Biomedical subjects

Louise Johansson

Publications and source records attributed to Louise Johansson.

4 recordsLinked to original sources

PubMed2006

Transcriptome analysis of a shikimic acid producing strain of Escherichia coli W3110 grown under carbon- and phosphate-limited conditions.

Shikimic acid, which is produced in the aromatic amino acid pathway in plants and microorganisms, is an industrially interesting chiral starting material for the synthesis of many chemical substances, e.g. the influenza medicine Tamiflu. When produced by genetically modified Escherichia coli it has previously been found that carbon-rich conditions (e.g. phosphate-limitation) favors production of shikimic acid over shikimate pathway by-products, whereas the situation is the opposite at carbon-(glucose-) limited conditions. In the present study, gene expression patterns of the shikimate producing strain W3110.shik1 (W3110 with aroL deletion and plasmid-overexpressed aroF) and the wild type strain W3110 grown under carbon- and phosphate-limited (carbon-rich) chemostat conditions (D=0.23h(-1)) were analyzed. The study suggests that the by-product formation under carbon-limitation is explained by a set of upregulated genes coupled to the shikimate pathway. The genes, ydiB, aroD and ydiN, were strongly induced only in carbon-limited W3110.shik1. Compared to W3110 the lg(2)-fold changes were: 6.25 (ydiB); 3.93 (aroD) and 8.18 (ydiN). In addition, the transcriptome analysis revealed a large change in the gene expression when comparing phosphate- to carbon-limitation, which to a large part could be explained by anabolic-catabolic uncoupling, which is present under phosphate-limitation but not under carbon-limitation. Interestingly, there was also a larger difference between the two strains under carbon-limitation than under phosphate-limitation. The reason for this difference is interpreted in terms of starvation for aromatic amino acids under carbon-limitation which is relieved under phosphate-limitation due to an upregulation of aroK and aroA.

Bioreactors↗

PubMed2005

Shikimic acid production by a modified strain of E. coli (W3110.shik1) under phosphate-limited and carbon-limited conditions.

Shikimic acid is one of several industrially interesting chiral starting materials formed in the aromatic amino acid pathway of plants and microorganisms. In this study, the physiology of a shikimic acid producing strain of Escherichia coli (derived from W3110) deleted in aroL (shikimic acid kinase II gene), was compared to that of a corresponding control strain (W3110) under carbon- and phosphate-limited conditions. For the shikimic acid producing strain (referred to as W3110.shik1), phosphate limitation resulted in a higher yield of shikimic acid (0.059 +/- 0.012 vs. 0.024 +/- 0.005 c-mol/c-mol) and a lower yield of by-products from the shikimate pathway, when compared to carbon-limited condition. The yield of the by-product 3-dehydroshikimic acid (DHS) decreased from 0.076 +/- 0.028 to 0.022 +/- 0.001 c-mol/c-mol. Several other by-products were only detected under carbon-limited conditions. The latter group included 3-dehydroquinic acid (0.021 +/- 0.021 c-mol/c-mol), quinic acid (0.012 +/- 0.005 c-mol/c-mol), and gallic acid (0.002 +/- 0.001 c-mol/c-mol). For both strains, more acetate was produced under phosphate than the carbon-limited case. Considerable cell lysis was found for both strains but was higher for W3110.shik1, and increased for both strains under phosphate limitation. The advantages of the latter condition in terms of an increased shikimic acid yield was thus counteracted by an increased cell lysis, which may make downstream processing more difficult.

Bioreactors↗

PubMedDate not supplied

MFA for overdetermined systems reviewed and compared with rna expression data to elucidate the difference in shikimate yield between carbon- and phosphate-limited continuous cultures of E. coli W3110.shik1.

The present contribution focuses on the mathematical techniques used to solve steady state metabolic models for the case of an overdetermined system. Even when parts of the system are underdetermined it is possible to solve the model partially and obtain statistically meaningful results. This is illustrated with data gathered from a set of E. coli W3110.shik1 phosphate- or carbon-limited continuous cultures. It is shown that the low yield in shikimate for C-limited cultures is not due to a lower flux going to the shikimate pathway but is caused by a high secretion of byproducts. Carbon-limited cultures could be better for shikimate production than carbon-abundant cultures provided the byproduct secretion is reduced. Finally, flux calculations are compared with RNA expression data.

Carbon↗

PubMedDate not supplied

A study of long-term effects on plasmid-containing Escherichia coli in carbon-limited chemostat using 2D-fluorescence spectrofluorimetry.

Strain stability of plasmid-containing recombinant organisms is clearly important for industrial applications. Stability is normally assessed by methods such as selective colony forming units or by simply measuring the recombinant product. These methods are typically performed off-line, are time-consuming, and do not give detailed information on the changes in the metabolism. In the current work, long-term stability of a plasmid-containing strain of Escherichia coli (W3110.shik1) capable of shikimic acid overproduction was studied by means of a 2D-fluorescence sensor (BioView) able to emit and detect light in ranges of 260-560 nm and 300-600 nm, respectively. Long-term carbon-limited chemostat experiments were made under both selective (tetracycline-containing medium) and nonselective conditions. It is shown that the fluorescence spectra provide information about metabolic changes at an earlier stage, thereby giving a noninvasive method for monitoring of strain stability. Further, the fluorescence measurements showed that (i) the metabolic changes in the strain W3110.shik1 with time were qualitatively different in selective and nonselective environment, (ii) plasmid recombination resulted primarily in increased biomass yield, and (iii) a change in metabolism probably involving FAD/FMN and pyridoxal-5-P occurred in all experiments. It was concluded that the strain was not stable in any growth condition for more than about 25 growth generations and even less if plasmid recombination took place.

Bacteriological Techniques↗