Activity of Exporters of Escherichia coli in Corynebacterium glutamicum, and Their Use to Increase L-Threonine Production

ABSTRACT The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by theilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and atdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.

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Characterization of salt-tolerant mutant for enhancement of l -threonine production in Escherichia coli

Applied Microbiology and Biotechnology ◽

10.1007/s002530000417 ◽

2000 ◽

Vol 54 (5) ◽

pp. 647-651 ◽

Cited By ~ 5

Author(s):

K.-H. Song ◽

H.-H. Lee ◽

H.-H. Hyun

Keyword(s):

Escherichia Coli ◽

Salt Tolerant ◽

Threonine Production ◽

Tolerant Mutant ◽

Salt Tolerant Mutant

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Formation and Metabolism of Methylmalonyl Coenzyme A in Corynebacterium glutamicum

Journal of Bacteriology ◽

10.1128/jb.01756-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2899-2901 ◽

Cited By ~ 4

Author(s):

Laure Botella ◽

Nic D. Lindley ◽

Lothar Eggeling

Keyword(s):

Escherichia Coli ◽

Corynebacterium Glutamicum ◽

Deletion Mutant ◽

Coenzyme A ◽

Growth Arrest ◽

Dry Weight ◽

Sequence Information ◽

Carboxylase Activity ◽

Genome Sequence Information ◽

Number Of Bacteria

ABSTRACT Genome sequence information suggests that B12-dependent mutases are present in a number of bacteria, including members of the suborder Corynebacterineae like Mycobacterium tuberculosis and Corynebacterium glutamicum. We here functionally identify a methylmalonyl coenzyme A (CoA) mutase in C. glutamicum that is retained in all of the members of the suborder Corynebacterineae and is encoded by NCgl1471, NCgl1472, and NCgl1470. In addition, we observe the presence of methylmalonate in C. glutamicum, reaching concentrations of up to 757 nmol g (dry weight)−1 in propionate-grown cells, whereas in Escherichia coli no methylmalonate was detectable. As demonstrated with a mutase deletion mutant, the presence of methylmalonate in C. glutamicum is independent of mutase activity but possibly due to propionyl-CoA carboxylase activity. During growth on propionate, increased mutase activity has severe cellular consequences, resulting in growth arrest and excretion of succinate. The physiological context of the mutase present in members of the suborder Corynebacterineae is discussed.

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Co-Expression of Threonine Dehydratase and Acetolactate Synthase in Escherichia Coli for L-Isoleucine Production

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.989-994.997 ◽

2014 ◽

Vol 989-994 ◽

pp. 997-1002 ◽

Cited By ~ 1

Author(s):

Jian Wang ◽

Jia Kai Sun ◽

Qing Yang Xu

Keyword(s):

Escherichia Coli ◽

Corynebacterium Glutamicum ◽

Carbon Flux ◽

Batch Fermentation ◽

Acetolactate Synthase ◽

Fed Batch ◽

Threonine Dehydratase ◽

E Coli ◽

Fed Batch Fermentation ◽

Fermentation Period

Metabolic engineering ofCorynebacterium glutamicumhas sought to divert carbon into L-isoleucine. However, the fermentation period of this strain is long. TheC.glutamicumYILW strain (LeuL, AHVr, SGr, Leu-MEr) was previously derived by repeated compound mutagenesis which could accumulate 20.2 g/L L-isoleucine in a 5-L jar fermentor. Overexpression of the threonine dehydratase gene (ilvA) fromCorynebacterium glutamicumYILW and coexpression of threonine dehydratase and acetolactate synthase (ilvBN) from it were employed to divert carbon flux toward L-isoleucine. The strainE. coliTRFC with the expression ofilvA could accumulate L-isoleucine of 6.8 g/L without accumulation of any L-threonine by fed-batch fermentation in a 5-L jar fermentor. However, the production of L-isoleucine by the strainE.coliTRFC with the co-expression ofilvA andilvBN was decreased by 19.1%, and the production of L-valine was increased by 40% compared with that ofE. coliTRFC with the expression ofilvA.

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Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production

Applied Microbiology and Biotechnology ◽

10.1007/s00253-010-2825-7 ◽

2010 ◽

Vol 88 (4) ◽

pp. 905-913 ◽

Cited By ~ 33

Author(s):

Jeong Wook Lee ◽

Sol Choi ◽

Jin Hwan Park ◽

Claudia E. Vickers ◽

Lars K. Nielsen ◽

...

Keyword(s):

Escherichia Coli ◽

Threonine Production ◽

K 12

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Overexpression of the ftsZ gene from Corynebacterium glutamicum (Brevibacterium lactofermentum) in Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/w04-105 ◽

2005 ◽

Vol 51 (1) ◽

pp. 85-89 ◽

Cited By ~ 1

Author(s):

María Pilar Honrubia-Marcos ◽

Angelina Ramos ◽

José A Gil

Keyword(s):

Escherichia Coli ◽

Corynebacterium Glutamicum ◽

Polyclonal Antibodies ◽

T7 Promoter ◽

Brevibacterium Lactofermentum ◽

E Coli ◽

Ftsz Protein ◽

Pure Protein ◽

Ftsz Gene ◽

Dna Deletions

Our goal in this work was to overexpress the essential cell division FtsZ protein from Corynebacterium glutamicum (Brevibacterium lactofermentum) (FtsZCG) in Escherichia coli to produce anti-FtsZCG polyclonal antibodies. Previous results from our laboratory showed that ftsZCG was not expressed in E. coli in a sufficient amount to purify FtsZCG. However, when ftsZCG (without upstream sequences) was transcriptionally fused to the T7 promoter, different truncated FtsZCG proteins (28–32 kDa) were overexpressed in E. coli, and in all cases, stop codons were created because of DNA deletions or rearrangements. Nevertheless, we were able to overexpress and purify an N-terminally hexa-His-tagged FtsZCG from uninduced E. coli cells carrying a pET-28a(+) derivative, yielding about 5 mg of 98% pure protein per 100-mL culture.Key words: Brevibacterium lactofermentum, Corynebacterium glutamicum, FtsZ overexpresssion, hexa-His-tagged FtsZ.

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