Recombinant Protein Expression System in Corynebacterium glutamicum and Its Application

Lee, Min Ju; Kim, Pil

doi:10.3389/fmicb.2018.02523

Cited by 39 publications

(21 citation statements)

References 104 publications

(65 reference statements)

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“…The C. glutamicum strain WT ATCC 13032 is an important microbial host for industrial amino acid production 31–33 , such as L-lysine 34 and L-glutamate 32 . In the first set of experiments, we investigated the microbial growth under oscillating (rectangular) feast and famine environmental conditions with different uniform oscillation times between 10 seconds and 1 hour (Fig.…”

Section: Resultsmentioning

confidence: 99%

dMSCC: A microfluidic platform for microbial single-cell cultivation under dynamic environmental medium conditions

Taeuber

Golze

et al. 2020

Preprint

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In nature and in technical systems, microbial cells are often exposed to rapidly fluctuating environmental conditions. These conditions can vary in quality, e.g., existence of a starvation zone, and quantity, e.g., average residence time in this zone. For strain development and process design, cellular response to such fluctuations needs to be systematically analysed. However, the existing methods for physically emulating rapidly changing environmental conditions are limited in spatio-temporal resolution. Hence, we present a novel microfluidic system for cultivation of single cells and small cell clusters under dynamic environmental conditions (dynamic microfluidic single-cell cultivation (dMSCC)). This system enables to control nutrient availability and composition between two media with second to minute resolution. We validate our technology using the industrially relevant model organism Corynebacterium glutamicum. The organism was exposed to different oscillation frequencies between nutrient excess (feasts) and scarcity (famine). Resulting changes in cellular physiology, such as the colony growth rate and cell morphology were analysed and revealed significant differences with growth rate and cell length between the different conditions. dMSCC also allows to apply defined but randomly changing nutrient conditions, which is important for reproducing more complex conditions from natural habitats and large-scale bioreactors. The presented system lays the foundation for the cultivation of cells under complex changing environmental conditions.

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Section: Resultsmentioning

confidence: 99%

dMSCC: A microfluidic platform for microbial single-cell cultivation under dynamic environmental medium conditions

Taeuber

Golze

et al. 2020

Preprint

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“…Because protein is the major macromolecule in cells, the rapidly reproducing JH41 strain was supposed to be able to produce a protein at an enhanced rate. The fact that no extracellular protease activity or economical purification of secretory protein was found in C. glutamicum host ( Lee and Kim, 2018 ) further encouraged us to hypothesize that the rapidly reproducing JH41 strain would become an improved C. glutamicum host for secretory protein production. To verify this hypothesis, a plasmid designed to secrete foreign agarase (pCG-H36A-agarase) was introduced into JH41 and PT strains.…”

Section: Resultsmentioning

confidence: 99%

“…The transcriptome pattern of the evolved JH41 strain revealed that the upregulation of genes in the tricarboxylic acid cycle (TCA) cycle and respiratory chain, and oxidative stress responses allowed the growth promotion. Since C. glutamicum is a useful host for recombinant protein expression ( Lee and Kim, 2018 ), it was proposed that the fast-reproducing JH41 strain might be an improved host for recombinant protein production and may also provide hidden genetic targets for reverse engineering of C. glutamicum hosts. In this report, the fast-reproducing JH41 was indeed verified as an improved host for recombinant protein production.…”

Section: Introductionmentioning

confidence: 99%

Reverse Engineering Targets for Recombinant Protein Production in Corynebacterium glutamicum Inspired by a Fast-Growing Evolved Descendant

Lee

Park

et al. 2020

Front. Bioeng. Biotechnol.

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We previously reported a Corynebacterium glutamicum JH41 strain with a 58% faster growth rate through application of adaptive laboratory evolution. To verify that the fast-reproducing strain was useful as a host for recombinant protein expression, we introduced a plasmid responsible for the secretory production of a recombinant protein. The JH41 strain harboring the plasmid indeed produced the secretory recombinant protein at a 2.7-fold greater rate than its ancestral strain. To provide the reverse engineering targets responsible for boosting recombinant protein production and cell reproduction, we compared the genome sequence of the JH41 strain with its ancestral strain. Among the 15 genomic variations, a point mutation was confirmed in the 14 bases upstream of NCgl1959 (encoding a presumed siderophore-binding protein). This mutation allowed derepression of NCgl1959, thereby increasing iron consumption and ATP generation. A point mutation in the structural gene ramA (A239G), a LuxR-type global transcription regulator involved in central metabolism, allowed an increase in glucose consumption. Therefore, mutations to increase the iron and carbon consumption were concluded as being responsible for the enhanced production of recombinant protein and cell reproduction in the evolved host.

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“…It is an important industrial bacterium that is widely used for amino acid production, including glutamate and lysine [ 1 , 2 ]. Complete genomic DNA sequencing, understanding of bacterial metabolism and its regulation, and development of genetic tools for metabolic engineering have expanded the portfolio of bioproducts by C. glutamicum and have made this bacterium a promising platform for microbial production of valuable products, including organic acids, biofuel, proteins, commodity chemicals, and healthcare products [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

The ldhA Gene Encoding Fermentative l-Lactate Dehydrogenase in Corynebacterium Glutamicum Is Positively Regulated by the Global Regulator GlxR

Toyoda

Inui

2021

Microorganisms

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Bacterial metabolism shifts from aerobic respiration to fermentation at the transition from exponential to stationary growth phases in response to limited oxygen availability. Corynebacterium glutamicum, a Gram-positive, facultative aerobic bacterium used for industrial amino acid production, excretes L-lactate, acetate, and succinate as fermentation products. The ldhA gene encoding L-lactate dehydrogenase is solely responsible for L-lactate production. Its expression is repressed at the exponential phase and prominently induced at the transition phase. ldhA is transcriptionally repressed by the sugar-phosphate-responsive regulator SugR and L-lactate-responsive regulator LldR. Although ldhA expression is derepressed even at the exponential phase in the sugR and lldR double deletion mutant, a further increase in its expression is still observed at the stationary phase, implicating the action of additional transcription regulators. In this study, involvement of the cAMP receptor protein-type global regulator GlxR in the regulation of ldhA expression was investigated. The GlxR-binding site found in the ldhA promoter was modified to inhibit or enhance binding of GlxR. The ldhA promoter activity and expression of ldhA were altered in proportion to the binding affinity of GlxR. Similarly, L-lactate production was also affected by the binding site modification. Thus, GlxR was demonstrated to act as a transcriptional activator of ldhA.

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Recombinant Protein Expression System in Corynebacterium glutamicum and Its Application

Cited by 39 publications

References 104 publications

dMSCC: A microfluidic platform for microbial single-cell cultivation under dynamic environmental medium conditions

dMSCC: A microfluidic platform for microbial single-cell cultivation under dynamic environmental medium conditions

Reverse Engineering Targets for Recombinant Protein Production in Corynebacterium glutamicum Inspired by a Fast-Growing Evolved Descendant

The ldhA Gene Encoding Fermentative l-Lactate Dehydrogenase in Corynebacterium Glutamicum Is Positively Regulated by the Global Regulator GlxR

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