scholarly journals A Four-Step Enzymatic Cascade for Efficient Production of L-Phenylglycine from Biobased L-Phenylalanine

2022 ◽  
Author(s):  
Yuling Zhu ◽  
Jifeng Yuan
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Industrial Applications ◽  
Efficient Production ◽  
E Coli ◽  
Amycolatopsis Orientalis ◽  
Enzyme Cascade ◽  
Pharmaceutical Industries ◽  
Rate Limiting

Enantiopure amino acids are of particular interest in the agrochemical and pharmaceutical industries. Here, we reported a multi-enzyme cascade for efficient production of L-phenylglycine (L-Phg) from biobased L-phenylalanine (L-Phe). We first attempted to engineer Escherichia coli for expressing L-amino acid deaminase (LAAD) from Proteus mirabilis, hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis, (S)-mandelate dehydrogenase (SMDH) from Pseudomonas putida, the endogenous aminotransferase (AT) encoded by ilvE and L-glutamate dehydrogenase (GluDH) from E. coli. However, 10 mM L-Phe only afforded the synthesis of 7.21 mM L-Phg. The accumulation of benzoylformic acid suggested that the transamination step might be rate-limiting. We next used leucine dehydrogenase (LeuDH) from Bacillus cereus to bypass the use of L-glutamate as amine donor, and 40 mM L-Phe gave 39.97 mM (6.04 g/L) L-Phg, reaching 99.9% conversion. In summary, this work demonstrated a concise four-step enzymatic cascade for the L-Phg synthesis from biobased L-Phe, with a potential for future industrial applications.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

scholarly journals Enzymic synthesis of N-acetyl-l-phenylalanine in Escherichia coli K12

1971 ◽  
Vol 124 (5) ◽  
pp. 905-913 ◽  
Author(s):  
R. V. Krishna ◽  
P. R. Krishnaswamy ◽  
D. Rajagopal Rao
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
E Coli ◽  
Enzymic Synthesis ◽  
Escherichia Coli K12

1. Cell-free extracts of Escherichia coli K12 catalyse the synthesis of N-acetyl-l-phenylalanine from acetyl-CoA and l-phenylalanine. 2. The acetyl-CoA–l-phenylalanine α-N-acetyltransferase was purified 160-fold from cell-free extracts. 3. The enzyme has a pH optimum of 8 and catalyses the acetylation of l-phenylalanine. Other l-amino acids such as histidine and alanine are acetylated at slower rates. 4. A transacylase was also purified from E. coli extracts and its substrate specificity studied. 5. The properties of both these enzymes were compared with those of other known amino acid acetyltransferases and transacylases.

scholarly journals Amino acid deprivation and its effect on mating ability inEscherichia coliK12

1966 ◽  
Vol 8 (1) ◽  
pp. 115-118 ◽  
Author(s):  
K. W. Fisher
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Deprivation ◽  
Mating Ability ◽  
Chromosome Transfer ◽  
E Coli ◽  
Donor Cells ◽  
Relationship Of ◽  
The Relationship

The conclusion by Suit, Matney, Doudney & Billen (1964) that Hfr donor cells ofEscherichia coliK12, starved of required amino acids can mate, has been re-examined. It appears that their conclusion is not valid and that apparent fertility of amino-acid starved cells is due to cross-feeding by the F−cells. The relationship of this result to the alternative mechanisms for chromosome transfer inE. coliis discussed.

scholarly journals Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli

1995 ◽  
Vol 309 (2) ◽  
pp. 411-417 ◽  
Author(s):  
K V Lu ◽  
M F Rohde ◽  
A R Thomason ◽  
W C Kenney ◽  
H S Lu
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Growth Factor ◽  
Translation Termination ◽  
Termination Codon ◽  
Platelet Derived Growth Factor ◽  
E Coli ◽  
Translation Termination Codon ◽  
Terminal Amino

The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids. In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis. Expression of this DNA via a plasmid vector in E. coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids. Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E. coli should be avoided when possible.

Gene cloning, protein purification, and enzymatic properties of multicopper oxidase, fromKlebsiellasp. 601

2008 ◽  
Vol 54 (9) ◽  
pp. 725-733 ◽  
Author(s):  
Yang Li ◽  
Jiao Yin ◽  
Guosheng Qu ◽  
Luchao Lv ◽  
Yadong Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Kinetic Studies ◽  
Multicopper Oxidase ◽  
Coli Strain ◽  
Gene Encoding ◽  
E Coli ◽  
C Terminus ◽  
Optimal Ph

A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2′-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat/Kmvalues of 0.49 mmol·L–1, 1.08 × 103s–1, and 2.23 × 103s–1·mmol–1·L, respectively, for DMP, 5.63 mmol·L–1, 6.64 × 103s–1, and 1.18 × 103s–1·mmol–1·L for ABTS, and 0.023 mmol·L–1, 11 s–1, and 4.68 × 102s–1·mmol–1·L for SGZ.

scholarly journals Effect of Drug Transporter Genes on Cysteine Export and Overproduction in Escherichia coli

2006 ◽  
Vol 72 (7) ◽  
pp. 4735-4742 ◽  
Author(s):  
Satoshi Yamada ◽  
Naoki Awano ◽  
Kyoko Inubushi ◽  
Eri Maeda ◽  
Shigeru Nakamori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Feedback Inhibition ◽  
Tetracycline Resistance ◽  
Industrial Applications ◽  
Genetically Engineered ◽  
Drug Transporter ◽  
E Coli ◽  
Important Amino Acid ◽  
Gene Assay

ABSTRACT l-Cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of l-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in l-cysteine degradation, increased l-cysteine productivity in E. coli. The use of an l-cysteine efflux system could be promising for breeding l-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as l-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on l-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by l-cysteine. We also found that overexpression of these eight genes reduces intracellular l-cysteine levels after cultivation in the presence of l-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes l-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more l-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in l-cysteine export and overproduction in genetically engineered E. coli cells.

scholarly journals Different Rifampin Sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA Polymerases Are Not Explained by the Difference in the β-Subunit Rifampin Regions I and II

2005 ◽  
Vol 49 (4) ◽  
pp. 1587-1590 ◽  
Author(s):  
N. Zenkin ◽  
A. Kulbachinskiy ◽  
I. Bass ◽  
V. Nikiforov
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Β Subunit ◽  
E Coli ◽  
Increased Sensitivity ◽  
The Difference

ABSTRACT Mycobacterium tuberculosis RNA polymerase is 1,000-fold more sensitive to rifampin than Escherichia coli RNA polymerase. Chimeric E. coli RNA polymerase in which the β-subunit segment encompassing rifampin regions I and II (amino acids [aa] 463 through 590) was replaced with the corresponding region from M. tuberculosis (aa 382 through 509) did not show an increased sensitivity to the antibiotic. Thus, the difference in amino acid sequence between the rifampin regions I and II of the two species does not account for the difference in rifampin sensitivity of the two polymerases.

scholarly journals A multi-enzyme cascade for efficient production of D-p-hydroxyphenylglycine from L-tyrosine

2021 ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Chemical Synthesis ◽  
Specific Activity ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

Abstract In this study, we designed and in vivo reconstructed a novel four-enzyme cascade pathway for the production of D-HPG, a valuable intermediate used to produce β-lactam antibiotics and for fine-chemical synthesis, from L-tyrosine. In this pathway, we identified catalytic conversion of the substrate 4-hydroxyphenylglyoxylic acid by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg− 1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50 g/L L-tyrosine in 24 h with 92.5% conversion and > 99% ee in a 3-L fermenter, representing the highest reported D-HPG titer to date. This four-enzyme cascade provides a novel and effective enzymatic approach to industrial production of D-HPG from cheap amino acids.

scholarly journals The rfaE Gene from Escherichia coli Encodes a Bifunctional Protein Involved in Biosynthesis of the Lipopolysaccharide Core Precursor ADP-l-glycero-d-manno-Heptose

2000 ◽  
Vol 182 (2) ◽  
pp. 488-497 ◽  
Author(s):  
Miguel A. Valvano ◽  
Cristina L. Marolda ◽  
Mauricio Bittner ◽  
Mike Glaskin-Clay ◽  
Tania L. Simon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Inner Core ◽  
Structural Features ◽  
Predicted Amino Acid Sequence ◽  
E Coli ◽  
K 12 ◽  
Domain I

ABSTRACT The intermediate steps in the biosynthesis of the ADP-l-glycero-d-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement therfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation ofSalmonella enterica SL1102. DNA sequencing of therfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation inR. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains ofrfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis ofd-glycero-d-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-d-glycero-d-manno-heptose.

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