Stéphane Delaunay

Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production

ABSTRACT Gene expression changes of glutamate-producing Corynebacterium glutamicum were identified in transcriptome comparisons by DNA microarray analysis. During glutamate production induced by a temperature shift, C. glutamicum strain 2262 showed significantly higher mRNA levels of the NCgl2816 and NCgl2817 genes than its non-glutamate-producing derivative 2262NP. Reverse transcription-PCR analysis showed that the two genes together constitute an operon. NCgl2816 putatively codes for a lactate permease, while NCgl2817 was demonstrated to encode quinone-dependent l-lactate dehydrogenase, which was named LldD. C. glutamicum LldD displayed Michaelis-Menten kinetics for the substrate l-lactate with a Km of about 0.51 mM. The specific activity of LldD was about 10-fold higher during growth on l-lactate or on an l-lactate-glucose mixture than during growth on glucose, d-lactate, or pyruvate, while the specific activity of quinone-dependent d-lactate dehydrogenase differed little with the carbon source. RNA levels of NCgl2816 and lldD were about 18-fold higher during growth on l-lactate than on pyruvate. Disruption of the NCgl2816-lldD operon resulted in loss of the ability to utilize l-lactate as the sole carbon source. Expression of lldD restored l-lactate utilization, indicating that the function of the permease gene NCgl2816 is dispensable, while LldD is essential, for growth of C. glutamicum on l-lactate.

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

ABSTRACT Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Kmr). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.

Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum

The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032DeltailvApJC1ilvBNCD. In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C. For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined. The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K(m)=8.3 mM). It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD). Moreover, the AHAS activity was inhibited by isoleucine. Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C. glutamicum.

Carnobacterium maltaromaticum: identification, isolation tools, ecology and technological aspects in dairy products.

Carnobacterium species constitute a genus of Lactic Acid Bacteria (LAB) present in different ecological niches. The aim of this article is to summarize the knowledge about Carnobacterium maltaromaticum species at different microbiological levels such as taxonomy, isolation and identification, ecology, technological aspects and safety in dairy products. Works published during the last decade concerning C. maltaromaticum have shown that this non-starter LAB (NSLAB) could present major interests in dairy product technology. Four reasons can be mentioned: i) it can grow in milk during the ripening period with no competition with starter LAB, ii) this species synthesizes different flavouring compounds e.g., 3-methylbutanal, iii) it can inhibit the growth of foodborne pathogens as Listeria monocytogenes due to its ability to produce bacteriocins, iv) it has never been reported to be involved in human diseases as no cases of human infection have been directly linked to the consumption of dairy products containing this species.

Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process

Kinetics and physiology of Corynebacterium glutamicum 2262 cultured for extended periods in continuous mode were investigated at 33, 39 and 41 degrees C. At 33 degrees C no glutamate production occurred whatever the dilution rates tested (ranging between 0.05 and 0.5 h(-1)). When the continuous culture was performed at 39 degrees C and D=0.05 h(-1), the glutamate was actively produced, while the activities of 2-oxoglutarate dehydrogenase complex (ODHC) and pyruvate dehydrogenase (PDH) were, respectively completely inhibited and 35% decreased. Simultaneously, the intracellular glutamate was 62% reduced compared to the level found at 33 degrees C and the co-metabolites lactate and trehalose were excreted. The decrease in PDH activity during the glutamate production was suggested to be responsible for the accumulation of by-products and for limiting the carbon flux required for glutamate synthesis. When the culture was prolonged for more than 100 h, a cell selection occurred, in favor of growth and to the detriment of glutamate production. In fact, these selected cells presented high levels of ODHC and PDH activities even at 39 degrees C, resulting in a complete inhibition of the glutamate production after 150 h of culture. A further temperature increase till 41 degrees C restored the glutamate production and abolished the ODHC activity of these selected cells.

Effect of surfactants, dispersion and temperature on solubility and biodegradation of phenanthrene in aqueous media

In the present study surfactant addition with the help of either a mechanical dispersion or a thermal treatment was applied in order to increase the solubility and the bioavailability of phenanthrene in aqueous media, and therefore to promote its biodegradation. Among four tested surfactants (Tween 80, Brij 30, sodium dodecyl sulphate and rhamnolipids), Brij 30 (0.5 gL(-1)) showed the best results allowing us to attain about 20 mgL(-1) of soluble phenanthrene. An additional thermal treatment at 60°C for 24h, 200 rpm permitted to increase the solubility of phenanthrene in the presence of Brij 30 (0.5 gL(-1)) to about 30 mgL(-1). Higher dispersions of phenanthrene particles as well as the reduction of their size were obtained using Ultra-Turrax and French press. The biodegradation of phenanthrene by Pseudomonas putida was then investigated. The reduction of size of phenanthrene particles by mechanical dispersion did not influence its biodegradation, suggesting that P. putida consumed only soluble phenanthrene. The addition of Brij 30 (0.5 gL(-1)) permitted to obtain more phenanthrene metabolized. The use of Brij 30 coupled with a transitory heating of phenanthrene-containing medium at 60°C led to an even more complete biodegradation. This might be a promising way to enhance biodegradation of PAHs.

OdhI dephosphorylation kinetics during different glutamate production processes involving Corynebacterium glutamicum

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase complex was shown to be controlled by the phosphorylation of a 15-kDa protein OdhI by different serine/threonine protein kinases. In this paper, the phosphorylation status and kinetics of OdhI dephosphorylation were assessed during glutamate producing processes triggered by either a biotin limitation or a temperature upshock from 33°C to 39°C. A dephosphorylation of OdhI in C. glutamicum 2262 was observed during the biotin-limited as well as the temperature-induced glutamate-producing process. Deletion of pknG in C. glutamicum 2262 did not affect the phosphorylation status of OdhI during growth and glutamate production phases triggered by a temperature upshock, though a 40% increase in the specific glutamate production rate was measured. These results suggest that, under the conditions analyzed, PknG is not the kinase responsible for the phosphorylation of OdhI in C. glutamicum 2262. The phosphorylation status of OdhI alone is, as expected, not the only parameter that determines the performance of a specific strain, as no clear relation between the specific glutamate production rate and OdhI phosphorylation level was demonstrated.

Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in Corynebacterium glutamicum

The glutamate-producing bacterium, Corynebacterium glutamicum is known to possess two anaplerotic enzymes: pyruvate carboxylase (Pc) and phosphoenolpyruvate carboxylase (PEPc). In vitro, this latter enzyme appeared to be inhibited by different glutamic acid salts, whereas ammonium-glutamate had no influence on Pc activity. To investigate the in vivo relevance of PEPc activity inhibition, the intracellular concentration of glutamate was determined throughout the glutamate-producing process. The intracellular concentration was then shown to be sufficient to induce a dramatic inhibition of PEPc activity during the process. As a consequence, intracellular accumulation of glutamate could be at least partially responsible for the weak participation of PEPc within the anaplerosis activity in amino-acid-producing strains of C. glutamicum.

Flexibility of the metabolism of Corynebacterium glutamicum 2262, a glutamic acid-producing bacterium, in response to temperature upshocks.

In order to test the temperature sensitivity of glutamate production metabolism, several temperature shifts, from 33 to 37, 38, 39, 40 or 41°C, were applied to the temperature-sensitive strain, Corynebacterium glutamicum 2262, cultivated in a 24-h fed-batch process. Whereas glucose was entirely dedicated to biomass synthesis when cells were grown at 33°C, applying temperature upshocks, whatever their range, triggered a redistribution of the carbon utilisation between glutamate, biomass and lactate production. Although increasing the culture temperature from 33 to 37, 38, 39 or 40°C resulted in final glutamate titers superior to 80 g/l, temperatures resulting in the best chanelling of the carbon flow towards glutamic acid synthesis were 39 and 40°C. Moreover, this study showed that the higher the temperature, the slower the growth rate and the higher the lactate accumulation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 333–337 DOI: 10.1038/sj/jim/7000251

Cell envelope fluidity modification for an effective glutamate excretion in Corynebacterium glutamicum 2262

Abstract1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to assess the cell envelope fluidity of Corynebacterium glutamicum 2262 during a temperature-triggered glutamate producing process. Because the fluorescence lifetime of TMA-DPH was shown to be constant all over the process, fluorescence anisotropy can be considered as a good index of cell envelope fluidity. When the temperature of the fed-batch culture was increased from 33 to 39°C to induce glutamate excretion, the fluorescence anisotropy values decreased from 0.212 ± 0.002 to 0.186 ± 0.002 (corresponding to an increase in the cell fluidity), while the specific glutamate production rate reached its maximal value. The increase in fluidity of the C. glutamicum cell envelope was not due to a physical effect related to the temperature elevation, but rather to an alteration of the composition of the cell envelope. Using a mutant devoid of corynomycolates, significant differences in fluorescence anisotropy values were obtained compared to the wild-type strain, suggesting that TMA-DPH is mainly anchored into the corynomycomembrane. Differences in fluorescence anisotropy were also observed when the bacteria were cultivated at 33, 36, 38, and 39°C in batch cultures, and a linear relationship was obtained between the maximum specific glutamate production rate and the measured fluidity. When using the glutamate non-producing variant of C. glutamicum 2262, the fluorescence anisotropy remained constant at 0.207 ± 0.003 whatever the applied temperature shift. This suggests that the fluidity of the Corynebacteria mycomembrane plays an important role in glutamate excretion during the temperature-triggered process.