Corynebacterium glutamicum
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PROVIDER: PRJNA342810 | ENA |
REPOSITORIES: ENA
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Project description:Corynebacterium glutamicum shows yellow pigmentation due to biosynthesis of the C50 carotenoid decaprenoxanthin and its glycosides. This bacterium has been engineered for production of various non-native cyclic C40 and C50 carotenoids such as β-carotene, astaxanthin or sarcinaxanthin. In this study, the effect of modulating gene expression more broadly by overexpression of sigma factor genes on carotenoid production by C. glutamicum was characterized. Overexpression of the primary sigma factor gene sigA improved lycopene production by recombinant C. glutamicum up to eight fold. In C. glutamicum wild type, overexpression of sigA led to two fold increased accumulation of the native carotenoid decaprenoxanthin in the stationary growth phase. Under these conditions, genes related to thiamine synthesis and aromatic compound degradation showed increased RNA levels and addition of thiamine and the aromatic iron chelator protocatechuic acid to the culture medium enhanced carotenoid production when sigA was overexpressed. Deletion of the gene for the alternative sigma factor SigB, which is expected to replace SigA in RNA polymerase holoenzymes during transition to the stationary growth phase, also increased carotenoid production. The strategy of sigA overexpression could be successfully transferred to production of the non-native carotenoids β-carotene and bisanhydrobacterioruberin. Production of the latter is the first demonstration that C. glutamicum may accumulate a non-native linear C50 carotenoid instead of the native cyclic C50 carotenoid decaprenoxanthin.
2016-09-14 | GSE86866 | GEO
Project description:The extracytoplasmic function sigma factor SigH is responsible for heat and oxidative stress response in a biotechnologically -important bacterium Corynebacterium glutamicum. Due to the hierarchical nature of the regulatory network, it has not been possible to fully determine the SigH regulon by previous transcriptome analyses. Here, we determined the direct genome-wide targets of SigH were determined by ChIP-chip analysis using a deletion mutant of rshA, encoding an anti-sigma factor of SigH. Seventy-five SigH-dependent promoters, including 39 new ones, were identified. Firstly, SigH-dependent heat induction was confirmed for several of the new targets, including two genes (ilvD and lipA) encoding Fe-S cluster containing enzymes. Internal SigH-dependent promoters were found in operon-like gene clusters involved in the pentose phosphate pathway, and riboflavin biosynthesis, and Zn uptake. Accordingly, deletion of rshA resulted in hyperproduction of riboflavin probably due to the coordinated upregulation of these genes. A SigH-dependent promoter was also found upstream of znuC1 in the znuA1C1B1 operon encoding an ABC-type transporter for Zn uptake. Overexpression of znuC1B1 by deletion of rshA and by an IPTG-inducible promoter and similarly affected expression of Zn-responsive genes, indicating that SigH-dependent upregulation of part of the operon potentiates Zn uptake. probably through Zn overload, indicating new physiological roles of SigH. Furthermore, reporter assays demonstrated that rshA in the sigH-rshA operon was heat-inducible under the control of an internal SigH-dependent promoters, identified upstream of rshA within the sigH-rshA operon, while the sigH promoter was independent of SigH, suggesting negative autoregulation of SigH activity. Finally, a SigH-dependent promoter identified and upstream of sigA encoding the primary sigma factor , wereas highly heat-inducible, but much weaker than the known SigA-dependent one.. The promoter upstream of sigH was independent of SigH but showed weak heat-shock response. The known SigA-dependent sigA promoter was much stronger than the SigH-dependent one. Taken together with the fact that SigH also regulates sigB encoding the primary-like sigma factor, Tthese findings uncovered a complicated regulatory network of the sigma factors.
2014-11-25 | GSE52040 | GEO
Project description:Background: Methanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes. Results: The methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by genetic adaption in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1. Conclusion: The methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity. The gene expression was analyzed in the methanol tolerant strain Tol1 in comparison to the C. glutamicumWT. Direct comparison in LB complex medium and analysis of expression response to methanol addition in mCGXII minimal medium with 100 mM glucose.
2015-08-01 | E-GEOD-71590 | biostudies-arrayexpress
Project description:Background: Methanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes. Results: The methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by genetic adaption in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1. Conclusion: The methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity.
2015-08-01 | GSE71590 | GEO
Project description:Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing all annotated sigma factor genes on C. glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. HPLC analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA Microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes of the pentose phosphate pathway and for enzymes dependent on FMN, FAD or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production. Endogenous sigma factor gene, sigH, was overexpressed in C. glutamicum ATCC13032 from IPTG inducible vector, pEKEx3. Two different concentration of IPTG (10 μM and 15 μM) was used for induction of SigH expression.
2015-06-20 | E-GEOD-70017 | biostudies-arrayexpress
Project description:Corynebacterium glutamicum shows a great potential for the production of gamma-aminobutyric acid (GABA) from glucose fermentation via putrescine. GABA, a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods and the biodegradable plastic polyamide 4. Here, the effect of GABA in the growth of C. glutamicum was evaluated. It was estimated that the presence 1.1 M of GABA in the medium reduces the maximum growth rate of C. glutamicum to half. It was also shown that the presence of GABA in the medium negatively affects the growth of C. glutamicum in ethanol as sole carbon source. Furthermore, a new route for the production of GABA in C. glutamicum was established. GABA production from glucose fermentation via putrescine was achieved by plasmid-based overexpression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) in a putrescine production strain. The resultant strain can produce 5.3 ± 0.1 g L-1 of GABA. GABA production was improved by avoiding the formation of N-acetylputrescine and by reducing the amount of nitrogen in CGXII medium. Deletion of the genes responsible for GABA catabolism and GABA re-uptake led to an increase in the GABA production of 21% achieving a titer 8.0 ± 0.3 g L-1 and an increase in the volumetric productivity of 41% reaching a productivity of 0.31 g L-1 h-1, the highest volumetric productivity achieved so far for GABA production in C. glutamicum from glucose fermentation in flasks fermentations. The results obtained hitherto are very promising and competitive compared to the traditional pathway for the production of GABA.
2015-10-22 | GSE74258 | GEO
Project description:The dicarboxylic acid glutarate is gaining attention in the chemical and pharmaceutical industry as promising building-block. Synthesis of glutarate via microbial fermentation is a desirable aim which will allow the production of biopolymers avoiding fossil raw materials. Here, by rational metabolic engineering of the biofactory microorganism Corynebacterium glutamicum the fermentative production of glutarate from glucose was established. Modifications focused on increase glucose consumption and reduce by-products formation together with the heterologous overexpression of the L-lysine decarboxylase, putrescine transaminase and putrescine dehydrogenase genes from E. coli in the L-lysine producer GRLys1 allowed production the glutarate precursor 5-aminovalerate. Additional heterologous overexpression of 5-aminovalerate amino transferase and glutarate-semialdehyde dehydrogenase genes from C. glutamicum and three Pseudomonas species enabled glutarate synthesis from glucose. By coupling glutarate production with the glutamate synthesis of C. glutamicum glutarate titer improved 10%. The final strain was tested in a glucose-based fed-batch fermentation
2018-11-20 | GSE117175 | GEO
Project description:Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our initial plasmid design iterations, we demonstrated an increased flux through the mevalonate-based bypass pathway to increase isoprenol production to the highest reported titers to date at nearly 1.5 g/L. These designs also evaluated the effects of backbone, promoter, and GPP synthase homolog source on product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 486.3 mg/L (± 54.6 mg/L) of geranoids, 557.3 mg/L of 1,8-cineole (± 12.3 mg/L), and 209.7 mg/L of linalool (±23.9 mg/L). Furthermore, we determined that C. glutamicum oxidizes geraniol through an aldehyde intermediate before asymmetrically reducing geraniol to citronellol.
2024-02-28 | PXD044193 | Pride
Project description:Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing all annotated sigma factor genes on C. glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. HPLC analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA Microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes of the pentose phosphate pathway and for enzymes dependent on FMN, FAD or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production.
2015-06-20 | GSE70017 | GEO
Project description:Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example
| PRJNA287495 | ENA