PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 9680637

High cell-density fermentation.

J Stratton·V Chiruvolu·M Meagher
View original source ↗Export metadata ↓

Abstract

The purpose of this chapter is to educate the reader about the basic equipment and strategies used in fermentations of P. pastoris in both bench-top and pilot-scale operations. A key element in expression of foreign proteins in this yeast is the need for sufficient aeration, which is achieved by proper mixing of the media and by blending gases to control dissolved oxygen content. Automatic pH control is essential for growth and expression in P. pastoris. Finally, fed-batch fermentations require the use of peristaltic pumps and tubing capable of low rates of delivery for the feeding of nutrients and base. Teflon tubing and peristaltic pump adapters are recommended for fed-batch operations. The information in this chapter should enable a reader with little or no experience to perform a high-cell density fermentation of a P. pastoris expression strain. Although most procedures described here are specifically for the BioFlo III (NBS), it should be possible to achieve high expression levels with almost any good-quality fermentor, modified to accommodate this organism.

Explore related subjects

Alcohol OxidoreductasesCloning, MolecularCulture MediaFermentationGene DeletionIndicators and ReagentsMicrobiological TechniquesPichiaRecombinant ProteinsTrace ElementsJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

J Stratton, V Chiruvolu, M Meagher. 1998. High cell-density fermentation.. https://doi.org/10.1385/0-89603-421-6%3A107

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2001

Association of COOH-terminal-binding protein (CtBP) and MEF2-interacting transcription repressor (MITR) contributes to transcriptional repression of the MEF2 transcription factor.

The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. We show that the amino-terminal regions of MITR and class II HDACs interact with the transcriptional corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the ability of MITR to inhibit MEF2-dependent transcription. The residual repressive activity of MITR mutants that fail to bind CtBP can be attributed to association with other HDAC family members. These findings reveal CtBP-dependent and -independent mechanisms for transcriptional repression by MITR and show that MITR represses MEF2 activity through recruitment of multicomponent corepressor complexes that include CtBP and HDACs.

Alcohol Oxidoreductases↗
PubMed2001

Induction of ppGpp synthesis in Streptomyces coelicolor A3(2) grown under conditions of nutritional sufficiency elicits actII-ORF4 transcription and actinorhodin biosynthesis.

Production of ppGpp in Streptomyces coelicolor A3(2) was achieved independently of amino acid limitation by placing N-terminal segments of the ppGpp synthetase gene, relA, under the control of a thiostrepton-inducible promoter (tipAp). S1 nuclease protection experiments indicated that induced ppGpp concentrations of 6-12 pmol mg(-1) dry weight in late-exponential phase cultures caused activation of transcription of actII-ORF4, the pathway-specific activator gene for actinorhodin production. This level of ppGpp had no effect on growth rate, implying a causal role for ppGpp in activating actII-ORF4 transcription. No effect was observed on the transcription of the corresponding and homologous activator gene for undecylprodigiosin production, redD, reflecting a requirement for additional regulatory factors for activation of its transcription. This work provides the most compelling evidence yet for the activation of an antibiotic biosynthetic pathway by the stringent factor ppGpp.

Alcohol Oxidoreductases↗
PubMed2001

DNA supercoiling-dependent transcriptional coupling between the divergently transcribed promoters of the ilvYC operon of Escherichia coli is proportional to promoter strengths and transcript lengths.

The twin-domain model of Liu and Wang suggested that high levels of DNA supercoiling generated in the region between closely spaced divergently transcribed promoters could serve to couple the activities of these promoters transcriptionally. In this report, we use topoisomer sets of defined superhelical densities as DNA templates in a purified in vitro transcription system to demonstrate transcriptional coupling between the divergently transcribed ilvY and ilvC promoters of the ilvYC operon of Escherichia coli. Current evidence for this type of DNA supercoiling-dependent transcriptional coupling, based largely on the in vivo activities of promoters contained in engineered DNA constructs, suggests that the transcription complex must be physically hindered to generate DNA supercoils and to prevent their diffusion throughout the DNA duplex. However, the in vitro results presented here demonstrate that (i) transcriptional coupling is observed between the divergent promoters of the ilvYC operon in the absence of transcript anchoring; (ii) the magnitude of the negative DNA supercoiling generated in the divergent promoter region is proportional to the sum of the global and transcription-induced superhelicity; and (iii) the magnitude of this transcription-induced superhelicity is proportional to promoter strengths and transcript lengths.

Alcohol Oxidoreductases↗