scholarly journals Incorporation of alternative amino acids into cyanophycin by different cyanophycin synthetases heterologously expressed in Corynebacterium glutamicum

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ramona Wördemann ◽  
Lars Wiefel ◽  
Volker F. Wendisch ◽  
Alexander Steinbüchel
Keyword(s):  
Amino Acids ◽  
Dry Weight ◽  
Growth Conditions ◽  
Amino Acid Biosynthesis ◽  
Water Soluble ◽  
Lysine Content ◽  
Polyaspartic Acid ◽  
Acid Biosynthesis ◽  
Cyanophycin Synthetase ◽  
Potential Precursor

AbstractCyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a biopolymer that could be used in various fields, for example, as a potential precursor for the synthesis of polyaspartic acid or for the production of CGP-derived dipeptides. To extend the applications of this polymer, it is therefore of interest to synthesize CGP with different compositions. A recent re-evaluation of the CGP synthesis in C. glutamicum has shown that C. glutamicum is a potentially interesting microorganism for CGP synthesis with a high content of alternative amino acids. This study shows that the amount of alternative amino acids can be increased by using mutants of C. glutamicum with altered amino acid biosynthesis. With the DM1729 mutant, the lysine content in the polymer could be increased up to 33.5 mol%. Furthermore, an ornithine content of up to 12.6 mol% was achieved with ORN2(Pgdh4). How much water-soluble or insoluble CGP is synthesized is strongly related to the used cyanophycin synthetase. CphADh synthesizes soluble CGP exclusively. However, soluble CGP could also be isolated from cells expressing CphA6308Δ1 or CphA6308Δ1_C595S in addition to insoluble CGP in all examined strains. The point mutation in CphA6308Δ1_C595S partially resulted in a higher lysine content. In addition, the CGP content could be increased to 36% of the cell dry weight under optimizing growth conditions in C. glutamicum ATCC13032. All known alternative major amino acids for CGP synthesis (lysine, ornithine, citrulline, and glutamic acid) could be incorporated into CGP in C. glutamicum.

Effects of an Electric Field of Sinusoidal Waves on the Amino Acid Biosynthesis by Azotobacter

1980 ◽  
Vol 35 (3-4) ◽  
pp. 258-261
Author(s):  
A. Martin Gonzalez ◽  
M. T. Izquierdo
Keyword(s):  
Amino Acids ◽  
Nitrogen Fixation ◽  
Amino Acid ◽  
Electric Field ◽  
Electric Fields ◽  
Culture Medium ◽  
Azotobacter Vinelandii ◽  
Amino Acid Biosynthesis ◽  
Medium Composition ◽  
Acid Biosynthesis

Abstract Electric Field Electric fields of sinusoidal waves have been applied in cultures of Azotobacter vinelandii, with potentials between 0 V and 10 V, intensities from 0 mA to 16 mA and frequencies between 5 Hz and 200 KHz. The influence of the electric field of sinusoidal waves on the nitrogen fixation on the post­ culture medium composition has a maximum at 5 V, 8 mA and 20 Hz. The rate of synthesis of specific amino acids by Azotobacter depends on the frequency and potential of the electric field applied. The concentration of each amino acid present in the post-culture medium is increased according to the electric field employed and the amino acid biosynthesis in culture medium is activated during the first days of incubation.

13C NMR analysis of Antarctic cryptogam extracts

1994 ◽  
Vol 6 (3) ◽  
pp. 295-305 ◽  
Author(s):  
B. E. Chapman ◽  
D. J. Roser ◽  
R. D. Seppelt
Keyword(s):  
Amino Acids ◽  
Dry Weight ◽  
Water Soluble ◽  
Soluble Carbohydrate ◽  
Common Amino Acid ◽  
13C Nmr Analysis ◽  
Prasiola Crispa ◽  
Wilkes Land ◽  
Snow Alga

Water soluble compounds were extracted from the dominant cryptogams of the Windmill Islands, Wilkes Land, and compared with standard polyols, sugars and amino acids using 13C nuclear magnetic resonance (NMR) spectroscopy. Previous findings for sugars and polyols from gas liquid chromatorgraphy were validated and extended. Arabitol, ribitol and mannitol were confirmed as the major soluble carbohydrate compounds in all lichen species examined. Sucrose, fructose and glucose, but no polyols were detected in two species of moss. Sorbitol was confirmed as a major component of the algae Prasiola crispa and Schizogonium murale. Mesotaenium bergrenii was confirmed to contain sucrose and glucose. No significant quantities of sugars or polyols or any other compound were found in extracts of the red snow alga Chloromonas sp.1. Amino acids were detected in the majority of cryptogam samples and were particularly abundant in the algae P. crispa and S. murale. In the latter species the total identified acids ranged from 13.5–66mg g-1 dry weight. In addition to the common amino acid components of proteins, betaine and γ-amino-butyric acid were detected, the latter being particularly abundant, being found widely in the moss, lichen and algae. Several unknown carbohydrates were characterized. Usnea sphacelata, U. antarctica and Pseudephebe minuscula contained a deoxy-hexitol, Grimmia antarctici contained resonance peaks consistent with a trisaccharide containing a sucrose moiety and Umbilicaria decussata possibly contained a glucose-arabitol dimer. 13C NMR was confirmed as a powerful tool for the characterization of low molecular weight constituents of Antarctic cryptogams.

The Gcn2–eIF2α pathway connects iron and amino acid homeostasis in Saccharomyces cerevisiae

2018 ◽  
Vol 475 (8) ◽  
pp. 1523-1534 ◽  
Author(s):  
Marcos Caballero-Molada ◽  
María D. Planes ◽  
Helena Benlloch ◽  
Sergio Atares ◽  
Miguel A. Naranjo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Iron Content ◽  
Amino Acid Biosynthesis ◽  
Initiation Factor ◽  
Acid Biosynthesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Iron Sulfur ◽  
Amino Acid Homeostasis

In eukaryotic cells, amino acid biosynthesis is feedback-inhibited by amino acids through inhibition of the conserved protein kinase Gcn2. This decreases phosphorylation of initiation factor eIF2α, resulting in general activation of translation but inhibition of translation of mRNA for transcription factor (TF) Gcn4 in yeast or ATF4 in mammals. These TFs are positive regulators of amino acid biosynthetic genes. As several enzymes of amino acid biosynthesis contain iron–sulfur clusters (ISCs) and iron excess is toxic, iron and amino acid homeostasis should be co-ordinated. Working with the yeast Saccharomyces cerevisiae, we found that amino acid supplementation down-regulates expression of genes for iron uptake and decreases intracellular iron content. This cross-regulation requires Aft1, the major TF activated by iron scarcity, as well as Gcn2 and phosphorylatable eIF2α but not Gcn4. A mutant with constitutive activity of Gcn2 (GCN2c) shows less repression of iron transport genes by amino acids and increased nuclear localization of Aft1 in an iron-poor medium, and increases iron content in this medium. As Aft1 is activated by depletion of mitochondrial ISCs, it is plausible that the Gcn2–eIF2α pathway inhibits the formation of these complexes. Accordingly, the GCN2c mutant has strongly reduced activity of succinate dehydrogenase, an iron–sulfur mitochondrial enzyme, and is unable to grow in media with very low iron or with galactose instead of glucose, conditions where formation of ISCs is specially needed. This mechanism adjusts the uptake of iron to the needs of amino acid biosynthesis and expands the list of Gcn4-independent activities of the Gcn2–eIF2α regulatory system.

Effect of sugars on amino acid biosynthesis in maize root tips

1970 ◽  
Vol 48 (1) ◽  
pp. 117-124 ◽  
Author(s):  
Ann Oaks ◽  
F. J. Johnson
Keyword(s):  
Amino Acids ◽  
Tricarboxylic Acid Cycle ◽  
Major Effect ◽  
Amino Acid Biosynthesis ◽  
Maize Root ◽  
Root Tips ◽  
Acid Biosynthesis ◽  
General Increase ◽  
Amino Butyric Acid ◽  
Protein Amino Acids

There is a general increase in incorporation of acetate carbon when excised maize root tips are aged for 3 h in a salts medium, but little further increase during a subsequent 3-h period. Addition of glucose or sucrose to the medium causes a further increase in the incorporation of acetate carbon into glutamic acid, glutamine, γ-amino butyric acid and into the protein amino acids, principal among these being glutamic, aspartic, proline, threonine, valine and the leucines. Sugars also cause a reduced incorporation of acetate carbon into asparagine. The results indicate that glucose (or sucrose) has a major effect in diverting carbon of the tricarboxylic acid cycle into glutamine and protein amino acids and away from asparagine.

scholarly journals GapMind: Automated Annotation of Amino Acid Biosynthesis

mSystems ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Morgan N. Price ◽  
Adam M. Deutschbauer ◽  
Adam P. Arkin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Building Blocks ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Alternative Pathways ◽  
Automated Annotation ◽  
A Genome ◽  
Minimal Media

ABSTRACT GapMind is a Web-based tool for annotating amino acid biosynthesis in bacteria and archaea (http://papers.genomics.lbl.gov/gaps). GapMind incorporates many variant pathways and 130 different reactions, and it analyzes a genome in just 15 s. To avoid error-prone transitive annotations, GapMind relies primarily on a database of experimentally characterized proteins. GapMind correctly handles fusion proteins and split proteins, which often cause errors for best-hit approaches. To improve GapMind’s coverage, we examined genetic data from 35 bacteria that grow in defined media without amino acids, and we filled many gaps in amino acid biosynthesis pathways. For example, we identified additional genes for arginine synthesis with succinylated intermediates in Bacteroides thetaiotaomicron, and we propose that Dyella japonica synthesizes tyrosine from phenylalanine. Nevertheless, for many bacteria and archaea that grow in minimal media, genes for some steps still cannot be identified. To help interpret potential gaps, GapMind checks if they match known gaps in related microbes that can grow in minimal media. GapMind should aid the identification of microbial growth requirements. IMPORTANCE Many microbes can make all of the amino acids (the building blocks of proteins). In principle, we should be able to predict which amino acids a microbe can make, and which it requires as nutrients, by checking its genome sequence for all of the necessary genes. However, in practice, it is difficult to check for all of the alternative pathways. Furthermore, new pathways and enzymes are still being discovered. We built an automated tool, GapMind, to annotate amino acid biosynthesis in bacterial and archaeal genomes. We used GapMind to list gaps: cases where a microbe makes an amino acid but a complete pathway cannot be identified in its genome. We used these gaps, together with data from mutants, to identify new pathways and enzymes. However, for most bacteria and archaea, we still do not know how they can make all of the amino acids.

scholarly journals Filling Gaps in Bacterial Amino Acid Biosynthesis Pathways with High-throughput Genetics

2017 ◽  
Author(s):  
Morgan N. Price ◽  
Grant M. Zane ◽  
Jennifer V. Kuehl ◽  
Ryan A. Melnyk ◽  
Judy D. Wall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
High Throughput ◽  
Heterotrophic Bacteria ◽  
Current Knowledge ◽  
Acid Synthesis ◽  
Amino Acid Biosynthesis ◽  
Desulfovibrio Vulgaris ◽  
Acid Biosynthesis ◽  
Amino Acid Synthesis

AbstractFor many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacteriumDesulfovibrio vulgarissynthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

scholarly journals The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Yasuyuki Yamamoto ◽  
Tamotsu Kanai ◽  
Tsuyoshi Kaneseki ◽  
Haruyuki Atomi
Keyword(s):  
Amino Acids ◽  
Transcriptional Regulation ◽  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Aromatic Amino Acid Biosynthesis

ABSTRACT TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcales aromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.

PATHWAYS FOR THE METABOLISM OF GLYOXYLATE AND ACETATE IN GERMINATING FATTY SEEDS

1965 ◽  
Vol 43 (9) ◽  
pp. 1531-1541 ◽  
Author(s):  
S. K. Sinha ◽  
E. A. Cossins
Keyword(s):  
Amino Acids ◽  
Castor Bean ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Amino Acid Biosynthesis ◽  
Acid Oxidation ◽  
Acid Biosynthesis ◽  
Tissue Slices ◽  
Acid Cycle ◽  
Pumpkin Seeds

Cotyledons of germinating sunflower, pumpkin, linseed, and watermelon seeds and the endosperm of germinating castor bean seeds have been examined for their ability to utilize glyoxylate-C14and acetate-C14for the biosynthesis of amino acids. All of the tissues examined readily utilized these acids when supplied in micromolar amounts to tissue slices. The chief products of this utilization included the organic acids of the glyoxylate and tricarboxylic acid cycles and a number of amino acids and amides. The results are interpreted as indicating that, in sunflower, watermelon, linseed, and pumpkin seeds, malate formed in the malate synthetase reaction is metabolized by the partial reactions of the tricarboxylic acid cycle. α-Ketoglutarate produced by these reactions is extensively utilized in the biosynthesis of glutamate, γ-aminobutyrate, and glutamine. In agreement with data already published, castor bean endosperm utilized acetate for the biosynthesis of sugars. This tissue also utilized glyoxylate for the formation of glycine, serine, glycollate, and malate. It is concluded that, with the exception of castor bean endosperm, acetyl CoA arising as a result of fatty acid oxidation might be utilized for amino acid biosynthesis via the partial reactions of the glyoxylate and tricarboxylic acid cycles.

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