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1.  Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris 
Background
Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. glucose) limitation.
Results
DNA microarray analysis of P. pastoris wild type cells growing in glycerol-based batch and glucose-based fed batch was used for the identification of genes with both, strong repression on glycerol and high-level expression in the feed phase. Six novel glucose-limit inducible promoters were successfully applied to express the intracellular reporter eGFP. The highest expression levels together with strong repression in pre-culture were achieved with the novel promoters PG1 and PG6.
Human serum albumin (HSA) was used to characterize the promoters with an industrially relevant secreted protein. A PG1 clone with two gene copies reached about 230% of the biomass specific HSA titer in glucose-based fed batch fermentation compared to a PGAP clone with identical gene copy number, while PG6 only achieved 39%. Two clones each carrying eleven gene copies, expressing HSA under control of PG1 and PG6 respectively were generated by post-transformational vector amplification. They produced about 1.0 and 0.7 g L-1 HSA respectively in equal fed batch processes. The suitability in production processes was also verified with HyHEL antibody Fab fragment for PG1 and with porcine carboxypeptidase B for PG6. Moreover, the molecular function of the gene under the control of PG1 was determined to encode a high-affinity glucose transporter and named GTH1.
Conclusions
A set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.
doi:10.1186/1475-2859-12-5
PMCID: PMC3615954  PMID: 23347568
Pichia pastoris; Heterologous protein production; Glucose-limited fed batch cultivation; Inducible promoter; High-affinity glucose transporter
2.  Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State of Pichia pastoris during the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures▿ †  
Applied and Environmental Microbiology  2010;76(13):4486-4496.
Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut+ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.
doi:10.1128/AEM.02475-09
PMCID: PMC2897410  PMID: 20472737
3.  Detection and Characterization of Conjugative Degradative Plasmids in Xenobiotic-Degrading Sphingomonas Strains 
Journal of Bacteriology  2004;186(12):3862-3872.
A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.
doi:10.1128/JB.186.12.3862-3872.2004
PMCID: PMC419928  PMID: 15175300
4.  Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by Sphingomonas xenophaga BN6 
During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.
doi:10.1128/AEM.68.9.4341-4349.2002
PMCID: PMC124094  PMID: 12200285

Results 1-4 (4)