scholarly journals Improvement of putrescine production through the arginine decarboxylase pathway in Escherichia coli K-12

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kullathida Thongbhubate ◽  
Kanako Irie ◽  
Yumi Sakai ◽  
Akane Itoh ◽  
Hideyuki Suzuki
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Batch Fermentation ◽  
Feedback Inhibition ◽  
Arginine Decarboxylase ◽  
Degradation Pathway ◽  
Gene Encoding ◽  
K 12 ◽  
Utilization Pathway

AbstractIn the bio-based polymer industry, putrescine is in the spotlight for use as a material. We constructed strains of Escherichia coli to assess its putrescine production capabilities through the arginine decarboxylase pathway in batch fermentation. N-Acetylglutamate (ArgA) synthase is subjected to feedback inhibition by arginine. Therefore, the 19th amino acid residue, Tyr, of argA was substituted with Cys to desensitize the feedback inhibition of arginine, resulting in improved putrescine production. The inefficient initiation codon GTG of argA was substituted with the effective ATG codon, but its replacement did not affect putrescine production. The essential genes for the putrescine production pathway, speA and speB, were cloned into the same plasmid with argAATG Y19C to form an operon. These genes were introduced under different promoters; lacIp, lacIqp, lacIq1p, and T5p. Among these, the T5 promoter demonstrated the best putrescine production. In addition, disruption of the puuA gene encoding enzyme of the first step of putrescine degradation pathway increased the putrescine production. Of note, putrescine production was not affected by the disruption of patA, which encodes putrescine aminotransferase, the initial enzyme of another putrescine utilization pathway. We also report that the strain KT160, which has a genomic mutation of YifEQ100TAG, had the greatest putrescine production. At 48 h of batch fermentation, strain KT160 grown in terrific broth with 0.01 mM IPTG produced 19.8 mM of putrescine.

scholarly journals Overproduction of l-Cysteine andl-Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase

1998 ◽  
Vol 64 (5) ◽  
pp. 1607-1611 ◽  
Author(s):  
Shigeru Nakamori ◽  
Shin-ichiro Kobayashi ◽  
Chitose Kobayashi ◽  
Hiroshi Takagi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Feedback Inhibition ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Serine Acetyltransferase ◽  
Methionine Residue ◽  
Cysteine Desulfhydrase ◽  
K 12

ABSTRACT Organisms that overproduced l-cysteine andl-cystine from glucose were constructed by usingEscherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg ofl-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production ofl-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production ofl-cysteine plus l-cystine was markedly increased compared to production in JM39.

scholarly journals Point Mutations in Transmembrane Helices 2 and 3 of ExbB and TolQ Affect Their Activities in Escherichia coli K-12

2004 ◽  
Vol 186 (13) ◽  
pp. 4402-4406 ◽  
Author(s):  
Volkmar Braun ◽  
Christina Herrmann
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Transmembrane Helices ◽  
The Third ◽  
Form Part ◽  
K 12

ABSTRACT Replacement of glutamate 176, the only charged amino acid in the third transmembrane helix of ExbB, with alanine (E176A) abolished ExbB activity in all determined ExbB-dependent functions of Escherichia coli. Combination of the mutations T148A in the second transmembrane helix and T181A in the third transmembrane helix, proposed to form part of a proton pathway through ExbB, also resulted in inactive ExbB. E176 and T148 are strictly conserved in ExbB and TolQ proteins, and T181 is almost strictly conserved in ExbB, TolQ, and MotA.

scholarly journals Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 880-890 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Toshiyuki Ishizuka ◽  
Shuhei Hotta ◽  
Michiko Aoki ◽  
Tomohiro Shimada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Cell Aggregation ◽  
Master Regulator ◽  
Gene Encoding ◽  
Content Type ◽  
Promoter Probe ◽  
K 12 ◽  
Specific Environmental Condition

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

scholarly journals Identification of the ubiD Gene on theEscherichia coli Chromosome

2000 ◽  
Vol 182 (21) ◽  
pp. 6243-6246 ◽  
Author(s):  
Haitao Zhang ◽  
George T. Javor
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Gene Product ◽  
Mutant Strain ◽  
Amino Acid Residue ◽  
Open Reading Frame ◽  
Reading Frame

ABSTRACT The open reading frame at 86.7 min on the Escherichia coli chromosome, “yigC,” complemented aubiD mutant strain, AN66, indicating that yigCis the ubiD gene. The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database.

scholarly journals Carboxy-Terminal Region Involved in Activity of Escherichia coli TolC

2001 ◽  
Vol 183 (23) ◽  
pp. 6961-6964 ◽  
Author(s):  
Hiroyasu Yamanaka ◽  
Hiroshi Izawa ◽  
Keinosuke Okamoto
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Outer Membrane ◽  
Amino Acid Residue ◽  
Terminal Region ◽  
Transport Activity ◽  
Carboxy Terminus ◽  
Partial Deletion ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

ABSTRACT The Escherichia coli TolC acts as a channel tunnel in the transport of various molecules across the outer membrane. Partial-deletion studies of tolC revealed that the region extending from the 50th to the 60th amino acid residue from the carboxy terminus plays an important role in this transport activity of TolC.

scholarly journals The Putrescine Importer PuuP of Escherichia coli K-12

2009 ◽  
Vol 191 (8) ◽  
pp. 2776-2782 ◽  
Author(s):  
Shin Kurihara ◽  
Yuichi Tsuboi ◽  
Shinpei Oda ◽  
Hyeon Guk Kim ◽  
Hidehiko Kumagai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Growth Phase ◽  
Exponential Growth ◽  
Minimal Medium ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Genes Encoding ◽  
K 12 ◽  
Utilization Pathway

ABSTRACT The Puu pathway is a putrescine utilization pathway involving gamma-glutamyl intermediates. The genes encoding the enzymes of the Puu pathway form a gene cluster, the puu gene cluster, and puuP is one of the genes in this cluster. In Escherichia coli, three putrescine importers, PotFGHI, PotABCD, and PotE, were discovered in the 1990s and have been studied; however, PuuP had not been discovered previously. This paper shows that PuuP is a novel putrescine importer whose kinetic parameters are equivalent to those of the polyamine importers discovered previously. A puuP + strain absorbed up to 5 mM putrescine from the medium, but a ΔpuuP strain did not. E. coli strain MA261 has been used in previous studies of polyamine transporters, but PuuP had not been identified previously. It was revealed that the puuP gene of MA261 was inactivated by a point mutation. When E. coli was grown on minimal medium supplemented with putrescine as the sole carbon or nitrogen source, only PuuP among the polyamine importers was required. puuP was expressed strongly when putrescine was added to the medium or when the puuR gene, which encodes a putative repressor, was deleted. When E. coli was grown in M9-tryptone medium, PuuP was expressed mainly in the exponential growth phase, and PotFGHI was expressed independently of the growth phase.

scholarly journals The arginine regulon of Escherichia coli: whole-system transcriptome analysis discovers new genes and provides an integrated view of arginine regulation

Microbiology ◽  
2006 ◽  
Vol 152 (11) ◽  
pp. 3343-3354 ◽  
Author(s):  
Marina Caldara ◽  
Daniel Charlier ◽  
Raymond Cunin
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Repression ◽  
Time Course ◽  
Feedback Inhibition ◽  
Uptake System ◽  
Mobility Shift ◽  
Real Time Quantitative Pcr ◽  
New Genes ◽  
Genes Encoding ◽  
K 12

Analysis of the response to arginine of the Escherichia coli K-12 transcriptome by microarray hybridization and real-time quantitative PCR provides the first coherent quantitative picture of the ArgR-mediated repression of arginine biosynthesis and uptake genes. Transcriptional repression was shown to be the major control mechanism of the biosynthetic genes, leaving only limited room for additional transcriptional or post-transcriptional regulation. The art genes, encoding the specific arginine uptake system, are subject to ArgR-mediated repression, with strong repression of artJ, encoding the periplasmic binding protein of the system. The hisJQMP genes of the histidine transporter (part of the lysine-arginine-ornithine uptake system) were discovered to be a part of the arginine regulon. Analysis of their control region with reporter gene fusions and electrophoretic mobility shift in the presence of pure ArgR repressor showed the involvement in repression of the ArgR protein and an ARG box 120 bp upstream of hisJ. No repression of the genes of the third uptake system, arginine-ornithine, was observed. Finally, comparison of the time course of arginine repression of gene transcription with the evolution of the specific activities of the cognate enzymes showed that while full genetic repression was achieved 2 min after arginine addition, enzyme concentrations were diluted at the rate of cell division. This emphasizes the importance of feedback inhibition of the first enzymic step in the pathway in controlling the metabolic flow through biosynthesis in the period following the onset of repression.

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