antarctica lipase B, Methanol-free, Pichia pastoris, Product formation kinetics, Secretion, Specific productivity,
• 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
• Názvy látek
• fungální proteiny MeSH
• glycerol MeSH
• lipasa MeSH
• lipase B, Candida antarctica MeSH Prohlížeč
• methanol MeSH
• rekombinantní proteiny MeSH