Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

2013 ◽  
Vol 40 (6) ◽  
pp. 643-651 ◽  
Author(s):  
Chuanzhi Zhang ◽  
Junli Zhang ◽  
Zhen Kang ◽  
Guocheng Du ◽  
Xiaobin Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Wild Type ◽  
Enhanced Production ◽  
Type Gene

scholarly journals Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production

1999 ◽  
Vol 65 (7) ◽  
pp. 3100-3107 ◽  
Author(s):  
S. Guillouet ◽  
A. A. Rodal ◽  
G.-H. An ◽  
P. A. Lessard ◽  
A. J. Sinskey
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Carbon Balance ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Original Activity ◽  
Threonine Dehydratase ◽  
Fourfold Increase ◽  
Overexpressing Strain

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.

scholarly journals Engineered Cyanophycin Synthetase (CphA) from Nostoc ellipsosporum Confers Enhanced CphA Activity and Cyanophycin Accumulation to Escherichia coli

2006 ◽  
Vol 72 (12) ◽  
pp. 7652-7660 ◽  
Author(s):  
Tran Hai ◽  
Kay M. Frey ◽  
Alexander Steinbüchel
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Dry Matter ◽  
Wild Type ◽  
Acinetobacter Baylyi ◽  
E Coli ◽  
Recombinant Cells ◽  
Hydrophobic Amino Acids ◽  
Type Gene ◽  
Nostoc Ellipsosporum

ABSTRACT The cyanophycin (CGP) synthetase gene (cphA NE1) of the transposon-induced argL mutant NE1 of the cyanobacterium Nostoc ellipsosporum, which exhibits a CGP-leaky phenotype during diazotrophical growth, was cloned and expressed in Escherichia coli strain TOP10. Its amino acid sequence exhibited high similarities to CphAs of other cyanobacteria. Recombinant cells of E. coli, which harbored a fragment comprising the complete cphA NE1 gene plus 400 bp of its downstream region in colinear orientation to the lacZ promoter, accumulated CGP up to 17 and 8.5% (wt/wt) of cellular dry matter (CDM) if cultivated in complex medium in the presence or absence of isopropyl-β-d-thiogalactopyranoside, respectively. Two truncated CphAs, lacking 31 (CphANE1del96) or 59 (CphANE1del180) amino acids of the C-terminal region, were derived from cphA NE1 by deleting 96 or 180 bp from its 3′ region through the introduction of stop codons. In comparison to the wild-type gene, cphA NE1del96 conferred about 2.1- to 2.2-fold-higher enzyme activity (up to 5.75 U/mg protein) on E. coli. Furthermore, these cells accumulated about twofold more CGP (up to 34.5% [wt/wt] of CDM) than cells expressing the wild-type gene. An engineered CphA possessing significantly enhanced activity and conferring the highest CGP content on E. coli is demonstrated. In contrast, CphANE1del180 was inactive and did not confer CGP accumulation on E. coli. Interestingly, a short conserved stretch of 4 to 5 hydrophobic amino acids is located in the protein region present in CphANE1del96 but absent in CphANE1del180. In addition, CphANE1 and CphANE1del96 are, besides CphA from Acinetobacter baylyi, the only CphAs exhibiting rigid substrate specificities that do not enable the incorporation of lysine instead of arginine into CGP.

scholarly journals Overexpression of the wild-type gene coding for Escherichia coli DNA adenine methylase (dam)

1992 ◽  
Vol 283 (3) ◽  
pp. 745-750 ◽  
Author(s):  
V U Nwosu
Keyword(s):  
Escherichia Coli ◽  
Cellular Protein ◽  
Wild Type ◽  
Recognition Sequence ◽  
Methyl Transfer ◽  
Gene Coding ◽  
Dna Adenine Methylase ◽  
Dam Methylase ◽  
Pcr Method ◽  
Type Gene

The gene coding for Escherichia coli dam methylase was isolated from a dam+ K12 strain by the PCR method. The gene was subcloned into an overexpression vector under the control of the strong lambda PL promoter. The resultant construct produced the dam methylase at about 20% of total cellular protein. Purification of the protein was achieved with two chromatography columns and yielded 6 mg of pure methylase per gram cell paste. The methylase readily methylates the synthetic dodecamer GACTGATCAGTC containing its recognition sequence (underlined). It also methylates a synthetic dodecamer containing the EcoRV recognition sequence GATATC. However, methyl transfer is to the second adenine in the EcoRV sequence.

scholarly journals Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4897-4903 ◽  
Author(s):  
Jong-il Choi ◽  
Sang Yup Lee ◽  
Kyuboem Han
Keyword(s):  
Escherichia Coli ◽  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Wild Type ◽  
High Productivity ◽  
E Coli ◽  
Alcaligenes Latus ◽  
Enhanced Production ◽  
Wide Range ◽  
High Level

ABSTRACT Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. RecombinantEscherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinantE. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. RecombinantE. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinantE. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.

scholarly journals Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

1998 ◽  
Vol 180 (11) ◽  
pp. 2931-2935 ◽  
Author(s):  
Toru Nakayashiki ◽  
Hachiro Inokuchi
Keyword(s):  
Escherichia Coli ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Suppressor Activity ◽  
Temperature Sensitive Mutants ◽  
Wobble Position ◽  
Group I ◽  
Group Ii ◽  
Type Gene

ABSTRACT Escherichia coli has only a single copy of a gene for tRNA6 Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O 2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4 Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O 2-methyluridine) as its anticodon, tRNA6 Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6 Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6 Leu. These tRNA6 Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaAgene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4 Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4 Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA6 Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6 Leu remains to be investigated.

scholarly journals Chaperone DnaJ Influences the Formation of Biofilm by Escherichia coli

2015 ◽  
Vol 64 (3) ◽  
pp. 279-283 ◽  
Author(s):  
Anna Grudniak ◽  
Jolanta Włodkowska ◽  
Krystyna Wolska
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Physiology ◽  
Wild Type ◽  
Type Gene ◽  
Biofilm Development

DnaJ chaperone, a member of the so called DnaK-DnaJ-GrpE chaperone machine plays an important role in cell physiology. The ability of Escherichia coli ΔdnaJ mutant to form biofilm was studied. It was shown that this mutant is impaired in biofilm development when exposed to 42°C for 2 h. The impairment in biofilm development was observed when the heat shock was applied either at the onset of biofilm formation or 2 h later. The biofilm formed was thinner and its structure was changed as compared to wild-type strain. This defect could be complemented by the introduction of a wild-type gene on a low-copy plasmid.

scholarly journals Membrane-Bound Division Proteins DivIB and DivIC ofBacillus subtilis Function Solely through Their External Domains in both Vegetative and Sporulation Division

1999 ◽  
Vol 181 (9) ◽  
pp. 2710-2718 ◽  
Author(s):  
Vittorio L. Katis ◽  
R. Gerry Wake
Keyword(s):  
Escherichia Coli ◽  
The Other ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Ofbacillus Subtilis ◽  
Transmembrane Segments ◽  
Terminal Domain ◽  
Division Site ◽  
Membrane Bound ◽  
Type Gene

ABSTRACT The Bacillus subtilis membrane-bound division proteins, DivIB and DivIC, each contain a single transmembrane segment flanked by a short cytoplasmic N-terminal domain and a larger external C-terminal domain. Both proteins become localized at the division site prior to septation. Mutagenesis of both divIB and divICwas performed whereby the sequences encoding the cytoplasmic domains were replaced by the corresponding sequence of the other gene. Finally, the cytoplasmic-plus-transmembrane-encoding domain of each protein was replaced by a totally foreign sequence not involved in division, that encodes the N-terminal-plus-transmembrane domains of theEscherichia coli TolR protein. B. subtilisstrains expressing the divIB and divIC hybrids, in the absence of the wild-type gene, were viable when grown under conditions in which the wild-type genes were found previously to be essential. Furthermore, these strains were able to sporulate to near normal levels. Thus, the cytoplasmic and transmembrane segments of DivIB and DivIC do not appear to have any specific functions other than to anchor these proteins correctly in the membrane. The implications of these findings are discussed.

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