Utilization of chymotrypsin as a sole carbon and (or) nitrogen source by Escherichia coli

1992 ◽  
Vol 38 (4) ◽  
pp. 290-295 ◽  
Author(s):  
Arthur S. Brecher ◽  
Timothy A. Moehlman ◽  
William D. Hann
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Nitrogen Source ◽  
Degradation Products ◽  
Defined Medium ◽  
Host Protein ◽  
Mammalian Host ◽  
Sole Nitrogen Source ◽  
E Coli ◽  
Nitrogen Nutrients

α-Chymotrypsin serves as a sole carbon source, sole nitrogen source, and as sole carbon plus nitrogen source for wild-type Escherichia coli in a totally defined medium. Hence, a mammalian host for E. coli may supply the necessary carbon and nitrogen nutrients for the microorganism. Growth is most rapid when chymotrypsin is a sole nitrogen source,and least rapid with chymotrypsin as a carbon source. The approximate doubling times for E. coli utilizing chymotrypsin as a nitrogen source, carbon plus nitrogen source, and carbon source are 1.6, 4.6, and 11.3 h, respectively. The activity of the residual enzyme in the culture supernates falls off asymptotically over the course of time, as followed by cleavage of glutaryl-L-phenylalanine-p-nitroanilide. Chymotrypsin hydrolyzes succinyl-L-ala-L-ala-L-ala-p-nitroanilide, the elastase substrate, to some extent. Peptidases do not appear to be secreted that hydrolyze such model substrates as benzoyl-DL-arginine-p-nitroanilide, the tryptic and cathepsin B substrate, L-leucine-p-nitroanilide, the leucine aminopeptidase substrate, or L-lysine-p-nitroanilide, the aminopeptidase B substrate. Growth of E. coli is generally directly related to the loss of chymotryptic activity in the medium. Hence, autolysis of chymotrypsin, i.e., self-degradation, is an important factor for the availability of degradation products of the enzyme to the bacterium for growth purposes. Accordingly, the degradation of a host protein by autolysis presents an opportunity for E. coli to survive during periods of host nutritional crisis by utilization of the degradation peptides that are produced during autolysis. Key words: chymotrypsin, Escherichia coli, growth, nutrition, peptide source.

scholarly journals Identification, Source, and Metabolism of N-Ethylglutamate in Escherichia coli

2010 ◽  
Vol 192 (20) ◽  
pp. 5437-5440 ◽  
Author(s):  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Yeast Extract ◽  
Sole Carbon Source ◽  
Isotope Dilution Analysis ◽  
Sole Nitrogen Source ◽  
Glucose Medium ◽  
Detectable Amount ◽  
Deuterated Water ◽  
E Coli

ABSTRACT N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-2H4]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.

scholarly journals Monomethylamine as a Nitrogen Source for a Nonmethylotrophic Bacterium, Agrobacterium tumefaciens

2010 ◽  
Vol 76 (12) ◽  
pp. 4102-4104 ◽  
Author(s):  
Yin Chen ◽  
Kathryn L. McAleer ◽  
J. Colin Murrell
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Carbon Source ◽  
Nitrogen Source ◽  
Sole Nitrogen Source ◽  
Key Enzymes ◽  
Genes Encoding

ABSTRACT Monomethylamine can be used by nonmethylotrophs as a sole nitrogen source but not as a carbon source; however, little is known about the genes and enzymes involved. The γ-glutamylmethylamide/N-methylglutamate pathway for monomethylamine utilization by methylotrophs has recently been resolved. We have identified genes encoding key enzymes of this pathway in nonmethylotrophs (e.g., Agrobacterium tumefaciens) and demonstrated that this pathway is also involved in the utilization of monomethylamine as a nitrogen source by nonmethylotrophs.

scholarly journals Mutants affecting histidine utilization inAspergillus nidulans

1975 ◽  
Vol 25 (2) ◽  
pp. 119-135 ◽  
Author(s):  
Meryl Polkinghorne ◽  
M. J. Hynes
Keyword(s):  
Carbon Source ◽  
Nitrogen Source ◽  
Sole Carbon Source ◽  
Nitrogen Sources ◽  
Limiting Factor ◽  
Sole Nitrogen Source ◽  
Wild Type ◽  
Exogenous Substrate ◽  
Growth Properties ◽  
Type Strains

SUMMARYWild-type strains ofAspergillus nidulansgrow poorly onL-histidine as a sole nitrogen source. The synthesis of the enzyme histidase (EC. 4.3.1.3) appears to be a limiting factor in the growth of the wild type, as strains carrying the mutantareA102 allele have elevated histidase levels and grow strongly on histidine as a sole nitrogen source.L-Histidine is an extremely weak sole carbon source for all strains.Ammonium repression has an important role in the regulation of histidase synthesis and the relief of ammonium repression is dependent on the availability of a good carbon source. The level of histidase synthesis does not respond to the addition of exogenous substrate.Mutants carrying lesions in thesarA orsarB loci (suppressor ofareA102) have been isolated. The growth properties of these mutants on histidine as a sole nitrogen source correlate with the levels of histidase synthesized. Mutation at thesarA andsarB loci also reduces the utilization of a number of other nitrogen sources. The data suggest that these two genes may code for regulatory products involved in nitrogen catabolism. No histidase structural gene mutants were identified and possible explanations of this are discussed.

scholarly journals Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material

2020 ◽  
Vol 47 (12) ◽  
pp. 1117-1132
Author(s):  
Katharina Novak ◽  
Juliane Baar ◽  
Philipp Freitag ◽  
Stefan Pflügl
Keyword(s):  
Escherichia Coli ◽  
Cheese Whey ◽  
Strain Improvement ◽  
Defined Medium ◽  
Raw Material ◽  
Efficient Production ◽  
Substrate Uptake ◽  
Chemically Defined Medium ◽  
E Coli ◽  
Plasmid Library

AbstractThe aim of this study was to establish isobutanol production on chemically defined medium in Escherichia coli. By individually expressing each gene of the pathway, we constructed a plasmid library for isobutanol production. Strain screening on chemically defined medium showed successful production in the robust E. coli W strain, and expression vector IB 4 was selected as the most promising construct due to its high isobutanol yields and efficient substrate uptake. The investigation of different aeration strategies in combination with strain improvement and the implementation of a pulsed fed-batch were key for the development of an efficient production process. E. coli W ΔldhA ΔadhE Δpta ΔfrdA enabled aerobic isobutanol production at 38% of the theoretical maximum. Use of cheese whey as raw material resulted in longer process stability, which allowed production of 20 g l−1 isobutanol. Demonstrating isobutanol production on both chemically defined medium and a residual waste stream, this study provides valuable information for further development of industrially relevant isobutanol production processes.

scholarly journals Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

Metabolites ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 228
Author(s):  
R. Axayacatl Gonzalez-Garcia ◽  
Lars K. Nielsen ◽  
Esteban Marcellin
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Structural Diversity ◽  
Heterologous Production ◽  
E Coli ◽  
Anticancer Properties ◽  
Defined Media ◽  
Extender Unit

Polyketides are a remarkable class of natural products with diverse functional and structural diversity. The class includes many medicinally important molecules with antiviral, antimicrobial, antifungal and anticancer properties. Native bacterial, fungal and plant hosts are often difficult to cultivate and coax into producing the desired product. As a result, Escherichia coli has been used for the heterologous production of polyketides, with the production of 6-deoxyerythronolide B (6-dEB) being the first example. Current strategies for production in E. coli require feeding of exogenous propionate as a source for the precursors propionyl-CoA and S-methylmalonyl-CoA. Here, we show that heterologous polyketide production is possible from glucose as the sole carbon source. The heterologous expression of eight genes from the Wood-Werkman cycle found in Propionibacteria, in combination with expression of the 6-dEB synthases DEBS1, DEBS2 and DEBS3 resulted in 6-dEB formation from glucose as the sole carbon source. Our results show that the Wood-Werkman cycle provides the required propionyl-CoA and the extender unit S-methylmalonyl-CoA to produce up to 0.81 mg/L of 6-dEB in a chemically defined media.

Carbon-source-dependent nitrogen regulation in Escherichia coli is mediated through glutamine-dependent GlnB signalling

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2163-2172 ◽  
Author(s):  
Mani Maheswaran ◽  
Karl Forchhammer
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Carbon Source ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Carbon Sources ◽  
Nitrogen Status ◽  
Low Concentrations ◽  
Signal Transduction Proteins

The PII signal transduction proteins GlnB and GlnK are uridylylated/deuridylylated in response to the intracellular glutamine level, the primary signal of the cellular nitrogen status. Furthermore, GlnB was shown to be allosterically regulated by 2-oxoglutarate, and thus GlnB was suggested to integrate signals of the cellular carbon and nitrogen status. Receptors of GlnB signal transduction in Escherichia coli are the NtrB/NtrC two-component system and GlnE, an enzyme which adenylylates/deadenylylates glutamine synthetase. In this study, the authors investigated the effect of different carbon sources on the expression of the NtrC-dependent genes glnA and glnK and on the uridylylation status of GlnB and GlnK. With glutamine as nitrogen source, high levels of glnA and glnK expression were obtained when glucose was used as carbon source, but expression was strongly decreased when the cells were grown with poor carbon sources or when cAMP was present. This response correlated with the uridylylation status of GlnB, suggesting that the carbon/cAMP effect was mediated through GlnB uridylylation, a conclusion that was confirmed by mutants of the PII signalling pathway. When glutamine was replaced by low concentrations of ammonium as nitrogen source, neither glnAglnK expression nor GlnB uridylylation responded to the carbon source or to cAMP. Furthermore, glutamine synthetase could be rapidly adenylylated in vivo by the external addition of glutamine; however, this occurred only when cells were grown in the presence of cAMP, not in its absence. Together, these results suggest that poor carbon sources, through cAMP signalling, favour glutamine uptake. The cellular glutamine signal is then transduced by uridylyltransferase and GlnB to modulate NtrC-dependent gene expression.

scholarly journals Comparison of methods for the identification and sub-typing of O157 and non-O157 Escherichia coli serotypes and their integration into a polyphasic taxonomy approach

2016 ◽  
Vol 55 (2) ◽  
pp. 81-90 ◽  
Author(s):  
M.A. Prieto-Calvo ◽  
M.K. Omer ◽  
O. Alvseike ◽  
M. López ◽  
A. Alvarez-Ordóñez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Carbon Sources ◽  
23S Rrna ◽  
Local Alignment ◽  
Taxonomic Structure ◽  
Sequencing Data ◽  
E Coli ◽  
Additional Information ◽  
Ft Ir

AbstractPhenotypic, chemotaxonomic and genotypic data from 12 strains ofEscherichia coli werecollected, including carbon source utilisation profiles, ribotypes, sequencing data of the 16S–23S rRNA internal transcribed region (ITS) and Fourier transform-infrared (FT-IR) spectroscopic profiles. The objectives were to compare several identification systems forE. coliand to develop and test a polyphasic taxonomic approach using the four methodologies combined for the sub-typing of O157 and non-O157E. coli. The nucleotide sequences of the 16S–23S rRNA ITS regions were amplified by polymerase chain reaction (PCR), sequenced and compared with reference data available at the GenBank database using the Basic Local Alignment Search Tool (BLAST) . Additional information comprising the utilisation of carbon sources, riboprint profiles and FT-IR spectra was also collected. The capacity of the methods for the identification and typing ofE. colito species and subspecies levels was evaluated. Data were transformed and integrated to present polyphasic hierarchical clusters and relationships. The study reports the use of an integrated scheme comprising phenotypic, chemotaxonomic and genotypic information (carbon source profile, sequencing of the 16S–23S rRNA ITS, ribotyping and FT-IR spectroscopy) for a more precise characterisation and identification ofE. coli. The results showed that identification ofE. colistrains by each individual method was limited mainly by the extension and quality of reference databases. On the contrary, the polyphasic approach, whereby heterogeneous taxonomic data were combined and weighted, improved the identification results, gave more consistency to the final clustering and provided additional information on the taxonomic structure and phenotypic behaviour of strains, as shown by the close clustering of strains with similar stress resistance patterns.

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flasks: Part 1

2016 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Yeast Extract ◽  
High Cell Density ◽  
Defined Medium ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Shake Flasks

Sufficient quantities of cells of consistent characteristics are needed for studying biological processes (at the population level) in many areas of applied microbiology. However, generating the requisite biomass by cell culture is usually the rate-limiting step of a project given the relatively low biomass yield of many commercial culture media in shake flasks. This work reports the formulation of a semi-defined medium that enabled aerobic high cell density cultivation of Escherichia coli DH5α (ATCC 53868) in shake flasks. The formulated medium (FM) comprises: a buffer system (K2HPO4: 12.54 g/L and KH2PO4: 2.31 g/L); vitamins and trace elements (yeast extract: 12.0 g/L); salts (NaCl: 5.0 g/L and MgSO4: 0.24 g/L); and carbon and nitrogen sources (D-Glucose: 6.0 g/L and NH4Cl: 1.5 g/L). Notable characteristics of this medium are: high buffer capacity (89 mM phosphate), 1:1 molar ratio between D-Glucose and NH4Cl, and yeast extract providing trace elements and a secondary source of carbon and nitrogen. Preliminary data revealed that an OD600nm of 9 was attained after 24 hours of cultivation at 37 oC, with glucose and NH4Cl as the main nutrients. At 48 hours, the OD600nm reached a maximum value of 11 with yeast extract providing the necessary nutrients for cell growth and biomass formation. The broth’s pH varied between 5.5 and 7.8 during cultivation. For comparison, the maximum OD600nm of E. coli grown in three commonly used complex media: Nutrient Broth, LB Lennox, and Tryptic Soy Broth (TSB) were 1.4, 3.2 and 9.2, respectively, under identical culture conditions. Finally, FM maintained the viability of a larger population of cells for three days - compared to a population collapse observed in TSB after one day. Collectively, the present findings suggested that the formulated medium might find use as a high cell density aerobic growth medium for E. coli in shake flasks. Part 2 of this work describes improvements in medium performance - specifically, higher cell yield as well as a shorter diauxic lag phase and total culture period – achieved through a small reduction in D-Glucose and NH4Cl concentrations in the medium composition. An abstract preprint of Part 2 is available at https://peerj.com/preprints/117/

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