scholarly journals Directed evolution of rRNA improves translation kinetics and recombinant protein yield

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fan Liu ◽  
Siniša Bratulić ◽  
Alan Costello ◽  
Teemu P. Miettinen ◽  
Ahmed H. Badran
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Bacterial Population ◽  
Protein Yield ◽  
Rrna Sequence ◽  
Evolutionary Trajectory ◽  
Noncanonical Amino Acids ◽  
Rate Limiting

AbstractIn bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

scholarly journals The effects of hyperphenylalaninaemia on the concentrations of aminoacyl-transfer ribonucleic acid in vivo. A mechanism for the inhibition of neural protein synthesis by phenylalanine

1977 ◽  
Vol 162 (3) ◽  
pp. 527-537 ◽  
Author(s):  
J V Hughes ◽  
T C Johnson
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Present Report ◽  
Periodate Oxidation ◽  
Essential Amino Acids ◽  
Acute Administration ◽  
Trna Species ◽  
Intracellular Concentrations ◽  
Rate Limiting

An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.

scholarly journals Aminoacyltransferase I-catalysed binding of phenylalanyl-transfer ribonucleic acid to muscle ribosomes from normal and diabetic rats

1971 ◽  
Vol 124 (3) ◽  
pp. 537-541 ◽  
Author(s):  
D. P. Leader ◽  
I. G. Wool ◽  
J. J. Castles
Keyword(s):  
Escherichia Coli ◽  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Ribonucleic Acid ◽  
Diabetic Rats ◽  
The Difference ◽  
Escherichia Coli B

The aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA (unfractionated Escherichia coli B tRNA acylated with radioactive phenylalanine and 19 non-radioactive amino acids) to skeletal-muscle ribosomes from diabetic rats was less than that to ribosomes from normal rats when the Mg2+ concentration was low (7.5mm); whereas just the reverse was true when the concentration of the cation was higher (15mm). Thus the Mg2+ dependency of aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA to ribosomes from normal and diabetic rats paralleled the effect of Mg2+ concentration on synthesis of polyphenylalanine reported before. During incubation at 7.5mm-Mg2+ phenylalanyl-tRNA was bound only to ribosomes bearing nascent peptidyl-tRNA. There are fewer such ribosomes in a preparation from the muscle of diabetic animals because diabetic animals synthesize less protein in vivo. Thus the difference in polyphenylalanine synthesis in vitro is adequately explained by the difference in enzyme-catalysed binding of phenylalanyl-tRNA to ribosomes, however, the basis of the difference in protein synthesis in vivo is still unknown.

scholarly journals LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

Genetics ◽  
1979 ◽  
Vol 91 (2) ◽  
pp. 215-227
Author(s):  
W Scott Champney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Chromosomal Locus ◽  
Ribosomal Subunits ◽  
Prolonged Incubation ◽  
Temperature Sensitive Mutants ◽  
Inhibition Of Growth ◽  
Localized Mutagenesis

ABSTRACT Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44°, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44°, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44°. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.

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 BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS

1972 ◽  
Vol 54 (2) ◽  
pp. 279-294 ◽  
Author(s):  
David C. Shephard ◽  
Wendy B. Levin
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Technical Problems ◽  
Hydrolyzed Protein ◽  
Protein Amino Acids ◽  
Amino Acid Labeling ◽  
Labeling Pattern

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with 14CO2 for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of 14CO2 into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with 14CO2 in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.

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